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1.
Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis 总被引:14,自引:0,他引:14
Integrable vectors were constructed based on the plasmid pHV60, which is essentially a pBR322 replicon carrying a chloramphenicol resistance marker, by inserting 1.3-kilobase chromosomal fragments of Lactococcus lactis subsp. lactis MG1363 into this plasmid. Three constructs as well as pHV60 were electroporated to strain MG1363. Transformants were obtained with all constructs, and also with pHV60 (albeit with low frequency). By using Southern hybridizations, it appeared that pHV60 showed homology with the chromosome of MG1363, and that it most probably uses this homology to integrate in a Campbell-like manner. The presence of chromosomal sequences in pHV60 stimulated insertion elsewhere in the chromosome by a factor of 5 to 100. In all cases the integrated plasmids were amplified, at a selective pressure of 5 micrograms of chloramphenicol per ml, to a level of approximately 15 copies per chromosome. Although the amplification was gradually lost under nonselective conditions, one copy remained stably integrated in the chromosome. The results show that a Campbell-like integration strategy can be used to improve the accessibility of the lactococcal chromosome for genetic analysis and is potentially useful in stabilizing unstable genes in lactococci. 相似文献
2.
Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis. 总被引:20,自引:11,他引:9
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Integrable vectors were constructed based on the plasmid pHV60, which is essentially a pBR322 replicon carrying a chloramphenicol resistance marker, by inserting 1.3-kilobase chromosomal fragments of Lactococcus lactis subsp. lactis MG1363 into this plasmid. Three constructs as well as pHV60 were electroporated to strain MG1363. Transformants were obtained with all constructs, and also with pHV60 (albeit with low frequency). By using Southern hybridizations, it appeared that pHV60 showed homology with the chromosome of MG1363, and that it most probably uses this homology to integrate in a Campbell-like manner. The presence of chromosomal sequences in pHV60 stimulated insertion elsewhere in the chromosome by a factor of 5 to 100. In all cases the integrated plasmids were amplified, at a selective pressure of 5 micrograms of chloramphenicol per ml, to a level of approximately 15 copies per chromosome. Although the amplification was gradually lost under nonselective conditions, one copy remained stably integrated in the chromosome. The results show that a Campbell-like integration strategy can be used to improve the accessibility of the lactococcal chromosome for genetic analysis and is potentially useful in stabilizing unstable genes in lactococci. 相似文献
3.
Cloning and expression of a Streptomyces fradiae neomycin resistance gene in Escherichia coli 总被引:2,自引:0,他引:2
A Streptomyces fradiae DNA sequence, which codes for a neomycin phosphotransferase, has been subcloned from the Streptomyces recombinant plasmid pIJ2 [a chimera between the Streptomyces plasmid SLP1.2 and chromosomal DNA containing a neomycin (Nm) resistance gene] into the BamHI restriction enzyme site of pHV14. Three different recombinant plasmids (pWHR1, pWHR2, pWHR3) have been isolated which transform Escherichia coli to Nm resistance. Southern transfer hybridization experiments show that the recombinant plasmids contain the cloned Streptomyces Nm resistance gene, and lysates of E. coli containing the recombinant plasmids were shown to have Nm phosphotransferase activity, demonstrating that a gene from Streptomyces can be expressed in E. coli. 相似文献
4.
Abstract By using an extension to group N streptococci of a contransformation procedure we have introduced 4 different-sized cryptic plasmids for Streptococcus lactis into the plasmid-free S. lactis IL1403. A mixture of 4 cryptic plasmids with an indicator plasmid (pHV1301) conferring erythromycin resistance was used for IL1403 protoplast transformation. Under such conditions, 41.5% of the erythromycin-resistant transformants were contransformed with one of the cryptic plasmids in addition to pHV1301. Indicator plasmid pHV1301 was later spontaneously segregated from doubly transformed cells. This protocol should be very useful for constructing lactic streptococcal strains bearing any phenotypically cryptic plasmid. 相似文献
5.
Twelve mecoprop-degrading bacteria were isolated from soil samples, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genus Sphingomonas. Ten different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 12 isolates. The isolates were found to be able to utilize the chiral herbicide mecoprop as a sole source of carbon and energy. While seven of the isolates were able to degrade both (R)- and (S)-mecoprop, four isolates exhibited enantioselective degradation of the (S)-type and one isolate could degrade only the (R)-enantiomer. All of the isolates were observed to possess plasmid DNAs. When certain plasmids were removed from isolates MP11, MP15, and MP23, those strains could no longer degrade mecoprop. This compelling result suggests that plasmid DNAs, in this case, conferred the ability to degrade the herbicide. The isolates MP13, MP15, and MP24 were identified as the same strain; however, they exhibited different plasmid profiles. This indicates that these isolates acquired different mecoprop-degradative plasmids in different soils through natural gene transfer. 相似文献
6.
Summary Small plasmids ofClostridium acetobutylicum and related strains were isolated and studied. Their restriction maps were established and different hybrid plasmids were constructed by ligation with plasmid pHV33. 相似文献
7.
Edda De Rossi Patrizia Brigidi Giovanna Riccardi Prof. Diego Matteuzzi 《Current microbiology》1989,19(1):13-19
Screening for the plasmid content of 94 strains belonging to ten species of thermophilic bacilli mainly referred toBacillus stearothermophilus was carried out. Twenty-seven of the strains tested were found to harbor one or more plasmids ranging in size from 1.7 to>50 kb. The physical map of the smallest plasmid, pBC1, is reported. Southern hybridization experiments showed that pBC1 hybridized strongly with the other small plasmids, weakly with medium-sized plasmids, and not at all with the largest plasmids and chromosomal DNA. pBC1 was cloned into pHV14 and pJH101 vectors, and the chimeric plasmids obtained were used to transformEscherichia coli andBacillus subtilis. 相似文献
8.
The plasmid pHV32, which replicates in Escherichia coli but not in Bacillus subtilis, transformed B. subtilis-competent cells efficiently when linked in vitro to EcoRI B. subtilis DNA segments. The transformed clones carried pHV32 inserted in their chromosomes, and often displayed a mutant phenotype. One of the transformed clones carried pHV32 inserted close to the thyB gene. We cleaved the DNA extracted from this clone with BglII restriction endonuclease, for which no sites exist on pHV32, ligated the released segments and used them to transform E. coli selecting for pHV32-carried genetic markers. The transformants harbored a hybrid plasmid which carried the B. subtilis thyB gene. Circular molecules composed of pHV32 joined to B. subtilis DNA inserted into the chromosome by a Campbell-like recombination event. Linear molecules, in which pHV32 was flanked by two non-adjacent DNA segments, underwent a double cross-over recombination with the chromosome. In this case the chromosomal sequences between the non-adjacent segments were deleted, and replaced by pHV32 sequences. 相似文献
9.
Twenty isolates of Rhizobium leguminosarum bv. viceae were isolated at random from one field and examined for symbiotic plasmid fragment length polymorphisms and for isoenzyme patterns. The latter are most probably chromosome markers. With one exception both methods separated the isolates into the same 13 different groups. The largest group was represented 7 times according to isoenzymes and 8 times according to RFLP. This fixed non-random association of plasmid and chromosomal genotypes is consistent with a clonal population structure; it indicates limited exchange of plasmids under natural conditions. Seventeen isolates of 11 groups were highly effective and 2 isolates in one group almost ineffective. 相似文献
10.
Genetic and plasmid diversity within natural populations of Pseudomonas syringae with various exposures to copper and streptomycin bactericides. 总被引:6,自引:4,他引:2
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We examined the genetic and plasmid diversity within natural populations of Pseudomonas syringae isolated from three ornamental pear nurseries in eastern Oklahoma. The bactericide spray regimen differed at each nursery; copper and streptomycin, only copper, and no bactericides were applied at nurseries I, II, and III respectively. Resistance to copper (Cur) and resistance to streptomycin (Smr) were determined for 1,938 isolates of P. syringae; isolates from nurseries I and II were generally Cur Sms; whereas most isolates from nursery III were Cus Sms. The plasmid profiles of 362 isolates were determined, and six, one, seven, and four plasmid profiles were obtained for Cur, Smr, Cur Smr, and Cus Sms isolates, respectively. All Smr plasmids contained sequences homologous to the strA and strB Smr genes from broad-host-range plasmid RSF1010 and were associated with Smr transposon Tn5393. Plasmids were placed into two groups on the basis of hybridization to the oriV and par sequences from pOSU900, a cryptic plasmid in P. syringae pv. syringae. A total of 100 randomly chosen P. syringae isolates from nurseries I and III were analyzed for genetic diversity by using the arbitrarily primed PCR (AP-PCR) technique. An analysis of chromosomal genotypes by AP-PCR revealed a high degree of genetic diversity among the isolates, and the results of this analysis indicated that the isolates could be clustered into two distinct groups. The plasmid profiles were specific to isolates belonging to particular AP-PCR groups. Within each AP-PCR group, identical plasmid profiles were produced by isolates that had different chromosomal genotypes, implying that plasmid transfer has played an important role in the dissemination of Cur and Smr within the populations studied. 相似文献
11.
Francisco José Ayala Dan E. Krane Daniel L. Hartl 《Journal of molecular evolution》1994,39(2):129-133
Nucleotide sequences of portions of three plasmid genes (cib, cir, and abi) present in IncI1-Co1Ib colicin plasmids obtained from strains of Salmonella typhimurium isolated in either 1974 (Barker strains) or between 1935 and 1941 (Murray strains) were examined along with sequences of the chromosomal gene for 6-phosphogluconate dehydrogenase (gnd). Our principal findings were: (1) The plasmid genes were virtually identical to those in IncI1-CoIIb plasmids from E. coli, suggesting that Salmonella and E. coli share overlapping pools of these plasmids. (2) The plasmid genes were much less polymorphic than gnd or any other known chromosomal gene from Salmonella, further suggesting horizontal transfer with rapid transmission and turnover. (3) No characteristic differences were found in either the plasmid genes or the chromosomal gene between the 1974 isolates and the Murray strains, indicating that these plasmids have been stable for at least several decades. (4) There was an excess of amino-acid replacement polymorphisms, relative to synonymous polymorphisms, in the plasmid genes, which is consistent with the hypothesis of diversifying selection among colicin-producing plasmid families. (5) The abi (abortive infection) gene present in each of the plasmids contained two single-nucleotide insertions relative to the published sequence. These result in a putative abi protein of 114 amino acids instead of 89. 相似文献
12.
Some Bacillus subtilis strains demonstrate a high degree of pHV14 and pP1251 plasmid integration into the chromosome (1.10(-1]. According to the data of genetical analysis the plasmids are integrated into several regions of the same chromosome. A significant plasmid amplification in the composition of chromosome is observed when breeding on an increasing chloramphenicol concentration (20-300 micrograms/ml). Both tandem repetitions and single plasmid DNA copies and their fragments are found in the bacterial chromosomal DNA. 相似文献
13.
Xiaomin Hu Izabela Swiecicka Sophie Timmery & Jacques Mahillon 《FEMS microbiology ecology》2009,70(3):12-23
Eighty soil-borne Bacillus cereus group isolates were collected from two neighbouring geographical sites in Belgium. Their genetic relationships and population structure were assessed using Multilocus sequence typing analysis of five chromosomal genes, while the contribution of extrachromosomal elements to the population dynamics was gauged by the presence, diversity and transfer capacity of pXO1- and pXO2-like plasmids. Globally, the bacterial population displayed a broad diversity, including an important subpopulation of psychrotolerant isolates related to Bacillus weihenstephanensis . pXO1- and pXO2-like replicons were present in 12% and 21% of the isolates, but no Bacillus anthracis -related toxin genes were found. Furthermore, only one of the isolates containing a pXO2-related plasmid was shown to be able to mobilize small non-self-conjugative plasmids. Interestingly, several B. cereus sensu lato isolates displaying the same sequence type were observed to have different plasmid contents, suggesting the occurrence of horizontal gene exchange. Similarly, a number of pXO2-like replicons with identical sequences were found in distinct bacterial isolates, therefore strongly arguing for lateral transfers among sympatric bacteria. 相似文献
14.
Cloning in Escherichia coli of the gene specifying the DNA-entry nuclease of Bacillus subtilis 总被引:6,自引:0,他引:6
With the aim of cloning genes involved in transformation of Bacillus subtilis, a set of transformation-deficient mutants was isolated by means of insertional mutagenesis with plasmid pHV60 (Vosman et al., 1986). Analysis of these mutants showed that those mapping in the aroI region lacked the DNA-entry nuclease activity. Plasmid pHV60 derivatives, containing flanking chromosomal DNA fragments, were isolated from these mutants and were used to screen a library of B. subtilis chromosomal DNA in phage lambda EMBL4. In Escherichia coli lysates, prepared with the phages that hybridized to the pHV60-based probe, a prominent nuclease activity could be detected. The nuclease encoded by the phage DNA had the same Mr as the B. subtilis DNA-entry nuclease and its activity was strongly stimulated by Mn2+, which is also characteristic for the B. subtilis DNA-entry nuclease. From these results it was concluded that the gene specifying the B. subtilis DNA-entry nuclease had been cloned. It was shown that the nuclease activity was specified by a 700-bp EcoRI-PstI fragment. 相似文献
15.
Replication functions of pC194 are necessary for efficient plasmid transduction by M13 phage. 总被引:19,自引:9,他引:10
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Escherichia coli plasmids pBR313 and pBR322 were transduced by phage M13 with low efficiency (10(-8) transductants/phage). Hybrid plasmids pHV12 or pHV33, composed of Staphylococcus aureus plasmid pC194 and pBR313 or pBR322, respectively, were transduced much more efficiently (10(-4) transductants/phage). Inactivation of either of the two zones necessary for pC194 replication, one coding for a protein, the other not, reduced the transforming efficiency of hybrids to the level of pBR322. Activity of the pC194 replication region was not necessary for the formation of chimeras between M13 and the transduced plasmid in the donor cells, but rather for the establishment of the plasmid in the recipient cells. 相似文献
16.
The Bacillus subtilis spoVE locus was isolated from a lambda clone bank and a 4.7 kbp EcoRV fragment subcloned into the shuttle vector pHV33. The resulting plasmid complemented chromosomal spoVE mutations. Its structure was stable in recE4 strains, but plasmid and chromosomal rearrangements occurred in rec+ strains. New spoVE mutations were obtained by mutagenesis of the plasmid; all the mutations tested mapped within three adjacent HindIII fragments of total length 1140 bp. 相似文献
17.
A 16.6-kb plasmid-pSG1-was isolated from Streptomyces griseus following transformation of protoplasts with unrelated plasmids. Southern hybridization experiments with radioactive probes prepared from pSG1 fragments and immobilized S. griseus DNA fragments indicated that the plasmid was present in the progenitor strain, in an integrated state. In the pSG1+ isolates plasmid sequences existed both as integrated sequences and as free plasmids. The integrated state of maintenance persisted in strains which have been cured of the free plasmid. The junction site on the plasmid was located on a 0.5-kb EcoRI-SalI fragment. The chromosomal integration site was demonstrated to be the same in all strains derived from S. griseus NRRL3851. The occurrence of both states of plasmid maintenance in the same clones indicates that an integrated pSG1 sequence does not interfere with free plasmid replication and partition. It suggests that the establishment of the free state may involve a replicative excision of pSG1 from the S. griseus chromosome. 相似文献
18.
A nuclear gene of Saccharomyces cerevisiae needed for stable maintenance of plasmids. 总被引:3,自引:0,他引:3
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We have isolated host mutants of Saccharomyces cerevisiae in which the 2 microns plasmid is poorly maintained. All the mutants tested constituted one complementation group, which was designated map1 (maintenance of plasmid). Minichromosomes carrying a chromosomal replication origin and a centromere were affected in the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS (autonomously replicating sequence) or a set of 2 microns inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication. 相似文献
19.
Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus,"
"T. brockianus,"
T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction
fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species
2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception
of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length.
Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14
strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were
found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging
to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical
taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains.
Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the
same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a
particular hot spring.
Received: 24 October 1994 / Accepted: 6 March 1995 相似文献
20.
M. van der Gaag A. J. M. Debets H. D. Osiewacz R. F. Hoekstra 《Molecular genetics and genomics : MGG》1998,258(5):521-529
A natural population of recently isolated Podospora anserina strains was screened for homologues of the linear longevity-inducing plasmid pAL2-1. Of the 78 wild-type isolates, 14 hybridised with a pAL2-1 specific probe, half of which contained a single plasmid and the other half multiple plasmid copies (plasmid family). All strains except one plasmid-containing strain, senesced normally. However, no inserted plasmid sequences were detected in the mitochondrial DNA, as was the case for the longevity-inducing pAL2-1 plasmid. Occasional loss of plasmids and of repeated plasmid sequences occurred during sexual transfer. Plasmid transmission was equally efficient for mono- and dikaryotic spores and was independent of the genetic background of the strains. Furthermore, horizontal transfer experiments showed that the linear plasmid could easily infect plasmid-free strains. Horizontal transfer was even observed between strains showing a clear vegetative incompatibility response (barrage). The linear plasmids are inherited maternally; however, paternal transmission was observed in crosses between confronted vegetative-incompatible strains. Paternal transmission of the plasmid was never observed using isolated spermatia for fertilisation, showing that mitochondrial plasmids can only gain access to maternal sexual reproductive structures following horizontal transfer. These findings have implications for both the function of vegetative incompatibility in fungi and for the mechanism of maintenance of linear plasmids. 相似文献