Thomas G. Kinscherf

A newly identified regulator is required for virulence and toxin production in Pseudomonas syringae

The 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 mutants 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.

Molecular analysis of the karyotype of Ustilago maydis

A molecular karyotype of the corn smut-inducing fungus Ustilago maydis was prepared using orthogonalfield-alternation gel electrophoresis (OFAGE). At least 20 chromosome-sized DNAs ranging from approximately 300 kb to the maximum limit of resolution of this system were identified in haploid cells of a widely used strain. Although general features of the banding pattern of chromosome-sized DNAs were conserved between strains, no two strains had identical karyotypes, indicating that considerable chromosome length polymorphism exists in this species. This polymorphism was seen in both laboratory strains as well as more recent isolates from nature. Length variation in apparently identical chromosomes was usually small, but was occasionally significant. In one strain Southern DNA hybridization analysis suggested the occurrence of a stable large-scale, inter-chromosomal exchange which had given rise to two novel chromosomes.

ESTIMATION OF RELATEDNESS BETWEEN PHYTOPHTHORA SPECIES BY ANALYSIS OF MITOCHONDRIAL DNA

The relatedness between four Phytophthora megasperma isolates [P. megasperma f. sp. glycinea (Pmg) from soybean, P. megasperma f. sp. medicaginis (Pmm) from alfalfa, and two broad host-range isolates from apple or alfalfa], P. cactorum, P. cryptogea, and P. parasitica var. nicotianae (Ppn) was investigated by a quantitative analysis of restriction fragment length polymorphisms of mitochondrial DNAs. Nucleotide sequence divergence values among P. megasperma isolates were within the same range as between the P. megasperma isolates and the three other Phytophthora species. An unrooted phylogenetic network based on nucleotide sequence divergence values indicated that several evolutionary lines exist for P. megasperma. These data are consistent with the observed variability of morphological, physiological and biochemical characteristics within this fungal group and support the contention that P. megasperma comprises several biological species which are the result of convergent evolution and which are not closely related. A consensus restriction map of the mtDNAs of Pmg, Pmm, and Ppn was constructed, and numerical analyses of conserved and variable restriction cleavage sites also support the separation of Pmg and Pmm into distinct species.

Global Regulation by gidA in Pseudomonas syringae

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 antibiotic-negative 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.

Genetic Evidence that Loss of Virulence Associated with gacS or gacA Mutations in Pseudomonas syringae B728a Does Not Result from Effects on Alginate Production

ABSTRACT Mutations in the global regulatory genes gacS andgacA render Pseudomonas syringae pv. syringae strain B728a completely nonpathogenic in foliar infiltration assays on bean plants. It had been previously demonstrated that gacgenes regulate alginate production in Pseudomonas species, while other published work indicated that alginate is involved in the pathogenic interaction of P. syringae on bean plants. Together, these results suggested that the effects of gacSand gacA mutations on virulence in B728a might stem directly from a role in regulating alginate. In this report, we confirm a role for gac genes in both algD expression and alginate production in B728a. However, B728a mutants completely devoid of detectable alginate were as virulent as the wild-type strain in our assay. Thus, factors other than, or in addition to, a deficiency of alginate must be involved in the lack of pathogenicity observed withgacS and gacA mutants.

Molecular analysis of the mitochondrial genome of Phytophthoraa

SummaryThe organization of the mitochondrial genomes from two morphologically similar Phytophthora isolates, P. megasperma f. sp. glycinea (Pmg) and P. megasperma f. sp. medicaginis (Pmm), and the morphologically different species, P. parasitica var. nicotianae (Ppn), has been studied. The mtDNAs are circular, and their estimated sizes are 45.3 kb, 41 kb, and 39.5 kb for Pmg, Pmm, and Ppn, respectively. Physical maps were constructed for restriction endonuclease sites. Four genes (l-rRNA, s-rRNA, oxi-2, and cob) were found to have the same order in the three mtDNAs.

The Biosynthetic Gene Cluster for the β-Lactam Antibiotic Tabtoxin in Pseudomonas syringae

DNA sequence analysis revealed that the biosynthetic genes of the unusual β-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 β-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.

Global Regulation in Pseudomonas Syringae

Two questions come rather quickly to mind when one is confronted with writing a chapter on global regulation in Pseudomonas syringae. The first of these is very basic, and goes to the eventual scope of the article: what is P. syringae? This is not a straightforward quest ion, as the species is something of a taxonomic mess. P. syringae has long been recognized as a member of the original “inner circle” of pseudomonads, eventually codified as the rRNA Group I47. This group was further refined by both general and molecular characteristics into two additional phylogenetic groupings recently described as the intrageneric clusters (IGC) I and II, with P. syringae being in IGC II63. Detailed analysis of the nucleotide sequences of the gyrB and rpoD genes indicated that the IGC II cluster could be reduced still further to three complexes, with one being the “P. syringae complex” containing the pathovars and nomenspecies traditionally associated with P. syringae 63, After this point, the taxonomic picture becomes less clear. Plant pathogenic pseudomonads are not readily differentiated by standard phenotypic characteristics and have often been classified largely by host range as pathovars or subspecies of P. syringae 53, This is an unusually heterologous grouping, with DNA hybridization studies, ribotyping, and general characteristics having defined at least six47, and possibly as many as nine17, discrete genomospecies within the complex.

Molecular Analysis of Pathogenesis in Ustilago maydis

We are isolating and studying genes required for pathogenicity of Ustilago maydis, the causative agent of corn smut disease (Christensen, 1963). This phytopathogenic basidiomycete offers an attractive system in which to gain a molecular understanding of host-parasite interactions. The organism grows as a haploid yeast on defined laboratory media, mutants are readily generated by UV or chemical mutagenesis, stable diploids can be constructed for mitotic recombination and complementation analysis, and the fungus is amenable to Mendelian genetic analysis (Holliday, 1974). These attributes are not found in combination in any other phytopathogenic fungus. Although this pathogen is no longer a production constraint in North America, where resistant hybrid corn is grown, it continues to be a problem in third world countries where susceptible varieties are still cultivated. Moreover, we hope that an understanding of mechanisms of pathogenesis in this host-parasite interaction will have application to more economically important and difficult to study fungal diseases such as the bunts and rusts. We have initiated a molecular genetic analysis of two gene systems thought to control pathogenic growth of U. maydis in maize. These include genes that program sexual development and genes involved in the high affinity, siderophore-mediated iron uptake system of the fungus. In order to conduct these analyses, one of our first goals has been to develop tools which enable us to clone, study, and transfer genes in U. maydis.

Identification and Isolation of a DNA Region Required for Tabtoxin Production: Apparent Deletion in Pseudomonas Syringae Pv Tabaci Variant ‘ Angulata’ and Tox − Mutants

Tabtoxin is a dipeptide pretoxin produced by several path-ovars and isolates of Pseudomonas syringae. Examples of such strains, their plant hosts, and the diseases they cause are listed in Table 1. While tabtoxin is synthesized in a biologically inactive form, it is readily cleaved by aminopep-tidases, present in either the bacteria or the plant, yielding threonine and the active moiety tabtoxinine-β-lactam (Figure 1). Cleavage of (2-serine)tabtoxin, a derivative form containing a serine molecule in place of threonine, yields T-β-L and serine. The accumulation of tabtoxinine-β-1actam (T-β-L) in planta results in a chlorotic halo surrounding the bacterial lesion and is thought to be a contributing factor in disease severity. T-β-L is an inhibitor of glutamine synthetase isolated from plants, bacteria, and fungi. The chlorosis caused by tabtoxin-producing phytobacteria has been attributed to ammonia accumulation resulting from the inhibition of glutamine synthetase by T-β-L in planta (Durbin, 1982).