Biovar Strain Research Articles

A study of Pseudomonas fluorescens biovars using the Automated Microbiology Identification System (AMBIS)

The AMBIS is a system which can determine the relationships between microbial strains by comparing the profiles produced after their radiolabelled intracellular proteins are subjected to SDS PAGE. This system was used to compare the profiles of strains representing the five biovars of Pseudomonas fluorescens, a species implicated in food spoilage. The three strains of biovar I, three strains of biovar III and two strains of biovar IV segregated into three distinct clusters with correlation coefficients (cc) of 0·85, 0·85 and 0·87 respectively. Although two of the biovar II strains studied clustered together (cc = 0·74) one of the remaining biovar II strains linked (cc = 0·83) with the cluster of biovar IV strains while the other was linked with biovar I and V strains (cc = 0·68). Biovar V strains (three in total) also failed to form a single cluster which was expected since this biovar is known to be heterogeneous. The findings are in substantial agreement with more comprehensive taxonomic studies of this species. AMBIS may be a useful tool in taxonomic studies of micro-organisms.

Occurrence of plasmids in Danish isolates of Vibrio anguillarum serovars O1 and O2 and association of plasmids with phenotypic characteristics

Two hundred and twenty-eight isolates of Vibrio anguillarum serovar O1 (125 isolates) and serovar O2 (103 isolates) have been characterized with regard to plasmid contents, biochemical properties, and in vitro hemagglutination and hydrophobic properties. Among 74 V. anguillarum isolates from diseased fish, 63 carried only a 67-kb plasmid (pJM1), 9 carried an additional 98-kb plasmid, and 1 isolate carried only the 98-kb plasmid. Only one isolate was without plasmids. In V. anguillarum serovar O1 from nondiseased fish (mucus and gills), plasmids of the same sizes were present in 29 isolates (58%), whereas 21 isolates (42%) were plasmid free. Based on hemagglutination and biochemical properties, V. anguillarum serovar O1 isolates were divided into eight biovars. The plasmid-carrying strains (102 isolates) all fell within biovars 1 and 2, whereas the 23 strains of biovars 3 to 8 were without plasmids. It was tentatively concluded there are two populations of V. anguillarum serovar O1. One population contains plasmid(s), is hemagglutination negative and trehalose negative, and does not form pellicles in broth cultures, whereas the other population is plasmid free and has the opposite characteristics. The former group is the one related to disease in fish. All 20 V. anguillarum serovar O2 isolates from the environment were without plasmids, whereas 54 (65%) of the isolates from fish (trout and cod) carried plasmids. The biochemical diversity within serovar O2 was pronounced; 13 different biovars were demonstrated. No correlation between the presence of plasmids and biochemical properties was observed.

Formation and structure of root nodules induced on Macroptilium atropurpureum inoculated with various species of Rhizobium

Fifteen strains of Rhizobium leguminosarum biovar trifolii formed ineffective nodules and (or) nodule-like structures (rhizobia were re-isolated from both structures) on Macroptilium atropurpureum grown in enclosed glass tubes. Bacteria were observed among the parenchyma cells surrounding the nodule-like structures. One variant of R. leguminosarum biovar trifolii (NGR66/ST) isolated from M. atropurpureum formed nodules on this host that exhibited abnormal intercellular and intracellular infection. The bacteria (NGR66/ST) were contained within threadlike structures, surrounded by matrix material. The identities of the Rhizobium strains were confirmed serologically after reisolation and in sections of nodule tissue using immunogold labelling. Rhizobium leguminosarum biovar phaseoli strain NGR76 isolated from Phaseolus vulgaris formed nodules on M. atropurpureum resembling those formed by effective Bradyrhizobium strains. The association was partially effective in nitrogen fixation, and this was reflected in the nodule structure. The percentage of cells infected was lower than that in fully effective nodules. There was a high frequency of infected cells showing degeneration; these were located throughout the nodule tissue and were often adjacent to healthy infected cells. The rhizobia appeared to infect new nodule cells via infection threads, which were abundant both intercellularly and intracellularly in young, mature, and degenerating host nodule cells. Strains of R. leguminosarum biovar viceae and Rhizobium meliloti were unable to induce nodule-like structures on M. atropurpureum. Key words: Macroptilium, Bradyrhizobium, Rhizobium, microscopy, nodule, structure.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of smooth-lipopolysaccharide heterogeneity among Brucella biovars related to A and M specificities

Smooth (S)-lipopolysaccharide (LPS) preparations from reference and field strains of several biovars of Brucella abortus, B. melitensis, and B. suis were prepared by (i) the hot phenol-water method, (ii) hot sodium dodecyl sulfate extraction and proteinase K digestion, or (iii) dimethyl sulfoxide extraction. These S-LPS-enriched fractions were further analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining after periodate oxidation. Immunoblots were developed by using either monoclonal antibodies specific for Brucella A or M antigens or polyclonal polyspecific or monospecific sera from rabbits, cattle, and goats. The specificity of monoclonal antibodies reactive with Brucella unique (A or M) epitopes was demonstrated by enzyme-linked immunosorbent assay, LPS latex agglutination, or agglutination inhibition. The most-represented subunits of S-LPS ranged in Mr from 30,000 to 70,000 relative to marker proteins. According to A or M immunodominance, two sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns were clearly distinguished among biovars, whatever the fraction tested: a close succession of regularly spaced narrow bands for A greater than M strains and regularly spaced triplets of bands including either (i) a first thin band followed by two thick bands for B. abortus M greater than A strains or (ii) one thick band between two thin bands for B. melitensis or B. suis M greater than A strains. Moreover, A and M specificities were reaffirmed by sandwich enzyme immunoassay and latex agglutination inhibition with monoclonal antibodies and polyclonal sera.

Comparison of dna:dna homology and enzymatic activity between Pasteurella haemolytica and related species

SUMMARY A commercially available microbiological identification system and dna:dna hybridization were used to determine relationships between and within serovars 1-13 of Pasteurella haemolytica, and between P haemolytica and P multocida and 4 species of Actinobacillus. All serovars of P haemolytica that belonged to biovar A were related with mean dna homology of 78%, whereas all serovars of P haemolytica that belonged to biovar T were related to each other with mean dna homology of 90%. The dna:dna hybridization between strains of biovars A and T ranged from 3 to 13%, indicating little or no genetic relationship between the 2 biovars of P haemolytica. The dna homology between all serovars of P haemolytica and other species of non-P haemolytica bacteria tested (P multocida and actinobacilli) was < 14%, suggestive of essentially no genetic relationship of P haemolytica with the atcc reference strains of the genus Pasteurella or the genus Actinobacillus. Enzymatic differences were observed between P haemolytica and the other non-P haemolytica bacteria tested; however, the microbiological identification system that uses enzymatic reactions could not distinguish among biovars of P haemolytica. Results of this research support other data that suggest that biovars A and T of P haemolytica should be classified as separate species, but do not support the inclusion of either biovar A or T within the genus Actinobacillus.

Variation in nitrate metabolism in biovars of Pseudomonas solanacearum

A collection of 327 strains of Pseudomonas solanacearum, representing five biovars, was divisible into three groups on the basis of differences in nitrate metabolism. Nine strains (2.8%), of which seven were biovar 2 from bacterial wilt of potato, were nitrate reduction‐deficient and failed to produce nitrite from nitrate by either of two methods of detection in five different media. A second group of 231 strains, comprising biovars 1 and 2 and a single biovar 3 strain, produced nitrite from nitrate and grew vigorously in the presence of nitrate under anaerobic conditions but were deficient in ability to denitrify. A third group comprising 57 strains of biovar 3, 28 of biovar 4 and one each of biovar 2 and 5 produced nitrite from nitrate and gave profuse growth and gas production from nitrate under anaerobic conditions. However, production of gas from nitrate (denitrification) was not a consistently reproducible property in some of the media tested. Gas production results were most reproducible when a semi‐solid succinate/nitrate or glycerol/nitrate medium was used. Serial passage of four nitrate reduction‐deficient isolates in nitrate medium did not restore ability to reduce nitrate.

The effect of temperature on the development of wilting and on progeny tuber infection of potatoes inoculated with South African strains of biovar 2 and 3 ofPseudomonas solanacearum

Potato plants, after inoculation with a South African strain of biovar 2 or a strain of biovar 3 ofPseudomonas solanacearum were grown in growth chambers at temperatures ranging from 14/16°C (dark/light) up to 25/30°C. When grown at 14/16°C or higher, plants inoculated with the biovar 2 strain developed wilting symptoms and internal symptoms on progeny tubers and the disease was transmitted by the progeny tubers. When the plants were inoculated with the biovar 3 strain, wilting symptoms developed from 18/20°C, internal symptoms on progeny tubers from 20/22°C and the disease was transmitted by progeny tubers from plants grown at 16/18°C or higher.

Recognition of Biovar C of Fusobacterium necrophorum (Flugge) Moore and Holdeman as Fusobacterium pseudonecrophorum sp. nov., nom. rev. (ex Prevot 1940)

The cellular morphology, colonial morphology, biochemical properties, DNA base compositions, and DNA-DNA homolgies of three biovars of Fusobacterium necrophorum were examined. Some differences were found among the three biovars in cellular morphology, colonial morphology, and biochemical properties. The guanine-plus-cytosine contents of DNAs from biovar C strains Fn521T (T = type strain), Fn522, and Fn520 were 30.4, 29.3, and 28.0 mol%, respectively, and the guanine-plus-cytosine contents of DNAs from strains VPI 2891 (biovar A) and VPI 6161 (biovar B) were 31.3 and 32.0 mol%, respectively. Labeled DNA from biovar C strain Fn521T exhibited 96 and 82% relatedness to DNAs from biovar C strains Fn522 and Fn520, respectively; however, it exhibited only about 10% relatedness to DNAs from strains of biovars A and B. Labeled DNAs from strains VPI 2891 and VPI 6161 exhibited more than 70% relatedness to each other, but about 6 to 20% relatedness to DNAs from biovar C strains. Therefore, Fusobacterium pseudonecrophorum sp. nov., nom. rev. (ex Prévot 1940) is proposed for Fusobacterium necrophorum biovar C. The type strain is strain Fn521 (= JCM 3722).

Spread and Persistence of Infection with a Trachoma Biovar Strain of Chlamydia trachomatis in Multiplying and Nonmultiplying McCoy Cells

Spread and Persistence of Infection with a Trachoma Biovar Strain of Chlamydia trachomatis in Multiplying and Nonmultiplying McCoy Cells

Spread and Persistence of Infection with a Trachoma Biovar Strain of Chlamydia trachomatis in Multiplying and Nonmultiplying McCoy Cells

The trachoma biovar of Chlamydia trachomatis enters host cells in culture with difficulty, and cell-to-cell spread resulting in amplification of the initial growth usually does not occur. An experimental model was devised to demonstrate that, by using conditions that more closely approximate those encountered in man, the trachoma biovar of C. trachomatis can readily achieve cell-to-cell passage. Fresh McCoy cells were sequentially added to monolayers that had been inoculated with a trachoma biovar strain of C. trachomatis 3, 6, and 9 days earlier. Subsequent incubation resulted in intercellular propagation, with an increase in the number of inclusions from 500 to 80,000 per coverslip. A second experiment demonstrated the reproducibility of this phenomenon and showed that cell-to-cell spread can occur at a low rate in overcrowded, not overlaid, cell layers; it also showed that, in multiplying cell layers, the infection tends to become persistent.

Acetylene reduction, hydrogen evolution and nodule respiration in Phaseolus vulgaris

In an experiment performed under greenhouse conditions, four cultivars of Phaseolus vulgaris L. (Venezuela-350; Aroana; Moruna; Carioca) were inoculated with three Rhizobium leguminosarum biovar phaseoli strains (C-05; C-40 = CIAT 255; C-89 = CIAT 55) and were fertilized with an N-free mineral nutrient solution. The plants were harvested 25, 40, and 55 days after emergence and the following paramenters were evaluated: Nitrogenase activity of nodulated roots, H2 evolution by the nodules; relative efficiency of nitrogenase; respiration rates of nodulated roots and detached nodules; dry weight and total N of stems, leaves, pods, roots, and nodules. Generally the bean cultivar, Rhizobium strain, had an effect and there was an interaction effect with both symbiotic partners, on all parameters. On average, nodules represented 23% of total root respiration but the best symbiotic combinations showed lower ratios of C respired to N fixed. The maximum N-assimilation rate (between 40 and 55 days after emergence) of 11.93 mg N plant−1 day−1 occurred with the symbiotic combination of Carioca × C-05, while the poorest rate of 0.55 mg N plant−1 day−1 was recorded with Venezuela-350 × C-89. The best symbiotic combinations always showed the highest relative nitrogenase efficiency, but the differences in N2-fixation rates cannot be explained solely in terms of conservation of energy by recycling of H2. This requires further investigation.

Detection of Surface-exposed Epitopes on Chlamydia trachomatis by Immune Electron Microscopy

The cell surfaces of two Chlamydia trachomatis serovars were explored by immune electron microscopy with monoclonal antibodies that recognize a number of chlamydial outer-membrane components. Species, subspecies and serovar-reactive epitopes on the major outer-membrane protein (MOMP) of a lymphogranuloma venereum biovar strain, L2/434/Bu, and a trachoma biovar strain, F/UW-6/Cx, were exposed on the surfaces of both elementary bodies (EBs) and reticulate bodies (RBs). Three epitopes on MOMP were inaccessible on EBs and RBs of both strains. These included a genus-reactive, species-reactive, and a subspecies-reactive epitope. In contrast, genus-specific epitopes on lipopolysaccharide (LPS) were not detected on the EB surface, but were clearly expressed on RBs of both L2/434/Bu and F/UW-6/Cx chlamydiae. Antibodies specific for the 60 kDa and 12 kDa 'cysteine-rich' outer-membrane proteins did not react with surface epitopes on either EBs or RBs. These data provide evidence that MOMP is a major surface antigen of both morphological forms, whereas some portions of the LPS molecule are exposed on the RB surface but become inaccessible to antibody after conversion to the infectious EB form.

DNA polymorphism in strains of the genus Brucella.

Preparations of DNA from 23 Brucella strains including 19 reference strains were compared by restriction endonuclease analysis. Pulsed-field gel electrophoresis resulted in optimal resolution of fragments generated by digestion with low-cleavage-frequency restriction enzymes such as XbaI. By this technique, five electrophoretypes were distinguished in five reference strains of the different species, i.e., B. abortus, B. melitensis, B. suis, B. canis, and B. ovis. Minor profile differences allowed us to discriminate between most biovars within a species. However, the differences in the DNA patterns of different field strains of biovar 2 of B. melitensis were not sufficient to serve as markers for epidemiological studies. From the XbaI fragments, we were able to estimate the size of the genomes of B. abortus 544T and B. melitensis 16 MT. This method revealed a relationship between DNA fingerprints, species, and pathovars which could shed light on problems concerning the classification and evolution of members of the genus Brucella.

Host Specific Nodulation of Plants of the Pea Cross-inoculation Group is Influenced by Genes in Fast Growing Rhizobium downstream nodC

Summary Tn5 induced mutants in nodI and nodJ of the Rhizobium leguminosarum biovar viceae Sym plasmid pRL1JI nodulated Vicia sativa and Vicia hirsuta plants slightly delayed. Complementation experiments showed that nodI,J were part of the nodA,B,C transcriptional unit. A Rhizobium strain in which two Sym plasmids were present, almost completely lost the ability to nodulate V sativa plants when nodI,J on both Sym plasmids were inactivated. Several other plants of the pea cross-inoculation group showed a likewise difference in their nodulation response. In addition we investigated the nodulation of Afghanistan peas. R. leguminosarum biovar viceae strains do not nodulate Afghanistan peas. When a Sym plasmid from such a strain was transferred to wild type R. leguminosarum biovar trifolii strains, the resulting strains nodulated Afghanistan peas. We provide evidence for the existence of a new gene, necessary for the nodulation of Afghanistan peas, which is located in the nodA,B,C,I,J operon of R. leguminosarum biovar trifolii strain ANU843.

Hydrophobicity ofFusobacterium necrophorumbiovars A and B

Biovar A strains of Fusobacterium necrophorum exhibited high hydrophobicity when examined by the method of Rosenberg et al. Biovar B strains showed a lower cell surface hydrophobicity than biovar A strains. Biovar B strains were divided into 2 groups according to their hydrophobic activity. The strains of biovar A and the first group of biovar B were increasingly removed from aqueous phase to octane phase by increasing the volume of octane, but the turbidity of the second group of biovar B was not significantly affected. The hydrophobicity of biovar A strains decreased on heating at 60 and 100°C for 30 min.

Hydrophobicity of Fusobacterium necrophorum biovars A and B

Biovar A strains of Fusobacterium necrophorum exhibited high hydrophobicity when examined by the method of Rosenberg et al. Biovar B strains showed a lower cell surface hydrophobicity than biovar A strains. Biovar B strains were divided into 2 groups according to their hydrophobic activity. The strains of biovar A and the first group of biovar B were increasingly removed from aqueous phase to octane phase by increasing the volume of octane, but the turbidity of the second group of biovar B was not significantly affected. The hydrophobicity of biovar A strains decreased on heating at 60 and 100°C for 30 min.

The low-molecular-mass, cysteine-rich outer membrane protein of Chlamydia trachomatis possesses both biovar- and species-specific epitopes.

We isolated, by hydroxylapatite high-performance liquid chromatography, 14- and 15-kilodalton (kDa), cysteine-rich outer membrane proteins from Chlamydia trachomatis TW-5/OT (serovar B) and LGV-434 (serovar L2), respectively. Monoclonal antibodies (MAbs) were generated against the purified proteins, and their specificities were determined by immunoblotting. MAb B-14k recognized an epitope located on the 14-kDa cysteine-rich protein of the TW-5/OT strain and was immunoreactive with a comigrating 14-kDa protein that was common to all trachoma biovar strains, but it did not react with the 15-kDa, cysteine-rich protein of LGV biovar strains. In contrast, MAb L2-15k, which recognized an epitope located on the 15-kDa protein of the LGV-434 strain, reacted with the 15- and 14-kDa, cysteine-rich proteins of both LGV and trachoma biovar strains, but did not react with related proteins of two Chlamydia psittaci strains. Thus, the low-molecular-mass, cysteine-rich outer membrane proteins of C. trachomatis possess antigenic determinants that are both biovar and species specific. Neither MAbB-14k nor MAb L2-15k was reactive by dot-blot assay when viable chlamydiae were used as test antigens, indicating that the cysteine-rich proteins are not accessible to antibody on the native chlamydial cell surface.

Characteristics of the yellow pigment from a strain of Yersinia enterocolitica biovar 1

Yersinia enterocolitica 195A14J, a milk-isolated strain of biovar 1, produces a non-diffusible yellow pigment and forms star-shaped colonies when grown on egg-yolk agar at 28°C. Solubility properties and in situ Raman spectrum of the pigment support evidence that it is not a carotenoid, although it contains a 9(±) double-bond polyenic chain. Pigmentless variants, also star-shaped appeared with a frequency of ca.10 −3 when bacteria were grown at 38°C. Agarose gel electrophoresis of DNA extracted from the pigmented strain revealed the presence of a 42-kb plasmid which was lost in pigmentless variants.

Differences in outer membrane proteins of the lymphogranuloma venereum and trachoma biovars of Chlamydia trachomatis

The lymphogranuloma venereum (LGV) and trachoma biovars of Chlamydia trachomatis exhibit differences in biological properties both in vivo and in vitro. To identify analogous biochemical differences, we studied the molecular charges of chlamydial outer membrane proteins (OMPs) by means of isoelectric focusing and nonequilibrium pH gradient electrophoresis. Analysis of proteins of whole elementary bodies biosynthetically labeled with L-[35S]cysteine revealed that most chlamydial proteins were neutral or acidic. The major OMPs (MOMPs) of all strains tested were acidic and had apparent isoelectric points (pIs) that varied within narrow limits (approximately 5.3 to 5.5) despite differences in molecular mass of up to 3,000 daltons (Da). However, a low-molecular-mass cysteine-rich OMP analogous to that previously described for Chlamydia psittaci varied consistently in molecular mass (12,500 versus 12,000 Da) and pI (5.4 versus 6.9) between LGV strains and trachoma strains, respectively. OMPs with a molecular mass of 60,000 Da in the trachoma biovar strains had pIs in the 7.3 to 7.7 range. However, analogous OMPs in the LGV strains existed as a doublet with a molecular mass of about 60,000 Da. Both members of the doublet were basic (pIs greater than 8.5). Both proteins of this basic doublet in LGV strains and the neutral analog in trachoma strains bound a species-specific monoclonal antibody in an immunoblot assay. These data indicate substantial differences in biochemical characteristics of analogous OMPs in the LGV and trachoma biovars. Such differences are the first structural differences described between LGV and trachoma strains which support their distinction into separate biovars and may be related to some of their biological differences.

Plasmid in Fusobacterium necrophorum.

Nine strains of Fusobacterium necrophorum biovars A, B, and C were examined for the presence of plasmid by agarose gel electrophoresis. Although none of biovars A and B possessed extrachromosomal deoxyribonucleic acids, all the strains of biovar C examined so far contained a single plasmid with a molecular weight of 118 megadaltons. This suggests the presence of plasmid may link to the biological variation in the F.necrophorum species.