A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria.

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.

Effect of host plant Xylem fluid on growth, aggregation, and attachment of Xylella fastidiosa.

In the Temecula Valley of California the proximity of citrus groves to vineyards influences the incidence and severity of Pierce's disease (PD) in grapes, a disease caused by the gram-negative bacterium, Xylella fastidiosa. Although the glassy-winged sharpshooter (GWSS), the major insect vector of the bacterium, feeds on and moves back and forth between citrus groves and vineyards, there are no visible symptoms of disease caused by X fastidiosa in citrus. Previous evidences suggested that while grapevines are susceptible to the PD strain of X. fastidiosa, citrus trees are resistant or tolerant but could be a reservoir to harbor the pathogen for the GWSS acquisition. We investigated the mechanisms of host plant resistance/susceptibility by examining the in vitro effect of xylem fluid from grapefruit, orange, lemon, and grape on the growth, aggregation, and attachment of a X. fastidiosa strain isolated from grape. Our results revealed that xylem fluid from grapefruit, orange, and lemon trees caused the bacterial cells to form aggregations of large whitish clumps, whereas the xylem fluid from grape vines created a visible thick biofilm. The densities of X. fastidiosa cells in grapefruit xylem fluid treatment were significantly higher at 6, 8, and 9 d postinoculation compared with those in grape xylem fluid treatment. The cell densities of X. fastidiosa cultures in orange or lemon xylem fluid were generally lower than those in grape xylem fluid treatment, whereas citrus xylem fluid significantly inhibited X. fastidiosa biofilm formation compared to grape xylem fluid.

Differentiation of Strains of Xylella fastidiosa Infecting Grape, Almonds, and Oleander Using a Multiprimer PCR Assay.

Xylella fastidiosa is a xylem-limited, nutritionally fastidious bacterium that causes several plant diseases including Pierce's disease (PD) in grape and leaf scorch in almond (ALS) and oleander (OLS). OLS strains belong to X. fastidiosa subsp. sandyi, PD strains belong to X. fastidiosa subsp. fastidiosa, and strains from almond designated as ALS strains are of two general types belonging either to X. fastidiosa subsp. multiplex or X. fastidiosa subsp. fastidiosa. The ALS strains assigned to X. fastidiosa subsp. multiplex belong to two different genotypes (ALSI and ALSII) below the subspecies level. The OLS strains do not infect grape or almond. PD strains produce diseases in grape, alfalfa, almond, and some weeds, but they do not infect oleander, oak, peach, or citrus. ALS strains that belong to X. fastidiosa subsp. multiplex do not produce disease on grape. In this study, a relatively simple polymerase chain reaction (PCR) based method was developed to distinguish among PD, OLS, and ALS strains. PCR performed with primers XF1968-L and XF1968-R amplified a 638-bp fragment from OLS strains but not from PD strains or ALS strains that belong to X. fastidiosa subsp. fastidiosa. PCR with primers XF2542-L and XF2542-R amplified a 412-bp fragment from PD strains, but not from OLS strains. PCR with primers ALM1 and ALM2 produced a fragment of 521 bp from strains isolated from almond that belong to X. fastidiosa subsp. multiplex. The combination of the three primer sets allowed the distinction of the two ALS genotypes of X. fastidiosa subsp. multiplex. These results are in agreement with those obtained from analysis of sequences of 16S-23S rDNA intergenic spacer regions sequence analysis and with previous results based on randomly amplified polymorphic DNA analysis.

Plant Hosts of Xylella fastidiosa In and Near Southern California Vineyards.

Xylella fastidiosa is a xylem-limited bacterium that causes Pierce's disease (PD) of grapevines. A variety of plant species found near a severe outbreak of PD in vineyards in the Temecula Valley of California were tested using enzyme-linked immunosorbent assay, culture on media, and polymerase chain reaction to identify potential inoculum sources in the area. Species that consistently tested positive for X. fastidiosa were the known hosts, grape, almond, and oleander, and two new hosts, Spanish broom (Spartium junceum) and wild mustard (Brassica spp). Sequence analysis of the 16S-23S rRNA spacer region found that strains isolated from grapevine, Spanish broom, wild mustard, and almond clustered with previously sequenced PD strains. Thus, these species could serve as sources of inoculum for infection of grapevines and should be removed or monitored for signs of infection. Sequences from oleander isolates from Temecula formed another cluster with a previously published oleander strain sequence. Oleander strains do not infect grapevines and thus do not appear to cause a direct threat to grapevines. Two additional isolates from almond were determined to be genetically different from PD strains, and the ability of these strains to infect grapevine is not known. Greenhouse transmission studies indicate that the glassy-winged sharpshooter was able to transmit a PD strain of X. fastidiosa to Spanish broom, black mustard, and other hosts.

Molecular characterization of global regulatory RNA species that control pathogenicity factors in Erwinia amylovora and Erwinia herbicola pv. gypsophilae.

rsmB(Ecc) specifies a nontranslatable RNA regulator that controls exoprotein production and pathogenicity in soft rot-causing Erwinia carotovora subsp. carotovora. This effect of rsmB(Ecc) RNA is mediated mostly by neutralizing the function of RsmA(Ecc), an RNA-binding protein of E. carotovora subsp. carotovora, which acts as a global negative regulator. To determine the occurrence of functional homologs of rsmB(Ecc) in non-soft-rot-causing Erwinia species, we cloned the rsmB genes of E. amylovora (rsmB(Ea)) and E. herbicola pv. gypsophilae (rsmB(Ehg)). We show that rsmB(Ea) in E. amylovora positively regulates extracellular polysaccharide (EPS) production, motility, and pathogenicity. In E. herbicola pv. gypsophilae, rsmB(Ehg) elevates the levels of transcripts of a cytokinin (etz) gene and stimulates the production of EPS and yellow pigment as well as motility. RsmA(Ea) and RsmA(Ehg) have more than 93% identity to RsmA(Ecc) and, like the latter, function as negative regulators by affecting the transcript stability of the target gene. The rsmB genes reverse the negative effects of RsmA(Ea), RsmA(Ehg), and RsmA(Ecc), but the extent of reversal is highest with homologous combinations of rsm genes. These observations and findings that rsmB(Ea) and rsmB(Ehg) RNA bind RsmA(Ecc) indicate that the rsmB effect is channeled via RsmA. Additional support for this conclusion comes from the observation that the rsmB genes are much more effective as positive regulators in a RsmA(+) strain of E. carotovora subsp. carotovora than in its RsmA(-) derivative. E. herbicola pv. gypsophilae produces a 290-base rsmB transcript that is not subject to processing. By contrast, E. amylovora produces 430- and 300-base rsmB transcripts, the latter presumably derived by processing of the primary transcript as previously noted with the transcripts of rsmB(Ecc). Southern blot hybridizations revealed the presence of rsmB homologs in E. carotovora, E. chrysanthemi, E. amylovora, E. herbicola, E. stewartii and E. rhapontici, as well as in other enterobacteria such as Escherichia coli, Salmonella enterica serovar Typhimurium, Serratia marcescens, Shigella flexneri, Enterobacter aerogenes, Klebsiella pneumoniae, Yersinia enterocolitica, and Y. pseudotuberculosis. A comparison of rsmB sequences from several of these enterobacterial species revealed a highly conserved 34-mer region which is predicted to play a role in positive regulation by rsmB RNA.

rsmC of the soft-rotting bacterium Erwinia carotovora subsp. carotovora negatively controls extracellular enzyme and harpin(Ecc) production and virulence by modulating levels of regulatory RNA (rsmB) and RNA-binding protein (RsmA).

Previous studies have shown that the production of extracellular enzymes (pectate lyase [Pel], polygalacturonase [Peh], cellulase [Cel], and protease [Prt]) and harpin(Ecc) (the elicitor of hypersensitive reaction) in Erwinia carotovora subsp. carotovora is regulated by RsmA, an RNA-binding protein, and rsmB, a regulatory RNA (Rsm stands for regulator of secondary metabolites) (Y. Liu et al., Mol. Microbiol. 29:219-234, 1998). We have cloned and characterized a novel regulatory gene, rsmC, that activates RsmA production and represses extracellular enzyme and harpin(Ecc) production, rsmB transcription, and virulence in E. carotovora subsp. carotovora. In an rsmC knockout mutant of E. carotovora subsp. carotovora Ecc71 carrying the chromosomal copy of the wild-type rsmA(+) allele, the basal levels of Pel, Peh, Cel, Prt, and harpin(Ecc) as well as the amounts of rsmB, pel-1, peh-1, celV, and hrpN(Ecc) transcripts are high, whereas the levels of rsmA transcripts and RsmA protein are low. Furthermore, the expression of an rsmA-lacZ gene fusion is lower in the RsmC(-) mutant than in the RsmC(+) parent. Conversely, the expression of an rsmB-lacZ operon fusion is higher in the RsmC(-) mutant than in the RsmC(+) parent. These observations establish that RsmC negatively regulates rsmB transcription but positively affects RsmA production. Indeed, comparative studies with an RsmC(-) mutant, an RsmA(-) mutant, and an RsmA(-) RsmC(-) double mutant have revealed that the negative effects on exoprotein production and virulence are due to the cumulative regulatory effects of RsmC on rsmA and rsmB. Exoprotein production by the RsmC(-) mutant is partially dependent on the quorum sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone. Southern blot data and analysis of PCR products disclosed the presence of rsmC sequences in E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, and E. carotovora subsp. carotovora. These findings collectively support the idea that rsmA and rsmB expression in these plant pathogenic Erwinia species is controlled by RsmC or a functional homolog of RsmC.

The RsmA- mutants of Erwinia carotovora subsp. carotovora strain Ecc71 overexpress hrpNEcc and elicit a hypersensitive reaction-like response in tobacco leaves.

Erwinia carotovora subsp. carotovora wild-type strain Ecc71 does not elicit the hypersensitive reaction (HR) in tobacco leaves. By mini-Tn5-Km and chemical mutagenesis we have isolated RsmA- mutants of Ecc71 that produce high basal levels of pectate lyases, polygalacturonase, cellulase, and protease; they also are hypervirulent. The RsmA- mutants, but not their parent strains, elicit an HR-like response in tobacco leaves. This reaction is characterized by the rapid appearance of water soaking followed by tissue collapse and necrosis. The affected areas remain limited to the region infiltrated with bacterial cells, and the symptoms closely resemble a typical HR, e.g., the reactions caused by Pseudomonas syringae pv. pisi. Moreover, low concentrations of cells of the mini-Tn5-Km insertion RsmA- mutant, AC5070, infiltrated into tobacco leaf tissue prevent elicitation of the rapid necrosis by AC5070 or by P. syringae pv. pisi. Elicitation of the HR-like response by the mutants is not affected by the deficiency of N-(3-oxohexanoyl)-L-homoserine lactone, the cell density (quorum) sensing signal. Cloning and sequence analysis have disclosed that E. carotovora subsp. carotovora strain Ecc71 possesses a homolog of E. chrysanthemi hrpN known to encode an elicitor of the HR; the corresponding Ecc71 gene is designated hrpNEcc. Northern (RNA) blot data show that the level of hrpNEcc mRNA is considerably higher in the RsmA- mutants than in the RsmA+ strains. Moreover, a low copy plasmid carrying the rsmA+ allele severely reduces the level of the hrpNEcc transcripts in the RsmA- mutants. These constructs, like the RsmA+ E. carotovora subsp. carotovora strains, do not elicit the HR-like response. These data taken along with the effects of rsmA on exoenzyme production and pathogenicity (A. Chatterjee et al., 1995, Appl. Environ. Microbiol. 61:1959-1967) demonstrate that this global regulator gene plays a critical role in plant interaction of E. carotovora subsp. carotovora.

Identification of a global repressor gene, rsmA, of Erwinia carotovora subsp. carotovora that controls extracellular enzymes, N-(3-oxohexanoyl)-L-homoserine lactone, and pathogenicity in soft-rotting Erwinia spp.

The production of extracellular enzymes such as pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt) is activated by the cell density (quorum)-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (HSL); plant signals; and aep genes during postexponential growth of Erwinia carotovora subsp. carotovora 71. Studies with mutants of E. carotovora subsp. carotovora 71 derepressed in exoenzyme production led to the identification of a negative regulator gene, rsmA (rsm, repressor of secondary metabolites). Nucleotide sequencing, transcript assays, and protein analysis established that a 183-bp open reading frame encodes the 6.8-kDa RsmA. rsmA has extensive homology with the csrA gene of Escherichia coli, which specifies a negative regulator of carbon storage. Moreover, the suppression of glycogen synthesis in E. coli by rsmA indicates that the Erwinia gene is functionally similar to csrA. Southern hybridizations revealed the presence of rsmA homologs in soft-rotting and non-soft-rotting Erwinia spp. and in other enterobacteria such as Enterobacter aerogenes, E. coli, Salmonella typhimurium, Shigella flexneri, Serratia marcescens, and Yersinia pseudotuberculosis. rsmA suppresses production of Pel, Peh, Cel, and Prt, plant pathogenicity, and synthesis of HSL in E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, and E. chrysanthemi. In the E. carotovora subsp. carotovora 71, rsmA reduces the levels of transcripts of hslI, a luxI homolog required for HSL biosynthesis. This specific effect and the previous finding that HSL is required for extracellular enzyme production and pathogenicity in soft-rotting Erwinia spp. support the hypothesis that rsmA controls these traits by modulating the levels of the cell density (quorum)-sensing signal.

Inactivation of rsmA leads to overproduction of extracellular pectinases, cellulases, and proteases in Erwinia carotovora subsp. carotovora in the absence of the starvation/cell density-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone.

The soft-rotting bacterium, Erwinia carotovora subsp. carotovora 71, produces extracellular enzymes such as pectate lyase isozymes (Pels), cellulase (Cel), polygalacturonase (Peh), and protease (Prt). While the extracellular levels of these enzymes are extremely low when the bacterium is grown in salts-yeast extract-glycerol (SYG) medium, the enzymatic activities are highly induced in SYG medium supplemented with celery extract. By transposon (mini-Tn5) mutagenesis, we isolated a RsmA- mutant, AC5070, which overproduces extracellular enzymes; the basal levels of Pel, Peh, and Cel in AC5070 are higher than the induced levels in the RsmA+ parent, AC5047. While Peh production is mostly constitutive in AC5070, Pel, Cel, and Prt production is still inducible with celery extract. The high basal levels of pel-1, pel-3, and peh-1 mRNAs in AC5070 demonstrate that overproduction of the pectolytic enzymes is due to the stimulation of transcription. Using chromosomal DNA flanking mini-Tn5 as a probe, we cloned the wild-type rsmA+ allele, which suppresses Pel, Peh, Cel, and Prt production in both RsmA+ and RsmA- strains. The RsmA- mutant, like its parent, produces N-(3-oxohexanoyl)-L-homoserine lactone (HSL), a starvation/cell density-sensing signal required for extracellular enzyme production. To examine the role of HSL, we constructed HSL-deficient strains by replacing hslI, a locus required for HSL production, with hslI::Tn3HoHo1-Spc. While the basal levels of Pel, Peh, Cel, and Prt are comparable in the RsmA- mutant and its HSL- derivative, these enzymes are barely detectable in the Hsl- derivative of the RsmA+ parent strain.(ABSTRACT TRUNCATED AT 250 WORDS)