Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
Shewanella Federation
36
Respiratory Pathways and Regulatory Networks of Shewanella oneidensis Involved in Energy Metabolism and Environmental Sensing
Alex Beliaev*1, Yuri Gorby1, Margie Romine1, Jeff McLean1, Grigoriy Pinchuk1, Eric Hill1, Jim Fredrickson1, Jizhong Zhou2, and Daad A. Saffarini3
1Pacific Northwest National Laboratory, Richland, WA; 2Oak Ridge National Laboratory, Oak Ridge, TN; and 3University of Wisconsin, Milwaukee, WI
Shewanella oneidensis MR-1 is a facultative γ-Proteobacterium with remarkable metabolic versatility in regards to electron acceptor utilization; it can utilize O2, nitrate, fumarate, Mn, Fe, and S0 as terminal electron acceptors during respiration. This versatility allows MR-1 to efficiently compete for resources in environments where electron acceptor type and concentration fluctuate in space and time. The ability to effectively reduce polyvalent metals and radionuclides, including solid phase Fe and Mn oxides, has generated considerable interest in the potential role of this organism in biogeochemical cycling and in the bioremediation of contaminant metals and radionuclides. The entire genome sequence of MR-1 has been determined and high throughput methods for measuring gene expression are being developed and applied. This project is part of the Shewanella Federation, a multi-investigator and cross-institutional consortium formed to achieve a systems level understanding of how S. oneidensis MR-1 senses and responds to its environment.
Electron Acceptor-Induced Shifts in S. oneidensis MR-1 Gene Expression Profiles. To define the repertoire of genes responding to both metal and non-metal electron acceptors and identify basic regulatory mechanisms governing anaerobic respiration in S. oneidensis, we compared mRNA expression patterns of anaerobic cultures incubated with fumarate to those exposed to nitrate, thiosulfate, DMSO, TMAO, ferric citrate, hydrous HFO, manganese dioxide, colloidal manganese, and cobalt using whole-genome arrays. The extent of S. oneidensis transcriptome response to metal electron acceptors was revealed by hierarchical clustering analysis, where a high degree of similarity in global expression profiles was exhibited throughout all metal-reducing conditions which resulted in metals grouping separately from non-metal electron acceptors and forming a tight, well-defined branch. In accordance with the results of a principal component analysis, we identified two major expression groups that displayed activation and repression in the presence of metals and accounted for over 60% of the differentially expressed genes. While genes encoding hypothetical and conserved hypothetical proteins dominated both clusters, there were several functional subgroups encoding putative components of electron transport chain, transcriptional regulators and detoxification/toxin resistance proteins that were characterized by their non-specific upregulation to all metal electron acceptors. Contrary to what was expected, the mtrCAB operon which encodes two deca-heme c-type cytochromes and an outer membrane protein essential for Fe(III) and Mn(IV) respiration in S. oneidensis showed 2- to 8- fold decrease in mRNA levels under metal-reducing conditions. In contrast, S. oneidensis demonstrated specific transcriptome responses to individual non-metal electron acceptors producing unique clusters of nitrate, thiosulfate and TMAO induced genes. While these observations undoubtedly reflect the nature of metal and non-metal electron acceptors, the diversification and tighter regulatory control of the non-metal respiratory systems may be indicative of different evolutionary pathways taken by these respiratory systems. Moreover, the absence of upregulation for known genes involved in metal reduction may be due to the low-specificity and the opportunistic nature of the metal reduction pathways in S. oneidensis. This work represents an important step towards understanding the anaerobic respiratory system of S. oneidensis MR-1 on a genomic scale and has yielded numerous candidate genes for more detailed functional analysis.
Autoaggregation of Shewanella oneidensis in Response to High Oxygen Concentrations. Despite the potential environmental importance of this phenomenon, little is known about the mechanisms inducing aggregate formation and subsequent impacts on cells inside the aggregates. Under aerobic conditions, S. oneidensis cells are highly adhesive to glass and in the presence of CaCl2 the cells aggregate into large multi-cellular structures. Microscopic analyses of these aggregates identified a presence of DNA, proteins and carbohydrate-like material in the extracellular matrix. In contrast, cells grown under suboxic conditions did not display any autoaggregation while their adhesion to surfaces was significantly reduced. Microarray expression analysis comparing samples of suboxically- vs. aerobically-grown cells identified a set of genes encoding cell-to-cell and cell-to-surface adhesion and colonization factors that positively responded to increased O2 concentrations. Of particular interest was the O2-dependent upregulation of S. oneidensis csgAB and csgDEF operons which are putatively involved in curli fimbrae formation. In other organisms, such Escherichia coli, these structures confer attachment to inert surfaces such as glass and have also been implicated in cell-cell attachment. Although, when compared to suboxic conditions, both flocculated and unflocculated cells displayed some similarities in gene expression in response to elevated levels of O2, autoaggregation had a significant impact on gene expression in S. oneidensis. Direct comparison of aggregated versus unagreggated cells grown under 50% dissolved O2 tension (DOT) revealed remarkable differences in mRNA patterns between these two states. Unflocculated cells displayed significant increase of mRNA levels of genes involved in aerobic energy metabolism, intermediary carbon metabolism and gluconeogenesis as well as chemotaxis and motility. In contrast, several genes putatively involved in anaerobic metabolism, gene cluster encoding outer membrane proteins and cytochromes, and transcriptional regulation were upregulated under 50% DOT aggregated conditions. Remarkably, the majority (~90%) of genes located on the 50-kb megaplasmid of MR-1 displayed substantial levels of upregulation in flocculated cells. It is currently unclear whether this phenomenon is due to a global regulatory effect or to an increase in plasmid copy number. Although further studies are required for resolution, we speculate that autoaggregation in S. oneidensis MR-1 may serve as a mechanism to facilitate reduced O2 tensions within aggregate, leading to the expression of anaerobic genes under bulk aerobic conditions.
Cyclic AMP Signaling and cAMP Receptor Protein-Dependent Regulation of Anaerobic Energy Metabolism in Shewanella oneidensis MR-1. Unlike many bacteria studied to date, the ability of S. oneidensis to grow anaerobically with several electron acceptors is regulated by the cAMP-receptor protein (CRP). CRP-deficient mutants of MR-1 are impaired in anaerobic reduction and growth with Fe(III), Mn(IV), fumarate, nitrate, and DMSO. Loss of anaerobic respiration in crp mutants is due to loss of terminal anaerobic reductases and not due to deficiency in carbon metabolism. To further elucidate the role of CRP and to understand the mechanisms of cAMP-dependent gene expression under anaerobic conditions in S. oneidensis, DNA microarray analyses were performed. Comparison of mRNA expression profiles of wild-type and crp mutant cells grown anaerobically with different electron acceptors indicated that CRP positively regulates the expression of genes involved in energy generation and transcriptional regulation. These include the periplasmic nitrate reductase, the polysulfide reductase, anaerobic DMSO reductase genes, as well as the nitrate/nitrite sensor protein narQ. To identify the mechanisms and proteins that lead to CRP activation under anaerobic conditions, genes predicted to encode adenylate cyclases were analyzed. The genome sequence of S. oneidensis contains three putative adenylate cyclase genes, designated cyaA, cyaB, and cyaC. Deletion of cyaA or cyaB did not affect anaerobic growth with any of the electron acceptors tested while deletion of cyaC resulted in growth deficiency with DMSO. Surprisingly, deletions of both cyaA and cyaC resulted in anaerobic growth deficiency with DMSO, nitrate, Fe(III), Mn(IV), and fumarate. These phenotypes are similar to the phenotypes of the CRP-deficient mutants. Our results indicate that both CyaA and CyaC are needed for the production of cAMP under anaerobic conditions, and for activation of CRP. Further work to identify the cAMP signaling pathways in S. oneidensis is underway.
* Presenting author
