RESUMO
Genes for seven putative serine proteases (ChpA-ChpG) belonging to the trypsin subfamily and homologous to the virulence factor pat-1 were identified on the chromosome of Clavibacter michiganensis subsp. michiganensis (Cmm) NCPPB382. All proteases have signal peptides indicating export of these proteins. Their putative function is suggested by two motifs and an aspartate residue typical for serine proteases. Furthermore, six cysteine residues are located at conserved positions. The genes are clustered in a chromosomal region of about 50 kb with a significantly lower G + C content than common for Cmm. The genes chpA, chpB and chpD are pseudogenes as they contain frame shifts and/or in-frame stop codons. The genes chpC and chpG were inactivated by the insertion of an antibiotic resistance cassette. The chpG mutant was not impaired in virulence. However, in planta the titre of the chpC mutant was drastically reduced and only weak disease symptoms were observed. Complementation of the chpC mutant by the wild-type allele restored full virulence. ChpC is the first chromosomal gene of Cmm identified so far that affects the interaction of the pathogen with the host plant.
Assuntos
Actinomycetales/enzimologia , Proteínas de Bactérias/metabolismo , Doenças das Plantas/microbiologia , Serina Endopeptidases/metabolismo , Solanum lycopersicum/microbiologia , Actinomycetales/genética , Actinomycetales/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Composição de Bases/genética , Cromossomos Bacterianos/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Doenças das Plantas/genética , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/genética , Virulência/genéticaRESUMO
Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete that causes bacterial wilt and canker of tomato. The nucleotide sequence of the genome of strain NCPPB382 was determined. The chromosome is circular, consists of 3.298 Mb, and has a high G+C content (72.6%). Annotation revealed 3,080 putative protein-encoding sequences; only 26 pseudogenes were detected. Two rrn operons, 45 tRNAs, and three small stable RNA genes were found. The two circular plasmids, pCM1 (27.4 kbp) and pCM2 (70.0 kbp), which carry pathogenicity genes and thus are essential for virulence, have lower G+C contents (66.5 and 67.6%, respectively). In contrast to the genome of the closely related organism Clavibacter michiganensis subsp. sepedonicus, the genome of C. michiganensis subsp. michiganensis lacks complete insertion elements and transposons. The 129-kb chp/tomA region with a low G+C content near the chromosomal origin of replication was shown to be necessary for pathogenicity. This region contains numerous genes encoding proteins involved in uptake and metabolism of sugars and several serine proteases. There is evidence that single genes located in this region, especially genes encoding serine proteases, are required for efficient colonization of the host. Although C. michiganensis subsp. michiganensis grows mainly in the xylem of tomato plants, no evidence for pronounced genome reduction was found. C. michiganensis subsp. michiganensis seems to have as many transporters and regulators as typical soil-inhabiting bacteria. However, the apparent lack of a sulfate reduction pathway, which makes C. michiganensis subsp. michiganensis dependent on reduced sulfur compounds for growth, is probably the reason for the poor survival of C. michiganensis subsp. michiganensis in soil.
Assuntos
Actinobacteria/genética , DNA Bacteriano/genética , Genoma Bacteriano , Solanum lycopersicum/microbiologia , Actinobacteria/patogenicidade , Composição de Bases/genética , DNA Bacteriano/química , Eletroforese em Gel de Campo Pulsado , Genes Bacterianos/genética , Ilhas Genômicas/genética , Modelos Genéticos , Dados de Sequência Molecular , Óperon/genética , Plasmídeos/genética , Análise de Sequência de DNA , Serina Endopeptidases/genéticaRESUMO
Hybridization of Clavibacter michiganensis subsp. michiganensis total DNA against the pathogenicity gene pat-1 indicated the presence of pat-1 homologous nucleotide sequences on the chromosome and on plasmid pCM2. Isolation of the corresponding DNA fragments and nucleotide sequence determination showed that there are three pat-1 homologous genes: chpA (chromosome) and phpA and phpB (plasmid pCM2). The gene products share common characteristics, i.e. a signal sequence for Sec-dependent secretion, a serine protease motif, and six cysteine residues at conserved positions. Gene chpA located on the chromosome is a pseudogene since it contains a translational stop codon after 97 of 280 amino acids. In contrast to pat-1, cloning of the plasmid encoded homologs phpA and phpB into the avirulent plasmid free Cmm strain CMM100 did not result in a virulent phenotype. So far, no proteolytic activity could be demonstrated for Pat-1, however, site specific mutagenesis of pat-1 showed that the serine residue in the motif GDSGG is required for the virulent phenotype of pat-1 and thus Pat-1 could be a functional protease.
Assuntos
Actinomycetales/enzimologia , Actinomycetales/genética , Serina Endopeptidases/genética , Actinomycetales/patogenicidade , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , Sequência Conservada , DNA Bacteriano/química , DNA Bacteriano/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Plasmídeos/genética , Sinais Direcionadores de Proteínas/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Virulência/genéticaRESUMO
Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete. It infects tomato, spreads through the xylem and causes bacterial wilt and canker. The wild-type strain NCPPB382 carries two plasmids, pCM1 and pCM2. The cured plasmid-free derivative CMM100 is still able to colonize tomato, but no disease symptoms develop indicating that all genes required for successful infection, establishment and growth in the plant reside on the chromosome. Both plasmids carry one virulence factor, a gene encoding a cellulase, CelA in case of pCM1 and a putative serine protease Pat-1 on pCM2. These genes can independently convert the non-virulent strain CMM100 into a pathogen causing wilt on tomatoes. Currently, genome projects for Cmm and the closely related potato-pathogen C. michiganensis subsp. sepedonicus have been initiated. The data from the genome project shall give clues on further genes involved in plant-microbe interaction that can be tested experimentally. Especially, identification of genes related to host-specificity through genome comparison of the two subspecies might be possible.
