Structural and functional similarities between the replication region of the Yersinia virulence plasmid and the RepFIIA replicons.

We sequenced the minimum replication region of the virulence plasmid pYVe439-80 from a serogroup O:9 Yersinia enterocolitica. This sequence is 68% homologous on a 1,873-nucleotide stretch to the sequence of the RepFIIA replicon of the resistance plasmid R100. The sequence contains two open reading frames, repA and repB, encoding proteins of 33,478 and 9,568 daltons, respectively. The amino acid sequences of the two proteins are 77 and 55% identical, respectively, to proteins RepA1 and RepA2 of the R100 replicon. Analysis of minicells transformed with a copy number mutant demonstrated that the replication region of pYVe439-80 directs the synthesis of a 33-kilodalton protein. Disruption of repA, encoding this protein, abolished replication. Two regions of pYVe439-80 are 76 and 70% homologous, respectively, to the copy number control antisense RNA and to the origin of replication region of R100. A mutation introduced in the pYVe439-80 DNA corresponding to the R100 sequence encoding the copy number control antisense RNA resulted in an increase in copy number, indicating a functional homology between the two replicons.     

Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within HupRhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H(2)-uptake hydrogenase positive (Hup) or negative (Hup) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H(2) oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H(2)-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H(2)-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup strains in this symbiotic species.     

Molecular characterization of Yersinia enterocolitica by pulsed-field gel electrophoresis and hybridization of DNA fragments to ail and pYV probes.

Sixty strains of Yersinia enterocolitica from five serogroups (O:3; O:9; O:8; O:5; and O:5,27) and eight non-Y. enterocolitica strains, recovered from diverse sources (humans, animals, food, and the environment) in Europe, Argentina, and the United States, were examined by the pulsed-field gel electrophoresis (PFGE) technique of contour clamped homogeneous electric field electrophoresis (CHEF) by using NotI and XbaI as restriction enzymes. NotI and XbaI generated 36 and 33 restriction endonuclease digestion profiles (REDP), respectively. By combining the results of both enzymes, 42 unique genomic groups were differentiated. DNA fragments were transferred to nylon membranes and hybridized with digoxigenin-labelled oligonucleotide probes to the ail gene and virulence plasmid to determine hybridization patterns and the potential virulence of the strains. The strains were tested for the presence of the plasmid by PFGE-CHEF and phenotypic characteristics encoded for by the virulence plasmid. Thirty of the 60 Y. enterocolitica strains tested harbored the virulence plasmid. The specificity of the ail and pYV probes was 100% when tested with 68 Yersinia strains and 19 different non-Yersinia strains. Sixteen selected Y. enterocolitica strains were tested for their virulence by lethality in iron- and desferrioxamine-sensitized mice. No correlation between REDP and the virulence of the strains was observed. The observed REDP and the hybridization patterns were very homogeneous within a serogroup and independent of the source of isolation. In addition, PFGE-CHEF was shown to be valuable in identifying and confirming serogroups. Principal component analysis of Dice similarity indices from REDP was an excellent tool for determining genetic relatedness among strains.     

Restriction map of virulence plasmid in Yersinia enterocolitica O:3

Restriction map of the 72-kb virulence plasmid isolated from Yersinia enterocolitica O:3 was generated using EcoRI, BamHI, HindIII, and XbaI restriction enzymes. The mapping was done after cloning all of the 13 BamHI fragments of the plasmid in Escherichia coli. In addition, the restriction enzyme analysis revealed two types of virulence plasmids (types I and II) in Y. enterocolitica O:3. No functional differences between the strains bearing type I or type II plasmid were observed.     

Second serogroup of Legionella quinlivanii isolated from two unrelated sources in the United Kingdom

R.J. BIRTLES, N. DOSHI, N.A. SAUNDERS AND T.G. HARRISON. 1991. A series of strains, presumptively identified as legionellas on the basis of their nutritional requirements and biochemical reactivity, were isolated from two unrelated environmental sources in the UK. Representatives of each of these series had a restriction endonuclease digest pattern indistinguishable from that of the Legionella quinlivanii type strain (1442-AUS-E) and the identity of these strains was confirmed by DNA homology studies. Serological examination of the two strains showed that they were distinct from the type strain 1442-AUS-E but indistinguishable from each other. A second serogroup, L. quinlivanii serogroup 2 (type strain LC870; NCTC 12434), is proposed to accommodate these strains.     

Purification of lipopolysaccharide from strains of Yersinia enterocolitica belonging to serogroups 03 and 09

Lipopolysaccharide (LPS) was purified from strains of Yersinia enterocolitica belonging to serogroups 03 and 09, by three methods, and analysed by SDS-PAGE and silver staining for carbohydrate. SDS-PAGE of LPS prepared from whole-cells by digestion with proteinase-K, produced profiles containing high molecular mass LPS and a lower molecular mass region migrating as discrete bands. LPS prepared from strains belonging to serogroup 03, using a hot-phenol procedure alone was found to contain cellular proteins, and LPS prepared from strains of serogroup 09, by this method, did not contain high molecular mass carbohydrate. A novel method of preparing LPS by digesting bacterial outer membranes with proteinase-K prior to hot-phenol extraction produced protein-free LPS from strain of Y. enterocolitica 03 and high molecular mass LPS from strains belonging to serogroup 09.     

Genetic organization of the duplicated vap region of the Dichelobacter nodosus genome.

The recombinant plasmid pJIR318 contains a fragment of the Dichelobacter nodosus genome which is associated with virulence. Sequence analysis of the pJIR318 insert has shown that it contains four vap (virulence-associated protein) genes which are homologous to open reading frames found on the Escherichia coli F plasmid and the Neisseria gonorrhoeae cryptic plasmid (M. E. Katz, R. A. Strugnell, and J. I. Rood, Infect. and Immun. 60:4586-4592, 1992). The plasmid pJIR318 hybridizes to three regions of the D. nodosus genome, each of which has now been isolated. Regions 1 and 3 were found to be adjacent in the genome of D. nodosus A198, and the order of the vap genes in vap regions 1 and 2 were shown to be identical. Partial sequence analysis and Southern blot analysis of the vap regions showed that the three regions probably arose by a duplication event(s) followed by insertions and/or deletions. A recombinant plasmid, pJIR749, was isolated from a library of a benign D. nodosus strain, 305. This plasmid contained sequences from both ends of vap region 2. Analysis of pJIR749 showed that the sequences on either side of vap region 2 were separated by 324 bp in the genome of benign strain 305 and that the orientations of the sequences were different. It is clear that a simple insertion or deletion event did not generate the benign and virulent strains studied. A model which describes the evolution of the duplicated vap regions in D. nodosus A198 is presented.     

Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids,outer membrane protein profiles and siderophore production

The virulence of 18 strains of Vibrio anguillarum serogroup O1 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only strains that carried the 67 kbp virulence plasmid or derivatives of it produced the outer membrane protein, OM2. All virulent strains harboured the 67 kbp plasmid or derivatives of it, indicating its importance for virulence. However, some strains carrying the virulence plasmid or a derivative of it, produced siderophores as well as OM2 but were non-pathogenic to fish. Likewise, among the virulent strains, considerable variation in LD50 values was recorded. Plasmid profiling and restriction analysis showed that the virulence plasmid existed in various molecular weights from 26 to 80 kbp, with 65-67 kbp being the most common, and that this plasmid displayed various restriction profiles. The presence of other plasmids did not seem to affect the pathogenic properties.     

Evaluation of the usefulness of selected virulence markers for identification of virulent strains of Yersinia enterocolitica strains. III. Chromosome markers of virulence

It is well known that virulent strains of Y. enterocolitica bear the virulence-associated plasmid pYV. Moreover some authors consider that the pathogenic strains of these bacteria have chromosome encoded phenotypic and genotypic features such as: genes ail and yst which could be used as virulence markers. The virulent strains of Y. enterocolitica do not produce pyrasinamidase and are not able to ferment salicin and cannot hydrolyse esculin. In addition these strains produce thermostable enterotoxin called YST and protein Ail (attachment invasion locus). In contrast to phenotypic virulence makers the biological function of proteins Ail, YST and nucleotide sequence of genes ail and yst is well described. In the presented study one hundred thirty virulence plasmid bearing Y. enterocolitica strains belonging to serogroup O3 were examined for the presence of genes ail, yst, and were tested for their inability to pyrasinamidase production, salicin fermentation and esculin hydrolysis. In addition forty pYV plasmid-cured isogenic strains were included in to the study. Genes ail and yst were detected by polymerase chain reaction (PCR). The obtained results indicate that all tested 130 pYV+ Y. enterocolitica strains as well as 40 plasmid-cured isogenic strains have carried ail and yst genes. All tested strains did not produce pyrasinamidase, hydrolyse esculin and ferment salicin. This generally was in agreement with the observations done by other authors and suggest that the chromosomal virulence markers, especially well described genes ail and yst, could be useful for excluding the potential virulence of Y. enterocolitica strains, which had lost pYV plasmid and have no ail or yst genes. Therefore, in clinical studies, Y. enterocolitica strains isolated directly from patients should be primarily tested for the presence of the virulence plasmid and secondarily, the negative ones could be examined for the presence of the chromosomal virulence markers.     

Plasmid profile of strains of various Legionella species

The plasmid profile of Legionella strains of different origin has been studied. 15 out of 32 Legionella cultures belonging to different strains have been found to contain plasmid DNA in an amount of 1 or 2 plasmids, with the exception of L. feelei having 6 plasmids. Only 1 out of 3 Legionella strains isolated in the USSR has been found to possess a plasmid with a molecular weight of about 80 MD. Plasmids with this molecular weight have been found in 13 Legionella strains under study, such plasmids in L. pneumophila strains of serogroup 1 (strains Flint 1 and Albuquerque 1) and serogroup 9 (strain No. 35282) having an exact molecular weight of 82.4 +/- 2.4 MD and being similar in molecular structure, which has been established as the result of their treatment with restrictases Pst 1 and Hind III.     

Molecular Comparison of Plasmids Encoding Heat-Labile Enterotoxin Isolated from Escherichia coli Strains of Human Origin

The molecular properties of enterotoxin (Ent) plasmids from 12 Escherichia coli strains of human origin were examined. Ten strains belonged to the O78 serogroup, and the remainder were of serogroup O7 or O159. Eleven plasmids coded for heat-labile enterotoxin (LT), and one coded for heat-stable enterotoxin (ST) and LT. The results of restriction enzyme digests and deoxyribonucleic acid reassociation experiments showed that all of the Ent plasmids were related, and supported the subdivision of the LT plasmids into three groups based on their genetic properties (M. M. McConnell et al., J. Bacteriol. 143: 158–167, 1980). Within group 1, two plasmids from South African strains were indistinguishable but differed in EcoRI and HindIII digests from the LT plasmid that originated from an Ethiopian strain. The three plasmids had >70% homology. The two non-autotransferring group 2 plasmids identified in O78.H11 strains from Bangladesh were indistinguishable. The group 3 plasmids were from strains belonging to serogroups O7 and O78 isolated in Bangladesh, India, and Thailand. They shared >95% homology but showed slight differences in fragment patterns when treated with EcoRI and HindIII. There was 60 to 70% homology between the plasmids of groups 1 and 3, and the group 2 plasmid had 40 to 50% homology with members of these two groups. The autotransferring Ent plasmids had up to 40% homology with R factors of incompatibility groups FI, FII, and FIV.     

限制和修饰系统LlaBIII在构建抗噬菌体菌株中的作用

限制和修饰 (ｒｅｓｔｒｉｃｔｉｏｎａｎｄｍｏｄｉｆｉｃａｔｉｏｎ ,Ｒ Ｍ)系统是指由限制性内切酶和甲基化酶组成的单亚基或多亚基复合酶系统 ,两者通常成对出现 ,具有相同的ＤＮＡ识别位点 ,其作用相反。Ｒ Ｍ系统在原核生物中普遍存在 ,在保护细胞免遭外源病毒侵害方面具有重要作用[1] 。作为发酵剂的乳酸乳球菌在乳制品发酵中具有重要作用 ,但这类菌株极易遭受噬菌体感染 ,导致菌株产酸力降低 ,甚至发酵失败 ,造成严重的经济损失。所以在乳制品发酵过程中防止噬菌体感染就成为十分重要的问题。通过自然筛选或诱变处理等手段筛选噬菌…     

限制和修饰系统LlaBⅢ在构建抗噬菌体菌株中的作用

限制和修饰(restriction and modification,R/M)系统是指由限制性内切酶和甲基化酶组成的单亚基或多亚基复合酶系统,两者通常成对出现,具有相同的DNA识别位点,其作用相反.R/M系统在原核生物中普遍存在,在保护细胞免遭外源病毒侵害方面具有重要作用[1].     

Spontaneous deletions in the TOL plasmid pWW20 which give rise to the B3 regulatory mutants of Pseudomonas putida MT20

The size of the TOL plasmid pWW20 from Pseudomonas putida MT20, as measured by analysis of agarose electrophoresis gels after restriction endonuclease hydrolysis, was 270-280 kilobase pairs (kb). During growth on benzoate, MT20 segregates strains carrying mutations in the plasmid regulatory gene xylS; these so-called B3 strains retain the ability to grow on m-xylene (Mxy+) but do not grow on its metabolite m-toluate (Mtol-) and have also lost the ability to transfer the plasmid (Tra-). Analysis of restriction digests of plasmid DNA from seven such segregants, independently isolated, showed that pWW20 had undergone extensive deletions of 90-100 kb. All the deleted plasmids had lost a common core of DNA, of about 72-80 kb, but in class A mutants the deletion extended at one end of this core and in class B mutants at the other end. Class A and B mutants also differed in their rate of growth on m-xylene as a result of differences in the level of expression of their plasmid-coded catabolic enzymes. This suggests that an additional gene, involved in regulating levels of gene expression, is located in the region uniquely deleted in the class B mutants.     

Arsenic and cadmium resistance in environmental isolates of Yersinia enterocolitica and Yersinia intermedia

Environmental strains of Yersinia enterocolitica representing biotype 1A lack virulence plasmid (pYV) and are regarded as non-pathogenic. Though these occupy a diverse range of environmental niches, nothing is known about their resistance to heavy metals. The minimal inhibitory concentrations (MICs) of various metal ions, namely Ag+, Cu2+, Zn2+, Cd2+, As5+, and As3+, for strains of Yersinia enterocolitica (biotype 1A) and Yersinia intermedia (biotypes 1, 2, and 4), isolated from sewage effluents or pork, were determined. All isolates were resistant (MICs 2.5-5 mM) to Cd2+. The MICs of arsenic varied with bacterial strain and the chemical species of the arsenic used. For the majority of the strains, however, it was between 5-10 mM of Na2HAsO4.7H2O and NaAsO2, and 0.625-2.5 mM of As2O3. Except for one isolate, MICs of Ag+, Cu2+, and Zn2+ for these strains were in the range of 0.3-0.625 mM.     

Conjugative R-plasmid resistance of the causative agents of Yersinia infection

Nature, structure, occurrence and drug resistance of 160 strains of Y. pseudotuberculosis and 60 strains of Y. enterocolitica isolated from various sources within 1986-1988 were studied. In the strains of Y. pseudotuberculosis, the cell composition with respect to the requirements in calcium ions as well as the plasmid profiles with determination of the molecular weights of the plasmids in the antibiotic sensitive and resistant pathogens and R(+)-transconjugants were investigated. Some molecular genetic properties of the Yersinia R plasmids were also investigated. Antibiotic polyresistant strains of Y. enterocolitica were the most frequent donors of the R plasmids while the strains of Y. pseudotuberculosis were less frequently the donors, in the resistance pattern of which there were more frequent streptomycin and tetracycline resistance determinants. The conjugative R plasmids of Y. pseudotuberculosis were characterized by strict control of replication, repressed frequency of transfers, and a molecular weight of about 47 MD. Their replicones as a rule contained streptomycin and tetracycline markers determining resistance to streptomycin and tetracycline at the levels of 1250 and 156 micrograms/ml, respectively.     

Low-frequency restriction fragment analysis of Frankia strains (Actinomycetales).

Low-frequency restriction fragment analysis of more than 100 strains of the genus Frankia showed that restriction enzyme DraI (recognition site, TTT'AAA) gave rise to large DNA fragments (200 to 1,500 kb), which, when they were subjected to cluster analysis, reflected the host plants from which the strains were isolated. Our results support the conclusions of Lalonde and his colleagues (M. Lalonde, L. Simon, J. Bousquet, and A. Seguin, p. 671-680, in H. Bothe, F. J. de Bruijn, and W. E. Newton, ed., Nitrogen Fixation: Hundred Years After, 1988; P. Normand, P. Simonet, and R. Bardin, Mol. Gen. Genet. 213:238-246, 1988) and Fernandez et al. (M. P. Fernandez, H. Meugnier, P. A. D. Grimont, and R. Bardin, Int. J. Syst. Bacteriol. 39:424-429, 1989) that various biochemical and genomic analyses can give rise to groupings of Frankia strains that are consistent with the host plants from which the strains are isolated and that the resulting groups may form a basis for defining Frankia species.     

Homology between clindamycin resistance plasmids in Bacteroides

Two different species of clindamycin-resistant Bacteroides were isolated from the same infection. One isolate contained a single 15-kb plasmid (pCP1) which encoded transferable clindamycin resistance. pCP1 appears similar to the Bacteroides clindamycin resistance plasmid pBFTM10 isolated independently by F.P. Tally, D.R. Snydman, M.J. Shimell, and M.H. Malamy (1982, J. Bacteriol. 151, 686-691). The second strain had a 10-kb plasmid (pCP2) but did not transfer resistance. DNA hybridization studies revealed that pCP1 shares a 5-kb region of homology with the B. fragilis R plasmid pBF4 studied by R.A. Welch and F.L. Macrina (1981, J. Bacteriol. 145, 867-872). This region in both plasmids was shown to be bounded by homologous direct repeats and contains the putative clindamycin resistance determinant. pCP1 and pCP2 were found to share extensive homology but sequences homologous to the clindamycin resistance region were missing from pCP2 and found instead in the whole cell DNA of the host strain. These results identify a transposon-like structure on Bacteroides clindamycin resistance plasmids.     

Virulence-associated plasmids from Yersinia enterocolitica and Yersinia pestis.

A 44-megadalton plasmid associated with virulence and Ca2+ dependence from Yersinia enterocolitica 8081 was compared at the molecular level with a 47-megadalton plasmid associated with Ca2+ dependence from Yersinia pestis EV76. The plasmids were found to share 55% deoxyribonucleic acid sequence homology distributed over approximately 80% of the plasmid genomes. One region in which the plasmids differed was found to contain sequences concerned with essential plasmid functions. Forty-five mutants of Y. pestis were isolated which had spontaneously acquired the ability to grow on calcium-free medium (Ca2+ independence). Of these mutants, 21 were cured of their 47-megadalton plasmid, whereas the remaining had either suffered a major deletion of the plasmid or had a 2.2-kilobase insertion located in one of two adjacent BamHI restriction fragments encompassing approximately 9 kilobases. The inserted sequence was found at numerous sites on the Y. pestis chromosome and on all three plasmids in the strain and may represent a Y. pestis insertion sequence element.