AnEscherichia colinitrogen starvation response is important for mutualistic coexistence withRhodopseudomonas 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’s 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 constitutively converts N2 into NH4+, providing E. coli with essential nitrogen. Using 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.ImportanceMutualistic 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.

Enhanced nutrient uptake is sufficient to drive emergent cross-feeding between bacteria in a synthetic community

“…Low formate yields could also be explained by decreased formate production by E. coli in favor of other fermentation products that R. palustris can readily consume. We previously observed low formate yields in slow-growing, nitrogen-limited NifA*-based cocultures (19, 20), suggesting that formate production by E. coli varies in response to growth rate. We have also not ruled out the possibility that R. palustris can consume some formate under certain conditions.…”

“…NtrC and the histidine kinase NtrB form a two-component system that senses and coordinates the nitrogen starvation response in E. coli (30, 31). Our lab previously found that the E. coli NtrBC-regulon is highly expressed in coculture with R. palustris NifA* (20). Thus E. coli NtrBC might be even more important in coculture with WT R. palustris wherein E. coli nitrogen starvation is expected to be intensified.…”

“…Based on the effects of NtrC mutations observed by others (32, 33), we hypothesized the NtrC S163R allele enables coculture growth with WT R. palustris by conferring constitutive expression of NtrBC-regulated genes important for NH 4 + acquisition. We previously determined that NtrC and AmtB were crucial gene products within the NtrBC regulon for growth and coexistence with R. palustris NifA* (18, 20). To test if the NtrC S163R allele increased amtB and ntrC expression, we measured transcript levels by reverse transcription quantitative PCR (RT-qPCR) in E. coli monocultures grown with 15 mM NH 4 Cl or subjected to complete nitrogen starvation (∼10 h with 0 mM NH 4 Cl).…”

“…To study the molecular mechanisms of nutrient cross-feeding, we previously developed a synthetic bacterial coculture in which Escherichia coli and Rhodopseudomonas palustris reciprocally exchange essential metabolites under anaerobic conditions (Fig.1A) (17-20). In this coculture, E. coli ferments glucose, a carbon source that R. palustris cannot consume, and excretes ethanol and organic acids as waste products.…”

Genome-wide analysis ofEscherichia colifitness determinants in a cross-feeding mutualism withRhodopseudomonas palustris

“…Recently, our lab used transcriptome sequencing (RNA-Seq) and proteomics to interrogate how cross-feeding impacts the physiology of each species (37). This study revealed that the E. coli NtrC-mediated nitrogen starvation response (NSR) is crucial for fitness in coculture (37). However, RNA-Seq does not necessarily identify genes that confer fitness under the tested conditions.…”