Physical and genetic analyses of streptococcal plasmid pAM beta 1 and cloning of its replication region.

Plasmid pAM beta 1, originally isolated from Streptococcus faecalis DS5, mediates resistance to the MLS (macrolide, lincosamide, and streptogramin B alpha) group of antibiotics. A restriction endonuclease map of the 26.5-kilobase (kb) pAM beta 1 molecule was constructed by using the enzymes AvaI, HpaII, EcoRI, PvuII, Kpn1, BstEII, HpaI, HhaI, and HindIII. A comparison of this map to those of four independently isolated deletion derivatives of pAM beta 1 located the MLS resistance determinant within a 2-kb DNA segment and at least one conjugative function within an 8-kb region. The 5.0-kb EcoRI-B fragment from pAM beta 1 was ligated onto the 4.0-kb Escherichia coli plasmid vector, pACKC1, and used to transform E. coli HB101. The 9.0-kb chimeric plasmid was then used to transform Streptococcus sanguis Challis with concurrent expression of the E. coli kanamycin resistance determinant. The 5.0-kb EcoRI-B fragment from pAM beta 1 was subsequently used as a vector to clone a streptomycin resistance determinant from a strain of Streptococcus mutans containing no detectable plasmid DNA. Subcloning experiments, using a HindIII partial digest of pAM beta 1 DNA, narrowed the replication region of this plasmid to a 2.95-kb fragment.     

Essential structure in the cloned transforming DNA that induces gene amplification of the Bacillus subtilis amyE-tmrB region.

Bacillus subtilis B7, a mutant which acquired gene amplification of the amyE-tmrB region, showed, as a result, hyperproductivity (about a 5- to 10-fold increase) of alpha-amylase and tunicamycin resistance. The mutational character was transferred to recipient cells by competence transformation. A 14-kilobase (kb) EcoRI chromosomal DNA fragment of strain B7 was found to have the transforming activity. We cloned a 6.4-kb EcoRI fragment on a phage vector lambda Charon 4A through a spontaneous deletion of 7.6 kb from the 14-kb fragment and subcloned a 1.6-kb HindIII fragment on pGR71. The cloned 6.4-kb EcoRI and 1.6-kb HindIII fragments retained the transforming activity of inducing gene amplification of the amyE-tmrB region. At the junction point (J) of the repeating units (16 kb), the tmrB gene was linked to a DNA region (M) located 4 kb upstream of amyE. The essential structure of the cloned, transforming (gene amplification-inducing) DNA was deduced to be that around J. The subcloned 1.6-kb HindIII fragment that retained the transforming activity was shown to be almost solely composed of the tmrB-J-M region. In addition, the DNA sequence around J was determined.     

A restriction map of IncFIV plasmid R124

A physical and genetic map of the 125.7-kb IncFIV plasmid R124 was constructed using the restriction enzymes Sal1 and EcoR1. Two discrete regions involved in plasmid replication were identified on the plasmid genome. One region was located on a 4.66-kb segment of an EcoR1 fragment at map coordinates 73.87 to 78.53 kb. Another was located within an 8.05-kb segment of an EcoR1 fragment at map coordinates 113.40 to 121.45. This region was very unstable but, when ligated to the 3.21-kb EcoR1 fragment E13 located at map coordinates 18.83 to 22.06 kb, replication was stable. Thus, at least three regions of R124 widely separated around the genome are associated with plasmid replication and stable maintenance. Each of these three regions expressed incompatibility with R124. The Tc resistance gene of R124 was located on the contiguous EcoR1 fragments E8 and E12 located at map coordinates 100.49 to 113.40.     

Physical and functional map of an amikacin-resistance plasmid isolated from a multiresistant strain of Serratia marcescens

pTK159, a multiresistance 40-kilobase (kb) plasmid, was isolated from a clinical strain of Serratia marcescens. pTK159 was nonconjugative and carried determinants for resistance to amikacin, streptomycin/spectinomycin, sulfamethoxazole and ampicillin. A physical and functional map of pTK159 was constructed. By cloning various fragments of pTK159 in pACYC184 or pBR322, genes for resistance to amikacin, streptomycin/spectinomycin, and sulfamethoxazole were found to be located on a 2.0-kb BamHI-HindIII fragment, a 1.4-kb HindIII fragment and a 2.1-kb HindIII fragment, respectively. The map of pTK159 was compared with published maps of amikacin-resistance determinants and transposons.     

Characterization of Bacteroides ovatus plasmid pBI136 and structure of its clindamycin resistance region.

Genetic and physical analyses were used to characterize the Bacteroides ovatus R plasmid pBI136. Results from restriction endonuclease cleavage studies were used to construct a physical map of the plasmid for the enzymes EcoRI, BamHI, ClaI, XbaI, SalI, and SmaI. Based on the sizes of restriction fragments generated in these studies, the plasmid was estimated to be 80.6 kilobase pairs (kb). A 7.2-kb region of the plasmid required for resistance to lincosamide and macrolide (LM) antibiotics was mapped by analysis of spontaneously occurring LM-sensitive deletion derivatives. Hybridization studies showed that this region and an adjoining 2.9-kb EcoRI fragment were responsible for the previously reported homology among Bacteroides plasmids pBF4, pBFTM10, and pBI136. Within this region of homology, 0.5 kb was attributed to a directly repeated sequence thought to bound the LM resistance determinant on pBF4 and pBFTM10. Two pBI136 EcoRI fragments spanning the putative LM resistance region were cloned in Escherichia coli, and heteroduplex analysis of these recombinant plasmids revealed the presence of a 1.2-kb directly repeated sequence. These results suggested that the pBI136 LM resistance determinant resides on an 8.4-kb segment of DNA containing 6.0 kb of intervening DNA sequences bounded by a 1.2-kb directly repeated sequence.     

Molecular cloning and expression of Rhizobium fredii USDA 193 nodulation genes: extension of host range for nodulation.

DNA hybridization with the cloned nodulation region of Rhizobium meliloti as a probe revealed DNA homology with four HindIII fragments, 12.5, 6.8, 5.2, and 0.3 kilobases (kb) in size, of the symbiotic plasmid pRjaUSDA193. Both hybridization and complementation studies suggest that the common nodulation genes nodABC and nodD of R. fredii USDA 193 are present on the 5.2-kb HindIII and 2.8-kb EcoRI fragments, respectively, of the Sym plasmid. Both fragments together could confer nodulation ability on soybeans when present in Sym plasmid-cured (Sym-) and wild-type (Sym+) Rhizobium strains or in a Ti plasmid-cured Agrobacterium tumefaciens strain. Furthermore, the 2.8-kb EcoRI fragment alone was able to form nodulelike structures on Glycine max L. cv. "Peking" (soybean). Microscopic examination of these nodules revealed bacterial invasion of the cells, probably via root hair penetration. Bacterial strains harboring plasmids carrying the 5.2- and 2.8-kb nod fragments elicited root-hair-curling responses on infection. These data suggest that the genes responsible for host range determination and some of the early events of nodulation may be coded for by the 5.2-kb HindIII and 2.8-kb EcoRI fragments.     

Cloning and expression of plasmid genes encoding resistances to chromate and cobalt in Alcaligenes eutrophus.

Resistances to chromate and cobalt were cloned on a 30-kilobase-pair (kb) DNA region from the large Alcaligenes eutrophus plasmid pMOL28 into the broad-host-range mobilizable cosmid vector pVK102. A restriction nuclease map of the 30-kb region was generated. The resistances expressed from the hybrid plasmids after transfer back into A. eutrophus were inducible and conferred the same degree of resistance as the parent plasmid pMOL28. Resistances were expressed in metal-sensitive Alcaligenes strains and related bacteria but not in Escherichia coli. Resistance to chromate was further localized on a 2.6-kb EcoRI fragment, and resistance to cobalt was localized on an adjoining 8.5-kb PstI-EcoRI fragment. When the 2.6-kb EcoRI fragment was expressed in E. coli under the control of a bacteriophage T7 promoter, three polypeptides with molecular masses of 31,500, 21,000, and 14,500 daltons were visible on autoradiograms. The 31,500- and 21,000-dalton polypeptides were membrane bound; the 14,500-dalton polypeptide was soluble.     

Nucleotide sequence of an incompatibility region of mini-Rts1 that contains five direct repeats.

The plasmid mini-Rts1, consisting of an EcoRI/HindIII fragment of about 1.8 kilobases (kb), contains an incompatibility determinant in its EcoRI/AccI region (0.5 kb). The nucleotide sequence of this incompatibility fragment was determined. A most striking feature of the sequence is the presence of five 24-base pair direct repeats. Four out of the five repeating units, which are contained in a 0.2-kb EcoRI/HincII fragment, were cloned en bloc in pACYC184 and found to express Rts1-specific incompatibility. In addition, the copy number of the mini-Rts1 plasmid appeared to be increased threefold upon removal of the 0.2-kb incompatibility region (incI) from the plasmid. This deletion derivative of mini-Rts1, as well as mini-Rts1, was maintained stably at 37 degrees C, but was cured at a high frequency at 42 degrees C. A possible role of the repeated nucleotide sequence was discussed. By subcloning the mini-Rts1 DNA, a second inc determinant (incII) was found on the AccI fragment, which is contiguous to the 0.5-kb EcoRI/AccI fragment.     

Physical and genetic organization of the plasmid R15

The conjugative IncN plasmid R15 (SmrSurHgr, 62.3 kb) is cleaved by the hexanucleotide-specific endonucleases BglII, HindIII, EcoRI, BamHI, SmaI, SalI, PstI and XhoI into 9, 9, 6, 5, 4, 4, 4 and 2 fragments, respectively. The restriction sites were located on the physical map of the R15 genome. Distribution of the cleavage sites is strongly asymmetric. 28 of 32 sites for BamHI, EcoRI, HindIII, SalI, SmaI and PstI were located close to or within the sequences of transposable elements Tn2353 and Tn2354. According to the results of analysis of R15::Tn1756 deletion derivatives and recombinant plasmids harboring fragments of R15, the genetic determinants for resistance to Sm, Su and Hg were mapped, as well as the regions necessary for EcoRII restriction--modification and for plasmid replication and conjugation. The features of physical and genetic structures of R15 and other IncN plasmids are discussed.     

Cloning of the DNA repair genes mtcA, mtcB, uvsC, uvsD, uvsE and the leuB gene from Deinococcus radiodurans

A gene library from Deinococcus radiodurans has been constructed in the cosmid pJBFH. A 51.5-kb hybrid cosmid, pUE40, that transduced Escherichia coli HB101 from leucine dependence to independence was selected, and a 6.9-kb fragment which carried the leuB gene from D. radiodurans was subcloned into the EcoRI site of pAT153. The DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE, which code for two D. radiodurans UV endonucleases were identified by transforming appropriate repair-deficient mutants of D. radiodurans to repair proficiency with DNA derived from the gene library. Hybrid cosmid pUE50 (37.9 kb) containing an insert carrying both the mtcA and mtcB genes was selected and 5.6- and 2.7-kb DNA fragments carrying mtcA and mtcB, respectively, i.e., the genes that code for UV endonuclease alpha, were subcloned into the EcoRI site of pAT153. The three genes uvsC, uvsD and uvsE, that code for UV endonuclease beta, were all present in the 46.0-kb hybrid cosmid pUE60. The uvsE gene in a 12.2-kb fragment was subcloned into the HindIII site of pAT153 and the size of the insert reduced to 6.1 kb by deletion of a 6.7-kb fragment from the hybrid plasmid pUE62. None of the uvs genes introduced into the UV-sensitive E. coli CSR603 (uvrA-) was able to complement its repair defect. The mtcA, uvsC, uvsD and uvsE genes were found in the 52.5-kb hybrid cosmid pUE70. It is concluded that the DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE are located within an 83.0-kb fragment of the D. radiodurans genome.     

Evidence that the clindamycin-erythromycin resistance gene of Bacteroides plasmid pBF4 is on a transposable element.

We constructed a shuttle vector, pE5-2, which can replicate in both Bacteroides spp. and Escherichia coli. pE5-2 contains a cryptic Bacteroides plasmid (pB8-51), a 3.8-kilobase (kb) EcoRI-D fragment from the 41-kb Bacteroides fragilis plasmid pBF4, and RSF1010, an IncQ E. coli plasmid. pE5-2 was mobilized by R751, an IncP E. coli plasmid, between E. coli strains with a frequency of 5 X 10(-2) to 3.8 X 10(-1) transconjugants per recipient. R751 also mobilized pE5-2 from E. coli donors to Bacteroides uniformis 0061RT and Bacteroides thetaiotaomicron 5482 with a frequency of 0.9 X 10(-6) to 2.5 X 10(-6). The Bacteroides transconjugants contained only pE5-2 and were resistant to clindamycin and erythromycin. Thus, the gene for clindamycin and erythromycin resistance must be located within the Eco RI-D fragment of BF4. A second recombinant plasmid, pSS-2, which contained 33 kb of pBF4 (including the EcoRI-D fragment and contiguous regions) could also be mobilized by R751 between E. coli strains. In some transconjugants, a 5.5-kb (+/- 0.3 kb) segment of the pBF4 portion of pSS2 was inserted into one of several sites on R751. In some other transconjugants this same 5.5-kb segment was integrated into the E. coli chromosome. This segment could transfer a second time onto R751. Transfer was RecA independent. The transferred segment included the entire EcoRI-D fragment, and thus the clindamycin-erythromycin resistance determinant, from pBF4.     

Physical map of the Rhodobacter sphaeroides bacteriophage phi RsG1 genome and location of the prophage on the host chromosome.

We constructed a physical map of the 50-kilobase-pair (kb) DNA of the temperate Rhodobacter sphaeroides bacteriophage phi RsG1, with the relative positions of the cleavage sites for the nine restriction endonucleases KpnI, HindIII, XbaI, ClaI, BclI, EcoRV, EcoRI, BglII, and BamHI indicated. Using biotinylated phi RsG1 DNA as a probe in hybridization studies, we detected homologies with virus DNA and fragments of restriction endonuclease-digested host chromosomal DNA but not with plasmid DNA. This indicates that the prophage is integrated into the host chromosome. In addition, the use of specific probes such as the 10.4-kb BglII A fragment and the 2.65-kb BamHI H fragment allowed the determination of the position of phage attachment site (attP).     

Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within HupRhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H(2)-uptake hydrogenase positive (Hup) or negative (Hup) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H(2) oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H(2)-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H(2)-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup strains in this symbiotic species.     

Transfer of the chromosomal bla gene from Enterobacter cloacae to Escherichia coli by RP4::mini-Mu

The resistance gene for beta-lactamase-stable cephalosporins from Enterobacter cloacae was transferred to Escherichia coli by the aid of RP4::mini-Mu. The R-prime plasmids generated carried 60 to 80 kilobases (kb) of E. cloacae DNA and coded for the chromosomal E. cloacae beta-lactamase. The gene was fully expressed in the recipient. Restriction endonuclease EcoRI fragments of the R-prime plasmid pBP100 were cloned into the vector pBP328, yielding the plasmid pBP102 with a size of 14 kb. A restriction map of this plasmid was constructed. By digesting pBP102 into seven PstI fragments, ligating the fragments, and looking for the smallest plasmid generated, pBP103 was isolated. It consisted of three PstI fragments, two of them (together 4.2 kb) necessary for resistance. During the experiment (performed in a recA+ background) the largest PstI fragment had undergone a substitution of a 0.3-kb segment of pBP102 by a 0.7-kb segment in pBP103 (as deduced by heteroduplex analysis). The bla gene of resistant E. cloacae strains was dominant over the gene of susceptible organisms.     

Construction of an Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp.

A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1(pMVS301) and transformed into Rhodococcus sp. strain AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. Thiostrepton-resistant transformants were also ampicillin resistant and were shown to contain pMVS301, which was subsequently isolated and transformed back into E. coli. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. Optimization of the transformation procedure resulted in transformation frequencies in the range of 10(5) transformants per micrograms of pMVS301 DNA in Rhodococcus sp. strain H13-A and derivative strains. The plasmid host range extends to strains of Rhodococcus erythropolis, R. globulerus, and R. equi, whereas stable transformants were not obtained with R. rhodochrous or with several coryneform bacteria tested as recipients. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and other actinomycetes. This is the first report of plasmid transformation and of heterologous gene expression in a Rhodococcus sp.     

Mapping polypeptide hormone genes in the mouse: somatostatin, glucagon, calcitonin, and parathyroid hormone

Mouse-Chinese hamster hybrids segregating mouse chromosomes were analyzed by Southern hybridization techniques to map the genes for somatostatin (Smst), glucagon (Gcg), calcitonin (Calc), and parathyroid hormone (Pth). The mouse gene for somatostatin, detected on a 20-kb EcoRI fragment, is located on mouse chromosome 16. Glucagon cDNA hybridized to a 14-kb EcoRI fragment residing on chromosome 2. Calcitonin and parathyroid hormone genes, detected on 7.8-kb HindIII and 6.0-kb BamHI fragments, respectively, were on mouse chromosome 7. The calcitonin and parathyroid hormone genes appear to be part of a larger linkage group which has been conserved in mouse and man.     

Highly mobile DNA segment of IncI alpha plasmid R64: a clustered inversion region.

When R64 DNA was digested with EcoRI, two DNA fragments not equimolar to the plasmid DNA were produced. A DNA region including these fragments was cloned (pKK009), and the pKK009 DNA sample was found to be a mixture of six or more DNA species with EcoRI, PstI, and AvaI cleavage sites at different positions, suggesting a complex rearrangement of DNA. When a part of the pKK009 DNA was removed by HindIII digestion, 33 different types of plasmids (pKK010-series plasmids) were obtained out of 58 clones tested, but no DNA rearrangement could be observed. On the basis of a comparison of the detailed restriction maps of these pKK010-series plasmids, we propose a model in which four DNA segments invert independently or in groups within the 1.95-kilobase region of R64, so that the arrangements of these four segments change randomly. The fixed pKK010-series plasmid DNA was again rearranged in the presence of R64, indicating that trans-acting gene function may be present to mediate the DNA rearrangement. The gene (tentatively designated as rci) was located on a 4.5-kilobase E9' fragment of R64.     

Construction of new cloning vehicles with genes of the tryptophan operon of Escherichia coli as genetic markers

In vitro recombination techniques were used to construct a series of new cloning vehicles with genes of the tryptophan (trp) operon of Escherichia coli as selective marker. To construct these plasmids we have made a restriction cleavage map of the trp operon for the enzymes AosI, AvaI, BglI, BglII, HindIII, HpaI, PvuII, SalI, SstI and XhoI. The constructed plasmids pHP39, pEP392, pEP3921 and pEP3923 are derived from the amplifiable plasmid pBR345 and carry two or more genes of the trp operon, which are controlled by the trp regulatory elements. Plasmid pEP3921 (7.0 kb) carries intact trpE and trpA genes and contains single BglII and SstI sites in trpE, a single HindIII site located between trpE and trpA, and single EcoRI, SalI and XhoI sites located outside the trp genes. Plasmid pEP121 (12 kb) is similar to pEP3921, but has an extra selective marker conferring bacterial resistance to ampicillin. Plasmid pEP3923 (7.4 kb) comprises intact trpB and trpA genes and single BglII, HindIII, EcoRI, SalI and XhoI sites. Plasmids pHP39 (9.8 kb) and pEP392 (9.8 kb) carry an intact trp operon and have two and one EcoRI site, respectively. Plasmid pHP3 (18 kb) carries an intact trp operon and markers for tetracycline and ampicillin resistance.     

Comparison of the transposon-like structures encoding clindamycin resistance in Bacteroides R-plasmids

The R-plasmids pBF4, pBFTM10, and pBI136 encode transmissible clindamycin resistance (Ccr) in Bacteroides spp. These plasmids are distinct replicons but the regions implicated in Ccr share some homology and appear to have a transposon-like structure. To better understand the mechanism of dissemination and to locate the Ccr determinant(s), the genetic and structural properties of the Ccr regions of each plasmid were compared and contrasted. For this work a single EcoRI restriction fragment containing the Ccr region from each plasmid was cloned into pBR322 in Escherichia coli. Results of restriction mapping and heteroduplex experiments showed that the pBF4 EcoRI-D and pBFTM10 EcoRI-B fragments shared more than 90% base sequence homology but that the EcoRI-C fragment of pBI136 had diverged significantly. The pBI136 fragment also did not confer tetracycline resistance in E. coli as shown for the pBF4 EcoRI-D fragment (D.G. Guiney, P. Hasegawa, and C. E. Davis, 1984, Plasmid 11, 248-252). Heteroduplex experiments showed that the pBI136 EcoRI-C and pBF4 EcoRI-D fragments shared a 1.2-kb region of homology attributed to a directly repeated sequence which bounds the Ccr region. Southern hybridization studies indicated that an additional 0.85 kb of the pBI136 EcoRI-C fragment was homologous to the EcoRI-D fragment of pBF4. This region was characterized by its sequential restriction endonuclease sites for HindIII, AvaII, and DdeI, and it is proposed that the Ccr gene(s) resides in this area.