EcoRI restriction endonuclease map of the composite R plasmid NR1.

A physical map of the composite R plasmid NR1 has been constructed using specific cleavage of deoxyribonucleic acid (DNA) by the restriction endonuclease EcoR-. Digestion of composite NR1 DNA by EcoRI yields thirteen fragments. The six largest fragments (designated A to F) are from the resistance transfer factor component that harbors the tetracycline resistance genes (RTF-TC). The seven smallest fragments (designated G to M) are from the r-determinants component that harbors the chloramphenicol (CM), streptomycin-spectinomycin (SM/SP), and sulfonamide (SA) resistance genes. The largest fragment of several RTF-TC segregants of NR1 that have deleted the r-determinants component is 0.8 X 10(6) daltons larger than fragment A of composite NR1. Only a part of fragment H of the r-determinants component is amplified in transitioned NR1 DNA in Proteus mirabilis, which consists of multiple, tandem sequences of r-determinants attached to a single copy of the RTF-TC component. Both of these changes can be explained by the locations of the excision sites at the RTF-TC: r-determinants junctions that are involved in the dissociation and reassociation of the RTF-TC and r-determinants components. The thirteen fragments of composite NR1 DNA produced by EcoRI have been ordered using partial digestion techniques. The order of the fragments is: A-D-C-E-F-B-H-I-L-K-G-M-J. The approximate locations of the TC, CM, SM/SP, and SA resistance genes on the EcoRI map were determined by analyzing several deletion mutants of NR1.     

R-Factor-Mediated Resistance to Tetracycline in Proteus mirabilis

The expression of R-factor-mediated resistance to tetracycline has been compared in Proteus mirabilis and Escherichia coli. Resistance to a range of concentrations of tetracycline was significantly lower in P. mirabilis than in E. coli in both induced and repressed states. Indirect evidence showed that conditions which result in a marked increase in the level of resistance of P. mirabilis harboring the R factor NR1 to chloramphenicol, streptomycin, and spectinomycin due to an amplification in the number of copies of r-determinants per cell do not detectably increase the level of resistance to tetracycline. Tetracycline resistance was inducible in early stationary-phase P. mirabilis NR1 although not after 5 h in this state. Double isotope labeling of control and tetracycline-induced P. mirabilis NR1 in early stationary phase revealed isotopic enrichment of certain peaks in extracts from induced cells subjected to polyacrylamide gel electrophoresis.     

Cloning of the Serratia marcescens recA gene and construction of a Serratia marcescens recA mutant

A recombinant plasmid, pSM2513, containing an 8.5 kb DNA insert was isolated from a genomic library of Serratia marcescens by using interspecific complementation. This plasmid conferred resistance to methyl methanesulphonate and UV irradiation upon recA mutants of Escherichia coli and enhanced recombination proficiency, as measured by Hfr-mediated conjugation, in recA mutants of E. coli. Furthermore, when recA mutants of E. coli harbouring pSM2513 were subjected to UV irradiation, filamentation of the cells was observed. This did not occur upon UV irradiation of the same mutants harbouring the cloning vector alone. These results imply that the S. marcescens recA gene on pSM2513 is functionally similar to the E. coli recA gene in several respects. Restriction enzyme analysis and subcloning studies revealed that the S. marcescens recA gene was located on a 2.7 kb Bg/II-KpnI fragment of pSM2513, and its gene product of approximately 39 kDa resembled the E. coli RecA protein in molecular mass. Using transformation-mediated marker rescue, a recA mutant of S. marcescens was successfully constructed; its proficiency both in homologous recombination and in DNA repair was abolished compared with its parent.     

Homologous sequences other than insertion elements can serve as recombination sites in plasmid drug resistance gene amplification.

A plasmid (pRR983) was constructed which has a gene coding for neomycin and kanamycin resistance flanked by direct repeats of regions of homology which contain no known insertion sequences. pRR983 does not have any homologous IS1 sequences. Growth of Proteus mirabilis harboring pRR983 in medium containing high concentration of neomycin resulted in cells which were highly resistant to both neomycin and kanamycin. Plasmid DNA was analyzed by using restriction endonucleases. In most cases the neomycin resistance gene had been tandemly duplicated by using the homologous DNA sequences flanking the resistance gene as recombination sites. This is analogous to tandem duplication of drug resistance genes on NR1 using the two direct repeats of IS1 as recombination sites. The amplified plasmid DNA returned to its original structure by the deletion of amplified neomycin resistance determinants when the host cells were cultured without selection for high resistance to neomycin.     

Transition of R factor NR1 in Proteus mirabilis: molecular structure and replication of NR1 deoxyribonucleic acid.

The structure of R factor NR1 DNA in Proteus mirabilis has been studied by using the techniques of CsCl density gradient centrifugation, sedimentation in neutral and alkaline sucrose gradients, and electron microscopy. It has been shown that the nontransitioned form of NR1 DNA isolated from P. mirabilis cultured in drug-free medium is a37-mum circular deoxyribonucleic acid (DNA) with a density of 1.712 g/ml in a neutral CsCl gradient. This circular molecule is a composite structure consisting of a 29-mum resistance transfer factor containing the tetracycline-resistance genes (RTF-TC) and an 8-mum r-determinants component conferring resistance to chloramphenicol (CM), streptomycin/spectinomycin, and the sulfonamides. There are one to two copies of NR1 per chromosome equivalent of DNA in exponential-phase cells cultured in Penassay broth. After growth of PM15/NR1 in medium containing 100 mug of CM per ml, the density of the NR1 DNA increased from 1.712 g/ml to approximately 1.718 g/ml and the proportion of NR1 DNA relative to the chromosome is amplified about 10-fold. The changes in R factor DNA structure which accompany this phenomenon (termed the transition) have been studied. DNA density profiles of the transitioned NR1 DNA consist of a 1.718 g/ml band which is skewed toward the less dense side. The transitioned NR1 DNA consists of molecules containing the RTF-TC element attached to multiple copies of r-determinants DNA (poly-r-determinant R factors) and multimeric and monomeric autonomous r-determinants structures. Poly-r-determinant R factors have a density intermediate between the basic composite structure (1.712 g/ml) and r-determinants DNA (1.718 g/ml). These species presumably account for the skewing of the 1.718-g/ml DNA band toward the less dense side. When transitioned cells are subsequently cultured in drug-free medium, poly-r-determinant R factors and autonomous poly-r-determinants undergo dissociation to form smaller structures containing fewer copies of r-determinants. This process continues until, after prolonged growth in drug-free medium the NR1 DNA returns to the nontransitioned state which consists of an RTF-TC and a single copy of r-determinants.     

Amplification of drug resistance genes flanked by inversely repeated IS1 elements: involvement of IS1-promoted DNA rearrangements before amplification.

Tn2653 contains one copy of the tet gene and two copies of the cat gene derived from plasmid pBR325 and is flanked by inverted repeats of IS1. Transposed onto the P1-15 prophage, it confers a chloramphenicol resistance phenotype to the Escherichia coli host. Because the prophage is perpetuated as a plasmid at about one copy per host chromosome, the host cell is still tetracycline sensitive even though P1-15 is carrying one copy of the tet gene. We isolated P1-15::Tn2653 mutants conferring a tetracycline resistance phenotype, in which the whole transposon and variable flanking P1-15 DNA segments were amplified. Amplification was most probably preceded by IS1-mediated DNA rearrangements which led to long direct repeats containing Tn2653 sequences and P1-15 DNA. Subsequent recombination events between these direct repeats led to amplification of a segment containing the tetracycline resistance gene in tandem arrays.     

Salmonella typhimurium LT2 mutation affecting the deletion of resistance determinants on R plasmids.

Plasmid Rms312, specifying resistance to tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), sulfonamide (Su), and mercury chloride (Mer), deletes both Tc and Cm Sm Su Mer determinants at a high frequency in Salmonella typhimurium LT2. S. typhimurium mutants that were stable carriers of Rms312 were isolated by alternate culture of R-bearing cells in a medium containing either tetracycline or chloramphenicol. In one of these mutants the deletion frequency of drug resistance determinants was decreased by about 100-fold not only Rms312, but also in R100, R1, and R6-5. This mutation caused a slight reduction of ultraviolet resistance but did not affect generalized genetic recombination, indicating that the mutation is different from recA. The mutation, designated dor (deletion of r-determinants), was mapped to a position near 57 units in the new linkage map of S. typhimurijm LT2 (K. E. Sanderson and P. E. Hartman, Microbiol. Rev. 42:471-519, 1978). The dor mutation had no effect on IS1-mediated illegitimate deletion, indicating that the dor mutation is different from the del mutation described by Nevers and Saedler (P. Nevers and H. Saedler, Mol. Gen. Genet. 160:209-214, 1978).     

Denaturation mapping of R factor deoxyribonucleic acid.

The R factor NR1 consists of two components: a resistance transfer factor which harbors the tetracycline resistance genes (RTF-TC) and the r-determinants component which harbors the other drug resistance genes. Using partial denaturation mapping it is possible to distinguish the RTF-TC region from the r-determinants region of the composite R factor NR1 DNA which has a contour length of 37 mum and a density of 1.712 g/ml. The r-determinants region was a relatively undenatured 8.5-mum segment of the molecule when the deoxyribonucleic acid was partially denatured at pH 10.7. An RTF-TC genetic segregant of NR1 which had lost the r-determinants component had a contour length of 28.7 mum and a density of 1.710 g/ml. Characterization of an RTF-TC using partial denaturation mapping at pH 10.7 confirmed that the relatively undenatured 8.5-mum r-determinants segment of the composite R factor had been deleted. Circular, transitioned NR1 DNA molecules (1.716 to 1.718 g/ml), whose contour lengths were consistent with an RTF-TC plus an integral number of tandem copies of r-determinants, were also characterized by denaturation mapping. The relatively undenatured region in these molecules had a length equal to an integral number of copies of r-determinants and was located at the same site in the partially denatured RTF-TC as the single copy of r-determinants in the 37-mum composite NR1. This indicates that there is a unique integration site for r-determinants in the RTF-TC component. The R factor UCR122, a TC deletion mutant of NR1, was also characterized by denaturation mapping. The translocation of the TC resistance gene(s) on the denaturation map permitted the alignment of the denaturation map with the heteroduplex map of Sharp et al. (u073). Linear and circular monomeric and presumed multimeric r-determinants DNA molecules (p = 1.718 g/ml) were partially denatured at a higher pH (11.10). The r-determinants multimers showed a repeating 8.3-mum (monomeric) partial denaturation pattern indicating a head-to-tail arrangement of monomers in these poly-r-determinant molecules.     

The recA gene of Chlamydia trachomatis: Cloning, sequence, and characterization in Escherichia coli

Abstract The recA gene of Chlamydia trachomatis was isolated by complementation of an Escherichia coli recA mutant. The cloned gene restored resistance to methyl methanesulfonate in E. coli recA mutants. The DNA sequence of the chlamydial gene was determined and the deduced protein sequence compared with other RecA proteins. In E. coli recA deletion mutants, the cloned gene conferred moderate recombinational activity as assayed by Hfr matings. The chlamydial recA gene was efficient in repairing alkylated DNA but less so in repairing of UV damage when compared with the E. coli homologue. As detected by an SOS gene fusion, a small but measurable amount of LexA co-cleavage was indicated.     

DNA Adenine Methylase Mutants of Salmonella Typhimurium and a Novel Dam-Regulated Locus

Mutants of Salmonella typhimurium lacking DNA adenine methylase were isolated; they include insertion and deletion alleles. The dam locus maps at 75 min between cysG and aroB, similar to the Escherichia coli dam gene. Dam(-) mutants of S. typhimurium resemble those of E. coli in the following phenotypes: (1) increased spontaneous mutations, (2) moderate SOS induction, (3) enhancement of duplication segregation, (4) inviability of dam recA and dam recB mutants, and (5) suppression of the inviability of the dam recA and dam recB combinations by mutations that eliminate mismatch repair. However, differences between S. typhimurium and E. coli dam mutants are also found: (1) S. typhimurium dam mutants do not show increased UV sensitivity, suggesting that methyl-directed mismatch repair does not participate in the repair of UV-induced DNA damage in Salmonella. (2) S. typhimurium dam recJ mutants are viable, suggesting that the Salmonella RecJ function does not participate in the repair of DNA strand breaks formed in the absence of Dam methylation. We also describe a genetic screen for detecting novel genes regulated by Dam methylation and a locus repressed by Dam methylation in the S. typhimurium virulence (or ``cryptic') plasmid.     

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.     

Gene copy number effects in the mer operon of plasmid NR1.

The level of resistance to Hg2+ determined by the inducible mer operon of plasmid NR1 was essentially the same for three gene copy number variants in Escherichia coli, less in Proteus mirabilis, and intermediate in P. mirabilis "transitioned" to a high r-determinant gene copy number. Cell-free volatilization rates of radioactive mercury indicated increasing levels of intracellular mercuric reductase enzyme from low- to high-gene copy number forms in P. mirabilis and from low- to high-copy number forms in E. coli, but the additional enzyme in E. coli was effectively cryptic.     

Transition of the R factor NR1 and Proteus mirabilis: level of drug resistance of nontransitioned and transitioned cells.

When Proteus mirabilis harboring the R factor NR1 is cultured in Penassay broth containing 100 mug of chloramphenicol (CM) per ml, there is an amplification in the number of copies of the r-determinants per cell. Under these conditions, R factors harboring multiple tandem sequences of r-determinants are formed. Autonomous poly-f-determinants consisting of multiple copies of r-determinants are also formed. This phenomenon has been referred to as the "transition". Transitioned cells have considerably higher levels of resistance to CM and streptomycin (SM), but not to tetracycline (TC), than do nontransitioned cells and grow more rapidly in medium containing either CM or SM. There is essentially no difference in growth rates between transitioned and nontransitioned cells in drug-free medium. The higher level of resistance of transitioned cells to SM has made it possible to investigate the mechanism of the transition. Using replica plating, it has been possible to isolate spontaneously occurring transitioned cells from a nontransitioned population which appear to outgrow the nontransitioned cells during growth in medium containing 100 mug of CM per ml. If transiitoned cells are subsequently cultured in drug-free medium, the cells return gradually to the nontransitioned state, which has been referred to as the "back-transition was monitored by examining the level of resisitance of the cells to SM. In both situations the cell populations were found to be heterogeneous, consisting of a mixture of nontransitioned and transitioned cells. Under the conditions of our experiments, the transition appeared to be due to the more rapid growth of a minor fraction of spontaneously occurring transitioned cells which outgrew the remainder of cells in the population. To obtain the transition, the drug resistance gene must reside on the r-determinants component of the R factor. The transition did not take place when the cells were cultured in medium containing high concentrations of TC. This indicates that the TC resistance genes reside on the resistance transfer factor component of the R factor, which is in agreement with physical studies on R factor deoxyribonucleic acid.     

Mapping of the resistance genes of the R plasmid NR1.

Summary The drug resistance genes on the r-determinants component of the composite R plasmid NR1 were mapped on the EcoRI restriction endonuclease fragments of the R plasmid by cloning the fragments using the plasmid RSF2124 as a vector. The sulfonamide (Su) and streptomycin/spectinomycin (Sm/Sp) resistance genes are located on EcoRI fragment G of NR1. The expression of resistance to mercuric ions (Mer) requires both EcoRI fragment H and I of NR1. The expression of chloramphenicol (Cm) and fusidic acid (Fus) resistance requires EcoRI fragments A and J of NR1. The kan fragment of the related R plasmid R6-5 can substitute for EcoRI fragment J of NR1 in the expression of Cm and Fus resistance. The structural genes for Cm and Fus resistance appear to be a part of an operon whose expression is controlled by the same promoter.     

Construction and characterization of a recA mutant of Thiobacillus ferrooxidans by marker exchange mutagenesis

To construct Thiobacillus ferrooxidans mutants by marker exchange mutagenesis, a genetic transfer system is required. The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from Escherichia coli to two private T. ferrooxidans strains (BRGM1 and Tf-49) and to two collection strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed. To knock out the T. ferrooxidans recA gene, a mobilizable suicide plasmid carrying the ATCC 33020 recA gene disrupted by a kanamycin resistance gene was transferred from E. coli to T. ferrooxidans ATCC 33020 by conjugation under the best conditions determined. The two kanamycin-resistant clones, which have retained the kanamycin-resistant phenotype after growth for several generations in nonselective medium, were shown to have the kanamycin resistance gene inserted within the recA gene, indicating that the recA::Omega-Km mutated allele was transferred from the suicide plasmid to the chromosome by homologous recombination. These mutants exhibited a slightly reduced growth rate and an increased sensitivity to UV and gamma irradiation compared to the wild-type strain. However, the T. ferrooxidans recA mutants are less sensitive to these physical DNA-damaging agents than the recA mutants described in other bacterial species, suggesting that RecA plays a minor role in DNA repair in T. ferrooxidans.     

Construction and characterization of Streptococcus suis-Escherichia coli shuttle cloning vectors

pSSU1, a native plasmid of Streptococcus suis DAT1, was used to construct pSET-series shuttle vectors. In addition to the replication function of pSSU1, these vectors contain the multiple cloning sites and lacZ' gene from pUC19, which means that X-gal screening can be used to select recombinants in Escherichia coli. pSET1, pSET2, and pSET3 carry cat, spc, and both of these genes, respectively, as selectable markers. These vectors could be introduced into S. suis, E. coli, Salmonella typhimurium, S. pneumoniae, and S. equi ssp. equi by electrotransformation. The recA gene was cloned from S. suis and sequenced, and this information was used in the construction of a recA mutant of S. suis. Transformation frequencies and/or plasmid stability of all pSET vectors tested were decreased in both S. suis and E. coli recA mutants compared with the parental strains. These results suggested that functional RecA protein improved the maintenance of pSET vectors in both S. suis and E. coli. Moreover, cloning of the functional S. suis recA gene into pSET2 and complementation analysis of the recA mutant were successful in S. suis but not in E. coli. These results showed that pSET vectors are useful tools for cloning and analyzing S. suis genes in S. suis strains directly.     

Generation of miniplasmids from copy number mutants of the R plasmid NR1.

Small, closed circular deoxyribonucleic acid molecules, called miniplasmids, were observed in Escherichia coli harboring copy number mutants of the R plasmid NR1 after growth in medium containing tetracycline. The level of tetracycline resistance conferred by the copy mutant plasmids was lower (3 to 6 microgram/ml) than that conferred by NR1 (100 MICROGRAM/ML). The presence of the miniplasmid enhanced the level of tetracycline resistance conferred by the copy mutant. Miniplasmids of molecular weights 4 X 10(6) to 13 X 10(6) were found. They carried no antibiotic resistance markers and could be eliminated by growth in the presence of chloramphenicol and/or streptomycin-spectinomycin. Studies with the restriction endonucleases EcoRI and Sal I indicated that the miniplasmids are derived from the region of the copy mutant plasmids that contains the origin for replication of the resistance transfer factor. There were approximately 12 copies of the miniplasmid per chromosome, compared with 3 and 6 copies of the copy mutants of NR1. The miniplasmids appeared to be incompatible with the copy mutant plasmids.