Characterization of a major cluster of nif, fix, and associated genes in a sugarcane endophyte, Acetobacter diazotrophicus.

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.

The endo-beta-1,4-glucanase CelA of Clavibacter michiganensis subsp. michiganensis is a pathogenicity determinant required for induction of bacterial wilt of tomato.

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.

Identification, sequencing and structural analysis of a nifA-like gene of Acetobacter diazotrophicus.

A recombinant plasmid, pAD101, containing a DNA fragment of Acetobacter diazotrophicus strain PAL5 was isolated by its ability to restore Nif+ phenotype to a nifA- ntrC- double mutant of Azotobacter vinelandii. Hybridization with the nifA genes of Azospirillum brasilense located the nifA gene more precisely to specific fragments of pAD101. DNA sequencing of appropriate subclones of pAD101 revealed that the nifA gene was adjacent to the nifB gene in A. diazotrophicus, and the 5' end of the nifB gene was located downstream of the nitrogenase MoFe subunit gene, nifK. The deduced aminoacid sequence of A. diazotrophicus nifA and nifB gene were most similar to the NifA and NifB proteins of Azorhizobium caulinodans and Rhodobacter capsulatus, respectively. In addition, nucleotide sequences upstream of the A. diazotrophicus nifA-encoding region indicate features similar to those in the A. caulinodans nifA promoter region involved in O2 and fixed N regulation of nifA expression.

Identification, sequencing and structural analysis of a nifA-like gene of Acetobacter diazotrophicus

A recombinant plasmid, pAD101, containing a DNA fragment of Acetobacter diazotrophicus strain PAL5 was isolated by its ability to restore Nif+ phenotype to a nifA- ntrC- double mutant of Azotobacter vinelandii. Hybridization with the nifA genes of Azospirillum brasilense located the nifA gene more precisely to specific fragments of pAD101. DNA sequencing of appropriate subclones of pAD101 revealed that the nifA gene was adjacent to the nifB gene in A. diazotrophicus, and the 5' end of the nifB gene was located downstream of the nitrogenase MoFe subunit gene, nifK. The deduced aminoacid sequence of A. diazotrophicus nifA and nifB gene were most similar to the NifA and NifB proteins of Azorhizobium caulinodans and Rhodobacter capsulatus, respectively. In addition, nucleotide sequences upstream of the A. diazotrophicus nifA-encoding region indicate features similar to those in the A. caulinodans nifA promoter region involved in O2 and fixed N regulation of nifA expression.

Characterization of the glnK-amtB operon of Azotobacter vinelandii.

To determine whether in Azotobacter vinelandii the PII protein influences the regulation of nif gene expression in response to fluxes in the ammonium supply, the gene encoding PII was isolated and characterized. Its deduced translation product was highly similar to PII proteins from other organisms, with the greatest degree of relatedness being exhibited to the Escherichia coli glnK gene product. A gene designated amtB was found downstream of and was contranscribed with glnK as in E. coli. The AmtB protein is similar to functionally characterized ammonium transport proteins from a few other eukaryotes and one other prokaryote. glnK and amtB comprise an operon. Attempts to isolate a stable glnK mutant strain were unsuccessful, suggesting that glnK, like glnA, is an essential gene in A. vinelandii. amtB mutants were isolated, and although growth on limiting amounts of ammonium was similar in the mutant and wild-type strains, the mutants were unable to transport [14C]methylammonium.

Characterization of the plasmid encoded virulence region pat-1 of phytopathogenic Clavibacter michiganensis subsp. michiganensis.

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.

Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors.

In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, and pHN216 were stably maintained without antibiotic selection in various strains of C. michiganensis subsp. sepedonicus. We observed that for a single plasmid, different strains of C. michiganensis subsp. sepedonicus showed significantly different transformation efficiencies. We also found unexplained strain-to-strain differences in stability with various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication. We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C. michiganensis subsp. sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained was 4.6 x 10(4) CFU/microgram of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels.

Evidence for plasmid-encoded virulence factors in the phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382.

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.

Transformation of the phytopathogenic bacterium Clavibacter michiganense subsp. michiganense by electroporation and development of a cloning vector.

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 pCM1 joined to a pBR325 derivative carrying the neomycin phosphotransferase of transposon Tn5 and the gentamicin acetyltransferase of Tn1696. 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 10(3) transfectants per microgram 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 10(3) transformants per microgram 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.