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An Escherichia coli Nitrogen Starvation Response Is Important for Mutualistic Coexistence with Rhodopseudomonas palustris.

ABSTRACT: Microbial mutualistic cross-feeding interactions are ubiquitous and can drive important community functions. Engaging in cross-feeding undoubtedly affects the physiology and metabolism of individual species involved. However, the nature in which an individual species' physiology is influenced by cross-feeding and the importance of those physiological changes for the mutualism have received little attention. We previously developed a genetically tractable coculture to study bacterial mutualisms. The coculture consists of fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris In this coculture, E. coli anaerobically ferments sugars into excreted organic acids as a carbon source for R. palustris In return, a genetically engineered R. palustris strain constitutively converts N2 into NH4+, providing E. coli with essential nitrogen. Using transcriptome sequencing (RNA-seq) and proteomics, we identified transcript and protein levels that differ in each partner when grown in coculture versus monoculture. When in coculture with R. palustris, E. coli gene expression changes resembled a nitrogen starvation response under the control of the transcriptional regulator NtrC. By genetically disrupting E. coli NtrC, we determined that a nitrogen starvation response is important for a stable coexistence, especially at low R. palustris NH4+ excretion levels. Destabilization of the nitrogen starvation regulatory network resulted in variable growth trends and, in some cases, extinction. Our results highlight that alternative physiological states can be important for survival within cooperative cross-feeding relationships.IMPORTANCE Mutualistic cross-feeding between microbes within multispecies communities is widespread. Studying how mutualistic interactions influence the physiology of each species involved is important for understanding how mutualisms function and persist in both natural and applied settings. Using a bacterial mutualism consisting of Rhodopseudomonas palustris and Escherichia coli growing cooperatively through bidirectional nutrient exchange, we determined that an E. coli nitrogen starvation response is important for maintaining a stable coexistence. The lack of an E. coli nitrogen starvation response ultimately destabilized the mutualism and, in some cases, led to community collapse after serial transfers. Our findings thus inform on the potential necessity of an alternative physiological state for mutualistic coexistence with another species compared to the physiology of species grown in isolation.

SUBMITTER: McCully AL

PROVIDER: S-EPMC6029099 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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An Escherichia coli Nitrogen Starvation Response Is Important for Mutualistic Coexistence with Rhodopseudomonas palustris.

McCully Alexandra L AL Behringer Megan G MG Gliessman Jennifer R JR Pilipenko Evgeny V EV Mazny Jeffrey L JL Lynch Michael M Drummond D Allan DA McKinlay James B JB

Applied and environmental microbiology 20180702 14

Microbial mutualistic cross-feeding interactions are ubiquitous and can drive important community functions. Engaging in cross-feeding undoubtedly affects the physiology and metabolism of individual species involved. However, the nature in which an individual species' physiology is influenced by cross-feeding and the importance of those physiological changes for the mutualism have received little attention. We previously developed a genetically tractable coculture to study bacterial mutualisms. ...[more]

PMID: 29728387

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Project description:To cope with fluctuations in their environment, bacteria have evolved multiple adaptive stress responses. One such response is the nitrogen regulation stress response, which allows bacteria, such as Escherichia coli, to cope with and overcome conditions of nitrogen limitation. This response is directed by the two-component system NtrBC, where NtrC acts as the major transcriptional regulator to activate the expression of genes to mount the response. Recently, my colleagues and I showed that NtrC directly regulates the expression of the relA gene, the major (p)ppGpp synthetase in E. coli, coupling the nitrogen regulation stress and stringent responses. As elevated levels of (p)ppGpp have been implicated in the formation of persister cells, here, I investigated whether nitrogen starvation promotes their formation and whether the NtrC-RelA regulatory cascade plays a role. The results reveal that nitrogen-starved E. coli synthesizes (p)ppGpp and forms a higher percentage of persister cells than nonstarved cells and that both NtrC and RelA are important for these processes. This study provides novel insights into how the formation of persisters can be promoted in response to a nutritional stress.IMPORTANCE Bacteria often reside in environments where nutrient availability is scarce; therefore, they have evolved adaptive responses to rapidly cope with conditions of feast and famine. Understanding the mechanisms that underpin the regulation of how bacteria cope with this stress is a fundamentally important question in the wider context of understanding the biology of the bacterial cell and bacterial pathogenesis. Two major adaptive mechanisms to cope with starvation are the nitrogen regulation (ntr) stress and stringent responses. Here, I describe how these bacterial stress responses are coordinated under conditions of nitrogen starvation to promote the formation of antibiotic-tolerant persister cells by elevating levels of the secondary messenger (p)ppGpp.

Project description:Rhodopseudomonas palustris, a nonsulphur purple photosynthetic bacteria, has been extensively investigated for its metabolic versatility including ability to produce hydrogen gas from sunlight and biomass. The availability of the finished genome sequences of six R. palustris strains (BisA53, BisB18, BisB5, CGA009, HaA2 and TIE-1) combined with online bioinformatics software for integrated analysis presents new opportunities to determine the genomic basis of metabolic versatility and ecological lifestyles of the bacteria species. The purpose of this investigation was to compare the functional annotations available for multiple R. palustris genomes to identify annotations that can be further investigated for strain-specific or uniquely shared phenotypic characteristics. A total of 2,355 protein family Pfam domain annotations were clustered based on presence or absence in the six genomes. The clustering process identified groups of functional annotations including those that could be verified as strain-specific or uniquely shared phenotypes. For example, genes encoding water/glycerol transport were present in the genome sequences of strains CGA009 and BisB5, but absent in strains BisA53, BisB18, HaA2 and TIE-1. Protein structural homology modeling predicted that the two orthologous 240 aa R. palustris aquaporins have water-specific transport function. Based on observations in other microbes, the presence of aquaporin in R. palustris strains may improve freeze tolerance in natural conditions of rapid freezing such as nitrogen fixation at low temperatures where access to liquid water is a limiting factor for nitrogenase activation. In the case of adaptive loss of aquaporin genes, strains may be better adapted to survive in conditions of high-sugar content such as fermentation of biomass for biohydrogen production. Finally, web-based resources were developed to allow for interactive, user-defined selection of the relationship between protein family annotations and the R. palustris genomes.

Dataset Information

An Escherichia coli Nitrogen Starvation Response Is Important for Mutualistic Coexistence with Rhodopseudomonas palustris.

Publications

An Escherichia coli Nitrogen Starvation Response Is Important for Mutualistic Coexistence with Rhodopseudomonas palustris.

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