@article {3150,
	title = {A genetic system for Clostridium ljungdahlii: a chassis for autotrophic production of biocommodities and a model homoacetogen.},
	journal = {Appl Environ Microbiol},
	volume = {79},
	year = {2013},
	month = {2013 Feb},
	pages = {1102-9},
	abstract = {<p>Methods for genetic manipulation of Clostridium ljungdahlii are of interest because of the potential for production of fuels and other biocommodities from carbon dioxide via microbial electrosynthesis or more traditional modes of autotrophy with hydrogen or carbon monoxide as the electron donor. Furthermore, acetogenesis plays an important role in the global carbon cycle. Gene deletion strategies required for physiological studies of C. ljungdahlii have not previously been demonstrated. An electroporation procedure for introducing plasmids was optimized, and four different replicative origins for plasmid propagation in C. ljungdahlii were identified. Chromosomal gene deletion via double-crossover homologous recombination with a suicide vector was demonstrated initially with deletion of the gene for FliA, a putative sigma factor involved in flagellar biogenesis and motility in C. ljungdahlii. Deletion of fliA yielded a strain that lacked flagella and was not motile. To evaluate the potential utility of gene deletions for functional genomic studies and to redirect carbon and electron flow, the genes for the putative bifunctional aldehyde/alcohol dehydrogenases, adhE1 and adhE2, were deleted individually or together. Deletion of adhE1, but not adhE2, diminished ethanol production with a corresponding carbon recovery in acetate. The double deletion mutant had a phenotype similar to that of the adhE1-deficient strain. Expression of adhE1 in trans partially restored the capacity for ethanol production. These results demonstrate the feasibility of genetic investigations of acetogen physiology and the potential for genetic manipulation of C. ljungdahlii to optimize autotrophic biocommodity production.</p>},
	keywords = {Clostridium, Electroporation, Gene Deletion, Genetic Complementation Test, Genetic Vectors, Genetics, Microbial, Metabolic Engineering, Molecular Biology, Plasmids, Transformation, Bacterial},
	issn = {1098-5336},
	doi = {10.1128/AEM.02891-12},
	author = {Leang, Ching and Ueki, Toshiyuki and Nevin, Kelly P 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 {558,
	title = {Cleaning up with genomics: applying molecular biology to bioremediation.},
	journal = {Nat Rev Microbiol},
	volume = {1},
	year = {2003},
	month = {2003 Oct},
	pages = {35-44},
	abstract = {Bioremediation has the potential to restore contaminated environments inexpensively yet effectively, but a lack of information about the factors controlling the growth and metabolism of microorganisms in polluted environments often limits its implementation. However, rapid advances in the understanding of bioremediation are on the horizon. Researchers now have the ability to culture microorganisms that are important in bioremediation and can evaluate their physiology using a combination of genome-enabled experimental and modelling techniques. In addition, new environmental genomic techniques offer the possibility for similar studies on as-yet-uncultured organisms. Combining models that can predict the activity of microorganisms that are involved in bioremediation with existing geochemical and hydrological models should transform bioremediation from a largely empirical practice into a science.},
	keywords = {Bacteria, Bacterial Physiological Phenomena, Biodegradation, Environmental, Ecology, Environmental Microbiology, Fungi, Genetic Vectors, Genome, Bacterial, Genome, Fungal, Genomics, Models, Biological, Molecular Biology},
	issn = {1740-1526},
	doi = {10.1038/nrmicro731},
	author = {Lovley, Derek R}
}
@article {595,
	title = {Development of a genetic system for Geobacter sulfurreducens.},
	journal = {Appl Environ Microbiol},
	volume = {67},
	year = {2001},
	month = {2001 Jul},
	pages = {3180-7},
	abstract = {Members of the genus Geobacter are the dominant metal-reducing microorganisms in a variety of anaerobic subsurface environments and have been shown to be involved in the bioremediation of both organic and metal contaminants. To facilitate the study of the physiology of these organisms, a genetic system was developed for Geobacter sulfurreducens. The antibiotic sensitivity of this organism was characterized, and optimal conditions for plating it at high efficiency were established. A protocol for the introduction of foreign DNA into G. sulfurreducens by electroporation was also developed. Two classes of broad-host-range vectors, IncQ and pBBR1, were found to be capable of replication in G. sulfurreducens. In particular, the IncQ plasmid pCD342 was found to be a suitable expression vector for this organism. When the information and novel methods described above were utilized, the nifD gene of G. sulfurreducens was disrupted by the single-step gene replacement method. Insertional mutagenesis of this key gene in the nitrogen fixation pathway impaired the ability of G. sulfurreducens to grow in medium lacking a source of fixed nitrogen. Expression of the nifD gene in trans complemented this phenotype. This paper constitutes the first report of genetic manipulation of a member of the Geobacter genus.},
	keywords = {Anti-Bacterial Agents, Bacterial Proteins, Blotting, Southern, Culture Media, Deltaproteobacteria, Electroporation, Fimbriae Proteins, Genetic Complementation Test, Genetic Vectors, Microbial Sensitivity Tests, Mutagenesis, Insertional, Nitrogen Fixation, Plasmids, Transformation, Bacterial},
	issn = {0099-2240},
	doi = {10.1128/AEM.67.7.3180-3187.2001},
	author = {Coppi, M V and Leang, C and Sandler, S J and Lovley, D R}
}
