RESUMO
R-tailocins are high-molecular-weight bacteriocins resembling bacteriophage tails. Pseudomonas chlororaphis 30-84 is a plant growth-promoting rhizobacterial (PGPR) strain that produces two distinct R-tailocin particles with different killing spectra. The two R-tailocins have different evolutionary histories but are released by the same lysis cassette. A previous study showed that both tailocins are important for pairwise competition with susceptible rhizosphere-colonizing strains; however, the broader role of tailocins in competition with the native rhizosphere microbiome was not tested. Genomic analysis of the P. chlororaphis 30-84 R-tailocin gene cluster uncovered the presence of three tail fiber genes in the tailocin 2 genetic module that could potentially result in tailocin 2 particles having different tail fibers and thus a wider killing spectrum. In this study, the tail fibers were found to incorporate onto different tailocin 2 particles, each with a distinct killing spectrum. A loss of production of one or both tailocins resulted in decreased P. chlororaphis 30-84 persistence within the wheat rhizosphere when in competition with the native microflora but not bulk soil. The capacity to produce three different versions of a single tailocin, each having one of three different types of tail fibers, is a previously unreported mechanism that leads to a broader R-tailocin killing spectrum. This study also provides evidence for the function of R-tailocins in competition with rhizosphere microbiome communities but not in bulk soil.IMPORTANCE Although R-tailocin gene clusters typically encode one tail fiber protein, three tail fiber-resembling genes were identified in association with one of the two sets of R-tailocin genes within the tailocin cluster of P. chlororaphis 30-84 and other sequenced P. chlororaphis strain genomes. This study confirmed that P. chlororaphis 30-84 not only produces two distinct tailocins, but that one of them is produced with three different types of tail fibers. This is a previously unreported strategy to increase the breadth of strains targeted by an R-tailocin. Our finding that R-tailocins produced by a PGPR Pseudomonas strain enhanced its persistence within the wheat rhizosphere microbiome confirms that R-tailocin production contributes to the population dynamics of rhizobacterial communities.
Assuntos
Antibiose , Bacteriocinas/genética , Pseudomonas chlororaphis/genética , Rizosfera , Bacteriocinas/metabolismo , Família Multigênica , Pseudomonas chlororaphis/metabolismoRESUMO
Many products of secondary metabolism are activated by quorum sensing (QS), yet even at cell densities sufficient for QS, their production may be repressed under suboptimal growth conditions via mechanisms that still require elucidation. For many beneficial plant-associated bacteria, secondary metabolites such as phenazines are important for their competitive survival and plant-protective activities. Previous work established that phenazine biosynthesis in Pseudomonas chlororaphis 30-84 is regulated by the PhzR/PhzI QS system, which in turn is regulated by transcriptional regulator Pip, two-component system RpeA/RpeB and stationary phase/stress sigma factor RpoS. Disruption of MiaA, a tRNA modification enzyme, altered primary metabolism and growth leading to widespread effects on secondary metabolism, including reduced phenazine production and oxidative stress tolerance. Thus, the miaA mutant provided the opportunity to examine the regulation of phenazine production in response to altered metabolism and growth or stress tolerance. Despite the importance of MiaA for translation efficiency, the most significant effect of miaA disruption on phenazine production was the reduction in the transcription of phzR, phzI and pip, whereas neither the transcription nor translation of RpeB, a transcriptional regulator of pip, was affected. Constitutive expression of rpeB or pip in the miaA mutant completely restored phenazine production, but it resulted in further growth impairment. Constitutive expression of RpoS alleviated sensitivity to oxidative stress resulting from RpoS translation inefficiency in the miaA mutant, but it did not restore phenazine production. Our results support the model that cells curtail phenazine biosynthesis under suboptimal growth conditions via RpeB/Pip-mediated regulation of QS.
Assuntos
Alquil e Aril Transferases/genética , Regulação Bacteriana da Expressão Gênica/genética , Estresse Oxidativo/fisiologia , Fenazinas/metabolismo , Pseudomonas chlororaphis/crescimento & desenvolvimento , Percepção de Quorum/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Peptídeo Sintases/genética , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo , Percepção de Quorum/fisiologia , Fator sigma/biossíntese , Fator sigma/genética , Transativadores/genética , Transcrição Gênica/genéticaRESUMO
R-type tailocins are high-molecular-weight bacteriocins that resemble bacteriophage tails and are encoded within the genomes of many Pseudomonas species. In this study, analysis of the P. chlororaphis 30-84 R-tailocin gene cluster revealed that it contains the structural components to produce two R-tailocins of different ancestral origins. Two distinct R-tailocin populations differing in length were observed in UV-induced lysates of P. chlororaphis 30-84 via transmission electron microscopy. Mutants defective in the production of one or both R-tailocins demonstrated that the killing spectrum of each tailocin is limited to Pseudomonas species. The spectra of pseudomonads killed by the two R-tailocins differed, although a few Pseudomonas species were either killed by or insusceptible to both tailocins. Tailocin release was disrupted by deletion of the holin gene within the tailocin gene cluster, demonstrating that the lysis cassette is required for the release of both R-tailocins. The loss of functional tailocin production reduced the ability of P. chlororaphis 30-84 to compete with an R-tailocin-sensitive strain within biofilms and rhizosphere communities. Our study demonstrates that Pseudomonas species can produce more than one functional R-tailocin particle sharing the same lysis cassette but differing in their killing spectra. This study provides evidence for the role of R-tailocins as determinants of bacterial competition among plant-associated Pseudomonas in biofilms and the rhizosphere.IMPORTANCE Recent studies have identified R-tailocin gene clusters potentially encoding more than one R-tailocin within the genomes of plant-associated Pseudomonas but have not demonstrated that more than one particle is produced or the ecological significance of the production of multiple R-tailocins. This study demonstrates for the first time that Pseudomonas strains can produce two distinct R-tailocins with different killing spectra, both of which contribute to bacterial competition between rhizosphere-associated bacteria. These results provide new insight into the previously uncharacterized role of R-tailocin production by plant-associated Pseudomonas species in bacterial population dynamics within surface-attached biofilms and on roots.
Assuntos
Bacteriocinas/metabolismo , Biofilmes , Raízes de Plantas/microbiologia , Pseudomonas chlororaphis/fisiologia , Antibiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Pseudomonas/fisiologia , Pseudomonas chlororaphis/genética , Rizosfera , Microbiologia do SoloRESUMO
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 genome's 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.
Assuntos
Adaptação Biológica , Biofilmes/crescimento & desenvolvimento , Genoma Bacteriano , Pseudomonas/fisiologia , Antibacterianos/metabolismo , Tolerância a Medicamentos , Perfilação da Expressão Gênica , Genômica , Locomoção , Dados de Sequência Molecular , Fenazinas/metabolismo , Raízes de Plantas/microbiologia , Pseudomonas/efeitos dos fármacos , Pseudomonas/crescimento & desenvolvimento , Pseudomonas/metabolismo , Análise de Sequência de DNA , Microbiologia do Solo , Triticum/microbiologiaRESUMO
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.
Assuntos
Genoma Bacteriano , Plantas , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo , Análise de Sequência de DNA , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Bacteriocinas/genética , Heterogeneidade Genética , Variação Genética , Interações Hospedeiro-Patógeno/genética , Insetos/genética , Família Multigênica , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Plantas/genética , Plantas/microbiologia , Sequências Repetitivas de Ácido Nucleico/genética , Resorcinóis/metabolismoRESUMO
BACKGROUND: The 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. RESULTS: In 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. CONCLUSION: Our 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.
Assuntos
Pseudomonas aeruginosa/genética , Percepção de Quorum/genética , Transcriptoma , Virulência/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Óperon , Pseudomonas aeruginosa/patogenicidade , Análise de Sequência de DNA , Transdução de SinaisRESUMO
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 (97 % 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 99 % 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.
Assuntos
Bactérias/classificação , Biodiversidade , Cavernas/microbiologia , Filogenia , Microbiologia do Solo , Arizona , Bactérias/genética , Bactérias/isolamento & purificação , DNA Bacteriano/genética , Biblioteca Gênica , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Fenazinas/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , Vias Biossintéticas/genética , Deleção de Genes , Perfilação da Expressão Gênica , Teste de Complementação Genética , Modelos Biológicos , Percepção de Quorum , Reação em Cadeia da Polimerase em Tempo Real , Transdução de SinaisRESUMO
Azotobacter vinelandii is a well-studied model system for nitrogen fixation in bacteria. Regulation of nitrogen fixation in A. vinelandii is independent of NtrB/NtrC, a conserved nitrogen regulatory system in proteobacteria. Previous work showed that an ntrC mutation in A. vinelandii resulted in a loss of induction of assimilatory nitrate and nitrite reductases encoded by the nasAB operon. In addition to NtrC, several other proteins, including NasT, a protein containing a potential RNA-binding domain ANTAR (AmiR and NasR transcription antitermination regulators), have been implicated in nasAB regulation. In this work, we characterize the sequence upstream of nasA and identify several DNA sequence elements, including two potential NtrC binding sites and a putative intrinsic transcriptional terminator upstream of nasA that are potentially involved in nasAB regulation. Our analyses confirm that the nasAB promoter, P(nasA), is under NtrC control. However, unlike NtrC-regulated promoters in enteric bacteria, P(nasA) shows high activity in the presence of ammonium; in addition, the P(nasA) activity is altered in the nifA gene mutation background. We discuss the implication of these results on NtrC-mediated regulation in A. vinelandii. Our study provides direct evidence that induction of nasAB is regulated by NasT-mediated antitermination, which occurs within the leader region of the operon. The results also support the hypothesis that NasT binds the promoter proximal hairpin of nasAB for its regulatory function, which contributes to the understanding of the regulatory mechanism of ANTAR-containing antiterminators.
Assuntos
Azotobacter vinelandii/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Nitrato Redutase/biossíntese , Óperon , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Sítios de Ligação , Nitrato Redutase/genética , Regiões Promotoras GenéticasRESUMO
Rhizosphere colonizing plant growth promoting bacteria (PGPB) increase their competitiveness by producing diffusible toxic secondary metabolites, which inhibit competitors and deter predators. Many PGPB also have one or more Type VI Secretion System (T6SS), for the delivery of weapons directly into prokaryotic and eukaryotic cells. Studied predominantly in human and plant pathogens as a virulence mechanism for the delivery of effector proteins, the function of T6SS for PGPB in the rhizosphere niche is poorly understood. We utilized a collection of Pseudomonas chlororaphis 30-84 mutants deficient in one or both of its two T6SS and/or secondary metabolite production to examine the relative importance of each T6SS in rhizosphere competence, bacterial competition, and protection from bacterivores. A mutant deficient in both T6SS was less persistent than wild type in the rhizosphere. Both T6SS contributed to competitiveness against other PGPB or plant pathogenic strains not affected by secondary metabolite production, but only T6SS-2 was effective against strains lacking their own T6SS. Having at least one T6SS was also essential for protection from predation by several eukaryotic bacterivores. In contrast to diffusible weapons that may not be produced at low cell density, T6SS afford rhizobacteria an additional, more immediate line of defense against competitors and predators.
RESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Genes Reguladores , Mutação , Pseudomonas/crescimento & desenvolvimento , Pseudomonas/genética , Fatores de Transcrição/genética , Animais , Biofilmes/crescimento & desenvolvimento , Variação Genética , Fenazinas/metabolismo , Seleção GenéticaRESUMO
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.
Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Fenômenos Fisiológicos Bacterianos , Plantas/microbiologia , Simbiose , Acetoína/metabolismo , Ácidos Indolacéticos/metabolismo , Fenazinas/metabolismo , Plantas/imunologia , Plantas/metabolismoRESUMO
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.
Assuntos
Bactérias/metabolismo , Fenazinas/metabolismo , Animais , Bactérias/genética , Bactérias/patogenicidade , Aderência Bacteriana , Infecções Bacterianas/microbiologia , Biofilmes/crescimento & desenvolvimento , Biotecnologia , Ecossistema , Transporte de Elétrons , Humanos , Ferro/metabolismo , Interações Microbianas , Oxirredução , Fenazinas/farmacologia , Plantas/metabolismo , Plantas/microbiologia , Transdução de Sinais/efeitos dos fármacos , Microbiologia do SoloRESUMO
LuxR solos are common in plant-associated bacteria and increasingly recognized for playing important roles in plant-microbe interkingdom signaling. Unlike the LuxR-type transcriptional regulators of prototype LuxR/LuxI quorum sensing systems, luxR solos do not have a LuxI-type autoinducer synthase gene associated with them. LuxR solos in plant-pathogenic bacteria are important for virulence and in plant endosymbionts contribute to symbiosis. In the present study, we characterized an atypical LuxR solo, PcsR2, in the biological control species Pseudomonas chlororaphis 30-84 that is highly conserved among sequenced P. chlororaphis strains. Unlike most LuxR solos in the plant-associated bacteria characterized to date, pcsR2 is not associated with a proline iminopeptidase gene and the protein has an atypical N-terminal binding domain. We created a pcsR2 deletion mutant and used quantitative RT-PCR to show that the expression of pcsR2 and genes in the operon immediately downstream was upregulated â¼10-fold when the wild type strain was grown on wheat roots compared to planktonic culture. PcsR2 was involved in upregulation. Using a GFP transcriptional reporter, we found that expression of pcsR2 responded specifically to root-derived substrates as compared to leaf-derived substrates but not to endogenous AHLs. Compared to the wild type, the mutant was impaired in the ability to utilize root carbon and nitrogen sources in wheat root macerate and to colonize wheat roots. Phenazine production and most biofilm traits previously shown to be correlated with phenazine production also were diminished in the mutant. Gene expression of several of the proteins in the phenazine regulatory network including PhzR, Pip (phenazine inducing protein) and RpeA/RpeB were reduced in the mutant, and overexpression of these genes in trans restored phenazine production in the mutant to wild-type levels, indicating PcsR2 affects the activity of the these regulatory genes upstream of RpeA/RpeB via an undetermined mechanism. Our results indicate PcsR2 upregulates the expression of the adjacent operon in response to unknown wheat root-derived signals and belongs to a novel subfamily of LuxR-type transcriptional regulators found in sequenced P. chlororaphis strains.
RESUMO
Application of plant growth promoting bacteria may induce plant salt stress tolerance, however the underpinning microbial and plant mechanisms remain poorly understood. In the present study, the specific role of phenazine production by rhizosphere-colonizing Pseudomonas in mediating the inhibitory effects of salinity on wheat seed germination and seedling growth in four different varieties was investigated using Pseudomonas chlororaphis 30-84 (wild type) and isogenic derivatives deficient or enhanced in phenazine production. The results showed that varieties differed in how they responded to the salt stress treatment and the benefits derived from colonization by P. chlororaphis 30-84. In all varieties, the salt stress treatment significantly reduced seed germination, and in seedlings, reduced relative water content, increased reactive oxygen species (ROS) levels in leaves, and in three of four varieties, reduced shoot and root production compared to the no salt stress treatment. Inoculation of seeds with Pseudomonas chlororaphis 30-84 wild type or derivatives promoted salt-stress tolerance in seedlings of the four commercial winter wheat varieties tested, but the salt-stress tolerance phenotype was not entirely due to phenazine production. For example, all P. chlororaphis derivatives (including the phenazine-producing mutant) significantly improved relative water content in two varieties, Iba and CV 1, for which the salt stress treatment had a large impact. Importantly, all P. chlororaphis derivatives enabled the salt inhibited wheat varieties studied to maintain above ground productivity in saline conditions. However, only phenazine-producing derivatives enhanced the shoot or root growth of seedlings of all varieties under nonsaline conditions. Notably, ROS accumulation was reduced, and antioxidant enzyme (catalase) activity enhanced in the leaves of seedlings grown in saline conditions that were seed-treated with phenazine-producing P. chlororaphis derivatives as compared to noninoculated seedlings. The results demonstrate the capacity of P. chlororaphis to improve salt tolerance in wheat seedlings by promoting plant growth and reducing osmotic stress and a role for bacterial phenazine production in reducing redox stress.
RESUMO
The specific role of phenazines produced by rhizosphere-colonizing Pseudomonas in mediating wheat seedling drought-stress tolerance and recovery from water deficit was investigated using Pseudomonas chlororaphis 30-84 and isogenic derivatives deficient or enhanced in phenazine production compared to wild type. Following a 7-day water deficit, seedlings that received no-inoculum or were colonized by the phenazine mutant wilted to collapse, whereas seedlings colonized by phenazine producers displayed less severe symptoms. After a 7-day recovery period, survival of seedlings colonized by phenazine-producing strains exceeded 80%, but was less than 60% for no-inoculum controls. A second 7-day water deficit reduced overall survival rates to less than 10% for no-inoculum control seedlings, whereas survival was â¼50% for seedlings colonized by phenazine-producers. The relative water content of seedlings colonized by phenazine-producers was 10-20% greater than for the no-inoculum controls at every stage of water deficit and recovery, resulting in higher recovery indices than observed for the no-inoculum controls. For 10-day water deficits causing the collapse of all seedlings, survival rates remained high for plants colonized by phenazine-producers, especially the enhanced phenazine producer (â¼74%), relative to the no-inoculum control (â¼25%). These observations indicate that seedlings colonized by the phenazine-producing strains suffered less from dehydration during water deficit and recovered better, potentially contributing to better resilience from a second drought/recovery cycle. Seedlings colonized by phenazine-producing strains invested more in root systems and produced 1.5 to 2 fold more root tips than seedlings colonized by the phenazine mutant or the no-inoculum controls when grown with or without water deficit. The results suggest that the presence of phenazine-producing bacteria in the rhizosphere provides wheat seedlings with a longer adjustment period resulting in greater drought-stress avoidance and resilience.
RESUMO
Disease caused by the bacterial pathogen "Candidatus Liberibacter solanacearum" (Lso) represents a serious threat to solanaceous crop production. Insecticide applications to control the psyllid vector, Bactericera cockerelli Sulc (Hemiptera: Triozidae) has led to the emergence of resistance in psyllids populations. Efforts to select natural resistant cultivars have been marginally successful and have been complicated by the presence of distinct Lso haplotypes (LsoA, LsoB) differing in symptoms severity on potato and tomato. A potentially promising management tool is to boost host resistance to the pathogen and/or the insect vector by promoting mycorrhization. Here we tested the hypothesis that mycorrhizal fungi can mitigate the effect of Lso infection on tomato plants. The presence of mycorrhizal fungi substantially delayed and reduced the incidence of Lso-induced symptoms on tomato as compared to non-mycorrhized plants. However, PCR with specific Lso primers revealed that mycorrhization did not prevent Lso transmission or translocation to newly formed leaves. Mycorrhization significantly reduced oviposition by psyllids harboring LsoA and survival of nymphs from these eggs. However, mycorrhization had no effect on oviposition by psyllids harboring LsoB or the survival of nymphs from parents harboring LsoB. These findings indicate the use of mycorrhizal fungi is a promising strategy for the mitigation of disease caused by both LsoA and LsoB and warrants additional field testing.
RESUMO
Pseudomonas fluorescens Pf-5 is a plant commensal bacterium that inhabits the rhizosphere and produces secondary metabolites that suppress soilborne plant pathogens. The complete sequence of the 7.1-Mb Pf-5 genome was determined. We analyzed repeat sequences to identify genomic islands that, together with other approaches, suggested P. fluorescens Pf-5's recent lateral acquisitions include six secondary metabolite gene clusters, seven phage regions and a mobile genomic island. We identified various features that contribute to its commensal lifestyle on plants, including broad catabolic and transport capabilities for utilizing plant-derived compounds, the apparent ability to use a diversity of iron siderophores, detoxification systems to protect from oxidative stress, and the lack of a type III secretion system and toxins found in related pathogens. In addition to six known secondary metabolites produced by P. fluorescens Pf-5, three novel secondary metabolite biosynthesis gene clusters were also identified that may contribute to the biocontrol properties of P. fluorescens Pf-5.
Assuntos
Genoma Bacteriano , Pseudomonas fluorescens/genética , Sequência de Bases , Transporte Biológico/genética , Genes Bacterianos , Dados de Sequência Molecular , Família Multigênica , Plantas/microbiologia , Pseudomonas fluorescens/metabolismo , Análise de Sequência de DNA , Sideróforos/biossíntese , Sideróforos/genéticaRESUMO
Pseudomonas chlororaphis 30-84 is a biological control agent selected for its ability to suppress diseases caused by fungal pathogens. P. chlororaphis 30-84 produces three phenazines: phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine-1-carboxylic acid (2OHPCA) and a small amount of 2-hydroxy-phenazine (2OHPHZ), and these are required for fungal pathogen inhibition and wheat rhizosphere competence. The two, 2-hydroxy derivatives are produced from PCA via the activity of a phenazine-modifying enzyme encoded by phzO. In addition to the seven biosynthetic genes responsible for the production of PCA, many other Pseudomonas strains possess one or more modifying genes, which encode enzymes that act independently or together to convert PCA into other phenazine derivatives. In order to understand the fitness effects of producing different phenazines, we constructed isogenic derivatives of P. chlororaphis 30-84 that differed only in the type of phenazines produced. Altering the type of phenazines produced by P. chlororaphis 30-84 enhanced the spectrum of fungal pathogens inhibited and altered the degree of take-all disease suppression. These strains also differed in their ability to promote extracellular DNA release, which may contribute to the observed differences in the amount of biofilm produced. All derivatives were equally important for survival over repeated plant/harvest cycles, indicating that the type of phenazines produced is less important for persistence in the wheat rhizosphere than whether or not cells produce phenazines. These findings provide a better understanding of the effects of different phenazines on functions important for biological control activity with implications for applications that rely on introduced or native phenazine producing populations.
RESUMO
Phenazines are bacterial secondary metabolites and play important roles in the antagonistic activity of the biological control strain P. chlororaphis 30-84 against take-all disease of wheat. The expression of the P. chlororaphis 30-84 phenazine biosynthetic operon (phzXYFABCD) is dependent on the PhzR/PhzI quorum sensing system located immediately upstream of the biosynthetic operon as well as other regulatory systems including Gac/Rsm. Bioinformatic analysis of the sequence between the divergently oriented phzR and phzX promoters identified features within the 5'-untranslated region (5'-UTR) of phzX that are conserved only among 2OHPCA producing Pseudomonas. The conserved sequence features are potentially capable of producing secondary structures that negatively modulate one or both promoters. Transcriptional and translational fusion assays revealed that deletion of 90-bp of sequence at the 5'-UTR of phzX led to up to 4-fold greater expression of the reporters with the deletion compared to the controls, which indicated this sequence negatively modulates phenazine gene expression both transcriptionally and translationally. This 90-bp sequence was deleted from the P. chlororaphis 30-84 chromosome, resulting in 30-84Enh, which produces significantly more phenazine than the wild-type while retaining quorum sensing control. The transcriptional expression of phzR/phzI and amount of AHL signal produced by 30-84Enh also were significantly greater than for the wild-type, suggesting this 90-bp sequence also negatively affects expression of the quorum sensing genes. In addition, deletion of the 90-bp partially relieved RsmE-mediated translational repression, indicating a role for Gac/RsmE interaction. Compared to the wild-type, enhanced phenazine production by 30-84Enh resulted in improvement in fungal inhibition, biofilm formation, extracellular DNA release and suppression of take-all disease of wheat in soil without negative consequences on growth or rhizosphere persistence. This work provides greater insight into the regulation of phenazine biosynthesis with potential applications for improved biological control.