Characterization of plasmids from plant pathogenic pseudomonads

Physical characterization of the resident plasmids from Pseudomonas tabaci, P. angulata, and P. coronafaciens strains indicated that they harbored five different plasmid DNA species. Two ATCC strains of P. tabaci contained indistinguishable plasmids that we have named pJP1 and pJP2. An isolate of one of these strains contained a spontaneous variant of pJP1, pJP1¹, which contains an insertion of 3.9 Mdal. This 3.9-Mdal region did not hybridize to pJP1 indicating that the region was foreign DNA and not a duplication of a segment of DNA already present in pJP1. Another P. tabaci strain, PT27881, contained a third plasmid species, pJP27, which had few similarities to pJP1 or pJP2, but was indistinguishable from the plasmids from all three P. angulata strains. pJP27 and pJP1 had a small region, 8.8 Mdal, of sequence homology. The one strain of P. coronafaciens examined contained a plasmid, pJP50, which was different from the P. tabaci plasmids, but had the 8.8-Mdal region and additional regions of sequence homology with pJP1 and pJP27 as well as homology with a portion of the pJP1¹ insertion. A fourth strain of P. tabaci, PTBR-2, a pathogen on beans, contained plasmid pBW, the only plasmid that lacked detectable regions of homology with the other plasmids.     

The Competitiveness of Pseudomonas chlororaphis Carrying pJP4 Is Reduced in the Arabidopsis thaliana Rhizosphere

The effect of the large catabolic IncP plasmid pJP4 on the competitiveness of Pseudomonas chlororaphis SPR044 and on its derivatives SPR244 (GacS deficient), SPR344 (phenazine-1-carboxamide overproducer), and SPR644 (phenazine-1-carboxamide deficient) in the Arabidopsis thaliana rhizosphere was assessed. Solitary rhizosphere colonization by the wild type, SPR244, and SPR644 was not affected by the plasmid. The size of the population of SPR344 carrying pJP4, however, was significantly reduced compared to the size of the population of the plasmid-free derivative. The abiotic stress caused by phenazine-1-carboxamide overproduction probably resulted in a selective disadvantage for cells carrying pJP4. Next, the effect of biotic stress caused by coinoculation of other bacteria was analyzed. Cells carrying pJP4 had a selective disadvantage compared to plasmid-free cells in the presence of the efficient colonizer Pseudomonas fluorescens WCS417r. This effect was not observed after coinoculation with a variety of other bacteria, and it was independent of quorum sensing and phenazine-1-carboxamide production. Thus, the presence of large catabolic plasmids imposes a detectable metabolic burden in the presence of biotic stress. Plasmid transfer in the A. thaliana rhizosphere from P. chlororaphis and its derivatives to Ralstonia eutropha was determined by using culture-dependent and culture-independent techniques. With the cultivation-independent technique we detected a significantly higher portion of exconjugants, but pJP4 transfer was independent of the quorum-sensing system and of phenazine-1-carboxamide production.     

Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus

Biophysical and genetic properties of six independently isolated plasmids encoding the degradation of the herbicides 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid are described. Four of the plasmids, pJP3, pJP4, pJP5, and pJP7, had molecular masses of 51 megadaltons, belonged to the IncP1 incompatibility group, and transferred freely to strains of Escherichia coli, Rhodopseudomonas sphaeroides, Rhizobium sp., Agrobacterium tumefaciens, Pseudomonas putida, Pseudomonas fluorescens, and Acinetobacter calcoaceticus. In addition, these four plasmids conferred resistance to merbromin, phenylmercury acetate, and mercuric ions, had almost identical restriction endonuclease cleavage patterns, and encoded degradation of m-chlorobenzoate. The two other plasmids, pJP2 and pJP9, did not belong to the IncP1 incompatibility group, had molecular masses of 37 megadaltons, encoded the degradation of phenoxyacetic acid, and possessed identical restriction endonuclease cleavage patterns.     

Indicator Technique for Antimetabolic Toxin Production by Phytopathogenic Species of Pseudomonas

A simple bacteriological technique involving inhibition zone production on a lawn of Escherichia coli was developed to detect antimetabolite toxin production by phytopathogenic species of Pseudomonas. It was established that the mechanism of E. coli inhibition paralleled that of phytotoxin-induced chlorosis of plant tissue. Derivatives of Pseudomonas tabaci and Pseudomonas phaseolicola which did not produce antimetabolite were readily identified by use of this technique. The presence of plasmid DNA in P. tabaci strains was demonstrated, but no physical evidence for plasmid involvement in tabtoxin production was found. The role of antimetabolite production in the pathogenicity of P. phaseolicola was also investigated. The method was extended to show that strains of P. maculicola, P. syringae, and P. coronofaciens also produce antimetabolites.     

Exogenous N‐acyl‐homoserine lactones enhance the expression of flagella of Pseudomonas syringae and activate defence responses in plants

In order to cope with pathogens, plants have evolved sophisticated mechanisms to sense pathogenic attacks and to induce defence responses. The N‐acyl‐homoserine lactone (AHL)‐mediated quorum sensing in bacteria regulates diverse physiological processes, including those involved in pathogenicity. In this work, we study the interactions between AHL‐producing transgenic tobacco plants and Pseudomonas syringae pv. tabaci 11528 (P. syringae 11528). Both a reduced incidence of disease and decrease in the growth of P. syringae 11528 were observed in AHL‐producing plants compared with wild‐type plants. The present data indicate that plant‐produced AHLs enhance disease resistance against this pathogen. Subsequent RNA‐sequencing analysis showed that the exogenous addition of AHLs up‐regulated the expression of P. syringae 11528 genes for flagella production. Expression levels of plant defence genes in AHL‐producing and wild‐type plants were determined by quantitative real‐time polymerase chain reaction. These data showed that plant‐produced AHLs activated a wide spectrum of defence responses in plants following inoculation, including the oxidative burst, hypersensitive response, cell wall strengthening, and the production of certain metabolites. These results demonstrate that exogenous AHLs alter the gene expression patterns of pathogens, and plant‐produced AHLs either directly or indirectly enhance plant local immunity during the early stage of plant infection.     

Virulence of Klebsiella pneumoniae Isolates Harboring bla KPC-2 Carbapenemase Gene in a Caenorhabditis elegans Model

Klebsiella pneumoniae carbapenemase (KPC) is a carbapenemase increasingly reported worldwide in Enterobacteriaceae. The aim of this study was to analyze the virulence of several KPC-2-producing K. pneumoniae isolates. The studied strains were (i) five KPC-2 clinical strains from different geographical origins, belonging to different ST-types and possessing plasmids of different incompatibility groups; (ii) seven transformants obtained after electroporation of either these natural KPC plasmids or a recombinant plasmid harboring only the bla _KPC-2 gene into reference strains K. pneumoniae ATCC10031/{"type":"entrez-protein","attrs":{"text":"CIP53153","term_id":"878514309","term_text":"CIP53153"}}CIP53153; and (iii) five clinical strains cured of plasmids. The virulence of K. pneumoniae isolates was evaluated in the Caenorhabditis elegans model. The clinical KPC producers and transformants were significantly less virulent (LT50: 5.5 days) than K. pneumoniae reference strain (LT50: 4.3 days) (p<0.01). However, the worldwide spread KPC-2 positive K. pneumoniae ST258 strains and reference strains containing plasmids extracted from K. pneumoniae ST258 strains had a higher virulence than KPC-2 strains belonging to other ST types (LT50: 5 days vs. 6 days, p<0.01). The increased virulence observed in cured strains confirmed this trend. The bla _KPC-2 gene itself was not associated to increased virulence.     

Transfer and expression of the herbicide-degrading plasmid pJP4 in aerobic autotrophic bacteria

Plasmid pJP4 encoding the ability to degrade the herbicide 2,4-dichlorophenoxyacetic acid (Tfd⁺) was transferred by conjugation from Escherichia coli JMP397 to various lithoautotrophic strains of Alcaligenes eutrophus and to the autotrophic bacterium Pseudomonas oxalaticus. The herbicide-degrading function of the plasmid was phenotypically expressed in all of the recipients. The majority of Tfd⁺ transconjugants also exhibited additional plasmid-encoded properties such as 3-chlorobenzoate degradation, resistance to mercuric ions, and sensitivity to the male-specific bacteriophage PR11. Furthermore, Tfd⁺ transconjugants were able to act as donors of plasmid pJP4. Physical evidence is presented by agarose gel electrophoresis showing that plasmid pJP4 coexisted with the resident plasmids widely distributed in this group of bacteria. However, in some of the hosts plasmid pJP4 was not stably maintained, had a reduced size and tended to form multimers.     

Conservation of plasmids among plant-pathogenic Pseudomonas syringae isolates of diverse origins

Thirty isolates of Pseudomonas syringae pv. tabaci, pv. angulata (pathogens on tobacco), pv. coronafaciens, and pv. striafaciens (pathogens on oats) were examined for plasmid DNAs. The strains were obtained from plants throughout the world, some over 50 years ago. Of the 22 tobacco pathogens, 16 contain predominantly one type of plasmid, the pJP27.00 type. The remaining six tobacco-specific strains do not harbor detectable plasmids. The oat pathogens contain one, two, or three plasmids. DNA homology studies indicate that the plasmid DNAs are highly conserved. More importantly, the plasmids harbored by strains isolated from one host plant are conserved most stringently; e.g., the plasmids from the tobacco pathogens are, with one exception, indistinguishable by restriction endonuclease digestion and Southern hybridization. There is also extensive homology among plasmids indigenous to the oat-specific P. syringae pv. coronafaciens and pv. striafaciens strains.     

Studies with RP1 deletion plasmids: Incompatibility properties,plasmid curing and the spontaneous loss of resistance

Four deletion plasmids, pHH301, pHH302, pHH303 and pHH401, obtained from RP1 DNA-transformed bacterial clones, were shown to be incompatible with three P plasmids inEscherichia coli K12 strains. Kinetic experiments and colony tests were used to verify the position of these R plasmids.Pseudomonas aeruginosa andE. coli strains, harbouring deletion plasmids, could be cured by using two mutagens, acriflavine and mitomycin C, which affect a percentage of the cell population. The deletion plasmid-positive strains could also be induced at an elevated temperature to spontaneously loose their plasmids.     

Isolation and characterization of a new plasmid from a Flavobacterium sp. which carries the genes for degradation of 2,4-dichlorophenoxyacetate.

A Flavobacterium sp. (strain 50001), capable of degrading 2,4-dichlorophenoxyacetate (2,4-D), 2-methyl-4-chlorophenoxyacetate, and 2-chlorobenzoate and imparting resistance to mercury, harbored a degradative plasmid, pRC10. Cured strains of the Flavobacterium sp. lost the plasmid as well as the ability to degrade these chlorinated compounds. Comparison of this plasmid with the well-characterized 2,4-D-degradative plasmid pJP4 from Alcaligenes eutrophus showed regions of homology between the two plasmids. Restriction fragments of plasmid pRC10 which shared homology with the regions conferring 2,4-D-degradative genes (tfd) of plasmid pJP4 were cloned into a broad-host-range plasmid and studied in Pseudomonas putida. From the results obtained, the cloned DNA fragment expressed the genes for 2,4-D monooxygenase (tfdA) and 2,4-dichlorophenol hydroxylase (tfdB). In spite of the similarity in function, the size (45 kilobases) and restriction pattern of plasmid pRC10 were considerably different from those of pJP4 (80 kilobases). This may be due to the difference in the microbial background during evolution of the two plasmids.     

An EFR-Cf-9 chimera confers enhanced resistance to bacterial pathogens by SOBIR1- and BAK1-dependent recognition of elf18

The transfer of well-studied native and chimeric pattern recognition receptors (PRRs) to susceptible plants is a proven strategy to improve host resistance. In most cases, the ectodomain determines PRR recognition specificity, while the endodomain determines the intensity of the immune response. Here we report the generation and characterization of the chimeric receptor EFR-Cf-9, which carries the ectodomain of the Arabidopsis thaliana EF-Tu receptor (EFR) and the endodomain of the tomato Cf-9 resistance protein. Both transient and stable expression of EFR-Cf-9 triggered a robust hypersensitive response (HR) upon elf18 treatment in tobacco. Co-immunoprecipitation and virus-induced gene silencing studies showed that EFR-Cf-9 constitutively interacts with SUPPRESSOR OF BIR1-1 (SOBIR1) co-receptor, and requires both SOBIR1 and kinase-active BRI1-ASSOCIATED KINASE1 (BAK1) for its function. Transgenic plants expressing EFR-Cf-9 were more resistant to the (hemi)biotrophic bacterial pathogens Pseudomonas amygdali pv. tabaci (Pta) 11528 and Pseudomonas syringae pv. tomato DC3000, and mounted an HR in response to high doses of Pta 11528 and P. carotovorum. Taken together, these data indicate that the EFR-Cf-9 chimera is a valuable tool for both investigating the molecular mechanisms responsible for the activation of defence responses by PRRs, and for potential biotechnological use to improve crop disease resistance.     

Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in soil inoculated with Pseudomonas cepacia DBO1(pRO101), Alcaligenes eutrophus AEO106(pRO101) and Alcaligenes eutrophus JMP134(pJP4): effects of inoculation level and substrate concentration

Mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) by two Alcaligenes eutrophus strains and one Pseudomonas cepacia strain containing the 2,4-D degrading plasmids pJP4 or pRO101 (=pJP4::Tn1721) was tested in 50 g (wet wt) samples of non-sterile soil. Mineralization was measured as ¹⁴C-CO₂evolved during degradation of uniformly-ring-labelled ¹⁴C-2,4-D. When the strains were inoculated to a level of approximately 10⁸ CFU/g soil, between 20 and 45% of the added 2,4-D (0.05 ppm, 10 ppm or 500 ppm) was mineralized within 72 h. Mineralization of 0.05 ppm and 10 ppm, 2,4-D by the two A. eutrophus strains was identical and rapid whereas mineralization by P. cepacia DBO1(pRO101) occurred more slowly. In contrast, mineralization of 500 ppm 2,4-D by the two A. eutrophus strains was very slow whereas mineralization by P. cepacia DBO1 was more rapid. Comparison of 2,4-D mineralization at different levels of inoculation with P. cepacia DBO1(pRO101) (6×10⁴, 6×10⁶ and 1×10⁸ CFU/g soil) revealed that the maximum mineralization rate was reached earlier with the high inoculation levels than with the low level. The kinetics of mineralization were evaluated by nonlinear regression analysis using five different models. The linear or the logarithmic form of a three-half-order model were found to be the most appropriate models for describing 2,4-D mineralization in soil. In the cases in which the logarithmic form of the three-half-order model was the most appropriate model we found, in accordance with the assumptions of the model, a significant growth of the inoculated strains.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - CFU colony forming units - PTYG peptone, tryptone, yeast & glucose - DPM disintegrations per minute     

Global genomic analysis of Pseudomonas savastanoi pv. savastanoi plasmids

Pseudomonas savastanoi pv. savastanoi strains harbor native plasmids belonging to the pPT23A plasmid family (PFPs) which are detected in all pathovars of the related species Pseudomonas syringae examined and contribute to the ecological and pathogenic fitness of their host. However, there is a general lack of information about the gene content of P. savastanoi pv. savastanoi plasmids and their role in the interaction of this pathogen with olive plants. We designed a DNA macroarray containing 135 plasmid-borne P. syringae genes to conduct a global genetic analysis of 32 plasmids obtained from 10 P. savastanoi pv. savastanoi strains. Hybridization results revealed that the number of PFPs per strain varied from one to four. Additionally, most strains contained at least one plasmid (designated non-PFP) that did not hybridize to the repA gene of pPT23A. Only three PFPs contained genes involved in the biosynthesis of the virulence factor indole-3-acetic acid (iaaM, iaaH, and iaaL). In contrast, ptz, a gene involved in the biosynthesis of cytokinins, was found in five PFPs and one non-PFP. Genes encoding a type IV secretion system (T4SS), type IVA, were found in both PFPs and non-PFPs; however, type IVB genes were found only on PFPs. Nine plasmids encoded both T4SSs, whereas seven other plasmids carried none of these genes. Most PFPs and non-PFPs hybridized to at least one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors and one or more insertion sequence transposase genes. These results indicate that non-PFPs may contribute to the virulence and fitness of the P. savastanoi pv. savastanoi host. The overall gene content of P. savastanoi pv. savastanoi plasmids, with their repeated information, mosaic arrangement, and insertion sequences, suggests a possible role in adaptation to a changing environment.     

The Bacterial Effector HopX1 Targets JAZ Transcriptional Repressors to Activate Jasmonate Signaling and Promote Infection in Arabidopsis

Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome.     

In silico analysis reveals multiple putative type VI secretion systems and effector proteins in Pseudomonas syringae pathovars

Type VI secretion systems (T6SS) of Gram-negative bacteria form injectisomes that have the potential to translocate effector proteins into eukaryotic host cells. In silico analysis of the genomes in six Pseudomonas syringae pathovars revealed that P. syringae pv. tomato DC3000, pv. tabaci ATCC 11528, pv. tomato T1 and pv. oryzae 1-6 each carry two putative T6SS gene clusters (HSI-I and HSI-II; HSI: Hcp secretion island), whereas pv. phaseolicola 1448A and pv. syringae B728 each carry one. The pv. tomato DC3000 HSI-I and pv. tomato T1 HSI-II possess a highly similar organization and nucleotide sequence, whereas the pv. tomato DC3000, pv. oryzae 1-6 and pv. tabaci 11528 HSI-II are more divergent. Putative effector orthologues vary in number among the strains examined. The Clp-ATPases and IcmF orthologues form distinct phylogenetic groups: the proteins from pv. tomato DC3000, pv. tomato T1, pv. oryzae and pv. tabaci 11528 from HSI-II group together with most orthologues from other fluorescent pseudomonads, whereas those from pv. phaseolicola, pv. syringae, pv. tabaci, pv. tomato T1 and pv. oryzae from HSI-I group closer to the Ralstonia solanacearum and Xanthomonas orthologues. Our analysis suggests multiple independent acquisitions and possible gene attrition/loss of putative T6SS genes by members of P. syringae.     

Recruitment of a chromosomally encoded maleylacetate reductase for degradation of 2,4-dichlorophenoxyacetic acid by plasmid pJP4.

When Pseudomonas aeruginosa PAO1c or P. putida PPO200 or PPO300 carry plasmid pJP4, which encodes enzymes for the degradation of 2,4-dichlorophenoxyacetic acid (TFD) to 2-chloromaleylacetate, cells do not grow on TFD and UV-absorbing material with spectral characteristics of chloromaleylacetate accumulates in the culture medium. Using plasmid pRO1727, we cloned from the chromosome of a nonfluorescent pseudomonad, Pseudomonas sp. strain PKO1, 6- and 0.5-kilobase BamHI DNA fragments which contain the gene for maleylacetate reductase. When carrying either of the recombinant plasmids, pRO1944 or pRO1945, together with pJP4, cells of P. aeruginosa or P. putida were able to utilize TFD as a sole carbon source for growth. A novel polypeptide with an estimated molecular weight of 18,000 was detected in cell extracts of P. aeruginosa carrying either plasmid pRO1944 or plasmid pRO1945. Maleylacetate reductase activity was induced in cells of P. aeruginosa or P. putida carrying plasmid pRO1945, as well as in cells of Pseudomonas strain PKO1, when grown on L-tyrosine, suggesting that the tyrosine catabolic pathway might be the source from which maleylacetate reductase is recruited for the degradation of TFD in pJP4-bearing cells of Pseudomonas sp. strain PKO1.     

Resistance of biofilms containing alginate‐producing bacteria to disintegration by an alginate degrading enzyme (Algl)

Pure culture biofilms of Pseudomonas aeruginosa (strains 8830 and ATCC 700829) and mixed population biofilms composed of Pseudomonas aeruginosa (ATCC 700829), Pseudomonas fluorescens (ATCC 700830), and Klebsiellapneumoniae (ATCC 700831) were treated with an alginate‐degrading enzyme (AlgL). The enzyme effectively depolymerized the mannuronic acid rich (92%), partially O‐acetylated bacterial alginate produced by P. aeruginosa (8830), both in dilute solution and in a gel‐like, concentrated state. However, both biofilms were unaffected by the presence of the enzyme. These findings suggest either that bacterial alginates do not contribute significantly to the cohesiveness of biofilms or that the alginate is protected from enzymatic degradation in biofilms.     

Role of tfdCIDIEIFI and tfdDIICIIEIIFII Gene Modules in Catabolism of 3-Chlorobenzoate by Ralstonia eutropha JMP134(pJP4)

The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC_ID_IE_IF_I (module I) and tfdD_IIC_IIE_IIF_II (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containing tfdR-tfdC_ID_IE_IF_I grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD_IIC_IIE_IIF_II. In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.