@article {3058, title = {Direct Observation of Electrically Conductive Pili Emanating from .}, journal = {mBio}, volume = {12}, year = {2021}, month = {2021 Aug 31}, pages = {e0220921}, abstract = {

Geobacter sulfurreducens is a model microbe for elucidating the mechanisms for extracellular electron transfer in several biogeochemical cycles, bioelectrochemical applications, and microbial metal corrosion. Multiple lines of evidence previously suggested that electrically conductive pili (e-pili) are an essential conduit for long-range extracellular electron transport in G. sulfurreducens. However, it has recently been reported that G. sulfurreducens does not express e-pili and that filaments comprised of multi-heme -type cytochromes are responsible for long-range electron transport. This possibility was directly investigated by examining cells, rather than filament preparations, with atomic force microscopy. Approximately 90\% of the filaments emanating from wild-type cells had a diameter (3 nm) and conductance consistent with previous reports of e-pili harvested from G. sulfurreducens or heterologously expressed in Escherichia coli from the G. sulfurreducens pilin gene. The remaining 10\% of filaments had a morphology consistent with filaments comprised of the -type cytochrome OmcS. A strain expressing a modified pilin gene designed to yield poorly conductive pili expressed 90\% filaments with a 3-nm diameter, but greatly reduced conductance, further indicating that the 3-nm diameter conductive filaments in the wild-type strain were e-pili. A strain in which genes for five of the most abundant outer-surface -type cytochromes, including OmcS, were deleted yielded only 3-nm-diameter filaments with the same conductance as in the wild type. These results demonstrate that e-pili are the most abundant conductive filaments expressed by G. sulfurreducens, consistent with previous functional studies demonstrating the need for e-pili for long-range extracellular electron transfer. Electroactive microbes have significant environmental impacts, as well as applications in bioenergy and bioremediation. The composition, function, and even existence of electrically conductive pili (e-pili) has been one of the most contentious areas of investigation in electromicrobiology, in part because e-pili offer a mechanism for long-range electron transport that does not involve the metal cofactors common in much of biological electron transport. This study demonstrates that e-pili are abundant filaments emanating from Geobacter sulfurreducens, which serves as a model for long-range extracellular electron transfer in direct interspecies electron transfer, dissimilatory metal reduction, microbe-electrode exchange, and corrosion caused by direct electron uptake from Fe(0). The methods described in this study provide a simple strategy for evaluating the distribution of conductive filaments throughout the microbial world with an approach that avoids artifactual production and/or enrichment of filaments that may not be physiologically relevant.

}, keywords = {Electric Conductivity, Electrons, Escherichia coli, Fimbriae Proteins, Fimbriae, Bacterial, Geobacter, Microscopy, Atomic Force, Oxidation-Reduction}, issn = {2150-7511}, doi = {10.1128/mBio.02209-21}, author = {Liu, Xinying and Walker, David J F and Nonnenmann, Stephen S and Sun, Dezhi and Lovley, Derek R} } @article {3070, title = {An Chassis for Production of Electrically Conductive Protein Nanowires.}, journal = {ACS Synth Biol}, volume = {9}, year = {2020}, month = {2020 Mar 20}, pages = {647-654}, abstract = {

pilin-based electrically conductive protein nanowires (e-PNs) are a revolutionary electronic material. They offer novel options for electronic sensing applications and have the remarkable ability to harvest electrical energy from atmospheric humidity. However, technical constraints limit mass cultivation and genetic manipulation of . Therefore, we designed a strain of to express e-PNs by introducing a plasmid that contained an inducible operon with genes for type IV pili biogenesis machinery and a synthetic gene designed to yield a peptide monomer that could be assembled into e-PNs. The e-PNs expressed in and harvested with a simple filtration method had the same diameter (3 nm) and conductance as e-PNs expressed in . These results, coupled with the robustness of for mass cultivation and the extensive toolbox for genetic manipulation, greatly expand the opportunities for large-scale fabrication of novel e-PNs.

}, keywords = {Electric Conductivity, Escherichia coli, Fimbriae Proteins, Fimbriae, Bacterial, Geobacter, Graphite, Microorganisms, Genetically-Modified, Microscopy, Atomic Force, Nanowires, Operon, Protein Engineering}, issn = {2161-5063}, doi = {10.1021/acssynbio.9b00506}, author = {Ueki, Toshiyuki and Walker, David J F and Woodard, Trevor L and Nevin, Kelly P and Nonnenmann, Stephen S and Lovley, Derek R} } @article {2620, title = {Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues.}, journal = {Chembiochem}, volume = {20}, year = {2019}, month = {2019 05 15}, pages = {1282-1291}, abstract = {

Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involves antigen 85 (Ag85)-catalyzed transfer of mycolic acids from the mycoloyl donor trehalose monomycolate (TMM) to acceptor carbohydrates and, in some organisms, proteins. We recently described an alkyne-modified TMM analogue (O-AlkTMM-C7) which, in conjunction with click chemistry, acted as a chemical reporter for mycoloylation in intact cells and allowed metabolic labeling of mycoloylated components of the mycomembrane. Here, we describe the synthesis and evaluation of a toolbox of TMM-based reporters bearing alkyne, azide, trans-cyclooctene, and fluorescent tags. These compounds gave further insight into the substrate tolerance of mycoloyltransferases (e.g., Ag85s) in a cellular context and they provide significantly expanded experimental versatility by allowing one- or two-step cell labeling, live cell labeling, and rapid cell labeling via tetrazine ligation. Such capabilities will facilitate research on mycomembrane composition, biosynthesis, and dynamics. Moreover, because TMM is exclusively metabolized by Corynebacterineae, the described probes may be valuable for the specific detection and cell-surface engineering of Mycobacterium tuberculosis and related pathogens. We also performed experiments to establish the dependence of probe incorporation on mycoloyltransferase activity, results from which suggested that cellular labeling is a function not only of metabolic incorporation (and likely removal) pathway(s), but also accessibility across the envelope. Thus, whole-cell labeling experiments with TMM reporters should be carefully designed and interpreted when envelope permeability may be compromised. On the other hand, this property of TMM reporters can potentially be exploited as a convenient way to probe changes in envelope integrity and permeability, facilitating drug development studies.

}, keywords = {Acyltransferases, Alkynes, Azides, Bacillus subtilis, Cell Engineering, Cell Membrane, Click Chemistry, Cord Factors, Corynebacterium, Escherichia coli, Fluorescent Dyes, Molecular Structure, Mycobacterium smegmatis, Mycobacterium tuberculosis}, issn = {1439-7633}, doi = {10.1002/cbic.201800687}, author = {Fiolek, Taylor J and Banahene, Nicholas and Kavunja, Herbert W and Holmes, Nathan J and Rylski, Adrian K and Pohane, Amol Arunrao and Siegrist, M Sloan and Swarts, Benjamin M} } @article {805, title = {Structure of the SSB-DNA polymerase III interface and its role in DNA replication.}, journal = {EMBO J}, volume = {30}, year = {2011}, month = {2011 Oct 19}, pages = {4236-47}, abstract = {Interactions between single-stranded DNA-binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB{\textquoteright}s association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. Here, the SSB-binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging-strand primer utilization is not supported. However, sequence changes in χ that block complex formation with SSB lead to salt-dependent uncoupling of leading- and lagging-strand DNA synthesis and to a surprising obstruction of the leading-strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature-dependent cell cycle defects that appear to arise from replisome instability.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Base Sequence, DNA Polymerase III, DNA Replication, DNA, Single-Stranded, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Holoenzymes, Molecular Sequence Data}, issn = {1460-2075}, doi = {10.1038/emboj.2011.305}, author = {Marceau, Aimee H and Bahng, Soon and Massoni, Shawn C and George, Nicholas P and Sandler, Steven J and Marians, Kenneth J and Keck, James L} } @article {734, title = {"Doubly selective" antimicrobial polymers: how do they differentiate between bacteria?}, journal = {Chemistry}, volume = {15}, year = {2009}, month = {2009 Nov 2}, pages = {11710-4}, abstract = {We have investigated how doubly selective synthetic mimics of antimicrobial peptides (SMAMPs), which can differentiate not only between bacteria and mammalian cells, but also between Gram-negative and Gram-positive bacteria, make the latter distinction. By dye-leakage experiments on model vesicles and complementary experiments on bacteria, we were able to relate the Gram selectivity to structural differences of these bacteria types. We showed that the double membrane of E. coli rather than the difference in lipid composition between E. coli and S. aureus was responsible for Gram selectivity. The molecular-weight-dependent antimicrobial activity of the SMAMPs was shown to be a sieving effect: while the 3000 g mol(-1) SMAMP was able to penetrate the peptidoglycan layer of the Gram-positive S. aureus bacteria, the 50000 g mol(-1) SMAMP got stuck and consequently did not have antimicrobial activity.}, keywords = {Anti-Infective Agents, Biomimetic Materials, Escherichia coli, Microbial Sensitivity Tests, Peptidoglycan, Polymers, Staphylococcus aureus}, issn = {1521-3765}, doi = {10.1002/chem.200802558}, author = {Lienkamp, Karen and Kumar, Kushi-Nidhi and Som, Abhigyan and N{\"u}sslein, Klaus and Tew, Gregory N} } @article {737, title = {Fast disinfecting antimicrobial surfaces.}, journal = {Langmuir}, volume = {25}, year = {2009}, month = {2009 Jan 20}, pages = {1060-7}, abstract = {Silicon wafers and glass surfaces were functionalized with facially amphiphilic antimicrobial copolymers using the "grafting from" technique. Surface-initiated atom transfer radical polymerization (ATRP) was used to grow poly(butylmethacrylate)-co-poly(Boc-aminoethyl methacrylate) from the surfaces. Upon Boc-deprotection, these surfaces became highly antimicrobial and killed S. aureus and E. coli 100\% in less than 5 min. The molecular weight and grafting density of the polymer were controlled by varying the polymerization time and initiator surface density. Antimicrobial studies showed that the killing efficiency of these surfaces was independent of polymer layer thickness or grafting density within the range of surfaces studied.}, keywords = {Anti-Bacterial Agents, Escherichia coli, Glass, Microbial Sensitivity Tests, Molecular Structure, Particle Size, Polymethacrylic Acids, Silicon, Staphylococcus aureus, Surface Properties}, issn = {0743-7463}, doi = {10.1021/la802953v}, author = {Madkour, Ahmad E and Dabkowski, Jeffery M and Nusslein, Klaus and Tew, Gregory N} } @article {809, title = {Single molecule analysis of a red fluorescent RecA protein reveals a defect in nucleoprotein filament nucleation that relates to its reduced biological functions.}, journal = {J Biol Chem}, volume = {284}, year = {2009}, month = {2009 Jul 10}, pages = {18664-73}, abstract = {Fluorescent fusion proteins are exceedingly useful for monitoring protein localization in situ or visualizing protein behavior at the single molecule level. Unfortunately, some proteins are rendered inactive by the fusion. To circumvent this problem, we fused a hyperactive RecA protein (RecA803 protein) to monomeric red fluorescent protein (mRFP1) to produce a functional protein (RecA-RFP) that is suitable for in vivo and in vitro analysis. In vivo, the RecA-RFP partially restores UV resistance, conjugational recombination, and SOS induction to recA(-) cells. In vitro, the purified RecA-RFP protein forms a nucleoprotein filament whose k(cat) for single-stranded DNA-dependent ATPase activity is reduced approximately 3-fold relative to wild-type protein, and which is largely inhibited by single-stranded DNA-binding protein. However, RecA protein is also a dATPase; dATP supports RecA-RFP nucleoprotein filament formation in the presence of single-stranded DNA-binding protein. Furthermore, as for the wild-type protein, the activities of RecA-RFP are further enhanced by shifting the pH to 6.2. As a consequence, RecA-RFP is proficient for DNA strand exchange with dATP or at lower pH. Finally, using single molecule visualization, RecA-RFP was seen to assemble into a continuous filament on duplex DNA, and to extend the DNA approximately 1.7-fold. Consistent with its attenuated activities, RecA-RFP nucleates onto double-stranded DNA approximately 3-fold more slowly than the wild-type protein, but still requires approximately 3 monomers to form the rate-limited nucleus needed for filament assembly. Thus, RecA-RFP reveals that its attenuated biological functions correlate with a reduced frequency of nucleoprotein filament nucleation at the single molecule level.}, keywords = {Cell Nucleus, DNA, DNA, Single-Stranded, Escherichia coli, Hydrogen-Ion Concentration, Kinetics, Luminescent Proteins, Nucleoproteins, Plasmids, Protein Binding, Rec A Recombinases, Recombination, Genetic, Sensitivity and Specificity, Ultraviolet Rays}, issn = {0021-9258}, doi = {10.1074/jbc.M109.004895}, author = {Handa, Naofumi and Amitani, Ichiro and Gumlaw, Nathan and Sandler, Steven J and Kowalczykowski, Stephen C} } @article {808, title = {Suppression of constitutive SOS expression by recA4162 (I298V) and recA4164 (L126V) requires UvrD and RecX in Escherichia coli K-12.}, journal = {Mol Microbiol}, volume = {73}, year = {2009}, month = {2009 Jul}, pages = {226-39}, abstract = {Sensing DNA damage and initiation of genetic responses to repair DNA damage are critical to cell survival. In Escherichia coli, RecA polymerizes on ssDNA produced by DNA damage creating a RecA-DNA filament that interacts with the LexA repressor inducing the SOS response. RecA filament stability is negatively modulated by RecX and UvrD. recA730 (E38K) and recA4142 (F217Y) constitutively express the SOS response. recA4162 (I298V) and recA4164 (L126V) are intragenic suppressors of the constitutive SOS phenotype of recA730. Herein, it is shown that these suppressors are not allele specific and can suppress SOS(C) expression of recA730 and recA4142 in cis and in trans. recA4162 and recA4164 single mutants (and the recA730 and recA4142 derivatives) are Rec(+), UV(R) and are able to induce the SOS response after UV treatment like wild-type. UvrD and RecX are required for the suppression in two (recA730,4164 and recA4142,4162) of the four double mutants tested. To explain the data, one model suggests that recA(C) alleles promote SOS(C) expression by mimicking RecA filament structures that induce SOS and the suppressor alleles mimic RecA filament at end of SOS. UvrD and RecX are attracted to these latter structures to help dismantle or destabilize the RecA filament.}, keywords = {Alleles, DNA Helicases, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Mutation, Missense, Rec A Recombinases, SOS Response (Genetics), Substrate Specificity}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2009.06765.x}, author = {Long, Jarukit E and Renzette, Nicholas and Sandler, Steven J} } @article {393, title = {Two functions of the C-terminal domain of Escherichia coli Rob: mediating "sequestration-dispersal" as a novel off-on switch for regulating Rob{\textquoteright}s activity as a transcription activator and preventing degradation of Rob by Lon protease.}, journal = {J Mol Biol}, volume = {388}, year = {2009}, month = {2009 May 8}, pages = {415-30}, abstract = {In Escherichia coli, Rob activates transcription of the SoxRS/MarA/Rob regulon. Previous work revealed that Rob resides in three to four immunostainable foci, that dipyridyl and bile salts are inducers of its activity, and that inducers bind to Rob{\textquoteright}s C-terminal domain (CTD). We propose that sequestration inactivates Rob by blocking its access to the transcriptional machinery and that inducers activate Rob by mediating its dispersal, allowing interaction with RNA polymerase. To test "sequestration-dispersal" as a new mechanism for regulating the activity of transcriptional activators, we fused Rob{\textquoteright}s CTD to SoxS and used indirect immunofluorescence microscopy to determine the effect of inducers on SoxS-Rob{\textquoteright}s cellular localization. Unlike native SoxS, which is uniformly distributed throughout the cell, SoxS-Rob is sequestered without an inducer, but is rapidly dispersed when cells are treated with an inducer. In this manner, Rob{\textquoteright}s CTD serves as an anti-sigma factor in regulating the co-sigma-factor-like activity of SoxS when fused to it. Rob{\textquoteright}s CTD also protects its N-terminus from Lon protease, since Lon{\textquoteright}s normally rapid degradation of SoxS is blocked in the chimera. Accordingly, Rob{\textquoteright}s CTD has novel regulatory properties that can be bestowed on another E. coli protein.}, keywords = {Decanoic Acids, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Reporter, Microscopy, Fluorescence, Models, Biological, Protease La, Protein Structure, Tertiary, Pyridines, Recombinant Fusion Proteins, Trans-Activators, Transcription, Genetic}, issn = {1089-8638}, doi = {10.1016/j.jmb.2009.03.023}, author = {Griffith, Kevin L and Fitzpatrick, M Megan and Keen, Edward F and Wolf, Richard E} } @article {742, title = {Antimicrobial polymers prepared by ROMP with unprecedented selectivity: a molecular construction kit approach.}, journal = {J Am Chem Soc}, volume = {130}, year = {2008}, month = {2008 Jul 30}, pages = {9836-43}, abstract = {Synthetic Mimics of Antimicrobial Peptides (SMAMPs) imitate natural host-defense peptides, a vital component of the body{\textquoteright}s immune system. This work presents a molecular construction kit that allows the easy and versatile synthesis of a broad variety of facially amphiphilic oxanorbornene-derived monomers. Their ring-opening metathesis polymerization (ROMP) and deprotection provide several series of SMAMPs. Using amphiphilicity, monomer feed ratio, and molecular weight as parameters, polymers with 533 times higher selectivitiy (selecitviy = hemolytic concentration/minimum inhibitory concentration) for bacteria over mammalian cells were discovered. Some of these polymers were 50 times more selective for Gram-positive over Gram-negative bacteria while other polymers surprisingly showed the opposite preference. This kind of "double selectivity" (bacteria over mammalian and one bacterial type over another) is unprecedented in other polymer systems and is attributed to the monomer{\textquoteright}s facial amphiphilicity.}, keywords = {Anti-Infective Agents, Antimicrobial Cationic Peptides, Biomimetic Materials, Erythrocytes, Escherichia coli, Hemolysis, Humans, Hydrophobic and Hydrophilic Interactions, Microbial Sensitivity Tests, Molecular Weight, Norbornanes, Polymers, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Staphylococcus aureus, Structure-Activity Relationship}, issn = {1520-5126}, doi = {10.1021/ja801662y}, author = {Lienkamp, Karen and Madkour, Ahmad E and Musante, Ashlan and Nelson, Christopher F and N{\"u}sslein, Klaus and Tew, Gregory N} } @article {470, title = {Comparative genomics of Geobacter chemotaxis genes reveals diverse signaling function.}, journal = {BMC Genomics}, volume = {9}, year = {2008}, month = {2008}, pages = {471}, abstract = {BACKGROUND: Geobacter species are delta-Proteobacteria and are often the predominant species in a variety of sedimentary environments where Fe(III) reduction is important. Their ability to remediate contaminated environments and produce electricity makes them attractive for further study. Cell motility, biofilm formation, and type IV pili all appear important for the growth of Geobacter in changing environments and for electricity production. Recent studies in other bacteria have demonstrated that signaling pathways homologous to the paradigm established for Escherichia coli chemotaxis can regulate type IV pili-dependent motility, the synthesis of flagella and type IV pili, the production of extracellular matrix material, and biofilm formation. The classification of these pathways by comparative genomics improves the ability to understand how Geobacter thrives in natural environments and better their use in microbial fuel cells. RESULTS: The genomes of G. sulfurreducens, G. metallireducens, and G. uraniireducens contain multiple (approximately 70) homologs of chemotaxis genes arranged in several major clusters (six, seven, and seven, respectively). Unlike the single gene cluster of E. coli, the Geobacter clusters are not all located near the flagellar genes. The probable functions of some Geobacter clusters are assignable by homology to known pathways; others appear to be unique to the Geobacter sp. and contain genes of unknown function. We identified large numbers of methyl-accepting chemotaxis protein (MCP) homologs that have diverse sensing domain architectures and generate a potential for sensing a great variety of environmental signals. We discuss mechanisms for class-specific segregation of the MCPs in the cell membrane, which serve to maintain pathway specificity and diminish crosstalk. Finally, the regulation of gene expression in Geobacter differs from E. coli. The sequences of predicted promoter elements suggest that the alternative sigma factors sigma28 and sigma54 play a role in regulating the Geobacter chemotaxis gene expression. CONCLUSION: The numerous chemoreceptors and chemotaxis-like gene clusters of Geobacter appear to be responsible for a diverse set of signaling functions in addition to chemotaxis, including gene regulation and biofilm formation, through functionally and spatially distinct signaling pathways.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Chemotaxis, Escherichia coli, Gene Expression Regulation, Bacterial, Genome, Bacterial, Genomics, Geobacter, Membrane Proteins, Molecular Sequence Data, Multigene Family, Promoter Regions, Genetic, Sequence Alignment, Sequence Homology, Amino Acid}, issn = {1471-2164}, doi = {10.1186/1471-2164-9-471}, author = {Tran, Hoa T and Krushkal, Julia and Antommattei, Frances M and Lovley, Derek R and Weis, Robert M} } @article {472, title = {Highly conserved genes in Geobacter species with expression patterns indicative of acetate limitation.}, journal = {Microbiology}, volume = {154}, year = {2008}, month = {2008 Sep}, pages = {2589-99}, abstract = {Analysis of the genome of Geobacter sulfurreducens revealed four genes encoding putative symporters with homology to ActP, an acetate transporter in Escherichia coli. Three of these genes, aplA, aplB and aplC, are highly similar (over 90 \% identical) and fell within a tight phylogenetic cluster (Group I) consisting entirely of Geobacter homologues. Transcript levels for all three genes increased in response to acetate limitation. The fourth gene, aplD, is phylogenetically distinct (Group II) and its expression was not influenced by acetate availability. Deletion of any one of the three genes in Group I did not significantly affect acetate-dependent growth, suggesting functional redundancy. Attempts to recover mutants in which various combinations of two of these genes were deleted were unsuccessful, suggesting that at least two of these three transporter genes are required to support growth. Closely related Group I apl genes were found in the genomes of other Geobacter species whose genome sequences are available. Furthermore, related genes could be detected in genomic DNA extracted from a subsurface environment undergoing in situ uranium bioremediation. The transporter genes recovered from the subsurface were most closely related to Group I apl genes found in the genomes of cultured Geobacter species that were isolated from contaminated subsurface environments. The increased expression of these genes in response to acetate limitation, their high degree of conservation among Geobacter species and the ease with which they can be detected in environmental samples suggest that Group I apl genes of the Geobacteraceae may be suitable biomarkers for acetate limitation. Monitoring the expression of these genes could aid in the design of strategies for acetate-mediated in situ bioremediation of uranium-contaminated groundwater.}, keywords = {Acetates, Biodegradation, Environmental, DNA, Bacterial, Escherichia coli, Escherichia coli Proteins, Gene Deletion, Gene Expression, Genes, Bacterial, Genome, Bacterial, Geobacter, Membrane Transport Proteins, Phylogeny, Uranium}, issn = {1350-0872}, doi = {10.1099/mic.0.2008/017244-0}, author = {Risso, Carla and Meth{\'e}, Barbara A and Elifantz, Hila and Holmes, Dawn E and Lovley, Derek R} } @article {394, title = {Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP.}, journal = {Mol Microbiol}, volume = {70}, year = {2008}, month = {2008 Nov}, pages = {1012-25}, abstract = {The ability to manipulate protein levels is useful for dissecting regulatory pathways, elucidating gene function and constructing synthetic biological circuits. We engineered an inducible protein degradation system for use in Bacillus subtilis based on Escherichia coli and Caulobacter crescentusssrA tags and SspB adaptors that deliver proteins to ClpXP for proteolysis. In this system, modified ssrA degradation tags are fused onto the 3{\textquoteright} end of the genes of interest. Unlike wild-type ssrA, these modified tags require the adaptor protein SspB to target tagged proteins for proteolysis. In the absence of SspB, the tagged proteins accumulate to near physiological levels. By inducing SspB expression from a regulated promoter, the tagged substrates are rapidly delivered to the B. subtilis ClpXP protease for degradation. We used this system to degrade the reporter GFP and several native B. subtilis proteins, including, the transcription factor ComA, two sporulation kinases (KinA, KinB) and the sporulation and chromosome partitioning protein Spo0J. We also used modified E. coli and C. crescentus ssrA tags to independently control the degradation of two different proteins in the same cell. These tools will be useful for studying biological processes in B. subtilis and can potentially be modified for use in other bacteria.}, keywords = {Alleles, Bacillus subtilis, Bacterial Proteins, Carrier Proteins, Caulobacter crescentus, DNA-Binding Proteins, Endopeptidase Clp, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genetic Vectors, Green Fluorescent Proteins, Plasmids, Promoter Regions, Genetic, Protein Engineering, RNA, Bacterial, Substrate Specificity}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2008.06467.x}, author = {Griffith, Kevin L and Grossman, Alan D} } @article {741, title = {Investigating the effect of increasing charge density on the hemolytic activity of synthetic antimicrobial polymers.}, journal = {Biomacromolecules}, volume = {9}, year = {2008}, month = {2008 Oct}, pages = {2805-10}, abstract = {The current study is aimed at investigating the effect of fine-tuning the cationic character of synthetic mimics of antimicrobial peptides (SMAMPs) on the hemolytic and antibacterial activities. A series of novel norbornene monomers that carry one, two, or three Boc-protected amine functionalities was prepared. Ring-opening metathesis polymerization (ROMP) of the monomers, followed by deprotection of the amine groups resulted in cationic antimicrobial polynorbornenes that carry one, two, and three charges per monomer repeat unit. Increasing the number of amine groups on the most hydrophobic polymer reduced its hemolytic activity significantly. To understand the membrane activity of these polymers, we conducted dye leakage experiments on lipid vesicles that mimic bacteria and red blood cell membranes, and these results showed a strong correlation with the hemolysis data.}, keywords = {Amines, Anti-Infective Agents, Antimicrobial Cationic Peptides, Chemistry, Pharmaceutical, Drug Design, Erythrocytes, Escherichia coli, Hemolysis, Humans, Lipids, Magnetic Resonance Spectroscopy, Models, Chemical, Peptides, Polymers, Staphylococcus aureus}, issn = {1526-4602}, doi = {10.1021/bm800569x}, author = {Al-Badri, Zoha M and Som, Abhigyan and Lyon, Sarah and Nelson, Christopher F and N{\"u}sslein, Klaus and Tew, Gregory N} } @article {812, title = {RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis.}, journal = {Mol Microbiol}, volume = {69}, year = {2008}, month = {2008 Sep}, pages = {1165-79}, abstract = {The Escherichia coli SOS response to DNA damage is modulated by the RecA protein, a recombinase that forms an extended filament on single-stranded DNA and hydrolyzes ATP. The RecA K72R (recA2201) mutation eliminates the ATPase activity of RecA protein. The mutation also limits the capacity of RecA to form long filaments in the presence of ATP. Strains with this mutation do not undergo SOS induction in vivo. We have combined the K72R variant of RecA with another mutation, RecA E38K (recA730). In vitro, the double mutant RecA E38K/K72R (recA730,2201) mimics the K72R mutant protein in that it has no ATPase activity. The double mutant protein will form long extended filaments on ssDNA and facilitate LexA cleavage almost as well as wild-type, and do so in the presence of ATP. Unlike recA K72R, the recA E38K/K72R double mutant promotes SOS induction in vivo after UV treatment. Thus, SOS induction does not require ATP hydrolysis by the RecA protein, but does require formation of extended RecA filaments. The RecA E38K/K72R protein represents an improved reagent for studies of the function of ATP hydrolysis by RecA in vivo and in vitro.}, keywords = {Adenosine Triphosphate, Amino Acid Substitution, Bacterial Proteins, DNA, Bacterial, DNA, Single-Stranded, Escherichia coli, Escherichia coli Proteins, Hydrolysis, Rec A Recombinases, Serine Endopeptidases, SOS Response (Genetics), Ultraviolet Rays}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2008.06341.x}, author = {Gruenig, Marielle C and Renzette, Nicholas and Long, Edward and Chitteni-Pattu, Sindhu and Inman, Ross B and Cox, Michael M and Sandler, Steven J} } @article {813, title = {Requirements for ATP binding and hydrolysis in RecA function in Escherichia coli.}, journal = {Mol Microbiol}, volume = {67}, year = {2008}, month = {2008 Mar}, pages = {1347-59}, abstract = {RecA is essential for recombination, DNA repair and SOS induction in Escherichia coli. ATP hydrolysis is known to be important for RecA{\textquoteright}s roles in recombination and DNA repair. In vitro reactions modelling SOS induction minimally require ssDNA and non-hydrolyzable ATP analogues. This predicts that ATP hydrolysis will not be required for SOS induction in vivo. The requirement of ATP binding and hydrolysis for SOS induction in vivo is tested here through the study of recA4159 (K72A) and recA2201 (K72R). RecA4159 is thought to have reduced affinity for ATP. RecA2201 binds, but does not hydrolyse ATP. Neither mutant was able to induce SOS expression after UV irradiation. RecA2201, unlike RecA4159, could form filaments on DNA and storage structures as measured with RecA-GFP. RecA2201 was able to form hybrid filaments and storage structures and was either recessive or dominant to RecA(+), depending on the ratio of the two proteins. RecA4159 was unable to enter RecA(+) filaments on DNA or storage structures and was recessive to RecA(+). It is concluded that ATP hydrolysis is essential for SOS induction. It is proposed that ATP binding is essential for storage structure formation and ability to interact with other RecA proteins in a filament.}, keywords = {Adenosine Triphosphate, Chromosomes, Bacterial, Escherichia coli, Escherichia coli Proteins, Hydrolysis, Mutation, Protein Binding, Rec A Recombinases, Ultraviolet Rays}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2008.06130.x}, author = {Renzette, Nicholas and Sandler, Steven J} } @article {818, title = {DinI and RecX modulate RecA-DNA structures in Escherichia coli K-12.}, journal = {Mol Microbiol}, volume = {63}, year = {2007}, month = {2007 Jan}, pages = {103-15}, abstract = {RecA plays a central role in recombination, DNA repair and SOS induction through forming a RecA-DNA helical filament. Biochemical observations show that at low ratios to RecA, DinI and RecX stabilize and destabilize RecA-DNA filaments, respectively, and that the C-terminal 17 residues of RecA are important for RecX function. RecA-DNA filament formation was assayed in vivo using RecA-GFP foci formation in log-phase and UV-irradiated cells. In log-phase cells, dinI mutants have fewer foci than wild type and that recX mutants have more foci than wild type. A recADelta17::gfp mutant had more foci like a recX mutant. dinI recX double mutants have the same number of foci as dinI mutants alone, suggesting that dinI is epistatic to recX. After UV treatment, the dinI, recX and dinI recX mutants differed in their ability to form foci. All three mutants had fewer foci than wild type. The dinI mutant{\textquoteright}s foci persisted longer than wild-type foci. Roles of DinI and RecX after UV treatment differed from those during log-phase growth and may reflect the different DNA substrates, population of proteins or amounts during the SOS response. These experiments give new insight into the roles of these proteins.}, keywords = {Bacterial Proteins, DNA, Bacterial, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Bacterial, Rec A Recombinases}, issn = {0950-382X}, doi = {10.1111/j.1365-2958.2006.05496.x}, author = {Renzette, Nicholas and Gumlaw, Nathan and Sandler, Steven J} } @article {816, title = {A hand-off mechanism for primosome assembly in replication restart.}, journal = {Mol Cell}, volume = {26}, year = {2007}, month = {2007 Jun 22}, pages = {781-93}, abstract = {Collapsed DNA replication forks must be reactivated through origin-independent reloading of the replication machinery (replisome) to ensure complete duplication of cellular genomes. In E. coli, the PriA-dependent pathway is the major replication restart mechanism and requires primosome proteins PriA, PriB, and DnaT for replisome reloading. However, the molecular mechanisms that regulate origin-independent replisome loading are not fully understood. Here, we demonstrate that assembly of primosome protein complexes represents a key regulatory mechanism, as inherently weak PriA-PriB and PriB-DnaT interactions are strongly stimulated by single-stranded DNA. Furthermore, the binding site on PriB for single-stranded DNA partially overlaps the binding sites for PriA and DnaT, suggesting a dynamic primosome assembly process in which single-stranded DNA is handed off from one primosome protein to another as a repaired replication fork is reactivated. This model helps explain how origin-independent initiation of DNA replication is restricted to repaired replication forks, preventing overreplication of the genome.}, keywords = {Binding Sites, DNA Helicases, DNA Replication, DNA, Bacterial, DNA, Single-Stranded, DNA-Binding Proteins, DNA-Directed DNA Polymerase, Escherichia coli, Escherichia coli Proteins, Genome, Bacterial, Models, Biological, Multienzyme Complexes, Protein Binding, Replication Origin}, issn = {1097-2765}, doi = {10.1016/j.molcel.2007.05.012}, author = {Lopper, Matthew and Boonsombat, Ruethairat and Sandler, Steven J and Keck, James L} } @article {524, title = {Characterization of metabolism in the Fe(III)-reducing organism Geobacter sulfurreducens by constraint-based modeling.}, journal = {Appl Environ Microbiol}, volume = {72}, year = {2006}, month = {2006 Feb}, pages = {1558-68}, abstract = {Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.}, keywords = {Amino Acids, Electron Transport, Escherichia coli, Fumarates, Geobacter, Iron, Models, Biological, Mutation, Oxidation-Reduction, Phenotype, Protons, Quinones, Species Specificity}, issn = {0099-2240}, doi = {10.1128/AEM.72.2.1558-1568.2006}, author = {Mahadevan, R and Bond, D R and Butler, J E and Esteve-Nu{\~n}ez, A and Coppi, M V and Palsson, B O and Schilling, C H and Lovley, D R} } @article {512, title = {Computational prediction of RpoS and RpoD regulatory sites in Geobacter sulfurreducens using sequence and gene expression information.}, journal = {Gene}, volume = {384}, year = {2006}, month = {2006 Dec 15}, pages = {73-95}, abstract = {RpoS, the sigma S subunit of RNA polymerase, is vital during the growth and survival of Geobacter sulfurreducens under conditions typically encountered in its native subsurface environments. We investigated the conservation of sites that may be important for RpoS function in G. sulfurreducens. We also employed sequence information and expression microarray data to predict G. sulfurreducens genome sites that may be related to RpoS regulation. Hierarchical clustering identified three clusters of significantly downregulated genes in the rpoS deletion mutant. The search for conserved overrepresented motifs in co-regulated operons identified likely -35 and -10 promoter elements upstream of a number of functionally important G. sulfurreducens operons that were downregulated in the rpoS deletion mutant. Putative -35/-10 promoter elements were also identified in the G. sulfurreducens genome using sequence similarity searches to matrices of -35/-10 promoter elements found in G. sulfurreducens and in Escherichia coli. Due to a sufficient degree of sequence similarity between -35/-10 promoter elements for RpoS, RpoD, and other sigma factors, both the sequence similarity searches and the search for conserved overrepresented motifs using microarray data may identify promoter elements for both RpoS and other sigma factors.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Base Sequence, Citrates, Computational Biology, Conserved Sequence, DNA-Directed RNA Polymerases, Escherichia coli, Escherichia coli Proteins, Gene Deletion, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genes, Bacterial, Genome, Bacterial, Geobacter, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Operon, Promoter Regions, Genetic, Sigma Factor, Transcription, Genetic}, issn = {0378-1119}, doi = {10.1016/j.gene.2006.06.025}, author = {Yan, Bin and N{\'u}{\~n}ez, Cinthia and Ueki, Toshiyuki and Esteve-N{\'u}{\~n}ez, Abraham and Puljic, Marko and Adkins, Ronald M and Meth{\'e}, Barbara A and Lovley, Derek R and Krushkal, Julia} } @article {819, title = {A novel dnaC mutation that suppresses priB rep mutant phenotypes in Escherichia coli K-12.}, journal = {Mol Microbiol}, volume = {60}, year = {2006}, month = {2006 May}, pages = {973-83}, abstract = {The loading of a replisome in prokaryotic and eukaryotic cells at an origin of DNA replication and during replication restart is a highly ordered and regulated process. During replication restart in Escherichia coli, the PriA, PriB, PriC, DnaT and Rep proteins form multiple pathways that bind to repaired replication forks. These complexes are then recognized by DnaC as sites to load DnaB, the replicative helicase. Several dnaC mutations have been isolated that suppress phenotypes of some replication restart mutants. A new dnaC mutation (dnaC824) is reported here that efficiently suppresses priB rep mutant phenotypes. Furthermore, it is shown that dnaC824 will suppress phenotypes of priB priA300, rep priA300 and priB priC strains. Unlike other dnaC suppressors, it can only weakly suppress the absence of priA. Others have reported a different type of dnaC mutation, dnaC1331, is able to mimic priB mutant phenotypes. This is supported herein by showing that like dnaC1331, a priB mutation is synthetically lethal with a dam mutation and this can be rescued by a mutH mutation. Furthermore, priB dam lethality can also be suppressed by dnaC824. Like a priB mutation, a dnaC1331 mutation causes a priA2::kan-like phenotype when combined with priA300. Lastly, we show that dnaC824 is dominant to wild type and that dnaC1331 is recessive to wild type. Several models are discussed for the action of these mutant dnaC proteins in replication restart.}, keywords = {DNA Replication, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Genes, Dominant, Genes, Lethal, Genes, Recessive, Mutant Proteins, Mutation, Phenotype, Suppression, Genetic}, issn = {0950-382X}, doi = {10.1111/j.1365-2958.2006.05147.x}, author = {Boonsombat, Ruethairat and Yeh, Su-Ping and Milne, Amy and Sandler, Steven J} } @article {718, title = {Simian virus 40 late proteins possess lytic properties that render them capable of permeabilizing cellular membranes.}, journal = {J Virol}, volume = {80}, year = {2006}, month = {2006 Jul}, pages = {6575-87}, abstract = {Many nonenveloped viruses have evolved an infectious cycle that culminates in the lysis or permeabilization of the host to enable viral release. How these viruses initiate the lytic event is largely unknown. Here, we demonstrated that the simian virus 40 progeny accumulated at the nuclear envelope prior to the permeabilization of the nuclear, endoplasmic reticulum, and plasma membranes at a time which corresponded with the release of the progeny. The permeabilization of these cellular membranes temporally correlated with late protein expression and was not observed upon the inhibition of their synthesis. To address whether one or more of the late proteins possessed an inherent capacity to induce membrane permeabilization, we examined the permeability of Escherichia coli that separately expressed the late proteins. VP2 and VP3, but not VP1, caused the permeabilization of bacterial membranes. Additionally, VP3 expression resulted in bacterial cell lysis. These findings demonstrate that VP3 possesses an inherent lytic property that is independent of eukaryotic signaling or cell death pathways.}, keywords = {Animals, Capsid Proteins, Cell Death, Cell Line, Cell Membrane Permeability, Cercopithecus aethiops, Endoplasmic Reticulum, Escherichia coli, Gene Expression Regulation, Viral, Nuclear Envelope, Permeability, Protein Biosynthesis, Simian virus 40, Virus Replication}, issn = {0022-538X}, doi = {10.1128/JVI.00347-06}, author = {Daniels, Robert and Rusan, Nasser M and Wilbuer, Anne-Kathrin and Norkin, Leonard C and Wadsworth, Patricia and Hebert, Daniel N} } @article {396, title = {Characterization of TetD as a transcriptional activator of a subset of genes of the Escherichia coli SoxS/MarA/Rob regulon.}, journal = {Mol Microbiol}, volume = {56}, year = {2005}, month = {2005 May}, pages = {1103-17}, abstract = {In Escherichia coli, SoxS, MarA and Rob form a closely related subset of the AraC/XylS family of positive regulators, sharing approximately 42\% amino acid sequence identity over the length of SoxS and the ability to activate transcription of a common set of target genes that provide resistance to redox-cycling compounds and antibiotics. On the basis of its approximately 43\% amino acid sequence identity with SoxS, MarA and Rob, TetD, encoded by transposon Tn10, appears to be a fourth member of the subset. However, although its expression has been shown to be negatively regulated by TetC and not inducible by tetracycline, the physiological function of TetD is unknown. Accordingly, in the work presented here, we initiate a molecular characterization of TetD. We show that expression of TetD activates transcription of a subset of the SoxS/MarA/Rob regulon genes and confers resistance to redox-cycling compounds and antibiotics. We show that mutations in the putative TetD binding site of a TetD-activatable promoter and a mutation in the protein{\textquoteright}s N-terminal DNA recognition helix interfere with transcription activation, thereby indicating that TetD directly activates target gene transcription. Finally, we show that TetD, like SoxS and MarA, is intrinsically unstable; however, unlike SoxS and MarA, TetD is not degraded by Lon or any of the cell{\textquoteright}s known cytoplasmic ATP-dependent proteases. Thus, we conclude that TetD is a bona fide member of the SoxS/MarA/Rob subfamily of positive regulators.}, keywords = {Amino Acid Sequence, Binding Sites, DNA, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Multigene Family, Promoter Regions, Genetic, Regulon, Tetracycline Resistance, Trans-Activators, Transcription Factors, Transcription, Genetic}, issn = {0950-382X}, doi = {10.1111/j.1365-2958.2005.04599.x}, author = {Griffith, Kevin L and Becker, Stephen M and Wolf, Richard E} } @article {820, title = {Localization of RecA in Escherichia coli K-12 using RecA-GFP.}, journal = {Mol Microbiol}, volume = {57}, year = {2005}, month = {2005 Aug}, pages = {1074-85}, abstract = {RecA is important in recombination, DNA repair and repair of replication forks. It functions through the production of a protein-DNA filament. To study the localization of RecA in live Escherichia coli cells, the RecA protein was fused to the green fluorescence protein (GFP). Strains with this gene have recombination/DNA repair activities three- to tenfold below wild type (or about 1000-fold above that of a recA null mutant). RecA-GFP cells have a background of green fluorescence punctuated with up to five foci per cell. Two types of foci have been defined: 4,6-diamidino-2-phenylindole (DAPI)-sensitive foci that are bound to DNA and DAPI-insensitive foci that are DNA-less aggregates/storage structures. In log phase cells, foci were not localized to any particular region. After UV irradiation, the number of foci increased and they localized to the cell centre. This suggested colocalization with the DNA replication factory. recA, recB and recF strains showed phenotypes and distributions of foci consistent with the predicted effects of these mutations.}, keywords = {Chromosomes, Bacterial, DNA Replication, DNA, Bacterial, Escherichia coli, Escherichia coli Proteins, Green Fluorescent Proteins, Mutation, Rec A Recombinases, Recombinant Fusion Proteins, Recombination, Genetic, Ultraviolet Rays}, issn = {0950-382X}, doi = {10.1111/j.1365-2958.2005.04755.x}, author = {Renzette, Nicholas and Gumlaw, Nathan and Nordman, Jared T and Krieger, Marlee and Yeh, Su-Ping and Long, Edward and Centore, Richard and Boonsombat, Ruethairat and Sandler, Steven J} } @article {821, title = {Requirements for replication restart proteins during constitutive stable DNA replication in Escherichia coli K-12.}, journal = {Genetics}, volume = {169}, year = {2005}, month = {2005 Apr}, pages = {1799-806}, abstract = {Constitutive stable DNA replication (cSDR) is a mechanism for replisome loading in Escherichia coli K-12. This occurs in a dnaA-independent fashion in an rnhA mutant. cSDR is dependent on recA, priA, and transcription. In this report, it is shown that dnaA rnhA mutants using cSDR for initiation of their DNA replication additionally require priB, but not priC, for viability. Two subtle priA missense mutations either eliminated the ability to grow using cSDR (priA301 C479Y) or resulted in very small colonies (priA300 K230R). DnaC809, a priA suppressor, failed to allow priA or priB mutants to grow using cSDR to initiate DNA replication. Furthermore, unlike dnaC(+) strains, dnaC809 strains require priC for cSDR. DnaC809,820, a priC-independent suppressor of priA2::kan phenotypes, allowed priA and priC (but not priB) mutants to grow using cSDR to initiate DNA replication. It is also shown that rep and rnhA mutations are synthetically lethal. DnaC809 and dnaC809,820 mutations suppress this lethality. Rep is further shown to be required for cSDR in a dnaC809 strain. A model whereby these different sets of replication restart proteins interact preferentially with substrates associated with either RecA or SSB during replication restart and cSDR, respectively, is proposed.}, keywords = {Bacterial Proteins, DNA, DNA Replication, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genome, Bacterial, Macromolecular Substances, Models, Genetic, Mutation, Mutation, Missense, Phenotype, Rec A Recombinases, RNA, SOS Response (Genetics)}, issn = {0016-6731}, doi = {10.1534/genetics.104.036962}, author = {Sandler, Steven J} } @article {397, title = {Genetic evidence for pre-recruitment as the mechanism of transcription activation by SoxS of Escherichia coli: the dominance of DNA binding mutations of SoxS.}, journal = {J Mol Biol}, volume = {344}, year = {2004}, month = {2004 Nov 12}, pages = {1-10}, abstract = {SoxS, the direct transcriptional activator of the Escherichia coli superoxide (SoxRS) regulon, displays several unusual characteristics which suggest that it is unlikely to activate transcription by the ususal recruitment mechanism. Thus, agents that generate superoxide endogenously and thereby provoke the defense response elicit the de novo synthesis of SoxS, and with the SoxS binding site being highly degenerate, the number of SoxS binding sites per cell far exceeds the number of SoxS molecules per cell. To account for these distinctive features of the SoxRS system, we proposed "pre-recruitment" as the mechanism by which SoxS activates transcription of the regulon{\textquoteright}s genes. In pre-recruitment, newly synthesized SoxS molecules form binary complexes with RNA polymerase in solution. These complexes provide the information content to allow the 2500 molecules of SoxS per cell to scan the 65,000 SoxS binding sites per cell for the 200 binding sites per cell that reside within SoxS-dependent promoters. As a test of whether SoxS activates transcription by recruitment or pre-recruitment, we determined the dominance relationships of SoxS mutations conferring defective DNA binding. We found that soxS DNA binding mutations are dominant to the wild-type allele, a result consistent with the pre-recruitment hypothesis, but opposite to that expected for an activator that functions by recruitment. Moreover, whereas positive control mutations of activators functioning by recruitment are usually dominant, a soxS positive control mutation was not. Lastly, with the SoxRS system as an example, we discuss the physiological requirement for stringent regulation of transcriptional activators that function by pre-recruitment.}, keywords = {Base Sequence, Binding Sites, DNA, Bacterial, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Bacterial, Genes, Dominant, Mutation, Regulon, Trans-Activators, Transcriptional Activation}, issn = {0022-2836}, doi = {10.1016/j.jmb.2004.09.007}, author = {Griffith, Kevin L and Wolf, Richard E} } @article {861, title = {Poly(hydroxyalkanoic acid) Biosynthesis in Ectothiorhodospirashaposhnikovii: Characterization and Reactivity of a Type III PHA Synthase.}, journal = {Biomacromolecules}, volume = {5}, year = {2004}, month = {2004 Jan-Feb}, pages = {40-8}, abstract = {Ectothiorhodospira shaposhnikovii is able to accumulate polyhydroxybutyrate (PHB) photoautotrophically during nitrogen-limited growth. The activity of polyhydroxyalkanoate (PHA) synthase in the cells correlates with PHB accumulation. PHA synthase samples collected during the light period do not show a lag phase during in vitro polymerization. Synthase samples collected in the dark period displays a significant lag phase during in vitro polymerization. The lag phase can be eliminated by reacting the PHA synthase with the monomer, 3-hydroxybutyryl-CoA (3HBCoA). The PHA synthase genes (phaC and phaE) were cloned by screening a genomic library for PHA accumulation in E. coli cells. The PHA synthase expressed in the recombinant E. coli cells was purified to homogeneity. Both sequence analysis and biochemical studies indicated that this PHA synthase consists of two subunits, PhaE and PhaC and, therefore, belongs to the type III PHA synthases. Two major complexes were identified in preparations of purified PHA synthase. The large complex appears to be composed of 12 PhaC subunits and 12 PhaE subunits (dodecamer), whereas the small complex appears to be composed of 6 PhaC and 6 PhaE subunits (hexamer). In dilute aqueous solution, the synthase is predominantly composed of hexamer and has low activity accompanied with a significant lag period at the initial stage of reaction. The percentage of dodecameric complex increases with increasing salt concentration. The dodecameric complex has a greatly increased specific activity for the polymerization of 3HBCoA and a negligible lag period. The results from in vitro polymerizations of 3HBCoA suggest that the PHA synthase from E. shaposhnikovii may catalyze a living polymerization and demonstrate that two PhaC and two PhaE subunits comprise a single catalytic site in the synthase complex.}, keywords = {Acyltransferases, Cloning, Molecular, Ectothiorhodospira shaposhnikovii, Escherichia coli, Hydroxybutyrates, Kinetics, Molecular Weight, Polyesters}, issn = {1525-7797}, doi = {10.1021/bm034171i}, author = {Zhang, Shiming and Kolvek, Steven and Goodwin, Steve and Lenz, Robert W} } @article {398, title = {Proteolytic degradation of Escherichia coli transcription activators SoxS and MarA as the mechanism for reversing the induction of the superoxide (SoxRS) and multiple antibiotic resistance (Mar) regulons.}, journal = {Mol Microbiol}, volume = {51}, year = {2004}, month = {2004 Mar}, pages = {1801-16}, abstract = {In Escherichia coli, the SoxRS regulon confers resistance to redox-cycling compounds, and the Mar regulon provides a defence against multiple antibiotics. The response regulators, SoxS and MarA, are synthesized de novo in response to their inducing signals and directly activate transcription of a common set of target genes. Although the mechanisms of transcription activation by SoxS and MarA have been well studied, little is known about how the systems are shut-off once the inducing stress has subsided, except that de novo synthesis of the regulators is known to cease almost immediately. Here, we induced the SoxRS regulon and determined that, upon removal of the inducer, expression of the regulon{\textquoteright}s genes quickly returns to the preinduced level. This rapid shut-off indicates that the system is reset by an active process. We found that SoxS is unstable and infer that SoxS degradation is responsible for the rapid return of the system to the ground state upon removal of the inducing signal. We also found that MarA is unstable and that the instability of both proteins is intrinsic and unregulated. We used null mutations of protease genes to identify the proteases involved in the degradation of SoxS and MarA. Among single protease mutations, only lon mutations increased the half-life of SoxS and MarA. In addition, SoxS appeared to be nearly completely stable in a lon ftsH double mutant. Using hexahistidine tags placed at the respective ends of the activators, we found that access to the amino-terminus is essential for the proteolytic degradation.}, keywords = {Base Sequence, Blotting, Western, DNA Primers, DNA-Binding Proteins, Drug Resistance, Bacterial, Drug Resistance, Multiple, Bacterial, Endopeptidases, Escherichia coli, Escherichia coli Proteins, Genes, Bacterial, Half-Life, Hydrolysis, Kinetics, Mutation, Oxidation-Reduction, Promoter Regions, Genetic, Regulon, Trans-Activators, Transcription, Genetic}, issn = {0950-382X}, author = {Griffith, Kevin L and Shah, Ishita M and Wolf, Richard E} } @article {774, title = {Quorum sensing in Staphylococci is regulated via phosphorylation of three conserved histidine residues.}, journal = {J Biol Chem}, volume = {279}, year = {2004}, month = {2004 Apr 9}, pages = {14665-72}, abstract = {Staphylococcus aureus cause infections by producing toxins, a process regulated by cell-cell communication (quorum sensing) through the histidine-phosphorylation of the target of RNAIII-activating protein (TRAP). We show here that TRAP is highly conserved in staphylococci and contains three completely conserved histidine residues (His-66, His-79, His-154) that are phosphorylated and essential for its activity. This was tested by constructing a TRAP(-) strain with each of the conserved histidine residues changed to alanine by site-directed mutagenesis. All mutants were tested for pathogenesis in vitro (expression of RNAIII and hemolytic activity) and in vivo (murine cellulitis model). Results show that RNAIII is not expressed in the TRAP(-) strain, that it is non hemolytic, and that it does not cause disease in vivo. These pathogenic phenotypes could be rescued in the strain containing the recovered traP, confirming the importance of TRAP in S. aureus pathogenesis. The phosphorylation of TRAP mutated in any of the conserved histidine residues was significantly reduced, and mutants defective in any one of these residues were non-pathogenic in vitro or in vivo, whereas those mutated in a non-conserved histidine residue (His-124) were as pathogenic as the wild type. These results confirm the importance of the three conserved histidine residues in TRAP activity. The phosphorylation pattern, structure, and gene organization of TRAP deviates from signaling molecules known to date, suggesting that TRAP belongs to a novel class of signal transducers.}, keywords = {Amino Acid Sequence, Animals, Blotting, Northern, Cell Communication, DNA, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Histidine, Mice, Molecular Sequence Data, Mutagenesis, Mutagenesis, Site-Directed, Mutation, Phenotype, Phosphorylation, Phylogeny, Plasmids, Protein Structure, Tertiary, RNA, Antisense, RNA, Bacterial, Sequence Homology, Amino Acid, Signal Transduction, Staphylococcus aureus, Time Factors}, issn = {0021-9258}, doi = {10.1074/jbc.M311106200}, author = {Gov, Yael and Borovok, Ilya and Korem, Moshe and Singh, Vineet K and Jayaswal, Radheshyam K and Wilkinson, Brian J and Rich, Stephen M and Balaban, Naomi} } @article {752, title = {Tuning the hemolytic and antibacterial activities of amphiphilic polynorbornene derivatives.}, journal = {J Am Chem Soc}, volume = {126}, year = {2004}, month = {2004 Dec 8}, pages = {15870-5}, abstract = {Amphiphilic cationic polynorbornene derivatives, soluble in water, were prepared from modular norbornene monomers, with a wide range of molecular weights (M(n) = 1600-137 500 g/mol) and narrow polydispersities (PDI = 1.1-1.3). The antibacterial activity determined by growth inhibition assays and the hemolytic activity against human red blood cells were measured and compared to determine the selectivity of the polymers for bacterial over mammalian cells. The effects of monomer repeat unit hydrophobicity and polymer molecular weight on antibacterial and hemolytic activities were determined. The hydrophobicity of the repeat unit was observed to have dramatic effects on antibacterial and hemolytic activities. Lipid membrane disruption activities of the polymers was confirmed by measuring polymer-induced dye leakage from large unilamellar vesicles. By tuning the overall hydrophobicity of the polymer through random copolymerizations of modular norbornene derivatives, highly selective, nonhemolytic antibacterial activities were obtained. For appropriate monomer composition, selectivity against bacteria versus human red blood cells was determined to be over 100.}, keywords = {Anti-Bacterial Agents, Bacillus subtilis, Cell Membrane, Cholesterol, Erythrocytes, Escherichia coli, Hemolysis, Humans, Liposomes, Microbial Sensitivity Tests, Molecular Weight, Norbornanes, Polymers, Structure-Activity Relationship}, issn = {0002-7863}, doi = {10.1021/ja045664d}, author = {Ilker, M Firat and N{\"u}sslein, Klaus and Tew, Gregory N and Coughlin, E Bryan} } @article {399, title = {A comprehensive alanine scanning mutagenesis of the Escherichia coli transcriptional activator SoxS: identifying amino acids important for DNA binding and transcription activation.}, journal = {J Mol Biol}, volume = {322}, year = {2002}, month = {2002 Sep 13}, pages = {237-57}, abstract = {SoxS is the direct transcriptional activator of the superoxide regulon. SoxS recognizes a highly degenerate "soxbox" DNA sequence, and activates transcription from class I and class II promoters. SoxS is the smallest member of the AraC/XylS family of transcription regulators whose hallmark is dual helix-turn-helix (HTH) DNA-binding motifs. Evidence suggests that the N-terminal HTH motif of SoxS interacts with a highly conserved region of the soxbox termed recognition element 1 (RE1), while the C-terminal HTH motif interacts with the less conserved recognition element 2 (RE2). In the work described here, we prepared a complete library of 101 SoxS mutants containing single alanine substitutions of SoxS, and we characterized the mutant proteins in vivo and in vitro. With SoxS being closely related to MarA, we analyzed the effects of the SoxS mutations in the context of the MarA-mar crystal structure and with respect to the NMR study of MarA-DNA complexes in solution. From the properties of the alanine substitutions, we conclude the following. (1) Surface-exposed residues of helix 3 and helix 6, the recognition helices of the dual HTH motifs, are important to DNA binding and transcription activation; however, substitutions of residues predicted from the MarA-mar crystal structure to make contact with the sugar-phosphate backbone are more detrimental to DNA binding than mutations predicted to make base-specific contacts. (2) Substitution of several residues within the recognition helix predicted to make base-specific contacts with RE2 have relatively little effect on DNA-binding, suggesting the possibility of alternative protein-DNA interactions than those inferred from the MarA-mar crystal structure. (3) DNA binding and transcription activation were reduced by substitution of conserved amino acid residues comprising the hydrophobic core, presumably because they disrupt the structural integrity of SoxS. (4) Mutant K30A appears to be a positive control mutant defective in a protein-protein interaction with RNA polymerase that is required for transcription activation at all SoxS-dependent promoters because it binds and bends DNA normally but fails to activate transcription from both classes of promoters. Alanine substitutions of surface-exposed residues H3, K5, D9, S31, and V45 confer a similar phenotype. Since these residues are near K30 on the surface of the protein, the surface formed by the six residues may be used to make protein-protein interactions with RNA polymerase that are required for transcription activation at both class I and class II SoxS-dependent promoters. (5) Mutants F74A, D75A, M78A, D79A and Q85A appear to define a surface required for protein-protein interaction with RNA polymerase specifically at class II promoters because these positive control mutants bind and bend DNA normally but are defective in activation of class II promoters but not class I promoters. These SoxS mutants that bind and bend DNA normally but are defective in transcription activation represent the first positive control mutants with putative defects in protein-protein interactions with RNA polymerase among the SoxS/MarA/Rob subset of the AraC/XylS family of transcription regulators.}, keywords = {Alanine, Amino Acid Substitution, Base Sequence, Binding Sites, DNA, DNA-Binding Proteins, Electrophoretic Mobility Shift Assay, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Lethal, Hydrophobic and Hydrophilic Interactions, Lac Operon, Models, Molecular, Mutagenesis, Nucleic Acid Conformation, Phenotype, Phosphates, Promoter Regions, Genetic, Protein Binding, Protein Structure, Tertiary, Structure-Activity Relationship, Trans-Activators, Transcriptional Activation}, issn = {0022-2836}, author = {Griffith, Kevin L and Wolf, Richard E} } @article {400, title = {Evidence for "pre-recruitment" as a new mechanism of transcription activation in Escherichia coli: the large excess of SoxS binding sites per cell relative to the number of SoxS molecules per cell.}, journal = {Biochem Biophys Res Commun}, volume = {291}, year = {2002}, month = {2002 Mar 8}, pages = {979-86}, abstract = {In response to the oxidative stress imposed by redox-cycling compounds like paraquat, Escherichia coli induces the synthesis of SoxS, which then activates the transcription of approximately 100 genes. The DNA binding site for SoxS-dependent transcription activation, the "soxbox," is highly degenerate, suggesting that the genome contains a large number of SoxS binding sites. To estimate the number of soxboxes in the cell, we searched the E. coli genome for SoxS binding sites using as query sequence the previously determined optimal SoxS binding sequence. We found approximately 12,500 sequences that match the optimal binding sequence under the conditions of our search; this agrees with our previous estimate, based on information theory, that a random sequence the size of the E. coli genome contains approximately 13,000 soxboxes. Thus, fast-growing cells with 4-6 genomes per cell have approximately 65,000 soxboxes. This large number of potential SoxS binding sites per cell raises the interesting question of how SoxS distinguishes between the functional soxboxes located within the promoters of target genes and the plethora of equivalent but nonfunctional binding sites scattered throughout the chromosome. To address this question, we treated cells with paraquat and used Western blot analysis to determine the kinetics of SoxS accumulation per cell; we also determined the kinetics of SoxS-activated gene expression. The abundance of SoxS reached a maximum of 2,500 molecules per cell 20 min after induction and gradually declined to approximately 500 molecules per cell over the next 1.5 h. Given that activation of target gene expression began almost immediately and given the large disparity between the number of SoxS molecules per cell, 2,500, and the number of SoxS binding sites per cell, 65,000, we infer that SoxS is not likely to activate transcription by the usual "recruitment" pathway, as this mechanism would require a number of SoxS molecules similar to the number of soxboxes. Instead, we propose that SoxS first interacts in solution with RNA polymerase and then the binary complex scans the chromosome for promoters that contain a soxbox properly positioned and oriented for transcription activation. We name this new pathway "pre-recruitment."}, keywords = {Bacterial Proteins, Binding Sites, Blotting, Western, Cell Division, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genome, Bacterial, Kinetics, Numerical Analysis, Computer-Assisted, Oxidative Stress, Paraquat, Protein Transport, Trans-Activators, Transcription Factors, Transcriptional Activation}, issn = {0006-291X}, doi = {10.1006/bbrc.2002.6559}, author = {Griffith, Kevin L and Shah, Ishita M and Myers, Todd E and O{\textquoteright}Neill, Michael C and Wolf, Richard E} } @article {401, title = {Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays.}, journal = {Biochem Biophys Res Commun}, volume = {290}, year = {2002}, month = {2002 Jan 11}, pages = {397-402}, abstract = {We describe a high-throughput procedure for measuring beta-galactosidase activity in bacteria. This procedure is unique because all manipulations, including bacterial growth and cell permeabilization, are performed in a 96-well format. Cells are permeabilized by chloroform/SDS treatment directly in the 96-well blocks and then transferred to 96-well microplates for standard colorimetric assay of beta-galactosidase activity as described by Miller [J. H. Miller (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Absorbance data are collected with a microplate reader and analyzed using a Microsoft Excel spreadsheet. The beta-galactosidase specific activity values obtained with the high-throughput procedure are identical to those obtained by the traditional single-tube method of Miller. Thus, values obtained with this procedure may be expressed as Miller units and compared directly to Miller units reported in the literature. The 96-well format for permeabilization and assay of enzyme specific activity together with the use of 12-channel and repeater pipettors enables efficient processing of hundreds of samples in an 8-h day.}, keywords = {Bacteria, beta-Galactosidase, Biochemistry, Cell Division, Chloroform, Escherichia coli, Polypropylenes, Sodium Dodecyl Sulfate, Software, Spectrophotometry, Time Factors}, issn = {0006-291X}, doi = {10.1006/bbrc.2001.6152}, author = {Griffith, Kevin L and Wolf, Richard E} } @article {778, title = {Production and characterization of protective human antibodies against Shiga toxin 1.}, journal = {Infect Immun}, volume = {70}, year = {2002}, month = {2002 Oct}, pages = {5896-9}, abstract = {Hemolytic-uremic syndrome (HUS) is a serious complication which is predominantly associated in children with infection by Shiga toxin-producing Escherichia coli (STEC). By using HuMAb-Mouse (Medarex) animals, human monoclonal antibodies (Hu-MAbs) were developed against Shiga toxin 1 (Stx1) for passive immunotherapy of HUS. Ten stable hybridomas comprised of fully human heavy- and light-chain immunoglobulin elements and secreting Stx1-specific Hu-MAbs (seven immunoglobulin M(kappa)() [IgM(kappa)] elements [one specific for the A subunit and six specific for the B subunit] and three IgG1(kappa) elements specific for subunit B) were isolated. Two IgM(kappa) Hu-MAbs (2D9 and 15G9) and three IgG1(kappa) Hu-MAbs (5A4, 10F4, and 15G2), all specific for subunit B, demonstrated marked neutralization of Stx1 in vitro and significant prolongation of survival in a murine model of Stx1 toxicosis.}, keywords = {Animals, Antibodies, Bacterial, Antibodies, Monoclonal, Child, Escherichia coli, Female, HeLa Cells, Hemolytic-Uremic Syndrome, Humans, Hybridomas, Immunization, Passive, Immunoglobulin G, Immunoglobulin M, Mice, Neutralization Tests, Shiga Toxin 1}, issn = {0019-9567}, author = {Mukherjee, Jean and Chios, Kerry and Fishwild, Dianne and Hudson, Deborah and O{\textquoteright}Donnell, Susan and Rich, Stephen M and Donohue-Rolfe, Arthur and Tzipori, Saul} } @article {826, title = {Effects of mutations involving cell division, recombination, and chromosome dimer resolution on a priA2::kan mutant.}, journal = {Proc Natl Acad Sci U S A}, volume = {98}, year = {2001}, month = {2001 Jul 17}, pages = {8203-10}, abstract = {Recombinational repair of replication forks can occur either to a crossover (XO) or noncrossover (non-XO) depending on Holliday junction resolution. Once the fork is repaired by recombination, PriA is important for restarting these forks in Escherichia coli. PriA mutants are Rec(-) and UV sensitive and have poor viability and 10-fold elevated basal levels of SOS expression. PriA sulB mutant cells and their nucleoids were studied by differential interference contrast and fluorescence microscopy of 4{\textquoteright},6-diamidino-2-phenylindole-stained log phase cells. Two populations of cells were seen. Eighty four percent appeared like wild type, and 16\% of the cells were filamented and had poorly partitioned chromosomes (Par(-)). To probe potential mechanisms leading to the two populations of cells, mutations were added to the priA sulB mutant. Mutating sulA or introducing lexA3 decreased, but did not eliminate filamentation or defects in partitioning. Mutating either recA or recB virtually eliminated the Par(-) phenotype. Filamentation in the recB mutant decreased to 3\%, but increased to 28\% in the recA mutant. The ability to resolve and/or branch migrate Holliday junctions also appeared crucial in the priA mutant because removing either recG or ruvC was lethal. Lastly, it was tested whether the ability to resolve chromosome dimers caused by XOs was important in a priA mutant by mutating dif and the C-terminal portion of ftsK. Mutation of dif showed no change in phenotype whereas ftsK1cat was lethal with priA2kan. A model is proposed where the PriA-independent pathway of replication restart functions at forks that have been repaired to non-XOs.}, keywords = {Adenosine Triphosphatases, Bacterial Proteins, Cell Division, Chromosomes, Bacterial, Dimerization, DNA Helicases, Endodeoxyribonucleases, Escherichia coli, Escherichia coli Proteins, Exodeoxyribonuclease V, Exodeoxyribonucleases, Gene Expression, Membrane Proteins, Mutagenesis, Rec A Recombinases, Recombination, Genetic, SOS Response (Genetics)}, issn = {0027-8424}, doi = {10.1073/pnas.121007698}, author = {McCool, J D and Sandler, S J} } @article {825, title = {PriA mutations that affect PriA-PriC function during replication restart.}, journal = {Mol Microbiol}, volume = {41}, year = {2001}, month = {2001 Aug}, pages = {697-704}, abstract = {In Escherichia coli, repair and restart of collapsed replication forks is thought to be essential for cell growth. The replication restart proteins, PriA, PriB, PriC, DnaB, DnaC, DnaG, DnaT and Rep, form redundant pathways that recognize repaired replication forks and restart them. Recognition, modulation of specific DNA structures and loading of the replicative helicase by the replication restart proteins, is likely to be important for replication restart. It has been hypothesized that PriB and PriC function with PriA in genetically separate and redundant PriA-PriB and PriA-PriC pathways. In this study, the del(priB)302 or priC303:kan mutations were used to isolate the PriA-PriB and PriA-PriC pathways genetically so that the effects of three priA missense mutations, priA300 (K230R), priA301 (C479Y) and priA306 (L557P), on these pathways could be assessed. In a wild-type background, the three priA mutations had little, if any, effect on the phenotypes of UV resistance, basal levels of SOS expression and cell viability. In the priB mutant, priA300 and priA301 caused dramatic negative changes in the three phenotypes listed above (and others), whereas the third priA mutant allele, priA306, showed very little negative effect. In the priC mutant, all three priA mutations behaved similarly, producing little, if any, changes in phenotypes. We conclude that priA300 and priA301 mostly affect the PriA-PriC pathway and do so more than priA306. We suggest that PriA{\textquoteright}s helicase activity is important for the PriA-PriC pathway of replication restart.}, keywords = {Bacterial Proteins, Bacteriophage mu, Cell Division, DNA Replication, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Genes, Lethal, Genotype, Models, Biological, Mutation, Missense, Phenotype, Replication Protein A, SOS Response (Genetics), Ultraviolet Rays}, issn = {0950-382X}, author = {Sandler, S J and McCool, J D and Do, T T and Johansen, R U} } @article {402, title = {Systematic mutagenesis of the DNA binding sites for SoxS in the Escherichia coli zwf and fpr promoters: identifying nucleotides required for DNA binding and transcription activation.}, journal = {Mol Microbiol}, volume = {40}, year = {2001}, month = {2001 Jun}, pages = {1141-54}, abstract = {SoxS is the direct transcriptional activator of at least 15 genes of the Escherichia coli superoxide regulon. SoxS is small (107 amino acids), binds DNA as a monomer and recognizes a highly degenerate DNA binding site, termed {\textquoteright}soxbox{\textquoteright}. Like other members of the AraC/XylS family, SoxS has two putative helix-turn-helix (HTH) DNA-binding motifs, and it has been proposed that each HTH motif recognizes a highly conserved recognition element of the soxbox. To determine which nucleotides are important for SoxS binding, we conducted a systematic mutagenesis of the DNA binding sites for SoxS in the zwf and fpr promoters and determined the effect of the soxbox mutations on SoxS DNA binding and transcription activation in vivo by measuring beta-galactosidase activity in strains with fusions to lacZ. We found that the sequences GCAC and CAAA, termed recognition elements 1 and 2 (RE 1 and RE 2), respectively, are critical for SoxS binding, as mutations within these elements severely hinder or eliminate SoxS-dependent transcription activation; substitutions within RE 2 (CAAA), however, are tolerated better than changes within RE 1 (GCAC). Although substitutions at the seven positions separating the two REs had only a modest effect on SoxS binding, AT basepairs were favoured within this {\textquoteright}spacer{\textquoteright} region, presumably because, by facilitating DNA bending, they help bring the two recognition elements into proper juxtaposition. We also found that the {\textquoteright}invariant A{\textquoteright} present at position 1 of 14/15 functional soxboxes identified thus far is important for SoxS binding, as a change to any other nucleotide at this position reduced SoxS-dependent transcription by approximately 50\%. In addition, positions surrounding the REs seem to show a context effect, in that certain substitutions there have little or no effect when the RE has the optimal binding sequence, but produce a pronounced effect when the RE has a suboptimal sequence. We propose that these nucleotides play an important role in effecting differential expression from the various promoters. Lastly, we used gel retardation assays to show that alterations in transcription activation in vivo are caused by effects on DNA binding. Based on this exhaustive mutagenesis, we propose the following optimal sequence for SoxS binding: AnVGCACWWWnKRHCAAAHn (n = A, C, G, T; V = A, C, G; W = A, T; K = G, T; R = A, G; H = A, C, T).}, keywords = {Amino Acid Sequence, Bacterial Proteins, Base Sequence, Binding Sites, Carrier Proteins, DNA, Bacterial, Electrophoresis, Escherichia coli, Escherichia coli Proteins, Molecular Sequence Data, Mutagenesis, Promoter Regions, Genetic, Trans-Activators, Transcription Factors, Transcriptional Activation}, issn = {0950-382X}, author = {Griffith, K L and Wolf, R E} } @article {863, title = {Enzymatic hydrolysis of oligomeric models of poly-3-hydroxybutyrate.}, journal = {Biomacromolecules}, volume = {1}, year = {2000}, month = {2000 Winter}, pages = {577-83}, abstract = {The mechanism of the enzymatic degradation of poly([R]-3-hydroxybutyrate) (PHB) was investigated by using well-defined model substrates, including both linear and cyclic [R]-3-hydroxybutyrate (3HB) and [R]-3-hydroxyvalerate (3HV) oligomers, with two different PHB depolymerases. The linear and cyclic oligomers containing from 2 to 10 repeating units were hydrolyzed in solutions of the depolymerase isolated from Aspergillus fumigatus and Alcaligenes faecalis, and the rates of hydrolysis and types of products formed were characterized. Both of the depolymerases catalyzed the hydrolysis of the cyclic oligomers (macrolides) which contained more than three 3HB and 3HV repeating units. The degradation reactions of the linear and cyclic 3HB oligomers with the A. fumigatus depolymerase gave similar ratios of monomer-to-dimer products, but PHB itself formed mostly monomer on hydrolysis, indicating that the enzymatic hydrolysis reactions occurred by different mechanisms for these different types of substrates. The results of this study conclusively show that at least the endo mode of polymer hydrolysis occurs with the two enzymes studied, while the A. fumigatus depolymerase was found to utilize both endo and exo modes of hydrolysis to efficiently degrade PHB and 3HB oligomers.}, keywords = {3-Hydroxybutyric Acid, Alcaligenes, Aspergillus fumigatus, Carboxylic Ester Hydrolases, Escherichia coli, Hydrolysis, Kinetics, Macrolides, Models, Chemical, Polymers}, issn = {1525-7797}, author = {Scherer, T M and Fuller, R C and Goodwin, S and Lenz, R W} } @article {828, title = {The importance of repairing stalled replication forks.}, journal = {Nature}, volume = {404}, year = {2000}, month = {2000 Mar 2}, pages = {37-41}, abstract = {The bacterial SOS response to unusual levels of DNA damage has been recognized and studied for several decades. Pathways for re-establishing inactivated replication forks under normal growth conditions have received far less attention. In bacteria growing aerobically in the absence of SOS-inducing conditions, many replication forks encounter DNA damage, leading to inactivation. The pathways for fork reactivation involve the homologous recombination systems, are nonmutagenic, and integrate almost every aspect of DNA metabolism. On a frequency-of-use basis, these pathways represent the main function of bacterial DNA recombination systems, as well as the main function of a number of other enzymatic systems that are associated with replication and site-specific recombination.}, keywords = {Bacteria, Bacterial Proteins, Chromosomes, Bacterial, DNA Replication, DNA, Bacterial, Escherichia coli, Recombination, Genetic, Replication Origin, SOS Response (Genetics)}, issn = {0028-0836}, doi = {10.1038/35003501}, author = {Cox, M M and Goodman, M F and Kreuzer, K N and Sherratt, D J and Sandler, S J and Marians, K J} } @article {864, title = {In vitro polymerization and copolymerization of 3-hydroxypropionyl-CoA with the PHB synthase from Ralstonia eutropha.}, journal = {Biomacromolecules}, volume = {1}, year = {2000}, month = {2000 Fall}, pages = {433-9}, abstract = {The poly(3-hydroxybutyrate) (PHB) synthase of Ralstonia eutropha, which was produced by a recombinant strain of Escherichia coli and purified in one step with a methyl-HIC column to a purity of more than 90\%, was used to polymerize 3-hydroxypropionyl-CoA (3HPCoA) and to copolymerize 3HPCoA with 3-hydroxybutyryl-CoA (3HBCoA). A Km of 189 microM and a kcat of 10 s-1 were determined for the activity of the enzyme in the polymerization reaction of 3HPCoA based on the assumption that the dimer form of PHB synthase was the active form. Free coenzyme A was found to be a very effective competitive inhibitor for the polymerization of 3HPCoA with a Ki of 85 microM. The maximum degree of conversion of 3HPCoA to polymer was less than 40\%. In the simultaneous copolymerization reactions of these two monomers, both the turnover number for the copolymerization reaction and the maximum degree of conversion of 3HPCoA and 3HBCoA to copolymers increased with an increase in the amount of 3HBCoA in the monomer mixture. However, the maximum conversion of 3HPCoA to copolymer was always less than 35\%, regardless of the ratio of 3HPCoA to 3HBCoA. Block copolymers were obtained by the sequential copolymerization of the two monomers and these copolymers had a much narrower molecular weight distribution than those obtained by the simultaneous copolymerization for the same molar ratio of 3HPCoA to 3HBCoA.}, keywords = {Acyltransferases, Coenzyme A, Escherichia coli, Kinetics, Magnetic Resonance Spectroscopy, Molecular Weight}, issn = {1525-7797}, author = {Song, J J and Zhang, S and Lenz, R W and Goodwin, S} } @article {827, title = {Multiple genetic pathways for restarting DNA replication forks in Escherichia coli K-12.}, journal = {Genetics}, volume = {155}, year = {2000}, month = {2000 Jun}, pages = {487-97}, abstract = {In Escherichia coli, the primosome assembly proteins, PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG, are thought to help to restart DNA replication forks at recombinational intermediates. Redundant functions between priB and priC and synthetic lethality between priA2::kan and rep3 mutations raise the possibility that there may be multiple pathways for restarting replication forks in vivo. Herein, it is shown that priA2::kan causes synthetic lethality when placed in combination with either Deltarep::kan or priC303:kan. These determinations were made using a nonselective P1 transduction-based viability assay. Two different priA2::kan suppressors (both dnaC alleles) were tested for their ability to rescue the priA-priC and priA-rep double mutant lethality. Only dnaC809,820 (and not dnaC809) could rescue the lethality in each case. Additionally, it was shown that the absence of the 3{\textquoteright}-5{\textquoteright} helicase activity of both PriA and Rep is not the critical missing function that causes the synthetic lethality in the rep-priA double mutant. One model proposes that replication restart at recombinational intermediates occurs by both PriA-dependent and PriA-independent pathways. The PriA-dependent pathways require at least priA and priB or priC, and the PriA-independent pathway requires at least priC and rep. It is further hypothesized that the dnaC809 suppression of priA2::kan requires priC and rep, whereas dnaC809,820 suppression of priA2::kan does not.}, keywords = {Base Sequence, DNA Primers, DNA Replication, Escherichia coli, Genes, Lethal, Mutation, Polymerase Chain Reaction}, issn = {0016-6731}, author = {Sandler, S J} } @article {829, title = {Role of PriA in replication fork reactivation in Escherichia coli.}, journal = {J Bacteriol}, volume = {182}, year = {2000}, month = {2000 Jan}, pages = {9-13}, keywords = {Bacterial Proteins, DNA Primase, DNA Repair, DNA Replication, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Models, Genetic, Replication Protein A}, issn = {0021-9193}, author = {Sandler, S J and Marians, K J} } @article {830, title = {dnaC mutations suppress defects in DNA replication- and recombination-associated functions in priB and priC double mutants in Escherichia coli K-12.}, journal = {Mol Microbiol}, volume = {34}, year = {1999}, month = {1999 Oct}, pages = {91-101}, abstract = {PriA, PriB and PriC were originally discovered as proteins essential for the PhiX174 in vitro DNA replication system. Recent studies have shown that PriA mutants are poorly viable, have high basal levels of SOS expression (SOSH), are recombination deficient (Rec-), sensitive to UV irradiation (UVS) and sensitive to rich media. These data suggest that priA{\textquoteright}s role may be more complex than previously thought and may involve both DNA replication and homologous recombination. Based on the PhiX174 system, mutations in priB and priC should cause phenotypes like those seen in priA2:kan mutants. To test this, mutations in priB and priC were constructed. We found that, contrary to the PhiX174 model, del(priB)302 and priC303:kan mutants have almost wild-type phenotypes. Most unexpectedly, we then found that the priBC double mutant had very poor viability and/or a slow growth rate (even less than a priA2:kan mutant). This suggests that priB and priC have a redundant and important role in Escherichia coli. The priA2:kan suppressor, dnaC809, partially suppressed the poor viability/slow growth phenotype of the priBC double mutant. The resulting triple mutant (priBC dnaC809 ) had small colony size, recombination deficiency and levels of SOS expression similar to a priA2:kan mutant. The priBC dnaC809 mutant, however, was moderately UVR and had good viability, unlike a priA2:kan mutant. Additional mutations in the triple mutant were selected to suppress the slow growth phenotype. One suppressor restored all phenotypes tested to nearly wild-type levels. This mutation was identified as dnaC820 (K178N) [mapping just downstream of dnaC809 (E176G)]. Experiments suggest that dnaC820 makes dnaC809 suppression of priA and or priBC mutants priB and or priC independent. A model is proposed for the roles of these proteins in terms of restarting collapsed replication forks from recombinational intermediates.}, keywords = {Bacterial Proteins, Bacteriophage mu, Cell Division, DNA Replication, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Mutation, Phenotype, Recombination, Genetic, Replication Protein A, SOS Response (Genetics), Substrate Specificity, Suppression, Genetic}, issn = {0950-382X}, author = {Sandler, S J and Marians, K J and Zavitz, K H and Coutu, J and Parent, M A and Clark, A J} } @article {403, title = {Interdependence of the position and orientation of SoxS binding sites in the transcriptional activation of the class I subset of Escherichia coli superoxide-inducible promoters.}, journal = {Mol Microbiol}, volume = {34}, year = {1999}, month = {1999 Nov}, pages = {414-30}, abstract = {SoxS is the direct transcriptional activator of the member genes of the Escherichia coli superoxide regulon. At class I SoxS-dependent promoters, e.g. zwf and fpr, whose SoxS binding sites ({\textquoteright}soxbox{\textquoteright}) lie upstream of the -35 region of the promoter, activation requires the C-terminal domain of the RNA polymerase alpha-subunit, while at class II SoxS-dependent promoters, e.g. fumC and micF, whose binding sites overlap the -35 region, activation is independent of the alpha-CTD. To determine whether SoxS activation of its class I promoters shows the same helical phase-dependent spacing requirement as class I promoters activated by catabolite gene activator protein, we increased the 7 bp distance between the 20 bp zwf soxbox and the zwf -35 promoter hexamer by 5 bp and 11 bp, and we decreased the 15 bp distance between the 20 bp fpr soxbox and the fpr -35 promoter hexamer by the same amounts. In both cases, displacement of the binding site by a half or full turn of the DNA helix prevented transcriptional activation. With constructs containing the binding site of one gene fused to the promoter of the other, we demonstrated that the positional requirements are a function of the specific binding site, not the promoter. Supposing that opposite orientation of the SoxS binding site at the two promoters might account for the positional requirements, we placed the zwf and fpr soxboxes in the reverse orientation at the various positions upstream of the promoters and determined the effect of orientation on transcription activation. We found that reversing the orientation of the zwf binding site converts its positional requirement to that of the fpr binding site in its normal orientation, and vice versa. Analysis by molecular information theory of DNA sequences known to bind SoxS in vitro is consistent with the opposite orientation of the zwf and fpr soxboxes.}, keywords = {Bacterial Proteins, Base Sequence, Binding Sites, Escherichia coli, Escherichia coli Proteins, Molecular Sequence Data, Plasmids, Promoter Regions, Genetic, Sequence Analysis, DNA, Superoxides, Trans-Activators, Transcription Factors, Transcription, Genetic, Transcriptional Activation}, issn = {0950-382X}, author = {Wood, T I and Griffith, K L and Fawcett, W P and Jair, K W and Schneider, T D and Wolf, R E} } @article {831, title = {Replication fork assembly at recombination intermediates is required for bacterial growth.}, journal = {Proc Natl Acad Sci U S A}, volume = {96}, year = {1999}, month = {1999 Mar 30}, pages = {3552-5}, abstract = {PriA, a 3{\textquoteright} --> 5{\textquoteright} DNA helicase, directs assembly of a primosome on some bacteriophage and plasmid DNAs. Primosomes are multienzyme replication machines that contribute both the DNA-unwinding and Okazaki fragment-priming functions at the replication fork. The role of PriA in chromosomal replication is unclear. The phenotypes of priA null mutations suggest that the protein participates in replication restart at recombination intermediates. We show here that PriA promotes replication fork assembly at a D loop, an intermediate formed during initiation of homologous recombination. We also show that DnaC810, encoded by a naturally arising intergenic suppressor allele of the priA2::kan mutation, bypasses the need for PriA during replication fork assembly at D loops in vitro. These findings underscore the essentiality of replication fork restart at recombination intermediates under normal growth conditions in bacteria.}, keywords = {Bacteria, Bacteriophage phi X 174, Base Sequence, DNA Polymerase III, DNA Replication, DNA-Binding Proteins, Escherichia coli, Molecular Sequence Data, Oligodeoxyribonucleotides, Open Reading Frames, Recombination, Genetic, Replication Protein A, Templates, Genetic}, issn = {0027-8424}, author = {Liu, J and Xu, L and Sandler, S J and Marians, K J} } @article {833, title = {RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange.}, journal = {Genes Dev}, volume = {12}, year = {1998}, month = {1998 May 1}, pages = {1248-53}, abstract = {With the discovery that the Saccharomyces cerevisiae Rad51 protein is both structurally and functionally similar to the Escherichia coli RecA protein, the RecA paradigm for homologous recombination was extended to the Eucarya. The ubiquitous presence of RecA and Rad51 protein homologs raises the question of whether this archetypal protein exists within the third domain of life, the Archaea. Here we present the isolation of a Rad51/RecA protein homolog from the archaeon Sulfolobus solfataricus, and show that this protein, RadA, possesses the characteristics of a DNA strand exchange protein: The RadA protein is a DNA-dependent ATPase, forms a nucleoprotein filament on DNA, and catalyzes DNA pairing and strand exchange.}, keywords = {Adenosine Triphosphatases, Archaeal Proteins, Bacterial Proteins, DNA Helicases, DNA, Archaeal, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Genes, Archaeal, Nucleoproteins, Rad51 Recombinase, Rec A Recombinases, Recombinant Proteins, Recombination, Genetic, Sulfolobus}, issn = {0890-9369}, author = {Seitz, E M and Brockman, J P and Sandler, S J and Clark, A J and Kowalczykowski, S C} } @article {836, title = {Differential suppression of priA2::kan phenotypes in Escherichia coli K-12 by mutations in priA, lexA, and dnaC.}, journal = {Genetics}, volume = {143}, year = {1996}, month = {1996 May}, pages = {5-13}, abstract = {First identified as an essential component of the phi X174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec-, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA{\textquoteright}s primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.}, keywords = {Bacterial Proteins, Bacteriophage phi X 174, beta-Galactosidase, Chromosomes, Bacterial, DNA Helicases, DNA Replication, DNA-Binding Proteins, Dose-Response Relationship, Radiation, Escherichia coli, Escherichia coli Proteins, Genes, Bacterial, Genetic Markers, Mutagenesis, Phenotype, Recombination, Genetic, Replication Protein A, Repressor Proteins, Serine Endopeptidases, Suppression, Genetic, Transduction, Genetic, Ultraviolet Rays}, issn = {0016-6731}, author = {Sandler, S J and Samra, H S and Clark, A J} } @article {796, title = {Mosaic structure of plasmids from natural populations of Escherichia coli.}, journal = {Genetics}, volume = {143}, year = {1996}, month = {1996 Jul}, pages = {1091-100}, abstract = {The distribution of plasmids related to the fertility factor F was examined in the ECOR reference collection of Escherichia coli. Probes specific for four F-related genes were isolated and used to survey the collection by DNA hybridization. To estimate the genetic diversity of genes in F-like plasmids, DNA sequences were obtained for four plasmid genes. The phylogenetic relationships among the plasmids in the ECOR strains is very different from that of the strains themselves. This finding supports the view that plasmid transfer has been frequent within and between the major groups of ECOR. Furthermore, the sequences indicate that recombination between genes in plasmids takes place at a considerably higher frequency than that observed for chromosomal genes. The plasmid genes, and by inference the plasmids themselves, are mosaic in structure with different regions acquired from different sources. Comparison of gene sequences from a variety of naturally occurring plasmids suggested a plausible donor of some of the recombinant regions as well as implicating a chi site in the mechanism of genetic exchange. The relatively high rate of recombination in F-plasmid genes suggests that conjugational gene transfer may play a greater role in bacterial population structure than previously appreciated.}, keywords = {Bacterial Proteins, Base Sequence, DNA Helicases, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Evolution, Molecular, Membrane Proteins, Molecular Sequence Data, Mosaicism, Nucleic Acid Hybridization, Phylogeny, Plasmids, Polymorphism, Genetic, Proteins, Recombination, Genetic, Repressor Proteins, RNA-Binding Proteins, Trans-Activators}, issn = {0016-6731}, author = {Boyd, E F and Hill, C W and Rich, S M and Hartl, D L} } @article {837, title = {Overlapping functions for recF and priA in cell viability and UV-inducible SOS expression are distinguished by dnaC809 in Escherichia coli K-12.}, journal = {Mol Microbiol}, volume = {19}, year = {1996}, month = {1996 Feb}, pages = {871-80}, abstract = {The recF and priA genes have roles in DNA repair and homologous recombination. Mutations in these genes also cause decreases in cell viability and alterations in UV-inducible sulAp-lacZ (SOS) expression. To find out if the two genes are in the same or different pathways for viability and SOS expression, the phenotypes of the double mutant strains were studied. The recF priA double mutant showed a lower viability and SOS expression level than either of the single mutants. In the case of cell viability, recF missense mutations decreased viability of a priA2::kan strain two to five-fold whereas recF null priA2::kan double mutants were not viable at all. dnaC809, a mutation that suppresses the UV-sensitive (UVs and Rec- phenotypes of priA2::kan, restored cell viability, but not UV-inducible SOS expression, to a priA recF strain. Since recF is epistatic with recO and recR (recOR) for UV resistance, recOR mutations were also tested with priA2::kan. No overlap was found between recOR and priA for viability and SOS expression. It is concluded that priA and recF have two different overlapping functions in viability and SOS expression that are distinguishable by the effects of dnaC809. The role of recF in a priA2::kan strain in cell viability is a new function for recF and unlike recF{\textquoteright}s other roles in DNA repair and recombination, is independent of recOR. A new role for priA in UV-inducible SOS expression in a recF mutant is also defined.}, keywords = {Bacterial Proteins, DNA Repair, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Genes, Bacterial, Lac Operon, Mutation, Recombinant Fusion Proteins, Recombination, Genetic, Replication Protein A, SOS Response (Genetics), Ultraviolet Rays}, issn = {0950-382X}, author = {Sandler, S J} } @article {835, title = {recA-like genes from three archaean species with putative protein products similar to Rad51 and Dmc1 proteins of the yeast Saccharomyces cerevisiae.}, journal = {Nucleic Acids Res}, volume = {24}, year = {1996}, month = {1996 Jun 1}, pages = {2125-32}, abstract = {The process of homologous recombination has been documented in bacterial and eucaryotic organisms. The Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins are the archetypal members of two related families of proteins that play a central role in this process. Using the PCR process primed by degenerate oligonucleotides designed to encode regions of the proteins showing the greatest degree of identity, we examined DNA from three organisms of a third phylogenetically divergent group, Archaea, for sequences encoding proteins similar to RecA and Rad51. The archaeans examined were a hyperthermophilic acidophile, Sulfolobus sofataricus (Sso); a halophile, Haloferax volcanii (Hvo); and a hyperthermophilic piezophilic methanogen, Methanococcus jannaschii (Mja). The PCR generated DNA was used to clone a larger genomic DNA fragment containing an open reading frame (orf), that we refer to as the radA gene, for each of the three archaeans. As shown by amino acid sequence alignments, percent amino acid identities and phylogenetic analysis, the putative proteins encoded by all three are related to each other and to both the RecA and Rad51 families of proteins. The putative RadA proteins are more similar to the Rad51 family (approximately 40\% identity at the amino acid level) than to the RecA family (approximately 20\%). Conserved sequence motifs, putative tertiary structures and phylogenetic analysis implied by the alignment are discussed. The 5{\textquoteright} ends of mRNA transcripts to the Sso radA were mapped. The levels of radA mRNA do not increase after treatment with UV irradiation as do recA and RAD51 transcripts in E.coli and S.cerevisiae. Hence it is likely that radA in this organism is a constitutively expressed gene and we discuss possible implications of the lack of UV-inducibility.}, keywords = {Amino Acid Sequence, Archaea, Base Sequence, Cell Cycle Proteins, DNA-Binding Proteins, Escherichia coli, Halobacteriaceae, Methanococcus, Models, Molecular, Molecular Sequence Data, Phylogeny, Rad51 Recombinase, Rec A Recombinases, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Alignment, Sulfolobus}, issn = {0305-1048}, author = {Sandler, S J and Satin, L H and Samra, H S and Clark, A J} } @article {838, title = {recO and recR mutations delay induction of the SOS response in Escherichia coli.}, journal = {Mol Gen Genet}, volume = {246}, year = {1995}, month = {1995 Jan 20}, pages = {254-8}, abstract = {RecF, RecO and RecR, three of the important proteins of the RecF pathway of recombination, are also needed for repair of DNA damage due to UV irradiation. recF mutants are not proficient in cleaving LexA repressor in vivo following DNA damage: therefore they show a delay of induction of the SOS response. In this communication, by measuring the in vivo levels of LexA repressor using anti-LexA antibodies, we show that recO and recR mutant strains are also not proficient in LexA cleavage reactions. In addition, we show that recO and recR mutations delay induction of beta-galactosidase activity expressed from a lexA-regulated promoter following exposure of cells to UV, thus further supporting the idea that recF, recO and recR gene products are needed for induction of the SOS response.}, keywords = {Bacterial Proteins, beta-Galactosidase, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Mutation, Promoter Regions, Genetic, Recombinant Fusion Proteins, Repressor Proteins, Serine Endopeptidases, SOS Response (Genetics), Ultraviolet Rays}, issn = {0026-8925}, author = {Hegde, S and Sandler, S J and Clark, A J and Madiraju, M V} } @article {842, title = {Homologous genetic recombination: the pieces begin to fall into place.}, journal = {Crit Rev Microbiol}, volume = {20}, year = {1994}, month = {1994}, pages = {125-42}, abstract = {One of the authors (AJC) acknowledges with gratitude the important role Fernando Bastarrachea played in the author{\textquoteright}s discovery that E. coli could carry out homologous genetic recombination by multiple pathways. This in turn led to the discovery of several genes, including recF, recO, and recR, whose role in recombination would not otherwise have been detected. Subsequent genetic and biochemical studies have led to a general formulation in which there are multiple nucleolytic ways to achieve a presynaptic intermediate bound to RecA protein. Postsynaptic events in the general formulation occur by means of multiple branch migration enzymes to form Holliday DNA structures and a specific nuclease to cleave them. The general formulation is built on synapsis catalyzed by RecA protein. A second RecA-independent synapsis catalyzed by RecT (and RecE?) protein is now under study and a third type independent of both RecA and RecT has apparently been discovered. How these will affect the general formulation remains to be seen. Some proteins, most prominently RecF, RecO, and RecR, have no role in the general formulation. The hypothesis is presented that these proteins act as a switch between replication and recombination by helping to convert replication to recombination intermediates. Universality of the general formulation is supported by the widespread occurrence of recA, recB, recC, and recD genes among bacteria. Recent discovery of recA-like genes in several eukaryotes further supports its universality. We have contributed additional support by sequencing a recA-like gene from an archaeal species, thus making it plausible that the mechanism of synapsis worked out for E. coli RecA protein will hold for all three organismal domains. The boundaries of the puzzle of homologous genetic recombination therefore seem complete and the pieces to the complex picture they encompass are falling into place.}, keywords = {DNA Replication, Escherichia coli, Genes, Bacterial, Rec A Recombinases, Recombination, Genetic}, issn = {1040-841X}, doi = {10.3109/10408419409113552}, author = {Clark, A J and Sandler, S J} } @article {840, title = {Mutational analysis of sequences in the recF gene of Escherichia coli K-12 that affect expression.}, journal = {J Bacteriol}, volume = {176}, year = {1994}, month = {1994 Jul}, pages = {4011-6}, abstract = {The level of translation of recF-lacZ fusions is reduced 20-fold by nucleotides 49 to 146 of recF. In this region of recF, we found a previously described ribosome-interactive sequence called epsilon and a hexapyrimidine tract located just upstream of the epsilon sequence. Mutational studies indicate that the hexapyrimidine sequence is involved in at least some of the reduced translation. When the hexapyrimidine sequence is mutant, mutating epsilon increases the level of translation maximally. We ruled out the possibility that ribosome frameshifting explains most of the effect of these two sequences on expression and suspect that multiple mechanisms may be responsible. In a separate report, we show that mutations in the hexapyrimidine tract and epsilon increase expression of the full-sized recF gene.}, keywords = {Bacterial Proteins, Base Sequence, DNA Mutational Analysis, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Bacterial, Half-Life, Lac Operon, Molecular Sequence Data, Protein Biosynthesis, Recombinant Fusion Proteins, Regulatory Sequences, Nucleic Acid, Ribosomes}, issn = {0021-9193}, author = {Sandler, S J and Clark, A J} } @article {841, title = {RecOR suppression of recF mutant phenotypes in Escherichia coli K-12.}, journal = {J Bacteriol}, volume = {176}, year = {1994}, month = {1994 Jun}, pages = {3661-72}, abstract = {The recF, recO, and recR genes form the recFOR epistasis group for DNA repair. recF mutants are sensitive to UV irradiation and fail to properly induce the SOS response. Using plasmid derivatives that overexpress combinations of the recO+ and recR+ genes, we tested the hypothesis that high-level expression of recO+ and recR+ (recOR) in vivo will indirectly suppress the recF mutant phenotypes mentioned above. We found that overexpression of just recR+ from the plasmid will partially suppress both phenotypes. Expression of the chromosomal recO+ gene is essential for the recR+ suppression. Hence we call this RecOR suppression of recF mutant phenotypes. RecOR suppression of SOS induction is more efficient with recO+ expression from a plasmid than with recO+ expression from the chromosome. This is not true for RecOR suppression of UV sensitivity (the two are equal). Comparison of RecOR suppression with the suppression caused by recA801 and recA803 shows that RecOR suppression of UV sensitivity is more effective than recA803 suppression and that RecOR suppression of UV sensitivity, like recA801 suppression, requires recJ+. We present a model that explains the data and proposes a function for the recFOR epistasis group in the induction of the SOS response and recombinational DNA repair.}, keywords = {Bacterial Proteins, Base Sequence, DNA Damage, DNA Repair, DNA-Binding Proteins, Epistasis, Genetic, Escherichia coli, Escherichia coli Proteins, Models, Genetic, Molecular Sequence Data, Phenotype, Plasmids, SOS Response (Genetics), Suppression, Genetic, Ultraviolet Rays}, issn = {0021-9193}, author = {Sandler, S J and Clark, A J} } @article {839, title = {Studies on the mechanism of reduction of UV-inducible sulAp expression by recF overexpression in Escherichia coli K-12.}, journal = {Mol Gen Genet}, volume = {245}, year = {1994}, month = {1994 Dec 15}, pages = {741-9}, abstract = {UV-inducible sulAp expression, an indicator of the SOS response, is reduced by recF+ overexpression in vivo. Different DNA-damaging agents and amounts of RecO and RecR were tested for their effects on this phenotype. It was found that recF+ overexpression reduced sulAp expression after DNA damage by mitomycin C or nalidixic acid, recO+ and recR+ overexpression partially suppressed the reduction of UV-induced sulAp expression caused by recF+ overexpression. The requirement for ATP binding to RecF to produce the phenotype was tested by genetically altering the putative phosphate binding cleft of recF in a way that should prevent the mutant recF protein from binding ATP. It was found that a change of lysine to glutamine at codon 36 results in a mutant recF protein (RecF4115) that is unable to reduce UV-inducible sulAp expression when overproduced. It is inferred from these results that recF overexpression may reduce UV-inducible sulAp expression by a mechanism that is sensitive to the ability of RecF to bind ATP and to the levels of RecO and RecR (RecOR) in the cell, but not to the type of DNA damage per se. Models are explored that can explain how recF+ overexpression reduces UV induction of sulAp and how RecOR overproduction might suppress this phenotype.}, keywords = {Bacterial Proteins, Base Sequence, DNA Primers, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genes, Bacterial, Molecular Sequence Data, Mutagenesis, Site-Directed, Restriction Mapping, RNA, Messenger, SOS Response (Genetics), Structure-Activity Relationship, Ultraviolet Rays}, issn = {0026-8925}, author = {Sandler, S J} } @article {843, title = {Use of high and low level overexpression plasmids to test mutant alleles of the recF gene of Escherichia coli K-12 for partial activity.}, journal = {Genetics}, volume = {135}, year = {1993}, month = {1993 Nov}, pages = {643-54}, abstract = {We showed that sufficient overexpression of the wild-type recF gene interfered with three normal cell functions: (1) UV induction of transcription from the LexA-protein-repressed sulA promoter, (2) UV resistance and (3) cell viability at 42 degrees. To show this, we altered a low-level overexpressing recF+ plasmid with a set of structurally neutral mutations that increased the rate of expression of recF. The resulting high-level overexpressing plasmid interfered with UV induction of the sulA promoter, as did the low-level overexpressing plasmid. It also reduced UV resistance more than its low level progenitor and decreased viability at 42 degrees, an effect not seen with the low-level plasmid. We used the high-level plasmid to test four recF structural mutations for residual activity. The structural alleles consisted of an insertion mutation, two single amino acid substitution mutations and a double amino acid substitution mutation. On the Escherichia coli chromosome the three substitution mutations acted similarly to a recF deletion in reducing UV resistance in a recB21 recC22 sbcB15 sbcC201 genetic background. By this test, therefore, all three appeared to be null alleles. Measurements of conjugational recombination revealed, however, that the three substitution mutations may have residual activity. On the high-level overexpressing plasmid all three substitution mutations definitely showed partial activity. By contrast, the insertion mutation on the high-level overexpressing plasmid showed no partial activity and can be considered a true null mutation. One of the substitutions, recF143, showed a property attributable to a leaky mutation. Another substitution, recF4101, may block selectively two of the three interference phenotypes, thus allowing us to infer a mechanism for them.}, keywords = {Alleles, Bacterial Proteins, Base Sequence, Chromosomes, Bacterial, DNA Repair, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Gene Expression, Genes, Bacterial, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Phenotype, Plasmids, Radiation Tolerance, Recombination, Genetic, Ultraviolet Rays}, issn = {0016-6731}, author = {Sandler, S J and Clark, A J} } @article {844, title = {Sequence and complementation analysis of recF genes from Escherichia coli, Salmonella typhimurium, Pseudomonas putida and Bacillus subtilis: evidence for an essential phosphate binding loop.}, journal = {Nucleic Acids Res}, volume = {20}, year = {1992}, month = {1992 Feb 25}, pages = {839-45}, abstract = {We have compared the recF genes from Escherichia coli K-12, Salmonella typhimurium, Pseudomonas putida, and Bacillus subtilis at the DNA and amino acid sequence levels. To do this we determined the complete nucleotide sequence of the recF gene from Salmonella typhimurium and we completed the nucleotide sequence of recF gene from Pseudomonas putida begun by Fujita et al. (1). We found that the RecF proteins encoded by these two genes contain respectively 92\% and 38\% amino acid identity with the E. coli RecF protein. Additionally, we have found that the S. typhimurium and P. putida recF genes will complement an E. coli recF mutant, but the recF gene from Bacillus subtilis [showing about 20\% identity with E. coli (2)] will not. Amino acid sequence alignment of the four proteins identified four highly conserved regions. Two of these regions are part of a putative phosphate binding loop. In one region (position 36), we changed the lysine codon (which is essential for ATPase, GTPase and kinase activity in other proteins having this phosphate binding loop) to an arginine codon. We then tested this mutation (recF4101) on a multicopy plasmid for its ability to complement a recF chromosomal mutation and on the E. coli chromosome for its effect on sensitivity to UV irradiation. The strain with recF4101 on its chromosome is as sensitive as a null recF mutant strain. The strain with the plasmid-borne mutant allele is however more UV resistant than the null mutant strain. We conclude that lysine-36 and possibly a phosphate binding loop is essential for full recF activity. Lastly we made two chimeric recF genes by exchanging the amino terminal 48 amino acids of the S. typhimurium and E. coli recF genes. Both chimeras could complement E. coli chromosomal recF mutations.}, keywords = {Amino Acid Sequence, Bacillus subtilis, Bacterial Proteins, Base Sequence, Binding Sites, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Genes, Bacterial, Genetic Complementation Test, Molecular Sequence Data, Nucleic Acid Conformation, Phosphates, Pseudomonas putida, Salmonella typhimurium, Sequence Alignment}, issn = {0305-1048}, author = {Sandler, S J and Chackerian, B and Li, J T and Clark, A J} } @article {845, title = {Factors affecting expression of the recF gene of Escherichia coli K-12.}, journal = {Gene}, volume = {86}, year = {1990}, month = {1990 Jan 31}, pages = {35-43}, abstract = {This report describes four factors which affect expression of the recF gene from strong upstream lambda promoters under temperature-sensitive cIAt2-encoded repressor control. The first factor was the long mRNA leader sequence consisting of the Escherichia coli dnaN gene and 95\% of the dnaA gene and lambda bet, N (double amber) and 40\% of the exo gene. When most of this DNA was deleted, RecF became detectable in maxicells. The second factor was the vector, pBEU28, a runaway replication plasmid. When we substituted pUC118 for pBEU28, RecF became detectable in whole cells by the Coomassie blue staining technique. The third factor was the efficiency of initiation of translation. We used site-directed mutagenesis to change the mRNA leader, ribosome-binding site and the 3 bp before and after the translational start codon. Monitoring the effect of these mutational changes by translational fusion to lacZ, we discovered that the efficiency of initiation of translation was increased 30-fold. Only an estimated two- or threefold increase in accumulated levels of RecF occurred, however. This led us to discover the fourth factor, namely sequences in the recF gene itself. These sequences reduce expression of the recF-lacZ fusion genes 100-fold. The sequences responsible for this decrease in expression occur in four regions in the N-terminal half of recF. Expression is reduced by some sequences at the transcriptional level and by others at the translational level.}, keywords = {Bacterial Proteins, Base Sequence, Cloning, Molecular, Escherichia coli, Gene Expression Regulation, Bacterial, Genes, Bacterial, Molecular Sequence Data, Peptide Chain Initiation, Translational, Recombinant Fusion Proteins, Recombination, Genetic, Transcription, Genetic}, issn = {0378-1119}, author = {Sandler, S J and Clark, A J} } @article {847, title = {Genes of the RecE and RecF pathways of conjugational recombination in Escherichia coli.}, journal = {Cold Spring Harb Symp Quant Biol}, volume = {49}, year = {1984}, month = {1984}, pages = {453-62}, keywords = {Chromosome Mapping, Conjugation, Genetic, Escherichia coli, Genes, Bacterial, Mutation, Protein Biosynthesis, Recombination, Genetic}, issn = {0091-7451}, author = {Clark, A J and Sandler, S J and Willis, D K and Chu, C C and Blanar, M A and Lovett, S T} } @article {846, title = {Molecular analysis of the recF gene of Escherichia coli.}, journal = {Proc Natl Acad Sci U S A}, volume = {81}, year = {1984}, month = {1984 Aug}, pages = {4622-6}, abstract = {We analyzed the nucleotide sequence of a 1.325-kilobase region of wild-type Escherichia coli containing a functional recF gene and six Tn3 mutations that inactivate recF. The analysis shows a potentially translatable reading frame of 1071 nucleotides, which is interrupted by all six insertions. A protein of 40.5 kilodaltons would result from translation of the open reading frame, and a radioactive band of protein of an apparent molecular weight of approximately 40 kilodaltons was seen by the maxicell method using a recF+ plasmid. Putative truncated peptides were seen when two recF::Tn3 mutant plasmids were used. Differential expression of dnaN and recF from a common promoter was noted. recF332::Tn3 was transferred to the chromosome where, in hemizygous condition, it produced UV sensitivity indistinguishable from that produced by two presumed recF point mutations.}, keywords = {Amino Acid Sequence, Bacterial Proteins, Base Sequence, DNA Repair, DNA Replication, Escherichia coli, Genes, Bacterial, Molecular Weight, Mutation, Plasmids, Recombination, Genetic}, issn = {0027-8424}, author = {Blanar, M A and Sandler, S J and Armengod, M E and Ream, L W and Clark, A J} } @article {1715, title = {Mechanistic studies of glutamine synthetase from Escherichia coli: kinetic evidence for two reaction intermediates in biosynthetic reaction.}, journal = {Proc Natl Acad Sci U S A}, volume = {73}, year = {1976}, month = {1976 Feb}, pages = {476-80}, abstract = {

Fast reaction techniques were used to study the kinetics of protein fluorescence intensity changes that are associated with the reactions of unadenylylated Escherichia coli glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] with its substrates. It was established that the synthesis of glutamine occurs by a stepwise mechanism. During the catalytic process two fluorometrically distinct intermediates were observed. Both forward and reverse rate constants which lead to the formation and consumption of these intermediates were evaluated. The catalytic rate constant, kc, which was calculated from these rate constants agrees well with the values of kc which were determined by direct measurement of the overall biosynthetic activities by means of stopped-flow technique or the steady-state assay method.

}, keywords = {Enzyme Activation, Escherichia coli, Glutamate-Ammonia Ligase, Kinetics, Magnesium}, issn = {0027-8424}, author = {Rhee, S G and Chock, P B} } @article {1714, title = {[Intermediate plateaux in kinetics of the reaction catalyzed by biodegradative L-threonine dehydratase from Escherichia coli].}, journal = {Biokhimiia}, volume = {40}, year = {1975}, month = {1975 May-Jun}, pages = {645-51}, abstract = {

It has been shown that for the reaction catalyzed by "biodegradative" L-threonine dehydratase from E. coli strains K-12 and 980 in 0.5 M phosphate-carbonate buffer, pH 8.4 and pH 9.5, the plots of initial reaction rate (v) versus the initial substrate concentration ([S]0 are characterized by several inflection points, i. e. an intermediate plateau. The plot of v versus the allosteric activator (AMP) concentration have very complicated shapes: there are several inflection points, and also the maximum at L-threonine concentration equal to 3-10(2) and 5-10(-2) M. High AMP concentrations inhibit the enzyme at high substrate concentrations. The reduced glutathion dose not influence the enzyme and does not alter the activating effect of AMP. On the basis of the data obtained it is proposed that the substrate and AMP shift the equilibrium between multiple oligomeric enzyme forms differing in catalytic activity and kinetic manifestations of allosteric interactions between the active and allosteric AMP-binding sites towards polymerization. Thus, the functioning the enzyme under study is discussed in the frames of the model of dissociating regulatory enzymes with multiple intermediate oligomeric forms.

}, keywords = {Adenosine Monophosphate, Allosteric Regulation, Allosteric Site, Binding Sites, Escherichia coli, Hydro-Lyases, Hydrogen-Ion Concentration, Kinetics, Protein Binding, Threonine Dehydratase}, issn = {0320-9725}, author = {Sinelnikova, E M and Dvoretskova, T V and Kagan, Z S} }