@article {3044, title = {Microbial nanowires with genetically modified peptide ligands to sustainably fabricate electronic sensing devices.}, journal = {Biosens Bioelectron}, volume = {226}, year = {2023}, month = {2023 Apr 15}, pages = {115147}, abstract = {

Nanowires have substantial potential as the sensor component in electronic sensing devices. However, surface functionalization of traditional nanowire and nanotube materials with short peptides that increase sensor selectivity and sensitivity requires complex chemistries with toxic reagents. In contrast, microorganisms can assemble pilin monomers into protein nanowires with intrinsic conductivity from renewable feedstocks, yielding an electronic material that is robust and stable in applications, but also biodegradable. Here we report that the sensitivity and selectivity of protein nanowire-based sensors can be modified with a simple plug and play genetic approach in which a short peptide sequence, designed to bind the analyte of interest, is incorporated into the pilin protein that is microbially assembled into nanowires. We employed a scalable Escherichia coli chassis to fabricate protein nanowires that displayed either a peptide previously demonstrated to effectively bind ammonia, or a peptide known to bind acetic acid. Sensors comprised of thin films of the nanowires amended with the ammonia-specific peptide had a ca. 100-fold greater response to ammonia than sensors made with unmodified protein nanowires. Protein nanowires with the peptide that binds acetic acid yielded a 4-fold higher response than nanowires without the peptide. The protein nanowire-based sensors had greater responses than previously reported sensors fabricated with other nanomaterials. The results demonstrate that protein nanowires with enhanced sensor response for analytes of interest can be fabricated with a flexible genetic strategy that sustainably eliminates the energy, environmental, and health concerns associated with other common nanomaterials.

}, keywords = {Acetic Acid, Ammonia, Biosensing Techniques, Electronics, Fimbriae Proteins, Ligands, Nanowires, Peptides}, issn = {1873-4235}, doi = {10.1016/j.bios.2023.115147}, author = {Lekbach, Yassir and Ueki, Toshiyuki and Liu, Xiaomeng and Woodard, Trevor and Yao, Jun and Lovley, Derek R} } @article {3048, title = {Microbial biofilms for electricity generation from water evaporation and power to wearables.}, journal = {Nat Commun}, volume = {13}, year = {2022}, month = {2022 Jul 28}, pages = {4369}, abstract = {

Employing renewable materials for fabricating clean energy harvesting devices can further improve sustainability. Microorganisms can be mass produced with renewable feedstocks. Here, we demonstrate that it is possible to engineer microbial biofilms as a cohesive, flexible material for long-term continuous electricity production from evaporating water. Single biofilm sheet (~40 {\textmu}m thick) serving as the functional component in an electronic device continuously produces power density (~1 μW/cm) higher than that achieved with thicker engineered materials. The energy output is comparable to that achieved with similar sized biofilms catalyzing current production in microbial fuel cells, without the need for an organic feedstock or maintaining cell viability. The biofilm can be sandwiched between a pair of mesh electrodes for scalable device integration and current production. The devices maintain the energy production in ionic solutions and can be used as skin-patch devices to harvest electricity from sweat and moisture on skin to continuously power wearable devices. Biofilms made from different microbial species show generic current production from water evaporation. These results suggest that we can harness the ubiquity of biofilms in nature as additional sources of biomaterial for evaporation-based electricity generation in diverse aqueous environments.

}, keywords = {Bioelectric Energy Sources, Biofilms, Electricity, Electrodes, Water, Wearable Electronic Devices}, issn = {2041-1723}, doi = {10.1038/s41467-022-32105-6}, author = {Liu, Xiaomeng and Ueki, Toshiyuki and Gao, Hongyan and Woodard, Trevor L and Nevin, Kelly P and Fu, Tianda and Fu, Shuai and Sun, Lu and Lovley, Derek R and Yao, Jun} } @article {3065, title = {Intrinsically Conductive Microbial Nanowires for {\textquoteright}Green{\textquoteright} Electronics with Novel Functions.}, journal = {Trends Biotechnol}, volume = {39}, year = {2021}, month = {2021 Sep}, pages = {940-952}, abstract = {

Intrinsically conductive protein nanowires, microbially produced from inexpensive, renewable feedstocks, are a sustainable alternative to traditional nanowire electronic materials, which require high energy inputs and hazardous conditions/chemicals for fabrication and can be highly toxic. Pilin-based nanowires can be tailored for specific functions via the design of synthetic pilin genes to tune wire conductivity or introduce novel functionalities. Other microbially produced nanowire options for electronics may include cytochrome wires, curli fibers, and the conductive fibers of cable bacteria. Proof-of-concept protein nanowire electronics that have been successfully demonstrated include biomedical sensors, neuromorphic devices, and a device that generates electricity from ambient humidity. Further development of applications will require interdisciplinary teams of engineers, biophysicists, and synthetic biologists.

}, keywords = {Electric Conductivity, Electronics, Nanowires, Proteins}, issn = {1879-3096}, doi = {10.1016/j.tibtech.2020.12.005}, author = {Lovley, Derek R and Yao, Jun} } @article {3061, title = {Self-sustained green neuromorphic interfaces.}, journal = {Nat Commun}, volume = {12}, year = {2021}, month = {2021 Jun 07}, pages = {3351}, abstract = {

Incorporating neuromorphic electronics in bioelectronic interfaces can provide intelligent responsiveness to environments. However, the signal mismatch between the environmental stimuli and driving amplitude in neuromorphic devices has limited the functional versatility and energy sustainability. Here we demonstrate multifunctional, self-sustained neuromorphic interfaces by achieving signal matching at the biological level. The advances rely on the unique properties of microbially produced protein nanowires, which enable both bio-amplitude (e.g., <100 mV) signal processing and energy harvesting from ambient humidity. Integrating protein nanowire-based sensors, energy devices and memristors of bio-amplitude functions yields flexible, self-powered neuromorphic interfaces that can intelligently interpret biologically relevant stimuli for smart responses. These features, coupled with the fact that protein nanowires are a green biomaterial of potential diverse functionalities, take the interfaces a step closer to biological integration.

}, keywords = {Biocompatible Materials, Electronics, Nanotechnology, Nanowires, Neural Networks, Computer, Proteins, Synapses}, issn = {2041-1723}, doi = {10.1038/s41467-021-23744-2}, author = {Fu, Tianda and Liu, Xiaomeng and Fu, Shuai and Woodard, Trevor and Gao, Hongyan and Lovley, Derek R and Yao, Jun} } @article {3062, title = {Stainless steel corrosion via direct iron-to-microbe electron transfer by Geobacter species.}, journal = {ISME J}, volume = {15}, year = {2021}, month = {2021 Oct}, pages = {3084-3093}, abstract = {

Microbial corrosion of iron-based materials is a substantial economic problem. A mechanistic understanding is required to develop mitigation strategies, but previous mechanistic studies have been limited to investigations with relatively pure Fe(0), which is not a common structural material. We report here that the mechanism for microbial corrosion of stainless steel, the metal of choice for many actual applications, can be significantly different from that~for Fe(0). Although H is often an intermediary electron carrier between the metal and microbes during Fe(0) corrosion, we found that H is not abiotically produced from stainless steel, making this corrosion mechanism unlikely. Geobacter sulfurreducens and Geobacter metallireducens, electrotrophs that are known to directly accept electrons from other microbes or electrodes, extracted electrons from stainless steel via direct iron-to-microbe electron transfer. Genetic modification to prevent H consumption did not negatively impact on stainless steel corrosion. Corrosion was inhibited when genes for outer-surface cytochromes that are key electrical contacts were deleted. These results indicate that a common model of microbial Fe(0) corrosion~by hydrogenase-positive microbes, in which H serves as an intermediary electron carrier between the metal surface and the microbe, may not apply to the microbial corrosion of stainless steel. However, direct iron-to-microbe electron transfer is a~feasible route for stainless steel corrosion.

}, keywords = {Corrosion, Electrons, Geobacter, Iron, Stainless Steel}, issn = {1751-7370}, doi = {10.1038/s41396-021-00990-2}, author = {Tang, Hai-Yan and Yang, Chuntian and Ueki, Toshiyuki and Pittman, Conor C and Xu, Dake and Woodard, Trevor L and Holmes, Dawn E and Gu, Tingyue and Wang, Fuhui and Lovley, Derek R} } @article {3069, title = {Bioinspired bio-voltage memristors.}, journal = {Nat Commun}, volume = {11}, year = {2020}, month = {2020 Apr 20}, pages = {1861}, abstract = {

Memristive devices are promising candidates to emulate biological computing. However, the typical switching voltages (0.2-2 V) in previously described devices are much higher than the amplitude in biological counterparts. Here we demonstrate a type of diffusive memristor, fabricated from the protein nanowires harvested from the bacterium Geobacter sulfurreducens, that functions at the biological voltages of 40-100 mV. Memristive function at biological voltages is possible because the protein nanowires catalyze metallization. Artificial neurons built from these memristors not only function at biological action potentials (e.g., 100 mV, 1 ms) but also exhibit temporal integration close to that in biological neurons. The potential of using the memristor to directly process biosensing signals is also demonstrated.

}, keywords = {Action Potentials, Biosensing Techniques, Electricity, Electronics, Equipment Design, Geobacter, Humans, Molecular Dynamics Simulation, Nanotechnology, Nanowires, Neural Networks, Computer, Neurons, Synapses, Wearable Electronic Devices}, issn = {2041-1723}, doi = {10.1038/s41467-020-15759-y}, author = {Fu, Tianda and Liu, Xiaomeng and Gao, Hongyan and Ward, Joy E and Liu, Xiaorong and Yin, Bing and Wang, Zhongrui and Zhuo, Ye and Walker, David J F and Joshua Yang, J and Chen, Jianhan and Lovley, Derek R and Yao, Jun} } @article {3071, title = {Power generation from ambient humidity using protein nanowires.}, journal = {Nature}, volume = {578}, year = {2020}, month = {2020 Feb}, pages = {550-554}, abstract = {

Harvesting energy from the environment offers the promise of clean power for self-sustained systems. Known technologies-such as solar cells, thermoelectric devices and mechanical generators-have specific environmental requirements that restrict where they can be deployed and limit their potential for continuous energy production. The ubiquity of atmospheric moisture offers an alternative. However, existing moisture-based energy-harvesting technologies can produce only intermittent, brief (shorter than 50 seconds) bursts of power in the ambient environment, owing to the lack of a sustained conversion mechanism. Here we show that thin-film devices made from nanometre-scale protein wires harvested from the microbe Geobacter sulfurreducens can generate continuous electric power in the ambient environment. The devices produce a sustained voltage of around 0.5~volts across a 7-micrometre-thick film, with a current density of around 17 microamperes per square centimetre. We find the driving force behind this energy generation to be a self-maintained moisture gradient that forms within the film when the film is exposed to the humidity that is naturally present in air. Connecting several devices linearly scales up the voltage and current to power electronics. Our results demonstrate the feasibility of a continuous energy-harvesting strategy that is less restricted by location or environmental conditions than other sustainable approaches.

}, issn = {1476-4687}, doi = {10.1038/s41586-020-2010-9}, author = {Liu, Xiaomeng and Gao, Hongyan and Ward, Joy E and Liu, Xiaorong and Yin, Bing and Fu, Tianda and Chen, Jianhan and Lovley, Derek R and Yao, Jun} } @article {3076, title = {Cryo-EM reveals the structural basis of long-range electron transport in a cytochrome-based bacterial nanowire.}, journal = {Commun Biol}, volume = {2}, year = {2019}, month = {2019}, pages = {219}, abstract = {

Electrically conductive pili from species, termed bacterial nanowires, are intensely studied for their biological significance and potential in the development of new materials. Using cryo-electron microscopy, we have characterized nanowires from conductive pili preparations that are composed solely of head-to-tail stacked monomers of the six-heme C-type cytochrome OmcS. The unique fold of OmcS - closely wrapped around a continuous stack of hemes that can serve as an uninterrupted path for electron transport - generates a scaffold that supports the unbranched chain of hemes along the central axis of the filament. We present here, at 3.4 {\r A} resolution, the structure of this cytochrome-based filament and discuss its possible role in long-range biological electron transport.

}, keywords = {Cryoelectron Microscopy, Cytochromes c, Electron Transport, Fimbriae, Bacterial, Geobacter, Nanowires}, issn = {2399-3642}, doi = {10.1038/s42003-019-0448-9}, author = {Filman, David J and Marino, Stephen F and Ward, Joy E and Yang, Lu and Mester, Zolt{\'a}n and Bullitt, Esther and Lovley, Derek R and Strauss, Mike} } @article {3106, title = {Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation.}, journal = {ISME J}, volume = {10}, year = {2016}, month = {2016 Feb}, pages = {310-20}, abstract = {

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.

}, keywords = {Amino Acid Sequence, Bacterial Proteins, Biodegradation, Environmental, Cytochrome c Group, Electron Transport, Ferric Compounds, Geobacter, Groundwater, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, Proteomics, Sequence Alignment, Uranium}, issn = {1751-7370}, doi = {10.1038/ismej.2015.113}, author = {Yun, Jiae and Malvankar, Nikhil S and Ueki, Toshiyuki and Lovley, Derek R} } @article {3116, title = {Visualization of charge propagation along individual pili proteins using ambient electrostatic force microscopy.}, journal = {Nat Nanotechnol}, volume = {9}, year = {2014}, month = {2014 Dec}, pages = {1012-7}, abstract = {

The nanoscale imaging of charge flow in proteins is crucial to understanding several life processes, including respiration, metabolism and photosynthesis. However, existing imaging methods are only effective under non-physiological conditions or are limited to photosynthetic proteins. Here, we show that electrostatic force microscopy can be used to directly visualize charge propagation along pili of Geobacter sulfurreducens with nanometre resolution and under ambient conditions. Charges injected at a single point into individual, untreated pili, which are still attached to cells, propagated over the entire filament. The mobile charge density in the pili, as well as the temperature and pH dependence of the charge density, were similar to those of carbon nanotubes and other organic conductors. These findings, coupled with a lack of charge propagation in mutated pili that were missing key aromatic amino acids, suggest that the pili of G. sulfurreducens function as molecular wires with transport via delocalized charges, rather than the hopping mechanism that is typical of biological electron transport.

}, keywords = {Fimbriae Proteins, Fimbriae, Bacterial, Geobacter, Microscopy, Electrochemical, Scanning}, issn = {1748-3395}, doi = {10.1038/nnano.2014.236}, author = {Malvankar, Nikhil S and Yalcin, Sibel Ebru and Tuominen, Mark T and Lovley, Derek R} } @article {935, title = {Critical roles for lipomannan and lipoarabinomannan in cell wall integrity of mycobacteria and pathogenesis of tuberculosis.}, journal = {MBio}, volume = {4}, year = {2013}, month = {2013}, abstract = {

ABSTRACT Lipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial glycolipids containing a long mannose polymer. While they are implicated in immune modulations, the significance of LM and LAM as structural components of the mycobacterial cell wall remains unknown. We have previously reported that a branch-forming mannosyltransferase plays a critical role in controlling the sizes of LM and LAM and that deletion or overexpression of this enzyme results in gross changes in LM/LAM structures. Here, we show that such changes in LM/LAM structures have a significant impact on the cell wall integrity of mycobacteria. In Mycobacterium smegmatis, structural defects in LM and LAM resulted in loss of acid-fast staining, increased sensitivity to \β-lactam antibiotics, and faster killing by THP-1 macrophages. Furthermore, equivalent Mycobacterium\ tuberculosis mutants became more sensitive to \β-lactams, and one mutant showed attenuated virulence in mice. Our results revealed previously unknown structural roles for LM and LAM and further demonstrated that they are important for the pathogenesis of tuberculosis. IMPORTANCE Tuberculosis (TB) is a global burden, affecting millions of people worldwide. Mycobacterium tuberculosis is a causative agent of TB, and understanding the biology of M.\ tuberculosis is essential for tackling this devastating disease. The cell wall of M.\ tuberculosis is highly impermeable and plays a protective role in establishing infection. Among the cell wall components, LM and LAM are major glycolipids found in all Mycobacterium species, show various immunomodulatory activities, and have been thought to play roles in TB pathogenesis. However, the roles of LM and LAM as integral parts of the cell wall structure have not been elucidated. Here we show that LM and LAM play critical roles in the integrity of mycobacterial cell wall and the pathogenesis of TB. These findings will now allow us to seek the possibility that the LM/LAM biosynthetic pathway is a chemotherapeutic target.

}, issn = {2150-7511}, doi = {10.1128/mBio.00472-12}, author = {Fukuda, Takeshi and Matsumura, Takayuki and Ato, Manabu and Hamasaki, Maho and Nishiuchi, Yukiko and Murakami, Yoshiko and Maeda, Yusuke and Yoshimori, Tamotsu and Matsumoto, Sohkichi and Kobayashi, Kazuo and Kinoshita, Taroh and Morita, Yasu S} } @article {1565, title = {Guided cobalamin biosynthesis supports Dehalococcoides mccartyi reductive dechlorination activity.}, journal = {Philos Trans R Soc Lond B Biol Sci}, volume = {368}, year = {2013}, month = {2013 Apr 19}, pages = {20120320}, abstract = {

Dehalococcoides mccartyi strains are corrinoid-auxotrophic Bacteria and axenic cultures that require vitamin B12 (CN-Cbl) to conserve energy via organohalide respiration. Cultures of D. mccartyi strains BAV1, GT and FL2 grown with limiting amounts of 1 {\textmu}g l(-1) CN-Cbl quickly depleted CN-Cbl, and reductive dechlorination of polychlorinated ethenes was incomplete leading to vinyl chloride (VC) accumulation. In contrast, the same cultures amended with 25 {\textmu}g l(-1) CN-Cbl exhibited up to 2.3-fold higher dechlorination rates, 2.8-9.1-fold increased growth yields, and completely consumed growth-supporting chlorinated ethenes. To explore whether known cobamide-producing microbes supply Dehalococcoides with the required corrinoid cofactor, co-culture experiments were performed with the methanogen Methanosarcina barkeri strain Fusaro and two acetogens, Sporomusa ovata and Sporomusa sp. strain KB-1, as Dehalococcoides partner populations. During growth with H2/CO2, M. barkeri axenic cultures produced 4.2 {\textpm} 0.1 {\textmu}g l(-1) extracellular cobamide (factor III), whereas the Sporomusa cultures produced phenolyl- and p-cresolyl-cobamides. Neither factor III nor the phenolic cobamides supported Dehalococcoides reductive dechlorination activity suggesting that M. barkeri and the Sporomusa sp. cannot fulfil Dehalococcoides{\textquoteright} nutritional requirements. Dehalococcoides dechlorination activity and growth occurred in M. barkeri and Sporomusa sp. co-cultures amended with 10 {\textmu}M 5{\textquoteright},6{\textquoteright}-dimethylbenzimidazole (DMB), indicating that a cobalamin is a preferred corrinoid cofactor of strains BAV1, GT and FL2 when grown with chlorinated ethenes as electron acceptors. Even though the methanogen and acetogen populations tested did not produce cobalamin, the addition of DMB enabled guided biosynthesis and generated a cobalamin that supported Dehalococcoides{\textquoteright} activity and growth. Guided cobalamin biosynthesis may offer opportunities to sustain and enhance Dehalococcoides activity in contaminated subsurface environments.

}, keywords = {Bacteriological Techniques, Benzimidazoles, Biodegradation, Environmental, Chloroflexi, Chromatography, High Pressure Liquid, Coculture Techniques, Culture Media, Dichloroethylenes, Halogenation, Oxidation-Reduction, Trichloroethylene, Vitamin B 12}, issn = {1471-2970}, doi = {10.1098/rstb.2012.0320}, author = {Yan, Jun and Im, Jeongdae and Yang, Yi and L{\"o}ffler, Frank E} } @article {3156, title = {Comparative genomic analysis of Geobacter sulfurreducens KN400, a strain with enhanced capacity for extracellular electron transfer and electricity production.}, journal = {BMC Genomics}, volume = {13}, year = {2012}, month = {2012 Sep 12}, pages = {471}, abstract = {

BACKGROUND: A new strain of Geobacter sulfurreducens, strain KN400, produces more electrical current in microbial fuel cells and reduces insoluble Fe(III) oxides much faster than the wildtype strain, PCA. The genome of KN400 was compared to wildtype with the goal of discovering how the network for extracellular electron transfer has changed and how these two strains evolved.

RESULTS: Both genomes were re-annotated, resulting in 14 fewer genes (net) in the PCA genome; 28 fewer (net) in the KN400 genome; and ca. 400 gene start and stop sites moved. 96\% of genes in KN400 had clear orthologs with conserved synteny in PCA. Most of the remaining genes were in regions of genomic mobility and were strain-specific or conserved in other Geobacteraceae, indicating that the changes occurred post-divergence. There were 27,270 single nucleotide polymorphisms (SNP) between the genomes. There was significant enrichment for SNP locations in non-coding or synonymous amino acid sites, indicating significant selective pressure since the divergence. 25\% of orthologs had sequence differences, and this set was enriched in phosphorylation and ATP-dependent enzymes. Substantial sequence differences (at least 12 non-synonymous SNP/kb) were found in 3.6\% of the orthologs, and this set was enriched in cytochromes and integral membrane proteins. Genes known to be involved in electron transport, those used in the metabolic cell model, and those that exhibit changes in expression during growth in microbial fuel cells were examined in detail.

CONCLUSIONS: The improvement in external electron transfer in the KN400 strain does not appear to be due to novel gene acquisition, but rather to changes in the common metabolic network. The increase in electron transfer rate and yield in KN400 may be due to changes in carbon flux towards oxidation pathways and to changes in ATP metabolism, both of which indicate that the overall energy state of the cell may be different. The electrically conductive pili appear to be unchanged, but cytochrome folding, localization, and redox potentials may all be affected, which would alter the electrical connection between the cell and the substrate.

}, keywords = {Bioelectric Energy Sources, Comparative Genomic Hybridization, Electron Transport, Gene Expression Regulation, Bacterial, Genome, Bacterial, Geobacter, Metabolic Networks and Pathways, Molecular Sequence Annotation, Polymorphism, Single Nucleotide}, issn = {1471-2164}, doi = {10.1186/1471-2164-13-471}, author = {Butler, Jessica E and Young, Nelson D and Aklujkar, Muktak and Lovley, Derek R} } @article {1567, title = {Characterization of a novel facultative Methylocystis species capable of growth on methane, acetate and ethanol.}, journal = {Environ Microbiol Rep}, volume = {3}, year = {2011}, month = {2011 Apr}, pages = {174-81}, abstract = {

A non-motile strain of Methylocystis, strain SB2, isolated from a spring bog in southeast Michigan, had a curved rod morphology with a typical type II intracytoplasmic membrane system. This organism expressed the membrane-bound or particulate methane monooxygenase (pMMO) as well as a chalkophore with high affinity for copper and did not express the cytoplasmic or soluble methane monooxygenase (sMMO). Strain SB2 was found to grow within the pH range of 6-9, with optimal growth at 6.8. Growth was observed at temperatures ranging between 10{\textdegree}C and 30{\textdegree}C, with no growth at 37{\textdegree}C. The DNA G+C content was 62.9 mol\%. Predominant fatty acids were 18:1ω7c (72.7\%) and 18:1ω9c (24\%) when grown on methane. Phylogenetic comparisons based on both pmoA and 16S rRNA sequences indicated that this organism belonged to the Methylocystis genus, and was closely related to Methylocystis rosea SV97(T) and Methylocystis echinoides IMET10491(T) (98\% 16S rRNA gene sequence similarity to both strains). DNA : DNA hybridizations indicated that strain SB2 had 70\% similarity with M. rosea SV97(T) . Unlike M. rosea SV97(T) , strain SB2 was able to utilize not only methane for growth, but also ethanol and acetate. Furthermore, the predominant fatty acids in strain SB2 were different from those found in M. rosea SV97(T) , i.e. 54.2\% and 39.7\% of fatty acids are 18:1ω8 and 18:1ω7 in M. rosea SV97(T) , while 18:1ω8 is completely absent in strain SB2.

}, issn = {1758-2229}, doi = {10.1111/j.1758-2229.2010.00204.x}, author = {Im, Jeongdae and Lee, Sung-Woo and Yoon, Sukhwan and Dispirito, Alan A and Semrau, Jeremy D} } @article {1566, title = {Constitutive expression of pMMO by Methylocystis strain SB2 when grown on multi-carbon substrates: implications for biodegradation of chlorinated ethenes.}, journal = {Environ Microbiol Rep}, volume = {3}, year = {2011}, month = {2011 Apr}, pages = {182-8}, abstract = {

The particulate methane monooxygenase (pMMO) in Methylocystis strain SB2 was found to be constitutively expressed in the absence of methane when the strain was grown on either acetate or ethanol. Real-time quantitative polymerase chain reaction (PCR) and reverse transcription-PCR showed that the expression of pmoA decreased by one to two orders of magnitude when grown on acetate as compared with growth of strain SB2 on methane. The capability of strain SB2 to degrade a mixture of chlorinated ethenes in the absence of methane was examined to verify the presence and activity of pMMO under acetate-growth conditions as well determine the effectiveness of such conditions for bioremediation. It was found that when strain SB2 was grown on acetate and exposed to 40 {\textmu}M each of trichloroethylene (TCE), trans-dichloroethylene (t-DCE) and vinyl chloride (VC), approximately 30\% of VC and t-DCE was degraded but no appreciable TCE removal was measured after 216 h of incubation. The ability to degrade VC and t-DCE was lost when acetylene was added, confirming that pMMO was responsible for the degradation of these chlorinated ethenes by Methylocystis strain SB2 when the strain was grown on acetate.

}, issn = {1758-2229}, doi = {10.1111/j.1758-2229.2010.00205.x}, author = {Yoon, Sukhwan and Im, Jeongdae and Bandow, Nathan and Dispirito, Alan A and Semrau, Jeremy D} } @article {687, title = {Inositol lipid metabolism in mycobacteria: biosynthesis and regulatory mechanisms.}, journal = {Biochim Biophys Acta}, volume = {1810}, year = {2011}, month = {2011 Jun}, pages = {630-41}, abstract = {BACKGROUND: The genus Mycobacterium includes a number of medically important pathogens. The cell walls of these bacteria have many unique features, including the abundance of various inositol lipids, such as phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). The biosynthesis of these lipids is believed to be prime drug targets, and has been clarified in detail over the past several years. SCOPE OF REVIEW: Here we summarize our current understanding of the inositol lipid metabolism in mycobacteria. We will highlight unsolved issues and future directions especially in the context of metabolic regulation. MAJOR CONCLUSIONS: Inositol is a building block of phosphatidylinositol (PI), which is further elaborated to become PIMs, LM and LAM. d-myo-inositol 3-phosphate is an intermediate of the de novo inositol synthesis, but it is also the starting substrate for mycothiol synthesis. Controlling the level of d-myo-inositol 3-phosphate appears to be important for maintaining the steady state levels of mycothiol and inositol lipids. Several additional control mechanisms must exist to control the complex biosynthetic pathways of PI, PIMs, LM and LAM. These may include regulatory proteins such as a lipoprotein LpqW, and spatial separation of enzymes, such as the amphipathic PimA mannosyltransferase and later enzymes in the PIMs/LM biosynthetic pathway. Finally, we discuss mechanisms that underlie control of LM/LAM glycan polymer elongation. GENERAL SIGNIFICANCE: Mycobacteria have evolved a complex network of inositol metabolism. Clarifying its metabolism will not only provide better understanding of bacterial pathogenesis, but also understanding of the evolution and general functions of inositol lipids in nature.}, keywords = {Inositol, Lipid Metabolism, Lipids, Models, Biological, Mycobacterium, Phosphatidylinositols}, issn = {0006-3002}, doi = {10.1016/j.bbagen.2011.03.017}, author = {Morita, Yasu S and Fukuda, Takeshi and Sena, Chubert B C and Yamaryo-Botte, Yoshiki and McConville, Malcolm J and Kinoshita, Taroh} } @article {423, title = {Monitoring the metabolic status of geobacter species in contaminated groundwater by quantifying key metabolic proteins with Geobacter-specific antibodies.}, journal = {Appl Environ Microbiol}, volume = {77}, year = {2011}, month = {2011 Jul}, pages = {4597-602}, abstract = {Simple and inexpensive methods for assessing the metabolic status and bioremediation activities of subsurface microorganisms are required before bioremediation practitioners will adopt molecular diagnosis of the bioremediation community as a routine practice for guiding the development of bioremediation strategies. Quantifying gene transcripts can diagnose important aspects of microbial physiology during bioremediation but is technically challenging and does not account for the impact of translational modifications on protein abundance. An alternative strategy is to directly quantify the abundance of key proteins that might be diagnostic of physiological state. To evaluate this strategy, an antibody-based quantification approach was developed to investigate subsurface Geobacter communities. The abundance of citrate synthase corresponded with rates of metabolism of Geobacter bemidjiensis in chemostat cultures. During in situ bioremediation of uranium-contaminated groundwater the quantity of Geobacter citrate synthase increased with the addition of acetate to the groundwater and decreased when acetate amendments stopped. The abundance of the nitrogen-fixation protein, NifD, increased as ammonium became less available in the groundwater and then declined when ammonium concentrations increased. In a petroleum-contaminated aquifer, the abundance of BamB, an enzyme subunit involved in the anaerobic degradation of mono-aromatic compounds by Geobacter species, increased in zones in which Geobacter were expected to play an important role in aromatic hydrocarbon degradation. These results suggest that antibody-based detection of key metabolic proteins, which should be readily adaptable to standardized kits, may be a feasible method for diagnosing the metabolic state of microbial communities responsible for bioremediation, aiding in the rational design of bioremediation strategies.}, keywords = {Acetates, Antibodies, Bacterial, Bacterial Proteins, Geobacter, Petroleum, Quaternary Ammonium Compounds, Soil Microbiology, Water Microbiology}, issn = {1098-5336}, doi = {10.1128/AEM.00114-11}, author = {Yun, Jiae and Ueki, Toshiyuki and Miletto, Marzia and Lovley, Derek R} } @article {443, title = {De Novo assembly of the complete genome of an enhanced electricity-producing variant of Geobacter sulfurreducens using only short reads.}, journal = {PLoS One}, volume = {5}, year = {2010}, month = {2010}, pages = {e10922}, abstract = {State-of-the-art DNA sequencing technologies are transforming the life sciences due to their ability to generate nucleotide sequence information with a speed and quantity that is unapproachable with traditional Sanger sequencing. Genome sequencing is a principal application of this technology, where the ultimate goal is the full and complete sequence of the organism of interest. Due to the nature of the raw data produced by these technologies, a full genomic sequence attained without the aid of Sanger sequencing has yet to be demonstrated.We have successfully developed a four-phase strategy for using only next-generation sequencing technologies (Illumina and 454) to assemble a complete microbial genome de novo. We applied this approach to completely assemble the 3.7 Mb genome of a rare Geobacter variant (KN400) that is capable of unprecedented current production at an electrode. Two key components of our strategy enabled us to achieve this result. First, we integrated the two data types early in the process to maximally leverage their complementary characteristics. And second, we used the output of different short read assembly programs in such a way so as to leverage the complementary nature of their different underlying algorithms or of their different implementations of the same underlying algorithm.The significance of our result is that it demonstrates a general approach for maximizing the efficiency and success of genome assembly projects as new sequencing technologies and new assembly algorithms are introduced. The general approach is a meta strategy, wherein sequencing data are integrated as early as possible and in particular ways and wherein multiple assembly algorithms are judiciously applied such that the deficiencies in one are complemented by another.}, keywords = {Algorithms, Electricity, Genome, Bacterial, Geobacter, Polymerase Chain Reaction}, issn = {1932-6203}, doi = {10.1371/journal.pone.0010922}, author = {Nagarajan, Harish and Butler, Jessica E and Klimes, Anna and Qiu, Yu and Zengler, Karsten and Ward, Joy and Young, Nelson D and Meth{\'e}, Barbara A and Palsson, Bernhard {\O} and Lovley, Derek R and Barrett, Christian L} } @article {449, title = {Evolution of electron transfer out of the cell: comparative genomics of six Geobacter genomes.}, journal = {BMC Genomics}, volume = {11}, year = {2010}, month = {2010}, pages = {40}, abstract = {BACKGROUND: Geobacter species grow by transferring electrons out of the cell--either to Fe(III)-oxides or to man-made substances like energy-harvesting electrodes. Study of Geobacter sulfurreducens has shown that TCA cycle enzymes, inner-membrane respiratory enzymes, and periplasmic and outer-membrane cytochromes are required. Here we present comparative analysis of six Geobacter genomes, including species from the clade that predominates in the subsurface. Conservation of proteins across the genomes was determined to better understand the evolution of Geobacter species and to create a metabolic model applicable to subsurface environments. RESULTS: The results showed that enzymes for acetate transport and oxidation, and for proton transport across the inner membrane were well conserved. An NADH dehydrogenase, the ATP synthase, and several TCA cycle enzymes were among the best conserved in the genomes. However, most of the cytochromes required for Fe(III)-reduction were not, including many of the outer-membrane cytochromes. While conservation of cytochromes was poor, an abundance and diversity of cytochromes were found in every genome, with duplications apparent in several species. CONCLUSIONS: These results indicate there is a common pathway for acetate oxidation and energy generation across the family and in the last common ancestor. They also suggest that while cytochromes are important for extracellular electron transport, the path of electrons across the periplasm and outer membrane is variable. This combination of abundant cytochromes with weak sequence conservation suggests they may not be specific terminal reductases, but rather may be important in their heme-bearing capacity, as sinks for electrons between the inner-membrane electron transport chain and the extracellular acceptor.}, keywords = {Acetates, Bacterial Proton-Translocating ATPases, Citric Acid Cycle, Cluster Analysis, Comparative Genomic Hybridization, Cytochromes, Electron Transport, Evolution, Molecular, Gene Duplication, Gene Expression Regulation, Bacterial, Gene Transfer, Horizontal, Genome, Bacterial, Genomics, Geobacter, NADH Dehydrogenase, Oxidation-Reduction, Phylogeny}, issn = {1471-2164}, doi = {10.1186/1471-2164-11-40}, author = {Butler, Jessica E and Young, Nelson D and Lovley, Derek R} } @article {434, title = {The genome of Geobacter bemidjiensis, exemplar for the subsurface clade of Geobacter species that predominate in Fe(III)-reducing subsurface environments.}, journal = {BMC Genomics}, volume = {11}, year = {2010}, month = {2010}, pages = {490}, abstract = {BACKGROUND: Geobacter species in a phylogenetic cluster known as subsurface clade 1 are often the predominant microorganisms in subsurface environments in which Fe(III) reduction is the primary electron-accepting process. Geobacter bemidjiensis, a member of this clade, was isolated from hydrocarbon-contaminated subsurface sediments in Bemidji, Minnesota, and is closely related to Geobacter species found to be abundant at other subsurface sites. This study examines whether there are significant differences in the metabolism and physiology of G. bemidjiensis compared to non-subsurface Geobacter species. RESULTS: Annotation of the genome sequence of G. bemidjiensis indicates several differences in metabolism compared to previously sequenced non-subsurface Geobacteraceae, which will be useful for in silico metabolic modeling of subsurface bioremediation processes involving Geobacter species. Pathways can now be predicted for the use of various carbon sources such as propionate by G. bemidjiensis. Additional metabolic capabilities such as carbon dioxide fixation and growth on glucose were predicted from the genome annotation. The presence of different dicarboxylic acid transporters and two oxaloacetate decarboxylases in G. bemidjiensis may explain its ability to grow by disproportionation of fumarate. Although benzoate is the only aromatic compound that G. bemidjiensis is known or predicted to utilize as an electron donor and carbon source, the genome suggests that this species may be able to detoxify other aromatic pollutants without degrading them. Furthermore, G. bemidjiensis is auxotrophic for 4-aminobenzoate, which makes it the first Geobacter species identified as having a vitamin requirement. Several features of the genome indicated that G. bemidjiensis has enhanced abilities to respire, detoxify and avoid oxygen. CONCLUSION: Overall, the genome sequence of G. bemidjiensis offers surprising insights into the metabolism and physiology of Geobacteraceae in subsurface environments, compared to non-subsurface Geobacter species, such as the ability to disproportionate fumarate, more efficient oxidation of propionate, enhanced responses to oxygen stress, and dependence on the environment for a vitamin requirement. Therefore, an understanding of the activity of Geobacter species in the subsurface is more likely to benefit from studies of subsurface isolates such as G. bemidjiensis than from the non-subsurface model species studied so far.}, keywords = {Aldehyde Oxidoreductases, Biodegradation, Environmental, Carbohydrate Metabolism, Carbon Dioxide, Cell Wall, Electrons, Environmental Microbiology, Fatty Acids, Frameshift Mutation, Fumarates, Genes, Bacterial, Genome, Bacterial, Geobacter, Glucose, Iron, Metabolic Networks and Pathways, Multienzyme Complexes, Multigene Family, Osmosis, Oxidation-Reduction, Oxo-Acid-Lyases, Propionic Acids, Pyruvic Acid, Species Specificity, Surface Properties}, issn = {1471-2164}, doi = {10.1186/1471-2164-11-490}, author = {Aklujkar, Muktak and Young, Nelson D and Holmes, Dawn and Chavan, Milind and Risso, Carla and Kiss, Hajnalka E and Han, Cliff S and Land, Miriam L and Lovley, Derek R} } @article {432, title = {Metabolic response of Geobacter sulfurreducens towards electron donor/acceptor variation.}, journal = {Microb Cell Fact}, volume = {9}, year = {2010}, month = {2010}, pages = {90}, abstract = {BACKGROUND: Geobacter sulfurreducens is capable of coupling the complete oxidation of organic compounds to iron reduction. The metabolic response of G. sulfurreducens towards variations in electron donors (acetate, hydrogen) and acceptors (Fe(III), fumarate) was investigated via (13)C-based metabolic flux analysis. We examined the (13)C-labeling patterns of proteinogenic amino acids obtained from G. sulfurreducens cultured with (13)C-acetate. RESULTS: Using (13)C-based metabolic flux analysis, we observed that donor and acceptor variations gave rise to differences in gluconeogenetic initiation, tricarboxylic acid cycle activity, and amino acid biosynthesis pathways. Culturing G. sulfurreducens cells with Fe(III) as the electron acceptor and acetate as the electron donor resulted in pyruvate as the primary carbon source for gluconeogenesis. When fumarate was provided as the electron acceptor and acetate as the electron donor, the flux analysis suggested that fumarate served as both an electron acceptor and, in conjunction with acetate, a carbon source. Growth on fumarate and acetate resulted in the initiation of gluconeogenesis by phosphoenolpyruvate carboxykinase and a slightly elevated flux through the oxidative tricarboxylic acid cycle as compared to growth with Fe(III) as the electron acceptor. In addition, the direction of net flux between acetyl-CoA and pyruvate was reversed during growth on fumarate relative to Fe(III), while growth in the presence of Fe(III) and acetate which provided hydrogen as an electron donor, resulted in decreased flux through the tricarboxylic acid cycle. CONCLUSIONS: We gained detailed insight into the metabolism of G. sulfurreducens cells under various electron donor/acceptor conditions using (13)C-based metabolic flux analysis. Our results can be used for the development of G. sulfurreducens as a chassis for a variety of applications including bioremediation and renewable biofuel production.}, keywords = {Acetic Acid, Acetyl Coenzyme A, Amino Acids, Carbon Isotopes, Citric Acid Cycle, Electrons, Ferric Compounds, Fumarates, Geobacter, Gluconeogenesis, Oxidation-Reduction, Phosphoenolpyruvate Carboxykinase (GTP), Pyruvates}, issn = {1475-2859}, doi = {10.1186/1475-2859-9-90}, author = {Yang, Tae Hoon and Coppi, Maddalena V and Lovley, Derek R and Sun, Jun} } @article {688, title = {Stress-induced synthesis of phosphatidylinositol 3-phosphate in mycobacteria.}, journal = {J Biol Chem}, volume = {285}, year = {2010}, month = {2010 May 28}, pages = {16643-50}, abstract = {Phosphoinositides play key roles in regulating membrane dynamics and intracellular signaling in eukaryotic cells. However, comparable lipid-based signaling pathways have not been identified in bacteria. Here we show that Mycobacterium smegmatis and other Actinomycetes bacteria can synthesize the phosphoinositide, phosphatidylinositol 3-phosphate (PI3P). This lipid was transiently labeled with [(3)H]inositol. Sensitivity of the purified lipid to alkaline phosphatase, headgroup analysis by high-pressure liquid chromatography, and mass spectrometry demonstrated that it had the structure 1,2-[tuberculostearoyl, octadecenoyl]-sn-glycero 3-phosphoinositol 3-phosphate. Synthesis of PI3P was elevated by salt stress but not by exposure to high concentrations of non-ionic solutes. Synthesis of PI3P in a cell-free system was stimulated by the synthesis of CDP-diacylglycerol, a lipid substrate for phosphatidylinositol (PI) biosynthesis, suggesting that efficient cell-free PI3P synthesis is dependent on de novo PI synthesis. In vitro experiments further indicated that the rapid turnover of this lipid was mediated, at least in part, by a vanadate-sensitive phosphatase. This is the first example of de novo synthesis of PI3P in bacteria, and the transient synthesis in response to environmental stimuli suggests that some bacteria may have evolved similar lipid-mediated signaling pathways to those observed in eukaryotic cells.}, keywords = {Cell-Free System, Chromatography, High Pressure Liquid, Leishmania, Lipids, Mass Spectrometry, Mycobacterium smegmatis, Nucleotides, Oxalic Acid, Phosphatidylinositol Phosphates, Phosphatidylinositols, Phospholipids, Phosphorylation, Salts, Signal Transduction}, issn = {1083-351X}, doi = {10.1074/jbc.M110.119263}, author = {Morita, Yasu S and Yamaryo-Botte, Yoshiki and Miyanagi, Kana and Callaghan, Judy M and Patterson, John H and Crellin, Paul K and Coppel, Ross L and Billman-Jacobe, Helen and Kinoshita, Taroh and McConville, Malcolm J} } @article {690, title = {Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle.}, journal = {J Exp Med}, volume = {206}, year = {2009}, month = {2009 Dec 21}, pages = {2879-88}, abstract = {Tuberculosis remains a fatal disease caused by Mycobacterium tuberculosis, which contains various unique components that affect the host immune system. Trehalose-6,6{\textquoteright}-dimycolate (TDM; also called cord factor) is a mycobacterial cell wall glycolipid that is the most studied immunostimulatory component of M. tuberculosis. Despite five decades of research on TDM, its host receptor has not been clearly identified. Here, we demonstrate that macrophage inducible C-type lectin (Mincle) is an essential receptor for TDM. Heat-killed mycobacteria activated Mincle-expressing cells, but the activity was lost upon delipidation of the bacteria; analysis of the lipid extracts identified TDM as a Mincle ligand. TDM activated macrophages to produce inflammatory cytokines and nitric oxide, which are completely suppressed in Mincle-deficient macrophages. In vivo TDM administration induced a robust elevation of inflammatory cytokines in sera and characteristic lung inflammation, such as granuloma formation. However, no TDM-induced lung granuloma was formed in Mincle-deficient mice. Whole mycobacteria were able to activate macrophages even in MyD88-deficient background, but the activation was significantly diminished in Mincle/MyD88 double-deficient macrophages. These results demonstrate that Mincle is an essential receptor for the mycobacterial glycolipid, TDM.}, keywords = {Animals, Cord Factors, Granuloma, Lectins, C-Type, Ligands, Lung Diseases, Macrophage Activation, Membrane Proteins, Mice, Mice, Inbred C57BL, Myeloid Differentiation Factor 88, Receptors, IgG}, issn = {1540-9538}, doi = {10.1084/jem.20091750}, author = {Ishikawa, Eri and Ishikawa, Tetsuaki and Morita, Yasu S and Toyonaga, Kenji and Yamada, Hisakata and Takeuchi, Osamu and Kinoshita, Taroh and Akira, Shizuo and Yoshikai, Yasunobu and Yamasaki, Sho} } @article {473, title = {Diversity of promoter elements in a Geobacter sulfurreducens mutant adapted to disruption in electron transfer.}, journal = {Funct Integr Genomics}, volume = {9}, year = {2009}, month = {2009 Feb}, pages = {15-25}, abstract = {The delta-proteobacterium, Geobacter sulfurreducens, can obtain energy by coupling the oxidation of organic matter to the reduction of insoluble Fe(III) or the anode of a microbial fuel cell. Because Fe(III) oxide or the anode surface, in contrast to oxygen, nitrate, or sulfate, is not soluble nor can it be reduced readily, Geobacter species have developed mechanisms which allow electrons to be delivered across outer membrane to the cell surface. OmcB is an outer-membrane c-type cytochrome important for G. sulfurreducens Fe(III) respiration. In the absence of OmcB, cells lost the ability to reduce soluble or insoluble Fe(III). However, the omcB deletion mutant can slowly adapt to growth on soluble Fe(III) over prolonged incubation in the medium with acetate as the electron donor. We discuss available information about predicted or experimentally validated promoters and transcription regulatory sites identified upstream of operons with transcriptional expression significantly changed in the adapted omcB mutant. DNA sequences of upstream regions of coregulated operons in the adapted mutant are divergent, suggesting the presence of recognition sites for different transcriptional regulators and indicating that adaptation of the omcB mutant to growth on soluble Fe(III) has shifted the relevant expression networks involved to a more diverse molecular basis.}, keywords = {Adaptation, Physiological, Electron Transport, Genetic Variation, Geobacter, Mutation, Promoter Regions, Genetic}, issn = {1438-7948}, doi = {10.1007/s10142-008-0094-7}, author = {Krushkal, Julia and Leang, Ching and Barbe, Jose F and Qu, Yanhua and Yan, Bin and Puljic, Marko and Adkins, Ronald M and Lovley, Derek R} } @article {463, title = {Evolution from a respiratory ancestor to fill syntrophic and fermentative niches: comparative fenomics of six Geobacteraceae species.}, journal = {BMC Genomics}, volume = {10}, year = {2009}, month = {2009}, pages = {103}, abstract = {BACKGROUND: The anaerobic degradation of organic matter in natural environments, and the biotechnical use of anaerobes in energy production and remediation of subsurface environments, both require the cooperative activity of a diversity of microorganisms in different metabolic niches. The Geobacteraceae family contains members with three important anaerobic metabolisms: fermentation, syntrophic degradation of fermentation intermediates, and anaerobic respiration. RESULTS: In order to learn more about the evolution of anaerobic microbial communities, the genome sequences of six Geobacteraceae species were analyzed. The results indicate that the last common Geobacteraceae ancestor contained sufficient genes for anaerobic respiration, completely oxidizing organic compounds with the reduction of external electron acceptors, features that are still retained in modern Geobacter and Desulfuromonas species. Evolution of specialization for fermentative growth arose twice, via distinct lateral gene transfer events, in Pelobacter carbinolicus and Pelobacter propionicus. Furthermore, P. carbinolicus gained hydrogenase genes and genes for ferredoxin reduction that appear to permit syntrophic growth via hydrogen production. The gain of new physiological capabilities in the Pelobacter species were accompanied by the loss of several key genes necessary for the complete oxidation of organic compounds and the genes for the c-type cytochromes required for extracellular electron transfer. CONCLUSION: The results suggest that Pelobacter species evolved parallel strategies to enhance their ability to compete in environments in which electron acceptors for anaerobic respiration were limiting. More generally, these results demonstrate how relatively few gene changes can dramatically transform metabolic capabilities and expand the range of environments in which microorganisms can compete.}, keywords = {Anaerobiosis, Bacteria, Anaerobic, Biological Evolution, Cluster Analysis, Deltaproteobacteria, DNA, Bacterial, Fermentation, Gene Transfer, Horizontal, Genome, Bacterial, Genomics, Multigene Family, Phylogeny, Sequence Analysis, DNA}, issn = {1471-2164}, doi = {10.1186/1471-2164-10-103}, author = {Butler, Jessica E and Young, Nelson D and Lovley, Derek R} } @article {460, title = {Selection of a variant of Geobacter sulfurreducens with enhanced capacity for current production in microbial fuel cells.}, journal = {Biosens Bioelectron}, volume = {24}, year = {2009}, month = {2009 Aug 15}, pages = {3498-503}, abstract = {Geobacter sulfurreducens produces current densities in microbial fuel cells that are among the highest known for pure cultures. The possibility of adapting this organism to produce even higher current densities was evaluated. A system in which a graphite anode was poised at -400 mV (versus Ag/AgCl) was inoculated with the wild-type strain of G. sulfurreducens, strain DL-1. An isolate, designated strain KN400, was recovered from the biofilm after 5 months of growth on the electrode. KN400 was much more effective in current production than strain DL-1. This was apparent with anodes poised at -400 mV, as well as in systems run in true fuel cell mode. KN400 had current (7.6A/m(2)) and power (3.9 W/m(2)) densities that respectively were substantially higher than those of DL1 (1.4A/m(2) and 0.5 W/m(2)). On a per cell basis KN400 was more effective in current production than DL1, requiring thinner biofilms to make equivalent current. The enhanced capacity for current production in KN400 was associated with a greater abundance of electrically conductive microbial nanowires than DL1 and lower internal resistance (0.015 versus 0.130 Omega/m(2)) and mass transfer limitation in KN400 fuel cells. KN400 produced flagella, whereas DL1 does not. Surprisingly, KN400 had much less outer-surface c-type cytochromes than DL1. KN400 also had a greater propensity to form biofilms on glass or graphite than DL1, even when growing with the soluble electron acceptor, fumarate. These results demonstrate that it is possible to enhance the ability of microorganisms to electrochemically interact with electrodes with the appropriate selective pressure and that improved current production is associated with clear differences in the properties of the outer surface of the cell that may provide insights into the mechanisms for microbe-electrode interactions.}, keywords = {Bioelectric Energy Sources, Electrochemistry, Equipment Design, Equipment Failure Analysis, Geobacter, Species Specificity}, issn = {1873-4235}, doi = {10.1016/j.bios.2009.05.004}, author = {Yi, Hana and Nevin, Kelly P and Kim, Byoung-Chan and Franks, Ashely E and Klimes, Anna and Tender, Leonard M and Lovley, Derek R} } @article {700, title = {Characterizing regulation of metabolism in Geobacter sulfurreducens through genome-wide expression data and sequence analysis.}, journal = {OMICS}, volume = {12}, year = {2008}, month = {2008 Mar}, pages = {33-59}, abstract = {Geobacteraceae are a family of metal reducing bacteria with important applications in bioremediation and electricity generation. G. sulfurreducens is a representative of Geobacteraceae that has been extensively studied with the goal of extending the understanding of this family of organisms for optimizing their practical applications. Here, we have analyzed gene expression data from 10 experiments involving environmental and genetic perturbations and have identified putative transcription factor binding sites (TFBS) involved in regulating key aspects of metabolism. Specifically, we considered data from both a subset of 10 microarray experiments (7 of 10) and all 10 experiments. The expression data from these two sets were independently clustered, and the upstream regions of genes and operons from the clusters in both sets were used to identify TFBS using the AlignACE program. This analysis resulted in the identification of motifs upstream of several genes involved in central metabolism, sulfate assimilation, and energy metabolism, as well as genes potentially encoding acetate permease. Further, similar TFBS were identified from the analysis of both sets, suggesting that these TFBS are significant in the regulation of metabolism in G. sulfurreducens. In addition, we have utilized microarray data to derive condition specific constraints on the capacity of key enzymes in central metabolism. We have incorporated these constraints into the metabolic model of G. sulfurreducens and simulated Fe(II)-limited growth. The resulting prediction was consistent with data, suggesting that regulatory constraints are important for simulating growth phenotypes in nonoptimal environments.}, keywords = {Gene Expression Regulation, Bacterial, Genome, Bacterial, Geobacter, Models, Genetic, Oligonucleotide Array Sequence Analysis, Sequence Analysis, DNA, Transcription, Genetic}, issn = {1536-2310}, doi = {10.1089/omi.2007.0043}, author = {Mahadevan, Radhakrishnan and Yan, Bin and Postier, Brad and Nevin, Kelly P and Woodard, Trevor L and O{\textquoteright}Neil, Regina and Coppi, Maddalena V and Meth{\'e}, Barbara A and Krushkal, Julia} } @article {1212, title = {Characterization of malate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum.}, journal = {Extremophiles}, volume = {11}, year = {2007}, month = {2007 Sep}, pages = {741-6}, abstract = {

Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins are the same. The apparent K ( m ) values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80 degrees C and pH 8.0) were 15 and 86 microM, respectively, with specific activity as high as 470 U mg(-1). Activity decreased more than 90\% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific activity of the P. islandicum MDH back-reaction also decreased more than 90\% when malate and NAD(+) were used as substrates and was not detected with NADP(+). A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines suggesting minimal lateral mdh transfer between Archaea.

}, keywords = {Citric Acid Cycle, Hydrogen-Ion Concentration, Kinetics, Malate Dehydrogenase, Malates, Molecular Weight, NAD, NADP, Oxaloacetic Acid, Phylogeny, Protein Conformation, Protein Subunits, Pyrobaculum, Recombinant Proteins, Sequence Analysis, Protein, Substrate Specificity, Temperature}, issn = {1431-0651}, doi = {10.1007/s00792-007-0081-2}, author = {Yennaco, Lynda J and Hu, Yajing and Holden, James F} } @article {771, title = {An expectation maximization approach to estimate malaria haplotype frequencies in multiply infected children.}, journal = {Stat Appl Genet Mol Biol}, volume = {6}, year = {2007}, month = {2007}, pages = {Article33}, abstract = {Characterizing genetic variability in the human pathogenic Plasmodium species, the group of parasites that cause Malaria, may have broad global health implications. Specifically, discerning the combinations of mutations that lead to viable parasites and the population level frequencies of these clonal sequences will allow for targeted vaccine development and individualized treatment choices. This presents an analytical challenge, however, since haplotypic phase (i.e. the alignment of bases on a single DNA strand) is generally unobservable in multiply infected individuals. This manuscript describes an expectation maximization (EM) approach to maximum likelihood estimation of haplotype frequencies in this missing data setting. The approach is applied to a cohort of N=341 malaria infected children in Uganda, Cameroon and Sudan to characterize regional differences. A simulation study is also presented to characterize method performance and assess sensitivity to distributional assumptions.}, keywords = {Animals, Child, Gene Frequency, Genotype, Humans, Likelihood Functions, Malaria, Malaria Vaccines, Mutation, Plasmodium}, issn = {1544-6115}, doi = {10.2202/1544-6115.1321}, author = {Li, Xiaohong and Foulkes, Andrea S and Yucel, Recai M and Rich, Stephen M} } @article {503, title = {Genome-wide expression profiling in Geobacter sulfurreducens: identification of Fur and RpoS transcription regulatory sites in a relGsu mutant.}, journal = {Funct Integr Genomics}, volume = {7}, year = {2007}, month = {2007 Jul}, pages = {229-55}, abstract = {Rel(Gsu) is the single Geobacter sulfurreducens homolog of RelA and SpoT proteins found in many organisms. These proteins are involved in the regulation of levels of guanosine 3{\textquoteright}, 5{\textquoteright} bispyrophosphate, ppGpp, a molecule that signals slow growth and stress response under nutrient limitation in bacteria. We used information obtained from genome-wide expression profiling of the rel(Gsu) deletion mutant to identify putative regulatory sites involved in transcription networks modulated by Rel(Gsu) or ppGpp. Differential gene expression in the rel(Gsu) deletion mutant, as compared to the wild type, was available from two growth conditions, steady state chemostat cultures and stationary phase batch cultures. Hierarchical clustering analysis of these two datasets identified several groups of operons that are likely co-regulated. Using a search for conserved motifs in the upstream regions of these co-regulated operons, we identified sequences similar to Fur- and RpoS-regulated sites. These findings suggest that Fur- and RpoS-dependent gene expression in G. sulfurreducens is affected by Rel(Gsu)-mediated signaling.}, keywords = {Bacterial Proteins, Base Sequence, Gene Deletion, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genes, Bacterial, Genome, Bacterial, Geobacter, Ligases, Mutation, Operon, Promoter Regions, Genetic, Regulatory Sequences, Nucleic Acid, Repressor Proteins, Sigma Factor, Transcription, Genetic}, issn = {1438-793X}, doi = {10.1007/s10142-007-0048-5}, author = {Krushkal, Julia and Yan, Bin and DiDonato, Laurie N and Puljic, Marko and Nevin, Kelly P and Woodard, Trevor L and Adkins, Ronald M and Meth{\'e}, Barbara A and Lovley, Derek R} } @article {509, title = {Genome-wide similarity search for transcription factors and their binding sites in a metal-reducing prokaryote Geobacter sulfurreducens.}, journal = {Biosystems}, volume = {90}, year = {2007}, month = {2007 Sep-Oct}, pages = {421-41}, abstract = {The knowledge obtained from understanding individual elements involved in gene regulation is important for reconstructing gene regulatory networks, a key for understanding cellular behavior. To study gene regulatory interactions in a model microorganism, Geobacter sulfurreducens, which participates in metal reduction and energy harvesting, we investigated the presence of 59 known Escherichia coli transcription factors and predicted transcription regulatory sites in its genome. The supplementary material, available at http://www.geobacter.org/research/genomescan/, provides the results of similarity comparisons that identified regulatory proteins of G. sulfurreducens and the genome locations of the predicted regulatory sites, including the list of putative regulatory elements in the upstream regions of every predicted operon and singleton open reading frame. Regulatory sequence elements, predicted using genome similarity searches to matrices of established transcription regulatory elements from E. coli, provide an initial insight into regulation of genes and operons in G. sulfurreducens. The predicted regulatory elements were predominantly located in the upstream regions of operons and singleton open reading frames. The validity of the predictions was examined using a permutation approach. Sequence similarity searches indicate that E. coli transcription factors ArgR, CytR, DeoR, FlhCD (both FlhC and FlhD subunits), FruR, GalR, GlpR, H-NS, LacI, MetJ, PurR, TrpR, and Tus are likely missing from G. sulfurreducens. Phylogenetic analysis suggests that one HU subunit is present in G. sulfurreducens as compared to two subunits in E. coli, while each of the two E. coli IHF subunits, HimA and HimD, have two homologs in G. sulfurreducens. The closest homolog of E. coli RpoE in G. sulfurreducens may be more similar to FecI than to RpoE. These findings represent the first step in the understanding of the regulatory relationships in G. sulfurreducens on the genome scale.}, keywords = {Bacterial Proteins, Binding Sites, False Positive Reactions, Gene Expression Regulation, Bacterial, Genome, Bacterial, Geobacter, Metals, Models, Biological, Models, Genetic, Models, Statistical, Operon, Phylogeny, Sigma Factor, Systems Biology, Transcription Factors, Transcription, Genetic}, issn = {0303-2647}, doi = {10.1016/j.biosystems.2006.10.006}, author = {Yan, Bin and Lovley, Derek R and Krushkal, Julia} } @article {495, title = {Steady state protein levels in Geobacter metallireducens grown with iron (III) citrate or nitrate as terminal electron acceptor.}, journal = {Proteomics}, volume = {7}, year = {2007}, month = {2007 Nov}, pages = {4148-57}, abstract = {Geobacter species predominate in aquatic sediments and submerged soils where organic carbon sources are oxidized with the reduction of Fe(III). The natural occurrence of Geobacter in some waste sites suggests this microorganism could be useful for bioremediation if growth and metabolic activity can be regulated. 2-DE was used to monitor the steady state protein levels of Geobacter metallireducens grown with either Fe(III) citrate or nitrate to elucidate metabolic differences in response to different terminal electron acceptors present in natural environments populated by Geobacter. Forty-six protein spots varied significantly in abundance (p<0.05) between the two growth conditions; proteins were identified by tryptic peptide mass and peptide sequence determined by MS/MS. Enzymes involved in pyruvate metabolism and the tricarboxylic acid (TCA) cycle were more abundant in cells grown with Fe(III) citrate, while proteins associated with nitrate metabolism and sensing cellular redox status along with several proteins of unknown function were more abundant in cells grown with nitrate. These results indicate a higher level of flux through the TCA cycle in the presence of Fe(III) compared to nitrate. The oxidative stress response observed in previous studies of Geobacter sulfurreducens grown with Fe(III) citrate was not seen in G. metallireducens.}, keywords = {Bacterial Proteins, Cell Proliferation, Electrons, Electrophoresis, Gel, Two-Dimensional, Ferric Compounds, Geobacter, Hydrogen-Ion Concentration, Nitrates, Oxidation-Reduction, Proteomics, Tandem Mass Spectrometry}, issn = {1615-9853}, doi = {10.1002/pmic.200600955}, author = {Ahrendt, Angela J and Tollaksen, Sandra L and Lindberg, Carl and Zhu, Wenhong and Yates, John R and Nevin, Kelly P and Babnigg, Gy{\"o}rgy and Lovley, Derek R and Giometti, Carol S} } @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 {702, title = {Differential protein expression in the metal-reducing bacterium Geobacter sulfurreducens strain PCA grown with fumarate or ferric citrate.}, journal = {Proteomics}, volume = {6}, year = {2006}, month = {2006 Jan}, pages = {632-40}, abstract = {Geobacter sulfurreducens, generally considered to be a strict anaerobe, is a predominant microbe in subsurface environments, where it utilizes available metals as electron acceptors. To better understand the metabolic processes involved in the metal-reduction capability of this microbe, the proteins expressed by cells grown anaerobically with either fumarate or ferric citrate as electron acceptor were compared. Proteins were separated by 2-DE under denaturing or nondenaturing conditions, and proteins varying in abundance with a high level of statistical significance (p<0.0001) were identified by peptide mass analysis. Denaturing 2-DE revealed significant differences in the relative abundance of the membrane proteins OmpA and peptidoglycan-associated lipoprotein, several metabolic enzymes, and, in addition, superoxide dismutase and rubredoxin oxidoreductase. Nondenaturing 2-DE revealed elevated catalase in cells grown with ferric citrate. These results suggest that, in addition to adjustments in membrane transport and specific metabolic pathways in response to these two different electron acceptors, distinct differences exist in the oxidative environment within the cell when fumarate or soluble ferric citrate is provided as electron acceptor. Although an anaerobe, G. sulfurreducens appears to have alternate mechanisms for dealing with reactive oxygen species in response to increased intracellular soluble iron.}, keywords = {Anaerobiosis, Bacterial Proteins, Electrophoresis, Gel, Two-Dimensional, Ferric Compounds, Fumarates, Geobacter, Oxidation-Reduction, Proteomics, Reactive Oxygen Species, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization}, issn = {1615-9853}, doi = {10.1002/pmic.200500137}, author = {Khare, Tripti and Esteve-N{\'u}{\~n}ez, Abraham and Nevin, Kelly P and Zhu, Wenhong and Yates, John R and Lovley, Derek and Giometti, Carol S} } @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 {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 {550, title = {Computational prediction of conserved operons and phylogenetic footprinting of transcription regulatory elements in the metal-reducing bacterial family Geobacteraceae.}, journal = {J Theor Biol}, volume = {230}, year = {2004}, month = {2004 Sep 7}, pages = {133-44}, abstract = {Members of the family Geobacteraceae are an important group of microorganisms from the delta subdivision of Proteobacteria that couple the oxidation of organic compounds to metal reduction. In order to uncover transcription regulatory interactions in these organisms, we used computational methods to identify conserved operons and putative cis-regulatory transcription elements. We identified 26 putative operons with gene order and function conserved among two species of Geobacteraceae, Geobacter sulfurreducens and Geobacter metallireducens. Most of these operons were also conserved in Desulfovibrio vulgaris, an additional metal reducing organism from family Desulfovibrionaceae of the delta subdivision of Proteobacteria. The predicted conserved operons were investigated for the presence of transcription factor binding sites by two different methods, (i) comparison of non-coding regions in conserved operons, and (ii) neural network promoter prediction. Predicted motifs were screened to identify most likely transcription factor binding sites and ribosome-binding sites. We provide information on motifs in Geobacteraceae similar to known transcription factor binding sites in Escherichia coli, conserved motifs in other bacterial species, putative palindromic sites, and predicted ribosome-binding sites. These predictions will aid in further elucidation of regulatory networks of gene interactions in Geobacteraceae.}, keywords = {Animals, Computational Biology, Conserved Sequence, DNA Footprinting, Genes, Regulator, Genome, Bacterial, Geobacter, Models, Genetic, Operon, Phylogeny}, issn = {0022-5193}, doi = {10.1016/j.jtbi.2004.04.022}, author = {Yan, Bin and Meth{\'e}, Barbara A and Lovley, Derek R and Krushkal, Julia} } @article {1215, title = {Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry.}, journal = {J Am Soc Mass Spectrom}, volume = {14}, year = {2003}, month = {2003 Sep}, pages = {957-70}, abstract = {

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100\% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97\% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50\% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50\% for approximately 70\% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.

}, keywords = {Amino Acid Sequence, Chromatography, Liquid, Databases, Protein, Electrophoresis, Gel, Two-Dimensional, Methanococcus, Molecular Sequence Data, Molecular Weight, Peptide Mapping, Proteins, Pyrococcus furiosus, Sensitivity and Specificity, Software, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Trypsin}, issn = {1044-0305}, author = {Lim, Hanjo and Eng, Jimmy and Yates, John R and Tollaksen, Sandra L and Giometti, Carol S and Holden, James F and Adams, Michael W W and Reich, Claudia I and Olsen, Gary J and Hays, Lara G} } @article {1211, title = {Identification of membrane proteins in the hyperthermophilic archaeon pyrococcus furiosus using proteomics and prediction programs.}, journal = {Comp Funct Genomics}, volume = {2}, year = {2001}, month = {2001}, pages = {275-88}, abstract = {

Cell-free extracts from the hyperthermophilic archaeon Pyrococcus furiosus were separated into membrane and cytoplasmic fractions and each was analyzed by 2D-gel electrophoresis. A total of 66 proteins were identified, 32 in the membrane fraction and 34 in the cytoplasmic fraction. Six prediction programs were used to predict the subcellular locations of these proteins. Three were based on signal-peptides (SignalP, TargetP, and SOSUISignal) and three on transmembrane-spanning alpha-helices (TSEG, SOSUI, and PRED-TMR2). A consensus of the six programs predicted that 23 of the 32 proteins (72\%) from the membrane fraction should be in the membrane and that all of the proteins from the cytoplasmic fraction should be in the cytoplasm. Two membrane-associated proteins predicted to be cytoplasmic by the programs are also predicted to consist primarily of transmembrane-spanning beta-sheets using porin protein models, suggesting that they are, in fact, membrane components. An ATPase subunit homolog found in the membrane fraction, although predicted to be cytoplasmic, is most likely complexed with other ATPase subunits in the membrane fraction. An additional three proteins predicted to be cytoplasmic but found in the membrane fraction, may be cytoplasmic contaminants. These include a chaperone homolog that may have attached to denatured membrane proteins during cell fractionation. Omitting these three proteins would boost the membrane-protein predictability of the models to near 80\%. A consensus prediction using all six programs for all 2242 ORFs in the P. furiosus genome estimates that 24\% of the ORF products are found in the membrane. However, this is likely to be a minimum value due to the programs{\textquoteright} inability to recognize certain membrane-related proteins, such as subunits associated with membrane complexes and porin-type proteins.

}, issn = {1531-6912}, doi = {10.1002/cfg.110}, author = {Holden, J F and Poole Ii, F L and Tollaksen, S L and Giometti, C S and Lim, H and Yates Iii, J R and Adams, M W} } @article {865, title = {Kinetic and mechanistic characterization of the polyhydroxybutyrate synthase from Ralstonia eutropha.}, journal = {Biomacromolecules}, volume = {1}, year = {2000}, month = {2000 Summer}, pages = {244-51}, abstract = {Purified Ralstonia eutropha polyhydroxybutyrate (PHB) synthase from recombinant cells can exist as monomer and dimer. The polymerization reaction catalyzed by this enzyme displays a lag phase, which causes difficulties for kinetic and mechanistic characterization of the enzymatic polymerization reaction. In this study, we developed a method to eliminate the lag phase of PHB synthase by physical means, i.e., adding multihydroxyl compounds to the enzyme solution. This method allows us to recognize the nature of the lag phase as a physical rather than a chemical process. With such lag-phase-free-enzyme, the kinetic properties of the enzyme were investigated. The results indicate that 3-hydroxybutyryl-CoA (3HBCoA) is the optimal substrate for the enzyme. A slower catalytic rate and lower binding ability account for a lower reactivity of 3-hydroxyvaleryl-CoA (3HVCoA) compared to that of 3HBCoA. The change of hydroxyl group from the beta to the gamma position causes dramatic decreases in the binding ability of 4-hydroxybutyryl-CoA (4HBCoA). By using a dilution strategy and size exclusion chromatographic technique, the active form of the enzyme was identified to be the dimeric form. The number of catalytic sites in the dimeric form of the enzyme was examined by comparing the molecular weight of polyhydroxybutyrate as a function of substrate-to-enzyme ratio. The results suggest that the dimeric enzyme has only one catalytic site. A revised model of polymerization reaction catalyzed by R. eutropha PHB synthase is described.}, keywords = {Acyltransferases, Catalysis, Chromatography, Gel, Coenzyme A, Culture Media, Hydrogen-Ion Concentration, Kinetics, Molecular Weight, Proteobacteria}, issn = {1525-7797}, author = {Zhang, S and Yasuo, T and Lenz, R W and Goodwin, S} }