Mode of replication, size and distribution of naturally occurring plasmids in Bacillus thuringiensis

Cloned replication origin regions, derived from both small (4.9-7.5 MDa) and large (43-60 MDa) plasmids of Bacillus thuringiensis subspecies kurstaki strains HD73 and HD263 were used as hybridization probes in a Southern-blot analysis to assess both the size and horizontal distribution of native plasmid replicon groups among different subspecies of B. thuringiensis. In general, resident plasmids hybridizing to the replication origin regions derived from strains HD263 and HD73 were more commonly found in kurstaki strains than in non-kurstaki strains, suggesting a non-random distribution of plasmid incompatibility groups. Replication origin regions derived from the large HD263 plasmids (43-60 MDa) hybridized almost exclusively with large plasmids (greater than 30 MDa) of widely varying sizes. In contrast, replication origin regions derived from small plasmids hybridized exclusively with small plasmids (less than 10 MDa) showing little size variation. These results are consistent with previous observations concerning the relationship between plasmid size, mode of replication, and structural stability.     

Plasmid composition of Yersinia pestis strains from different natural foci]

The study of the plasmid composition of 246 Y. pestis strains from different natural foci in the USSR and other countries revealed that 173 strains (70%) carried three known plasmids with a molecular weight of about 6, 45-50 and 60 megadaltons (MD) respectively. In 20 strains (8%) obtained from different sources additional cryptic plasmids were detected. In some cases the absence of one or two typical plasmids was observed. Replicon pPst was shown to have quite constant molecular weight (6 MD), whereas plasmids pCad and especially pFra exhibited certain variations of their molecular weight (45-49 MD and 60-149 MD respectively) in strains of different origin.     

Identification of contemporary plasmid virulence genes in ancestral isolates of Salmonella enteritidis and Salmonella typhimurium

Six epidemiologically distinct ancestral strains of Salmonella enteritidis and 5 of S. typhimurium from the pre-antibiotic era were examined for plasmid content, and for presence of plasmid genes implicated in mouse-virulence. Five sizes of plasmid were detected in S. enteritidis varying from 1 to 60 MDa. Two sizes of plasmid were found in S. typhimurium, 28 and 60 MDa. Plasmids of the same size were not common to both serovars. The HindIII restriction patterns of 3 of the ancestral S. enteritidis plasmids were identical to the modern 38 MDa plasmid, while all contained identical bands of 3.5, 2.7 and 1.9 kb. All the 60-MDa S. typhimurium plasmids, ancestral and contemporary, had an identical restriction pattern. Three different sized S. enteritidis plasmids and one size S. typhimurium plasmid contained a 3.5-kb DNA fragment carrying the virulence locus VirA. The VirB virulence locus was located on a 2.7-kb DNA fragment in S. enteritidis and on a 2.5-kb fragment in S. typhimurium. Both loci were precisely conserved between the ancestral strains and the modern representatives of both serovars.     

Genetic and molecular characterization of an epidemic plasmid coding for multidrug resistance in Salmonella typhimurium of human origin

All 201 multidrug resistant Salmonella typhimurium strains isolated from epidemics in India contained nonconjugative (157 strains) or conjugative (44 strains) Inc F1me multiresistance plasmids. Two small R-plasmids of 7 MDa which coded for resistance to either ampicillin or streptomycin and sulfamethoxazole were also detected along with other plasmids. The small plasmids were members of group 1 and group 2 incompatibility groups. Restriction endonuclease analysis of conjugative (96 MDa) and nonconjugative (88 MDa) Inc F1me plasmids showed considerable similarity except for the presence of unique fragments among both the groups and the loss of fragments corresponding to the smaller size of the nonconjugative plasmid. A single Inc F1me plasmid appears responsible for various outbreaks of multiresistant S. typhimurium in different parts of India.     

Virulence and immunogenicity in experimental animals of Bacillus anthracis strains harbouring or lacking 110 MDa and 60 MDa plasmids

A comparative study was made of the virulence and immunogenicity in mice or guinea pigs of Bacillus anthracis strains harbouring 110 MDa and/or 60 MDa plasmids. Strains cured of the 110 MDa or the 60 MDa plasmid were more than 100-fold less virulent to mice than were the parental strains harbouring these plasmids. Guinea-pigs immunized with plasmid-free derivatives of the non-encapsulated vaccine strain 34F2 showed no resistance to challenge with strain 17JB, which harbours both 110 MDa and 60 MDa plasmids, suggesting that the derivative strains had lost their immunizing ability against anthrax.     

Plasmids in Listeria monocytogenes and other Listeria species.

One hundred and twenty-two food, clinical, and veterinary strains of Listeria monocytogenes were examined for the presence of plasmids. Twenty-five (20%) contained plasmids, which varied from 1.3 to 66 MDa in size. Of 10 strains of other Listeria species (L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi, and L. murrayi) examined, seven (70%) contained plasmids, varying from 38 to 53 MDa. No strains with multiple plasmids were found. Plasmids of identical size were isolated from related strains in some, although not all, cases. The presence of a plasmid in a strain was not related to phenotypic characters of known extrachromosomal inheritance.     

Plasmid deoxyribonucleic acid in strains of Bacillus sphaericus and in Bacillus moritai

Bacillus moritai and six strains of Bacillus sphaericus pathogenic to dipteran larvae were examined for the presence of covalently closed circular (CCC) DNA. The plasmid profiles of the bacteria were analyzed using a cleared lysate electrophoresis technique. Four of the six strains of B. sphaericus examined contained CCC DNA. Strain SSII-1 contained two plasmids (pKA1, pKA2) having molecular weights of about 8.4 and 2.0 megadaltons (MDa). Strains 1404 and 1881 each contained one plasmid, pKA3 and pKA4, respectively. pKA3 had a molecular weight of about 8.2 MDa. pKA4 had a relatively large plasmid with a molecular weight of about 33.5 MDa. Strain K contained five size classes of CCC DNA. The plasmids pKA5, pKA6, pKA7, pKA8, and pKA9 had molecular weights of about 11.4, 10.9, 7.4, 7.0, and 6.4 MDa, respectively. Strains 1593-4 and 1691 were plasmidless and could not be distinguished from each other based on their plasmid profiles. B. moritai ATCC 21042 contained two size classes of CCC duplex DNA; pRF100 had a molecular weight of about 4.6 MDa and pRF101 had a molecular weight of about 2.1 MDa. No phenotype association with any of the isolated plasmids has been determined.     

Prevalence, biotypes, plasmid profile and antimicrobial resistance of Campylobacter isolated from wild and domestic animals from northeast Portugal.

The incidence of Campylobacter jejuni and Campylobacter coli in wild and producing animals has been studied to evaluate their importance as potential reservoirs of campylobacter infection. These organisms were isolated from: 59 chicken (60.2%), 65 swine (59.1%), 31 black rats (57.4%), 61 sparrows (45.5%), 21 ducks (40.5%), 32 cows (19.5%) and 27 sheep (15.3%). Biotypes, plasmid and resistance profiles were studied in order to characterize the isolates. Biotypes I and II of C. jejuni were predominant in all reservoirs except swine, where C. coli I was more frequent. Plasmid prevalence was higher in strains isolated from swine (53.8%) and rats (45.5%). The size of the plasmids ranged from 1.3 to 82 MDa. A 2.3 MDa plasmid was the most frequent, detected in all the reservoirs except ducks. Antimicrobial susceptibility testing revealed that 5.5% of the strains were resistant to ampicillin, 5.5% to tetracycline, 12.6% to erythromycin and 23.5% to streptomycin. Resistance to erythromycin (26.2%) and to streptomycin (58.4%) was particularly high in isolates from swine. Tetracycline resistance was encoded by a 33 or a 41 MDa plasmid and transferred by conjugation.     

Prevalence, biotypes, plasmid profile and antimicrobial resistance of Campylobacter isolated from wild and domestic animals from Northeast Portugal

The incidence of Campylobacter jejuni and Campylobacter coli in wild and producing animals has been studied to evaluate their importance as potential reservoirs of campylobacter infection. These organisms were isolated from: 59 chicken (60.2%), 65 swine (59.1%), 31 black rats (57.4%), 61 sparrows (45.5%), 21 ducks (40.5%), 32 cows (19.5%) and 27 sheep (15.3%). Biotypes, plasmid and resistance profiles were studied in order to characterize the isolates. Biotypes I and II of C. jejuni were predominant in all reservoirs except swine, where C. coli I was more frequent. Plasmid prevalence was higher in strains isolated from swine (53.8%) and rats (45.5%). The size of the plasmids ranged from 1.3 to 82 MDa. A 2.3 MDa plasmid was the most frequent, detected in all the reservoirs except ducks. Antimicrobial susceptibility testing revealed that 5.5% of the strains were resistant to ampicillin, 5.5% to tetracycline, 12.6% to erythromycin and 23.5% to streptomycin. Resistance to erythromycin (26.2%) and to streptomycin (58.4%) was particularly high in isolates from swine. Tetracycline resistance was encoded by a 33 or a 41 MDa plasmid and transferred by conjugation.     

Conjugative co-transfer of lactose and bacteriophage resistance plasmids from Streptococcus cremoris UC653

Abstract When conjugative transfer of lactose-fermenting ability (Lac) was observed between Streptococcus cremoris UC653 (donor) and S. lactis MG1363 Sm (recipient), 70% of the Lac⁺ transconjugants had acquired total resistance to phage 712 and propagated phage C2 at a lower efficiency and with a reduced plaque size. Plasmid analysis of transconjugants combined with curing experiments showed that the Lac and phage resistance markers were associated with plasmids of 26 and 50 MDa, respectively. Some transconjugants contained a large plasmid of either 77 or 83 MDa which coded for both Lac and phage resistance. The phage resistance mechanism did not act at the adsorption stage and was not affected by incubation at 37°C.     

Genetic and physical characterization of trimethoprim resistance plasmids from Shigella sonnei and Shigella flexneri

Analysis of six Shigella flexneri and four S. sonnei isolates with trimethoprim (Tp) resistance from clinical cases in Ontario has shown that, in all isolates, the Tp resistance is mediated by gene(s) on conjugative, multiple antibiotic-resistance plasmids. The physical and genetic characterization of these plasmids revealed that there are three different Tp resistance plasmids. One group, composed of all six S. flexneri plasmids, consists of plasmids which are about 70 megadaltons (MDa) and inhibit the fertility of an Escherichia coli Hfr strain (Fi+). A representative member of this group, pPT4, demonstrates a weak incompatibility reaction with IncFl plasmid R455-2. Another group, three of the four S. sonnei plasmids, contains plasmids which are about 43 MDa, Fi-, and mediate propagation of phage PRD1. The third group, the remaining S. sonnei plasmid, is 53 MDa, fi+, mediates propagation of phages fd and MS2, and is incompatible with IncFII plasmid R100. These plasmids also have been differentiated by restriction endonuclease fragment profiles. Analysis of pPT4 has revealed that the Tp resistance of this plasmid is transposable. The transposon, Tn536, is different from previously described Tp resistance transposons; it is 16 MDa, and in addition to Tp, it encodes resistance to mercuric chloride ions, spectinomycin, streptomycin, and sulfonamides.     

Distribution of virulence plasmids within Salmonellae

The virulence region of the Salmonella dublin 50 MDa plasmid shared homology with 678 of 1021 salmonellae tested in colony hybridization experiments. The majority of S. dublin, S. typhimurium and S. enteritidis isolates tested hybridized with the region whereas, with the exception of S. hessarek, S. pullorum and S. gallinarum, other serotypes did not. Homologous virulence regions were plasmid encoded. In S. typhimurium a common 60 MDa plasmid was present in all phage types tested but not in DT4, DT37 and DT170. Smaller plasmids showing partial homology were found in DT12, DT18, DT193 and DT204C. In S. enteritidis a distinct plasmid profile for each of eight phage types was observed. Hybridizing plasmids were found in DT3, DT4, DT8, DT9 and DT11 whereas DT7, which was plasmid free, and DT10 and DT14, which harboured plasmids, did not hybridize. The extent of homology shared between S. dublin, S. typhimurium and S. enteritidis virulence plasmids was about 10 MDa and appeared conserved. Virulence plasmids from S. typhimurium and S. enteritidis did not show homology with a region of the S. dublin 50 MDa plasmid which was not associated with virulence functions whereas plasmids of about 24 MDa and 38 MDa in some S. typhimurium phage types did. The association of conserved virulence regions upon differing plasmids within salmonellae is discussed with reference to possible mechanisms of distribution and evolution of virulence genes.     

Plasmid analysis of bacteria of Bacillus genus used in the development of probiotics

Presence of plasmid-contained strains in probiotics prepared on the basis of Bacillus bacteria was assessed. Plasmid analysis was performed by the method based on neutralization of bacterial suspension after alkaline treatment with lithium chloride. Presence of 4-6 plasmids with molecular weight from 1 to 75 MDa was revealed in 4 commercially available probiotics. Since plasmids can facilitate transfer of genes of the antimicrobial resistance to pathogenic microorganisms it was recommended to perform thorough search of probiotic microorganisms without plasmids or ensure the elimination of R-plasmids in start cultures for the most promising biopreparations.     

Protein composition of Saccharomyces cerevisiae mitochondrial ribosomes

Protein composition of mitochondrial ribosomes of the yeast Saccharomyces cerevisiae was analysed by two-dimensional electrophoresis. The small (37S) mitoribosomal subunit contains 36 different polypeptides with molecular weights ranging from 10,000 to 60,000. The large (50S) subunit is composed of 41 proteins with molecular weights from 10,000 to 43,000. The molecular weights of mitoribosomal small and large subunits are 1.85 MDa and 2.35 MDa, respectively. Proteins represent 60-62% and 42-45% of the total mass of 37S and 50S subunits respectively. On the basis of the protein content and molecular weights of individual proteins we conclude that all mitoribosomal proteins are present in the mitoribosome in equimolar proportions.     

Comparison of the Deoxyribonucleic Acid Molecular Weights and Homologies of Plasmids Conferring Linked Resistance to Streptomycin and Sulfonamides

Bacterial strains showing linked resistance to streptomycin (Sm) and sulfonamides (Su) were chosen representing a wide taxonomic and geographical range. Their SmSu resistances were transferred to Escherichia coli K-12 and then plasmid deoxyribonucleic acid (DNA) was isolated by ethidium bromide CsCl centrifugation. The plasmid DNA was examined by electron microscopy and analyzed by sedimentation through 5 to 20% neutral sucrose gradients. Plasmid DNA from strains having transmissible SmSu resistance consisted of two or three molecular species, one of which had a molecular mass of about 5.7 Mdal (10(6) daltons), the others varying between 20 to 60 Mdal. By using transformation or F' mobilization, we isolated the SmSu-resistance determinant from any fellow resident plasmids in each strain and again isolated the plasmid DNA. Cosedimentation of each of these with a differently labeled reference plasmid DNA (R300B) showed 9 out of 12 of the plasmids to have a molecular mass not significantly different from the reference (5.7 Mdal); two others were 6.3 and 9.2 Mdal, but PB165 consisted of three plasmids of 7.4, 14.7, and 21.4 Mdal. Three separate isolations of the SmSu determinant from PB165 gave the same three plasmids, which we conclude may be monomer, dimer, and trimer, respectively. DNA-DNA hybridizations at 75 C demonstrated 80 to 93% homology between reference R300B DNA and each isolated SmSu plasmid DNA, except for the 9.2-Mdal plasmid which had 45% homology and PB165 which had 35%. All the SmSu plasmids were present as multiple copies (about 10) per chromosome. The conjugative plasmid of R300 (present as 1.3 copies per chromosome) has been shown to have negligible effect on the number of copies of its accompanying SmSu plasmid R300B. We conclude that the SmSu plasmids are closely related and probably have a common evolutionary origin.     

Plasmids controlling biodegradation of epsilon-caprolactam

Bacterial strains (190) capable of growth on epsilon-caprolactam as a sole source of carbon and nitrogen were isolated from epsilon-caprolactam industrial sewage. Most of the strains (90%) were found to contain plasmids. Some of the strains (36.8%) assigned provisionally to the genus Pseudomonas contained plasmids controlling epsilon-caprolactam catabolism. The plasmids had a molecular mass from 50 to 300 MDa. Certain plasmids differed in the frequency of conjugation transfer and in the presence of other genetic determinants (resistance against heavy metal ions) and also determined the different character of bacterial growth on epsilon-caprolactam and on its intermediate catabolites.     

Plasmid profiles of lactic acid bacteria associated with vacuum-packaged vienna sausage manufacture and spoilage

Plasmid profiles of lactic acid bacteria from spoiled vacuum-packaged vienna sausages (61) and the corresponding manufacturing environment (50) were determined to assess the use of this technique for pinpointing sources of spoilage populations. Although plasmids were present in >60% of the strains, no profiles were common to these two groups. The occurrence of specific individual plasmids in several isolates from the spoilage population may, however, be useful in diagnosing the spoilage potential of individual strains.     

Stability of plasmids in lactococci during extended incubation in growth media

The stability of plasmids in Lactococcus lactis ssp. lactis strains C2 and ML3, and L. lactis ssp. cremoris strains ML1 and SC607, was investigated by extended incubation of bacterial cells in low nutrient media under acidic conditions. Strains were grown overnight (16-18 h) in skim milk and unbuffered medium (M17-) at 32 degrees C and subsequently held at that temperature for extended periods (greater than or equal to 96 h). Lac- variants were obtained from each strain in milk and (M17-) broth. The plasmid profiles of Lac- variants when compared with their parental Lac+ strains showed loss of one or more plasmid bands. None of the Lac- mutants showed loss of smaller plasmids (less than 5 MDa) indicating that smaller plasmids in lactococci are more stable under these conditions than larger plasmids (greater than 10 MDa). Concomitant loss of the Lac+ phenotype and plasmids by the method used in the present investigation may have application for isolating mutants devoid of one or more plasmids.     

Plasmid Heterogeneity in Spanish Isolates of Agrobacterium tumefaciens from Thirteen Different Hosts

Plasmid DNA was isolated from 80 Spanish isolates of Agrobacterium tumefaciens from 13 hosts of several geographical and temporal origins. One to five plasmids occurred in all of the isolates studied. Plasmid sizes varied between 5 and greater than 1,000 MDa. Generally, there was no correlation between plasmid number or size and geographical origin, host, biovar, sensitivity to agrocin 84, or opine-catabolizing ability of the different isolates.     

Occurrence of antibiotic and metal resistance and plasmids in Bacillus strains isolated from marine sediment

Eleven hundred Bacillus strains isolated from marine sediment from the Minas Basin, Nova Scotia, Canada, were purified on LB agar supplemented with ampicillin, chloramphenicol, erythromycin, streptomycin, tetracycline, or mercuric chloride. Seventy-seven isolates were examined for plasmid DNA, and for resistance to 11 antibiotics, HgCl2, and phenylmercuric acetate. Minimum inhibitory concentrations of Ag, Cd, Co, Cu, and Zn were also determined. Forty-three percent of antibiotic- and mercury-resistant strains contained one or more plasmids ranging from 1.9 to 210 MDa. Fifty-four percent carried plasmids greater than 20 MDa, and 97% were resistant to two or more metals. There was no correlation between plasmid content and resistance either to antibiotics or to mercurial compounds in these strains. Mercury-resistant isolates were unable to transform Hg2+ to volatile Hg0 by virtue of a mercuric reductase enzyme system (mer). Strains resistant to Hg2+ were investigated for their ability to produce H2S and intracellular acid-labile sulfide when grown in the absence and presence of HgCl2. Lower levels of H2S and intracellular sulfide were detected only in metal-resistant strains grown in the presence of HgCl2, suggesting that cellular sulfides complexed with Hg2+ in these strains.