Reductive tricarboxylic acid cycle enzymes and reductive amino acid synthesis pathways contribute to electron balance in a Rhodospirillum rubrum Calvin-cycle mutant.

Purple non-sulfur bacteria (PNSB) use light for energy and organic substrates for carbon and electrons when growing photoheterotrophically. This lifestyle generates more reduced electron carriers than are required for biosynthesis, even during consumption of some of the most oxidized organic substrates like malate and fumarate. Reduced electron carriers not used in biosynthesis must still be oxidized for photoheterotrophic growth to occur. Diverse PNSB commonly rely on the CO2-fixing Calvin cycle to oxidize reduced electron carriers. Some PNSB also produce H2 or reduce terminal electron acceptors as alternatives to the Calvin cycle. Rhodospirillum rubrum Calvin-cycle mutants defy this trend by growing phototrophically on malate or fumarate without H2 production or access to terminal electron acceptors. We used 13C-tracer experiments to examine how a Rs. rubrum Calvin-cycle mutant maintains electron balance under such conditions. We detected the reversal of some tricarboxylic acid cycle enzymes, carrying reductive flux from malate or fumarate to αKG. This pathway and the reductive synthesis of αKG-derived amino acids are likely important for electron balance, as supplementing the growth medium with αKG-derived amino acids prevented Rs. rubrum Calvin-cycle-mutant growth unless a terminal electron acceptor was provided. Flux estimates also suggested that the Calvin-cycle mutant preferentially synthesized isoleucine using the reductive threonine-dependent pathway instead of the less-reductive citramalate-dependent pathway. Collectively, our results suggest that alternative biosynthetic pathways can contribute to electron balance within the constraints of a relatively constant biomass composition.

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

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.

Pantothenate auxotrophy in Zymomonas mobilis ZM4 is due to a lack of aspartate decarboxylase activity.

The bacterium Zymomonas mobilis naturally produces ethanol at near theoretical maximum yields, making it of interest for industrial ethanol production. Zymomonas mobilis requires the vitamin pantothenate for growth. Here we characterized the genetic basis for the Z. mobilis pantothenate auxotrophy. We found that this auxotrophy is due to the absence of a single gene, panD, encoding aspartate-decarboxylase. Heterologous expression of Escherichia coli PanD in Z. mobilis or supplementation of the growth medium with the product of PanD activity, ß-alanine, eliminated the need for exogenous pantothenate. We also determined that Z. mobilis IlvC, an enzyme better known for branched-chain amino acid synthesis, is required for pantothenate synthesis in Z. mobilis, as it compensates for the absence of PanE, another pantothenate synthesis pathway enzyme. In addition to contributing to an understanding of the nutritional requirements of Z. mobilis, our results have led to the design of a more cost-effective growth medium.