Genetic diversity of a natural population of Sinorhizobium meliloti, detected during analysis of a cryptic plasmid and ISRm2011-2 fingerprints]

Fifty-six natural strains of alfalfa nodule bacteria were isolated from samples of the soil under wild legume and alfalfa in two different field sites of Irkutsk oblast. Based on the results of analysis of plasmid profile, 11 different types of strains were detected, and 43 types were identified based on the results of hybridization with the insertion sequence element ISRm2011-2. Significant differences were found in the plasmid profile and IS fingerprints between strains isolated from the soil under alfalfa and the soil under legume. In contrast, strains growing at some distance from each other differed only in the IS fingerprints. From a comparison of results obtained in the assessment of plasmid profile and in analysis of IS fingerprints with results of RFLP analysis in strains, the conclusion about the transference of cryptic plasmids between strains and genetic rearrangements in strains of this population was drawn.     

Relative genetic structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of alfalfa (Medicago sativa) and sweet clover (Melilotus alba)

Insertion sequence (IS) hybridization was used to define the structure of a population of Rhizobium meliloti isolated directly from soil and from nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown under controlled conditions and inoculated with a suspension of the same soil. The detection of R. meliloti isolated from soil on agar plates was facilitated by use of a highly species specific DNA probe derived from ISRm5. All R. meliloti obtained directly from soil proved to be symbiotic (i.e. nodulated and fixed nitrogen with alfalfa). Analysis of 293 R. meliloti isolates revealed a total of 17 distinct IS genotypes of which 9, 9 and 15 were from soil, M. alba and M. sativa, respectively; 8 genotypes were common to soil and both plant species. The frequency of R. meliloti genotypes from soil differed markedly from that sampled from nodules of both legume species: 5 genotypes represented about 90% of the isolates from soil whereas a single genotype predominated among isolates from nodules accounting for more than 55% of the total. The distribution of genotypes differed between M. sativa and M. alba indicating species variation in nodulation preferences for indigenous R. meliloti. The data are discussed in the context of competition for nodulation of the host plant and the selection of Rhizobium strains for use in legume inoculants. This study has ecological implications and suggests that the composition of R. meliloti populations sampled by the traditionally used host legume may not be representative of that actually present in soil.     

Distribution of Symbiotic Genotypes in Rhizobium leguminosarum biovar viciae Populations Isolated Directly from Soils

The distribution of symbiotic (Sym) plasmid types across background genotypes was investigated in two field populations of Rhizobium leguminosarum biovar viciae isolated directly from soils. PCR-based methods were used to characterize the background genotypes and the Sym gene types. Identical Sym gene types were associated with a variable range of background genotypes, while the same background genotype could harbor distinct Sym gene types. Random distributions of Sym gene types in the background genotypes were observed in the two soil populations. These results suggest that Sym plasmid transfer is less restricted than previously thought on the basis of the analysis of strains isolated from legume nodules.     

Characterization of Salmonella virchow phage types by plasmid profile and IS200 distribution

The type strains of the 57 phage types of Salmonella virchow have been characterized by plasmid profile and by distribution of the insertion sequence IS 200 . Thirty-two strains carried plasmids and 21 profile types were identified; 17 strains were resistant to antimicrobial agents. In contrast only six of the type strains carried IS 200 elements and three patterns were identified. Within Salm. virchow phage type 31, five of 10 wild-type isolates carried plasmids and two plasmid profiles were identified; in contrast, an IS 200 element was identified in the genome of only one of these strains. It is concluded that for Salm. virchow , IS 200 is unlikely to significantly extend the degree of discrimination achieved by phage typing which may be supplemented when appropriate by plasmid profile typing.     

Identification of nodule-dominant Rhizobium meliloti strains carrying pRmeGR4b-type plasmid within indigenous soil populations by PCR using primers derived from specific DNA sequences

Abstract: Rhizobium meliloti strain GR4 is a highly infective and competitive bacteria which was isolated in 1975 from a field site in Granada (Spain) and which has a high potential as an inoculant. R. meliloti isolates from alfalfa plants grown in this field site were characterized using polymerase chain reaction. Characterization was based on primers derived from insertion sequence elements (IS Rm3 and IS Rm4 ), plasmid origin of replication (pRmeGR4a repC locus) and plasmid pRmeGR4b specific DNA sequences. Soil isolates harbouring plasmid type pRmeGR4b represented the major infective population in this field site. A direct correlation between the presence of pRmeGR4b-like plasmid and the competitiveness of the strains was found. In addition, four different R. meliloti field populations isolated from Spanish soils were analyzed for the presence of pRmeGR4b related plasmids. Our results indicate that this plasmid type is widespread among R. meliloti field populations and that its frequency within the infective isolates depends on the host plant.     

Restriction Fragment Length Polymorphism Analysis of Epidemiologically Related Mycobacterium tuberculosis Isolates

Restriction fragment length polymorphism (RFLP) analysis of a large number of Japanese isolates of Mycobacterium tuberculosis, containing isolates from small outbreaks of M. tuberculosis infection, and clinical isolates of M. bovis BCG, was carried out using a DNA probe derived from the insertion sequence IS986. Clinical isolates of M. tuberculosis had a high degree of RFLP. The occurrences of the IS element varied from 1 to 19, the majority of isolates having 8 to 15 copies. Very similar fingerprints, however, were seen among strains isolated in the Kanto district. In particular, 3 strains were of the same pattern with or without an additional band. Similarity of the banding patterns of strains islated in the same district was observed in other areas. Six groups of strains, each group arising from a suspected common source of infection, were analyzed. Of these, 5 showed identical fingerprints within each group, but one showed different fingerprints. RFLP patterns of three strains isolated from individuals with lymphadenitis developed about two months after BCG vaccination, and one strain isolated from a bladder cancer patient with BCG instillation therapy were identical to those of BCG-Tokyo which had been used for the vaccination and therapy. These results confirm that RFLP analysis using IS986 is a suitable tool for epidemiology of tuberculosis.     

Symbiotic competence, genetic diversity and plasmid profiles of Egyptian isolates of a Rhizobium species from Leucaena leucocephala (Lam.) Dewit

L.D. KUYKENDALL, D.M. SWELIM, F.M. HASHEM, S.M. ABDEL-WAHAB AND N.I. HEGAZI. 1996. There is considerable interest in improving nitrogen fixation in tropical legume trees to increase soil fertility, particularly in developing countries. To provide information needed for the development of improved strains, characterization of strains of Leucaena -nodulating Rhizobium was performed. Thirteen strains were isolated from root nodules of Leucaena leucocephala grown in different geographical regions in Egypt. Plasmid DNA profile groups, including identification of symbiosis-controlling plasmids, were defined. Symbiotic competence was determined in plant tests and some strains were perhaps more symbiotically proficient at fixing nitrogen with L. leucocephala than were reference strains. The genetic diversity of these strains was determined by RFLP analysis of total DNAs using, as probes, six arbitrarily selected cosmid clones from a gene library of strain TAL 1145, a well-characterized reference strain. There were four plasmid profile groups which did not correlate with either symbiotic competence or RFLP analysis. RFLP analysis, unlike plasmid profiles, permitted a determination that all 13 Egyptian strains were evidently homogeneous and quite distinct from previously studied Leucaena -nodulating Rhizobium as represented by reference strains.     

Strain typing with IS200 fingerprints in Salmonella abortusovis.

A collection of Salmonella abortusovis isolates was examined for the presence of insertion element IS200. All proved to contain three or four copies of the element. One IS200 hybridization band of approximately 9 kb was found in all isolates, indicating that all S. abortusovis strains carry an IS200 element in similar or identical locations; this band can be potentially useful for serovar identification. S. abortusovis collection isolates from distinct geographic areas were highly polymorphic, suggesting that IS200 fingerprints might provide information on the geographic origin of S. abortusovis strains. Isolates obtained from the same geographic area (the island of Sardinia, Italy) were less polymorphic: all shared three constant IS200 hybridization bands, indicating that they derive from a single ancestor. Most strains analyzed contained an additional copy of IS200 in the variable region of the virulence plasmid. Certain Sardinian flocks proved to be infected by only one S. abortusovis strain, while others harbored two strains. Strain typing with IS200 fingerprints proved to be more reliable than plasmid analysis, because the latter yielded a high degree of polymorphism, even among isolates from the same flock.     

Comparison of Parental and Transgenic Alfalfa Rhizosphere Bacterial Communities Using Biolog GN Metabolic Fingerprinting and Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR)

Abstract Rhizosphere bacterial communities of parental and two transgenic alfalfa (Medicago sativa L.) of isogenic background were compared based on metabolic fingerprinting using Biolog GN microplates and DNA fingerprinting of bacterial communities present in Biolog GN substrate wells by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR). The two transgenic alfalfa expressed either bacterial (Bacillus licheniformis) genes for alpha-amylase or fungal (Phanerochaete chrysosporium) genes for Mn-dependent lignin peroxidase (Austin S, Bingham ET, Matthews DE, Shahan MN, Will J, Burgess RR, Euphytica 85:381–393). Cluster analysis and principal components analysis (PCA) of the Biolog GN metabolic fingerprints indicated consistent differences in substrate utilization between the parental and lignin peroxidase transgenic alfalfa rhizosphere bacterial communities. Cluster analysis of ERIC-PCR fingerprints of the bacterial communities in Biolog GN substrate wells revealed consistent differences in the types of bacteria (substrate-specific populations) enriched from the rhizospheres of each alfalfa genotype. Comparison of ERIC-PCR fingerprints of bacterial strains obtained from substrate wells to substrate community ERIC-PCR fingerprints suggested that a limited number of populations were responsible for substrate oxidation in these wells. Results of this study suggest that transgenic plant genotype may affect rhizosphere microorganisms and that the methodology used in this study may prove a useful approach for the comparison of bacterial communities. Received: 1 June 1998; Accepted: 20 October 1998     

Attenuation of Symbiotic Effectiveness by Rhizobium meliloti SAF22 Related to the Presence of a Cryptic Plasmid

Several wild-type strains of Rhizobium meliloti isolated from alfalfa nodules exhibited different plasmid profiles, yet did not differ in growth rate in yeast-mannitol medium, utilization of 43 different carbon sources, intrinsic resistance to 14 antibiotics, or detection of 16 enzyme activities. In contrast, three measures of effectiveness in symbiotic nitrogen fixation with alfalfa (shoot length, dry weight, and nitrogen content) indicated that R. meliloti SAF22, whose plasmid profile differs from those of the other strains tested, is significantly less effective than other wild-type strains in symbiotic nitrogen fixation. Light microscopy of nodules infected with strain SAF22 showed an abnormal center of nitrogen fixation zone III, with bacteria occupying a smaller portion of the infected host cells and vacuoles occupying a significantly larger portion of adjacent uninfected host cells. In contrast, the effective nodules infected with other wild types or plasmid pRmSAF22c-cured segregants of SAF22 did not display this cytological abnormality.     

Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors.

On the basis of an RSF1010-derived broad-host-range vector, three different systems which enable positive detection and isolation of insertion sequence (IS) elements from gram-negative bacteria were constructed. Vectors pSUP104-pheS, pSUP104-rpsL, and pSUP104-sac were used successfully in a number of Rhizobium strains and in Xanthomonas campestris. More than 20 different IS elements were isolated and characterized. The 16 IS elements from Rhizobium meliloti were further used to characterize various R. meliloti strains by hybridization. The resulting hybridization patterns were different for every strain and gave a clear and definite IS fingerprint of each strain. These IS fingerprints can be used to identify and characterize R. meliloti strains rapidly and unequivocally, as they proved to be relatively stable. Some of the IS elements were found to be identical when the IS fingerprints from a given strain were compared. This method of IS fingerprinting can also establish whether IS elements are the same, related, or different.     

Distribution of two Agrobacterium tumefaciens insertion elements in natural isolates: Evidence for stable association between Ti plasmids and their bacterial hosts

Summary A large number of Agrobacterium tumefaciens strains of the biotype III group carry two to ten copies of two related IS elements, IS866 and IS867. A study of the distribution and localization of these elements in 54 strains showed that one IS866 and two IS867 copies are always found at characteristic sites on the octopine/cucumopine and vitopine Ti plasmids, whereas varying amounts of IS866 and IS867 copies occur at different positions on the chromosome. By comparison of the IS patterns, an evolutionary tree could be deduced which shows the phylogenetic relationships between 23 different types of Agrobacterium strains. The structures of the T-regions of the different strains were also compared. Within the octopine/cucumopine group, eight T-region patterns could be defined. These patterns were found to be correlated with the chromosomal IS patterns. This strongly suggests that the IS866 and IS867 containing Ti plasmids are stably associated with their bacterial hosts. The possible role of the IS866 and IS867 elements in Ti plasmid evolution is discussed.     

Instability of Cryptic Plasmids in Strain Sinorhizobium meliloti P108 in the Course of Symbiosis with Alfalfa Medicago sativa

Instability of cryptic plasmids in Sinorhizobium meliloti laboratory strains SXM1, DM7-R, and P108 as well as in their clones isolated from nodules of alfalfa grown during a long-term microvegetation experiment (120 days) was studied. The isolated clones of strains SXM1 and DM7-R manifested stable inheritance of plasmids, whereas 12.7–14.0% of clones with changed plasmid profile were detected in a population of clones from strain P108. These segregants were designated as P108c. Segregants P108c exhibited significantly decreased symbiotic effectiveness, nitrogenase activity, and the competitiveness with respect to alfalfa, compared to the original strain P108. It was established that a 80-kb deletion occurred in a larger of two cryptic plasmids (240 and 230 kb) of segregants P108c. It was concluded that genetic rearrangements are possible in rhizobial clones that did not undergo structural transformation and retained viability in the nodule during the natural vegetation period of alfalfa.     

Molecular genetic analysis of the response of three soil microbial communities to the application of 2, 4-D

The responses of three different soil microbial communities to the experimental application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) were evaluated with a variety of molecular genetic techniques. Two of the three soil communities had histories of prior direct exposure to 2, 4-D, and one had no prior direct application of any herbicide. Dominant 2, 4-D degrading strains isolated from these soils the previous year were screened for hybridization with three catabolic genes (tfdA, tfdAII, and tfdB) cloned from the well-studied 2, 4-D degradative plasmid, pJP4, revealing varying degrees of similarity with the three genes. Hybridization of total community DNA from the three soils with the tfd gene probes also indicated that pJP4-like tfd genes were not harboured by a significant percentage of the community. Community level response was evaluated by the comparison of different treatments by Random Amplified Polymorphic DNA (RAPD) fingerprints and by community DNA cross-hybridization. No differences between treatments within the same soil were detected in any of the RAPD fingerprints generated with 17 primers. Community DNA cross-hybridization also indicated that the application of 2, 4-D at the applied rates did not quantitatively affect the structure of the soil microbial communities present in the three soils during the time-frame studied.     

Genetic diversity of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> associated with alfalfa in Chilean volcanic soils and their symbiotic effectiveness under acidic conditions

A study was conducted with the aim of evaluating the genetic diversity of alfalfa rhizobia isolated from volcanic soils in southern Chile and their ability to establish an effective symbiosis with alfalfa. Rhizobial strains isolated from nodules were identified and selected based on PCR analyses and acid tolerance. Symbiotic effectiveness (nodulation and shoot dry weight) of acid-tolerant rhizobia was evaluated in glasshouse experiments under acidic conditions. The results revealed that Sinorhizobium meliloti is the dominant species in alfalfa nodules with a high genetic diversity at strain level grouped in three major clusters. There was a close relationship (r ² = 0.895, P ≤ 0.001, n = 40) between soil pH and the size of rhizobial populations. Representative isolates from major cluster groups showed wide variation in acid tolerance expressed on buffered agar plates (pH 4.5–7.0) and symbiotic effectiveness with alfalfa. One isolate (NS11) appears to be suitable as an inoculant for alfalfa according to its acid tolerance and symbiotic effectiveness at low pH (5.5). The isolation and selection of naturalized S. meliloti strains with high symbiotic effectiveness under acidic conditions is an alternative approach to improving the productivity of alfalfa and for reducing the application of synthetic fertilizers in Chile.     

哈尔滨市绿脓杆菌质粒图谱分析

本文对近年来从哈尔滨市五所临床医院分离的109株绿脓杆菌作了质粒图谱分析,结果发现在哈尔滨市流行的绿脓杆菌呈六种质粒图谱型别,其中77.1％的菌株呈两种相似的质粒图谱型别,且均含有一个共同的75Kb质粒。另外,还证实绿脓杆菌质粒图谱型别与血清型无明显相关性,而与耐药谱有一定的对应关系。     

A decade of land use contributes to changes in the chemistry,biochemistry and bacterial community structures of soils in the Cerrado

The bacterial community structures (BCSs) of Cerrado soils cultivated under conventional tillage (CT), no-tillage (NT) and under native Cerrado (NC) vegetation were evaluated using PCR/DGGE of bacterial 16S rRNA (rrs) and rpoB genes and of Pseudomonas group genes. Soil chemical analysis, microbial biomass and the enzyme activities were also evaluated and correlated with the BCS measurements. The multivariate ordinations of DGGE profiles showed differences between the BCS of the NC area and those from cultivated areas. The BCSs of the CT and NT areas also differed in all DGGE fingerprints, including changes in the profile of Pseudomonas populations, indicating that agricultural systems can also be responsible for changes within specific microbial niches, although the clearest differences were found in the rpoB profiles. The MRPP analysis demonstrated significant differences between the BCSs from different soil layers of NT areas based on all gene fingerprints and those of NC areas based on bacterial 16S rRNA and rpoB genes fingerprints. No differences were observed in the microbial fingerprints of CT samples from different depths, indicating that ploughing affected the original BCS stratification. The BCS from NC areas, based on all gene fingerprints, could be related to higher levels of soil acidity and higher amounts of MBC and of phosphatase activity. In contrast, the BCSs from cultivated areas were related to higher levels of Ca + Mg, P and K, likely as a result of a history of chemical fertilisation in these areas. The relationships between rpoB and Pseudomonas BCSs and all chemical and biochemical properties of soils were significant, according to a Mantel test (P < 0.05), indicating that the different changes in soil properties induced by soil use or management may drive the formation of the soil BCS.     

Combined Use of Ribotyping,PFGE Typing and IS431 Typing in the Discrimination of Nosocomial Strains of Methicillin-Resistant Staphylococcus aureus

We have previously reported the phenotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) clinical strains isolated in Malaya University Hospital in the period 1987 to 1989 using antibiogram, coagulase typing, plasmid profiles, and phage typing. Here, we report the analysis of the same strains with three genotyping methods; ribotyping, pulsed-field gel electrophoresis (PFGE) typing, and IS431 typing (a restriction enzyme fragment length polymorphism analysis using an IS431 probe). Ribotyping could discriminate 46 clinical MRSA strains into 5 ribotypes, PFGE typing into 22 types, and IS431 typing into 15 types. Since the differences of the three genotyping patterns from strain to strain were quite independent from one another, the combined use of the three genotyping methods could discriminate 46 strains into 39 genotypes. Thus, the powerful discriminatory ability of the combination was demonstrated.     

Characterization of a new Agrobacterium tumefaciens strain from alfalfa (Medicago sativa L.)

Agrobacterium tumefaciens strain 1D1609 is reported here as the first field isolate from alfalfa (Medicago sativa L.). Unlike well-characterized A. tumefaciens strains such as C58 and Ach5, strain 1D1609 is highly virulent on alfalfa and has a distinctive host range. Interestingly, strain 1D1609 is naturally resistant to kanamycin and spectinomycin. The Ti plasmid in strain 1D1609 is an octopine-type; thus, tumors formed by strain 1D1609 synthesize octopine, which is utilized by the bacterium as a sole carbon source. Reciprocal exchange of Ti plasmids between strains 1D1609 and C58 showed that both chromosomal and Ti plasmid genes in strain 1D1609 contribute specifically to tumor formation on alfalfa. In addition, the nondormant CUF101 alfalfa cultivar from which strain 1D1609 was isolated was significantly more susceptible to all Agrobacterium strains tested than was the dormant Agate cultivar. Received: 17 October 1997 / Accepted: 8 December 1997     

Genotyping of Staphylococcus epidermidis by Small-Fragment Restriction Endonuclease Analysis and Pulsed-Field Gel Electrophoresis of Genomic Restriction Fragments

Small-fragment restriction endonuclease analysis (SF-REA) was established as a typing tool for Staphylococcus epidermidis. A total of 60 isolates comprising 48 epidemiologically nonrelated strains and 12 putatively linked isolates from 7 patients in 2 wards were analyzed. Nonrelated isolates were characterized by unique fingerprints when DNA was cleaved with EcoRI or ClaI, electrophoretically separated in a polyacrylamide gel, and silver stained. Three blood culture isolates from one patient in an intensive care unit, 4 isolates obtained from a child over a span of 2 weeks, and 5 isolates from 5 newborns in the same ward were grouped into 3 DNA pattern types, indicating identity of sequential isolates from 2 patients and nosocomial transmission of one Staphylococcus epidermidis strain between 5 babies. Results from pulsed-field gel electrophoresis of SmaI and SacII DNA digests and conventional marker systems such as antibiogram and plasmid profile were in accordance with these interpretations, whereas slight variation was observed in the biotypes of several strains. From the results of this study, we conclude that SF-REA is a precise and efficient method for the genotypic characterization of Staphylococcus epidermidis strains that can be used as a rapid and reliable typing tool.