Bacterial adenine cross-feeding stems from a purine salvage bottleneck.

Diverse ecosystems host microbial relationships that are stabilized by nutrient cross-feeding. Cross-feeding can involve metabolites that should hold value for the producer. Externalization of such communally valuable metabolites is often unexpected and difficult to predict. Previously, we discovered purine externalization by Rhodopseudomonas palustris by its ability to rescue an Escherichia coli purine auxotroph. Here we found that an E. coli purine auxotroph can stably coexist with R. palustris due to purine cross-feeding. We identified the cross-fed purine as adenine. Adenine was externalized by R. palustris under diverse growth conditions. Computational modeling suggested that adenine externalization occurs via diffusion across the cytoplasmic membrane. RNAseq analysis led us to hypothesize that adenine accumulation and externalization stem from a salvage pathway bottleneck at the enzyme encoded by apt. Ectopic expression of apt eliminated adenine externalization, supporting our hypothesis. A comparison of 49 R. palustris strains suggested that purine externalization is relatively common, with 16 strains exhibiting the trait. Purine externalization was correlated with the genomic orientation of apt, but apt orientation alone could not always explain purine externalization. Our results provide a mechanistic understanding of how a communally valuable metabolite can participate in cross-feeding. Our findings also highlight the challenge in identifying genetic signatures for metabolite externalization.

Bacterial quorum sensing controls carbon metabolism to optimize growth in changing environmental conditions.

Bacteria sense population density via the cell-cell communication system called quorum sensing (QS). Some QS-regulated phenotypes ( e.g. , secreted enzymes, chelators), are public goods exploitable by cells that stop producing them. We uncovered a phenomenon in which Vibrio cells optimize expression of the methionine and tetrahydrofolate (THF) synthesis genes via QS. Strains that are genetically 'locked' at high cell density grow slowly in minimal glucose media and suppressor mutants accumulate via inactivating-mutations in metF (methylenetetrahydrofolate reductase) and luxR (the master QS transcriptional regulator). Methionine/THF synthesis genes are repressed at low cell density when glucose is plentiful and are de-repressed by LuxR at high cell density as glucose becomes limiting. In mixed cultures, QS mutant strains initially co-exist with wild-type, but as glucose is depleted, wild-type outcompetes the QS mutants. Thus, QS regulation of methionine/THF synthesis is a fitness benefit that links private and public goods within the QS regulon, preventing accumulation of QS-defective mutants.

Nitrous oxide reduction by two partial denitrifying bacteria requires denitrification intermediates that cannot be respired.

Denitrification is a form of anaerobic respiration wherein nitrate (NO3-) is sequentially reduced via nitrite (NO2-), nitric oxide, and nitrous oxide (N2O) to dinitrogen gas (N2) by four reductase enzymes. Partial denitrifying bacteria possess only one or some of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here, we tested the denitrifying capabilities of two purple nonsulfur bacteria, Rhodopseudomonas palustris CGA0092 and Rhodobacter capsulatus SB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO2- to N2, and SB1003 reduced N2O to N2. For each bacterium, N2O reduction could be used both for electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in darkness. However, N2O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO3- served as a stable, non-catalyzable molecule that was sufficient to activate N2O reduction. Using a ß-galactosidase reporter, we found that NO3- acted, at least in part, by stimulating N2O reductase gene expression. In SB1003, NO2- but not NO3- activated N2O reduction, but NO2- was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use.IMPORTANCEDenitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N2O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here, we found that at least some partial denitrifying N2O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions.

Better off breathing: an explanation for the seemingly detrimental impact of aerobic respiration on Zymomonas mobilis.

The bacterium Zymomonas mobilis is best known for fermentatively producing more ethanol than yeast. However, Z. mobilis has also puzzled researchers for decades with the counterintuitive observation that disrupting aerobic respiration benefits aerobic growth, implying that fermentation remains favorable. Retention of detrimental respiration genes seemed to defy natural selection. New findings by Felczak et al. help clarify the importance of respiration for Z. mobilis and the factors that led to the confusing prior results (M. M. Felczak, M. P. Bernard, and M. A. TerAvest, 2023, mBio 14:e02043-23, https://doi.org/10.1128/mbio.02043-23). The team overcame redundancy from multiple genome copies to delete what turned out to be a key terminal oxidase. Unlike previous studies, wherein mutants exhibited low respiration rates and had improved aerobic growth, this mutant was incapable of respiration and had poor aerobic growth. Thus, respiration is important but surprisingly exceeds what is optimal under lab conditions. Respiration likely protects against toxic effects of oxygen, ensuring retention of respiration genes in the Z. mobilis genome.

A purine salvage bottleneck leads to bacterial adenine cross-feeding.

Diverse ecosystems host microbial relationships that are stabilized by nutrient cross-feeding. Cross-feeding can involve metabolites that should hold value for the producer. Externalization of such communally valuable metabolites is often unexpected and difficult to predict. Previously, we fortuitously discovered purine externalization by Rhodopseudomonas palustris by its ability to rescue growth of an Escherichia coli purine auxotroph. Here we found that an E. coli purine auxotroph can stably coexist with R. palustris due to purine cross-feeding. We identified the cross-fed purine as adenine. Adenine was externalized by R. palustris under diverse growth conditions. Computational models suggested that adenine externalization occurs via passive diffusion across the cytoplasmic membrane. RNAseq analysis led us to hypothesize that accumulation and externalization of adenine stems from an adenine salvage bottleneck at the enzyme encoded by apt. Ectopic expression of apt eliminated adenine externalization, supporting our hypothesis. A comparison of 49 R. palustris strains suggested that purine externalization is relatively common, with 15 of the strains exhibiting the trait. Purine externalization was correlated with the genomic orientation of apt orientation, but apt orientation alone could not explain adenine externalization in some strains. Our results provide a mechanistic understanding of how a communally valuable metabolite can participate in cross-feeding. Our findings also highlight the challenge in identifying genetic signatures for metabolite externalization.

Are Bacteria Leaky? Mechanisms of Metabolite Externalization in Bacterial Cross-Feeding.

The metabolism of a bacterial cell stretches beyond its boundaries, often connecting with the metabolism of other cells to form extended metabolic networks that stretch across communities, and even the globe. Among the least intuitive metabolic connections are those involving cross-feeding of canonically intracellular metabolites. How and why are these intracellular metabolites externalized? Are bacteria simply leaky? Here I consider what it means for a bacterium to be leaky, and I review mechanisms of metabolite externalization from the context of cross-feeding. Despite common claims, diffusion of most intracellular metabolites across a membrane is unlikely. Instead, passive and active transporters are likely involved, possibly purging excess metabolites as part of homeostasis. Re-acquisition of metabolites by a producer limits the opportunities for cross-feeding. However, a competitive recipient can stimulate metabolite externalization and initiate a positive-feedback loop of reciprocal cross-feeding.

Complete Genome Sequence of Rhodopseudomonas palustris CGA0092 and Corrections to the R. palustris CGA009 Genome Sequence.

Rhodopseudomonas palustris CGA009 is a versatile model purple nonsulfur bacterium used for both fundamental and applied research. Here, we present a new genome sequence for the derivative strain CGA0092. We further present an improved CGA009 genome assembly that differs from the original CGA009 sequence at three positions.

Nitric oxide stimulates type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA.

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. The dynamic extension and retraction of pili are often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To address this question, we study the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we use a combination of genetic and cell biological approaches to describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. Specifically, we show that V. cholerae cells retract MSHA pili and detach from a surface in a diffusion-limited, enclosed environment. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP to induce MSHA pilus retraction. CdpA contains a putative nitric oxide (NO)-sensing NosP domain, and we demonstrate that NO is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that the endogenous production of NO (or an NO-like molecule) in V. cholerae stimulates the retraction of MSHA pili. These results extend our understanding of how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

Extracellular Metabolism Sets the Table for Microbial Cross-Feeding.

The transfer of nutrients between cells, or cross-feeding, is a ubiquitous feature of microbial communities with emergent properties that influence our health and orchestrate global biogeochemical cycles. Cross-feeding inevitably involves the externalization of molecules. Some of these molecules directly serve as cross-fed nutrients, while others can facilitate cross-feeding. Altogether, externalized molecules that promote cross-feeding are diverse in structure, ranging from small molecules to macromolecules. The functions of these molecules are equally diverse, encompassing waste products, enzymes, toxins, signaling molecules, biofilm components, and nutrients of high value to most microbes, including the producer cell. As diverse as the externalized and transferred molecules are the cross-feeding relationships that can be derived from them. Many cross-feeding relationships can be summarized as cooperative but are also subject to exploitation. Even those relationships that appear to be cooperative exhibit some level of competition between partners. In this review, we summarize the major types of actively secreted, passively excreted, and directly transferred molecules that either form the basis of cross-feeding relationships or facilitate them. Drawing on examples from both natural and synthetic communities, we explore how the interplay between microbial physiology, environmental parameters, and the diverse functional attributes of extracellular molecules can influence cross-feeding dynamics. Though microbial cross-feeding interactions represent a burgeoning field of interest, we may have only begun to scratch the surface.

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

Interactive microbial communities are ubiquitous, influencing biogeochemical cycles and host health. One widespread interaction is nutrient exchange, or cross-feeding, wherein metabolites are transferred between microbes. Some cross-fed metabolites, such as vitamins, amino acids, and ammonium (NH4+), are communally valuable and impose a cost on the producer. The mechanisms that enforce cross-feeding of communally valuable metabolites are not fully understood. Previously we engineered a cross-feeding coculture between N2-fixing Rhodopseudomonas palustris and fermentative Escherichia coli. Engineered R. palustris excretes essential nitrogen as NH4+ to E. coli, while E. coli excretes essential carbon as fermentation products to R. palustris. Here, we sought to determine whether a reciprocal cross-feeding relationship would evolve spontaneously in cocultures with wild-type R. palustris, which is not known to excrete NH4+. Indeed, we observed the emergence of NH4+ cross-feeding, but driven by adaptation of E. coli alone. A missense mutation in E. coli NtrC, a regulator of nitrogen scavenging, resulted in constitutive activation of an NH4+ transporter. This activity likely allowed E. coli to subsist on the small amount of leaked NH4+ and better reciprocate through elevated excretion of fermentation products from a larger E. coli population. Our results indicate that enhanced nutrient uptake by recipients, rather than increased excretion by producers, is an underappreciated yet possibly prevalent mechanism by which cross-feeding can emerge.

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.

Microbial energy management-A product of three broad tradeoffs.

Wherever thermodynamics allows, microbial life has evolved to transform and harness energy. Microbial life thus abounds in the most unexpected places, enabled by profound metabolic diversity. Within this diversity, energy is transformed primarily through variations on a few core mechanisms. Energy is further managed by the physiological processes of cell growth and maintenance that use energy. Some aspects of microbial physiology are streamlined for energetic efficiency while other aspects seem suboptimal or even wasteful. We propose that the energy that a microbe harnesses and devotes to growth and maintenance is a product of three broad tradeoffs: (i) economic, trading enzyme synthesis or operational cost for functional benefit, (ii) environmental, trading optimization for a single environment for adaptability to multiple environments, and (iii) thermodynamic, trading energetic yield for forward metabolic flux. Consideration of these tradeoffs allows one to reconcile features of microbial physiology that seem to opposingly promote either energetic efficiency or waste.

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.

Fermentative Escherichia coli makes a substantial contribution to H2 production in coculture with phototrophic Rhodopseudomonas palustris.

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.

Cell Aggregation and Aerobic Respiration Are Important for Zymomonas mobilis ZM4 Survival in an Aerobic Minimal Medium.

Zymomonas mobilis produces ethanol from glucose near the theoretical maximum yield, making it a potential alternative to the yeast Saccharomyces cerevisiae for industrial ethanol production. A potentially useful industrial feature is the ability to form multicellular aggregates called flocs, which can settle quickly and exhibit higher resistance to harmful chemicals than single cells. While spontaneous floc-forming Z. mobilis mutants have been described, little is known about the natural conditions that induce Z. mobilis floc formation or about the genetic factors involved. Here we found that wild-type Z. mobilis forms flocs in response to aerobic growth conditions but only in a minimal medium. We identified a cellulose synthase gene cluster and a single diguanylate cyclase that are essential for both floc formation and survival in a minimal aerobic medium. We also found that NADH dehydrogenase 2, a key component of the aerobic respiratory chain, is important for survival in a minimal aerobic medium, providing a physiological role for this enzyme, which has previously been found to be disadvantageous in a rich aerobic medium. Supplementation of the minimal medium with vitamins also promoted survival but did not inhibit floc formation.IMPORTANCE The bacterium Zymomonas mobilis is best known for its anaerobic fermentative lifestyle, in which it converts glucose into ethanol at a yield surpassing that of yeast. However, Z. mobilis also has an aerobic lifestyle, which has confounded researchers with its attributes of poor growth, accumulation of toxic acetic acid and acetaldehyde, and respiratory enzymes that are detrimental for aerobic growth. Here we show that a major Z. mobilis respiratory enzyme and the ability to form multicellular aggregates, called flocs, are important for survival, but only during aerobic growth in a medium containing a minimum set of nutrients required for growth. Supplements, such as vitamins or yeast extract, promote aerobic growth and, in some cases, inhibit floc formation. We propose that Z. mobilis likely requires aerobic respiration and floc formation in order to survive in natural environments that lack protective factors found in supplements such as yeast extract.

Phototrophic Lactate Utilization by Rhodopseudomonas palustris Is Stimulated by Coutilization with Additional Substrates.

The phototrophic purple nonsulfur bacterium Rhodopseudomonas palustris is known for its metabolic versatility and is of interest for various industrial and environmental applications. Despite decades of research on R. palustris growth under diverse conditions, patterns of R. palustris growth and carbon utilization with mixtures of carbon substrates remain largely unknown. R. palustris readily utilizes most short-chain organic acids but cannot readily use lactate as a sole carbon source. Here we investigated the influence of mixed-substrate utilization on phototrophic lactate consumption by R. palustris We found that lactate was simultaneously utilized with a variety of other organic acids and glycerol in time frames that were insufficient for R. palustris growth on lactate alone. Thus, lactate utilization by R. palustris was expedited by its coutilization with additional substrates. Separately, experiments using carbon pairs that did not contain lactate revealed acetate-mediated inhibition of glycerol utilization in R. palustris This inhibition was specific to the acetate-glycerol pair, as R. palustris simultaneously utilized acetate or glycerol when either was paired with succinate or lactate. Overall, our results demonstrate that (i) R. palustris commonly employs simultaneous mixed-substrate utilization, (ii) mixed-substrate utilization expands the spectrum of readily utilized organic acids in this species, and (iii) R. palustris has the capacity to exert carbon catabolite control in a substrate-specific manner.IMPORTANCE 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.

Characterization of a Glycyl Radical Enzyme Bacterial Microcompartment Pathway in Rhodobacter capsulatus.

Bacterial microcompartments (BMCs) are large (∼100-nm) protein shells that encapsulate enzymes, their substrates, and cofactors for the purposes of increasing metabolic reaction efficiency and protecting cells from toxic intermediates. The best-studied microcompartment is the carbon-fixing carboxysome that encapsulates ribulose-1,5-bisphosphate carboxylase and carbonic anhydrase. Other well-known BMCs include the Pdu and Eut BMCs, which metabolize 1,2-propanediol and ethanolamine, respectively, with vitamin B12-dependent diol dehydratase enzymes. Recent bioinformatic analyses identified a new prevalent type of BMC, hypothesized to utilize vitamin B12-independent glycyl radical enzymes to metabolize substrates. Here we use genetic and metabolic analyses to undertake in vivo characterization of the newly identified glycyl radical enzyme microcompartment 3 (GRM3) class of microcompartment clusters. Transcriptome sequencing analyses showed that the microcompartment gene cluster in the genome of the purple photosynthetic bacterium Rhodobacter capsulatus was expressed under dark anaerobic respiratory conditions in the presence of 1,2-propanediol. High-performance liquid chromatography and gas chromatography-mass spectrometry analyses showed that enzymes coded by this cluster metabolized 1,2-propanediol into propionaldehyde, propanol, and propionate. Surprisingly, the microcompartment pathway did not protect these cells from toxic propionaldehyde under the conditions used in this study, with buildup of this intermediate contributing to arrest of cell growth. We further show that expression of microcompartment genes is regulated by a two-component system located downstream of the microcompartment cluster.IMPORTANCE BMCs are protein shells that are designed to compartmentalize enzymatic reactions that require either sequestration of a substrate or the sequestration of toxic intermediates. Due to their ability to compartmentalize reactions, BMCs have also become attractive targets for bioengineering novel enzymatic reactions. Despite these useful features, little is known about the biochemistry of newly identified classes of BMCs. In this study, we have undertaken genetic and in vivo metabolic analyses of the newly identified GRM3 gene cluster.

Restricted Localization of Photosynthetic Intracytoplasmic Membranes (ICMs) in Multiple Genera of Purple Nonsulfur Bacteria.

In bacteria and eukaryotes alike, proper cellular physiology relies on robust subcellular organization. For the phototrophic purple nonsulfur bacteria (PNSB), this organization entails the use of a light-harvesting, membrane-bound compartment known as the intracytoplasmic membrane (ICM). Here we show that ICMs are spatially and temporally localized in diverse patterns among PNSB. We visualized ICMs in live cells of 14 PNSB species across nine genera by exploiting the natural autofluorescence of the photosynthetic pigment bacteriochlorophyll (BChl). We then quantitatively characterized ICM localization using automated computational analysis of BChl fluorescence patterns within single cells across the population. We revealed that while many PNSB elaborate ICMs along the entirety of the cell, species across as least two genera restrict ICMs to discrete, nonrandom sites near cell poles in a manner coordinated with cell growth and division. Phylogenetic and phenotypic comparisons established that ICM localization and ICM architecture are not strictly interdependent and that neither trait fully correlates with the evolutionary relatedness of the species. The natural diversity of ICM localization revealed herein has implications for both the evolution of phototrophic organisms and their light-harvesting compartments and the mechanisms underpinning spatial organization of bacterial compartments.IMPORTANCE Many bacteria organize their cellular space by constructing subcellular compartments that are arranged in specific, physiologically relevant patterns. The purple nonsulfur bacteria (PNSB) utilize a membrane-bound compartment known as the intracytoplasmic membrane (ICM) to harvest light for photosynthesis. It was previously unknown whether ICM localization within cells is systematic or irregular and if ICM localization is conserved among PNSB. Here we surveyed ICM localization in diverse PNSB and show that ICMs are spatially organized in species-specific patterns. Most strikingly, several PNSB resolutely restrict ICMs to regions near the cell poles, leaving much of the cell devoid of light-harvesting machinery. Our results demonstrate that bacteria of a common lifestyle utilize unequal portions of their intracellular space to harvest light, despite light harvesting being a process that is intuitively influenced by surface area. Our findings therefore raise fundamental questions about ICM biology and evolution.

Differential carbohydrate utilization and organic acid production by honey bee symbionts.

The honey bee worker gut hosts a community of bacteria that comprises 8-10 core bacterial species, along with a set of more transient environmental microbes. Collectively, these microbes break down and ferment saccharides present in the host's diet, based on analyses of metagenomes, and metatranscriptomes from this environment. As part of this metabolism, the bacteria produce short-chain fatty acids that may serve as a food source for the host bee, stimulating biological processes that may contribute to host weight gain. To identify metabolic contributions of symbionts within the honey bee gut, we utilized a combination of molecular and biochemical approaches. We show significant variation in the metabolic capabilities of honey bee-associated taxa, highlighting the fact that honey bee gut microbiota members of the same clade are highly variable in their ability to use specific carbohydrates and produce organic acids. Finally, we confirm that the honey bee core microbes are active in vivo, expressing key enzymatic genes critical for utilizing plant-derived molecules and producing organic acids (i.e. acetate and lactate). These results suggest that core taxa may contribute significantly to weight gain in the honey bee, specifically through the production of organic acids.