Competitive Colonization Research Articles

A Genetically Adaptable Strategy for Ribose Scavenging in a Human Gut Symbiont Plays a Diet-Dependent Role in Colon Colonization

Efficient nutrient acquisition in the competitive human gut is essential for microbial persistence. While polysaccharides have been well-studied nutrients for the gut microbiome, other resources such as co-factors and nucleic acids have been less examined. We describe a series of ribose utilization systems (RUSs) that are broadly represented in Bacteroidetes and appear to have diversified to allow access to ribose from a variety of substrates. One Bacteroides thetaiotaomicron RUS variant is critical for competitive gut colonization in a diet-specific fashion. Using molecular genetics, we probed the nature of the ribose source underlying this dietspecific phenotype, revealing that hydrolytic functions in RUS (e.g., to cleave ribonucleosides) are present but dispensable. Instead, ribokinases that are activated in vivo and participate in cellular ribose-phosphate metabolism are essential. Our results underscore the extensive mechanisms that gut symbionts have evolved to access nutrients and how metabolic context determines the impact of these functions in vivo

Import of Aspartate and Malate by DcuABC Drives H 2/Fumarate Respiration to Promote Salmonella Gut-Luminal Colonization

Enteropathogen growth in the microbiota-colonized gut is poorly understood. Salmonella Typhimurium is metabolically plastic and can harvest energy by anaerobic respiration using hydrogen (H2), a product of microbiota metabolism, as an electron donor and fumarate as an electron acceptor. As fumarate is scarce in the gut, the source of this electron acceptor is unclear. Transposon sequencing analysis along the colonization-trajectory of S.Typhimurium implicated the C4-dicarboxylate exchanger DcuABC in murine gut colonization. In competitive colonization assays, DcuABC and enzymes converting the C4-dicarboxylates aspartate and malate into fumarate (AspA, FumABC), were required for fumarate/H2-dependent growth. Thus, S.Typhimurium obtains fumarate by DcuABC-mediated import and the subsequent conversion of L-malate and L-aspartate into fumarate. Fumarate reduction yields succinate, which is exported by DcuABC in exchange for L-aspartate and L-malate. This mechanism allows S.Typhimurium to harvest energy by H2/fumarate respiration in the microbiota-colonized gut. It might be relevant for other enteropathogens encoding the requisite genes.

Three new serine-protease autotransporters of Enterobacteriaceae (SPATEs) from extra-intestinal pathogenic Escherichia coli and combined role of SPATEs for cytotoxicity and colonization of the mouse kidney

ABSTRACTSerine protease autotransporters of Enterobacteriaceae (SPATEs) are secreted proteins that contribute to virulence and function as proteases, toxins, adhesins, and/or immunomodulators. An extra-intestinal pathogenic E. coli (ExPEC) O1:K1 strain, QT598, isolated from a turkey, was shown to contain vat, tsh, and three uncharacterized SPATE-encoding genes. Uncharacterized SPATEs: Sha (Serine-protease hemagglutinin autotransporter), TagB and TagC (tandem autotransporter genes B and C) were tested for activities including hemagglutination, autoaggregation, and cytotoxicity when expressed in E. coli K-12. Sha and TagB conferred autoaggregation and hemagglutination activities. TagB, TagC, and Sha all exhibited cytopathic effects on a bladder epithelial cell line. In QT598, tagB and tagC are tandemly encoded on a genomic island, and were present in 10% of UTI isolates and 4.7% of avian E. coli. Sha is encoded on a virulence plasmid and was present in 1% of UTI isolates and 20% of avian E. coli. To specifically examine the role of SPATEs for infection, the 5 SPATE genes were deleted from strain QT598 and tested for cytotoxicity. Loss of all five SPATEs abrogated the cytopathic effect on bladder epithelial cells, although derivatives producing any of the 5 SPATEs retained cytopathic activity. In mouse infections, sha gene-expression was up-regulated a mean of sixfold in the bladder compared to growth in vitro. Loss of either tagBC or sha did not reduce urinary tract colonization. Deletion of all 5 SPATEs, however, significantly reduced competitive colonization of the kidney supporting a cumulative role of SPATEs for QT598 in the mouse UTI model.

Preferential Colonization of Osteoblasts Over Co-cultured Bacteria on a Bifunctional Biomaterial Surface

Implant-related infection is a devastating complication in clinical trauma and orthopedics. The aim of this study is to use a bifunctional biomaterial surface in order to investigate the competitive colonization between osteoblasts and bacteria, which is the cause of implant-related infection. A bone-engineering material capable of simultaneously facilitating osteoblast adhesion and inhibiting the growth of Staphylococcus aureus (S. aureus) was prepared. Then, three different co-cultured systems were developed in order to investigate the competitive colonization between the two cohorts on the surface. The results suggested that while the pre-culturing of either cohort compromised the subsequent adhesion of the other according to the ‘race for the surface’ theory, the synergistic effect of preferential cell adhesion and antibacterial activity of the bifunctional surface led to the predominant colonization and survival of osteoblasts, effectively inhibiting the bacterial adhesion and biofilm formation of S. aureus in the co-culture systems with both cohorts. This research offers new insight into the investigation of competitive surface-colonization between osteoblasts and bacteria for implant-related infection.

Genome-Based Discovery of Polyketide-Derived Secondary Metabolism Pathways in the Barley Pathogen Ramularia collo-cygni.

Ramularia collo-cygni causes Ramularia leaf spot (RLS) disease of barley. The fungus develops asymptomatically within its host until late in the growing season, when necrotic lesions become visible on upper leaves. Fungal secondary metabolites (SM) have been proposed as important factors in RLS lesion formation but the biosynthetic pathways involved remain largely unknown. Mining the R. collo-cygni genome revealed the presence of 10 polyketide synthases (PKS), 10 nonribosomal peptide synthetases (NRPS), and 3 hybrid PKS-NRPS (HPS) identified within clusters of genes with predicted functions associated with secondary metabolism. SM core genes along with their predicted transcriptional regulators exhibited transcriptional coexpression during infection of barley plants. Moreover, their expression peaked during early stages of host colonization and preceded or overlapped with the appearance of disease symptoms, suggesting that SM may manipulate the host to promote colonization or protect R. collo-cygni from competing organisms. Accordingly, R. collo-cygni inhibited the growth of several fungi in vitro, indicating that it synthesized and excreted antifungal agents. Taken together, these findings demonstrate that the R. collo-cygni genome contains the genetic architecture to synthesize a wide range of SM and suggests that coexpression of PKS and HPS is associated with competitive colonization of the host and early symptom development.

Rodent seed predators and a dominant grass competitor affect coexistence of co‐occurring forb species that vary in seed size

Abstract Propagule size and number often vary by several orders of magnitude among co‐occurring plant species. Explaining the maintenance of this variation and understanding how propagule size contributes to coexistence remain a central challenge for community ecologists. The dominant paradigm is that a competition–colonization trade‐off maintains interspecific variation in seed size, but empirical support is limited and other coexistence mechanisms, such as size‐dependent seed predation, have not been examined. We examined how seed size, fecundity, and other functional traits of 18 co‐occurring perennial forbs trade‐off with both vulnerability to rodent seed predation and competitive response to a community‐dominant perennial bunchgrass. We added seeds of these species to 1 m2 plots at 10 sites where we factorially manipulated rodent seed predation and competition from the community dominant, Festuca campestris. For a given plot, seeds of each species were added at densities that reflected the fecundity (i.e., per capita seed production) for each species. Moreover, we varied total seed density among plots by adding seeds at one of five relative densities (0, 0.25. 0.5, 1.0, and 1.5 times each species’ fecundity) to examine how fecundity affected overall recruitment for each species. There was a trade‐off between seed size and fecundity, as expected, but larger seed size was also associated with greater plant height, lower C/N ratios, and lower water use efficiency, suggesting that seed size represented a “resource acquisitive” trait syndrome (as quantified by trait principal component scores). In the field experiment, the suppressive effects of seed predation on seed recruitment rate increased with increasing seed size. In contrast, smaller seeded species with less resource‐acquisitive traits were more negatively affected by competition from F. campestris than were species with more resource acquisitive traits. Synthesis. While the competition–colonization trade‐off has been the predominant mechanism thought to maintain coexistence among species that vary in fecundity and seed size, our work suggests that susceptibility to rodent seed predation and competitive response to community dominants represent alternative mechanisms that can differentially influence plant recruitment of species based on their seed size and associated traits.

Identification and Characterization of a Phase-Variable Element That Regulates the Autotransporter UpaE in Uropathogenic Escherichia coli.

ABSTRACTUropathogenic Escherichia coli (UPEC) is the most common etiologic agent of uncomplicated urinary tract infection (UTI). An important mechanism of gene regulation in UPEC is phase variation that involves inversion of a promoter-containing DNA element via enzymatic activity of tyrosine recombinases, resulting in biphasic, ON or OFF expression of target genes. The UPEC reference strain CFT073 has five tyrosine site-specific recombinases that function at two previously characterized promoter inversion systems, fimS and hyxS. Three of the five recombinases are located proximally to their cognate target elements, which is typical of promoter inversion systems. The genes for the other two recombinases, IpuA and IpuB, are located distal from these sites. Here, we identified and characterized a third phase-variable invertible element in CFT073, ipuS, located proximal to ipuA and ipuB. The inversion of ipuS is catalyzed by four of the five CFT073 recombinases. Orientation of the element drives transcription of a two-gene operon containing ipuR, a predicted LuxR-type regulator, and upaE, a predicted autotransporter. We show that the predicted autotransporter UpaE is surface located and facilitates biofilm formation as well as adhesion to extracellular matrix proteins in a K-12 recombinant background. Consistent with this phenotype, the ipuS ON condition in CFT073 results in defective swimming motility, increased adherence to human kidney epithelial cells, and a positive competitive kidney colonization advantage in experimental mouse UTIs. Overall, the identification of a third phase switch in UPEC that is regulated by a shared set of recombinases describes a complex phase-variable virulence network in UPEC.

Neonatal selection by Toll-like receptor 5 influences long-term gut microbiota composition.

Alterations in enteric microbiota are associated with several highly prevalent immune-mediated and metabolic diseases1-3, and experiments involving faecal transplants have indicated that such alterations have a causal role in at least some such conditions4-6. The postnatal period is particularly critical for the development of microbiota composition, host-microbe interactions and immune homeostasis7-9. However, the underlying molecular mechanisms of this neonatal priming period have not been defined. Here we report the identification of a host-mediated regulatory circuit of bacterial colonization that acts solely during the early neonatal period but influences life-long microbiota composition. We demonstrate age-dependent expression of the flagellin receptor Toll-like receptor 5 (TLR5) in the gut epithelium of neonate mice. Using competitive colonization experiments, we demonstrate that epithelial TLR5-mediated REG3γ production is critical for the counter-selection of colonizing flagellated bacteria. Comparative microbiota transfer experiments in neonate and adult wild-type and Tlr5-deficient germ-free mice reveal that neonatal TLR5 expression strongly influences the composition of the microbiota throughout life. Thus, the beneficial microbiota in the adult host is shaped during early infancy. This might explain why environmental factors that disturb the establishment of the microbiota during early life can affect immune homeostasis and health in adulthood.

In vitro anti-inflammatory activity among probiotic Lactobacillus species isolated from fermented foods

Probiotic bacteria have the ability to alter immune response conferring treatment to inflammatory diseases. In the current study, probiotic Lactobacillus strains were subjected to adhesion to epithelial Caco-2 cells. Subsequently, gene expression of pro-and anti-inflammatory genes were studied under three stimulation conditions (which includes LPS-challenge, stress induction and pathogens invasion). Herein, immunomodulatory activity of native Lactobacillus strains (n = 3) were compared to L. rhamnosus GG. The studied strains had down-regulated the gene expression of pro-inflammatory cytokines (CKs) (like TNF-α, IL-1α, IL-1β, IL-6 and IL-8) and up-regulated the anti-inflammatory CKs like TGFβ1, IL-4, and IL-10. Gene expression of TLR2 has also been noted. Strain-specific interactions were noticed during the different probiotic treatment. Further, phylogenetic dendrogram of mucin adhesion domain suggested the interaction of Lactobacillus and Listeria species, explaining competitive colonization and exclusion mechanism in an ecological niche. Overall, isolate L. paraplantarum MTCC 9483 has effectively modulated induced conditions by its anti-inflammatory activity.

Few Differences in Metabolic Network Use Found Between Salmonella enterica Colonization of Plants and Typhoidal Mice.

The human enteric pathogen Salmonella enterica leads a cross-kingdom lifestyle, actively colonizing and persisting on plants in between animal hosts. One of the questions that arises from this dual lifestyle is how S. enterica is able to adapt to such divergent hosts. Metabolic pathways required for S. enterica animal colonization and virulence have been previously identified, but the metabolism of this bacterium on plants is poorly understood. To determine the requirements for plant colonization by S. enterica, we first screened a library of metabolic mutants, previously examined in a systemic mouse typhoidal model, for competitive plant colonization fitness on alfalfa seedlings. By comparing our results to those reported in S. enterica-infected murine spleens, we found that the presence of individual nutrients differed between the two host niches. Yet, similar metabolic pathways contributed to S. enterica colonization of both plants and animals, such as the biosynthesis of amino acids, purines, and vitamins and the catabolism of glycerol and glucose. However, utilization of at least three metabolic networks differed during the bacterium's plant- and animal-associated lifestyles. Whereas both fatty acid biosynthesis and degradation contributed to S. enterica animal colonization, only fatty acid biosynthesis was required during plant colonization. Though serine biosynthesis was required in both hosts, S. enterica used different pathways within the serine metabolic network to achieve this outcome. Lastly, the metabolic network surrounding manA played different roles during colonization of each host. In animal models of infection, O-antigen production downstream of manA facilitates immune evasion. We discovered that manA contributed to S. enterica attachment, to seeds and germinated seedlings, and was essential for growth in early seedling exudates, when mannose is limited. However, only seedling attachment was linked to O-antigen production, indicating that manA played additional roles critical for plant colonization that were independent of surface polysaccharide production. The integrated view of S. enterica metabolism throughout its life cycle presented here provides insight on how metabolic versatility and adaption of known physiological pathways for alternate functions enable a zoonotic pathogen to thrive in niches spanning across multiple kingdoms of life.

Crayfish plague in Japan: A real threat to the endemic Cambaroides japonicus

Global introductions of aquatic species and their associated pathogens are threatening worldwide biodiversity. The introduction of two North American crayfish species, Procambarus clarkii and Pacifastacus leniusculus, into Japan in 1927 seems to have negatively affected native Japanese crayfish populations of Cambaroides japonicus. Several studies have shown the decline of these native populations due to competition, predation and habitat colonization by the two invasive North American crayfish species. Here, we identify an additional factor contributing to this decline. We report the first crayfish plague outbreaks in C. japonicus populations in Japan, which were diagnosed using both histological and molecular approaches (analyses of the internal transcribed spacer region). Subsequent analyses of the mitochondrial ribosomal rnnS and rnnL regions of diseased specimens indicate that these outbreaks originated from a P. clarkii population and identify a novel haplotype of Aphanomyces astaci, d3-haplotype, hosted by P. clarkii. Overall, our findings demonstrate the first two cases of crayfish plague in Japan, and the first case in a non-European native crayfish species, which originated from the red swamp crayfish P. clarkii. This finding is a matter of concern for the conservation of the native freshwater species of Japan and also highlights the risk of introducing crayfish carrier species into biogeographic regions harboring species susceptible to the crayfish plague.

Regulation of Hydroxycinnamic Acid Degradation Drives Agrobacterium fabrum Lifestyles.

Regulatory factors are key components for the transition between different lifestyles to ensure rapid and appropriate gene expression upon perceiving environmental cues. Agrobacterium fabrum C58 (formerly called A. tumefaciens C58) has two contrasting lifestyles: it can interact with plants as either a rhizosphere inhabitant (rhizospheric lifestyle) or a pathogen that creates its own ecological niche in a plant tumor via its tumor-inducing plasmid (pathogenic lifestyle). Hydroxycinnamic acids are known to play an important role in the pathogenic lifestyle of Agrobacterium spp. but can be degraded in A. fabrum species. We investigated the molecular and ecological mechanisms involved in the regulation of A. fabrum species-specific genes responsible for hydroxycinnamic acid degradation. We characterized the effectors (feruloyl-CoA and p-coumaroyl-CoA) and the DNA targets of the MarR transcriptional repressor, which we named HcaR, which regulates hydroxycinnamic acid degradation. Using an hcaR-deleted strain, we further revealed that hydroxycinnamic acid degradation interfere with virulence gene expression. The HcaR deletion mutant shows a contrasting competitive colonization ability, being less abundant than the wild-type strain in tumors but more abundant in the rhizosphere. This supports the view that A. fabrum C58 HcaR regulation through ferulic and p-coumaric acid perception is important for the transition between lifestyles.

A Chemotaxis-Like Pathway of Azorhizobium caulinodans Controls Flagella-Driven Motility, Which Regulates Biofilm Formation, Exopolysaccharide Biosynthesis, and Competitive Nodulation.

The genome of the Azorhizobium caulinodans ORS571 contains a unique chemotaxis gene cluster (che) including five chemotaxis genes: cheA, cheW, cheY1, cheB, and cheR. Analysis of the role of the chemotaxis cluster of A. caulinodans using deletion mutant strains revealed that CheA or the Che signaling pathway controls chemotaxis behavior and flagella-driven motility and plays important roles in formation of biofilms and production of extracellular polysaccharides (EPS). Furthermore, the deletion mutants (ΔcheA and ΔcheA-R) were defective in competitive adsorption and colonization on the root surface of host plants. In addition, a functional CheA or Che pathway promoted competitive nodulation on roots and stems. Interestingly, a nonflagellated mutant, ΔfliM, displayed a phenotype highly similar to that of the ΔcheA or ΔcheA-R mutant strains. These findings suggest that through controlling flagella-driven motility behavior, the chemotaxis signaling pathway in A. caulinodans coordinates biofilm formation, EPS, and competitive colonization and nodulation.

AmrZ is a major determinant of c-di-GMP levels in Pseudomonas fluorescens F113

The transcriptional regulator AmrZ is a global regulatory protein conserved within the pseudomonads. AmrZ can act both as a positive and a negative regulator of gene expression, controlling many genes implicated in environmental adaption. Regulated traits include motility, iron homeostasis, exopolysaccharides production and the ability to form biofilms. In Pseudomonas fluorescens F113, an amrZ mutant presents a pleiotropic phenotype, showing increased swimming motility, decreased biofilm formation and very limited ability for competitive colonization of rhizosphere, its natural habitat. It also shows different colony morphology and binding of the dye Congo Red. The amrZ mutant presents severely reduced levels of the messenger molecule cyclic-di-GMP (c-di-GMP), which is consistent with the motility and biofilm formation phenotypes. Most of the genes encoding proteins with diguanylate cyclase (DGCs) or phosphodiesterase (PDEs) domains, implicated in c-di-GMP turnover in this bacterium, appear to be regulated by AmrZ. Phenotypic analysis of eight mutants in genes shown to be directly regulated by AmrZ and encoding c-di-GMP related enzymes, showed that seven of them were altered in motility and/or biofilm formation. The results presented here show that in P. fluorescens, AmrZ determines c-di-GMP levels through the regulation of a complex network of genes encoding DGCs and PDEs.

Comparative genome analysis of Streptococcus iniae DX09 reveals new insights into niche adaptation and competitive host colonisation ability

// Tao Liu 1, 2, * , Yajun Wang 1, 4, * and Kaiyu Wang 1, 3 1 Department of Basic Veterinary, Veterinary Medicine College, Sichuan Agricultural University, Sichuan, Chengdu 611134, P.R. China 2 Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Sichuan, Chengdu 611134, P.R. China 3 Department of Aquaculture, College of Animal Science & Technology, Sichuan Agricultural University, Sichuan 611134, Cheng’du, P.R. China 4 Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou 510380, Guangdong, P.R. China * These authors contributed equally to this work Correspondence to: Kaiyu Wang, email: kywang1955@126.com Keywords: Streptococcus iniae; whole-genome sequencing; comparative genome analysis; pathogenic Received: September 22, 2017      Accepted: December 01, 2017      Published: December 08, 2017 ABSTRACT Streptococcus iniae is a significant pathogen in a variety of marine and freshwater cultured fish species. Previous investigations on S. iniae have been limited to a single virulence gene or genome. However, different strains are associated with varying pathogenicity and niche adaptation properties. For comprehensive characterization of the genetic variations in S. iniae, whole-genome sequencing of S. iniae DX09 (isolated from diseased catfish) was performed and comparative genome analysis with eight S. iniae strains conducted to determine the virulence evolution patterns. Comparative analysis of all sequenced S. iniae revealed genome-genome variations, mainly in two plasticity zones, within genes encoding specific functions, such as the Ess/type VII secretion system and phosphoenolpyruvate-carbohydrate phosphotransferase system, reflecting adaptation to colonisation of specific host habitats. The plasticity zones analyzed in the S. iniae genome may be a paradigm rather than a unique combination of horizontal gene transfer and underlie the emergence of pathogenic Gram-positive bacteria.

Comparison of colonization ability of O1 and O139 Vibrio cholerae strains on soft-shelled turtle's surface

Objective: To study the preferred colonization sites of O1 Vibrio cholerae (V.cholerae) and the colonization ability difference for O1 and O139 V. cholerae on soft-shelled turtle's surface. Methods: 8 O1 and O139 V. cholerae strains were obtained from branch of diarrheal diseases, Chinese center for disease control and prevention. 63 soft-shelled turtles weighing 150 g and 9 cm in length (diameter of calipash) were selected for use in the study. The preferred colonization sites and proliferation trend were studied by using bioluminescent imaging method. The colonization factors for O1 V. cholerae strains were studied by constructing colonization gene mutant strains (VC1897dmshA, VC1897dgbpA and VC1897dtcpA), performing competition colonization assays and analyzing the competitive indexes. After pairing off O1 and O139 strains respectively to perform 16 competition groups, the colonization difference of these two strains were studied by competition colonization assays. Results: The colonization sites by V. cholerae on soft-shelled turtles surfaces was clustered. More V. cholerae strains colonized on turtle's calipash and carapace on dorsal side and less strain colonized on ventral side. The competition colonization assays showed that colonization ability of O1 serogroup mshA mutant strains were 7.26 times lower than VC1897dlacZ. Besides, the CI value (O139/O1) of 11 out of the 16 competition groups were greater than 2 (between 2.07 and 59.84). Two groups showed values of 1.43 and 0.93 respectively and 3 groups lower than 0.7. Conclusion: The preferred colonization sites for O1 V. cholerae strains on body surface were observed.MSHA was one of the main colonization factors for its colonization. Our study suggested that in general, O139 V. cholerae strains have stronger colonization ability than O1 strains. Besides, strains isolated from soft-shelled turtles tend to have stronger colonization ability than strains isolated from patients.

A Subset of Polysaccharide Capsules in the Human Symbiont Bacteroides thetaiotaomicron Promote Increased Competitive Fitness in the Mouse Gut

Capsular polysaccharides (CPSs) play multiple roles in protecting bacteria from host and environmental factors, and many commensal bacteria can produce multiple capsule types. To better understand the roles of different CPSs in competitive intestinal colonization, we individually expressed the eight different capsules of the human gut symbiont Bacteroides thetaiotaomicron. Certain CPSs were most advantageous invivo, and increased anti-CPS immunoglobulin A correlated with increased fitness of a strain expressing one particular capsule, CPS5, suggesting that it promotes avoidance of adaptive immunity. Astrain with the ability to switch between multiple capsules was more competitive than those expressing any single capsule except CPS5. After antibiotic perturbation, only the wild-type, capsule-switching strain remained in the gut, shifting to prominent expression of CPS5 only in mice with intact adaptive immunity. These data suggest that different capsules equip mutualistic gut bacteria with the ability to thrive in various niches, including those influenced by immune responses and antibiotic perturbations.

Genome-wide identification of bacterial plant colonization genes.

Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system. Among the genes we identified were some with obvious colonization-related roles in motility and carbon metabolism, as well as 44 other genes that had no or vague functional predictions. Independent validation assays of individual genes confirmed colonization functions for 20 of 22 (91%) cases tested. To further characterize genes identified by our screen, we compared the functional contributions of P. simiae genes to growth in 90 distinct in vitro conditions by RB-TnSeq, highlighting specific metabolic functions associated with root colonization genes. Our analysis of bacterial genes by sequence-driven saturation mutagenesis revealed a genome-wide map of the genetic determinants of plant root colonization and offers a starting point for targeted improvement of the colonization capabilities of plant-beneficial microbes.

Azorhizobium caulinodans Transmembrane Chemoreceptor TlpA1 Involved in Host Colonization and Nodulation on Roots and Stems.

Azorhizobium caulinodans ORS571 is a motile soil bacterium that interacts symbiotically with legume host Sesbania rostrata, forming nitrogen-fixing root and stem nodules. Bacterial chemotaxis plays an important role in establishing this symbiotic relationship. To determine the contribution of chemotaxis to symbiosis in A. caulinodans ORS571-S. rostrata, we characterized the function of TlpA1 (transducer-like protein in A. caulinodans), a chemoreceptor predicted by SMART (Simple Modular Architecture Research Tool), containing two N-terminal transmembrane regions. The tlpA1 gene is located immediately upstream of the unique che gene cluster and is transcriptionally co-oriented. We found that a ΔtlpA1 mutant is severely impaired for chemotaxis to various organic acids, glycerol and proline. Furthermore, biofilm forming ability of the strain carrying the mutation is reduced under certain growth conditions. Interestingly, competitive colonization ability on S. rostrata root surfaces is impaired in the ΔtlpA1 mutant, suggesting that chemotaxis of the A. caulinodans ORS571 contributes to root colonization. We also found that TlpA1 promotes competitive nodulation not only on roots but also on stems of S. rostrata. Taken together, our data strongly suggest that TlpA1 is a transmembrane chemoreceptor involved in A. caulinodans-S. rostrata symbiosis.

Transcriptomic Analysis of the Swarm Motility Phenotype of Salmonella enterica Serovar Typhimurium Mutant Defective in Periplasmic Glucan Synthesis.

Movement of food-borne pathogens on moist surfaces enables them to migrate towards more favorable niches and facilitate their survival for extended periods of time. Salmonella enterica serovar Typhimurium mutants defective in Osmoregulated periplasmic glucans (OPG) synthesis are unable to exhibit motility on moist surfaces (swarming); however, their mobility in liquid (swim motility) remains unaffected. In order to understand the role of OPG in swarm motility, transcriptomic analysis was performed using cells growing on a moist agar surface. In opgGH deletion mutant, lack of OPG significantly altered transcription of 1039 genes out of total 4712 genes (22%). Introduction of a plasmid-borne copy of opgGH into opgGH deletion mutant restored normal expression of all but 30 genes, indicating a wide-range influence of OPG on gene expression under swarm motility condition. Major pathways that were differentially expressed in opgGH mutants were motility, virulence and invasion, and genes related to the secondary messenger molecule, cyclic di-GMP. These observations provide insights and help explain the pleiotropic nature of OPG mutants such as sub-optimal virulence and competitive organ colonization in mice, biofilm formation, and sensitivity towards detergent stress.