Search

Xylella fastidiosa is an aerobic, Gram-negative bacterium that is responsible for many plant diseases. The bacterium is the causal agent of Pierce's disease in grapes and is also responsible for citrus variegated chlorosis, peach phony disease, olive quick decline syndrome and leaf scorches of various species. The production of biofilm is intrinsically linked with persistence and transmission in X. fastidiosa . Biofilm formation is regulated by members of the Diffusible Signal Factor (DSF) quorum sensing signalling family which are comprised of a series of long chain cis -unsaturated fatty acids. This article describes the evaluation of a library of N -acyl sulfonamide bioisosteric analogues of BDSF, Xf DSF1 and Xf DSF2 for their ability to control biofilm production in X. fastidiosa . The compounds were screened against both the wild-type strain Temecula and an rpfF * mutant which can perceive but not produce Xf DSF. Planktonic cell abundance was measured via OD600 while standard crystal violet assays were used to determine biofilm biomass. Several compounds were found to be effective biofilm inhibitors depending on the nature of the sulfonamide substituent. The findings reported here may provide future opportunities for biocontrol of this important plant pathogen.

We sequenced and comprehensively analysed the genomic architecture of 98 fluorescent pseudomonads isolated from different symptomatic and asymptomatic tissues of almond and a few other Prunus spp. Phylogenomic analyses, genome mining, field pathogenicity tests, and in vitro ice nucleation and antibiotic sensitivity tests were integrated to improve knowledge of the biology and management of bacterial blast and bacterial canker of almond. We identified Pseudomonas syringae pv. syringae, P. cerasi, and P. viridiflava as almond canker pathogens. P. syringae pv. syringae caused both canker and foliar (blast) symptoms. In contrast, P. cerasi and P. viridiflava only caused cankers, and P. viridiflava appeared to be a weak pathogen of almond. Isolates belonging to P. syringae pv. syringae were the most frequently isolated among the pathogenic species/pathovars, composing 75% of all pathogenic isolates. P. cerasi and P. viridiflava isolates composed 8.3 and 16.7% of the pathogenic isolates, respectively. Laboratory leaf infiltration bioassays produced results distinct from experiments in the field with both P. cerasi and P. syringae pv. syringae, causing significant necrosis and browning of detached leaves, whereas P. viridiflava conferred moderate effects. Genome mining revealed the absence of key epiphytic fitness-related genes in P. cerasi and P. viridiflava genomic sequences, which could explain the contrasting field and laboratory bioassay results. P. syringae pv. syringae and P. cerasi isolates harboured the ice nucleation protein, which correlated with the ice nucleation phenotype. Results of sensitivity tests to copper and kasugamycin showed a strong linkage to putative resistance genes. Isolates harbouring the ctpV gene showed resistance to copper up to 600 μg/ml. In contrast, isolates without the ctpV gene could not grow on nutrient agar amended with 200 μg/ml copper, suggesting ctpV can be used to phenotype copper resistance. All isolates were sensitive to kasugamycin at the label-recommended rate of 100μg/ml., Competing Interests: The authors have declared that no competing interests exist, (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Microbial competition within plant tissues affects invading pathogens' fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa , a Gram-negative bacterium causing Pierce's disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography-mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa . Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans . These findings suggest that P. phytofirmans -mediated disease suppression involves modulation of the exometabolome of X. fastidiosa , impacting plant immunity.

In disease ecology, pathogen transmission among conspecific versus heterospecific hosts is known to shape pathogen specialization and virulence, but we do not yet know if similar effects occur at the microbiome level. We tested this idea by experimentally passaging leaf-associated microbiomes either within conspecific or across heterospecific plant hosts. Although conspecific transmission results in persistent host-filtering effects and more within-microbiome network connections, heterospecific transmission results in weaker host-filtering effects but higher levels of interconnectivity. When transplanted onto novel plants, heterospecific lines are less differentiated by host species than conspecific lines, suggesting a shift toward microbiome generalism. Finally, conspecific lines from tomato exhibit a competitive advantage on tomato hosts against those passaged on bean or pepper, suggesting microbiome-level host specialization. Overall, we find that transmission mode and previous host history shape microbiome diversity, with repeated conspecific transmission driving microbiome specialization and repeated heterospecific transmission promoting microbiome generalism., Competing Interests: Author contributions K.M.M., S.E.L., C.J.E.M., and B.K. conceptualized and designed the experiments. K.M.M., A.L.S.V., J.K.S., and I.E.M. performed the experiments and processed the samples. K.M.M. performed data processing, analysis, and writing of the initial draft. All authors contributed to the writing and editing of the paper. Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Leaves harbor distinct microbial communities that can have an important impact on plant health and microbial ecosystems worldwide. Nevertheless, the ecological processes that shape the composition of leaf microbial communities remain unclear, with previous studies reporting contradictory results regarding the importance of bacterial dispersal versus host selection. This discrepancy could be driven in part because leaf microbiome studies typically consider the upper and lower leaf surfaces as a single entity despite these habitats possessing considerable anatomical differences. We characterized the composition of bacterial phyllosphere communities from the upper and lower leaf surfaces across 24 plant species. Leaf surface pH and stomatal density were found to shape phyllosphere community composition, and the underside of leaves had lower richness and higher abundances of core community members than upper leaf surfaces. We found fewer endemic bacteria on the upper leaf surfaces, suggesting that dispersal is more important in shaping these communities, with host selection being a more important force in microbiome assembly on lower leaf surfaces. Our study illustrates how changing the scale in which we observe microbial communities can impact our ability to resolve and predict microbial community assembly patterns on leaf surfaces. IMPORTANCE Leaves can harbor hundreds of different bacterial species that form unique communities for every plant species. Bacterial communities on leaves are really important because they can, for example, protect their host against plant diseases. Usually, bacteria from the whole leaf are considered when trying to understand these communities; however, this study shows that the upper and lower sides of a leaf have a very different impact on how these communities are shaped. It seems that the bacteria on the lower leaf side are more closely associated with the plant host, and communities on the upper leaf side are more impacted by immigrating bacteria. This can be really important when we want to treat, for example, crops in the field with beneficial bacteria or when trying to understand host-microbe interactions on the leaves., Competing Interests: The authors declare no conflict of interest.

The genes encoding the phosphate uptake system in Xanthomonas citri pv. glycines 12-2 were previously found to be upregulated when in soybean leaves. This study thus explored the role of the phosphate uptake system on its virulence to soybean. While phoB and pstSCAB mutants were greatly impaired in both inciting disease symptoms and growth in soybean, the virulence and growth in soybean of a phoU mutant was not reduced when compared with the wild-type strain. The expression of phoB and pstSCAB was highly induced in phosphate-deficient media. In addition, the expression of phoB , assessed with a fusion to a promoterless ice nucleation reporter gene, was greatly increased in soybean leaves, confirming that the soybean apoplast is a phosphorus-limited habitat for X. citri pv. glycines . Global gene expression profiles of phoB and phoU mutants of X. citri pv. glycines conducted under phosphate-limitation conditions in vitro, using RNA-seq, revealed that PhoB positively regulated genes involved in signal transduction, the xcs cluster type II secretion system, cell motility, and chemotaxis, while negatively regulating cell wall and membrane biogenesis, DNA replication and recombination and repair, and several genes with unknown function. PhoU also positively regulated the same genes involved in cell motility and chemotaxis. The severity of bacterial pustule disease was decreased in soybean plants grown under high phosphate fertilization conditions, demonstrating that high phosphate availability in soybean plants can affect infection by X. citri pv. glycines by modulation of the expression of phosphate uptake systems. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Surface adhesion strategies are widely employed by bacterial pathogens during establishment and systemic spread in their host. A variety of cell-surface appendages such as pili, fimbriae, and afimbrial adhesins are involved in these processes. The phytopathogen Xylella fastidiosa employs several of these structures for efficient colonization of its insect and plant hosts. Among the adhesins encoded in the X. fastidiosa genome, three afimbrial adhesins, XadA1, Hsf/XadA2, and XadA3, are predicted to be trimeric autotransporters with a C-terminal YadA-anchor membrane domain. We analyzed the individual contributions of XadA1, XadA2, and XadA3 to various cellular behaviors both in vitro and in vivo. Using isogenic X. fastidiosa mutants, we found that cell-cell aggregation and biofilm formation were severely impaired in the absence of XadA3. No significant reduction of cell-surface attachment was found with any mutant under flow conditions. Acquisition by insect vectors and transmission to grapevines were reduced in the XadA3 deletion mutant. While the XadA3 mutant was hypervirulent in grapevines, XadA1 or XadA2 deletion mutants conferred lower disease severity than the wild-type strain. This insight of the importance of these adhesive proteins and their individual contributions to different aspects of X. fastidiosa biology should guide new approaches to reduce pathogen transmission and disease development. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Efflux transporters such as MexAB-OprM contribute to bacterial resistance to diverse antimicrobial compounds. Here, we show that MexB contributes to epiphytic and late-stage apoplastic growth of Pseudomonas syringae strain B728a, as well as lesion formation in common bean ( Phaseolus vulgaris ). Although a ∆ mexB mutant formed fewer lesions after topical application to common bean, these lesions contain the same number of cells (10 5 to 10 7 cells) as those caused by the wild-type strain. The internalized population size of both the wild-type and the ∆ mexB mutant within small samples of surface-sterilized asymptomatic portions of leaves varied from undetectably low to as high as 10 5 cells/cm 2 . Localized bacterial populations within individual lesions consistently exceeded 10 5 cells/cm 2 . Strain B728a was capable of moderate to extensive apoplastic growth in diverse host plants, including lima bean ( P. lunatus ), fava bean ( Vicia faba ), pepper ( Capsicum annuum ), Nicotiana benthamiana , sunflower ( Helianthus annuus ), and tomato ( Solanum lycopersicum ), but MexB was not required for growth in a subset of these plant species. A model is proposed that MexB provides resistance to as-yet-unidentified antimicrobials that differ between plant species. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Microbial communities associated with plant leaf surfaces (i.e., the phyllosphere) are increasingly recognized for their role in plant health. While accumulating evidence suggests a role for host filtering of its microbiota, far less is known about how community composition is shaped by dispersal, including from neighboring plants. We experimentally manipulated the local plant neighborhood within which tomato, pepper, or bean plants were grown in a 3-month field trial. Focal plants were grown in the presence of con- or hetero-specific neighbors (or no neighbors) in a fully factorial combination. At 30-day intervals, focal plants were harvested and replaced with a new age- and species-matched cohort while allowing neighborhood plants to continue growing. Bacterial community profiling revealed that the strength of host filtering effects (i.e., interspecific differences in composition) decreased over time. In contrast, the strength of neighborhood effects increased over time, suggesting dispersal from neighboring plants becomes more important as neighboring plant biomass increases. We next implemented a cross-inoculation study in the greenhouse using inoculum generated from the field plants to directly test host filtering of microbiomes while controlling for directionality and source of dispersal. This experiment further demonstrated that focal host species, the host from which the microbiome came, and in one case the donor hosts' neighbors, contribute to variation in phyllosphere bacterial composition. Overall, our results suggest that local dispersal is a key factor in phyllosphere assembly, and that demographic factors such as nearby neighbor identity and biomass or age are important determinants of phyllosphere microbiome diversity., (© 2022. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Trans-acyltransferase polyketide synthases (trans-AT PKSs) are bacterial multimodular enzymes that biosynthesize diverse pharmaceutically and ecologically important polyketides. A notable feature of this natural product class is the existence of chemical hybrids that combine core moieties from different polyketide structures. To understand the prevalence, biosynthetic basis, and evolutionary patterns of this phenomenon, we developed transPACT, a phylogenomic algorithm to automate global classification of trans-AT PKS modules across bacteria and applied it to 1782 trans-AT PKS gene clusters. These analyses reveal widespread exchange patterns suggesting recombination of extended PKS module series as an important mechanism for metabolic diversification in this natural product class. For three plant-associated bacteria, i.e., the root colonizer Gynuella sunshinyii and the pathogens Xanthomonas cannabis and Pseudomonas syringae, we demonstrate the utility of this computational approach for uncovering cryptic relationships between polyketides, accelerating polyketide mining from fragmented genome sequences, and discovering polyketide variants with conserved moieties of interest. As natural combinatorial hybrids are rare among the more commonly studied cis-AT PKSs, this study paves the way towards evolutionarily informed, rational PKS engineering to produce chimeric trans-AT PKS-derived polyketides.

Production of the biosurfactant syringafactin by the plant pathogen Pseudomonas syringae B728a is a surface contact-dependent trait. Expression of syfA, as measured using a gfp reporter gene fusion was low in planktonic cells in liquid cultures but over 4-fold higher in cells immobilized on surfaces as varied as glass, plastic, paper, parafilm, agar, membrane filters, and leaves. Induction of syfA as measured by GFP fluorescence was rapid, occurring within two hours after immobilization of cells on surfaces. Comparison of the global transcriptome by RNA sequencing of planktonic cells in a nutrient medium with that of cells immobilized for 2 hours on filters placed on this solidified medium revealed that, in addition to syfA, 3156 other genes were differentially expressed. Genes repressed in immobilized cells included those involved in quaternary ammonium compound (QAC) metabolism and transport, compatible solute production, carbohydrate metabolism and transport, organic acid metabolism and transport, phytotoxin synthesis and transport, amino acid metabolism and transport, and secondary metabolism. Genes induced in immobilized cells included syfA plus those involved in translation, siderophore synthesis and transport, nucleotide metabolism and transport, flagellar synthesis and motility, lipopolysaccharide (LPS) synthesis and transport, energy generation, transcription, chemosensing and chemotaxis, replication and DNA repair, iron-sulfur proteins, peptidoglycan/cell wall polymers, terpenoid backbone synthesis, iron metabolism and transport, and cell division. That many genes are rapidly differentially expressed upon transfer of cells from a planktonic to an immobilized state suggests that cells experience the two environments differently. It seems possible that surface contact initiates anticipatory changes in P. syringae gene expression, which enables rapid and appropriate physiological responses to the different environmental conditions such as might occur in a biofilm. Such responses could help cells survive transitions from aquatic habitats fostering planktonic traits to attachment on surfaces, conditions that alternatively occur on leaves., Competing Interests: The authors have declared that no competing interests exist.

A variety of traits are necessary for bacterial colonization of the interior of plant hosts, including well-studied virulence effectors as well as other phenotypes contributing to bacterial growth and survival within the apoplast. High-throughput methods such as transposon sequencing (TnSeq) are powerful tools to identify such genes in bacterial pathogens. However, there is little information as to the distinctiveness of traits required for bacterial colonization of different hosts. Here, we utilize randomly barcoded TnSeq (RB-TnSeq) to identify the genes that contribute to the ability of Pseudomonas syringae strain B728a to grow within common bean (Phaseolus vulgaris), lima bean (Phaseolus lunatus), and pepper (Capsicum annuum); species representing two different plant families. The magnitude of contribution of most genes to apoplastic fitness in each of the plant hosts was similar. However, 50 genes significantly differed in their fitness contributions to growth within these species. These genes encoded proteins in various functional categories including polysaccharide synthesis and transport, amino acid metabolism and transport, cofactor metabolism, and phytotoxin synthesis and transport. Six genes that encoded unannotated, hypothetical proteins also contributed differentially to growth in these hosts. The genetic repertoire of a relatively promiscuous pathogen such as P. syringae may thus be shaped, at least in part, by the conditional contribution of some fitness determinants., Competing Interests: The authors have declared that no competing interests exist.

Bacteria express numerous efflux transporters that confer resistance to diverse toxicants present in their environment. Due to a high level of functional redundancy of these transporters, it is difficult to identify those that are of most importance in conferring resistance to specific compounds. The r esistance- n odulation- d ivision (RND) protein family is one such example of redundant transporters that are widespread among Gram-negative bacteria. Within this family, the MexAB-OprM protein complex is highly expressed and conserved among Pseudomonas species. We exposed barcoded transposon mutant libraries in isogenic wild-type and Δ mexB backgrounds in P. syringae B728a to diverse toxic compounds in vitro to identify mutants with increased susceptibility to these compounds. Mutants with mutations in genes encoding both known and novel redundant transporters but with partially overlapping substrate specificities were observed in a Δ mexB background. Psyr_0228, an uncharacterized member of the major facilitator superfamily of transporters, preferentially contributes to tolerance of acridine orange and acriflavine. Another transporter located in the inner membrane, Psyr_0541, contributes to tolerance of acriflavine and berberine. The presence of multiple redundant, genomically encoded efflux transporters appears to enable bacterial strains to tolerate a diversity of environmental toxins. This genome-wide screen performed in a hypersusceptible mutant strain revealed numerous transporters that would otherwise be dispensable under these conditions. Bacterial strains such as P. syringae that likely encounter diverse toxins in their environment, such as in association with many different plant species, probably benefit from possessing multiple redundant transporters that enable versatility with respect to toleration of novel toxicants. IMPORTANCE Bacteria use protein pumps to remove toxic compounds from the cell interior, enabling survival in diverse environments. These protein pumps can be highly redundant, making their targeted examination difficult. In this study, we exposed mutant populations of Pseudomonas syringae to diverse toxicants to identify pumps that contributed to survival in those conditions. In parallel, we examined pump redundancy by testing mutants of a population lacking the primary efflux transporter responsible for toxin tolerance. We identified partial substrate overlap for redundant transporters, as well as several pumps that appeared more substrate specific. For bacteria that are found in diverse environments, having multiple, partially redundant efflux pumps likely allows flexibility in habitat colonization.

The foliar plant pathogen Pseudomonas syringae can establish large epiphytic populations on leaf surfaces before apoplastic colonization. However, the bacterial genes that contribute to these lifestyles have not been completely defined. The fitness contributions of 4,296 genes in P. syringae pv. syringae B728a were determined by genome-wide fitness profiling with a randomly barcoded transposon mutant library that was grown on the leaf surface and in the apoplast of the susceptible plant Phaseolus vulgaris Genes within the functional categories of amino acid and polysaccharide (including alginate) biosynthesis contributed most to fitness both on the leaf surface (epiphytic) and in the leaf interior (apoplast), while genes involved in type III secretion system and syringomycin synthesis were primarily important in the apoplast. Numerous other genes that had not been previously associated with in planta growth were also required for maximum epiphytic or apoplastic fitness. Fourteen hypothetical proteins and uncategorized glycosyltransferases were also required for maximum competitive fitness in and on leaves. For most genes, no relationship was seen between fitness in planta and either the magnitude of their expression in planta or degree of induction in planta compared to in vitro conditions measured in other studies. A lack of association of gene expression and fitness has important implications for the interpretation of transcriptional information and our broad understanding of plant-microbe interactions., Competing Interests: The authors declare no conflict of interest.

The epiphytic bacterium Pseudomonas syringae strain B728a produces the biosurfactant syringafactin, which is hygroscopic. The water-absorbing potential of syringafactin is high. Syringafactin attracts 250% of its weight in water at high relative humidities but is less hygroscopic at lower relative humidities. This finding suggests that the benefit of syringafactin to the producing cells is strongly context dependent. The contribution of syringafactin to the water availability around cells on different matrices was assessed by examining the water stress exhibited by biosensor strains expressing gfp via the water-stress-activated proU promoter. Wild-type cells exhibited significantly less green fluorescent protein (GFP) fluorescence than a syringafactin-deficient strain on dry filters in atmospheres of high water saturation, as well as on leaf surfaces, indicating greater water availability. When infiltrated into the leaf apoplast, wild-type cells also subsequently exhibited less GFP fluorescence than the syringafactin-deficient strain. These results suggest that the apoplast is a dry but humid environment and that, just as on dry but humid leaf surfaces, syringafactin increases liquid water availability and reduces the water stress experienced by P. syringae IMPORTANCE Many microorganisms, including the plant pathogen Pseudomonas syringae , produce amphiphilic compounds known as biosurfactants. While biosurfactants are known to disperse hydrophobic compounds and to reduce water tension, they have other properties that can benefit the cells that produce them. Leaf-colonizing bacteria experience frequent water stress, since liquid water is present only transiently on or in leaf sites that they colonize. The demonstration that syringafactin, a biosurfactant produced by P. syringae , is sufficiently hygroscopic to increase water availability to cells, thus relieving water stress, reveals that P. syringae can modify its local habitat both on leaf surfaces and in the leaf apoplast. Such habitat modification may be a common role for biosurfactants produced by other bacterial species that colonize habitats (such as soil) that are not always water saturated., (Copyright © 2019 American Society for Microbiology.)

Xylella fastidiosa releases outer membrane vesicles (OMVs) known to play a role in the systemic dissemination of this pathogen. OMVs inhibit bacterial attachment to xylem wall and traffic lipases/esterases that act on the degradation of plant cell wall. Here, we extended the characterization of X. fastidiosa OMVs by identifying proteins and metabolites potentially associated with OMVs produced by Temecula1, a Pierce's disease strain, and by 9a5c and Fb7, two citrus variegated chlorosis strains. These results strengthen that one of the OMVs multiple functions is to carry determinants of virulence, such as lipases/esterases, adhesins, proteases, porins, and a pectin lyase-like protein. For the first time, we show that the two citrus variegated chlorosis strains produce X. fastidiosa diffusible signaling factor 2 (DSF2) and citrus variegated chlorosis-DSF (likewise, Temecula1) and most importantly, that these compounds of the DSF ( X. fastidiosa DSF) family are associated with OMV-enriched fractions. Altogether, our findings widen the potential functions of X. fastidiosa OMVs in intercellular signaling and host-pathogen interactions.

Enhancing the aerobic stability of silage is very important in silage production. The objective of this study was to compare the roles of a new bacterial strain Lactobacillus parafarraginis ZH1 with Lactobacillus plantarum Chikuso-1 (LP) and Lactobacillus buchneri NCIMB 40788 (LB) in improving the aerobic stability of oat silage and identify the anti-yeast substances produced by them. After ensiled for 45 days, either inoculated silages or control silage were of pH values lower than 4.2. The control and LP inoculated silage had poorer aerobic stability than LB and ZH1 inoculated ones (p < 0.05). ZH1 inoculated silage produced more acetic acid, benzoic acid and hexadecanoic acid than LB inoculated one and had the best aerobic stability (p < 0.05). In MRS medium, strain ZH1 produced more hexadecanoic acid than LP and LB strains, more benzoic acid than strain LP (p < 0.05), and the equal amount of benzoic acid to LB (p > 0.05). In addition, benzoic acid and hexadecanoic acid had low minimal inhibitory concentrations to target yeasts in the pure culture. In conclusion, L. parafarraginis ZH1 had stronger anti-yeast potential and more effectively improved the aerobic stability of silage than other strains used in this study., (© 2018 Japanese Society of Animal Science.)

Knowledge of the factors controlling the diverse chemical emissions of common environmental bacteria and fungi is crucial because they are important signal molecules for these microbes that also could influence humans. We show here not only a high diversity of mVOCs but that their abundance can differ greatly in different environmental contexts. Microbial volatiles exhibit dynamic changes across microbial growth phases, resulting in variance of composition and emission rate of species-specific and generic mVOCs. In vitro experiments documented emissions of a wide range of mVOCs (>400 different chemicals) at high time resolution from diverse microbial species grown under different controlled conditions on nutrient media, or residential structural materials ( N = 54, N control = 23). Emissions of mVOCs varied not only between microbial taxa at a given condition but also as a function of life stage and substrate type. We quantify emission factors for total and specific mVOCs normalized for respiration rates to account for the microbial activity during their stationary phase. Our VOC measurements of different microbial taxa indicate that a variety of factors beyond temperature and water activity, such as substrate type, microbial symbiosis, growth phase, and lifecycle affect the magnitude and composition of mVOC emission.

The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

Background: Microorganisms influence the chemical milieu of their environment, and chemical metabolites can affect ecological processes. In built environments, where people spend the majority of their time, very little is known about how surface-borne microorganisms influence the chemistry of the indoor spaces. Here, we applied multidisciplinary approaches to investigate aspects of chemical microbiology in a house., Methods: We characterized the microbial and chemical composition of two common and frequently wet surfaces in a residential setting: kitchen sink and bathroom shower. Microbial communities were studied using culture-dependent and independent techniques, including targeting RNA for amplicon sequencing. Volatile and soluble chemicals from paired samples were analyzed using state-of-the-art techniques to explore the links between the observed microbiota and chemical exudates., Results: Microbial analysis revealed a rich biological presence on the surfaces exposed in kitchen sinks and bathroom shower stalls. Microbial composition, matched for DNA and RNA targets, varied by surface type and sampling period. Bacteria were found to have an average of 25× more gene copies than fungi. Biomass estimates based on qPCR were well correlated with measured total volatile organic compound (VOC) emissions. Abundant VOCs included products associated with fatty acid production. Molecular networking revealed a diversity of surface-borne compounds that likely originate from microbes and from household products., Conclusions: Microbes played a role in structuring the chemical profiles on and emitted from kitchen sinks and shower stalls. Microbial VOCs (mVOCs) were predominately associated with the processing of fatty acids. The mVOC composition may be more stable than that of microbial communities, which can show temporal and spatial variation in their responses to changing environmental conditions. The mVOC output from microbial metabolism on kitchen sinks and bathroom showers should be apparent through careful measurement, even against a broader background of VOCs in homes, some of which may originate from microbes in other locations within the home. A deeper understanding of the chemical interactions between microbes on household surfaces will require experimentation under relevant environmental conditions, with a finer temporal resolution, to build on the observational study results presented here.

Xylella fastidiosa colonizes the xylem network of host plant species as well as the foregut of its required insect vectors to ensure efficient propagation. Disease management strategies remain inefficient due to a limited comprehension of the mechanisms governing both insect and plant colonization. It was previously shown that X. fastidiosa has a functional chitinase (ChiA), and that chitin likely serves as a carbon source for this bacterium. We expand on that research, showing that a chiA mutant strain is unable to grow on chitin as the sole carbon source. Quantitative PCR assays allowed us to detect bacterial cells in the foregut of vectors after pathogen acquisition; populations of the wild-type and complemented mutant strain were both significantly larger than the chiA mutant strain 10 days, but not 3 days, post acquisition. These results indicate that adhesion of the chiA mutant strain to vectors may not be impaired, but that cell multiplication is limited. The mutant was also affected in its transmission by vectors to plants. In addition, the chiA mutant strain was unable to colonize host plants, suggesting that the enzyme has other substrates associated with plant colonization. Lastly, ChiA requires other X. fastidiosa protein(s) for its in vitro chitinolytic activity. The observation that the chiA mutant strain is not able to colonize plants warrants future attention to be paid to the substrates for this enzyme.

We report here the draft genome sequences of eight bacterial strains of the genera Staphylococcus , Microbacterium, Mycobacterium, Plantibacter, and Pseudomonas . These isolates were obtained from aerosol sampling of bathrooms of five residences in the San Francisco Bay area. Taxonomic classifications as well as the genome sequence and gene annotation of the isolates are described. As part of the "Built Environment Reference Genome" project, these isolates and associated genome data provide valuable resources for studying the microbiology of the built environment.

Unlabelled: To better characterize the bacterial community members capable of biosurfactant production on leaves, we distinguished culturable biosurfactant-producing bacteria from nonproducers and used community sequencing to compare the composition of these distinct cultured populations with that from DNA directly recovered from leaves. Communities on spinach, romaine, and head lettuce leaves were compared with communities from adjacent samples of soil and irrigation source water. Soil communities were poorly described by culturing, with recovery of cultured representatives from only 21% of the prevalent operational taxonomic units (OTUs) (>0.2% reads) identified. The dominant biosurfactant producers cultured from soil included bacilli and pseudomonads. In contrast, the cultured communities from leaves are highly representative of the culture-independent communities, with over 85% of the prevalent OTUs recovered. The dominant taxa of surfactant producers from leaves were pseudomonads as well as members of the infrequently studied genus Chryseobacterium The proportions of bacteria cultured from head lettuce and romaine leaves that produce biosurfactants were directly correlated with the culture-independent proportion of pseudomonads in a given sample, whereas spinach harbored a wider diversity of biosurfactant producers. A subset of the culturable bacteria in irrigation water also became enriched on romaine leaves that were irrigated overhead. Although our study was designed to identify surfactant producers on plants, we also provide evidence that most bacteria in some habitats, such as agronomic plant surfaces, are culturable, and these communities can be readily investigated and described by more classical culturing methods., Importance: The importance of biosurfactant production to the bacteria that live on waxy leaf surfaces as well as their ability to be accurately assessed using culture-based methodologies was determined by interrogating epiphytic populations by both culture-dependent and culture-independent methods. Biosurfactant production was much more frequently observed in cultured communities on leaves than in other nearby habitats, such as soil and water, suggesting that this trait is important to life on a leaf by altering either the leaf itself or the interaction of bacteria with water. While pseudomonads were the most common biosurfactant producers isolated, this habitat also selects for taxa, such as Chryseobacterium, for which this trait was previously unrecognized. The finding that most epiphytic bacterial taxa were culturable validates strategies using more classical culturing methodologies for their study in this habitat., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Pierce's disease (PD) is a deadly disease of grapevines caused by the Gram-negative bacterium Xylella fastidiosa. Though disease symptoms were formerly attributed to bacteria blocking the plant xylem, this hypothesis is at best overly simplistic. Recently, we used a proteomic approach to characterize the secretome of X. fastidiosa, both in vitro and in planta, and identified LesA as one of the pathogenicity factors of X. fastidiosa in grapevines that leads to leaf scorching and chlorosis. Herein, we characterize another such factor encoded by PD0956, designated as an antivirulence secreted protease "PrtA" that displays a central role in controlling in vitro cell proliferation, length, motility, biofilm formation, and in planta virulence. The mutant in X. fastidiosa exhibited reduced cell length, hypermotility (and subsequent lack of biofilm formation) and hypervirulence in grapevines. These findings are supported by transcriptomic and proteomic analyses with corresponding plant infection data. Of particular interest, is the hypervirulent response in grapevines observed when X. fastidiosa is disrupted for production of PrtA, and that PD-model tobacco plants transformed to express PrtA exhibited decreased symptoms after infection by X. fastidiosa.

Unlabelled: Cell density-dependent regulation of gene expression in Xylella fastidiosa that is crucial to its switching between plant hosts and insect vectors is dependent on RpfF and its production of 2-enoic acids known as diffusible signal factor (DSF). We show that X. fastidiosa produces a particularly large variety of similar, relatively long-chain-length 2-enoic acids that are active in modulating gene expression. Both X. fastidiosa itself and a Pantoea agglomerans surrogate host harboring X. fastidiosa RpfF (XfRpfF) is capable of producing a variety of both saturated and unsaturated free fatty acids. However, only 2-cis unsaturated acids were found to be biologically active in X. fastidiosa X. fastidiosa produces, and is particularly responsive to, a novel DSF species, 2-cis-hexadecanoic acid that we term XfDSF2. It is also responsive to other, even longer 2-enoic acids to which other taxa such as Xanthomonas campestris are unresponsive. The 2-enoic acids that are produced by X. fastidiosa are strongly affected by the cellular growth environment, with XfDSF2 not detected in culture media in which 2-tetradecenoic acid (XfDSF1) had previously been found. X. fastidiosa is responsive to much lower concentrations of XfDSF2 than XfDSF1. Apparently competitive interactions can occur between various saturated and unsaturated fatty acids that block the function of those agonistic 2-enoic fatty acids. By altering the particular 2-enoic acids produced and the relative balance of free enoic and saturated fatty acids, X. fastidiosa might modulate the extent of DSF-mediated quorum sensing., Importance: X. fastidiosa, having a complicated lifestyle in which it moves and multiplies within plants but also must be vectored by insects, utilizes DSF-based quorum sensing to partition the expression of traits needed for these two processes within different cells in this population based on local cellular density. The finding that it can produce a variety of DSF species in a strongly environmentally context-dependent manner provides insight into how it coordinates the many genes under the control of DSF signaling to successfully associate with its two hosts. Since the new DSF variant XfDSF2 described here is much more active than the previously recognized DSF species, it should contribute to plant disease control, given that the susceptibility of plants can be greatly reduced by artificially elevating the levels of DSF in plants, creating "pathogen confusion," resulting in lower virulence., (Copyright © 2016 Ionescu et al.)

Unlabelled: Given that epiphytic microbes are often found in large population sizes on plants, we tested the hypothesis that plants are quantitatively important local sources of airborne microorganisms. The abundance of microbial communities, determined by quantifying bacterial 16S RNA genes and the fungal internal transcribed spacer (ITS) region, in air collected directly above vegetation was 2- to 10-fold higher than that in air collected simultaneously in an adjacent nonvegetated area 50 m upwind. Nonmetric multidimensional scaling revealed that the composition of airborne bacteria in upwind air samples grouped separately from that of downwind air samples, while communities on plants and downwind air could not be distinguished. In contrast, fungal taxa in air samples were more similar to each other than to the fungal epiphytes. A source-tracking algorithm revealed that up to 50% of airborne bacteria in downwind air samples were presumably of local plant origin. The difference in the proportional abundances of a given operational taxonomic unit (OTU) between downwind and upwind air when regressed against the proportional representation of this OTU on the plant yielded a positive slope for both bacteria and fungi, indicating that those taxa that were most abundant on plants proportionally contributed more to downwind air. Epiphytic fungi were less of a determinant of the microbiological distinctiveness of downwind air and upwind air than epiphytic bacteria. Emigration of epiphytic bacteria and, to a lesser extent, fungi, from plants can thus influence the microbial composition of nearby air, a finding that has important implications for surrounding ecosystems, including the built environment into which outdoor air can penetrate., Importance: This paper addresses the poorly understood role of bacterial and fungal epiphytes, the inhabitants of the aboveground plant parts, in the composition of airborne microbes in outdoor air. It is widely held that epiphytes contribute to atmospheric microbial assemblages, but much of what we know is limited to qualitative assessments. Elucidating the sources of microbes in outdoor air can inform basic biological processes seen in airborne communities (e.g., dispersal and biogeographical patterns). Furthermore, given the considerable contribution of outdoor air to microbial communities found within indoor environments, the understanding of plants as sources of airborne microbes in outdoor air might contribute to our understanding of indoor air quality. With an experimental design developed to minimize the likelihood of other-than-local plant sources contributing to the composition of airborne microbes, we provide direct evidence that plants are quantitatively important local sources of airborne microorganisms, with implications for the surrounding ecosystems., (Copyright © 2016 Lymperopoulou et al.)

To better understand the behavior of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule of soybean within its host, its global transcriptome within soybean leaves was compared with that in a minimal medium in vitro, using deep sequencing of mRNA. Of 5,062 genes predicted from a draft genome of X. axonopodis pv. glycines, 534 were up-regulated in the plant, while 289 were down-regulated. Genes encoding YapH, a cell-surface adhesin, as well as several others encoding cell-surface proteins, were down-regulated in soybean. Many genes encoding the type III secretion system and effector proteins, cell wall-degrading enzymes and phosphate transporter proteins were strongly expressed at early stages of infection. Several genes encoding RND multidrug efflux pumps were induced in planta and by isoflavonoids in vitro and were required for full virulence of X. axonopodis pv. glycines, as well as resistance to soybean phytoalexins. Genes encoding consumption of malonate, a compound abundant in soybean, were induced in planta and by malonate in vitro. Disruption of the malonate decarboxylase operon blocked growth in minimal media with malonate as the sole carbon source but did not significantly alter growth in soybean, apparently because genes for sucrose and fructose uptake were also induced in planta. Many genes involved in phosphate metabolism and uptake were induced in planta. While disruption of genes encoding high-affinity phosphate transport did not alter growth in media varying in phosphate concentration, the mutants were severely attenuated for growth in soybean. This global transcriptional profiling has provided insight into both the intercellular environment of this soybean pathogen and traits used by X. axonopodis pv. glycines to promote disease.

Pseudomonas syringae pv. syringae cell densities fluctuate regularly during host plant colonization. Previously we identified nine genes dependent on the quorum-sensing-associated luxR homolog ahlR during epiphytic and apoplastic stages of host colonization. Yet their contributions to host colonization remain obscure, despite ahlR regulon presence within and beyond the P. syringae pan-genome. To elucidate AhIR regulon member functions, we characterized their regulation, interactions with each other, and contributions to the metabolome. We report Psyr&#95;1625, encoding a functional pyruvate deydrogenase-E1 subunit PdhQ, is required to prevent the accumulation of pyruvate in rich media. Furthermore it is exquisitely regulated by both repression of its own promoter by QrpR within a novel clade of the MarR regulator family, and co-transcription on a 5kb transcript originating from the AhlR-driven ahlI promoter, that reads over ahlR and qrpR. Metabolites accumulated during expression of the second AhlR-driven operon (Psyr&#95;1620-1616, paoABCDE), only in a pdhQ mutant background, in addition to pyruvate, are herein associated with derepression of QrpR-repressed pdhQ. AHL signaling, QrpR, and transcriptional read-through events integrate to ensure AHL-dependent expression of a novel metabolism in anticipation of environmental stress, while minimizing endogenously generated cytotoxicity., (© 2015 John Wiley & Sons Ltd.)

Background: The study of the microbial communities in the built environment is of critical importance as humans spend the majority of their time indoors. While the microorganisms in living spaces, especially those in the air, can impact health and well-being, little is known of their identity and the processes that determine their assembly. We investigated the source-sink relationships of airborne bacteria in 29 homes in the San Francisco Bay Area. Samples taken in the sites expected to be source habitats for indoor air microbes were analyzed by 16S rRNA-based pyrosequencing and quantitative PCR. The community composition was related to the characteristics of the household collected at the time of sampling, including the number of residents and pets, activity levels, frequency of cooking and vacuum cleaning, extent of natural ventilation, and abundance and type of vegetation surrounding the building., Results: Indoor air harbored a diverse bacterial community dominated by Diaphorobacter sp., Propionibacterium sp., Sphingomonas sp., and Alicyclobacillus sp. Source-sink analysis suggested that outdoor air was the primary source of indoor air microbes in most homes. Bacterial phylogenetic diversity and relative abundance in indoor air did not differ statistically from that in outdoor air. Moreover, the abundance of bacteria in outdoor air was positively correlated with that in indoor air, as would be expected if outdoor air was the main contributor to the bacterial community in indoor bioaerosols. The number of residents, presence of pets, and local tap water also influenced the diversity and size of indoor air microbes. The bacterial load in air increased with the number of residents, activity, and frequency of natural ventilation, and the proportion of bacteria putatively derived from skin increased with the number of residents. Vacuum cleaning increased the signature of pet- and floor-derived bacteria in indoor air, while the frequency of natural ventilation decreased the relative abundance of tap water-derived microorganisms in air., Conclusions: Indoor air in residences harbors a diverse bacterial community originating from both outdoor and indoor sources and is strongly influenced by household characteristics.

Human occupants are an important source of microbes in indoor environments. In this study, we used DNA sequencing of filter samples to assess the fungal and bacterial composition of air in an environmental chamber under different levels of occupancy, activity, and exposed or covered carpeting. In this office-like, mechanically ventilated environment, results showed a strong influence of outdoor-derived particles, with the indoor microbial composition tracking that of outdoor air for the 2-hour sampling periods. The number of occupants and their activity played a significant but smaller role influencing the composition of indoor bioaerosols. Human-associated taxa were observed but were not particularly abundant, except in the case of one fungus that appeared to be transported into the chamber on the clothing of a study participant. Overall, this study revealed a smaller signature of human body-associated taxa than had been expected based on recent studies of indoor microbiomes, suggesting that occupants may not exert a strong influence on bioaerosol microbial composition in a space that, like many offices, is well ventilated with air that is moderately filtered and moderately occupied.