Identification and cloning of genes involved in phaseolotoxin production by Pseudomonas syringae pv. "phaseolicola"

Genes involved in the production of phaseolotoxin by Pseudomonas syringae pv. "phaseolicola" NPS3121 were identified by Tn5 mutagenesis and cosmid cloning. A total of 5,180 kanamycin-resistant colonies were screened for the loss of phaseolotoxin production by a microbiological assay. Six independent, prototrophic, Tox- mutants were isolated that had Tn5 insertions in five different EcoRI fragments. All six mutants had Tn5 inserted in the same KpnI fragment, which had a length of ca. 28 kilobases including Tn5. The mutants produced residual toxin in vitro. An EcoRI fragment containing Tn5 and flanking sequences from mutant NPS4336 was cloned and used to probe a wild-type genomic library by colony hybridization. Seven recombinant plasmids showing homology to this probe were identified. Each Tox- mutant was restored in OCTase-specific toxin production by two or more of the recombinant plasmids. The data suggest that at least some of the genes involved in phaseolotoxin production were clustered in a large KpnI fragment. No homology was detected between the Tn5 target fragment cloned from mutant NPS4336 and the total genomic DNA from closely or distantly related bacteria that do not produce phaseolotoxin.     

Genetic organization of a cluster of genes involved in the production of phaseolotoxin, a toxin produced by Pseudomonas syringae pv. phaseolicola.

Phaseolotoxin [N delta(N'-sulfo-diaminophosphinyl)-ornithyl-alanyl- homoarginine] produced by Pseudomonas syringae pv. phaseolicola, the bean halo blight pathogen, is a potent inhibitor of ornithine carbamoyltransferase (OCT). Inhibition of OCT in infected plants leads to chlorosis and growth inhibition. A genomic cosmid clone, pHK120, containing a 25-kb fragment of DNA from a wild-type strain of P. syringae pv. phaseolicola restores toxin production in Tox- mutants. Tn5 mutagenesis of pHK120 and marker exchange of pHK120::Tn5 plasmids in the wild-type strain resulted in the isolation of 39 chromosomal mutants that harbor Tn5 insertions at known positions. Toxin bioassays revealed that 28 of the mutants, with Tn5 insertions distributed throughout the insert of pHK120, were Tox-, indicating that a functional locus for toxin production in each mutant was inactivated. Complementation analysis was done by testing for toxin production strains that carried a genomic Tn5 at one location and a plasmid-borne Tn5 at another location (pair complementation). Pair complementation analysis of nine marker exchange mutants and a random genomic Tn5 mutant revealed that there are a minimum of eight toxin loci (phtA through phtH) in pHK120. Mutants carrying Tn5 insertions in the phtA, phtD, and phtF loci were complemented by deletion subclones containing fragments from pHK120; mutants carrying Tn5 insertions in the phtC locus were partially complemented by a subclone, and mutants carrying Tn5 insertions in the phtB, phtE, phtG, and phtH loci were not complemented by any of the available subclones. A comparison of the insert from pHK120 with that from pRCP17, a clone reported previously (R. C. Peet, P. B. Lindgren, D. K. Wills, and N. J. Panopoulos, J. Bacteriol. 166:1096-1105, 1986) by another laboratory to contain some of the phaseolotoxin genes and the phaseolotoxin-resistant OCT gene, revealed that the inserts in these two cosmids overlap but differ in important respects.     

Isolation and characterization of the gene coding for the amidinotransferase involved in the biosynthesis of phaseolotoxin in Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola is the causal agent of the "halo blight" disease of beans. A key component in the development of the disease is a nonhost-specific toxin, Ndelta-(N'-sulphodiaminophosphinyl)-ornithyl-alanyl-homoarginine, known as phaseolotoxin. The homoarginine residue in this molecule has been suggested to be the product of L-arginine:lysine amidinotransferase activity, previously detected in extracts of P. syringae pv. phaseolicola grown under conditions of phaseolotoxin production. We report the isolation and characterization of an amidinotransferase gene (amtA) from P. syringae pv. phaseolicola coding for a polypeptide of 362 residues (41.36 kDa) and showing approximately 40% sequence similarity to L-arginine:inosamine-phosphate amidinotransferase from three species of Streptomyces spp. and 50.4% with an L-arginine:glycine amidinotransferase from human mitochondria. The cysteine, histidine, and aspartic acid residues involved in substrate binding are conserved. Furthermore, expression of the amtA and argK genes and phaseolotoxin production occurs at 18 degrees C but not at 28 degrees C. An amidinotransferase insertion mutant was obtained that lost the capacity to synthesize homoarginine and phaseolotoxin. These results show that the amtA gene isolated is responsible for the amidinotransferase activity detected previously and that phaseolotoxin production depends upon the activity of this gene.     

In Pseudomonas syringae pv. phaseolicola, expression of the argK gene, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase, is regulated indirectly by temperature and directly by a precursor resembling carbamoylphosphate

Pseudomonas syringae pv. phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin. ROCT is encoded by argK, which is expressed coordinately with phaseolotoxin synthesis at 18 degrees C. To investigate the regulatory mechanisms of this system, null mutants were constructed for argK, argF (encoding the phaseolotoxin-sensitive OCTase [SOCT]), and amtA (encoding an amidinotransferase involved in phaseolotoxin synthesis). The argF mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28 degrees C, because under this condition SOCT was replaced by ROCT. This loss of thermoregulation of argK was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT. Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type P. syringae pv. phaseolicola and was shown to be able to induce argK expression in M9 medium at 28 degrees C. These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.     

Cloning and expression of the tabtoxin biosynthetic region from Pseudomonas syringae.

Pseudomonas syringae BR2, a causal agent of bean wildfire, was subjected to Tn5 mutagenesis in an effort to isolate mutants unable to produce the beta-lactam antibiotic tabtoxin. Three of the tabtoxin-minus (Tox-) mutants generated appeared to have physically linked Tn5 insertions and retained their resistance to the active toxin form, tabtoxnine-beta-lactam (T beta L). The wild-type DNA corresponding to the mutated region was cloned and found to restore the Tn5 mutants to toxin production. The use of cloned DNA from the region as hybridization probes revealed that the region is highly conserved among tabtoxin-producing pathovars of P. syringae and that the region deletes at a relatively high frequency (10(-3)/CFU) in BR2. The Tox- deletion mutants also lost resistance to tabtoxinine-beta-lactam. A cosmid designated pRTBL823 restored toxin production and resistance to BR2 deletion mutants. This cosmid also converted the tabtoxin-naive P. syringae epiphyte Cit7 to toxin production and resistance, indicating that pRTBL823 contains a complete set of biosynthetic and resistance genes. Tox- derivatives of BR2 did not produce disease symptoms on bean. Clones that restored toxin production to both insertion and deletion mutants also restored the ability to cause disease. However, tabtoxin-producing Cit7 derivatives remained nonpathogenic on bean and tobacco, suggesting that tabtoxin production alone is not sufficient to cause disease.     

Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola.     

Pathovar-specific requirement for the Pseudomonas syringae lemA gene in disease lesion formation.

The lemA gene is conserved among strains and pathovars of Pseudomonas syringae. In P. syringae pv. syringae B728a, a causal agent of bacterial brown spot disese of bean, the lemA gene is required for lesion formation on leaves and pods. Using lemA-containing DNA as a probe, we determined that 80 P. syringae pv. syringae strains isolated from bean leaves could be grouped into seven classes based on restriction fragment length polymorphism. Marker exchange mutagenesis showed that the lemA gene was required for lesion formation by representative strains from each restriction fragment length polymorphism class. Hybridization to the lemA locus was detected within six different P. syringae pathovars and within Pseudomonas aeruginosa. Interestingly, a lemA homolog was present and functional within the nonpathogenic strain P. syringae Cit7. We cloned a lemA homolog from a genomic library of P. syringae pv. phaseolicola NPS3121, a causal agent of halo blight of bean, that restored lesion formation to a P. syringae pv. syringae lemA mutant. However, a lemA mutant P. syringae pv. phaseolicola strain retained the ability to produce halo blight disease symptoms on bean plants. Therefore, the lemA gene played an essential role in disease lesion formation by P. syringae pv. syringae isolates, but was not required for pathogenicity of a P. syringae pv. phaseolicola strain.     

A novel L-amino acid ligase is encoded by a gene in the phaseolotoxin biosynthetic gene cluster from Pseudomonas syringae pv. phaseolicola 1448A

In the phaseolotoxin biosynthetic gene cluster of Pseudomonas syringae pv. phaseolicola 1448A, the PSPPH_4299 gene encodes a novel L-amino acid ligase. The PSPPH_4299 protein synthesized various hetero-dipeptides containing basic amino acids in an ATP-dependent manner, and also synthesized alanyl-homoarginine, part of the phaseolotoxin scaffold.     

Phaseolotoxin-insensitive ornithine carbamoyltransferase of Pseudomonas syringae pv. phaseolicola: basis for immunity to phaseolotoxin.

Cell-free extracts from phaseolotoxin-producing strains of Pseudomonas syringae pv. phaseolicola grown at 18 degrees C, the optimum temperature for phaseolotoxin production, contain an ornithine carbamoyltransferase activity that is insensitive to phaseolotoxin. Extracts from the same strains grown at 30 degrees C, a temperature at which little or no detectable phaseolotoxin is produced, and from phaseolotoxin-nonproducing strains contain a phaseolotoxin-sensitive ornithine carbamoyltransferase activity. The phaseolotoxin-insensitive ornithine carbamoyltransferase activity is also less senstive to N delta-(phosphonacetyl)-L-ornithine than the phaseolotoxin-sensitive ornithine carbamoyltransferase activity of the corresponding strain.     

The global arginine regulator ArgR controls expression of argF in Pseudomonas syringae pv. phaseolicola but is not required for the synthesis of phaseolotoxin or for the regulated expression of argK

In Pseudomonas syringae pv. phaseolicola the enzyme ornithine carbamoyltransferase (OCTase), encoded by argF, is negatively regulated by argR, similar to what has been reported for Pseudomonas aeruginosa. However, production of the phaseolotoxin-resistant OCTase encoded by argK, synthesis of phaseolotoxin, and infectivity for bean pods occur independently of the ArgR protein.     

Construction of a cosmid clone library of Pseudomonas syringae pv. phaseolicola and isolation of genes by functional complementation

A genomic library constructed from a wild-type strain of Pseudomonas syringae pv. phaseolicola in the broad-host-range cosmid vector pVK102 was used to isolate wild-type genes by complementation of Tn5-induced auxotrophic mutants. Selection pressure was required for maintenance of the vector and members of the library in strains of P. syringae.     

Characterization of genes required for pilus expression in Pseudomonas syringae pathovar phaseolicola.

Nonpiliated, phage phi 6-resistant mutants of Pseudomonas syringae pv. phaseolicola were generated by Tn5 transposon mutagenesis. A P. syringae pv. phaseolicola LR700 cosmid library was screened with Tn5-containing EcoRI fragments cloned from nonpiliated mutants. The cosmid clone pVK253 complemented the nonpiliated mutant strain HB2.5. A 3.8-kb sequenced region spanning the Tn5 insertion site contained four open reading frames. The transposon-inactivated gene, designated pilP, is 525 bp long, potentially encoding a 19.1-kDa protein precursor that contains a typical membrane lipoprotein leader sequence. Generation of single mutations in each of the three remaining complete open reading frames by marker exchange also resulted in a nonpiliated phenotype. Expression of this gene region by the T7 expression system in Escherichia coli resulted in four polypeptides of approximately 39, 26, 23, and 18 kDa, in agreement with the sizes of the open reading frames. The three genes upstream of pilP were designated pilM (39 kDa), pilN (23 kDa), and pilO (26 kDa). The processing of the PilP precursor into its mature form was shown to be inhibited by globomycin, a specific inhibitor of signal peptidase II. The gene region identified shows a high degree of homology to a gene region reported to be required for Pseudomonas aeruginosa type IV pilus production.     

Construction of a cosmid clone library of Pseudomonas syringae pv. phaseolicola and isolation of genes by functional complementation.

A genomic library constructed from a wild-type strain of Pseudomonas syringae pv. phaseolicola in the broad-host-range cosmid vector pVK102 was used to isolate wild-type genes by complementation of Tn5-induced auxotrophic mutants. Selection pressure was required for maintenance of the vector and members of the library in strains of P. syringae.     

Phaseolotoxin transport in Escherichia coli and Salmonella typhimurium via the oligopeptide permease.

Phaseolotoxin [(N delta-phosphosulfamyl)ornithylalanylhomoarginine], a phytotoxic tripeptide produced by Pseudomonas syringae pv. phaseolicola that inhibits ornithine carbamoyltransferase, is transported into Escherichia coli and Salmonella typhimurium via the oligopeptide transport system (Opp). Mutants defective in oligopeptide permease (Opp-) were resistant to phaseolotoxin. Spontaneous phaseolotoxin-resistant mutants (Toxr) lacked the Opp function as evidenced by their cross-resistance to triornithine and failure to utilize glycylhistidylglycine as a source of histidine. Growth inhibition by phaseolotoxin was prevented by peptides known to be transported via the Opp system and by treatment of the toxin with L-aminopeptidase. In both E. coli and S. typhimurium, Toxr mutations were cotransducible with trp, suggesting that the opp locus occupies similar positions in genetic maps of these bacteria.     

Ethylene Production by Pseudomonas syringae Pathovars In Vitro and In Planta

Significant amounts of ethylene were produced by Pseudomonas syringae pv. glycinea, pv. phaseolicola (which had been isolated from viny weed Pueraria lobata [Willd.] Ohwi [common name, kudzu]), and pv. pisi in synthetic medium. On the other hand, the bean strains of P. syringae pv. phaseolicola and strains of 17 other pathovars did not produce ethylene. P. syringae pv. glycinea and P. syringae pv. phaseolicola produced nearly identical levels of ethylene (about 5 x 10(sup-7) nl h(sup-1) cell(sup-1)), which were about 10 times higher than the ethylene level of P. syringae pv. pisi. Two 22-bp oligonucleotide primers derived from the ethylene-forming enzyme (efe) gene of P. syringae pv. phaseolicola PK2 were investigated for their ability to detect ethylene-producing P. syringae strains by PCR analysis. PCR amplification with this primer set resulted in a specific 0.99-kb fragment in all ethylene-producing strains with the exception of the P. syringae pv. pisi strains. Therefore, P. syringae pv. pisi may use a different biosynthetic pathway for ethylene production or the sequence of the efe gene is less conserved in this bacterium. P. syringae pv. phaseolicola isolated from kudzu and P. syringae pv. glycinea also produced ethylene in planta. It could be shown that the enhanced ethylene production in diseased tissue was due to the production of ethylene by the inoculated bacteria. Ethylene production in vitro and in planta was strictly growth associated.     

Molecular analysis of thermoregulation of phaseolotoxin-resistant ornithine carbamoyltransferase (argK) from Pseudomonas syringae pv. phaseolicola

The phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) and phaseolotoxin are produced by Pseudomonas syringae pv. phaseolicola at 18 degrees C but not at 28 degrees C. At 28 degrees C, the pathogen produces a protein(s) that binds (in vitro) to a 485-bp fragment (thermoregulatory region, TRR) from a heterologous clone from the pathogen genomic library, which in multiple copies overrides thermoregulation of phaseolotoxin production in wild-type cells (K. B. Rowley, D. E. Clements, M. Mandel, T. Humphreys, and S. S. Patil, Mol. Microbiol. 8:625-635, 1993). We report here that DNase I protection analysis of the 485-bp fragment shows that a single site is protected from cleavage by the protein in the 28 degrees C extract and that this site contains two repeats of a core motif G/C AAAG separated by a 5-bp spacer. Partially purified binding protein forms specific complexes with a synthetic oligonucleotide containing four tandem repeats of this motif. A 492-bp upstream fragment from argK encoding ROCT also forms specific complexes with the protein in the 28 degrees C crude extract, and a 260-bp subfragment from the TRR containing the binding site cross competes with the argk fragment, indicating that the same protein binds to nucleotides in both fragments. DNase I protection analysis of the fragment from argK revealed four separate protected sequence elements, with element III containing half of the core motif sequence (CTTTG), and the other elements containing similar sequences. Gel shift assays were done with DNA fragments from which one or all of the sites were removed as competitor DNAs against the argK probe. The results of these experiments confirmed that the binding sites (in argK) are necessary for the protein to bind to the argK fragment in a specific manner. Taken together, the results of studies presented here suggest that in cells of P. syringae pv. phaseolicola grown at high temperature argK may be negatively regulated by the protein produced at this temperature.     

Stimulation of exopolysaccharide production by fluorescent pseudomonads in sucrose media due to dehydration and increased osmolarity

Abstract Exopolysaccharides produced by plant pathogenic bacteria are thought to play an important role in both the general ecology and the virulence of the producing organism. The environmental factors affecting exopolysaccharide production in planta by Pseudomonas syringae pathovars are not known. We tested the effect of increased medium osmolarity and dehydration on exopolysaccharide production in a sucrose-containing medium by three P. syringae pathovars, one ( P. syringae pv. phaseolicola ) capable of levan and alginate production and two ( P. syringae pv. papulans and pv. savastanoi ) capable of only alginate production. Addition of NaCl and ethanol to the medium led to increased accumulation of alginate by all three pathovars as well as increased levan production by P. syringae pv. phaseolicola . Culture fluids of the two non-levan producers also contained increased amounts of neutral carbohydrate which was not levan. Based on sugar compostion this material may have originated from outer membrane lipopolysaccharide. In addition, the ratio of neutral material (levan or not) to alginate varied dependent on culture conditions.