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 and phototrophic In this coculture, anaerobically ferments sugars into excreted organic acids as a carbon source for In return, a genetically engineered strain constitutively converts N into NH, providing 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, gene expression changes resembled a nitrogen starvation response under the control of the transcriptional regulator NtrC. By genetically disrupting NtrC, we determined that a nitrogen starvation response is important for a stable coexistence, especially at low NH 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. 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 and growing cooperatively through bidirectional nutrient exchange, we determined that an nitrogen starvation response is important for maintaining a stable coexistence. The lack of an 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. 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.…”

“…To study the molecular mechanisms of nutrient cross-feeding, we previously developed a bacterial coculture in which Escherichia coli and Rhodopseudomonas palustris reciprocally exchange essential metabolites under anaerobic conditions (Fig. 1A) [17][18][19][20]. In this coculture, E. coli ferments glucose, a carbon source that R. palustris cannot consume, and excretes ethanol and organic acids, namely acetate, lactate, succinate, and formate, as waste products.…”

Covert Cross-Feeding Revealed by Genome-Wide Analysis of Fitness Determinants in a Synthetic Bacterial Mutualism

“…These data demonstrate that coculture with R. palustris can restore E. coli purine auxotroph growth to wild-type levels. In support of this conclusion, E. coli pur gene transcription is downregulated in coculture with R. palustris (10). Although this downregulation was originally ascribed to slower E. coli growth (10), de novo purine synthesis is repressed by the presence of purines in the medium (57), as could occur if provided by R. palustris.…”

“…E. coli nac expression is upregulated Ͼ40-fold in coculture compared to monoculture (10). However, only four Nac-regulated genes are differentially expressed in coculture versus monoculture (10), and none of these met our thresholds for strongly impacting fitness in coculture (Table S4). Thus, although the low NH 4…”

“…Nac is the second main transcriptional regulator of the NSR, behind NtrC, and of the 23 operons within the NtrC regulon, which includes nac, nine are regulated indirectly via Nac (22). E. coli nac expression is upregulated Ͼ40-fold in coculture compared to monoculture (10). However, only four Nac-regulated genes are differentially expressed in coculture versus monoculture (10), and none of these met our thresholds for strongly impacting fitness in coculture (Table S4).…”