An <i>Escherichia coli</i> nitrogen starvation response is important for mutualistic coexistence with <i>Rhodopseudomonas palustris</i>

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 N 2 into NH 4 + , 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 NH 4 + 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.