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Interactions between commensal microbes and invading pathogens are understudied, despite their likely effects on pathogen population structure and infection processes. We describe the population structure and genetic diversity of a broad range of co-occurring Pseudomonas syringae isolated from infected and uninfected kiwifruit during an outbreak of bleeding canker disease caused by P. syringae pv. actinidiae (Psa) in New Zealand. Overall population structure was clonal and affected by ecological factors including infection status and cultivar. Most isolates are members of a new clade in phylogroup 3 (PG3a), also present on kiwifruit leaves in China and Japan. Stability of the polymorphism between pathogenic Psa and commensal P. syringae PG3a isolated from the same leaf was tested using reciprocal invasion from rare assays in vitro and in planta. P. syringae G33C (PG3a) inhibited Psa NZ54, while the presence of Psa NZ54 enhanced the growth of P. syringae G33C. This effect could not be attributed to virulence activity encoded by the Type 3 secretion system of Psa. Together our data contribute toward the development of an ecological perspective on the genetic structure of pathogen populations., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Horizontal gene transfer can precipitate rapid evolutionary change. In 2010 the global pandemic of kiwifruit canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) reached New Zealand. At the time of introduction, the single clone responsible for the outbreak was sensitive to copper, however, analysis of a sample of isolates taken in 2015 and 2016 showed that a quarter were copper resistant. Genome sequences of seven strains showed that copper resistance - comprising czc/cusABC and copABCD systems - along with resistance to arsenic and cadmium, was acquired via uptake of integrative conjugative elements (ICEs), but also plasmids. Comparative analysis showed ICEs to have a mosaic structure, with one being a tripartite arrangement of two different ICEs and a plasmid that were isolated in 1921 (USA), 1968 (NZ) and 1988 (Japan), from P. syringae pathogens of millet, wheat and kiwifruit respectively. Two of the Psa ICEs were nearly identical to two ICEs isolated from kiwifruit leaf colonists prior to the introduction of Psa into NZ. Additionally, we show ICE transfer in vitro and in planta, analyze fitness consequences of ICE carriage, capture the de novo formation of novel recombinant ICEs, and explore ICE host-range., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Food security depends on enhancing production and reducing loss to pests and pathogens. A promising alternative to agrochemicals is the use of plant growth-promoting rhizobacteria (PGPR), which are commonly associated with many, if not all, plant species. However, exploiting the benefits of PGPRs requires knowledge of bacterial function and an in-depth understanding of plant-bacteria associations. Motility is important for colonization efficiency and microbial fitness in the plant environment, but the mechanisms employed by bacteria on and around plants are not well understood. We describe and investigate an atypical mode of motility in Pseudomonas fluorescens SBW25 that was revealed only after flagellum production was eliminated by deletion of the master regulator fleQ. Our results suggest that this 'spidery spreading' is a type of surface motility. Transposon mutagenesis of SBW25ΔfleQ (SBW25Q) produced mutants, defective in viscosin production, and surface spreading was also abolished. Genetic analysis indicated growth-dependency, production of viscosin, and several potential regulatory and secretory systems involved in the spidery spreading phenotype. Moreover, viscosin both increases efficiency of surface spreading over the plant root and protects germinating seedlings in soil infected with the plant pathogen Pythium. Thus, viscosin could be a useful target for biotechnological development of plant growth promotion agents., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Pseudomonas proliferate in a wide spectrum of harsh and variable environments. In many of these environments, amino acids, such as histidine, are a valuable source of carbon, nitrogen and energy. Here, we demonstrate that the histidine uptake and utilization (hut) pathway of Pseudomonas aeruginosa PAO1 contains two branches from the intermediate formiminoglutamate to the product glutamate. Genetic analysis revealed that the four-step route is dispensable as long as the five-step route is present (and vice versa). Mutants with deletions of either the four-step (HutE) or five-step (HutFG) branches were competed against each other and the wild-type strain to test the hypothesis of ecological redundancy; that is, that the presence of two pathways confers no benefit beyond that delivered by the individual pathways. Fitness assays performed under several environmental conditions led us to reject this hypothesis; the four-step pathway can provide an advantage when histidine is the sole carbon source. An IclR-type regulator (HutR) was identified that regulates the four-step pathway. Comparison of sequenced genomes revealed that P.aeruginosa strains and P.fluorescens Pf-5 have branched hut pathways. Phylogenetic analyses suggests that the gene encoding formiminoglutamase (hutE) was acquired by horizontal gene transfer from a Ralstonia-like ancestor. Potential barriers to inter-species transfer of the hutRE module were explored by transferring it from P.aeruginosa PAO1 to P.fluorescens SBW25. Transfer of the operon conferred the ability to utilize histidine via the four-step pathway in a single step, but the fitness cost of acquiring this new operon was found to be environment dependent., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Phenotypic variation is a fundamental requirement for evolution by natural selection. While evidence of phenotypic variation in natural populations abounds, its genetic basis is rarely understood. Here we report variation in the ability of plant-colonizing Pseudomonas to utilize histidine, and its derivative, urocanate, as sole sources of carbon and nitrogen. From a population of 164 phyllosphere-colonizing Pseudomonas strains, 77% were able to utilize both histidine and urocanate (His(+) , Uro(+) ) as growth substrates, whereas the remainder could utilize histidine, but not urocanate (His(+) , Uro(-) ), or vice versa (His(-) , Uro(+) ). An in silico analysis of the hut locus, which determines capacity to utilize both histidine and urocanate, from genome-sequenced Pseudomonas strains, showed significant variation in the number of putative transporters. To identify transporter genes specific for histidine and urocanate, we focused on a single genotype of Pseudomonas fluorescens, strain SBW25, which is capable of utilizing both substrates. Site-directed mutagenesis, combined with [(3) H]histidine transport assays, shows that hutT(u) encodes a urocanate-specific transporter; hutT(h) encodes the major high-affinity histidine transporter; and hutXWV encodes an ABC-type transporter that plays a minor role in histidine uptake. Introduction of cloned copies of hutT(h) and hutT(u) from SBW25 into strains incapable of utilizing either histidine, or urocanate, complemented the defect, demonstrating a lack of functional transporters in these strains. Taken together our data show that variation in transport systems, and not in metabolic genes, explains a naturally occurring phenotypic polymorphism., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

CbrB is a global sigma(54)-dependent regulator required for nutrient acquisition in Pseudomonas. Located downstream of cbrB on the Pseudomonas fluorescens SBW25 chromosome is pcnB, a putative poly(A) polymerase gene. Presence of a sigma(54) promoter in the intergenic region of cbrB and pcnB led to the hypothesis that CbrB regulates pcnB expression in a sigma(54)-dependent manner. Here we show that transcription of pcnB is CbrB dependent. However, 5'-RACE analysis of the pcnB transcript using primers located in the pcnB coding region shows that transcription starts immediately upstream of the putative ATG site at a sigma(70)-like promoter. Deletion of pcnB caused approximately 80% decrease of ployadenylated 23S rRNA; growth of the pcnB mutant was compromised in a range of laboratory media and on sugar beet seedlings. Further 5'-RACE analysis confirmed the existence of the predicted sigma(54) promoter. Genetic analysis showed that the sigma(54) promoter drives expression of crcZ, a homologue of the recently described small RNA from Pseudomonas aeruginosa, in a CbrB-dependent manner. Taken together, our data show that both pcnB and crcZ are part of the CbrB regulon. Moreover, the data draw further attention to the central regulatory role of CbrB and provides a link between mRNA degradation and cellular catabolism.

Copper is an essential element for life, but too much copper is harmful: copper homeostasis must therefore be carefully regulated. When growing on plant surfaces, the plant growth-promoting bacterium Pseudomonas fluorescens SBW25 activates expression of a copper-transporting P1-type ATPase (CueA). Using a combination of transcriptional gene fusions and site-directed mutants, we show that copper-induced transcription of cueA is controlled by the MerR-type regulator, CueR; CueR is also required for activation of the copper chaperone protein encoded by cueZ (pflu0660). The promoters of cueA and cueZ are also responsive to the metal salts of gold, silver and mercury. In each case, CueR transduces the stimulus. Resistance to exogenously applied copper sulfate shows that cueA and cueR mutants are significantly less resistant than the wild type. This is consistent with the role of CueA as a copper efflux system and a general role for the CueR regulon in copper resistance. A search of the SBW25 genome for orthologues of genes predicted to play additional roles in copper homeostasis identified copCD of the known four-component copABCD system (unusually, copAB are absent from the SBW25 genome). Genetic studies showed that expression of copCD is controlled by copper and mediated by the CopRS two-component regulatory system. Mutants devoid of copCD or copS displayed an increased tolerance to copper and overexpression of copCD caused increased sensitivity. This is consistent with CopCD encoding a copper uptake system. Taken together, we suggest that the Cue and Cop systems are integral to copper homeostasis in P. fluorescens SBW25 with one (Cop) being active at low-copper environments and bringing copper into the cell, and the other (Cue) being active in high-copper environments and serving to export excess copper.

Saprophytic Pseudomonas are common root-colonizing bacteria that can improve plant health. Efficient exploitation of these bacteria in agriculture requires knowledge of traits that enhance ecological performance in the rhizosphere. Here, I describe the development and application of a promoter-trapping technology (IVET) that enables the isolation of Pseudomonas fluorescens genes that show elevated levels of expression in the rhizosphere. Using IVET, 20 P. fluorescens genes were identified that are induced during rhizosphere colonization, and their patterns of expression were analysed in laboratory media and in the rhizosphere. Fourteen genes showed significant homology to sequences in GenBank that are involved in nutrient acquisition, stress response, or secretion; six showed no homology. Seven of the rhizosphere-induced (rhi) genes have homology to known non-Pseudomonas genes. One of the rhi genes (hrcC) is a component of a type III secretion pathway, not previously known in non-parasitic bacteria. Together, these genes provide a view of the rhizosphere environment as perceived by a rhizosphere colonist, and suggest that the nature of the association between P. fluorescens and the plant root may be more complex and intimate than previously thought.