A Rhizobiales-Specific Unipolar Polysaccharide Adhesin Contributes to Rhodopseudomonas palustris Biofilm Formation across Diverse Photoheterotrophic Conditions

Post-print, accepted manuscript versionBacteria predominantly exist as members of surfaced-attached communities known as biofilms. Many bacterial species initiate biofilms and adhere to each other using cell surface adhesins. This is the case for numerous ecologically diverse Alphaprotebacteria, which use polar exopolysaccharide adhesins for cell-cell adhesion and surface attachment. Here, we show that Rhodopseudomonas palustris, a metabolically versatile member of the alphaproteobacterial order Rhizobiales, contains a functional unipolar polysaccharide (UPP) biosynthesis gene cluster. Deletion of genes predicted to be critical for UPP biosynthesis and export abolished UPP production. We also found that R. palustris uses UPP to mediate biofilm formation across diverse photoheterotrophic growth conditions, wherein light and organic substrates are used to support growth. However, UPP was less important for biofilm formation during photoautotrophy, where light and CO2 support growth, and during aerobic respiration with organic compounds. Expanding our analysis beyond R. palustris, we examined the phylogenetic distribution and genomic organization of UPP gene clusters among Rhizobiales species that inhabit diverse niches. Our analysis suggests that UPP is a conserved ancestral trait of the Rhizobiales but that it has been independently lost multiple times during the evolution of this clade, twice coinciding with adaptation to intracellular lifestyles within animal hosts

Two Group A Streptococcal Peptide Pheromones Act through Opposing Rgg Regulators to Control Biofilm Development

Streptococcus pyogenes (Group A Streptococcus, GAS) is an important human commensal that occasionally causes localized infections and less frequently causes severe invasive disease with high mortality rates. How GAS regulates expression of factors used to colonize the host and avoid immune responses remains poorly understood. Intercellular communication is an important means by which bacteria coordinate gene expression to defend against host assaults and competing bacteria, yet no conserved cell-to-cell signaling system has been elucidated in GAS. Encoded within the GAS genome are four rgg-like genes, two of which (rgg2 and rgg3) have no previously described function. We tested the hypothesis that rgg2 or rgg3 rely on extracellular peptides to control target-gene regulation. We found that Rgg2 and Rgg3 together tightly regulate two linked genes encoding new peptide pheromones. Rgg2 activates transcription of and is required for full induction of the pheromone genes, while Rgg3 plays an antagonistic role and represses pheromone expression. The active pheromone signals, termed SHP2 and SHP3, are short and hydrophobic (DI[I/L]IIVGG), and, though highly similar in sequence, their ability to disrupt Rgg3-DNA complexes were observed to be different, indicating that specificity and differential activation of promoters are characteristics of the Rgg2/3 regulatory circuit. SHP-pheromone signaling requires an intact oligopeptide permease (opp) and a metalloprotease (eep), supporting the model that pro-peptides are secreted, processed to the mature form, and subsequently imported to the cytoplasm to interact directly with the Rgg receptors. At least one consequence of pheromone stimulation of the Rgg2/3 pathway is increased biogenesis of biofilms, which counteracts negative regulation of biofilms by RopB (Rgg1). These data provide the first demonstration that Rgg-dependent quorum sensing functions in GAS and substantiate the role that Rggs play as peptide receptors across the Firmicute phylum

Phototrophic Lactate Utilization by <i>Rhodopseudomonas palustris</i> Is Stimulated by Coutilization with Additional Substrates

Bacterial carbon source utilization is frequently assessed using cultures provided single carbon sources. However, the utilization of carbon mixtures by bacteria (i.e., mixed-substrate utilization) is of both fundamental and practical importance; it is central to bacterial physiology and ecology, and it influences the utility of bacteria as biotechnology. Here we investigated mixed-substrate utilization by the model organism Rhodopseudomonas palustris . Using mixtures of organic acids and glycerol, we show that R. palustris exhibits an expanded range of usable carbon substrates when provided substrates in mixtures. Specifically, coutilization enabled the prompt consumption of lactate, a substrate that is otherwise not readily used by R. palustris . Additionally, we found that R. palustris utilizes acetate and glycerol sequentially, revealing that this species has the capacity to use some substrates in a preferential order. These results provide insights into R. palustris physiology that will aid the use of R. palustris for industrial and commercial applications. </jats:p

Growth-independent cross-feeding modifies boundaries for coexistence in a bacterial mutualism

AbstractNutrient cross-feeding can stabilize microbial mutualisms, including those important for carbon cycling in nutrient-limited anaerobic environments. It remains poorly understood how nutrient limitation within natural environments impacts mutualist growth, cross-feeding levels, and ultimately mutualism dynamics. We examined the effects of nutrient limitation within a mutualism using theoretical and experimental approaches with a synthetic anaerobic coculture pairing fermentativeEscherichia coliand phototrophicRhodopseudomonas palustris. In this coculture,E. coliandR. palustrisresemble an anaerobic food web by cross-feeding essential carbon (organic acids) and nitrogen (ammonium), respectively. Organic acid cross-feeding stemming fromE. colifermentation can continue in a growth-independent manner during nutrient limitation, while ammonium cross-feeding byR. palustrisis growth-dependent. When ammonium cross-feeding was limited, coculture trends changed yet coexistence persisted under both homogenous and heterogenous conditions. Theoretical modeling indicated that growth-independent fermentation was crucial to sustain cooperative growth under conditions of low nutrient exchange. We also show that growth-independent fermentation sets the upperE. colicell density at which this mutualism is supported. Thus, growth-independent fermentation can conditionally stabilize or destabilize a mutualism, indicating the potential importance of growth-independent metabolism for nutrient-limited mutualistic communities.Conflict of interestThe authors declare no conflict of interest.</jats:sec

Recipient-biased competition for a cross-fed nutrient is required for coexistence of microbial mutualists

AbstractMany mutualistic microbial relationships are based on nutrient cross-feeding. Traditionally, cross-feeding is viewed as being unidirectional from the producer to the recipient. This is likely true when a producer’s metabolic waste, such as fermentation products, provides carbon for a recipient. However, in some cases the cross-fed nutrient holds value for both the producer and the recipient. In such cases, there is potential for nutrient reacquisition by producer cells in a population, leading to competition against recipients. Here we investigate the consequences of inter-partner competition for cross-fed nutrients on mutualism dynamics using an anaerobic coculture pairing fermentativeEscherichia coliand phototrophicRhodopseudomonas palustris. In this coculture,E. coliexcretes waste organic acids that provide carbon forR. palustris. In return,R. palustriscross-feedsE. coliammonium (NH4+), a valuable nitrogen compound that both species prefer. To explore the potential for inter-partner competition, we first used a kinetic model to simulate cocultures with varied affinities for NH4+in each species. The model predicted that inter-partner competition for cross-fed NH4+could profoundly impact population dynamics. We then experimentally tested the predictions by culturing mutants lacking NH4+transporters in both NH4+competition assays and cooperative cocultures. Both theoretical and experimental results indicated that the recipient must have a competitive advantage in acquiring valuable cross-fed NH4+to avoid collapse of the mutualism. Thus, the very metabolites that form the basis for cooperative cross-feeding can also be subject to competition between mutualistic partners.SignificanceMutualistic relationships, particularly those based on nutrient cross-feeding, promote stability of diverse ecosystems and drive global biogeochemical cycles. Cross-fed nutrients within these systems can be either waste products valued only by one partner or nutrients that both partners value. Here, we explore how inter-partner competition for a communally-valuable cross-fed nutrient impacts mutualism dynamics. We discovered that mutualism stability necessitates that the recipient have a competitive advantage against the producer in obtaining the cross-fed nutrient. We propose that the requirement for recipient-biased competition is a general rule for mutualistic coexistence based on the transfer of communally valuable resources, microbial or otherwise.</jats:sec

N ₂ gas is an effective fertilizer for bioethanol production by <i>Zymomonas mobilis</i>

Significance Recently there has been a surge in ethanol biofuel production from cellulosic feedstocks, offering more environmental benefits than conventional ethanol production from food crops. However, cellulosic feedstocks are low in nitrogen, requiring that millions of dollars be spent on nitrogen supplements to grow the ethanol-producing microbes. Zymomonas mobilis is a bacterium that has long been viewed as a potential rival to Baker’s yeast as an ethanol producer. Contrary to published remarks, we discovered that Z. mobilis can use the most abundant gas in the atmosphere, N 2 , as a nitrogen source and does so without detriment to the high ethanol yield. Using N 2 -utilizing Z. mobilis could offset much of the monetary and environmental costs of current industrial nitrogen supplements. </jats:p

Fermentative <i>Escherichia coli</i> makes a substantial contribution to H2 production in coculture with phototrophic <i>Rhodopseudomonas palustris</i>

ABSTRACT Individual species within microbial communities can combine their attributes to produce services that benefit society, such as the transformation of renewable resources into valuable chemicals. Under defined genetic and environmental conditions, fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris exchange essential carbon and nitrogen, respectively, to establish a mutualistic relationship. In this relationship, each species produces H2 biofuel as a byproduct of its metabolism. However, the extent to which each species contributes to H2 production and the factors that influence their relative contributions were previously unknown. By comparing H2 yields in cocultures pairing R. palustris with either wild-type E. coli or a formate hydrogenlyase mutant that is incapable of H2 production, we determined the relative contribution of each species to total H2 production. Our results indicate that E. coli contributes between 32 and 86% of the H2 produced in coculture depending on the level of ammonium excreted by the R. palustris partner. The level of ammonium excretion influenced the time over which E. coliwas exposed to formate, the types of E. colifermentation products available to R. palustris, and the pH of the medium, all of which affected the contribution of each species to H2 production.</jats:p

Fermentative<i>Escherichia coli</i>makes a substantial contribution to H₂production in coculture with phototrophic<i>Rhodopseudomonas palustris</i>

AbstractIndividual species within microbial communities can combine their attributes to produce services that benefit society, such as the transformation of renewable resources into valuable chemicals. Under defined genetic and environmental conditions, fermentativeEscherichia coliand phototrophicRhodopseudomonas palustrisexchange essential carbon and nitrogen, respectively, to establish a mutualistic relationship. In this relationship, each species produces H2biofuel as a byproduct of their metabolism. However, the extent to which each species contributes to H2production and the factors that influence their relative contributions were previously unknown. By comparing H2yields in cocultures pairingR. palustriswith either wild-typeE. colior a formate hydrogenlyase mutant that is incapable of H2production, we determined the relative contribution of each species to total H2production. Our results indicate thatE. colicontributes between 32% and 86% of the H2produced in coculture depending on the level of ammonium excreted by theR. palustrispartner. AnR. palustrisstrain that stimulated rapidE. coligrowth through a high level of ammonium excretion resulted in earlier accumulation of formate and acidic conditions that allowedE. colito be the major contributor to H2production.</jats:p

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

Microbial interactions abound in natural ecosystems and shape community structure and function. Substantial attention has been given to cataloging mechanisms by which microbes interact, but there is a limited understanding of the genetic landscapes that promote or hinder microbial interactions. We previously developed a mutualistic coculture pairing Escherichia coli and Rhodopseudomonas palustris, wherein E. coli provides carbon to R. palustris in the form of glucose fermentation products and R. palustris fixes N2 gas and provides nitrogen to E. coli in the form of NH4+ The stable coexistence and reproducible trends exhibited by this coculture make it ideal for interrogating the genetic underpinnings of a cross-feeding mutualism. Here, we used random barcode transposon sequencing (RB-TnSeq) to conduct a genome-wide search for E. coli genes that influence fitness during cooperative growth with R. palustris RB-TnSeq revealed hundreds of genes that increased or decreased E. coli fitness in a mutualism-dependent manner. Some identified genes were involved in nitrogen sensing and assimilation, as expected given the coculture design. The other identified genes were involved in diverse cellular processes, including energy production and cell wall and membrane biogenesis. In addition, we discovered unexpected purine cross-feeding from R. palustris to E. coli, with coculture rescuing growth of an E. coli purine auxotroph. Our data provide insight into the genes and gene networks that can influence a cross-feeding mutualism and underscore that microbial interactions are not necessarily predictable a prioriIMPORTANCE Microbial communities impact life on Earth in profound ways, including driving global nutrient cycles and influencing human health and disease. These community functions depend on the interactions that resident microbes have with the environment and each other. Thus, identifying genes that influence these interactions will aid the management of natural communities and the use of microbial consortia as biotechnology. Here, we identified genes that influenced Escherichia coli fitness during cooperative growth with a mutualistic partner, Rhodopseudomonas palustris Although this mutualism centers on the bidirectional exchange of essential carbon and nitrogen, E. coli fitness was positively and negatively affected by genes involved in diverse cellular processes. Furthermore, we discovered an unexpected purine cross-feeding interaction. These results contribute knowledge on the genetic foundation of a microbial cross-feeding interaction and highlight that unanticipated interactions can occur even within engineered microbial communities

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

Microbial communities impact life on Earth in profound ways, including driving global nutrient cycles and influencing human health and disease. These community functions depend on the interactions that resident microbes have with the environment and each other. Thus, identifying genes that influence these interactions will aid the management of natural communities and the use of microbial consortia as biotechnology. Here, we identified genes that influenced Escherichia coli fitness during cooperative growth with a mutualistic partner, Rhodopseudomonas palustris . Although this mutualism centers on the bidirectional exchange of essential carbon and nitrogen, E. coli fitness was positively and negatively affected by genes involved in diverse cellular processes. Furthermore, we discovered an unexpected purine cross-feeding interaction. These results contribute knowledge on the genetic foundation of a microbial cross-feeding interaction and highlight that unanticipated interactions can occur even within engineered microbial communities. </jats:p