An integrative plasmid and multiple-sized plasmids of Pseudomonas syringae pv. phaseolicola have extensive homology

Summary Plasmids isolated from five strains of the bean pathogen Pseudomonas syringae pv. phaseolicola were characterized by restriction endonuclease and filter hybridization analyses. BamHI and EcoRI restriction patterns revealed that total plasmid DNA from each strain had a high level of sequence homology with pMC7105, a 148 kbp integrative plasmid found in a sixth strain. Only six BamHI fragments from the eight plasmids in these strains failed to hybridize with pMC7105 probe. Four of these fragments, three from pPP6520 and one from pPP6525 of strain PP652, hybridized strongly to plasmid DNA from a closely-related pathovar, P. syringae pv. glycinea. BamHI fragment 8, which is involved in the integration of pMC7105 into the host chromosome, contains a repeat sequence that was present on all the plasmids except pPP6120 (6.8 kbp), pPP6310 (40 kbp) and pPP6520 (45 kbp). Every plasmid but pPP6520 had fragments that showed weak hybridization to the small plasmid, pPP6120. This homology suggests that a second repetitive sequence is common to these plasmids. The large plasmids (148 to 151 kbp) were essentially identical to pMC7105. The intermediate plasmids (122 to 128 kbp) appeared to be derived mainly from pMC7105 or a related plasmid, whereas the smaller plasmids (6.8 to 45 kbp) appear to have been derived in part from sequences not present in pMC7105.     

Structural characterization of an O-linked tetrasaccharide from Pseudomonas syringae pv. tabaci flagellin

The flagellin of Pseudomonas syringae pv. tabaci is a glycoprotein that contains O-linked oligosaccharides composed of rhamnosyl and 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methylglucosyl residues. These O-linked glycans are released by hydrazinolysis and then labeled at their reducing ends with 2-aminopyridine (PA). A PA-labeled trisaccharide and a PA-labeled tetrasaccharide are isolated by normal-phase high-performance liquid chromatography. These oligosaccharides are structurally characterized using mass spectrometry and NMR spectroscopy. Our data show that P. syringae pv. tabaci flagellin is glycosylated with a tetrasaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap-(1→2)-α-l-Rha-(1→, as well a trisaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1→3)-α-l-Rhap-(1→2)-α-l-Rha-(1→, which was identified in a previous study.     

Location of the O-methyl groups in the O polysaccharide of Pseudomonas syringae pv. phaseolicola

The O-methylation pattern of the O polysaccharide (OPS) of the lipopolysaccharide of Pseudomonas syringae pv. phaseolicola GSPB 1552 was revealed by methylation (CD₃I) analysis, Smith degradation, and NMR spectroscopy. Together with the major O repeats consisting of -rhamnopyranose ( -Rhap) and -fucofuranose ( -Fucf), there are minor repeats (30%) containing 3-O-methyl- -rhamnose ( -acofriose), which is 2-substituted in the interior repeats and occupies the terminal non-reducing end of the OPS. It was suggested that the methylated O repeats are linked to each other nearby the non-reducing end of the OPS and that the ‘biological’ O repeat of the OPS has the following structure:

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Transcriptional profile of Pseudomonas syringae pv. phaseolicola NPS3121 in response to tissue extracts from a susceptible Phaseolus vulgaris L. cultivar

Background  

Pseudomonas syringae pv. phaseolicola is a Gram-negative plant-pathogenic bacterium that causes "halo blight" disease of beans (Phaseolus vulgaris L.). This disease affects both foliage and pods, and is a major problem in temperate areas of the world. Although several bacterial genes have been determined as participants in pathogenesis, the overall process still remains poorly understood, mainly because the identity and function of many of the genes are largely unknown. In this work, a genomic library of P. syringae pv. phaseolicola NPS3121 was constructed and PCR amplification of individual fragments was carried out in order to print a DNA microarray. This microarray was used to identify genes that are differentially expressed when bean leaf extracts, pod extracts or apoplastic fluid were added to the growth medium.     

Deletion mutagenesis of the ice nucleation gene from Pseudomonas syringae S203

Summary The ice nucleation gene inaZ, from Pseudomonas syringae S203, was manipulated to produce a series of defined rearrangements in its coding sequence without changing the reading frame. The effects of these mutations on the ice nucleation phenotype were determined in a heterologous host, Escherichia coli K12. Deletions which disrupted the periodicity of 16 codons, in a repetitive region of inaZ, caused the frequencies of ice nuclei in the bacterial population to be significantly depressed; the nuclei with thresholds at warmer temperatures were most affected. In contrast, when the periodicity was left intact, deletions and duplications in the same region had only slight effects on nucleation activity. Deletions removing part or all of one of the nonrepetitive regions (that encoding the amino-terminal domain of the InaZ protein) did not abolish nucleation activity, but caused it to be limited to cooler threshold temperatures. In contrast, the non-repetitive carboxy-terminal domain of the InaZ protein was shown to be essential for ice nucleation at all temperatures. The differential requirements (for periodicity, and for the amino-terminus) in forming nuclei with different thresholds may be significant for understanding what determines the threshold temperature of an ice nucleus.     

Characterization of Plasmids Encoding the Phytotoxin Coronatine in Pseudomonas syringae

Coronatine (COR) is a nonhost-specific phytotoxin that substantially contributes to the virulence of several pathovars (pvs.) of Pseudomonas syringae. The COR gene cluster in P. syringae is generally plasmid-encoded in pvs. atropurpurea, glycinea, morsprunorum, and tomato but chromosomally encoded in pv. maculicola. In the present study, we investigated whether the COR plasmids in four pathovars shared other traits including self-transmissibility, conserved oriV/par loci, and insertion sequences (ISs) known to reside on other plasmids in P. syringae. Three COR plasmids were shown to be self-transmissible, and all COR plasmids shared a related oriV/par region. Two COR plasmids hybridized to IS801, an IS element widely distributed in P. syringae. Further analysis of p4180A, a 90-kb COR plasmid in P. syringae pv. glycinea, indicated that multiple copies of IS801 were present on this plasmid, and all copies mapped outside the COR gene cluster. Sequence analysis of the region adjacent to the COR gene cluster in p4180A indicated the presence of additional IS elements including IS870, IS51, and IS1240. The IS elements borne on p4180A may have contributed to horizontal transfer of the COR gene cluster and the evolution of the COR biosynthetic pathway.     

The genetic characterization of Pseudomonas syringae pv. tagetis based on the 16S–23S rDNA intergenic spacer regions

Pseudomonas syringae pv. tagetis, a plant pathogen being considered as a biological control agent of Canada thistle (Cirsium arvense), produces tagetitoxin, an inhibitor of RNA polymerase which results in chlorosis of developing shoot tissues. Although the bacterium is known to affect several plant species in the Asteraceae and has been reported in several countries, little is known of its genetic diversity. The genetic relatedness of 24 strains of P. syringae pv. tagetis with respect to each other and to other P. syringae and Pseudomonas savastanoi pathovars was examined using 16S–23S rDNA intergenic spacer (ITS) sequence analysis. The size of the 16S–23S rDNA ITS regions ranged from 508 to 548 bp in length for all 17 P. syringae and P. savastanoi pathovars examined. The size of the 16S–23S rDNA ITS regions for all the P. syringae pv. helianthi and all the P. syringae pv. tagetis strains examined were 526 bp in length. Furthermore, the 16S–23S rDNA ITS regions of both P. syringae pv. tagetis and P. syringae pv. helianthi had DNA signatures at specific nucleotides that distinguished them from the 15 other P. syringae and P. savastanoi pathovars examined. These results provide strong evidence that P. syringae pv. helianthi is a nontoxigenic form of P. syringae pv. tagetis. The results also demonstrated that there is little genetic diversity among the known strains of P. syringae pv. tagetis. The genetic differences that do exist were not correlated with differences in host plant, geographical origin, or the ability to produce toxin.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

The indoleacetic acid-lysine synthetase gene of Pseudomonas syringae subsp. savastanoi induces developmental alterations in transgenic tobacco and potato plants

Summary The iaaL gene of Pseudomonas syringae subsp. savastanoi encodes an indoleacetic acid-lysine synthetase that conjugates lysine to indoleacetic acid. A chimaeric gene consisting of the iaaL coding region under the control of the 35S RNA promoter from cauliflower mosaic virus (35SiaaL) has been used to test if iaaL gene expression leads to morphological alterations in tobacco and potato. Transgenic tobacco plantlets bearing this construct have been shown to synthesize IAA-[¹⁴C]lysine when fed with [¹⁴C]lysine. In late stages of development, their leaves show an increased nastic curvature (epinasty) of the petiole and midvein, a finding suggestive of an abnormal auxin metabolism. The alteration is transmitted to progeny as a dominant Mendelian trait cosegregating with the kanamycin resistance marker. Transgenic potato plants harbouring the construct are also characterised by petiole epinasty. Moreover, 35SiaaL transgenic plants have an increased internode length in potato and decreased root growth in both tobacco and potato. An increased content of IAA-conjugates in leaf blade was found to correlate with the epinastic alterations caused by iaaL gene expression in tobacco leaves. These data provide evidence that IAA conjugation is able to modulate hormone action, suggesting that the widespread endogenous auxin-conjugating activities are of physiological importance.     

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.     

A bifunctional plasmid carrying the recA gene of E. coli

Summary To investigate the effect of an active, plasmid-carried recA gene on the stability and/or the expression of plasmid genes in different genetic backgrounds, we have constructed a bifunctional plasmid (able to replicate in Escherichia coli and in Bacillus subtilis). Chimeric plasmids were obtained by inserting pC194 (Ehrlich 1977) into pDR1453 (Sancar and Rupp 1979). pDR1453 is a 12.9 Kbp plasmid constructed by inserting an E. coli chromosome fragment carrying the recA gene into pBR322. The expected bifunctional recombinant (pMR22/1) (15.7 Kbp) was easily obtained but surprisingly the Cm resistance was expressed only at a very low level in E. coli (as compared, for example, to pHV14, pHV15). We attribute this effect to the presence of multiple recA genes in the cell. On the contrary, Cm^r E. coli transformants bear a recombinant plasmid (pMR22/n) containing tandemly repeated copies of pC194 in equilibrium with excised free pC194. Such amplification has never been observed in a Rec^- background and is therefore mediated by the recA genes. Growth of these clones in the absence of Cm causes the loss of the extra copies, yielding a plasmid with a single copy of pC194, indistingishable from pMR22/1. Interestingly, we have observed that deletions occur at high frequency in pC194, which drastically increase Cm^r in E. coli containing plasmids with a single copy of pC194. Two types of such deletions were detected: (a) large 1050 bp deletions covering about onethird of pC194 and (b) small 120–150 bp deletions (near the MspI site) in the region containing the replicative functions of pC194 (Horinouchi and Weisblum 1982). Both types of deletion render the recombinant plasmid unable to replicate in B. subtilis. pM22/1 replicates, although with a low copy-number, and is stable in B. subtilis wild type; the recA gene of E. coli does not complement any of the rec ^- mutations of B. subtilis. A strong instability, mainly of the E. coli and pBR322 sequences, was observed in many dna and rec mutants of B. subtilis yielding smaller plasmid with a much higher copy-number.     

Transposable elements ofXanthomonas campestris pv.citri originating from indigenous plasmids

Summary After introduction of the broad host range plasmid RP4 inXanthomonas campestris pv.citri strain XAS4501 twoXanthomonas transposable elements, ISXC4 and ISXC5, were isolated. These elements were found to be capable of transposition inEscherichia coli. Restriction analysis, DNA hybridization and heteroduplex experiments revealed that ISXC4 and ISXC5 are about 5.55 and 6.95 kb in size, respectively, possess inverted repeats about 50±18 bp in length and share DNA homology in their left (5.0 kb) and right (0.6 kb) ends. ISXC4 and ISXC5 were found to originate from plasmids pXW45N and pXW45J, which are indigenous replicons inX. campestris pv.citri strain XW45.     

Homology of the root adhesin of Pseudomonas fluorescens OE 28.3 with porin F of P. neruginosa and P. syringae

Summary A transformation system for the thermophilic cellulolytic fungus Talaromyces sp. CL240 has been developed, using the phleomycin resistance gene from Streptoalloteichus hindustanus (Sh ble) as a dominant selectable marker. The plasmids (pAN8-1 and pUT720) carrying the Sh ble gene under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter, allowed selection of phleomycin-resistant transformants. A new promoter sequence cloned from chromosomal DNA of Trichoderma reesei (pUT737) was also able to drive efficient expression of the Sh ble gene in Talaromyces sp. CL240, resulting in the selection of transformants that were highly resistant to phleomycin.     

Genes required for pathogenicity and hypersensitivity are conserved and interchangeable among pathovars of Pseudomonas syringae

Summary A group of pathogenicity genes was previously identified in Pseudomonas syringae pv. phaseolicola which controls the ability of the pathogen to cause disease on bean and to elicit the hypersensitive response on non-host plants. These genes, designated hrp, are located in a ca. 20 kb region which was referred to as the hrp cluster. Homologous sequences to DNA segments derived from this region were detected in several pathovars of P. syringae but not in symbiotic, saprophytic or other phytopathogenic bacteria. A Tn5-induced Hrp^- mutation was transferred from P. syringae pv. phaseolicola to P. syringae pv. tabaci and to three races of P. syringae pv. glycinea by marker exchange mutagenesis. The resulting progeny were phenotypically Hrp^-, i.e. no longer pathogenic on their respective hosts and unable to elicit the hypersensitive response on non-host plants. These mutants were restored to wild-type phenotype upon introduction of a recombinant plasmid carrying the corresponding wild-type locus from P. syringae pv. phaseolicola. The marker exchange mutants of P. syringae pv. glycinea psg0 and Psg5 which carry different avr genes for race specific avirulence did not elicit a hypersensitive response on incompatible soybean cultivars. It appears, therefore, that P. syringae pathovars possess common genes for pathogenicity which also control their interaction with non-host plants. Furthermore, the expression of race/cultivar specific incompatibility of P. syringae pv. glycinea requires a fully functional hrp region in addition to the avr genes which determine avirulence on single-gene differential cultivars of soybean.     

Identification of a gene, pilF, required for type 4 fimbrial biogenesis and twitching motility in Pseudomonas aeruginosa

Many bacterial pathogens produce a class of surface structures called type 4 fimbriae. In Pseudomonas aeruginosa these fimbriae are responsible for adhesion and translocation across host epithelial surfaces. We have identified a novel gene involved in the complex process of type 4 fimbrial biogenesis. This gene, termed pilF, is located on SpeI fragment S at 30 min on the P. aeruginosa genomic map, which is the sixth region on the chromosome shown to contain a fimbrial-associated gene. The PilF protein has a predicted M_r of 22 402, and together with a highly homologous upstream ORF shares a chromosomal arrangement similar to that found in Haemophilus influenzae. A pilF mutant is blocked in the export/assembly of the fimbrial subunit PilA, and accumulates this protein in the membrane fraction. Complementation studies indicate that the cloned pilF gene is able to restore the expression of surface fimbriae, twitching motility and susceptibility to fimbrial-specific bacteriophage