Bacterial γ-glutamyltranspeptidases: Comparison of Escherichia coli and Pseudomonasγ-glutamyltranspeptidase

Serpulina (Treponema) hyodysenteriae strain A-1 partially purified rRNA, labelled with photobiotin, was used as a non-radioactive probe to identify the rRNA gene restriction patterns of S. hyodysenteriae strains and other spirochetes. Sau3A restriction enzyme digests resulted in similar rRNA gene restriction patterns in S. hyodysenteriae strains from five different countries. Some S. hyodysenteriae strains could be differentiated by variations in their rRNA gene restriction patterns after cleavage of DNA by restriction enzymes SspI or BglII. S. innocens and Treponema succinifaciens, non-pathogenic pig intestinal spirochetes, had rRNA gene restriction patterns that differed markedly from the S. hyodysenteriae patterns, and from each other.     

Identification of Azospirillum strains by restriction fragment length polymorphism of the 16S rDNA and of the histidine operon

Abstract DNA fingerprints of several Azospirillum strains, belonging to the five known species A. amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum , were obtained by restriction analysis of the amplified 16S rDNA and by restriction fragment length polymorphism of the histidine biosynthetic genes. Data obtained showed that amplified rDNA restriction analysis is an easy, fast, reproducible and reliable tool for identification of Azospirillum strains, mainly at the species level, whereas restriction fragment length polymorphism could, in some cases, differentiate strains belonging to the same species. Moreover, both analyses gave congruent results in grouping strains and in the assignment of new strains to a given species.     

Fluorescent Pseudomonas species categorized by using polymerase chain reaction (PCR)/restriction fragment analysis of 16S rDNA

A rapid procedure for the identification of fluorescent pseudomonads, based on the polymerase chain reaction (PCR) and restriction fragment analysis of 16S rDNA genes is described. Thirty-one strains belonging to 10 different Pseudomonas species of the Pseudomonas fluorescens rRNA branch were characterized. Amplified rDNA was digested with 13 different restriction endonucleases. The combined data from restriction analysis enabled the definition of 17 different 16S rDNA genotypes. All type strains belonging to different species were differentiated. The good correlation between grouping obtained using restriction analysis with other molecular classification criteria demonstrates the value of the described method to characterize rapidly fluorescent Pseudomonas strains at the species level.     

Restriction fragment length polymorphism of Azospirillum strains

Abstract: Total DNA of various Azospirillum strains representing different species was digested with restriction enzymes, Southern blotted and hybridized with four A. brasilense probes. Pairwise comparison of the conserved hybridization fragments was applied to calculate sequence divergence and to group the strains using the unweighted pair group method. The resulting dendrogram grouped the strains according to the known species indicating that the analysis of the restriction fragment length polymorphism is an useful tool for characterizing Azospirillum isolates.     

Rapid DNA fingerprinting of pathogens by flow cytometry

BACKGROUND: A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. METHODS: Bacterial genomic DNA was isolated and digested with a rare-cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. RESULTS: Five different strains of gram-negative Escherichia coli and six different strains of gram-positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were approximately 100 times faster and approximately 200,000 times more sensitive than pulsed-field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. CONCLUSIONS: DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)-based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains.     

A rapid purification method of restriction endonucleases from Haemophilus strains.

A simple and rapid method of purification of restriction endonucleases from different Haemophilus strains is presented. By this method highly purified and stable enzymes can be obtained. Separation of different restriction activities present in the same strain is possible. This method was so far successfully used with Haemophilus influenzae, Haemophilus parainfluenzae and Haemophilus aegyptius strains. The main advantages over previously published procedures reside in the simplication of certain purification steps (for instance the BioGel A 0.5 M filtration is replaced by a hydroxyapatite batch step), elimination of exonuclease activity by fractionation with (NH4) 2SO4, separation of different restriction activities by phosphocellulose chromatography, application of this method to various strains and high purification degree of enzymes.     

Identification and characterization of Streptococcus thermophilus strains by pulsed-field gel electrophoresis

The chromosomal DNA of a number of strains of the lactic acid bacterium Streptococcus thermophilus was analysed with the aim of rapidly differentiating and assessing the characteristics of each strain. Pulsed-field gel electrophoresis was used to separate large DNA fragments formed by the restriction enzymes Sma I, Sfi I or Apa I. Hybridization with a non-radioactive DNA probe confirmed the identification of strains as Strep. thermophilus and analysis of the electrophoretic patterns differentiated some strains from others. A more extensive study of the pulsed-field electrophoresis restriction patterns of new isolates of Strep. thermophilus may facilitate assessment of their technological properties by comparison of their restriction patterns with those of reference strains.     

High-resolution alignment of a 1-megabase-long genome region of three strains of Rhodobacter capsulatus.

A detailed restriction map of the genome of Rhodobacter capsulatus SB1003 was constructed recently by using an ordered set of overlapping cosmids. Pulsed-field gel electrophoresis-generated restriction patterns of the chromosomes of 14 other R. capsulatus strains were compared. Two of them, St. Louis and 2.3.1, were chosen for high-resolution alignment of their genomes with that of SB1003. A 1-Mb segment of the R. capsulatus SB1003 cosmid set was used as a source of ordered probes to group cosmids from the other strains. Selected cosmids were linked into one 800-kb contig and two smaller contigs of 100 kb each. EcoRV and BamHI restriction maps of the newly ordered cosmids were constructed by using lambda terminase. Long-range gene order in the new strains was mainly conserved for the regions studied. However, one large genome rearrangement inverted a 470-kb DNA fragment of the St. Louis strain between the rrnA and rrnB operons. A 50-kb deletion covering three SB1003 probes was found in strain 2.3.1 near rrnB. Conservation of about 50% of the positions of restriction sites in all these strains and nearly 80% for the pair 2.3.1- St. Louis made it possible to produce high-resolution alignment of the contiguous 800-kb genome segment. Ten deletions of 2 to 27 kb, one 30-kb inversion, and three translocations were found in this region. Strong clustering of the positions of polymorphic restriction sites was observed. For a 50-kb size interval, two patterns of the distribution of restriction sites were found, one with about 90% and the other with 5 to 30% conservation of sites. This structure may be explained by independent acquisition of these divergent regions from other Rhodobacter strains.     

Re-identification of Gluconobacter strains based on restriction analysis of 16S-23S rDNA internal transcribed spacer regions

Thirty Gluconobacter strains maintained at Culture Collection NBRC were re-identified at the species level on the basis of restriction analysis of 16S-23S rDNA internal transcribed spacer (ITS) regions by digestion with two restriction endonucleases MboII and Bsp1286I. The strains examined were divided into seven groups, designated as Group I and Group III-VIII, by the combination of the restriction patterns obtained with the two restriction endonucleases. Group I included seven strains, which gave "G. oxydans patterns" with the two restriction endonucleases and were re-identified as G. oxydans. Group III included 12 strains, which gave "G. frateurii patterns" and were re-identified as G. frateurii. Group IV included six strains, which gave "G. cerinus pattern" with MboII and "G. frateurii pattern" with Bsp1286I and were re-identified as G. frateurii. Group V included one strain (NBRC 3274), which gave respectively "G. frateurii pattern" and "G. cerinus pattern" and was re-identified as G. cerinus. Group VI included one strain (NBRC 3990), which gave respectively "G. oxydans pattern" and an unidentified restriction pattern and was re-identified temporarily as G. oxydans. Group VII included two strains (NBRC 3250 and NBRC 3273), which gave respectively an unidentified restriction pattern and "G. oxydans pattern." Group VIII included one strain (NBRC 3266), which gave unidentified restriction patterns. The three strains of Group VII and Group VIII were suggested to constitute new taxa by sequencing of 16S-23S rDNA ITS regions.     

Molecular analysis of 16S–23S spacer regions ofAcetobacter species

16S-23S rDNA internal transcribed spacer regions (ITS) similarities were determined in 8 Acetobacter and 1 Gluconacetobacter strains. ITS-PCR amplification of the 16S-23S spacers showed 2 products of similar size in 7 strains; only 1 product of similar size was found in the 2 remaining strains. Analysis of the PCR products using restriction endonucleases HaeIII, HpaII and AluI revealed 3 different restriction groups of A. pasteurianus for AluI and HaeIII, and 4 restriction groups for HpaII. ITS nucleotide sequences of all studied strains exhibited a 52-98% similarity.     

Plasmid DNA from methanogenic bacteria

In total, 73 strains of methanogen isolates from our laboratory and 6 from culture collections were examined for the presence of plasmid DNA. Five strains were found to contain detectable plasmids. Multiple plasmids were found in two isolates, while three strains contained only one plasmid each. A physical map of the plasmid pT3 was constructed by use of six different restriction endonucleases. All sites were aligned with a single BgII site, and the position of the restriction sites was determined by double or sequential digestion of the plasmid DNA.     

Search for producers of restriction endonucleases with a given specificity]

Six site-specific restriction endonucleases were isolated from Bacillus thuringiensis strains chosen of 58 strains ought purposefully for production of restriction enzymes. All six strains produce isoschisomers of Sau3A.     

Novel restriction fragment length polymorphism of the growth hormone gene in inbred rats

A novel restriction fragment length polymorphism in inbred rats was detected by Southern blot analysis with rat growth hormone cDNA as a probe. Four alleles, characterized by PstI fragments of 1.2, 1.1, 0.9, and 0.7 kb, respectively, were detected in 27 strains examined. The same distribution of polymorphisms was observed on digestion of DNAs of these strains with three other enzymes, PvuII, HindIII, and BamHI. Moreover, the same differences in length of allelic restriction fragments were obtained with these restriction enzymes as with PstI. These findings suggested that the polymorphism was caused by insertion or deletion of variable DNA segments in the second intron of the growth hormone gene. Linkage analyses using backcross progeny provided no evidence for close linkage between the restriction fragment length polymorphism locus and 10 other loci examined.     

Plasmid-mediated chloramphenicol resistance in Staphylococcus hyicus

A small plasmid of 3.95 kb, encoding resistance to chloramphenicol (Cm) was detected in three of 33 Staphylococcus hyicus strains. The plasmid in each of the three strains was indistinguishable by Southern-blot hybridization and restriction enzyme analysis. It was shown by curing and by transformation to specify resistance to Cm. A preliminary restriction map of the plasmid, designated pSC2, is presented. Chloramphenicol acetyltransferase was demonstrated by enzyme assay and by SDS-PAGE of cell-free lysates of pSC2 transformants.     

中国钩端螺旋体rRNA基因多态性分析

以ＤｉｇｄＵＴＰ标记的１６ＳｒＲＮＡ及２３ＳｒＲＮＡ基因为探针,分析了八个血清群５４个血清型６４株国内外致病性钩端螺旋体参考株和２７株野生株染色体经限制性内切酶ＥｃｏＲⅠ消化后的ｒＲＮＡ基因限制性图谱。结果发现,９１株菌中共有５６个核糖核酸型（Ｒｉｂｏｔｙｐｅ,简称ＲＴ）,除部分血清群中少数不同的血清型有相同的ＲＴ型外,大部分血清型都有独特的ＲＴ型,同一血清群往往拥有共同的核心片段;除黄疸出血群的黄疸出血型外,同一血清型的国内和国际参考株的ＲＴ型不相同;大多数野生株的ＲＴ和相应血清型国内参考株相同,差异也只表现为谱形上个别带型的缺少和增加,所研究的波摩那型野生株的ＲＴ型和国际参考株相同而和国内参考株不同     

Molecular and phenotypic characterization of Aspergillus japonicus and Aspergillus aculeatus strains with special regard to their mitochondrial DNA polymorphisms

Forty Aspergillus japonicus and A. aculeatus strains, most of them wild-type isolates, were examined using various molecular and phenotypic techniques. The rDNAs proved to be invariable (even strains of the species A. aculeatus exhibited the same restriction profile), while the strains could be classified into seven different mtDNA RFLP groups. Hybridisation data suggest that six of these mtDNA types have certain common restriction sites, while mtDNA type 7, which was exhibited by some A. aculeatus strains, probably has quite different mtDNA organisation and their size was smallest among the strains studied. The RAPD technique and isoenzyme analysis revealed some variabilities within these RFLP groups and strain specific features could also be recognised. Carbon source assimilation spectra were found to be very distinctive for strains of A. japonicus, A. aculeatus and A. niger, providing a useful tool for pre-characterising new wild-type isolates of black Aspergilli. Only a limited correlation was observed between the dendrograms based on genotypic and phenotypic characters.     

Identification and differentiation of species and strains of Arthrobacter and Microbacterium barkeri isolated from smear cheeses with Amplified Ribosmal DNA Restriction Analysis (ARDRA) and pulsed field gel electrophoresis (PFGE)

ARDRA (Amplified Ribosomal-DNA Restriction Analysis) was used to differentiate among species and genera of Arthrobacter and Microbacteria. Species-specific restriction patterns of PCR-products were obtained with NciI for Arthrobacter citreus (DSM 20133T), A. sulfureus (DSM 20167T), A. globiformis (DSM 20124T) and A. nicotianae strains (DSM 20123T, MGE 10D, CA13, CA14, isolate 95293, 95294, and 95299), A. rhombi CCUG 38813T, and CCUG 38812, and Microbacterium barkeri strains (DSM 30123T, MGE 10D, CA12 and CA15, isolate 95292, and isolate 95207). All yellow pigmented coryneforme bacteria isolated from the smear of surface ripened cheeses were identified as either A. nicotianae or M. barkeri strains. Using pulsed field gel electrophoresis (PFGE) strain specific restriction pattern for all Arthrobacter species and Microbacteria tested were obtained with restriction enzymes AscI and SpeI.     

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.     

Bacteria associated with hazelnut (Corylus avellana L.) decline are of two groups: Pseudomonas avellanae and strains resembling P. syringae pv. syringae

A total of 118 fluorescent pseudomonads associated with hazelnut decline, which has been occurring for many years in different areas of northern Greece and Italy, were assessed by performing a repetitive PCR analysis with enterobacterial repetitive intergenic consensus, box element, and repetive extragenic palindromic primer sets, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein extracts, a carbon compound utilization analysis, and an analysis to determine the presence of the syrB gene. A subset of 53 strains was also characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA) by using nine restriction endonucleases. The virulence of 40 representative strains was assessed by using serial doses. The pathogenic specificities of the strains were also verified. ARDRA carried out with HinfI revealed two main groups of strains, groups A and B, which exhibited a level of similarity of 57%. The other eight restriction endonucleases used did not separate the strains. In addition, a cluster analysis performed by the unweighted pair group method using arithmetic averages after repetitive PCR and SDS-PAGE of protein extracts also revealed the same two groups. Furthermore, the differential utilization of some carbon compounds made it possible to differentiate the groups. Virulence assessment clearly indicated that the group A strains are very virulent, whereas the group B strains proved to be mildly virulent for hazelnut. Group A included the strains isolated in northern Greece and central Italy (i.e., the province of Viterbo); these strains do not have the syrB gene, are pathogenically restricted to Corylus avellana, and belong to Pseudomonas avellanae. Group B includes the other strains obtained from hazelnut cultivated in Piedmont, Campania, Latium, Sicily, and Sardinia. They represent a distinct taxon closely related to Pseudomonas syringae pv. syringae.