Biology subjects

LaSarre, B.

Publications and source records attributed to LaSarre, B..

13 recordsLinked to original sources

bioRxiv2026

The bacterial pathogen causing cucurbit yellow vine disease associates with the specialized extrafascicular phloem unique to cucurbits

Cucurbit yellow vine disease (CYVD) is a phloem disease caused by a group of Serratia ureilytica bacteria within the S. marcescens complex. The CYVD pathogen is distinct from other phloem pathogens in two fundamental ways: i) unlike other bacterial phloem pathogens, the CYVD pathogen readily grows on standard laboratory media; and ii) whereas most bacterial phloem diseases are transmitted solely by hemipteran insects, CYVD can be transmitted by both hemipteran (squash bug) and non-hemipteran (cucumber beetle) vectors. The phloem of cucurbits is also distinct, with cucurbits having a unique secondary phloem system, the extrafascicular phloem (EFP), for which a role in CYVD has not been investigated. The distinctive features of this pathosystem prompted a re-evaluation of pathogen localization in planta using a green fluorescent protein-tagged CYVD strain during infection of squash (Cucurbita pepo). In epifluorescence and confocal microscopy images of transverse and longitudinal sections of stem and petiole tissues, the fluorescence patterns of the CYVD pathogen were consistent with pathogen colonization of all four known EFP tissues, namely the peripheral, entocyclic, ectocyclic, and commissural sieve tubes. The CYVD pathogen may enter the EFP during herbivory by pathogen-infected vectors that cause mechanical damage, as the EFP exhibits much slower blockage following injury than the conventional fascicular (bundle) phloem. Moreover, although EFP has been proposed to have an antimicrobial function, EFP exudate collected from the stem and petioles of C. pepo supported rapid pathogen growth. These results provide evidence for a unique tissue tropism of the newly emerged plant pathogen S. ureilytica.

plant biology↗

bioRxiv2026

A Bioinformatic Pipeline for Consensus Taxonomic Classification of Long-Read Amplicons

Characterizing community composition is fundamental to understanding microbial community function. Recent advances in Oxford Nanopore Technology (ONT) long-read sequencing now allow community profiling using full-length gene amplicons, affording better taxonomic resolution than standard short-amplicon Illumina sequencing. However, robust ONT-compatible profiling workflows are lacking. To address this, we have created the Amplicon Consensus Taxonomy (ACT) pipeline for classifying long-read amplicons. ACT combines output from three existing pipelines - Emu, Sintax, and LACA - to leverage the strengths of each while offsetting their individual limitations. We also developed the ACT database (ACT-DB), a sequence-similarity-aware reference database that clusters highly similar sequences into multi-taxa groups to reduce overclassification. We benchmarked ACT performance against Emu and Sintax using a defined simple mock community, simulated datasets, and a complex rhizosphere community supplemented with novel species. While ACT exhibited generally comparable or superior performance across datasets, ACT demonstrated a marked advantage over Emu and Sintax in identifying novel and low-abundance taxa in both simple and complex communities, resulting in significantly higher species-richness estimates that better reflected those observed in prior Illumina amplicon studies. Furthermore, by clustering ambiguous reference sequences, ACT-DB allowed ACT to resolve reads to meaningful multi-species groups, improving resolution without coercing artificial precision. Together, ACT and ACT-DB form a robust long-read amplicon profiling workflow that confidently identifies known species while reducing overclassification and preserving low-abundance and unknown taxa. IMPORTANCEMicrobial communities are frequently characterized by amplicon sequencing of marker genes, such as the bacterial 16S rRNA gene and fungal ITS region. Historically, the standard profiling method has been Illumina sequencing of 200-300 bp amplicons, but improved accuracy of ONT long-read sequencing means it is now possible to sequence amplicons spanning full genes of any size, prompting the need for tools optimized for long amplicons. Here, we describe the ACT bioinformatic pipeline for assigning taxonomy to amplicons of any length. We evaluated ACT performance using full-length 16S amplicon data relative to that of two commonly used pipelines. Additionally, we developed a sequence ambiguity-aware ACT database (ACT-DB) of 16S rRNA sequences to further improve classification accuracy and resolution.

microbiology↗

bioRxiv2025

Evolved bacterial formate assimilation is likely potentiated by a rudimentary CO2 concentrating mechanism

Formate is a single-carbon compound that is challenging to assimilate, including when assimilation involves a CO2 intermediate that can diffuse from the cell. Mutations that overcome such challenges can be identified through adaptive laboratory evolution. We evolved the anoxygenic phototrophic bacterium Rhodopseudomonas palustris to use formate as the sole carbon source. Through gene deletions, we determined that formate is assimilated via oxidation to CO2 by formate dehydrogenase followed by CO2 fixation by the Calvin cycle. However, this pathway had no clear link to three genes that were commonly mutated in evolved isolates: (i) ribB, a flavin synthesis enzyme, (ii) ppsR2, a repressor of light-harvesting genes, and (iii) RPA0893, a regulator of unknown function. A RibB mutation was necessary and sufficient for formate assimilation and improved formate oxidation. PpsR2 mutations occurred early, facilitated formate assimilation, and caused elevated pigmentation. Pigment production generates CO2 and alkaline conditions that, along with intracellular chromatophore membranes, could represent a rudimentary but important CO2-concentrating mechanism. RPA0983 mutations emerged late and facilitated formate assimilation. RPA0983s proximity to a CO2-liberating pigment synthesis gene suggests a similar effect as PpsR2 mutations. Our findings reveal unintuitive pathway intersections that could have broad implications for formate and CO2-utilizing organisms.

microbiology↗

bioRxiv2023

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.

microbiology↗

bioRxiv2022

Non-catalyzable denitrification intermediates induce nitrous oxide reduction in two purple nonsulfur bacteria

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 for both electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in the dark. 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 {beta}-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.

microbiology↗

bioRxiv2020

Genome-wide analysis of Escherichia coli fitness determinants in a cross-feeding mutualism with Rhodopseudomonas palustris

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. Additionally, 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 priori. IMPORTANCEMicrobial 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, Rhodospeudomonas 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.

microbiology↗

bioRxiv2019

The reverse TCA cycle and reductive amino acid synthesis pathways contribute to electron balance in a Rhodospirillum rubrum Calvin cycle mutant

Purple nonsulfur 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. Excess reduced electron carriers must be oxidized for photoheterotrophic growth to occur. Diverse PNSB commonly rely on the CO2-fixing Calvin cycle to oxidize excess 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 TCA cycle enzymes, which carried reductive flux from malate or fumarate to -ketoglutarate. This pathway and the reductive synthesis of amino acids derived from -ketoglutarate are likely important for electron balance, as supplementing the growth medium with -ketoglutarate-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.

microbiology↗

bioRxiv2019

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 their 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. An R. palustris strain that stimulated rapid E. coli growth through a high level of ammonium excretion resulted in earlier accumulation of formate and acidic conditions that allowed E. coli to be the major contributor to H2 production.

microbiology↗

bioRxiv2018

Phototrophic lactate utilization by Rhodopseudomonas palustris is stimulated by co-utilization 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 co-utilization 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.\n\nIMPORTANCEBacterial 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 in mixtures. Specifically, co-utilization 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.

microbiology↗

bioRxiv2018

Diverse spatial organization of photosynthetic membranes among purple nonsulfur bacteria

In diverse bacteria, proper cellular physiology requires the utilization of protein- or membrane-bound compartments that afford specific metabolic capabilities. One such compartment is the light-harvesting intracytoplasmic membrane (ICM) of purple nonsulfur bacteria (PNSB). Here we reveal that ICMs are subject to differential spatial organization among PNSB. We visualized ICMs in live cells of fourteen PNSB species by exploiting the natural autofluorescence of the photosynthetic machinery. We then quantitatively characterized ICM localization using automated computational analysis of autofluorescence patterns within single cells across the population. Our studies revealed that ICMs are localized in distinct subcellular patterns that differ between species; some PNSB elaborate ICMs throughout the cell, while others spatially restrict ICM to varying degrees. The most highly-restricted ICMs were localized in a specific pattern corresponding to progression of cell growth and division. An identical pattern of ICM restriction was conserved across at least two genera. 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. This discovery of new diversity in bacterial cell organization has implications for understanding both the mechanisms underpinning spatial arrangement of bacterial compartments and the potential benefits of adopting different spatiotemporal patterns.

microbiology↗

bioRxiv2017

Recipient-Biased Competition For A Cross-Fed Nutrient Is Required For Coexistence Of Microbial Mutualists

Many 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 producers 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 fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris. In this coculture, E. coli excretes waste organic acids that provide carbon for R. palustris. In return, R. palustris cross-feeds E. coli ammonium (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.\n\nSignificanceMutualistic 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.

microbiology↗

bioRxiv2016

A Rhizobiales-specific unipolar polysaccharide adhesin contributes to Rhodopseudomonas palustris biofilm formation across diverse photoheterotrophic conditions

Bacteria 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 -proteobacteria, which use polar exopolysaccharide adhesins for cell-cell adhesion and surface attachment. Here, we show that Rhodopseudomonas palustris, a metabolically versatile member of the -proteobacterial order Rhizobiales, encodes 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.\n\nIMPORTANCEBacteria are ubiquitously found as surface-attached communities and cellular aggregates in nature. Here, we address how bacterial adhesion is coordinated in response to diverse environments using two complementary approaches. First, we examined how Rhodopseudomonas palustris, one of the most metabolically versatile organisms ever described, varies its adhesion to surfaces in response to different environmental conditions. We identified 2 critical genes for the production of a unipolar polysaccharide (UPP) and showed that UPP is important for adhesion when light and organic substrates are used for growth. Looking beyond R. palustris, we performed the most comprehensive survey to date on the conservation of UPP biosynthesis genes among a group of closely related bacteria that occupy diverse niches. Our findings suggest that UPP is important for free-living and plant-associated lifestyles but dispensable for animal pathogens. Additionally, we propose guidelines for classifying the adhesins produced by various -proteobacteria, facilitating future functional and comparative studies.

microbiology↗

bioRxiv2016

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

Nutrient 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 fermentative Escherichia coli and phototrophic Rhodopseudomonas palustris. In this coculture, E. coli and R. palustris resemble an anaerobic food web by cross-feeding essential carbon (organic acids) and nitrogen (ammonium), respectively. Organic acid cross-feeding stemming from E. coli fermentation can continue in a growth-independent manner during nutrient limitation, while ammonium cross-feeding by R. palustris is 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 upper E. coli cell 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.\n\nConflict of interestThe authors declare no conflict of interest.

microbiology↗