The phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt and canker of tomato, harbors two plasmids, pCM1 (27.35 kb) and pCM2 (72 kb), encoding genes involved in virulence (D. Meletzus, A. Bermpohl, J. Dreier, and R. Eichenlaub, 1993, J. Bacteriol. 175:2131-2136; J. Dreier, D. Meletzus, and R. Eichenlaub, 1997, Mol. Plant-Microbe Interact. 10:195-206). The region of pCM1 carrying the endoglucanase gene celA was mapped by deletion analysis and complementation. RNA hybridization identified a 2.4-knt (kilonucleotide) transcript of the celA structural gene and the transcriptional initiation site was mapped. The celA gene encodes CelA, a protein of 78 kDa (746 amino acids) with similarity to endo-beta-1,4-glucanases of family A1 cellulases. CelA has a three-domain structure with a catalytic domain, a type IIa-like cellulose-binding domain, and a C-terminal domain. We present evidence that CelA plays a major role in pathogenicity, since wilt induction capability is obtained by endoglucanase expression in plasmid-free, nonvirulent strains and by complementation of the CelA- gene-replacement mutant CMM-H4 with the wild-type celA gene.

The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt, harbors two plasmids pCM1 (27.5 kb) and pCM2 (72 kb). After curing of the plasmids, bacterial derivatives were still proficient in the ability to colonize the host plant and in the production of exopolysaccharides but exhibited a reduced virulence. When one of the two plasmids is lost, there is a significant delay in the development of wilting symptoms after infection and a plasmid-free derivative is not able to induce disease symptoms. By cloning of restriction fragments of both plasmids in the plasmid-free strain CMM100, two DNA fragments which restored the virulent phenotype were identified. Further analysis suggested that a fragment of plasmid pCM1 encodes an endocellulase which is involved in the expression of the pathogenic phenotype.

A major 30.5-kb cluster of nif and associated genes of Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus), a nitrogen-fixing endophyte of sugarcane, was sequenced and analyzed. This cluster represents the largest assembly of contiguous nif-fix and associated genes so far characterized in any diazotrophic bacterial species. Northern blots and promoter sequence analysis indicated that the genes are organized into eight transcriptional units. The overall arrangement of genes is most like that of the nif-fix cluster in Azospirillum brasilense, while the individual gene products are more similar to those in species of Rhizobiaceae or in Rhodobacter capsulatus.Biological nitrogen fixation occurs in species of more than 100 genera distributed among several of the major phylogenetic divisions of prokaryotes (Eubacteria and Archaea) (25). Sequence and mutational analyses of the genes necessary for nitrogen fixation (nif) in many diazotrophs indicate that their products have common structures and functions, while the degree of linkage and arrangement of specific nif and associated genes vary considerably (5,8,17). In addition, nif genes and genes involved in plant invasion and nitrogen fixation effectiveness, such as nod and fix in species of Rhizobiaceae, are often linked.The identification of nitrogen-fixing bacteria with endophytic habitats raises the possibility of a new classification of symbiosis (3). The relationship of a proteobacterial ␣ group member, Acetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus), with sugarcane represents a promising model system for the study of an association between a monocot and an endophytic nitrogen-fixing bacterium (13,22). The ability of A. diazotrophicus to enhance sugarcane growth has been documented, and while the benefit to plant growth might be due at least in part to the transfer of bacterially fixed N, another plant growth-stimulating factor(s) is indicated, possibly auxin production by A. diazotrophicus (22). Because of its potential agronomic use and unique status as the only diazotrophic species of Acetobacter so far identified, it was of interest to isolate and characterize genes that are involved in nitrogen fixation and regulation.Identification of a major cluster of nif and associated genes. A genomic library of A. diazotrophicus constructed in the widehost-range cosmid pLAFR3 (Tet r ) (4) was transferred by conjugation from Escherichia coli to several different nif mutants of Azotobacter vinelandii (12). Two mutants that yielded Nif ϩ Tet r transconjugants were DJ71 (nifV) and DJ35 (nifE), and several transconjugants of both strains carried the same cosmid, pAD71, the 22-kb insert fragment of which carries nifD through mcpA (Fig.

The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, causing bacterial wilt and canker, harbors two plasmids, pCM1 (27.5 kb) and pCM2 (72 kb), carrying genes involved in virulence. The region of plasmid pCM2 encoding the pathogenicity locus pat-1 was mapped by deletion analysis and complementation studies to a 1.5-kb Bg/II/SmaI DNA fragment. Introduction of the pat-1 region into endophytic, plasmid-free isolates of C. michiganensis subsp. michiganensis converted these bacteria into virulent pathogens. Based on the nucleotide sequence of the pat-1 region, an open reading frame (ORF1) can be predicted, coding for a protein of 280 amino acids and 29.7 kDa with homology to serine proteases. Introduction of a frame-shift mutation in ORF1 leads to a loss of the pathogenic phenotype. Northern (RNA) hybridizations identified an 1.5-knt transcript of the pat-1 structural gene. The site of transcription initiation was mapped by primer extension and a typical -10/-35 region was located with significant homology to the consensus Escherichia coli sigma 70 and Bacillus subtilis sigma 43 promoters. Downstream of the pat-1 structural gene, a peculiar repetitive sequence motif (pat-1rep) is located, consisting of 20 direct tandem repeats preceded by a run of 14 guanosine residues. DNA sequences homologous to pat-1rep were isolated and characterized from four virulent C. michiganensis subsp. michiganensis strains exhibiting a high extent of structural conservation. The deletion of this repetitive sequence reduced virulence significantly but did not lead to a complete loss of the virulence phenotype.

We constructed a cloning vector for use in the plant pathogenic bacterium Clavibacter michiganense subsp. michiganense. The vector pDM100 consists of a 3.2-kb restriction fragment of the Clavibacter plasmid pCMl joined to a pBR325 derivative carrying the neomycin phosphotransferase of transposon TnS and the gentamicin acetyltransferase of Tnl696. Both antibiotic resistance genes are efficiently expressed in C. michiganense subsp. michiganense. Although polyethylene glycol-mediated transfection of spheroplasts with the DNA of the C. michiganense subsp. michiganense-specific bacteriophage CMP1 yielded about 3 x 103 transfectants per ,ug of DNA, in transformations with plasmid DNA only a very few transformants were obtained. However, the transformation efficiency could be improved by electroporation of intact cells, giving about 2 x 103 transformants per ,ug of plasmid DNA. Since a transformation procedure and a cloning vector are now available, pathogenicity in C. michiganense subsp. michiganense can now be analyzed genetically.