@article {458, title = {Genome-wide analysis of the RpoN regulon in Geobacter sulfurreducens.}, journal = {BMC Genomics}, volume = {10}, year = {2009}, month = {2009}, pages = {331}, abstract = {BACKGROUND: The role of the RNA polymerase sigma factor RpoN in regulation of gene expression in Geobacter sulfurreducens was investigated to better understand transcriptional regulatory networks as part of an effort to develop regulatory modules for genome-scale in silico models, which can predict the physiological responses of Geobacter species during groundwater bioremediation or electricity production. RESULTS: An rpoN deletion mutant could not be obtained under all conditions tested. In order to investigate the regulon of the G. sulfurreducens RpoN, an RpoN over-expression strain was made in which an extra copy of the rpoN gene was under the control of a taclac promoter. Combining both the microarray transcriptome analysis and the computational prediction revealed that the G. sulfurreducens RpoN controls genes involved in a wide range of cellular functions. Most importantly, RpoN controls the expression of the dcuB gene encoding the fumarate/succinate exchanger, which is essential for cell growth with fumarate as the terminal electron acceptor in G. sulfurreducens. RpoN also controls genes, which encode enzymes for both pathways of ammonia assimilation that is predicted to be essential under all growth conditions in G. sulfurreducens. Other genes that were identified as part of the RpoN regulon using either the computational prediction or the microarray transcriptome analysis included genes involved in flagella biosynthesis, pili biosynthesis and genes involved in central metabolism enzymes and cytochromes involved in extracellular electron transfer to Fe(III), which are known to be important for growth in subsurface environment or electricity production in microbial fuel cells. The consensus sequence for the predicted RpoN-regulated promoter elements is TTGGCACGGTTTTTGCT. CONCLUSION: The G. sulfurreducens RpoN is an essential sigma factor and a global regulator involved in a complex transcriptional network controlling a variety of cellular processes.}, keywords = {Bacterial Proteins, DNA, Bacterial, Gene Expression Profiling, Gene Expression Regulation, Bacterial, Genome-Wide Association Study, Geobacter, Multigene Family, Oligonucleotide Array Sequence Analysis, Promoter Regions, Genetic, Regulon, RNA Polymerase Sigma 54}, issn = {1471-2164}, doi = {10.1186/1471-2164-10-331}, author = {Leang, Ching and Krushkal, Julia and Ueki, Toshiyuki and Puljic, Marko and Sun, Jun and Ju{\'a}rez, Katy and N{\'u}{\~n}ez, Cinthia and Reguera, Gemma and DiDonato, Raymond and Postier, Bradley and Adkins, Ronald M and Lovley, Derek R} } @article {701, title = {Benefits of in-situ synthesized microarrays for analysis of gene expression in understudied microorganisms.}, journal = {J Microbiol Methods}, volume = {74}, year = {2008}, month = {2008 Jul}, pages = {26-32}, abstract = {Although the genome sequences of many microorganisms are now known, whole-genome DNA microarray platforms consisting of PCR amplicon, or oligonucleotide elements printed onto glass slides have been readily available for only a relatively few, highly studied microorganisms. For those microorganisms more recently cultured or studied by fewer investigators it has been difficult to justify the initial time and expense of developing such array platforms especially if only a limited number of gene expression studies are envisioned. However, in-situ synthesized oligonucleotide (ISO) arrays can be inexpensively fabricated on an {\textquoteright}as needed{\textquoteright} basis with a reduced initial investment in time, personnel, resources, and costs. To evaluate the performance of one ISO array platform, gene expression patterns in Geobacter sulfurreducens under nitrogen-fixing conditions were compared with results from quantitative reverse transcriptase PCR (qRT-PCR) and previously published data from a similar experiment using spotted PCR amplicon arrays. There were strong correlations between the results of the ISO arrays and the results from qRT-PCR (r(2)=0.762) and spotted array (r(2)=0.744) analyses. After initial use the ISO arrays could be successfully stripped and reused. The increased flexibility in array design and reusability coupled with a lower initial investment in terms of fabrication time and cost for the ISO arrays suggest that they may be the preferred approach when investigating gene expression in microorganisms, especially when only a few expression studies are required.}, keywords = {Bacterial Proteins, Computational Biology, Gene Expression, Geobacter, Nitrogen Fixation, Oligonucleotide Array Sequence Analysis, Oligonucleotides, Polymerase Chain Reaction}, issn = {0167-7012}, doi = {10.1016/j.mimet.2007.07.004}, author = {Postier, Bradley and DiDonato, Raymond and Nevin, Kelly P and Liu, Anna and Frank, Bryan and Lovley, Derek and Methe, Barbara A} } @article {476, title = {Geobacter sulfurreducens strain engineered for increased rates of respiration.}, journal = {Metab Eng}, volume = {10}, year = {2008}, month = {2008 Sep}, pages = {267-75}, abstract = {Geobacter species are among the most effective microorganisms known for the bioremediation of radioactive and toxic metals in contaminated subsurface environments and for converting organic compounds to electricity in microbial fuel cells. However, faster rates of electron transfer could aid in optimizing these processes. Therefore, the Optknock strain design methodology was applied in an iterative manner to the constraint-based, in silico model of Geobacter sulfurreducens to identify gene deletions predicted to increase respiration rates. The common factor in the Optknock predictions was that each resulted in a predicted increase in the cellular ATP demand, either by creating ATP-consuming futile cycles or decreasing the availability of reducing equivalents and inorganic phosphate for ATP biosynthesis. The in silico model predicted that increasing the ATP demand would result in higher fluxes of acetate through the TCA cycle and higher rates of NADPH oxidation coupled with decreases in flux in reactions that funnel acetate toward biosynthetic pathways. A strain of G. sulfurreducens was constructed in which the hydrolytic, F(1) portion of the membrane-bound F(0)F(1) (H(+))-ATP synthase complex was expressed when IPTG was added to the medium. Induction of the ATP drain decreased the ATP content of the cell by more than half. The cells with the ATP drain had higher rates of respiration, slower growth rates, and a lower cell yield. Genome-wide analysis of gene transcript levels indicated that when the higher rate of respiration was induced transcript levels were higher for genes involved in energy metabolism, especially in those encoding TCA cycle enzymes, subunits of the NADH dehydrogenase, and proteins involved in electron acceptor reduction. This was accompanied by lower transcript levels for genes encoding proteins involved in amino acid biosynthesis, cell growth, and motility. Several changes in gene expression that involve processes not included in the in silico model were also detected, including increased expression of a number of redox-active proteins, such as c-type cytochromes and a putative multicopper outer-surface protein. The results demonstrate that it is possible to genetically engineer increased respiration rates in G. sulfurreducens in accordance with predictions from in silico metabolic modeling. To our knowledge, this is the first report of metabolic engineering to increase the respiratory rate of a microorganism.}, keywords = {Adenosine Triphosphate, Bacterial Proteins, Biodegradation, Environmental, Citric Acid Cycle, Electron Transport, Geobacter, Metals, Models, Biological, NADH Dehydrogenase, NADP, Oxygen Consumption, Phosphates, Proton-Translocating ATPases, Radioactive Pollutants}, issn = {1096-7184}, doi = {10.1016/j.ymben.2008.06.005}, author = {Izallalen, Mounir and Mahadevan, Radhakrishnan and Burgard, Anthony and Postier, Bradley and DiDonato, Raymond and Sun, Jun and Schilling, Christopher H and Lovley, Derek R} }