Association of the strA-strB genes with plasmids and transposons in the present-day bacteria and in bacterial strains from permafrost

Transposons closely related to the streptomycin resistance transposon of modern bacteria, Tn5393, were detected in the bacterial isolates from permafrost resistant to streptomycin. Many transposons studied were located on the medium-size plasmids with a narrow host range. None of the streptomycin-resistant strains isolated from permafrost contained small plasmids carrying the strA-strB genes and related to the broad host range plasmid RSF1010.     

Comparison of Ti plasmids from three different biotypes of Agrobacterium tumefaciens isolated from grapevines.

Twenty-six plasmids from grapevine isolates of Agrobacterium tumefaciens were analyzed by SmaI fingerprinting and by hybridization of nick-translated DNA to DNA of another plasmid. These experiments established that octopine Ti plasmids are not highly conserved, although octopine Ti plasmids from biotype 1 A. tumefaciens strains appeared to be very similar. Octopine Ti plasmids from biotype 3 strains are more variable in terms of host range and SmaI fingerprints, but share extensive DNA homology. Fingerprints of nopaline Ti plasmids from strains of a given biotype resemble each other but not fingerprints of Ti plasmids from strains of the other two biotypes. The wide host range octopine Ti plasmid from the biotype 3 strain Ag86 shares more DNA homology with narrow host range Ti plasmids, nopaline Ti plasmids, and octopine catabolism plasmids than with the wide host range octopine Ti plasmid from biotype 1 strain 20/1. pTiAg86 does share homology with the portion of pTi20/1 integrated and expressed in plant tumor cells. Since all wide host range Ti plasmids studied contain these sequences, we suggest that natural selection for a wide host range resulted in the presence of the common sequences in distantly related plasmids. The lack of homology between this "common DNA" and limited host range Ti plasmids shows that the DNA sequences per se are not required for tumorigenesis.     

Comparative analysis of the structure of incompatibility plasmids N, P and W

Evolutionary relationships of the IncN plasmid R15 and other broad host range plasmids (IncN plasmids N3 and R46, IncP plasmids RP4 and R906, IncW plasmids Sa and R388) were studied by Southern blot hybridization technique. The IncN plasmids were shown to harbour homologous determinants for replication and conjugation. No homology was found between the rep and tra genes in R15 and in the IncW and IncP plasmids, respectively. The second rep region of the N3 plasmid is distinctive from the corresponding determinants in the IncN plasmids. Homology was demonstrated for the plasmid genes that mediate restriction and modification in R15 and N3, mercury resistance in R15 and R906, sulfanilamide resistance in R15, N3, R46, Sa, R388, and R906, streptomycin resistance in R15, R46 and Sa. The latter genes are different from the R906 SmR gene. In addition to the three known mobile elements in the plasmid R15, the fourth one (IS46) that is a part of the transposon Tn2353 was identified in this study. Besides, the third copy of this insertion sequence was found in the N3 plasmid.     

The complete genome sequences of four new IncN plasmids from wastewater treatment plant effluent provide new insights into IncN plasmid diversity and evolution

The dissemination of antibiotic resistance genes among bacteria often occurs by means of plasmids. Wastewater treatment plants (WWTP) were previously recognized as hot spots for the horizontal transfer of genetic material. One of the plasmid groups that is often associated with drug resistance is the incompatibility group IncN. The aim of this study was to gain insights into the diversity and evolutionary history of IncN plasmids by determining and comparing the complete genome sequences of the four novel multi-drug resistance plasmids pRSB201, pRSB203, pRSB205 and pRSB206 that were exogenously isolated from the final effluent of a municipal WWTP. Their sizes range between 42,875 bp and 56,488 bp and they share a common set of backbone modules that encode plasmid replication initiation, conjugative transfer, and plasmid maintenance and control. All plasmids are transferable at high rates between Escherichia coli strains, but did not show a broad host range. Different genes conferring resistances to ampicillin, streptomycin, spectinomycin, sulfonamides, tetracycline and trimethoprim were identified in accessory modules inserted in these plasmids. Comparative analysis of the four WWTP IncN plasmids and IncN plasmids deposited in the NCBI database enabled the definition of a core set of backbone genes for this group. Moreover, this approach revealed a close phylogenetic relationship between the IncN plasmids isolated from environmental and clinical samples. Phylogenetic analysis also suggests the existence of host-specific IncN plasmid subgroups. In conclusion, IncN plasmids likely contribute to the dissemination of resistance determinants between environmental bacteria and clinical strains. This is of particular importance since multi-drug resistance IncN plasmids have been previously identified in members of the Enterobacteriaceae that cause severe infections in humans.     

Plasmids and transposons and their stability and mutability in bacteria isolated during an outbreak of hospital infection.

In an outbreak of hospital infection caused by Klebsiella aerogenes type K-16 isolates over a 3-month period carried, apparently unaltered, a cryptic 90-Megadalton (Md) plasmid (unclassified) and a multiple-resistance 65-Md plasmid of IncM. The IncM plasmid, identified in environmentally related strains of Citrobacter koseri and Escherichia coli, showed minor variations from that in the klebsiella vector. The IncM plasmids, as well as all wild host strains cured of the IncM plasmids, carried a transposable DNA sequence, encoding trimethoprim and, in every case but one, streptomycin resistance. This transposon appeared identical with Tn7, previously identified in unrelated plasmids in bacteria from different environments.     

Relationship between the limited and wide host range octopine-type Ti plasmids of Agrobacterium tumefaciens.

The relationship between the limited host range octopine Ti plasmids and the wide host range octopine Ti plasmids pTiB6806 and pTiA6 was studied. The limited host range Ti plasmids shared extensive deoxyribonucleic acid homology; pTiAg63 and pTiAg162 were essentially completely homologous with pTiAg158 while pTiAg57 shared approximately 64% homology with pTiAg158. In contrast, the limited host range octopine Ti plasmids only shared 6 to 15% homology with the wide host range octopine Ti plasmid pTiB6806. Thus, limited and wide host range octopine Ti plasmids comprise distinct families of plasmids. The deoxyribonucleic acid homology shared between the limited host range Ti plasmids and pTiB6806, however, was distributed over some 50% of pTiB6806, suggesting that both families of plasmids evolved from a common progenitor plasmid. The limited host range Ti plasmids showed relatively strong homology with pTiB6806 HpaI fragment 7, a region which codes for octopine utilization by the bacterium, but showed only weak homology with pTiB6806 HpaI fragment 12, a region required for virulence. In addition, homology between the limited host range octopine Ti plasmids and the "common deoxyribonucleic acid," sequences shown to have a central role in plant cell transformation, was barely detectable when stringent hybridization conditions were used. We therefore conclude that a highly conserved version of the common deoxyribonucleic acid is not required for crown gall tumorigenesis on all plant species.     

Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10.

This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals. Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings. Optimal temperatures for mobilization of pD10 were between 15 and 25 degrees C. Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of beta- and gamma-purple bacteria. In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies. Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species.     

Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10.

This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals. Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings. Optimal temperatures for mobilization of pD10 were between 15 and 25 degrees C. Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of beta- and gamma-purple bacteria. In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies. Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species.     

Monitoring the spread of broad host and narrow host range plasmids in soil microcosms

Abstract: Escherichia coli recipient and E. coli donor strains carrying streptothricin-resistance genes were inoculated together into different soil microcosms. These genes were localized on the narrow host range plasmids of incompatibility (Inc) groups FII, Il, and on the broad host range plasmids of IncP1, IncN, IncW3, and IncQ. The experiments were intended to study the transfer of these plasmids in sterile and non-sterile soil with and without antibiotic selective pressure and in planted soil microcosms. Transfer of all broad host range plasmids from the introduced E. coli donor into the recipient was observed in all microcosm experiments. These results indicate that broad host range plasmids encoding short and rigid pili might spread in soil environments by conjugative transfer. In contrast, transfer of the narrow host range plasmids of IncFII and IncI1, into E. coli recipients was not found in sterile or non-sterile soil. These plasmids encoded flexible pili or flexible and rigid pili, respectively. In all experiments highest numbers of transconjugants were detected for the IncP1-plasmid (pTH16). There was evidence with plasmids belonging to IncP group transferred by conjugation into a variety of indigenous soil bacteria at detectable frequencies. Significantly higher numbers of indigenous transconjugants were obtained for the IncP-plasmid under antibiotic selection pressure, and a greater diversity of transconjugants was detected. Availability of nutrients and rhizosphere exudates stimulated transfer in soil. Furthermore, transfer of the IncN-plasmid (pIE1037) into indigenous bacteria of the rhizosphere community could be detected. The transconjugants were determined by BIOLOG as Serratia liquefaciens . Despite the known broad host range of IncW3 and IncQ-plasmids, transfer into indigenous soil bacteria could not be detected.     

Characterization of sulfonamide resistance determinants and relatedness of Bordetella avium R plasmids.

Five plasmids, varying in size from 16 to 51.5 kb, were isolated from virulent strains of Bordetella avium and compared by restriction endonuclease digestion and DNA-DNA hybridization. These plasmids confer resistance to streptomycin and sulfonamides, and three of the five also confer resistance to tetracycline, but they are not closely related. Four of the plasmids, pRL100, p4093, pCW, and pWAM, carried determinants related to the heat-labile type I plasmid-mediated dihydropteroate synthase of the plasmid R388, while one plasmid, p4168, carried a determinant related to the heat-stable type II dihydropteroate synthase of pGS05.     

Incompatibility and molecular relationships between small staphylococcal plasmids carrying the same resistance marker

Incompatibility relationships between staphylococcal plasmids carrying the same, single resistance marker were studied by means of appropriate recombinant plasmids. Naturally occurring plasmids encoding streptomycin, tetracycline, or chloramphenicol resistance, respectively, were used in this study, four of each phenotype. The plasmids responsible for tetracycline resistance proved to belong to a single incompatibility set. Similarly, the four streptomycin resistance plasmids fall in the same incompatibility set. On the other hand, plasmids encoding chloramphenicol resistance were divided in four distinct incompatibility sets, three of them being newly defined. Study of the molecular relationships between these plasmids by DNA-DNA hybridization and restriction endonuclease cleavage supported the conclusions from genetic tests that the four Tc^r and the four Sm^r plasmids are essentially identical, whereas the four Cm^r plasmids are diverse.     

Transposition of the carbenicillin-hydrolyzing beta-lactamase gene

We isolated a new transposon Tn2101, from plasmid Rms433 in Enterobacter cloacae. Tn2101 encoded the formation of type IV (carbenicillin-hydrolyzing) beta-lactamase and multiple resistance to streptomycin, sulfanilamide, spectinomycin, and mercury in addition to ampicillin. Tn2101 was transposable between conjugative (or nonconjugative) plasmids and the host chromosome. Transposition occurred independently of the general recombination ability of the host cell. Tn2101 had a molecular size of 9.5 x 10(6) and contained short inverted repeat terminal sequences.     

Molecular structure and translocation of a multiple antibiotic resistance region of a Psychrobacter psychrophilus permafrost strain

A Psychrobacter psychrophilus strain resistant to tetracycline and streptomycin was isolated from a 15 000–35 000-year-old permafrost subsoil sediment sampled from the coast of the Eastern-Siberian Sea. The genes conferring antibiotic resistance were localized on an c . 30-kb pKLH80 plasmid. It was shown that the antibiotic resistance region of this plasmid has a mosaic structure and contains closely linked streptomycin resistance ( strA-strB ) and tetracycline resistance [ tetR-tet (H)] genes, followed by a novel IS element (IS Ppy1 ) belonging to the IS 3 family. Both the strA-strB and tetR-tet (H) genes of pKLH80 were highly similar to those found in modern clinical bacterial isolates. It was shown that the IS Ppy1 element of pKLH80 can direct translocation of the adjacent antibiotic resistance genes to different target plasmids, either by one-ended transposition or by formation of a composite transposon resulting from the insertion of the IS Ppy1 second copy at the other side of the antibiotic resistance region. Thus, our data demonstrate that clinically important antibiotic resistance genes originated long before the introduction of antibiotics into clinical practice and confirm an important role of horizontal gene transfer in the distribution of these genes in natural bacterial populations.     

Spreading of streptomycin antibiotic resistance gene in Escherichia coli plasmids demonstrated by Southern blot analysis

Plasmids from E. coli strains of 38 donors were transconjugated to common recipient SY663 Escherichia coli K12. The restriction patterns of the isolated plasmids were highly heterogenous. However, the streptomycin (Sm) resistance genes of the plasmids were identical or closely homologous in 29 of the 33 plasmids conferring Sm resistance. These data were based on Southern blot analysis, using the Sm resistance gene (encoding aminoglycoside phosphoryl transferase) as probe cut out from pBP1 plasmid. Our data suggest an extensive spreading of streptomycin resistance gene of this type.     

Physicochemical properties of the pKMR plasmids controlling antibiotic resistance and the capacity to produce colonization antigen

KMR plasmids controlling antibiotic resistance and the capacity for production of the colonization antigen were identified in wild strains of E. coli (026, 0126, 0124) and S. sonnei isolated from patients with acute intestinal diseases. The strains of E. coli 026 and E. coli 0126 carried p KMR207-1 plasmid determining resistance to chloramphenicol and tetracycline and the adhesive properties. The molecular weight of the plasmid is 98 mD. The strain of S. sonnei carried p KMR 208-1 plasmid responsible for resistance to streptomycin, chloramphenicol and tetracycline and the adhesive properties. The molecular weight of this plasmid is 98 mD. The resistance to streptomycin and tetracycline and the capacity for the synthesis of the colonization antigen in E. coli 0214 was controlled by p KMR209 plasmid with the molecular weight of 2.66 mD. The restriction analysis suggests that p KMR207a-1 and p KMR 207b-1 plasmids detected in E. coli of different serotypes were identical, since they could be broken with BamH1 endonuclease into equal numbers of fragments similar by their molecular weights. p KMR207-1 and p KMR208-1 plasmids differed in their sensitivity to BamH-1 endonuclease. However, they were broken into 6 fragments similar by their molecular weights. p KMR207-1 and p KMR208-1 plasmids are probably closely related but not identical.     

Streptomyces griseus streptomycin phosphotransferase: expression of its gene in Escherichia coli and sequence homology with other antibiotic phosphotransferases and with eukaryotic protein kinases

The aphD gene of Streptomyces griseus, encoding a streptomycin 6-phosphotransferase (SPH), was sub-cloned in the pBR322-based expression vector pRK9 (which contains the Serratia marcescens trp promoter) with selection for expression of streptomycin resistance in Escherichia coli. Two hybrid plasmids, pCKL631 and pCKL711, were isolated which conferred resistance. Both contained a approximately 2 kbp fragment already suspected to include aphD. The properties of in vitro deletion derivatives of these plasmids were consistent with the presumed location of aphD. In vitro deletion of a sequence including most of the trp promoter largely, but not quite completely, abolished the ability of the plasmid to confer streptomycin resistance, confirming that expression was indeed principally from the trp promoter. A polypeptide of approximately 34.5 kDa was present in minicells containing plasmids that conferred streptomycin resistance, but was absent when the plasmids contained in vitro deletions removing streptomycin resistance. Part of the fragment was sequenced and an open reading frame corresponding to aphD identified. A computer-assisted comparison of the deduced SPH sequence with those of other antibiotic phosphotransferases suggested a common structure A-B-C-D-E, where B and D were conserved between all sequences compared while A, C and E divided between the streptomycin and hygromycin B phosphotransferases on one hand and kanamycin/neomycin ones on the other. A composite sequence data base was searched for homologues to consensus matrices constructed from five approximately 12-residue subsequences within blocks B and D. For one subsequence, corresponding to the N-terminal portion of block D, those sequences from the database that yielded the highest homology scores comprised almost entirely either antibiotic phosphotransferases or eukaryotic protein kinases. Possible evolutionary implications of this homology, previously described by other groups, are discussed.     

Corrected Version Distribution of Heterogeneous and Homologous Plasmids in Bacillus spp

A total of 75 strains (including 5 reference strains) of Bacillus amyloliquefaciens, B. cereus, B. circulans, B. licheniformis, B. megaterium, B. pumilus, B. sphaericus, B. subtilis, and B. thuringiensis and 36 species-unidentified Bacillus strains were surveyed for plasmids by cesium chloride-ethidium bromide equilibrium centrifugation of cell lysates in a study of antibiotic resistance in host cells. Of the 111 strains, 13 (including 3 reference strains) were found to harbor plasmids, and 5 of the 13 showed antibiotic resistance. This antibiotic resistance appeared not to be due to the plasmids, however, because the trait was not cured by cultivation of cells in nutrient medium containing ethidium bromide (1 mug/ml), sodium dodecyl sulfate (0.2 mug/ml), or novobiocin (1 mug/ml), except in one strain, in which kanamycin and streptomycin resistances were cured by novobiocin. One strain of B. amyloliquefaciens, S294, was found to harbor a plasmid, pFTB14, which differed from the plasmid species of classes 1 to 6 in B. subtilis and B. amyloliquefaciens, as determined by restriction analysis and DNA contour length determination. However, in DNA-DNA hybridization on a filter after Southern blotting from an agarose gel, the pFTB14 DNA hybridized with plasmids of classes 1 to 5. Three strains of B. thuringiensis each carried at least 4 to 11 plasmid species, whereas no plasmids were detected in four strains of B. cereus, which, in relation to B. thuringiensis, is closely related taxonomically and has highly homologous DNA sequences. The plasmid DNAs prepared from species other than B. subtilis and B. amyloliquefaciens did not hybridize with that of pFTB14.     

Transposition of a duplicate antibiotic resistance gene and generation of deletions in plasmid R6K.

Transformation experiments showed that spontaneous deletions which result in loss of streptomycin resistance and an increase in conjugal transfer efficiency are present at a frequency of about 10(-4) in plasmid molecules of R6K. Similar deletions were thus readily selected by conjugal transfer of R6K, and their appearance was dependent upon recA+ activity in either donor or recipient host. The deoxyribonucleic acid segment deleted in four mutants examined was concluded to extend from the same terminus of the transposon, TnA, in the same direction, but to different extents, and to retain the TnA region intact. Insertions of a duplicate TnA element were found in R6K plasmids isolated from strains selected for increased ampicillin resistance, which were unstable in recA+ strains. In four plasmids examined after transfer to a recA host, an inverted repeat of the preexisting TnA element was shown to have been inserted at a similar location and was in two instances associated with deletions which extended from the same direction as those described above. The deletions are ascribed to the result of recA+-dependent recombination between direct repeats of TnA.     

Inheritance of biodegradation plasmids in the cells of homo- and heterologous hosts

A systematic analysis of the inheritance of D plasmids of the IncP-9 group (alpha-, beta-, gamma-, delta-, epsilon-, zeta-, eta-, theta-subgroups), IncP-7, as well as of those of undefined systematic affiliation in the cells of homologous (Pseudomonas putida) and heterologous (Escherichia coli) hosts was performed for the first time. For this purpose, mini-Tn5 transposons determining resistance to kanamycin (or streptomycin) were introduced into all the D plasmids under study. It has been established that all IncP-9 plasmids can be transmitted to the cells of a heterologous host E. coli (with the exception of plasmid pSVS15 from theta-subgroup). IncP-7 plasmids and those of undefined systematic affiliation do not possess this property and can be transmitted and stably inherited only in P. putida. The distinctive feature of most IncP-9 plasmids (alpha-, beta-, gamma-, delta-, epsilon-, and zeta-subgroups) is strict dependence of their inheritance on the temperature factor. At 37 degrees C, the plasmids of delta-, zeta-, and theta-subgroups are unstable in P. putida cells, while in E. coli nearly all plasmids of this systematic group are unstable. The exceptions are the plasmids of eta- and gamma-subgroups. Inheritance of these plasmids does not depend on temperature. At 28 degrees C and 37 degrees C, the eta plasmid is not maintained stably (inheritance stability is 2%), while the gamma plasmid has almost 100% stability under the same conditions.     

Streptomycin and tetracycline resistance plasmids in Staphylococcus hyicus and other staphylococci.

Plasmids associated with resistance to streptomycin, to streptomycin plus chloramphenicol or to tetracycline in Staphylococcus hyicus isolated from the skin of pigs have been compared, by restriction endonuclease digest patterns, with similar staphylococci from human sources and with published DNA base sequences. Several plasmids from Staph. hyicus have proved to have a very similar structure to those described from Staph. aureus but others appeared very dissimilar. This confirms the opinion that staphylococci from animal skin share a pool of plasmids with those from human skin but may also possess some of quite different structure.