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Coryneform bacteria are frequently isolated from bovine mastitis with the lipophilic species, and Corynebacterium bovis is the most frequently isolated organism of this group. However, previous studies on the phylogeny of corynebacteria have incorporated only a single reference strain. We examined the phylogeny of C. bovis using 47 strains isolated from bovine mammary glands. Phylogenetic studies were performed by direct sequencing of the 16S ribosomal RNA and comparison to sequences of reference strains. All strains identified as C. bovis demonstrated similarity of 98% or higher to the ribosomal RNA gene sequences of the type strain of C. bovis. Phylogenetic analyses indicated that all strains tested clustered with members of the Corynebacterium urealyticum group confirming that C. bovis is a legitmate member of the genus Corynebacterium. Further investigation into the diversity within the species using repetitive element palindrome PCR indicated only minor differences between the strains tested. Corynebacterium bovis ATCC 13722 demonstrated the highest similarity (95%) with Brevibacterium helvolum, indicating that this organism does not belong in the genus Corynebacterium.

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Coryneform bacteria are frequently isolated from bovine mastitis and are associated with economic losses. Generally, the MICs of the 15 antimicrobial agents tested at which 90% of the isolates tested are inhibited for 46 Corynebacterium bovis and 13 Corynebacterium amylocolatum strains were low. These are the first quantitative antimicrobial susceptibility data available for coryneforms from bovine mastitis. Data from this study suggest that comparable corynebacteria from humans have a much higher level of antimicrobial resistance to a variety of antimicrobial agents.

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Bovine mastitis remains the most economically important disease in dairy cows. Corynebacterium bovis, a lipid-requiring Corynebacterium spp., is frequently isolated from the milk of infected mammary glands of dairy cows and is associated with reduced milk production. A total of 212 coryneform bacteria isolated from the milk of dairy cows were obtained from mastitis reference laboratories in the United States and Canada. All isolates had been presumptively identified as Corynebacterium bovis based on colony morphology and growth in the presence of butterfat. Preliminary identification of the isolates was based on Gram stain, oxidase, catalase, and growth on unsupplemented trypticase soy agar (TSA), TSA supplemented with 5% sheep blood, and TSA supplemented with 1% Tween 80. Of the 212 isolates tested, 183 were identified as Corynebacterium spp. based on preliminary characteristics. Of the strains misidentified, one was identified as a yeast, two as Bacillus spp., 11 as Enterobacteriaceae, 18 as staphylococci, one as a Streptococcus spp., and one as an Enterococcus spp. Eighty-seven coryneforms were selected for identification to the species level by direct sequencing of the 16S rRNA gene, the Biolog system and the API Coryne system. Fifty strains were identified as C. bovis by 16S rRNA gene similarity studies: the Biolog and API Coryne systems correctly identified 54.0 and 88.0% of these strains, respectively. The other coryneforms were identified as other Corynebacterium spp., Rhodococcus spp., or Microbacterium spp. These data indicate that the coryneform bacteria isolated from bovine mammary glands are a heterogeneous group of organisms. Routine identification of C. bovis should include Gram-stain, cell morphology, catalase production, nitrate reduction, stimulated growth on 1% Tween 80 supplemented media, and beta-galactosidase production as the minimum requirements.

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Zinc-regulated genes were analyzed in Pseudomonas fluorescens employing mutagenesis with a reporter gene transposon. Six mutants responded with increased gene expression to elevated concentrations of zinc. Genetic and biochemical analysis revealed that in four of the six mutants the transposon had inserted into genes essential for the biosynthesis of the siderophore pyoverdine. The growth of one of the mutants was severely impaired in the presence of elevated concentrations of cadmium and zinc ions. In this mutant, the transposon had inserted in a gene with high similarity to P-type ATPases involved in zinc and cadmium ion transport. Four mutants reacted with reduced gene expression to elevated concentrations of zinc. One of these mutants was sensitive to zinc, cadmium and copper ions. The genetic region targeted in this mutant did not show similarity to any known gene.

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To study the mechanisms soil bacteria use to cope with elevated concentrations of heavy metals in the environment, a mutagenesis with the lacZ-based reporter gene transposon Tn5B20 was performed. Random gene fusions in the genome of the common soil bacterium Pseudomonas fluorescens strain ATCC 13525 were used to create a bank of 5,000 P. fluorescens mutants. This mutant bank was screened for differential gene expression in the presence of the toxic metal cadmium. Fourteen mutants were identified that responded with increased or reduced gene expression to the presence of cadmium. The mutants were characterized with respect to their metal-dependent gene expression and their metal tolerance. Half the identified mutants reacted with differential gene expression specifically to the metal cadmium, whereas some of the other mutants also responded to elevated concentrations of copper and zinc ions. One of the mutants, strain C8, also showed increased gene expression in the presence of the solvent ethanol, but otherwise no overlap between cadmium-induced gene expression and general stress response was detected. Molecular analysis of the corresponding genetic loci was performed using arbitrary polymerase chain reaction (PCR), DNA sequencing and comparison of the deduced protein products with sequences deposited in genetic databases. Some of the genetic loci targeted by the transposon did not show any similarities to any known genes; thus, they may represent 'novel' loci. The hypothesis that genes that are differentially expressed in the presence of heavy metals play a role in metal tolerance was verified for one of the mutants. This mutant, strain C11, was hypersensitive to cadmium and zinc ions. In mutant C11, the transposon had inserted into a genetic region displaying similarity to genes encoding the sensor/regulator protein pairs of two-component systems that regulate gene expression in metal-resistant bacteria, including czcRS of Ralstonia eutropha, czrRS of Pseudomonas aeruginosa and copRS of Pseudomonas syringae. Although the P. fluorescens strain used in this study had not been isolated from a metal-rich environment, it nevertheless contained at least one genetic region enabling it to cope with elevated concentrations of heavy metals.

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Rhizopine (L-3-O-methyl-scyllo-inosamine; 3-O-MSI) synthesis (mos) and catabolism (moc) genes were originally isolated from Rhizobium meliloti strain L5-30 (Murphy et al., Proc. Natl. Acad. Sci. U.S.A., 84:493, 1987). These genes have been postulated to give a competitive advantage to this strain in the rhizosphere, since the ability to utilize the unusual nutritional mediator rhizopine as nitrogen and carbon source appears to be correlated with the ability of Moc+ bacteria to efficiently infect alfalfa plants. This study examines the distribution of rhizopine catabolism (moc) genes among different soil bacteria. By using oligonucleotide primers homologous to the moc genes and the polymerase chain reaction (PCR), moc genes were shown to be absent from a random collection of 100 different soil isolates. However, screening 50 different electrophoretic type strains of a worldwide R. meliloti collection (Eardly et al., Appl. Environ. Microbiol. 56:187, 1990) revealed the presence of moc genes in three additional strains, S33, 102F51, and 74B3. These three strains were found to be able to synthesize rhizopine in planta (Mos+) and to catabolize it (Moc+). To determine the relatedness of the Mos+/Moc+ strains to each other and to other R. meliloti strains, we used the rep-PCR method to generate genomic fingerprints, and to create a phylogenetic tree with the help of an optical imaging system and data analysis program (AMBIS). Because of the apparent infrequent occurrence of moc genes among soil bacteria, we suggest that the use of moc genes as a selectable marker trait for tracking genetically manipulated organisms is feasible.

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Bradyrhizobium japonicum possesses three soluble c-type cytochromes, c550, c552, and c555. The genes for cytochromes c552 (cycB) and c555 (cycC) were characterized previously. Here we report the cloning, sequencing, and mutational analysis of the cytochrome c550 gene (cycA). A B. japonicum mutant with an insertion in cycA failed to synthesize a 12-kDa c-type cytochrome. This protein was detectable in the cycA mutant complemented with cloned cycA, which proves that it is the cycA gene product. The cycA mutant, a cycB-cycC double mutant, and a cycA-cycB-cycC triple mutant elicited N2-fixing root nodules on soybean (Nod+ Fix+ phenotype); hence, none of these three cytochromes c is essential for respiration supporting symbiotic N2 fixation. However, cytochrome c550, in contrast to cytochromes c552 and c555, was shown to be essential for anaerobic growth of B. japonicum, using nitrate as the terminal electron acceptor.

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Rhizopine (L-3-O-methyl-scyllo-inosamine, 3-O-MSI) is a symbiosis-specific compound, which is synthesized in nitrogen-fixing nodules of Medicago sativa induced by Rhizobium meliloti strain L5-30. 3-O-MSI is thought to function as an unusual growth substrate for R. meliloti L5-30, which carries a locus (mos) responsible for its synthesis closely linked to a locus (moc) responsible for its degradation. Here, the essential moc genes were delimited by Tn5 mutagenesis and shown to be organized into two regions, separated by 3 kb of DNA. The DNA sequence of a 9-kb fragment spanning the two moc regions was determined, and four genes were identified that play an essential role in rhizopine catabolism (mocABC and mocR). The analysis of the DNA sequence and the amino acid sequence of the deduced protein products revealed that MocA resembles NADH-dependent dehydrogenases. MocB exhibits characteristic features of periplasmic-binding proteins that are components of high-affinity transport systems. MocC does not share significant homology with any protein in the database. MocR shows homology with the GntR class of bacterial regulator proteins. These results suggest that the mocABC genes are involved in the uptake and subsequent degradation of rhizopine, whereas mocR is likely to play a regulatory role.

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We report the cloning and nucleotide sequence analysis of the cytochrome c552 gene (cycB) of Bradyrhizobium japonicum strain 110. The gene was identified with help of an oligonucleotide that was designed on the basis of the amino acid sequence determined for purified cytochrome c552 of B. japonicum strain CC705. The cycB gene product has an N-terminal 23-amino acid signal peptide that is missing in the mature cytochrome c552 protein. A B. japonicum cycB insertion mutant was constructed which had no observable phenotypic defects in denitrification and symbiotic nitrogen fixation. Thus, the function of c552 remains unknown.

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A Bradyrhizobium japonicum Tn5 mutant (strain 3160) induced numerous, tiny, white nodules which were dispersed over the whole root system of its natural host plant, soybean (Glycine max). These ineffective, nitrogen non-fixing pseudonodules were disturbed at a very early step of bacteroid and nodule development. Subsequent cloning and sequencing of the DNA region mutated in strain 3160 revealed that the Tn5 insertion mapped in a gene that had 60% homology to the Escherichia coli glyA gene coding for serine hydroxymethyltransferase (SHMT; E.C.2.1.2.1.). SHMT catalyses the biosynthesis of glycine from serine and the transfer of a one-carbon unit to tetrahydrofolate. The B. japonicum glyA region was able to fully complement the glycine auxotrophy of an E. coli glyA deletion strain. Although the Tn5 insertion in B. japonicum mutant 3160 disrupted the glyA coding sequence, this strain was only a bradytroph (i.e. a leaky auxotroph). Thus, B. japonicum may have an additional pathway for glycine biosynthesis. Nevertheless, the glyA mutation was responsible for the drastic symbiotic phenotype visible on plants. It may be possible, therefore, that a sufficient supply with glycine and/or a functioning C1-metabolism are indispensable for the establishment of a fully effective, nitrogen-fixing root nodule symbiosis.

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We have cloned and characterized three distinct Rhizobium meliloti loci involved in glutamine biosynthesis (glnA, glnII, and glnT). The glnA locus shares DNA homology with the glnA gene of Klebsiella pneumoniae, encodes a 55,000-dalton monomer subunit of the heat-stable glutamine synthetase (GS) protein (GSI), and complemented an Escherichia coli glnA mutation. The glnII locus shares DNA homology with the glnII gene of Bradyrhizobium japonicum and encodes a 36,000-dalton monomer subunit of the heat-labile GS protein (GSII). The glnT locus shares no DNA homology with either the glnA or glnII gene and complemented a glnA E. coli strain. The glnT locus codes for an operon encoding polypeptides of 57,000, 48,000, 35,000, 29,000, and 28,000 daltons. glnA and glnII insertion mutants were glutamine prototrophs, lacked the respective GS form (GSI or GSII), grew normally on different nitrogen sources (Asm+), and induced normal, nitrogen-fixing nodules on Medicago sativa plants (Nod+ Fix+). A glnA glnII double mutant was a glutamine auxotroph (Gln-), lacked both GSI and GSII forms, but nevertheless induced normal Fix+ nodules. glnT insertion mutants were prototrophs, contained both GSI and GSII forms, grew normally on different N sources, and induced normal Fix+ nodules. glnII and glnT, but not glnA, expression in R. meliloti was regulated by the nitrogen-regulatory genes ntrA and ntrC and was repressed by rich N sources such as ammonium and glutamine.

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The ntrC locus of Agrobacterium tumefaciens C58 has been cloned using the Azorhizobium sesbaniae ORS571 ntrC gene as a DNA hybridization probe. Transposon Tn5 mutagenesis of the cloned ntrC locus was carried out and one Tn5 insertion within the region of highest DNA homology with A. sesbaniae ORS571 ntrC was used for gene replacement of the wild-type C58 ntrC gene. The A. tumefaciens ntrC::Tn5 mutant was found to be unable to grow on nitrate as sole nitrogen (N) source, to lack glutamine synthetase (GSII) activity and to be unable to use arginine (or ornithine) as sole N source, unless the Ti-encoded arginine catabolism pathway was induced with small amounts of nopaline. Thus the A. tumefaciens ntrC regulatory gene is essential for (transcriptional) activation of the GSII and nitrate reductase genes, as well as for the chromosomal but not the Ti-borne arginine catabolism pathways.

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