Restriction endonuclease analysis of four Borrelia burgdorferi strains

Abstract A restriction endonuclease analysis was performed on four strains of Borrelia burgdorferi : one isolated from man (SF), one from Ixodes dammini (B31) and two form I. ricinus (BITS in Italy and B45 in Germany). Digestion by Taq I and Hae III gave the best resolution of the DNA fragments. Three different restriction patterns were obtained: BITS and B45 showed only one band difference. These results correlate with the reactivity of the four strains with monoclonal antibodies.     

DNA homology comparison between American and European Borrelia burgdorferi strains

Seven strains of Borrelia burgdorferi isolated from ticks and from human beings in Europe and U.S.A. were analyzed for DNA restriction patterns with several enzymes and for DNA homology in Southern blot hybridizations. The restriction patterns showed a moderately high variability. In Southern blot hybridization, strain B31 (U.S.A.) DNA gave a strong signal with itself, strain Bsf (U.S.A.) and Alcaide (isolated in Italy but presumably contracted in Venezuela). Strain B45 (F.R.G.) hybridized to itself, strain BITS (Italy) and to strain D.A. (Italy). Strain Nancy (Italy) gave a signal only when hybridized to itself, although it was classified as Borrelia on the basis of the clinical manifestations, SDS-PAGE protein pattern and antigenic determinants. No hybridization differences were observed for strains isolated from different hosts in the same continental geographical area.     

Restriction endonucleases from Bifidobacteria

The sitespecific restriction endonucleases were found in four strains among the twelve strains of anaerobic bacteria of generum Bifidobacterium. Two of the restriction endonucleases studied, BadI from B. adolescentis LVA1 and BbfI from B. bifidum LVA3, are isoshizomers of XhoI and recognize the nucleotide sequence CTCGAG. The restriction endonucleases Bbf7411I from B. bifidum 7411 and Bla7920I from B. lactentis 7920 recognize and hydrolize the nucleotide sequence TCCGGA having the specifity analogous to the one of restriction endonuclease CauB3I. Like CauB3I, these restriction endonucleases are unable to hydrolyize DNA if the adenine residues in the recognition site are methylated.     

PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells

Strains of species in the Bacillus cereus group are potentially enterotoxic. Thus, the detection of all B. cereus group strains is important. As 16S ribosomal DNA sequence analysis cannot adequately differentiate species of the B. cereus group, we explored the potential of the groEL gene as a phylogenetic marker. A phylogenetic analysis of the groEL sequences of 78 B. cereus group strains revealed that the B. cereus group strains were split into two major clusters, one including six B. mycoides and one B. pseudomycoides (cluster II) and the other including two B. mycoides and the rest of the B. cereus group strains (cluster I). Cluster I was further differentiated into two subclusters, Ia and Ib. The sodA gene sequences of representative strains from different clusters were also compared. The phylogenetic tree constructed from the sodA sequences showed substantial similarity to the tree constructed from the groEL sequences. Based on the groEL sequences, a PCR assay for detection and identification of B. cereus group strains was developed. Subsequent restriction fragment length polymorphism (RFLP) analysis verified the PCR amplicons and the differentiation of the B. cereus group strains. RFLP with MboI was identical for all the B. cereus group strains analyzed, while RFLP with MfeI or PstI classified all B. cereus and B. thuringiensis strains into two groups. All cluster II B. mycoides and B. pseudomycoides strains could be discriminated from other B. cereus group bacteria by restriction analysis with TspRI.     

Biological specificity of bottled natural mineral waters: characterization by ribosomal ribonucleic acid gene restriction patterns

The organisms from heterotrophic plate counts of four brands of bottled non-carbonated mineral waters were analysed by ribosomal ribonucleic acid gene restriction patterns in addition to identification by customary techniques. Five commercialized bottles of each brand were examined once in the year 1989–90. The total bacterial flora was divided into two main groups: the pseudomonads and the unidentified strains. Whereas phenotypic results did not reveal any significant differences between the four brands, rRNA gene restriction patterns were discriminatory. Among the 73 strains studied, 45 distinct patterns were observed, with only three common to several springs. These results reveal, at one determined time, a remarkable biological specificity of each brand of water.     

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.     

Differences in chromosome number and genome rearrangements in the genus Brucella

We have studied the genomic structure and constructed the Spe I, Pac I and I- Ceu I restriction maps of the four biovars of the pathogenic bacterium Brucella suis . B . suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B . melitensis , B . abortus , B . ovis and B . neotomae . Two chromosomes were also observed in the genome of B . suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B . suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome-containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B . suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.     

Detection of aquatic microorganisms from the Black Sea--producers of restriction endonucleases

300 clones of microorganisms isolated at different stations and from different depths in the Black Sea were screened for restriction endonucleases production. The production of restriction endonucleases was found in 17 clones screened. Three of them were identified to be Alteromonas haloplanktis B1. Restriction endonuclease AhaB1 is an isoshizomer of Sau961. An identified Alteromonas haloplanktis clone B8 produces AhaB8I restriction endonuclease the prototype to which is KpnI. Of the clones isolated three are Moraxella species B4 producing MapB4I restriction endonuclease analogous to BanI, three are Bacillus species producing BspB2I and one is Micrococcus lylae 113 producing Mly1131 analogue of NarI, six Moraxella species B6 produce MspB6I. The isolated producer strains may be used for isolation of above mentioned restriction endonucleases.     

Localization of prophages of serological group B and F on restriction fragments defined in the restriction map of Staphylococcus aureus NCTC 8325

Abstract Several Staphylococcus aureus strains were lysogenized by the phages of serological group B (phages φ53, φ85) as well as by some of serological group F (phages φ77, φ84) and macrorestriction fragment patterns of genomic DNA were estimated in the lysogenized, non-lysogenic and delysogenized (cured of prophages) strains. It was shown that the integration of phage DNA into chromosome of S. aureus leads to specific changes in restriction fragment pattern in all the lysogenized strains. These changes correlate well with the Sma I restriction map of S. aureus NCTC 8325 since they concern the restriction fragments defined in this map. Phages φ53 and φ85 integrate into Sma I fragment B. On the other hand, phages φ77 and φ84 integrate into Smal fragment E of the S. aureus restriction map. The prophages of strain NCTC 8511 have their integration sites, as follows: the phage designated by us φM integrates in fragment A, whereas the integration site for phage φJ lies in fragment E. Phage φM was estimated to be genetically related to phages of serological group A and phage φJ to those of serological group F. Evidence was given that lysogenization of S. aureus strains by at least four prophages does not cast any doubt upon the estimation of their genetic relatedness based on their similarity in restriction pattern.     

Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

Genotypic characteristics of the rrn operon and genome of indigenous soybean Bradyrhizobia in cropping zones of China

Four genetic assays, 16S rRNA restriction fragment length polymorphism (RFLP), 16S rRNA sequencing, 16S-23S rRNA intergenetic spacer (IGS) RFLP, and amplified fragment length polymorphism (AFLP), were conducted to determine the genotypic characteristics of 44 indigenous strains of Bradyrhizobium from soybean (Glycine max L.) cropping zones of China. The results generated from different assays showed that soybean bradyrhizobial isolates comprised four genomic groups. Group I was composed of strains mainly isolated from the North and Northeast plains of China. All four assays confirmed this group as phylogenetically divergent from all the reference strains. Strains of the group may represent a new species. Strains in Group II isolated from a variety of geographic regions were ascribed to B. liaoningense. Strains in Group III, mainly isolated from Central and East China, were closely related to the reference strains of B. japonicum. Strains in Group IV belonged to B. elkanii.     

Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis.

DNA from 1008 strains of Mycobacterium avium subspecies paratuberculosis, digested by restriction endonucleases PstI and BstEII, was hybridised with a standard IS900 probe prepared by PCR and labelled non-radioactively by ECL. DNA fingerprints were scanned by CCD camera and analysed using the software Gel Compar (Applied Maths, Kortrijk, Belgium). Thirteen restriction fragment length polymorphism (RFLP) (PstI) types were detected, which where designated as A, B, C, D, E, F, G, H, I, J, K, L and M in accordance with the study of Pavlik et al. (1995) [Pavlik, I., Bejckova, L., Pavlas, M., Rozsypalova, V., Koskova, S., 1995. Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities. Vet. Microbiol. 45, 311-318]. Twenty RFLP (BstEII) types were detected and designated as C1-3, C5, C7-20, S1 and I1 in accordance with the study by Collins et al. 1990 [Collins, D.M., Gabric, D.M., de Lisle, G.W., 1990. Identification of two groups of Mycobacterium paratuberculosis strains by restriction endonuclease analysis and DNA hybridization. J. Clin. Microbiol. 28, 1591-1596]. A combination of both RFLP (PstI) and RFLP (BstEII) results revealed a total of 28 different RFLP types. All the RFLP types and detailed protocols are available at Intemet web site WWW...: http:/ /www.vri.cz/wwwrflptext.htm.     

Recognition of Leuconostoc oenos strains by the use of DNA restriction profiles

The chromosome of 41 Leuconostoc oenos strains obtained from collections in different countries was analysed with the aim of differentiating the strains. Pulsed field electrophoresis (TAFE) was used to separate large DNA fragments created by the restriction enzymes NotI, SfiI and ApaI, which specifically recognize guanines or cytosines. The genomic DNA of 11 strains was analysed initially with NotI and only four different restriction profiles were observed. The genome size ranged from 1.8 to 2.1 megabase pairs (Mbp). Constant field electrophoresis applied to DNA treatment with 19 different restriction enzymes showed that the size of the fragments obtained increased proportionally to the percentage G+C present at the site of restriction. EcoRI and HindIII profiles revealed that the zone between 9 and 23 kbp allowed differentiation of the strains tested. Thus, the 41 strains fell into 30 restriction groups using only two enzymes. Hybridization with a non-radioactive DNA probe coding for 16S rRNA revealed that there were two 16S genes on the chromosome. Correspondence to: C. Diviès     

Identification of thermophilic and marine bacilli from shallow thermal vents by restriction analysis of their amplified 16S rDNA

AIMS: In order to identify 73 thermophilic isolates from shallow, marine thermal vents of Eolian Islands, we compared their restriction patterns of amplified 16S rDNA with those of nine well described Bacillus species and eight Eolian Bacillus strains. METHODS AND RESULTS: This study allowed to assign 57 (78%) isolates to different Bacillus species. Nineteen field strains were recognised as representatives of four described species, namely B. thermodenitrificans, "B. caldolyticus", B. vulcani and B. stearothermophilus. The profiles of 38 isolates matched instead, those of seven Eolian strains (B. thermodenitrificans strain A2, B. licheniformis strain B3-15, and five novel species, represented by Bacillus strain 1bw, Bacillus strain 4-1, Bacillus strain 5-2, Bacillus strain 10-1, Bacillus strain 1as). Among the 16 unidentified isolates, seven restriction patterns were recognised. CONCLUSIONS: This study showed that restriction analysis of amplified 16S rDNA is useful for a rapid and reliable identification of strains belonging to described species as well as for recognition of new species. SIGNIFICANCE AND IMPACT OF THE STUDY: This work revealed a high taxonomic diversity among the thermophilic bacilli isolated from Eolian Islands and a distinct distribution of the species within the Eolian hydrothermal vent system.     

Characterization of Borrelia afzelii isolated from Ixodes nipponensis and Apodemus agrarius in Chungju,Korea, by PCR-rFLP analyses of ospC gene and rrf (5S)-rrl (23S) intergenic spacer

Nine Borrelia afzelii strains, which had been isolated from two vectors, Ixodes nipponensis and Apodemus agrarius in Chungju, Korea, were characterized by PCR-RFLP analyses of ospC genes and rrf (5S)-rrl (23S) intergenic spacer. DraI restriction patterns of Chungju strains were identical to those of B. afzelii VS461. But MseI restriction patterns of rrf (5S)-rrl (23S) intergenic spacer genes of KK2, KM4, KK5 differed from those of previously reported B. burgdorferi sensu lato strains. Nine Chungju strains were classified with four distinct ospC RFLP patterns, which differed from the eight ospC RFLP patterns (A-1 to A-8) of previously reported B. afzelii. Moreover, five additional restriction patterns were deduced from published ospC sequences of reference strains. These results suggest that Chungju strains are very heterogeneous.     

伊氏锥虫动基体DNA微环的研究

采用限制性内切酶消化、琼脂糖凝胶电泳及分子杂交技术对８株中国伊氏锥虫动基体ＤＮＡ微环进行了比较研究。结果显示,我国伊氏锥虫株之间的ｋＤＮＡ微环序列具有较高的同源性,仅限制酶ＡｌｕＩ,ＨｉｎｆＩ及ＭｂｌＩ的酶解结果显示少数虫株的ｋＤＮＡ微环存在异源序列。这种异源性可以作为伊氏锥虫种内分类的遗传学标志。     

Restriction fragment length polymorphism of 16S-23S rDNA intergenic spacer of Aeromonas spp

We analyzed restriction fragment length polymorphism (RFLP) of 16S-23S rDNA intergenic spacer region (ISR) of Aeromonas species. A total of 69 isolates belonging to 18 DNA hybridization groups (HG; equivalent of genomic species) were used in this study. ISRs were amplified by PCR and the products were digested with four restriction endonucleases: Hin6I, Csp6I, TaqI, and TasI. The RFLP patterns obtained after digesting by particular enzymes revealed ISR polymorphism of isolates allocated to individual genomic species. The combined Hin6I, Csp6I, TaqI, and TasI restriction profiles were examined by Dice coefficient (SD) and unweighted pair group method of clustering (UPGMA). The isolates were allocated into 15 groups, three strains were unclustered. The strains belonging to the following genomic species: A. hydrophila, A. bestiarum, A. salmonicida, A. caviae, A. media, A. schubertii, A. allosaccharophila, A. popoffii, and A. culicicola formed distinct clusters. Strains belonging to HG 6, HG 7, HG 11, and HG 16 revealed similar combined RFLP patterns and constituted one group. Similarly, the strains of A. jandaei (HG 9) and the type strain of A. trota were allocated into one cluster. Two isolates of HG 14 formed distinct cluster. We noticed a genetic diversity among A. veronii isolates, the strains were clustered in two groups. Our study showed that combined ISR-RFLP analysis may be used for identification of some species of Aeromonas.     

Strategy for identification of Bacillus cereus and Bacillus thuringiensis strains closely related to Bacillus anthracis

Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these "near neighbors" are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.     

Polymorphism of Nopaline-type T-DNAs from Agrobacterium tumefaciens.

The structure of several T-DNAs of Agrobacterium tumefaciens was determined by molecular cloning and Southern hybridization. The T-DNAs cloned in Escherichia coli vectors from four different nopaline type strains (PyTE1, PO31, PO22, and AKE10) showed various sizes of restriction enzyme fragments. Comparative analysis of the restriction maps revealed that the T-DNAs were composed of three distinct structural domains: (1) the region proximal to the right border (Domain I) containing the portion essential for tumorigenicity, (2) the proximity to the left border (Domain II), and (3) the region between the two domains (Domain III) to both of which no functional assignments have yet been made. The restriction map indicated that the Domains I and II were conserved in the most clones, including the well-characterized T37 T-DNA. The only exception was AKK1 (obtained from AKE10) which differed in Domain I. In the Domain III, insertions of 1.5- or 1.6-kb DNA were found in four clones, whereas an additional 2.5-kb insertion was found in one clone (PO22P1). The individual T-DNAs including Domain III with insertions was demonstrated in petunia and poplar tumors induced by the referred A. tumefaciens strains. However, resulting tumors differed in morphology and growth. These results suggest that the length polymorphism of the nopaline type T-DNA can be accounted by DNA insertions, and that diverse T-DNAs reflect their different roles in tumorigenicity.     

Genetic Fingerprinting of Brevibacterium linens by Pulsed-Field Gel Electrophoresis and Ribotyping

Members of Brevibacterium linens display physiological features that are relevant for cheese production. The genomes of five B. linens strains deposited on culture collections were compared by examining large restriction fragments on pulsed-field gel electrophoresis and detection of polymorphism at the level of 16S rRNA genes. Pulsed-field analysis with the endonucleases DraI and AsnI showed a characteristic restriction profile for each strain and allowed the calculation of genome sizes ranging between 3.2 and 3.9 Mbp. No linear genomic elements were detected. Polymorphisms at the level of 16S rRNA genes were revealed by hybridization with an oligonucleotide probe complementary to a universal domain of the 16S genes. An EcoRI fragment of 1.4 kb was identified as common to all strains under study. According to the number of positive bands detected by the probe, at least four rRNA operons must be present on the genome of the B. linens strains here studied. Received: 13 January 2000 / Accepted: 9 February 2000