Leland S. Pierson

Comparative Genomics of Plant-Associated Pseudomonas spp.: Insights into Diversity and Inheritance of Traits Involved in Multitrophic Interactions

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45–52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire.

Metabolism and function of phenazines in bacteria: impacts on the behavior of bacteria in the environment and biotechnological processes

Phenazines constitute a large group of nitrogen-containing heterocyclic compounds produced by a diverse range of bacteria. Both natural and synthetic phenazine derivatives are studied due their impacts on bacterial interactions and biotechnological processes. Phenazines serve as electron shuttles to alternate terminal acceptors, modify cellular redox states, act as cell signals that regulate patterns of gene expression, contribute to biofilm formation and architecture, and enhance bacterial survival. Phenazines have diverse effects on eukaryotic hosts and host tissues, including the modification of multiple host cellular responses. In plants, phenazines also may influence growth and elicit induced systemic resistance. Here, we discuss emerging evidence that phenazines play multiple roles for the producing organism and contribute to their behavior and ecological fitness.

The Multifactorial Basis for Plant Health Promotion by Plant-Associated Bacteria

ABSTRACT On plants, microbial populations interact with each other and their host through the actions of secreted metabolites. However, the combined action of diverse organisms and their different metabolites on plant health has yet to be fully appreciated. Here, the multifactorial nature of these interactions, at the organismal and molecular level, leading to the biological control of plant diseases is reviewed. To do so, we describe in detail the ecological significance of three different classes of secondary metabolites and discuss how they might contribute to biological control. Specifically, the roles of auxin, acetoin, and phenazines are considered, because they represent very different but important types of secondary metabolites. We also describe how studies of the global regulation of bacterial secondary metabolism have led to the discovery of new genes and phenotypes related to plant health promotion. In conclusion, we describe three avenues for future research that will help to integrate these complex and diverse observations into a more coherent synthesis of bacterially mediated biocontrol of plant diseases.

Roles of the Gac-Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30-84

The GacS/GacA two‐component regulatory system activates the production of secondary metabolites including phenazines crucial for biological control activity in Pseudomonas chlororaphis 30‐84. To better understand the role of the Gac system on phenazine regulation, transcriptomic analyses were conducted by comparing the wild‐type strain to a gacA mutant. RNA‐seq analysis identified 771 genes under GacA control, including many novel genes. Consistent with previous findings, phenazine biosynthetic genes were significantly downregulated in a gacA mutant. The transcript abundances of phenazine regulatory genes such as phzI, phzR, iopA, iopB, rpoS, and pip also were reduced. Moreover, the transcript abundance of three noncoding RNAs (ncRNAs) including rsmX, rsmY, and rsmZ was significantly decreased by gacA mutation consistent with the presence of consensus GacA‐binding sites associated with their promoters. Our results also demonstrated that constitutive expression of rsmZ from a non‐gac regulated promoter resulted in complete restoration of N‐acyl‐homoserine lactone (AHL) and phenazine production as well as the expression of other gac‐dependent secondary metabolites in gac mutants. The role of RsmA and RsmE in phenazine production also was investigated. Overexpression of rsmE, but not rsmA, resulted in decreased AHL and phenazine production in P. chlororaphis, and only a mutation in rsmE bypassed the requirement for GacA in phenazine gene expression. In contrast, constitutive expression of the phzI/phzR quorum sensing system did not rescue phenazine production in the gacA mutant, indicating the direct posttranscriptional control by Gac on the phenazine biosynthetic genes. On the basis of these results, we propose a model to illustrate the hierarchic role of phenazine regulators modulated by Gac in the control of phenazine production. The transcriptomic analysis also was used to identify additional genes regulated by GacA that may contribute to the biological control capability of strain 30‐84.

Profiling Bacterial Diversity and Taxonomic Composition on Speleothem Surfaces in Kartchner Caverns, AZ

Caves are relatively accessible subterranean habitats ideal for the study of subsurface microbial dynamics and metabolisms under oligotrophic, non-photosynthetic conditions. A 454-pyrotag analysis of the V6 region of the 16S rRNA gene was used to systematically evaluate the bacterial diversity of ten cave surfaces within Kartchner Caverns, a limestone cave. Results showed an average of 1,994 operational taxonomic units (97xa0% cutoff) per speleothem and a broad taxonomic diversity that included 21 phyla and 12 candidate phyla. Comparative analysis of speleothems within a single room of the cave revealed three distinct bacterial taxonomic profiles dominated by either Actinobacteria, Proteobacteria, or Acidobacteria. A gradient in observed species richness along the sampling transect revealed that the communities with lower diversity corresponded to those dominated by Actinobacteria while the more diverse communities were those dominated by Proteobacteria. A 16S rRNA gene clone library from one of the Actinobacteria-dominated speleothems identified clones with 99xa0% identity to chemoautotrophs and previously characterized oligotrophs, providing insights into potential energy dynamics supporting these communities. The robust analysis conducted for this study demonstrated a rich bacterial diversity on speleothem surfaces. Further, it was shown that seemingly comparable speleothems supported divergent phylogenetic profiles suggesting that these communities are very sensitive to subtle variations in nutritional inputs and environmental factors typifying speleothem surfaces in Kartchner Caverns.

Spontaneous Gac Mutants of Pseudomonas Biological Control Strains: Cheaters or Mutualists?

ABSTRACT Bacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit plant or animal hosts. The GacS/GacA two-component regulatory system positively controls the expression of many of these beneficial external products in pseudomonad bacteria. Natural populations often contain variants with defective Gac systems that do not produce most external products. These mutants benefit from a decreased metabolic load but do not appear to displace the wild type in nature. How could natural selection maintain the wild type in the presence of a mutant with enhanced growth? One hypothesis is that Gac mutants are “cheaters” that do not contribute to the public good, favored within groups but selected against between groups, as groups containing more mutants lose access to ecologically important external products. An alternative hypothesis is that Gac mutants have a mutualistic interaction with the wild type, so that each variant benefits by the presence of the other. In the biocontrol bacterium Pseudomonas chlororaphis strain 30-84, Gac mutants do not produce phenazines, which suppress competitor growth and are critical for biofilm formation. Here, we test the predictions of these alternative hypotheses by quantifying interactions between the wild type and the phenazine- and biofilm-deficient Gac mutant within growing biofilms. We find evidence that the wild type and Gac mutants interact mutualistically in the biofilm context, whereas a phenazine-defective structural mutant does not. Our results suggest that the persistence of alternative Gac phenotypes may be due to the stabilizing role of local mutualistic interactions.

Transcriptome profiling reveals links between ParS/ParR, MexEF-OprN, and quorum sensing in the regulation of adaptation and virulence in Pseudomonas aeruginosa

BackgroundThe ParS/ParR two component regulatory system plays critical roles for multidrug resistance in Pseudomonas aeruginosa. It was demonstrated that in the presence of antimicrobials, ParR enhances bacterial survival by distinct mechanisms including activation of the mexXY efflux genes, enhancement of lipopolysaccharide modification through the arn operon, and reduction of the expression of oprD porin.ResultsIn this study, we report on transcriptomic analyses of P. aeruginosa PAO1 wild type and parS and parR mutants growing in a defined minimal medium. Our transcriptomic analysis provides the first estimates of transcript abundance for the 5570 coding genes in P. aeruginosa PAO1. Comparative transcriptomics of P. aeruginosa PAO1 and par mutants identified a total of 464 genes regulated by ParS and ParR. Results also showed that mutations in the parS/parR system abolished expression of the mexEF-oprN operon by down-regulating the regulatory gene mexS. In addition to the known effects on drug resistance genes, transcript abundances of the quorum sensing genes (rhlIR and pqsABCDE-phnAB) were higher in both parS and parR mutants. In accordance with these results, a significant portion of the ParS/ParR regulated genes belonged to the MexEF-OprN and quorum sensing regulons. Deletion of the par genes also led to increased phenazine production and swarming motility, consistent with the up-regulation of the phenazine and rhamnolipid biosynthetic genes, respectively.ConclusionOur results link the ParS/ParR two component signal transduction system to MexEF-OprN and quorum sensing systems in P. aeruginosa. These results expand our understanding of the roles of the ParS/ParR system in the regulation of gene expression in P. aeruginosa, especially in the absence of antimicrobials.

Adaptation Genomics of a Small-Colony Variant in a Pseudomonas chlororaphis 30-84 Biofilm

ABSTRACT The rhizosphere-colonizing bacterium Pseudomonas chlororaphis 30-84 is an effective biological control agent against take-all disease of wheat. In this study, we characterize a small-colony variant (SCV) isolated from a P. chlororaphis 30-84 biofilm. The SCV exhibited pleiotropic phenotypes, including small cell size, slow growth and motility, low levels of phenazine production, and increased biofilm formation and resistance to antimicrobials. To better understand the genetic alterations underlying these phenotypes, RNA and whole-genome sequencing analyses were conducted comparing an SCV to the wild-type strain. Of the genomes 5,971 genes, transcriptomic profiling indicated that 1,098 (18.4%) have undergone substantial reprograming of gene expression in the SCV. Whole-genome sequence analysis revealed multiple alterations in the SCV, including mutations in yfiR (cyclic-di-GMP production), fusA (elongation factor), and cyoE (heme synthesis) and a 70-kb deletion. Genetic analysis revealed that the yfiR locus plays a major role in controlling SCV phenotypes, including colony size, growth, motility, and biofilm formation. Moreover, a point mutation in the fusA gene contributed to kanamycin resistance. Interestingly, the SCV can partially switch back to wild-type morphologies under specific conditions. Our data also support the idea that phenotypic switching in P. chlororaphis is not due to simple genetic reversions but may involve multiple secondary mutations. The emergence of these highly adherent and antibiotic-resistant SCVs within the biofilm might play key roles in P. chlororaphis natural persistence.

Differential regulation of phenazine biosynthesis by RpeA and RpeB in Pseudomonas chlororaphis 30-84

RpeA is a two-component sensor protein that negatively controls biosynthesis of phenazines, which are required for biological control activity by Pseudomonas chlororaphis 30-84. In this study, we identified the cognate response regulator RpeB and investigated how RpeA and RpeB interact with the PhzR/PhzI quorum sensing system and other known regulatory genes to control phenazine production. Quantitative real-time PCR revealed that, in contrast with an rpeA mutant, expression of the phenazine biosynthetic genes as well as the pip and phzR genes were significantly reduced in an rpeB mutant, suggesting positive control of phenazines by RpeB. Complementation assays showed that overexpression of pip in trans rescued phenazine production in an rpeB mutant, whereas multiple copies of rpeB genes were unable to restore phenazine production in a pip or phzR mutant. These results indicate that RpeA and RpeB differentially regulate phenazine production and act upstream of Pip and PhzR in the phenazine regulatory network. The differential regulatory functions for RpeA and RpeB also affected the capacity of 30-84 for fungal inhibition. Based on these results, a model is proposed to illustrate the relationship of RpeA/RpeB to other regulatory genes controlling phenazine biosynthesis in P. chlororaphis 30-84, a regulatory hierarchy that may be conserved in other pseudomonads and may play a role in stress response.

Factors Influencing Observed Variations in the Structure of Bacterial Communities On Calcite Formations in Kartchner Caverns, AZ, USA

Kartchner Caverns is an oligotrophic subterranean environment that hosts a wide diversity of actively growing calcite speleothems (secondary mineral deposits). In a previous study, we demonstrated that bacterial communities extracted from these surfaces are quite complex and vary between formations. In the current study, we evaluated the influence of several environmental variables on the superficial bacterial community structure of 10 active formations located in close proximity to one another in a small room of Kartchner Caverns State Park, Arizona, USA. Physical (color, dimensions) and chemical (elemental profile and organic carbon concentration) properties, as well as the DGGE-based bacterial community structure of the formations were analyzed. While elemental concentration was found to vary among the formations, the differences in the community structure could not be correlated with concentrations of either organic carbon or any of the elements evaluated. In contrast, the locations of formations within a distinct region of the cave as well as the relative location of specific formations within a single room were found to have a significant influence on the bacterial community structure of the formations evaluated. Interestingly, Canonical Correspondence Analysis suggests an association between the observed drip pathways (drip lines) feeding the formations (as determined by the patterns of soda straws and small stalactites that reveal water flow patterns) and the bacterial community structure of the respective formations. The results presented here indicate that a broad range of formations fed by a diversity of drip sources must be sampled to fully characterize the community composition of bacteria present on the surfaces of calcite formations in carbonate caves.