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1.
The molecular relatedness among alpha-hemolytic plasmids from various incompatibility groups 总被引:3,自引:0,他引:3
Deoxyribonucleic acid (DNA) reassociation studies among α-hemolytic (Hly) plasmids from FVI and FIII–IV incompatibility groups showed a close similarity between the nucleotide sequences of plasmids from the same group. With respect to R plasmids from the F overgroup, they have 20–26 Mdal in common, an amount of DNA close to the amount involved in the traF operon. No more extensive sequence homology was found between pSU316 (IncFIII–IV) and the incompatible plasmids ColB-K98 (IncFIII) or R124 (IncFIV). The IncIα I2 plasmid pSU5 has only the α-hemolytic region (5 Mdal) in common with plasmid pSU316 but it is much more closely related to IncFVI plasmids where the DNA in common amounts to 22 Mdal. Finally, the genetically unrelated plasmid pSU233 shares 66% of its nucleotide sequences (40 Mdal) with the IncFVI plasmids and has 16–23 Mdal in common with various F-like plasmids. 相似文献
2.
Relationship of the replication and incompatibility genes of an IncFVI haemolysin plasmid with other F-like haemolysin plasmids 总被引:1,自引:0,他引:1
The replication and incompatibility region of the IncFVI plasmid pSU502 has been isolated by in vitro DNA manipulation as part of a 12.6 kb plasmid, denominated pSU503. Plasmid pSU503 was strongly incompatible with its parental plasmid, pSU1, but was fully compatible with the haemolytic plasmids pSU316 (IncFIII/IV), pHly152 (IncI2) and pSU233 (Inc-pSU233). Furthermore, the 6.9 kb EcoRI fragment of pSU503 which carries the replication and incompatibility determinants of pSU1 did not show any detectable homology (less than 70%) with any of the haemolysin-determining plasmids with which it is compatible. Thus, homologous haemolysin determinants have become linked to apparently unrelated replicons. 相似文献
3.
Incompatibility properties of the IncFIII/FIV haemolytic plasmid pSU316 when integrated in the Escherichia coli chromosome 总被引:1,自引:0,他引:1
J Navas F de la Cruz J C Rodriguez I Andrés G Pulgar J M Ortiz 《Journal of general microbiology》1983,129(7):2277-2283
The haemolytic plasmid pSU316 is incompatible with members of the IncFIII and IncFIV incompatibility groups. Plasmid pSU307 (pSU316 hlyC::Tn5) was inserted by integrative suppression into the chromosome of JW112, a temperature-sensitive dnaA mutant of Escherichia coli. The incompatibility properties of this strain (SU51) were studied and it was found that: (1) plasmid pSU306 (pSU316 hlyA::Tn802) was rapidly lost from strain SU51 both at 30 degrees C and 42 degrees C; (2) the IncFIII plasmid pSU397 (ColB-K98::Tn802) was lost from strain SU51 and at 42 degrees C but not at 30 degrees C; and (3) the IncFIV plasmid R124 was stably maintained in strain SU51 at both temperatures. Revertants of pSU307 to the autonomous state could be obtained from SU51. These revertants exerted incompatibility towards the prototype plasmids pSU306, pSU397 and R124 in the same way as pSU307 itself. Thus, strain SU51 provided a suitable method for distinguishing the three different incompatibility determinants of plasmid pSU316. 相似文献
4.
Incompatibility among alpha-hemolytic plasmids studied after inactivation of the alpha-hemolysin gene by transposition of Tn802. 总被引:12,自引:0,他引:12
Five α-hemolytic plasmids were studied with respect to their molecular and genetic properties. Their molecular weights ranged from 48 to 93 Mdal. Digestion with HindIII restriction endonuclease indicated that they were all clearly different plasmids although similarities in their band patterns were observed. Plasmids pSU1, pSU105, and pSU316 produce F-like pili. Incompatibility studies between Hly plasmids were prevented by lack of markers other than α-hemolysin production. In order to overcome this problem, the inactivating properties of the transposable element Tn802 were used. Several Hly plasmids that have lost the ability to produce α-hemolysin were isolated after insertion of the ampicillin transposon Tn802. Incompatibility between the parental plasmids and their Tn802 derivatives suggests that α-hemolytic plasmids have spread over, at least, four incompatibility groups. Plasmids pSU1 and pSU105 were found to be incompatible with Hly-P212, the only representative, so far, of IncFVI. Plasmid pSU316 was incompatible both with ColB-K98 and R124, which suggests the existance of a FIII-FIV incompatibility complex. In addition, pSU5 and pSU233 were compatible with each other and with pSU316, pSU1, pSU105, and Hly-P212. They also produce a different type of pili from this test plasmids. 相似文献
5.
Five α-hemolytic plasmids were studied with respect to their molecular and genetic properties. Their molecular weights ranged from 48 to 93 Mdal. Digestion with HindIII restriction endonuclease indicated that they were all clearly different plasmids although similarities in their band patterns were observed. Plasmids pSU1, pSU105, and pSU316 produce F-like pili. Incompatibility studies between Hly plasmids were prevented by lack of markers other than α-hemolysin production. In order to overcome this problem, the inactivating properties of the transposable element Tn802 were used. Several Hly plasmids that have lost the ability to produce α-hemolysin were isolated after insertion of the ampicillin transposon Tn802. Incompatibility between the parental plasmids and their Tn802 derivatives suggests that α-hemolytic plasmids have spread over, at least, four incompatibility groups. Plasmids pSU1 and pSU105 were found to be incompatible with Hly-P212, the only representative, so far, of IncFVI. Plasmid pSU316 was incompatible both with ColB-K98 and R124, which suggests the existance of a FIII-FIV incompatibility complex. In addition, pSU5 and pSU233 were compatible with each other and with pSU316, pSU1, pSU105, and Hly-P212. They also produce a different type of pili from this test plasmids. 相似文献
6.
Identification and expression of a copy number control gene in the IncFIII hemolytic plasmid pSU316. 总被引:2,自引:0,他引:2
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A DNA fragment carrying both the IncFIII determinant and a copy number control gene of the hemolytic plasmid pSU316 has been cloned in pBR322. Deletion derivatives of the hybrid plasmid generated by Bal 31 digestion, which no longer exhibit the IncFIII phenotype, fall into two complementation groups when tested against a pSU316 miniplasmid derivative. Type 1 mutants exhibit the copy number control (Cop+) phenotype whereas type 2 mutants do not. Restriction analysis of type 1 and type 2 mutants allowed us to locate the cop gene of pSU316 in a 700-base-pair fragment adjacent to the IncFIII determinant. Plasmid expression in a minicell system suggests that the product of the cop gene of pSU316 could be a 13,000-dalton protein. 相似文献
7.
Hemolysis determinant common to Escherichia coli hemolytic plasmids of different incompatibility groups 总被引:22,自引:12,他引:10
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By using cloned deoxyribonucleic acid fragments from the hemolysis determinant of the hemolytic plasmid pHly152 as hybridization probes, a deoxyribonucleic acid segment of about 3.8 megadaltons was identified as a common sequence in several hemolytic (Hly) plasmids of Escherichia coli belonging in four different incompatibility groups. This segment contained the genetic information for the synthesis and secretion of the extracellular toxin alpha-hemolysin of E. coli. With the exception of pSU5, representing a composite plasmid, one part of which seems to be very similar to pHly152, the overall sequence homology of these Hly plasmids with pHly152 seems to be rather restricted. However, the Hly plasmid pSU316 showed sequence homology with pHly152 that did not extend beyond the hemolysis determinant. The two other plasmids, pSU233 and pSU105, also shared homology with pHly152 in the hemolysis determinant as well as in various other parts of this plasmid which did not seem to be directly linked to the hemolysis determinant. This suggests that the hemolysis determinant has spread to presumably unrelated plasmids of E. coli. 相似文献
8.
A restriction endonuclease analysis of the hemolytic plasmid pSU316 has allowed location of the cleavage sites for the endonucleases BamHI, XbaI, KpnI, BglII, SalGI, EcoRI, and HindIII. Hybridization experiments between pSU316 and pED100 have shown that the tra region of pSU316 lies in a segment comprising part of SalGI fragments S-1 and S-3 and the entire fragment S-4. The positions of other plasmid coded functions, namely the replication functions and α-hemolysin production, have been determined in the physical map. 相似文献
9.
Genetics of the replication and maintenance functions of the hemolytic plasmid pSU316. Cloning of an IncFIII determinant 总被引:1,自引:0,他引:1
Two miniplasmids have been constructed from pSU306, a Tn802 insertion derivative of the IncFIII-IncFIV hemolytic plasmid pSU316. One of these, pSU3027, is a low copy number plasmid expressing both IncFIII and IncFIV incompatibilities, but is rather unstable, and probably lacks a putative par gene. The other, pSU3025, is maintained in about 340 copies per genome equivalent and expresses only IncFIII incompatibility. Most of the PstI-generated fragments from pSU3027 have been cloned in pBR322. One of the resulting plasmids, pSU3135, contains an insertion of 0.5 kb in the vector molecule, and expresses IncFIII, but not the IncFIV incompatibility. These results allowed us to identify and locate several genes involved in the control of pSU316 replication and stable plasmid maintenance. 相似文献
10.
Cookson AL Noel S Hussein H Perry R Sang C Moon CD Leahy SC Altermann E Kelly WJ Attwood GT 《FEMS microbiology letters》2011,316(2):144-151
The rumen bacterium Butyrivibrio proteoclasticus B316(T) has a 4.4-Mb genome composed of four replicons (approximately 3.55 Mb, 361, 302 and 186 kb). Mutagenesis of B316(T) was performed with the broad host-range conjugative transposon Tn916 to screen for functionally important characteristics. The insertion sites of 123 mutants containing a single copy of Tn916 were identified and corresponded to 53 different insertion points, of which 18 (34.0%), representing 39 mutants (31.7%), were in ORFs and 12 were where transposition occurred in both directions (top and bottom DNA strand). Up to eight mutants from several independent conjugation experiments were found to have the same integration site. Although transposition occurred in all four replicons, the number of specific insertion sites, transposition frequency and the average intertransposon distance between insertions varied between the four replicons. In silico analysis of the 53 insertion sites was used to model a target consensus sequence for Tn916 integration into B316(T) . A search of the B316(T) genome using the modelled target consensus sequence (up to two mismatches) identified 39 theoretical Tn916 insertion sites (19 coding, 20 noncoding), of which nine corresponded to Tn916 insertions identified in B316(T) mutants during our conjugation experiments. 相似文献
11.
The isolation and characteristics of plasmids derived from the insertion of MupAp1 into pML2: their behavior during transposition 总被引:1,自引:0,他引:1
Three independent insertions of the phage Mu variant MupAp1 into the ColE1 derivative pML2 have been isolated. From one of these hybrid plasmids (pSU1), two mini-Mu plasmids have been generated. These have lost internal regions of the Mu genome but retain the ends of the prophage. In pSU17 all but one kilobase pair of the early region and most of the late region have been deleted whereas in pSU123 most of the early region is still present but the deletion covers nearly all the late region. Mu immunity, host killing, and transposition functions are located in the DNA present in pSU123 but absent from pSU17. Transposition of Mu and the mini-Mu from the hybrid plasmids to the sex factor R388 is usually associated with the formation of cointegrates between the two parental plasmids. 相似文献
12.
The integrative vectors pSU 475 and pSU 476 with variable numbers of copies per genome were developed for antibiotic producing actinomycetes. For this, the amplifying sequence AUD-Sr 1 of Streptomyces rimosus and the BamHIB fragment of the eSA 1 genetic element from Streptomyces antibioticus were used. The eSA 1 fragment was an element required for integration of a vector to the actinomycete chromosomes since it was homologous with the chromosomal DNAs of S. lividans, S. erythraeus and S. antibioticus. At the first stage the AUD-Sr 1 sequence within the actinomycete plastid pSU 23 was cloned by the vector pUC 19 to E coli. In that experiment the 12.4-kb plasmid pSU 449 was isolated. At the second stage the BamHIB-fragment of the eSA 1 element was incorporated into the resultant hybrid plasmid pSU 449. The 16.5-kb hybrid plasmids pSU 475 and pSU 476 were isolated. In these plasmids the BamHIB fragment of eSA 1 was present in two orientations. The developed vectors were useful in cloning DNA to S. lividans and S. erythraeus. 相似文献
13.
14.
The genome organization of Ralstonia eutropha strain H16 and related species of the Burkholderiaceae
Fricke WF Kusian B Bowien B 《Journal of molecular microbiology and biotechnology》2009,16(1-2):124-135
Ralstonia eutropha strain H16 is a facultatively chemolithoautotrophic, hydrogen-oxidizing bacterium belonging to the family Burkholderiaceae of the Betaproteobacteria. The genome of R. eutropha H16 consists of two chromosomes (Chr1, Chr2) and one megaplasmid (pHG1), and thus shows a multi-replicon architecture, which is characteristic for all members of the Burkholderiaceae sequenced so far. The genes for housekeeping cell functions are located on Chr1. In contrast, many characteristic traits of R. eutropha H16 such as the ability to switch between alternative lifestyles and to utilize a broad variety of growth substrates are primarily encoded on the smaller replicons Chr2 and pHG1. The latter replicons also differ from Chr1 by carrying a repA-associated origin of replication typically found on plasmids. Relationships between the individual replicons from various Burkholderiaceae genomes were studied by multiple sequence alignments and whole-replicon protein comparisons. While strong conservation of gene content and order among the largest replicons indicate a common ancestor, the resemblance between the smaller replicons is considerably lower, suggesting a species-specific origin of Chr2. The megaplasmids, however, in most cases do not show any taxonomically related similarities. Based on the results of the comparative studies, a hypothesis for the evolution of the multi-replicon genomes of the Burkholderiaceae is proposed. 相似文献
15.
CS1 is the prototype of a class of pili of enterotoxigenic Escherichia coli (ETEC) associated with diarrheal disease in humans. The genes encoding this pilus are carried on a large plasmid, pCoo. We report the sequence of the complete 98,396-bp plasmid. Like many other virulence plasmids, pCoo is a mosaic consisting of regions derived from multiple sources. Complete and fragmented insertion sequences (IS) make up 24% of the total DNA and are scattered throughout the plasmid. The pCoo DNA between these IS elements has a wide range of G+C content (35 to 57%), suggesting that these regions have different ancestries. We find that the pCoo plasmid is a cointegrate of two functional replicons, related to R64 and R100, which are joined at a 1,953-bp direct repeat of IS100. Recombination between these repeats in the cointegrate generates the two smaller replicons which coexist with the cointegrate in the culture. Both of the smaller replicons have plasmid stability genes as well as genes that may be important in pathogenesis. Examination by PCR of 17 other unrelated CS1 ETEC strains with a variety of serotypes demonstrated that all contained at least parts of both replicons of pCoo and that strains of the O6 genotype appear to contain a cointegrate very similar to pCoo. The results suggest that this family of CS1-encoding plasmids is evolving rapidly. 相似文献
16.
Nucleotide sequences at the ends of the mercury resistance transposon, Tn501. 总被引:16,自引:3,他引:13
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N L Brown C L Choi J Grinsted M H Richmond P R Whitehead 《Nucleic acids research》1980,8(9):1933-1945
The nucleotide sequences at the ends of the mercury-resistance transposon, Tn501, have been determined. The terminal sequences are inverted repeated sequences 38 nucleotide pairs in length, which differ in 3 nucleotide pairs. The transposon is flanked by directly repeated sequences of 5 nucleotide pairs, originating from a single pentanucleotide sequence in the recipient replicon. There is no obvious homology between recipient replicons at the site of insertion of the transposon. The structures of the ends of Tn501 are compared with those of other transposons and insertion sequences. The use of Tn501 to locate an EcoRI site within a genetically defined sequence of interest is discussed. 相似文献
17.
A hot spot in plasmid F for site-specific recombination mediated by Tn21 integron integrase. 总被引:2,自引:0,他引:2
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Integron In2 integrase (IntI1)-mediated site-specific recombination between two primary sites occurs at a high frequency, while that between a primary and a secondary site occurs at frequencies around 10,000 times lower. Secondary sites consist of a pentanucleotide with only two fully conserved residues (GWTMW). The analysis of IntI1-mediated recombinants in the plasmid pOX38 revealed the existence in this plasmid of a site used at a frequency intermediate between those of primary and secondary sites. Analysis of this site showed two potentially relevant structural features: first, a set of two consensus pentanucleotides, separated by 5 bp and in opposite orientations, forming what will be called a double site; and second, a longer sequence with some extent of sequence symmetry with the double site at its 3' end. A recombinant plasmid, pSU18P, containing a double site was constructed. Examination of R388-pSU18P recombinants showed that double sites were used preferentially over single pentanucleotides by IntI1. Comparisons of the nucleotide sequences of known 59-bp elements showed that in most cases there was a double site at each element end. Mutagenesis of the F hot spot was carried out to make it look more like the consensus 59-bp element. The improved sites showed recombination frequencies and specificities almost comparable to those observed at IntI1 primary sites. 相似文献
18.
The S. rimosus amplifying sequence AUD-Sr1 encodes kanamycin and neomycin resistance, defined in the case of neomycin by aminoglycoside phosphotransferase. Its cloning on plasmid SLP1.2 makes possible the co-amplification of the obtained hybrid plasmids in S. lividans. In our study the regions responsible for resistance to aminoglycoside antibiotics and the capacity for amplification the two hybrid plasmids pSU10 and pSU3 were determined. Experiments on subcloning of the AUD-Sr1 sequence fragments on vector pIJ702 revealed localization of kanamycin and neomycin resistance determinants between PvuII(6) and BglII(7) on the AUD-Sr1 sequence fragments of 2.0 kb length. Two regions responsible for amplification of the hybrid plasmids were detected with deletion and insertion mapping. The first region is localized in the region of the plasmid SLP1.2 BamHI site and the second region is localized on the PstI(4)-PvuII(6) of the AUD-Sr1 sequence fragment of 1.1 kb length. 相似文献
19.
Isolation and location on the R27 map of two replicons and an incompatibility determinant specific for IncHI1 plasmids. 总被引:6,自引:2,他引:4
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Two replicons were isolated independently from different IncHI1 plasmids. One was isolated from R27, and a second was isolated from pIP522. We demonstrate, by DNA-DNA hybridization experiments, that these maintenance regions are different and that they are specific to, and carried by, all IncHI1 plasmids tested. In view of this specificity we decided to designate the replicon isolated from R27 as RepHI1A and the replicon isolated from pIP522 as RepHI1B. These two autoreplicative regions are not related to a third replicon present in all IncHI1 plasmids that bears homology with RepFIA and that expresses the characteristic incompatibility of IncHI1 subgroup plasmids toward F factor (D. Saul, D. Lane, and P. L. Bergquist, Mol. Microbiol. 2:219-225, 1988; D. E. Taylor, R. W. Hedges, and P. L. Bergquist, J. Gen. Microbiol. 131:1523-1530, 1985). These results demonstrate that all IncHI1 plasmids tested contain at least three replicons. An incompatibility (Inc) region that hybridizes specifically to all the IncHI1 plasmids was previously isolated (M. Couturier, F. Bex, P. L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Although this Inc locus is not located in an autoreplicative region of IncHI1 plasmids, we observed that this locus stabilizes a low-copy-number replicon. This Inc locus is probably a component of an active partition locus involved in the maintenance of IncHI1 plasmids. The nucleotide sequence of the Inc region contains direct repeats of 31 bp. In addition, this incompatibility determinant hybridizes specifically with IncHI1 plasmids but expresses incompatibility toward plasmids of both IncHI subgroups (IncHI1 and IncHI2). In this communication, we present the mapping of these maintenance elements on the R27 genome. 相似文献
20.
Lam AM Espiritu C Bansal S Micolochick Steuer HM Zennou V Otto MJ Furman PA 《Journal of virology》2011,85(23):12334-12342
PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methylguanosine-5'-monophosphate. Both compounds are metabolized to the same active 5'-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2'-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both wild-type and S282T NS5B polymerases. In order to identify mutations that confer resistance to these compounds, in vitro selection studies were performed using HCV replicon cells. While no resistant genotype 1a or 1b replicons could be selected, cells containing genotype 2a JFH-1 replicons cultured in the presence of PSI-352938 or PSI-353661 developed resistance to both compounds. Sequencing of the NS5B region identified a number of amino acid changes, including S15G, R222Q, C223Y/H, L320I, and V321I. Phenotypic evaluation of these mutations indicated that single amino acid changes were not sufficient to significantly reduce the activity of PSI-352938 and PSI-353661. Instead, a combination of three amino acid changes, S15G/C223H/V321I, was required to confer a high level of resistance. No cross-resistance exists between the 2'-F-2'-C-methylguanosine prodrugs and other classes of HCV inhibitors, including 2'-modified nucleoside/-tide analogs such as PSI-6130, PSI-7977, INX-08189, and IDX-184. Finally, we determined that in genotype 1b replicons, the C223Y/H mutation failed to support replication, and although the A15G/C223H/V321I triple mutation did confer resistance to PSI-352938 and PSI-353661, this mutant replicated at only about 10% efficiency compared to the wild type. 相似文献