Plasmids for biodegradation of 2,6-dimethylpyridine, 2,4-dimethylpyridine, and pyridine in strains of Arthrobacter]

Arthrobacter crysallopoietes strain KM-4 degrading 2,6-dimethylpyridine and strain KM-4a degrading both 2,6-dimethylpyridine and pyridine, Arthrobacter sp. KM-4b degrading 2,4-dimethylpyridine were isolated from soil. Arthrobacter crystallopoietes KM-4 and Arthrobacter sp. KM-4b contain 100 Md plasmids pBS320 and pBS323. Arthrobacter crystallopietes KM-4a harbours a 100 Md and 80 Md plasmids. Plasmid curing and conjugation transfer results confirm that these plasmids are involved in degradation of 2,6-dimethylpyridine, 2,4-dimethylpyridine and pyridine. A mutant with lost ability to degrade 2,6-dimethylpyridine was isolated during the growth of strain KM-4 rifR at 42 degrees C. Electrophoretic analysis of the plasmid from temperature sensitive mutant revealed the deletion the size of 26 Md from pBS320 plasmid.     

New plasmidovars of Yersinia pestis isolated in Mongolia]

The plasmid spectres of 122 strains of Yersinia pestis isolated in Mongolia from patients, wild mammals and arthropods were studied. The populations of three plasmidovars of Yersinia pestis were found to be circulating in the natural foci of plague in Mongolia. The first plasmidovar harbours three plasmids with mol masses 6, 47, 65 Md. The second and third plasmidovars contain the plasmids with mol masses 6, 16, 47, 65 Md and 8, 47, 75-80 Md.     

Plasmid profiles of Neisseria gonorrhoeae in Peninsular Malaysia

The plasmid profiles of 160 strains of Neisseria gonorrhoeae isolated in Peninsular Malaysia, comprising 80 penicillinase-producing (PPNG) and 80 non-penicillinase-producing (non-PPNG) isolates, were determined. The 80 PPNG isolates were divided into two plasmid groups. All of them harbored two common plasmid species, a 4.4 megadalton (Md) R plasmid previously associated with beta-lactamase production in PPNG strains from the Far East and a 2.6 Md multicopy plasmid of unknown function. In addition to these two plasmids, 60 (75%) PPNG isolates also carried a large 24.5 Md conjugative plasmid. In contrast, the 80 non-PPNG strains were divided into three plasmid groups. All of them possessed the 2.6 Md cryptic plasmid, and 35 (44%) isolates also harbored the 24.5 Md transfer plasmid. Besides these two plasmids, one non-PPNG isolate carried an additional 7.8 Md cryptic plasmid.     

Plasmids of the cyanobacterium Synechocystis sp. 6803

Three cryptic plasmids have been isolated from cyanobacterium Synechocystis sp. 6803::pSS2 (1.4 Md), pSS3 (36 Md), pSS4 (60 Md). Plasmid DNA was isolated in Cs-Cl-EB density gradient and analyzed by gel electrophoresis and electron microscopy by gel electrophoresis and electron microscopy techniques. The restriction map is constructed for plasmid pSS2 having the cleavage sites for Sau3a, HincII, HindIII, MspI restriction endonucleases. The plasmid may be used to construct the recombinant vector DNAs capable of autonomous replication in cyanobacterium Synechocystis sp. 6803. cells.     

The effect of Yersinia pestis EV76 6 MD plasmid on the composition of outer membrane proteins of Yersinia pseudotuberculosis YPIII]

On the basis of Yersinia pseudotuberculosis strain YPIII the isogenic variants containing the different combinations of 47 Md plasmids from Yersinia pestis or Yersinia pseudotuberculosis cells with the 6 Md pYP plasmid from Yersinia pestis EV (intact or having impaired the pla gene determining the synthesis of plasmocoagulase). The degradation of the secreted proteins encoded by the 47 Md plasmids of Yersinia pestis and Yersinia pseudotuberculosis in the cells harbouring the 6Md pYP plasmid has been registered. Yersinia pseudotuberculosis strain YPIII carrying its own 47Md and pYP plasmids also contained no YOP1 protein, in contract to the parent strain. The damage of the pla gene eliminated the destructive effect on the outer membrane proteins. Imposition of the 47Md and 6Md plasmids from Yersinia pestis in Yersinia pseudotuberculosis cells may be used for obtaining and study of the physiological role of low molecular mass proteins resulting from proteolysis of proteins encoded by the 47Md virulence plasmid of Yersinia.     

Polyresistant Enterobacteriaceae strains and their plasmids in a hospital. Medical and theoretical aspects

The properties and origin of multiple resistant strains of Enterobacteriaceae found in the intestine and nasopharynx of infants admitted to the hospital for premature infants were studied. The strains of E. coli of different serovars isolated at various periods contained similar conjugative R plasmids with a molecular weight of 80 Md belonging to the O incompatibility group controlling resistance to kanamycin and physically independent small plasmids controlling resistance to ampicillin (7 Md) and streptomycin-sulfanilamides (4 Md). Multiple drug resistance in the strains of K. pneumoniae was controlled by single large (100-120 Md) plasmid cointegrates with 6-8 resistance markers. Such cointegrates consisted of several potentially independent plasmids, sometimes dividing on transformation of plasmid DNA of the recipient strains of E. coli K12. The small plasmids controlling resistance to ampicillin and streptomycin-sulfanilamides similar to the respective plasmids of E. coli were the constant components of the plasmids cointegrates. The multiple drug resistance in the above strains was combined with high capacity for colonization in premature infants. The medical staff and mothers were the sources of bacterial strains with single plasmids controlling definite types of resistance. It is suggested that the multiple resistant strains of Enterobacteriaceae are formed in hospital as a result of accumulation of the plasmids or plasmid markers and selection. One of the conditions for successive acquisition of new plasmid markers by definite bacterial strains was their high capacity for colonization in patients, which provided constant contacts and genetic exchange of such strains with a wide range of immigrant strains during colonization in the newly admitted patients.     

The R-factors of multiple antibiotic resistant faecal coliforms isolated from a domestic dog

The antibiotic resistant faecal flora of a domestic dog suffering from an acute enteric infection was examined. The flora exhibited overall resistance to a wide variety of antibiotics. However, following restoration of the animal to normal health, overall resistance to ampicillin (Ap), tetracycline (Tc), chloramphenicol (Cm) and streptomycin (Sm) was lost, although low numbers of bacteria resistant to these four antimicrobial agents could still be isolated up to one year later. A total of 11 strains were purified for further study. All 11 were positively identified as Escherichia coli and shown to be resistant to various combinations of the above antibiotics, and additionally to kanamycin (Km). Each strain harboured from one to five plasmids, although only four proved capable of transferring antibiotic resistance to Escherichia coli K-12. One of the strains was found to harbour two conjugal plasmids pNJ101 (60 Md) and pNJ102 (133 Md) which coded for resistance to Cm, Tc, Ap and Cm, Tc, Km respectively. A third plasmid pNJ103 (29 Md) remains cryptic. The possession of the two plasmids pNJ101 and pNJ102 appears to be an unstable situation as variants arose harbouring one or other of the plasmids.     

杭州市淋病奈瑟菌质粒酶切图谱分析研究

目的 :了解杭州市淋病奈瑟菌质粒携带及质粒谱型分布情况。方法 :采用碱裂解法对门诊 2 0 0 0年 1月～ 2 0 0 1年 10月分离的 2 0 7株淋病奈瑟菌进行了质粒抽提及质粒谱分型研究 ,并对菌株的青霉素耐药现象和耐药性质粒的关系进行探讨。结果 :2 0 7株淋病奈瑟菌中 194株 (93 .7% )检见质粒带 ,其中含一条质粒带的 112株 (5 4.11% ) ,含两条质粒带的 12株 (5 .8% ) ,含三条带的 70株 (3 3 .82 % ) ,尚有 13株(6.2 8% )未检测到质粒。以 E.coli V5 17细菌质粒作分子量标准 ,测得这些分子量分别为 2 .6、4.5、和 2 4.5Md。质粒谱型以 2 .6 4.5 2 4.5 Md(3 3 .82 % )多见。结论 :杭州地区质粒酶切图谱的分析研究有助于淋病的治疗和防治 ,这将对该地区淋病奈瑟菌的分子流行病学调查和淋病监控提供依据     

Screening of plasmids in museum strains of Yersinia pestis isolated from various natural foci

92 strains of Yersinia pestis isolated from different natural foci and stored for 3-40 years in the museum of live cultures have been studied. The strains having three typical plasmids, their different combinations, plasmidless strains or the strains carrying nontypical plasmids with the molecular masses 9, 15, 55, 80, 90 and 150 Md were found. The old museum strains are proposed to be used as a source of plasmids for the genetical research. The current control of plasmid contents in the museum strains is suggested by the plasmid changes in course of storage.     

Partial characterization of plasmids from rabbit isolates of Pasteurella multocida.

Plasmids have not been reported for isolates of Pasteurella multocida from rabbits. We assayed 28 isolates of rabbit P. multocida for plasmids and sought to determine whether or not plasmid presence correlated with clinical or pathologic findings, serotype, toxin production, possession of pili, or biochemical characteristics. Fourteen isolates bore a single 1.6 Md (covalently closed circular form in 0.7% agarose gels) plasmid. An additional isolate had two plasmids which migrated as a closely-spaced doublet, centered around 1.6 Md. Eleven isolates appeared to have identical plasmids, according to Hae III and Hinf I digests. The apparent linear size of this common plasmid in 2% agarose gels was 2.1 Md, as calculated from the sums of the sizes of Hae III or Hinf I digestion fragments. Linearization of the common plasmid with Msp I produced an apparent size of 2.5 Md in 0.7% agarose gels. No correlations between presence of the common plasmid and somatic serotype, toxigenicity, presence of pili, antimicrobial resistance, selected biochemical characteristics, anatomic site from which the bacteria were cultured, or disease status of the host were found.     

Characterization of conjugative plasmids belonging to a new incompatibility group (IncZ)

More than 30 conjugative R plasmids between 60 and 70 Md in size were identified in wild type strains of Enterobacteriaceae isolated in German Democratic Republic, Bulgaria, Mongolia, Poland, Ethiopia, Iraque, and Soviet Union. They have been characterized by means of several genetic and molecular techniques as members of a new incompatibility group, termed IncZ. The properties of these plasmids including digestion pattern after EcoRI treatment demonstrate a phylogenetic relatedness.     

Genetic determination of the vibriocinogenicity trait in Vibrio cholerae of the El Tor biotype

In two different strains of cholera vibrios two recA-dependent plasmids, pVib I (1.9-2.2 Md) and pVib II (5.2-5.8 Md), have been detected. These plasmids determine the synthesis of vibriocin, coagulase and fibrinolysin, which has been established by the cotransformation of the DNA of plasmids pVib I and pBR322 and by the transfer mobilization with the use of plasmid RP4.     

Functional characteristics of plasmids of Shigella sonnei 47 strains

The phenotypic characteristics of Shigella sonnei strain 47 containing 7 plasmids of low molecular weight and 2 plasmids 60-100 Md large have been studied. The strains of Escherichia coli containing the single plasmids or plasmid groups from Shigella sonnei have been obtained by transformation and conjugation. The comparison of phenotypes of the obtained strains has helped to find the plasmid location of the determinants for streptomycin resistance (P7), genes for colicinogenicity and colicin immunity (P5), the enzymes of host cell specificity system Sso47I (P6), Sso47II (P4), and the genes for the conjugative DNA transfer (P9). Escherichia coli strains producing individual restriction enzymes SsoI and SsoII have been isolated.     

Use of genetic and molecular characteristics of R plasmids as an epidemiological marker in an outbreak of hospital infection

Antibiotic sensitivity of 38 strains of enteric bacteria, such as Serratia marcescens Klebsiella pneumoniae and others and Ps. aeruginosa isolated during an outbreak of meningitis in a premature infant resuscitation department was studied. It was shown that all the isolates were multiple resistant, most frequently to 7 antibiotics. All the resistance markers were transferred on conjugation, segregation of some markers being observed. Investigation of the plasmid composition of the clinical strains and transconjugants of E. coli revaled the presence of 2 plasmids with the molecular weights of 40 and 60 Md or one of them. The restriction analysis demonstrated that the plasmids with the same molecular weights isolated from different strains were identical. It was suggested that such plasmids originated from the same source and were distributed by conjugation. The possible part of R plasmids in epidemiological analysis of hospital infections is discussed: the possible part as an additional marker in determination of the infection source and the possible part through its ability to change the host cell phenotype, including the phage and bacteriocin types.     

Construction of plasmid vectors for gene cloning in Escherichia coli and Bacillus subtilis

The construction and some properties of new hybrid plasmids which are able to replicate in both Escherichia coli and Bacillus subtilis are presented. A 5.5 Md hybrid plasmid pJP9 was constructed from pBR322 (Tc, Ap) and pUB110 (Nm) plasmids. pIM1 (7.0 Md) and pIM3 (7.7 Md) plasmids are its different erythromycin resistant derivatives. Tetracycline, ampicillin, neomycin and possibly erythromycin resistance genes are expressed in E. coli while neomycin and erythromycin resistance genes are expressed in B. subtilis. Insertional inactivation of only one gene is possible using the pJP9 plasmid as a vector in B. subtilis. However, insertional inactivation of at least two different genes can be achieved and monitored in E. coli and B. subtilis transformants in cloning experiments with PIM1 and pIM3 plasmids. Insertional inactivation of antibiotic resistance genes present in pJP9 plasmid was achieved by cloning of Streptococcus sanguis DNA fragments generated by appropriate restriction endonucleases. The pJP9 plasmid and its derivatives were found to be stable in both hosts cells.     

Plasmids encoding trimethoprim resistance in bacterial isolates from man and pigs

Trimethoprim (Tp) resistant Gram negative bacteria were isolated from humans and pigs. The bacterial hosts were characterized by their resistance pattern and biotype. The presence of transferable Tp plasmids was demonstrated in 86% of 59 porcine isolates and 37% of 49 human isolates. The Tp R-plasmids carried a diversity of resistance determinants such as Tc, Cm, Sp, Sm and Su. Incompatibility tests distinguished two major groups, Inc FIV and Inc N. Thirty of 99 Tp R-plasmids isolated from humans were grouped as Inc FIV and eight as Inc N. The results of molecular weight determination of Tp R-plasmids performed by agarose gel electrophoresis were consistent with the existence of two groups--larger R-plasmids (76 to 104 Md) belonging to Inc FIV and lower molecular weight R-plasmids (25 to 35 Md) belonging to Inc N. Results from this study indicate that the Tp R-plasmids isolated in Perth have evolved independently from those described in Europe and the United Kingdom. There is also evidence for their local spread between Escherichia, Klebsiella, Enterobacter, Citrobacter and Acinetobacter from man and animals.     

Analysis of the plasmid composition of Yersinia pseudotuberculosis strains and its use for typing pseudotuberculosis pathogens

Electrophoresis in agarose gel has been used to study the plasmid spectra of 854 Yersinia pseudotuberculosis strains isolated from different sources. The plasmids found in the microbial strains are represented by the elements with molecular masses 82; 57; 45; 5.5; 4.4; 3.5; 2.7; 2.4; 2.3 Md. The variable spectra of plasmids is peculiar only for serovar I of Yersinia pseudotuberculosis. Plasmids p45 and p82 are classified as the main, while other plasmids as auxiliary ones. In accord with the classification all plasmid containing strains are divided into 8 plasmid strains. Using the proposed method for intraspecific typing of Yersinia pseudotuberculosis permits one to perfect the epidemiological analysis of pseudotuberculosis infection and make concrete the direction of prophylactic and antiepidemic measures.     

Plasmids encoding trimethoprim resistance in bacterial isolates from man and pigs

Trimethoprim (Tp) resistant Gram negative bacteria were isolated from humans and pigs. The bacterial hosts were characterized by their resistance pattern and biotype. The presence of transferable Tp plasmids was demonstrated in 86% of 59 porcine isolates and 37% of 49 human isolates. The Tp R-plasmids carried a diversity of resistance determinants such as Tc, Cm, Sp, Sm and Su. Incompatibility tests distinguished two major groups, Inc FIV and Inc N. Thirty of 99 Tp R-plasmids isolated from pigs were grouped as Inc FIV and three as Inc N. Eleven of 26 Tp R-plasmids isolated from humans were grouped as Inc FIV and eight as Inc N. The results of molecular weight determination of Tp R-plasmids performed by agarose gel electrophoresis were consistent with the existence of two groups—larger R-plasmids (76 to 104 Md) belonging to Inc FIV and lower molecular weight R-plasmids (25 to 36 Md) belonging to Inc N. Results from this study indicate that the Tp R-plasmids isolated in Perth have evolved independently from those described in Europe and the United Kingdom. There is also evidence for their local spread between Escherichia, Klebsiella, Enterobacter, Citrobacter and Acinetobacter from man and animals.     

Melanin production encoded by a cryptic plasmid in a Rhizobium leguminosarum strain

Rhizobium leguminosarum strain VF39, isolated from nodules of field-grown faba beans in the Federal Republic of Germany, was shown to contain six plasmids ranging in molecular weight from 90 to 400 Md. Hybridisation to nif gene probes, plasmid curing, and mobilisation to other strains of Rhizobium and to Agrobacterium showed that the third largest plasmid, pRleVF39d (220 Md), carried genes for nodulation and nitrogen fixation. This plasmid was incompatible with pRL10JI, the Sym plasmid of R. leguminosarum strain JB300. Of the other plasmids, the two smallest (pRleVF39a and pRleVF39b, 90 and 160 Md respectively) were shown to be self-transmissible at a low frequency. Although melanin production is as yet unreported in strains of R. leguminosarum biovar viceae, strain VF39 produced a dark pigment, which, since it was not produced on minimal media and its production was greatly enhanced by the presence of tyrosine in the media, is probably melanin-like. Derivatives of VF39 cured of pRleVF39a no longer produced this pigment, but regained the ability to produce it when this plasmid was transferred into them. Strains of Agrobacterium tumefaciens, R. meliloti, and some strains of R. leguminosarum carrying pRleVF39a did not produce this pigment, indicating perhaps that some genes elsewhere on the VF39 genome are also involved in pigment production. Plasmid pRleVF39a appeared to be incompatible with the cryptic Rhizobium plasmids pRle336b and pRL8JI (both ca. 100 Md), but was compatible with the R. leguminosarum biovar phaseoli Sym plasmids pRP1JI, pRP2JI and pRph51a, all of which also code for melanin production. The absence of pRleVF39a in cured derivatives of VF39 had no effect on the symbiotic performance or competitive ability of this strain.     

The role of plasmids in the regulation of delta-endotoxin synthesis in Bacillus thuringiensis H14

The role of plasmids in regulation of delta-endotoxin synthesis by Bacillus thuringiensis H14 was studied. The derivatives of strain Is-1 H14 containing a 4Md plasmid integrated into the chromosome synthesize small crystals and are not toxic for the gnat larvae. The transceptional transfer into this strain of a plasmid coding for crystal synthesis from the strain 69-6 serotype H5 results in restoration of insecticidal activity to the level of the parental strain Is-1. Transcipients activity is increased 10-15 fold in case of 4Md plasmid excision from the chromosome and autonomous functioning. Evidently, 4Md plasmid from the strain Is-1 as well as a plasmid coding for crystal synthesis from the strain 69-6 contains the regulatory elements participating in the expression of crystalline protein genes localized on other plasmids. The existence of two cellular regulatory groups is supposed to result in the significant increase in crystalline protein synthesis.