Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
University of Massachusetts, Amherst
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Novel Regulatory Systems and Adaption of Some Well-Known Systems Controlling Respiration, Growth, and Chemotaxis of Geobactor Species
Maddalena Coppi1* (mcoppi@microbio.umass.edu), Byoung-Chan Kim1, Laurie DiDonato1, Julia Krushkal2, Bin Yan2, Richard Glaven1, Regina O’ Neil1, Suphan Bakkal1, Allen Tsang1, Hoa Tran1, Abraham Esteve-Nunez1, Cinthia Nunez1, Ching Leang1, Kuk-Jeong Chin1, Barbara Methe3, Robert Weis1, Pablo Pomposiello1, Kelly Nevin1, and Derek Lovley1
1University of Massachusetts, Amherst, MA; 2University of Tennessee Health Science Center, Memphis, TN; and 3The Institute for Genomic Research, Rockville, MD
The goal of our Genomics:GTL project is to be able to predicatively model the growth and activity of the Geobacter species that predominate during in situ bioremediation of uranium and on the surface of energy-harvesting electrodes in order to better understand these processes and to have the ability to predict the likely outcome of optimization strategies. This requires an understanding not only of the physiological capabilities of Geobacter species, but also of how the expression of those physiological capabilities is regulated under various environmental conditions. At last year’s meeting we reported on elucidation of global transcriptional regulatory systems in G. sulfurreducens, such as the RpoS, RpoE, and Fur regulons. In the past year studies have focused on genome-scale computational and microarray analysis of transcriptional regulation as well as more in depth studies on the regulation of expression of genes known to encode for proteins important in extracellular electron transfer to metals and electrodes.
One of the most surprising findings was that some of the c-type cytochromes in G. sulfurreducens have a regulatory function and play a role in regulating the production of other c-type cytochromes via either transcriptional or post-translation regulatory functions. For example, the omcF gene is predicted to encode for a small outer-membrane mono-heme c-type cytochrome. Thus, its function as a potential electron transfer protein was evaluated. An OmcF-deficient mutant was deficient in its ability to reduce Fe(III) and this was associated with an absence of the outer-membrane c-type cytochrome, OmcB, which is known to be required for Fe(III) reduction. Reverse transcriptase PCR and northern blot analysis revealed that the omcB was not transcribed in the OmcF-deficient mutant. Expression of omcF in trans restored the expression of omcB as well as the ability of the mutant to reduce Fe(III). These results suggest that OmcF may play a role in transcriptional regulation of omcB. Deletion of another outer-membrane c-type cytochrome gene, designated omcG, which is predicted to contain 13 hemes, also greatly diminished levels of OmcB. However, unlike the omcF mutant, omcB transcription was not affected. These results indicate that OmcG is specifically involved in either the modification, stabilization, or maturation of OmcB. These are the first reports of cytochromes that are necessary for electron transfer to metals, but not directly involved in electron transfer process. Their more likely role is to serve as sensors that regulate cytochrome expression.
Expression of omcB is also controlled by RpoS and RelGsu, the G. sulfurreducens homolog of RelA, which are important in response to growth under nutrient-limited or stressful conditions. Another transcriptional regulator of omcB expression appears to be the product of the gene ofrR, which is immediately upstream of the operon that includes omcB. Levels of omcB transcripts increased orders of magnitude in response to a limitation in electron-acceptor availability or as rates of growth on Fe(III) increased. In a similar manner, expression of omcS, which encodes for an outer-membrane c-type cytochrome that is required for electricity production is regulated via multiple mechanisms. Microarray studies have identified two-component systems that subsequent genetic studies have demonstrated control cytochrome production in response to changing environmental conditions. These results demonstrate that extracellular electron transfer is highly regulated in G. sulfurreducens.As outlined in a companion abstract, it has recently been determined that the pili of G. sulfurreducens function as nanowires that are required for electron transfer to Fe(III) oxides. Genome-scale studies of the regulation of pilin formation suggested that expression of pilA, the gene for the structural pilin protein, is regulated in response to electron acceptor availability, as well as redox and nutrient status. For example, levels of pilA transcripts were significantly higher in mutants in which one of the two Fnr-like genes was deleted or when cells were grown under electron-acceptor limiting conditions. Deleting the relGsu lowered pilA transcript levels. Genome analysis suggests that pilA expression is also controlled by a two component regulatory system and the sigma factor, RpoN. Additional mechanisms for pilin production will be reported.
It is expected that regulation of cell behavior in the form of chemotaxis plays an important role in the predominance of Geobacter species in subsurface environments. Previous studies have demonstrated that chemotaxis to iron is an important aspect of the reduction of Fe(III) oxides by Geobacter species. The genome sequence of G. sulfurreducens, contains multiple homologs of chemotaxis genes, including cheW (10), cheA (4), cheY (7), cheR (5), cheB (3), cheC (3), cheD (3) and cheV (1). This contrasts with the genome of E. coli which only contains a single copy of a subset of these genes. In order to elucidate factors controlling chemotaxis in Geobacter species the gene for a methyl-accepting chemotaxis protein (MCP), from Geobacter metallireducens was expressed in a strain of E. coli (HCB429) that lacks MCPs. This restored chemotaxis-like in the E. coli strain grown in soft agar. Evaluating the function of Geobacter chemotaxis proteins in E. coli shows promise as a versatile high throughput approach.
Computational analyses that integrated whole genome analyses, comparative genomics, and gene expression microarray data have identified thousands of sites potentially related to gene regulation in Geobacter species. A comprehensive resource has been developed that provides the predicted operon organization of the genomes and contig assemblies of Geobacter species, potential transcriptional regulatory motifs in the upstream regions of every predicted operon, the results of bi-directional similarity comparisons between E. coli regulatory proteins and proteins of G. sulfurreducens, and the genome locations of predicted transcription regulatory elements, palindromic motifs, and conserved bacterial elements. This is an invaluable resource for ongoing experimental studies of regulation of respiration and more recently initiated studies on regulation of central metabolism. As will be detailed at the meeting, many of the binding sites for transcriptional regulators that were predicted from analysis of multiple whole-genome gene expression studies with microarrays are in agreement with sequence-based predictions and with experimental results.
Details will also be provided on studies of other forms of regulation such as the response to oxidative stress and the coordinated regulation of the expression of central metabolism and respiratory genes under conditions simulating those expected during in situ uranium bioremediation as well as continued studies of the global regulatory systems first described at last years meeting.
* Presenting author
