Comparative study of Agrobacterium biotypes 1, 2 and 3 by electrophoresis and serological methods

A comparative study by electrophoresis and serology of strains representing the three Agrobacterium biotypes was carried out. Thirteen Spanish isolates and strains from international collections were included. Ten antisera were prepared by using strains from the three biotypes and different types of antigens. The strains were studied by immunodiffusion, indirect immunofluorescence and indirect ELISA. Serological relationship among all the strains was observed, although serological heterogeneity within each of the biotypes occurred. Biotype 3 appears as serologically related to biotypes 1 and 2, having an intermediate position. This observation is in agreement with their biochemical characteristics. Electrophoretic analysis of the three biotypes showed that there was high variability. Three main bands appeared in the six strains studied. One specific band occurred in the biotype 1 strains and another in the biotype 3 strains.     

Identification of oral Neisseria species of animals

Ninety-seven strains of presumptive Neisseria spp. obtained from the dental plaque of a wide variety of animals and 21 strains from culture collections were compared by physiological tests, enzyme electrophoresis and isoelectric focusing of proteins. Three physiological groups based on the fermentation of maltose and production of extracellular polysaccharide were established. The ability of strains within these groups to reduce nitrate and nitrite differed. The electrophoretic mobility of dehydrogenases and isoelectric focusing patterns of proteins were not, however, characteristic of species or physiological groups. It is difficult, therefore, to identify new isolates of Neisseria because the criteria for describing species overlap. A rapid spot test was devised to distinguish Branhamella catarrhalis from other Neisseria by the absence of glucose-6-phosphate dehydrogenase.     

RAPD fingerprinting for the distinction of Prevotella intermedia sensu stricto from Prevotella nigrescens

A collection of 70 oral strains including reference strains and clinical isolates identified as Prevotella intermedia sensu lato was constituted to cover a large clinical and geographical diversity. Electrophoresis of the enzyme malate dehydrogenase allowed the identification of the 70 study strains as Prevotella intermedia sensu stricto (n= 36), Prevotella nigrescens (n= 31) and three unclassified strains. By using four primers, DNA fingerprints were generated from 20 strains as random amplified polymorphic DNA (RAPD). Matching co-migrating amplicon positions by pairwise comparison allowed the clustering of the fingerprints as two groups coincident with the P. intermedia/P. nigrescens assignment by enzyme electrophoresis of malate dehydrogenase. Our data suggest that isolates identified asP. intermedia sensu lato by conventional criteria can be speciated asP. intermedia sensu stricto or P. nigrescens by RAPD fingerprinting.     

Purification and properties of formate dehydrogenase from Moraxella sp. strain C-1

NAD+-dependent formate dehydrogenase was screened in various bacterial strains. Facultative methanol-utilizing bacteria isolated from soil samples, acclimated to a medium containing methanol and formate at pH 9.5, were classified as members of the genus Moraxella. From a crude extract of Moraxella sp. strain C-1, formate dehydrogenase was purified to homogeneity, as judged by disc gel electrophoresis. The enzyme has an isoelectric point of 3.9 and a molecular weight of approximately 98,000. The enzyme is composed of two identical subunits with molecular weights of about 48,000. The apparent Km values for sodium formate and NAD+ were calculated to be 13 mM and 0.068 mM, respectively.     

Occurrence of megaplasmids in halobacteria

Sixty-five halobacteria, including culture collection and freshly isolated strains from widely differing geographical areas, were examined for the presence of high molecular weight plasmids by agarose gel electrophoresis. Seventy-five per cent of all the strains were shown to harbour at least one plasmid. In the majority of strains three or four megaplasmids were detected. Approximate molecular weights of the plasmids were in the range < 100 to 300 megadaltons (Mdal). In most culture collection strains, two or three plasmids were demonstrated, except in two in which no plasmid was detected, and in two Haloarcula strains which were found to contain five and eight plasmids; four and six of the latter were more than 100 Mdal. No relationship between the plasmid profile of each strain and its taxonomic assignation nor its isolation source was found. Evidence is presented for the first time on the occurrence of megaplasmids in halobacteria.     

Reproductive biology of the Pteridophyta IV. An experimental study of mating systems in Ceratopteris thalictroides (L.) Brongán

Gametophytes of the fern Ceratopteris thalictroides originating from four different geographic areas were studied morphologically and genetically. Intraspecific variability was found among the collections for gametophyte morphology and the frequency of lethal sporophytic genotypes. The differences in gametophyte morphology were shown to result in a different mating system for each collection.     

Karyotype polymorphism correlates with intraspecific infertility in the homothallic ascomycete Sordaria macrospora

The homothallic fungus Sordaria macrospora produces perithecia with meiotically derived ascospores. In most cases, intraspecies crosses between strains from different culture collections generate fertile hybrid perithecia in the contact zone of two mycelia. However, in some of these crosses we observed a significant decrease in the fertility of the hybrid perithecia when strains of different origin were used for mating. Since we assumed that chromosome variability between the culture collection strains might contribute to this reduction in fertility, we performed pulsed‐field gel electrophoresis. In the course of our study, we were able to identify two major groups of electrophoretic karyotypes in S. macrospora culture collection strains. A quantitative analysis revealed that polymorphic karyotypes contribute to a reduction of fertility in forced crosses between strains carrying differently sized chromosomes. The observed intraspecific chromosome length polymorphism might have consequences on the speciation process of a homothallic fungus capable of sexual but not of asexual spore formation.     

Structural heterogeneity of the lipopolysaccharides of the Neisseriaceae

Lipopolysaccharides from 5 different genera of the Neisseriaceae were analyzed on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and visualized by silver staining. Significant heterogeneity in the banding patterns was observed with some of the strains producing only low molecular mass molecules and others producing O-repeating units. All genera examined except Branhamella contained strains that were able to produce an O-repeating side chain on their lipopolysaccharides. The ability to produce the repeating subunit did not correlate with the presence of plasmids.     

Characterization of Enterobacteria by Starch-Gel Electrophoresis of Glucose-6-Phosphate Dehydrogenase and Phosphogluconate Dehydrogenase

Specific activities and electrophoretic mobilities of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase were determined in 38 isolates of the family Enterobacteriaceae and in 10 isolates of the related Pasteurella. The deficiency of glucose-6-phosphate dehydrogenase in P. pestis was verified. Enzymes obtained from different strains of the same species exhibited an unexpected degree of heterogeneity. For example, 8 and 11 apparent variants of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase, respectively, were found in 14 strains of Escherichia coli. Although similar frequencies of heterogeneity were noted in 7 strains of P. pseudotuberculosis, 5 species of Shigella, and 8 species of Salmonella, differences in mobility were generally small in comparison with those observed between strains of E. coli. Values obtained for the pasteurellae, shigellae, and salmonellae, thus fell within narrow ranges that may prove typical for the genera. However, most of these ranges, as well as many values observed for single species of other genera, were overlapped by the wide range recorded for E. coli. The significance of this observation was discussed with respect to the relative age and taxonomic position of the organisms in question. The method could be used to distinguish between most wild-type strains of the same species and should thus facilitate investigations of genetic transfer and epidemiology.     

Chromosome sizes and phylogenetic relationships between serotypes of Actinobacillus pleuropneumoniae

The genome size of Actinobacillus pleuropneumoniae was determined by pulsed field gel electrophoresis of AscI and ApaI digested chromosomal DNA. The genome size of the type strain 4074^T (serotype 1) was determined to be 2404±40 kb. The chromosome sizes for the reference strains of the other serotypes range between 2.3 and 2.4 Mb. The restriction pattern profiles of AscI, ApaI and NheI digested chromosomes showed a high degree of polymorphism among the different serotype reference strains and allowed their discrimination. The analysis of the macrorestriction pattern polymorphism revealed phylogenetic relationships between the different serotype reference strains which reflect to some extent groups of serotypes known to cross-react serologically. In addition, different pulsed fields gel electrophoresis patterns also revealed heterogeneity in the chromosomal structure among different field strains of serotypes 1, 5a, and 5b, while strains of serotype 9 originating from most distant geographical places showed homogeneous ApaI patterns in pulsed field gel electrophoresis.     

Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reductase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonas fluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition.     

Genetic Diversity of Xanthomonas arboricola pv. juglandis (synonyms: X. campestris pv. juglandis;X. juglandis pv. juglandis) Strains from Different Geographical Areas shown by Repetitive Polymerase Chain Reaction Genomic Fingerprinting

A world-wide collection of 61 Xanthomonas arboricola pv. juglandis strains, isolated from Persian walnut ( Juglans regia L.) or obtained from international culture collections and bacterial plant diseases laboratories, were studied by means of repetitive polymerase chain reaction (PCR) genomic fingerprinting using ERIC, BOX and REP primer sets and polyacrylamide gel electrophoresis. Cluster analyses were performed by UPGMA . Copper resistance, ability to hydrolize starch and quinate metabolism of the strains was also assessed. Pathogenicity was tested by inoculating leaves and nuts of Persian walnut seedlings. Polyacrylamide gel electrophoresis allowed very clear and reproducible differentiation of the PCR products. Cluster analysis showed the existence of three major groups of strains. The first two groups were 85% genetically similar, whereas the third clustered at 78% similarity with the other two. Each group could be divided into two subgroups which clustered according to the geographical origin of the isolates. In some cases, different genomic profiles were shown by strains from one country. This is possibly due to Persian walnut cultivation being mainly based on ecotypes and/or local seedlings that have become adapted to particular environments and so have allowed selection of different X.a . pv. juglandis populations. All strains were pathogenic and positive in starch hydrolysis and quinate metabolism tests. This is the first record of copper-resistant strains occurring outside California, USA.     

Diversity and effectiveness of rhizobial strains from <Emphasis Type="Italic">Gliricidia sepium</Emphasis> native to Reunion Island,Kenya and New Caledonia

The aim of this study was to use several approaches to assess the diversity occuring in a collection of rhizobial strains that had been isolated from Gliricidia sepium (Jacq.) Walp. originating from Kenya, Reunion Island and New Caledonia. Results showed that G. sepium establishes an effective symbiosis only with fast-growing rhizobia. This suggests that this legume nodulates preferentially with this type of rhizobia, although ineffective nodules were observed on root systems of plants inoculated with slow-growing rhizobia. It was possible to distinguish several groups of strains which have the same behaviour in terms of utilization of amino acids and sources of carbon and intrinsic resistance to antibiotics and salinity. Six molecular groups were distinguished by profiles obtained using PCR/RFLP techniques. No relationship was demonstrated between phenotypic groups and molecular groups. Neither was it possible to find a relationship between the geographic origins of the strains and their distribution in the several phenotypic and/or molecular groups. In an experiment carried out under glasshouse conditions, we observed that all strains from the collection significantly improved the growth of G. sepium cultivated in a substrate totally devoid of nitrogen. The strain GsK5 induced the greatest stimulation of plant growth.     

Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria.

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reductase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonas fluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition.     

Identification of Paramecium bursaria syngens through molecular markers--comparative analysis of three loci in the nuclear and mitochondrial DNA

This is the first attempt to resolve the phylogenetic relationship between different syngens of Paramecium bursaria and to investigate at a molecular level the intraspecific differentiation of strains originating from very distant geographical locations. Herein we introduce a new collection of five P. bursaria syngens maintained at St Petersburg State University, as the international collection of syngens was lost in the 1960s. To analyze the degree of speciation within Paramecium bursaria, we examined 26 strains belonging to five different syngens from distant and geographically isolated localities using rDNA (ITS1-5.8S-ITS2-5'LSU) fragments, mitochondrial cytochrome c oxidase subunit I (COI), and H4 gene fragments. It was shown that P. bursaria strains of the same syngens cluster together in all three inferred molecular phylogenies. The genetic diversity among the studied P. bursaria strains based on rDNA sequences was rather low. The COI divergence of Paramecium bursaria was also definitely lower than that observed in the Paramecium aurelia complex. The nucleotide sequences of the H4 gene analyzed in the present study indicate the extent of genetic differences between the syngens of Paramecium bursaria. Our study demonstrates the diagnostic value of molecular markers, which are important tools in the identification of Paramecium bursaria syngens.     

On the detection of functional and structural enzyme mutants by coordinated affinity chromatography and isoelectric focusing

A method of studying structural and functional heterogeneity of enzymes has been developed and tested on chymotrypsin. The enzyme, prepared from single mouse pancreata, has been fractionated with respect to function and charge content by a combination of affinity chromatography and isoelectric focusing. By comparing chymotrypsin isolated from isogenic strains, chymotrypsinogen of strains A/Sn and NZB was found to be genetically heterogeneous, thus not revealed as different chymotrypsin forms of a single zymogen. Chymotrypsinogen originating from two loci was investigated, and structural and functional differences of the corresponding enzymes were determined. At both loci, structural allelomorphism was indicated. At one locus, the structural heterogeneity was also found to be reflected in functional heterogeneity of the corresponding enzymes. By mating the two strains and fractionating the enzyme of the cross, the differences were shown to be inherited.     

Compositional analysis of Helicobacter pylori rough-form lipopolysaccharides.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the macromolecular heterogeneity of lipopolysaccharides (LPS) from seven fresh clinical isolates and three culture collection strains of the human pathogen Helicobacter pylori. All the clinical isolates produced smooth-form LPS with O side chains of relatively homogeneous chain length, whereas the culture collection strains yielded rough-form LPS. A better yield of the latter LPS was obtained when combined protease pretreatment and hot phenol-water extraction were used than when the conventional phenol-water technique alone was used for extraction. The LPS of the three culture collection strains (S-24, C-5437, and NCTC 11637) were chemically characterized. Constituents common to all the LPS were fucose, D-mannose, D-glucose, D-galactose, D-glycero-D-manno-heptose, L-glycero-D-manno-heptose, and 3-deoxy-D-manno-2-octulosonic acid. The molar ratios of the hexoses differed between different strains, thereby reflecting structural differences. Phosphate, phosphorylethanolamine, and pyrophosphorylethanolamine were present also. Free lipid A contained D-glucosamine and fatty acids, with phosphate and a minor amount of ethanolamine. The major fatty acids were ester- and amide-bound 3-hydroxyoctadecanoic acid and ester-bound octadecanioc and 3-hydroxyhexadecanoic acids, with minor amounts of ester-bound tetradecanoic and hexadecanoic acids. In addition to the uncommonly long 3-hydroxy fatty acids, an unusual phosphorylation pattern was deduced to be present in the lipid A.     

Charge heterogeneity of heat-labile enterotoxins from human enterotoxigenic Escherichia coli

Abstract We purified heat-labile enterotoxins (LThs) from YT3, H-10407 and YT240 strains isolated from human diarrheal patients. These LThs were immunologically identical to each other. The molecular weights of their A and B subunits were also the same by means of SDS-polyacrylamide gel electrophoresis. However, the ionic charges of the molecular surfaces of these LThs were different as shown by polyacrylamide gel isoelectric focusing. Though the p I points of B subunits of the LThs were identical to each other, the p I points of A subunits were found to be different. These data suggest that the ionic charge differences among A subunits cause differences in holo LThs in their charge, and that there is heterogeneity among A subunits produced by strains of human enterotoxigenic Escherichia coli .     

Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Morocco

AIMS: To determine the biodiversity of rhizobial strains nodulating Cicer arietinum L. in representative soils from various areas of Morocco. METHODS AND RESULTS: Symbiotic traits, utilization of 49 carbohydrate sources, resistance to antibiotics and heavy metals, tolerance to salinity, to extreme temperatures and pH were studied as phenotypic markers. In addition, restriction fragment length polymorphism (RFLP) of PCR-amplified 16S rDNAs were compared with those of reference strains. Numerical analysis of the phenotypic characteristics showed that the 48 strains studied fell into three distinct groups. RFLP analysis of 16S rRNA genes revealed an additional heterogeneity and four ribotypes were identified. CONCLUSIONS: Chickpea rhizobia isolated from Moroccan soils are both phenotypically and genetically diverse. Most of these rhizobia belong to the Mesorhizobium genus. However, some strains originating from a particular soil appeared to have 16S rRNA genes similar to Sinorhizobium as well as very distinct auxanographic characteristics compared with Mesorhizo- bium isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: A well characterized collection of chickpea-nodulating rhizobia in representative soils of Morocco has been generated, which can be used to develop efficient inoculants for this crop. This is the first report evidencing that chickpeas may be nodulated by bacteria from the Sinorhizobium genus.