SummaryThe genes lemA (which we here redesignate gacS) and gacA encode members of a widely conserved two-component regulatory system. In Pseudomonas syringae strain B728a, gacS and gacA are required for lesion formation on bean, as well as for the production of protease and the toxin syringomycin. A gene, designated salA, was discovered that restored syringomycin production to a gacS mutant when present on a multiple-copy plasmid. Disruption of chromosomal salA resulted in loss of syringomycin production and lesion formation in laboratory assays. Sequence analysis of salA suggests that it encodes a protein with a DNA-binding motif but without other significant similarity to proteins in current databases. Chromosomal reporter fusions revealed that gacS and gacA positively regulate salA, that salA upregulates its own expression and that salA positively regulates the expression of a syringomycin biosynthetic gene, syrB. Loss of syringomycin production does not account for the salA mutant's attenuated pathogenicity, as a syrB mutant was found to retain full virulence. The salA gene did not similarly suppress the protease deficient phenotype of gacS mutants, nor were salA mutants affected for protease production. A gacS/gacA-dependent homoserine lactone activity as detected by bioassay was also unaffected by the disruption of salA. Thus, salA appears to encode a novel regulator that activates the expression of at least two separate genetic subsets of the gacS/gacA regulon, one pathway leading to syringomycin production and the other resulting in plant disease.
Mutational analysis of the bean-pathogenic Pseudomonas syringae pv. syringae strain B728a has led to the genetic identification of the gacA gene as encoding the response regulator for the unlinked lemA sensor kinase. The analysis of a collection of spontaneous mutants of P. syringae pv. syringae suggested that the gacA gene was involved in lesion formation and the production of protease and syringomycin. The gacA gene originally was identified as a regulator of extracellular antibiotic production by Pseudomonas fluorescens, and the predicted GacA protein is a member of the FixJ family of bacterial response regulators. The sequence of the putative B728a GacA protein revealed 92% identity with the P. fluorescens GacA protein. An insertional mutation within the P. syringae pv. syringae gacA gene abrogated lesion formation on beans, production of extracellular protease, and production of the toxin syringomycin, the same phenotypes affected by a lemA mutation. DNA sequence analysis identified the P. syringae pv. syringae uvrC gene immediately downstream of the gacA gene, an arrangement conserved in P. fluorescens and Escherichia coli. The gacA insertional mutant was sensitive to UV, presumably because of polarity on transcription of the downstream uvrC gene. Southwestern (DNA-protein) analysis revealed that the lemA and gacA genes were required for the full expression of a DNA binding activity.
Pseudomonas syringae BR2, a causal agent of bean wildfire, was subjected to Tn5 mutagenesis in an effort to isolate mutants unable to produce the I8-lactam antibiotic tabtoxin. Three of the tabtoxin-minus (Tox-) mutants generated appeared to have physically linked TnS insertions and retained their resistance to the active toxin form, tabtoxinine-,-lactam (T,IlL). The wild-type DNA corresponding to the mutated region was cloned and found to restore the TnS 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-p-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.
Analysis of two virulence mutants of Pseudomonas syringae B728a revealed that the Tn 5 sites of insertion were within the gidA open reading frame (ORF). These mutations were pleiotropic, affecting diverse phenotypic traits, such as lipodepsipeptide (syringomycin and syringopeptin) antibiotic production, swarming, presence of fluorescent pigment, and virulence. Site-specific recombination of a disrupted gidA gene into the chromosome resulted in the same phenotypic pattern as transposon insertion. Mutant phenotypes were restored by the gidA ORF on a plasmid. The salA gene, a copy number suppressor of the syringomycin-deficient phenotype in gacS and gacA mutants, was also found to suppress the antibioticnegative phenotypes of gidA mutants, suggesting that gidA might play some role in salA regulation. Reporter studies with chromosomal salA-lacZ translational fusions confirmed that salA reporter expression decreased approximately fivefold in a gidA mutant background, with a concurrent decrease in the expression of the syringomycin biosynthetic reporter fusion syrB-lacZ. Wild-type levels of reporter expression were restored by supplying an intact gidA gene on a plasmid. Often described as being involved in cell division, more recent evidence suggests a role for gidA in moderating translational fidelity, suggesting a mechanism by which global regulation might occur. The gidA gene is essentially universal in the domains Bacteria and Eucarya but has no counterparts in Archaea, probably reflecting specific differences in the translational machinery between the former and latter domains.
DNA sequence analysis revealed that the biosynthetic genes of the unusual b -lactam antibiotic tabtoxin reside at the att site adjacent to the lysC tRNA gene in Pseudomonas syringae BR2. ORFs encoded within the region included ones with similarity to b -lactam synthase and clavaminic acid synthase, as well as amino acid synthesis enzymes. Novel ORFs were present in a portion of the biosynthetic region associated with a toxin hypersensitivity phenotype. Tabtoxin resistance was associated with a fragment containing a major facilitator superfamily (MFS) transporter gene. (Fig. 1). The antibiotic is usually associated with bacteria of the Pseudomonas syringae species group causal to several types of chlorotic plant disease. While most b -lactam antibiotics exert their antimicrobial influence through the inhibition of bacterial cell wall synthesis, TBL appears to irreversibly inhibit the enzyme glutamine synthetase, causing cells to become intoxicated by high levels of their own unprocessed ammonia [3,4]. The genes encoding the pathway for both tabtoxin production and host resistance were originally cloned as a single cosmid insert from BR2, a Pseudomonas syringae strain causal to wildfire disease on bean [5]. Here we report the analysis of the DNA sequence of this cloned region.Subcloned fragments of the cosmid pRTBL823 were sequenced using a combination of primer walking with synthetic primers derived from endogenous sequence within the insert DNA and transposon priming (GPS-1, New England Biolabs). Sequence of plasmid miniprep DNA (Qiagen) was generated using dye terminator kits (ABI) analyzed by capillary electrophoresis (GeneAnalyser 310, ABI). The sequence of overlaps between subclones was confirmed by sequencing with the original cosmid
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