Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium species using the Sherlock Microbial Identification System.

Previous studies have demonstrated that cellular fatty acid analysis is a useful tool for identifying unknown strains of rhizobia and establishing taxonomic relationships between the species. In this study, the fatty acid profiles of over 600 strains belonging to the genera Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium were evaluated using the gaschromatography-based Sherlock Microbial Identification System (MIS). Data collected with the MIS showed that the three phylogenetically defined biovars of the genus Agrobacterium formed discrete clusters, whilst species belonging to the genus Mesorhizobium formed three subclusters which were easily distinguished. These three subclusters contained Mesorhizobium ciceri and Mesorhizobium mediterraneum, Mesorhizobium tianshanense fatty acid group I and Mesorhizobium plurifarium, and Mesorhizobium huakuii and Mesorhizobium loti. The genus Sinorhizobium was composed of an individual position for Sinorhizobium meliloti and a large cluster comprising Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium terangae, Sinorhizobium kostiense and Sinorhizobium arboris. S. meliloti contained significantly higher levels of the fatty acid 19:0 cyclo omega 8 cis and clustered with Rhizobium sp. (Hedysarum coronarium). However, discrimination between the species of genera Sinorhizobium and Rhizobium was a function of the concentration of 16:0 3-OH. The genus Rhizobium contained a single cluster containing Rhizobium sp. (Hedysarum coronarium), Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium etli, along with individual positions for Rhizobium giardinii, Rhizobium tropici, Rhizobium galegae and Rhizobium hainanense. R. tropici and R. hainanense exhibited similarity to Agrobacterium biovar 2, whilst R. galegae was similar to Agrobacterium biovar 1. R. giardinii appeared unique, with comparatively little similarity to the other species. Analysis of the genus Bradyrhizobium revealed large differences from the other genera studied. Two subgroups of Bradyrhizobium elkanii were detected and easily distinguished from Bradyrhizobium japonicum. Bradyrhizobium liaoningense and Bradyrhizobium sp. (Arachis hypogaea), a group isolated from Chinese peanut plants, showed similarities to B. japonicum, whilst a subgroup of M. tianshanense appeared identical to Bradyrhizobium sp. (Arachis hypogaea).

Isozyme variation of Microsporum canis and M. cookei from New Zealand.

Fifty-four isolates of Microsporum canis (Arthroderma otae) from humans, cats and dogs obtained from Auckland, Palmerston North and Wellington, New Zealand and 18 M. cookei and two Diheterospori spp. from soils were examined for variation using eight isozyme loci. M. canis isolates were from infected and non-infected cases. Isozyme analysis separated the three species which were further subdivided into electrophoretic types (ETs). Clustering analysis using normalized percentage disagreement (PTC) average linkage method revealed two clusters for M. cookei with two subclusters in cluster 2. M. canis had three main divisions (clusters 3, 4 and 5) and Diheterospora formed a separate division. The presence of isolates from different sources in the same clusters and lack of statistical significance as measured by confidence intervals suggests the existence of isolates with common lineage.

Isozyme variation within and among populations of Microsporum species.

Isozyme variation among 54 isolates of Microsporum canis, 18 Microsporum cookei isolates and two Diheterospora isolates were studied using starch gel electrophoresis. Of eight enzymes examined, four were polymorphic (EST, G6P, MDH and PEP), having from two to four electrophoretic forms. Within each species, consistent and reproducible isozyme patterns of the eight enzyme systems were obtained. Phenotypic diversity (H) in M. canis was higher than in M. cookei (H = 0.459 and H = 0.408 respectively), but phenotypic differentiation of M. canis isolates from different geographical regions (Auckland, Wellington and Palmerston North, New Zealand) was low, with a proportion of total diversity (Gst) of 0.151 found among the localities. The results suggest that the isolates of M. canis from different geographical regions are closely related, supporting the theory of a common lineage.

Identification of soil bacteria expressing a symbiotic plasmid from Rhizobium leguminosarum bv. trofolii.

A hundred strains of non-nodulating, Gram-negative, rod-shaped bacteria were isolated from clover-ryegrass pastures on three different soil types and from a sandy loam under lupins. When crossed with Escherichia coli PN200 containing the cointegrate plasmid pPN1, 11 transconjugants gained the ability to form nodules on the roots of white clover (Trifolium repens cv. Grasslands Huia). A nodA probe indicated that they had gained nodulation genes. The identities of these 11 strains and 4 others derived from earlier work on non-nodulating root nodule bacteria, were determined by ribotyping, DNA-DNA hybridization, and partial 16S rRNA sequencing. Good agreement was obtained between the three methods, and 11 of the strains were identified as Rhizobium leguminosarum (6), Rhizobium loti (2), Rhizobium etli (1), Rhizobium tropici (1), and Sinorhizobium meliloti (1). DNA-DNA hybridization indicated that the remaining four strains were related to the Rhizobium leguminosarum reference strains. The existence of several species of non-nodulating rhizobia in pasture soil, including species for which the normal host plant was absent, is discussed in relation to the fate of symbiotic plasmids from Rhizobium seed inoculants. It is also suggested that new species should be named for the geographical region from which they are first isolated rather than the host plant.

Molecular biology of lactococcal bacteriophage c2.

The 22163 bp genome of the lytic prolate-headed lactococcal phage c2 was fully sequenced. Mapping of restriction sites and RNA transcripts demonstrated the presence of early and late genes. Early and late promoters were identified. The early region contained 21 ORFs, with predicted protein products of 4.4-32.9 kDA, all reading right to left. Significant similarity was found between a putative protein encoded by an early region ORF and the erf (essential recombination function) gene product of Salmonella phage P22. The late genes for which a function has been identified, all of which read from left to right, included a possible holin gene, and genes encoding three major and six minor phage structural proteins. Analysis of the cohesive termini revealed complementary, non-symmetrical, 9-base single-stranded 3' extended DNAs. The exploitation of phage sequence data and analysis of phage genomes to find ways of inhibiting phage replication is discussed.

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2.

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3' extended DNAs, with the following sequence: 5'-GTTAGGCTT-3' 3'-CAATCCGAA-5'. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3' extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5' extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.

Expression of the symbiotic plasmid from Rhizobium leguminosarum biovar trifolii in Sphingobacterium multivorum.

An inoculant strain of Rhizobium leguminosarum biovar trifolii containing a Tn5 marked symbiotic plasmid transferred this plasmid by conjugation to Sphingobacterium multivorum, an organism that can be found in soil. The transconjugant bacteria nodulated the roots of white clover (Trifolium repens) seedlings but did not fix atmospheric nitrogen. Microscopic examination revealed abnormal nodule structures. Bacteria isolated from the nodules were shown to be closely related to the recipient S. multivorum and Southern blots of genomic digests probed with nodA DNA confirmed that the transconjugants contained symbiotic genes. This is the first report of the spontaneous transfer, by conjugation, of a symbiotic plasmid from R. leguminosarum biovar trifolii to S. multivorum.

Sequence analysis of the lysin gene region of the prolate lactococcal bacteriophage c2.

Approximately 80% of the genome of the prolate-headed lactococcal bacteriophage c2 was cloned into shuttle vectors pSA3 and pFX3 in Escherichia coli and transferred to Lactococcus lactis. A 1.67-kilobase EcoRV fragment containing the gene for the phage lysin was identified and the position and orientation of the phage lysin gene in the physical map of the phage were determined. The phage lysin was expressed in E. coli and its sequence was determined and compared with the sequences of other bacteriophage lytic genes. The sequence was similar, but not identical, to that of the related lactococcal phage m13, having a number of silent substitutions and an apparent deletion that altered the carboxy terminus of the protein. Possible alternative translation initiation codons for the lysin gene and two possible alternative mechanisms for access of the lysin enzyme to the cell wall are discussed. An open reading frame upstream of the putative lysin gene was found to be 177 base pairs longer than that reported for phage m13. A codon usage table for the lysin genes of several phages as well as for reported gene sequences from L. lactis and lactococcal bacteriophages is presented.

Genetic characterization of pathogenic Leptospira species by DNA hybridization.

A total of 66 serovars of potentially pathogenic Leptospira species were examined by slot blot hybridization, and 57 of these serovars were classified in six DNA homology groups. In cases in which common serovars were studied, the results were in general agreement with the results of previous workers, who used different DNA homology methods. However, we propose a new species, Leptospira kirschneri, comprising the following serovars: bulgarica, butembo, cynopteri, dania, grippotyphosa, kabura, kambale, ramisi, and tsaratsovo. Seven of these serovars have not had their DNAs studied by other workers.

Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.

Species differentiation of Leptospira interrogans serovar hardjo strain Hardjobovis from strain Hardjoprajitno by DNA slot blot hybridisation.

Slot blot hybridisation studies with total genomic DNA probes were used to compare Leptospira interrogans serovar hardjo strain Hardjoprajitno, strain Hardjobovis and a number of other Leptospira interrogans serovars. Strains Hardjoprajitno and Hardjobovis were found to have little genetic relationship with each other when compared to some of the other serovars tested. Hardjoprajitno is closely related to serovar icterohaemorrhagiae and not to Hardjobovis whereas Hardjobovis is closely related to serovars vietnam, balcanica and javanica but not to serovar icterohaemorrhagiae; this places strain Hardjoprajitno in the species L interrogans and strain Hardjobovis in the species L borgpetersoni. Because of this lack of genetic relatedness between strains Hardjoprajitno and Hardjobovis, it is proposed to remove the prefix Hardjo from the strain name Hardjobovis and call it L borgpetersoni serovar hardjo strain Bovis.

DNA relatedness among strains of Leptospira biflexa.

The slot blot method of DNA hybridization was used to study 38 strains of Leptospira biflexa belonging to 38 serovars. Fifteen of these serovars were placed into six groups. The remaining 23 serovars were generally too diverse to show significant DNA relatedness either to these groups or to one another. Serovar thracia was related to Group 5, but it was not included in this group because its percent relatedness was too low. We found that genetically related organisms were antigenically dissimilar. The absence of any significant genetic relationship between Leptonema illini and the Leptospira biflexa serovars tested supports the placement of the former species in a separate genus.

Isolation and characterization of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus.

A Rhizobium loti gene required for effective invasion of the host Lotus pedunculatus has been identified by transposon Tn5 mutagenesis. Cosmids that complemented a previously isolated mutation (239) at this invasion (inv) locus were identified by in planta complementation and used to construct a physical map of the gene region. The insertion site of Tn5 in PN239 was mapped to a 7.5-kb EcoRI fragment, which complemented the mutation when subcloned into pLAFR1. Further Tn5 mutagenesis of the 7.5-kb fragment was carried out in Escherichia coli using bacteriophage lambda 467, and the mutations homogenotized into R. loti NZP2037. Three additional Fix- mutations were isolated, and these were found to map adjacent to the position of the original mutation in strain PN239. All the other Tn5 insertions isolated in the 7.5-kb fragment gave a Fix+ phenotype on L. pedunculatus. Electron microscopic examination of the L. pedunculatus nodules induced by the isolated Fix- mutants showed that bacteria were either blocked in release from the infection threads or were unable to undergo normal bacteroid development. The inv locus as defined by the Tn5 insertions was sequenced, and a single open-reading frame (ORF) of 576 bp, corresponding to a polypeptide of 21.3 kDa, was identified. The position and orientation of this ORF were consistent with those of the isolated Tn5 Fix- insertions.

Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii.

Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr514a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.

Analysis of the Symbiotic Performance of Bradyrhizobium japonicum USDA 110 and Its Derivative I-110 and Discovery of a New Mannitol-Utilizing, Nitrogen-Fixing USDA 110 Derivative.

Previously, Bradyrhizobium japonicum USDA 110 was shown to contain colony morphology variants which differed in nitrogen-fixing ability. Mannitol-utilizing derivatives L1-110 and L2-110 have been shown to be devoid of symbiotic nitrogen fixation ability, and non-mannitol-utilizing derivatives I-110 and S-110 have been shown to be efficient at nitrogen fixation. The objectives of this study were to determine the effect of media carbon sources on the symbiotic N(2)-fixing ability of strain USDA 110 and to compare the effectiveness of strain USDA 110 and derivative I-110. Based on acetylene reduction activity and the nitrogen content of 41-day-old soybean plants, neither derivative I-110 nor cultures of USDA 110 grown in media favoring non-mannitol-using derivatives had symbiotic nitrogen fixation that was statistically superior to that of cultures of USDA 110 grown in media favoring mannitol-using derivatives. In another experiment 200 individual nodules formed by strain USDA 110 grown in yeast extract gluconate were screened for colony morphology of occupying variant(s) and acetylene reduction activity. Nodules occupied by mannitol-using derivatives (large colony type on 0.1% yeast extract-0.05% K(2)HPO(4)-0.08% MgSO(4) . 7H(2)O-0.02% NaCl-0.001% FeCl(3) . 6H(2)O [pH 6.7] with 1% mannitol [YEM] plates) had a mean acetylene reduction activity equal to that of nodules occupied by non-mannitol-using derivatives (small colony type on YEM plates). A total of 20 large colonial derivatives and 10 small colonial derivatives (I-110-like) were isolated and purified by repeated culture in YEM and YEG (same as YEM except 1% gluconate instead of 1% mannitol) media, respectively, followed by dilution in solutions containing 0.05% Tween 40. After 25 days of growth, soybean plants inoculated with the large colony isolates had mean whole-plant acetylene reduction activity, whole-plant dry weight, and whole-plant nitrogen contents equal to or better than those of plants inoculated with either the small colony isolates (I-110-like) or the I-110 (non-mannitol-using) derivative. Hence, the existence of a mannitol-utilizing derivative that fixes nitrogen in a culture of strain USDA 110 obtained from the U.S. Department of Agriculture, Beltsville, Md., was established. This new USDA 110 derivative was designated as MN-110 because it was a mannitol-utilizing nitrogen-fixing USDA 110 derivative. This derivative was morphologically indistinguishable from the non-nitrogen-fixing derivative L2-110 found in cultures obtained earlier from the U.S. Department of Agriculture, Beltsville. DNA-DNA homology and restriction enzyme analyses indicated that MN-110 is genetically related to other USDA 110 derivatives that have been characterized previously.

Isolation and characterization of transposon Tn5-induced symbiotic mutants of Rhizobium loti.

Rhizobium loti NZP2037 and NZP2213, each cured of its single large indigenous plasmid, formed effective nodules on Lotus spp., suggesting that the symbiotic genes are carried on the chromosome of these strains. By using pSUP1011 as a vector for introducing transposon Tn5 into R. loti NZP2037, symbiotic mutants blocked in hair curling (Hac), nodule initiation (Noi), bacterial release (Bar), and nitrogen fixation (Nif/Cof) on Lotus pedunculatus were isolated. Cosmids complementing the Hac, Noi, and Bar mutants were isolated from a pLAFR1 gene library of NZP2037 DNA by in planta complementation and found to contain EcoRI fragments of identical sizes to those into which Tn5 had inserted in the mutants. The cosmids that complemented the mutants of these phenotypic classes did not share common fragments, nor did cosmids that complemented four mutants within the Noi class, suggesting that these symbiotically important regions are not tightly linked on the R. loti chromosome.