Construction of a Fibrobacter succinogenes Genomic Map and Demonstration of Diversity at the Genomic Level

The genomic cleavage map of the type strain Fibrobacter succinogenes S85 was constructed. The restriction enzymes AscI, AvrII, FseI, NotI, and SfiI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis (PFGE). An average genome size of 3.6 Mb was obtained by summing the total fragment sizes. The linkages between the 15 AscI fragments of the genome were determined by combining two approaches: isolation of linking clones and cross-hybridization of restriction fragments. The genome of F. succinogenes was found to be represented by the single circular DNA molecule. Southern hybridization with specific probes allowed the eight genetic markers to be located on the restriction map. The genome of this bacterium contains at least three rRNA operons. PFGE of the other three strains of F. succinogenes gave estimated genome sizes close to that of the type strain. However, RFLP patterns of these strains generated by AscI digestion are completely different. Pairwise comparison of the genomic fragment distribution between the type strain and the three isolates showed a similarity level in the region of 14.3% to 31.3%. No fragment common to all of these F. succinogenes strains could be detected by PFGE. A marked degree of genomic heterogeneity among members of this species makes genomic RFLP a highly discriminatory and useful molecular typing tool for population studies. Received: 23 October 1996 / Accepted: 31 December 1996     

Analysis of DNA restriction fragments greater than 5.7 Mb in size from the centromeric region of human chromosomes

Pulsed electrophoresis was used to study the organization of the human centromeric region. Genomic DNA was digested with rare-cutting enzymes. DNA fragments from 0.2 to greater than 5.7 Mb were separated by electrophoresis and hybridized with alphoid and simple DNA repeats. Rare-cutting enzymes (Mlu I, Nar I, Not I, Nru I, Sal I, Sfi I, Sst II) demonstrated fewer restriction sites at centromeric regions than elsewhere in the genome. The enzyme Not I had the fewest restriction sites at centromeric regions. As much as 70% of these sequences from the centromeric region are present in Not I DNA fragments greater than 5.7 and estimated to be as large as 10 Mb in size. Other repetitive sequences such as short interspersed repeated segments (SINEs), long interspersed repeated segments (LINEs), ribosomal DNA, and minisatellite DNA that are not enriched at the centromeric region, are not enriched in Not I fragments of greater than 5.7 Mb in size.     

Comparison of Leuconostoc oenos Strains by Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis of chromosomal DNA digested with NotI or SfiI was used to differentiate individual strains of Leuconostoc oenos. L. oenos isolates with 13 different restriction digest patterns were detected in New Zealand wines undergoing malolactic fermentation. The average genome size was estimated to be 1,800 kb.     

Genomic Diversity within the Genus Pediococcus as Revealed by Randomly Amplified Polymorphic DNA PCR and Pulsed-Field Gel Electrophoresis

The genomic diversity of 33 previously assigned strains from six species within the genus Pediococcus was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes ApaI, NotI, and AscI produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the NotI and AscI macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with AscI, all Pediococcus parvulus strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three P. damnosus and two P. pentosaceus strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped.     

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.     

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     

An improved physical and genetic map of the genome of alkaliphilic Bacillus sp. C-125

Among alkaliphilic bacteria reported so far, Bacillus sp. C-125 is the strain most thoroughly characterized physiologically, biochemically, and genetically. A physical map of the chromosome of this strain was constructed to facilitate further genome analysis, and the genome size was revised from 3.7 to 4.25 Mb. Complete digestion of the chromosomal DNA with two rare cut restriction endonucleases, AscI and Sse8387I, each yielded 20 fragments ranging in size from 20 to 600 kb. Seventeen linking clones were isolated in each instance to join the adjacent AscI or Sse8387I fragments in the chromosomal map. All AscI linking clones isolated were sequenced and analyzed by comparison with the BSORF database to map the genes in the chromosome of strain C-125. Several ORFs showing significant similarities to those of B. subtilis in the AscI linking clones were positioned on the physical map. The oriC region of the C-125 chromosome was identified by southern blot analysis with a DNA probe containing the gyrB region. Received: May 6, 1998 / Accepted: May 26, 1998     

A Physical Map of the Genome of a Unicellular Cyanobacterium Synechocystis sp. Strain PCC6803

An accurate physical map of the genome of a cyanobacterium,Synechocystis sp. strain PCC6803, was constructed on the basisof restriction and linking clone analysis. The genome contained6 recognition sites for AscI, 25 sites for MluI, and 31 sitesfor SplI, and the entire genome size was estimated to be 3.6Mb. Sixteen genes or gene clusters, including those involvedin the photosynthetic systems, were localized on the physicalmapof the genome by hybridization. In the course of the above analysis,two extra chromosomal units with approximate sizes of 110 kband 125 kb were identified.     

Genome Analysis of Several Marine, Magnetotactic Bacterial Strains by Pulsed-Field Gel Electrophoresis

The genomic DNA of three strains of marine magnetotactic bacteria, including two facultatively anaerobic vibrios, strains MV-1 and MV-2, and the microaerophilic coccus, strain MC-1, was analyzed by pulsed-field gel electrophoresis (PFGE). Digestion of the genomic DNA of strain MV-1 by the restriction endonucleases AvrII, BamHI, HindIII, NheI, SalI, SfiI, SgfI, SgrAI, and XbaI resulted in a large number of fragments below 400 kb that were difficult to resolve by PFGE. Digestion of MV-1 DNA with NotI and RsrII resulted in no fragments. Treatment of genomic DNA of strains MV-1 and MV-2 with PacI, PmeI, and SpeI yielded a manageable number of fragments (ca. 20) that were relatively easily resolved with PFGE, while PacI and SpeI were effective for strain MC-1. There was no evidence for the presence of plasmids and linear chromosomes in any of the strains, and strains MV-1 and MV-2 appear to contain a single, circular chromosome. Genome sizes of strains MV-1, MV-2, and MC-1 were estimated to be between 3.6 and 3.9 Mb (mean ± SD; 3.7 ± 0.2), 3.3 and 3.7 Mb (3.6 ± 0.2), and 4.3 and 4.7 Mb (4.5 ± 0.3), respectively. The restriction fragment patterns of the vibrioid strains MV-1 and MV-2 were extremely similar, suggesting that the strains are closely related. Received: 30 March 1999 / Accepted: 17 May 1999     

Genome Analysis of Bacteroides by Pulsed-field Gel Electrophoresis: Chromosome Sizes and Restriction Patterns

The chromosomal DNAs of nine strains of seven Bacteroides speciesincluding B. fragilis, the type species of the genus Bacteroides,were digested with rare-cutting restriction enzymes I-Ceu I,Not I, and Asc I and analysed by pulsed-field gel electrophoresis.The genome sizes of B. fragilis, B. distasonis, B. eggerthii,B. ovatus, B. thetaiotaomicron, B. uniformis, and B. vulgatuswere determined to be 5.3, 4.8, 4.4, 6.9, 4.8, 4.6, and 5.1Mbp, respectively. B. distasonis and B. vulgatus, and also B.uniformis and B. eggerthii, showed simillar I-Ceu I restrictionprofiles. I-Ceu I cut B. uniformis and B. eggerthii genomesinto four, B. ovatus into five, B. fragilis and B. thetaiotaomicroninto six, and B. distasonis and B. vulgatus into seven fragments.On the basis of genome size, restriction profile, and I-CeuI fragment number, a phylogenetic tree of the Bacteroides specieswas proposed. This was in overall agreement with the previousphylogenetic tree obtained by 16S rRNAdata, with the exceptionsof B. distasonis and B. ovatus.     

Determination of genome size and restriction pattern polymorphism of Rickettsia prowazekii and Rickettsia typhi by pulsed field gel electrophoresis

Abstract Pulsed field gel electrophoresis (PFGE) of Sma I, Mlu I and Sal I digested DNA was used to estimate genome size and perform restriction fragment length polymorphism analysis for Rickettsia prowazekii and Rickettsia typhi . We concluded that the genome of R. prowazekii and R. typhi consisted of a single chromosomal DNA. The total length of DNA of R. prowazekii was 1,106±54 kb and of R. typhi was 1,133±44kb. It was possibleto differentiate two strains of R. prowazekii , Breinl and EVir, by PFGE analysis after Sal I digestion. Restriction fragment length polymorphism analysis did not reveal intraspecies differences between three human isolates and one Xenopsilla cheopis isolate of R. typhi .     

Differentiation of Citrobacter strains using chromosomal DNA restriction patterns

The aim of this study was checking of the usefulness of chromosomal DNA restriction patterns in differentiation of Citrobacter strains. Molecular characterization of total 56 isolates of Citrobacter from Poland and Czech Republic, was performed by pulsed-field gel electrophoresis after digestion of chromosomal DNA with restriction endonuclease Xba I (5'-TCTAGA-3'). Chromosomal DNA of all tested Citrobacter strains gave after electrophoresis 12 to 21 bands and patterns consisting of 12 to 21 fragments ranging in size from 790 kb to 48.5 kb and smaller, which where not distinguishable. Pulsed-field gel electrophoresis patterns were useful for comparing Citrobacter strains. Identical restriction patterns generated by PFGE were observed in the case of selected strains e.g. strains C. sedlakii studied in this study, coming from an outbreak, having the some phenotype. In addition, PFGE patterns can be used to evaluate the clonal relatedness among bacterial isolates. PFGE can be helpful for assessing genetic relatedness among strains epidemiologicaly unrelated e.g. C. werkmanii strains tested in this study. The sum of DNA fragments after Xba I digestion indicates the genome size of Citrobacter strains. This suggests that PFGE should be useful for epidemiological investigations of Citrobacter strains.     

Genotypic Diversity among Bacillus cereus and Bacillus thuringiensis Strains

Twenty-four strains of Bacillus cereus were analyzed by pulsed-field gel electrophoresis (PFGE) and compared with 12 Bacillus thuringiensis strains. In addition, the 36 strains were examined for variation in 15 chromosomal genes encoding enzymes (by multilocus enzyme electrophoresis [MEE]). The genome of each strain had a distinct NotI restriction enzyme digestion profile by PFGE, and the 36 strains could be assigned to 27 multilocus genotypes by MEE. However, neither PFGE nor MEE analysis could distinguish between the two species. Two of the B. cereus strains contained extrachromosomal DNA that hybridized to a cryIA insecticidal toxin probe, and seven strains contained DNA with homology to a Tn4430 transposon probe derived from B. thuringiensis. The results strongly indicate that B. cereus and B. thuringiensis should be regarded as one species.     

Integrated map of the Schizosaccharomyces pombe genome

A restriction map of the entire Schizosaccharomyces pombe genome was constructed using two restriction enzymes (BamHI and PstI) that recognize 6 bp. The restriction map contains 420 minimally overlapping clones (miniset) and has 22 gaps. We located 126 genes, marker fragments of DNA (NotI and SfiI linking clones), and 36 transposable elements by hybridization to unique restriction fragments. Received: 21 November 1996; in revised form: 3 March 1997 / Accepted: 27 March 1997     

Physical and genetic chromosomal maps of Streptococcus agalactiae, serotypes II and III; rRNA operon organization

A detailed analysis of two Streptococcus agalactiae (group B streptococcus, GBS) strains was performed by pulsed field gel electrophoresis (PFGE). Digestion of the chromosomal DNA with SmaI and SgrAI endonucleases, followed by separation and analysis of fragments by PFGE was carried out. Physical chromosomal maps of serotype II/(α+β) and III/α strains of S. agalactiae were constructed. The GBS genome size was estimated to be 2200 kb. Sixteen GBS genes were used as probes and were located on the restriction maps of both strains by DNA-DNA hybridization. Six copies of ribosomal operons were found in the genome of the analyzed strains. Significant differences in the restriction patterns of chromosomal DNA and DNA-DNA hybridization between the two analyzed strains were detected so that DNA restriction patterns may be used to trace outbreaks of disease. The overall GBS chromosomal organization as determined is fairly conserved.     

A physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome

A combined physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome was constructed using pulsed-field gel electrophoresis (PFGE) and hybridizations with cloned gene probes. Total genomic DNA was digested with the meganucleasesSwaI (5′-ATTTAAAT-3′),PacI (5′-TTAATTAA-3′), andPmeI (5′-GTTTAAAC-3′) yielding 26, 27, and 23 fragments, respectively. The chromosomal restriction fragments were then separated by PFGE. By summing up the lengths of the fragments generated with each of the three enzymes, a genome size of 3082 +/- 20 kb was determined. To identify adjacentSwaI fragments, a genomic cosmid library ofC. glutamicum was screened for chromosomal inserts containingSwaI sites. Southern blots of the PFGE gels were hybridized with these linking clones to connect theSwaI fragments in their natural order. By this method, about 90% of the genome could be ordered into three contigs. Two of the remaining gaps were closed by cross-hybridization of blottedSwaI digests using as probesPacI andPmeI fragments isolated from PFGE gels. The last gap in the chromosomal map was closed by hybridization experiments using partialSwaI digestions, thereby proving the circularity of the chromosome. By hybridization of gene probes toSwaI fragments separated by PFGE about 30 genes, including rRNA operons, IS element and transposon insertions were localized on the physical map.     

Genetic Characterization of a New Thermotolerant Bacillus licheniformis Strain

A potentially new thermotolerant B. licheniformis strain (code name I89), producer of an antibiotic active against Gram-positive bacteria, was genetically characterized and compared with the type strain B. licheniformis ATCC 10716, producer of bacitracin. Studies on DNA base composition (G + C content) and DNA reassociation revealed that the two strains show around 76% homology. Nevertheless, results obtained by rRNA hybridization, with a heterologous probe coding for most of the 16S region of the rRNA operon of Bacillus subtilis, revealed differences in the number of copies for that gene and in the hybridization pattern. Additionally, a different restriction digestion pattern was obtained when DNA was digested with the enzymes NotI, SmaI and analyzed by PFGE. The I89 strain holds a 7.6-kb plasmid not present in the reference strain. The existence of various unique restriction sites and also the stability of this plasmid make it ideal for the future development of a cloning and expression vector. Received: 29 June 1999 / Accepted: 1 September 1999     

Genomic Analysis of Clostridium botulinum Group II by Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) was optimized for genomic analyses of Clostridium botulinum (nonproteolytic) group II. DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. A rapid (4-h) in situ DNA isolation method was also assessed and gave indistinguishable results. Genomic DNA from 21 strains of various geographical and temporal origins was digested with 15 rare-cutting restriction enzymes. Of these, ApaI, MluI, NruI, SmaI, and XhoI gave the most revealing PFGE patterns, enabling strain differentiation. Twenty strains yielded PFGE patterns containing 13 pulsotypes. From summation of MluI, SmaI, and XhoI restriction fragments, the genome size of C. botulinum group II was estimated to be 3.6 to 4.1 Mb (mean ± standard deviation = 3,890 ± 170 kb). The results substantiate that after problems due to DNases are overcome, PFGE analysis will be a reproducible and highly discriminating epidemiological method for studying C. botulinum group II at the molecular level.     

Molecular Typing by Pulsed-Field Gel Electrophoresis of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> from Root Voles

The root voles intestinal strains of Bacillus thuringiensis, were characterised by pulsed-field gel electrophoresis (PFGE). For 14 isolates, three pulsotypes were found, with the use of SmaI or NotI as restriction enzymes. Strains in each pulsotypes presented identical DNA patterns, indicating that the population structure of B. thuringiensis from root voles is clonal. The similarities in banding patterns were estimated at 56% and 33% for SmaI and NotI digests, respectively. The strains under study differed significantly in the size of their entire genome, which varied between 2.4 and 4.2 Mb. No significant differences were detected among the isolates subjected to biochemical properties determined by API tests. Present study showed that genomic diversity is a common feature of B. thuringiensis originating from one ecological niche. PFGE appears to be a useful technique for use in studies on the spread of B. thuringiensis in the environment. Received: 14 May 2002 / Accepted: 21 June 2002     

Physical map of the linear chromosome of Streptomyces hygroscopicus 10-22 deduced by analysis of overlapping large chromosomal deletions

The chromosomal DNA of Streptomyces hygroscopicus 10-22, a derivative of strain 5102-6, was digested with several restriction endonucleases and analyzed by pulsed-field gel electrophoresis (PFGE). Digestions with AseI gave 11 fragments with a total length of ca. 7.36 Mb. The AseI sites were mapped by analysis of overlapping chromosomal deletions in different mutants and confirmed by Southern hybridizations using partially digested genome fragments and linking cosmids as probes. PFGE analysis of DNA with and without proteinase K treatment, together with the hybridization results, suggested a linear organization with terminal proteins and large terminal inverted repeats. Some deletion mutants had circular chromosomes.