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1.
Plasmids that confer resistance to cadmium (MIC > or = 125 microM), were found in 18 out of 30 independent Staphylococcus lugdunensis strains from clinical specimens. Variants that were cured of their plasmid were cadmium sensitive. Restriction endonuclease sites of a 3.2-kb cadmium-resistance plasmid of S. lugdunensis, designated pLUG10, were similar to those of pOX6, a S. aureus cadmium-resistance plasmid containing the cadB gene. Southern-blot hybridisation was performed with a probe intragenic to cadB. Hybridisation was found with the cadB probe in the cadmium-resistant S. lugdunensis isolates to the 2.9-, 3.2- and 3.7-kb plasmids. These findings suggest that cadmium-resistance in some S. lugdunensis strains is due to a gene sharing homology with the cadB gene of S. aureus.  相似文献   

2.
Exfoliative toxin type B is produced by bacteriophage group II strains of Staphylococcus aureus and is a causative agent of staphylococcal scalded-skin syndrome. In addition to exfoliative toxin B, most isolates also produce a bacteriocin and are immune to the action of the bacteriocin. These phenotypes, as well as resistance to cadmium, were lost after elimination of a 37.5-kilobase plasmid, pRW001, from S. aureus UT0007. Transduction and transformation showed that pRW001 carries the structural genes for four phenotypic characteristics of S. aureus UT0007: (i) exfoliative toxin B production, (ii) bacteriocin production, (iii) bacteriocin immunity, and (iv) resistance to Cd(NO3)2. The exfoliative toxin B structural gene (etb), which is located on a 1.7-kilobase HindIII fragment of pRW001, was cloned in the plasmid pDH5060 and transformed into phage group III S. aureus RN4220. Transformant clones produced extracellular exfoliative toxin B that was biologically active in the neonatal mouse assay. In the Escherichia coli genetic background, the exfoliative toxin B gene was expressed only after being cloned into the positive selection-expression vector pSCC31. The structural gene for cadmium resistance was also isolated on an HindIII fragment of pRW001 cloned in pDH5060. The loci for the exfoliative toxin B gene and the cadmium resistance gene(s) were identified on a restriction map of plasmid pRW001.  相似文献   

3.
The genetic basis of cadmium resistance conferred by three penicillinase plasmids, PI(524), PI(258), and PII(147), of Staphylococcus aureus was examined by mutation, recombination, and deletion analysis. Three separate loci were identified: cadA, responsible for high-level resistance; cadB, giving a low-level resistance, nonadditive to cadA; and mad, a locus marginally decreasing the cadmium resistance of plasmid-positive staphylococci. The loci cadA and mad were present on all three plasmids, but cadB was only found on PII(147). Spontaneous deletions of mad involved up to three-fourths of the plasmid genome, which allowed derivation of a partial deletion map of PII(147), a plasmid with a contour length of 10.9 mum, corresponding to a molecular weight of 20.4 x 10(6).  相似文献   

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Two open reading frames on a 3.7-kb BglII-XbaI fragment which encodes the Staphylococcus aureus cadA cadmium (and zinc) resistance determinant of plasmid pI258 were identified (G. Nucifora, L. Chu, T. K. Misra, and S. Silver, Proc. Natl. Acad. Sci. USA 86:3544-3548, 1989). The [35S]methionine-labelled protein products of the 727-amino-acid CadA ATPase and of the 122-amino-acid CadC polypeptide in Escherichia coli were identified by using the T7 RNA polymerase-promoter expression system. A truncated CadA polypeptide (402 amino acids) did not confer resistance in S. aureus but was expressed in E. coli under control of the T7 RNA polymerase-promoter. Removal of 678 nucleotides from the 5' end of the published sequence (which includes the cadA promoter) abolished resistance to cadmium, whereas a 146-nucleotide-shorter deletion was without effect. The cadC gene is needed in addition to cadA for full resistance to cadmium in S. aureus and Bacillus subtilis. cadC functions both in cis and in trans.  相似文献   

7.
Borderline methicillin-susceptible Staphylococcus aureus strains are a rather homogeneous group, characterized by MICs of penicillinase-resistant penicillins (PRPs) at or just below the susceptibility breakpoint. Other features unique to this group include the presence of a pBW15-like beta-lactamase plasmid, the association with phage complex 94/96, and the production of a PRP-hydrolyzing beta-lactamase activity in addition to the classical penicillinase activity. The four HindIII fragments of pBORa53, a pBW15-like plasmid from the well-studied borderline S. aureus strain a53, were cloned in Escherichia coli, sequenced and analyzed. The plasmid (17,334 bp in size) contains 14 open reading frames (ORFs) and a complete copy of transposon Tn552, which harbors the three genes of the bla complex (blaZ, blaR1, and blaI) necessary for penicillinase production. Among the other 11 ORFs identified, two were homologous to cadmium resistance determinants of Staphylococcus lugdunensis and to the cadD and cadX genes recently detected in S. aureus. Consistent with this, strain a53 was found to be cadmium resistant. From a collection of 30 S. aureus isolates with borderline PRP MIC levels, 27 matched strain a53 in the positive amplification reactions with all of the four primer pairs targeting the cadD-cadX region, the presence of the 17.3-kb plasmid, and the level of cadmium resistance. The well-established S. aureus laboratory strain ATCC 29213 was also found to express cadD-cadX-mediated cadmium resistance. pBORa53 could be re-isolated from transformants obtained by transferring it into a PRP-susceptible recipient. However, while the transformants demonstrated levels of cadmium and penicillin resistance similar to those of strain a53, they remained fully susceptible to PRPs.  相似文献   

8.
pLm74 is the smallest known plasmid in Listeria monocytogenes. It confers resistance to the toxic divalent cation cadmium. It contains a 3.1-kb EcoRI fragment which hybridizes with the cadAC genes of plasmid pI258 of Staphylococcus aureus. When introduced into cadmium-sensitive L. monocytogenes or Bacillus subtilis strains, this fragment conferred cadmium resistance. The DNA sequence of the 3.1-kb EcoRI fragment contains two open reading frames, cadA and cadC. The deduced amino acid sequences are similar to those of the cad operon of plasmid pI258 of S. aureus, known to prevent accumulation of Cd2+ in the bacteria by an ATPase efflux mechanism. The cadmium resistance determinant of L. monocytogenes does not confer zinc resistance, in contrast to the cadAC determinant of S. aureus, suggesting that the two resistance mechanisms are slightly different. Slot blot DNA-RNA hybridization analysis showed cadmium-inducible synthesis of L. monocytogenes cadAC RNA.  相似文献   

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The effect of Cd2+ on aerobic and anaerobic growth was studied in the Cd2+-resistant Staphylococcus aureus 17810R which harbours the cadA and cadB markers on a penicillinase plasmid pII17810. Also the effect of Cd2+ on growth of the plasmidless strain 17810S, sensitive to Cd2+ was investigated. The results indicate that under all growth conditions the Cd2+-resistant S. aureus 17810R is protected against Cd2+ toxicity up to 100 microM Cd2+ by the 2H+/Cd2+ antiporter, the product of the cadA gene. Energetics of growth of both strains under various conditions is also discussed.  相似文献   

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A 25.9-kb plasmid, pXU5, encoding high level cadmium resistance was isolated from Staphylococcus aureus strain ATCC25923. A labelled cadA probe from plasmid pI258 hybridised to a 2.3-kb EcoRI fragment of pXU5. pXU5 was incompatible with an S. aureus incompatibility group 1 plasmid.  相似文献   

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Lysine decarboxylase of Escherichia coli has been the subject of enzymological studies, and the gene encoding lysine decarboxylase (cadA) and a regulatory gene (cadR) have been mapped. This enzyme is induced at low pH in the presence of lysine and achieves maximal level under anaerobic conditions. The induction of lysine decarboxylase increases the pH of the extracellular medium and provides a distinctive marker in tests of clinical strains. We report the sequence of the cad operon encoding lysine decarboxylase, a protein of 715 amino acids, and another protein, CadB, of 444 amino acids. The amino acid sequence of lysine decarboxylase showed high homology to that of the lysine decarboxylase of Hafnia alvei with less homology to the sequence of speC, which encodes the biosynthetic ornithine decarboxylase of E. coli. The cadA and cadB genes were separately cloned and placed under the control of lac and tac promoters, respectively, to facilitate independent study of their physiological effects. The cadB gene product had a mobility characteristic of a smaller protein on protein gels, analogous to that found for some other membrane proteins. The CadB sequence showed homology to that of ArcD of Pseudomonas aeruginosa, encoding an arginine/ornithine antiporter. Excretion studies of various strains, the coinduction of cadB and cadA, and the attractive physiological role for an antiport system led to a model for the coupled action of cadA and cadB in uptake of lysine, the reduction of H+ concentration, and excretion of cadaverine.  相似文献   

15.
Cadmium and zinc are removed from cells of Ralstonia metallidurans by the CzcCBA efflux pump and by two soft-metal-transporting P-type ATPases, CadA and ZntA. The czcCBA genes are located on plasmid pMOL30, and the cadA and zntA genes are on the bacterial chromosome. Expression of zntA from R. metallidurans in Escherichia coli predominantly mediated resistance to zinc, and expression of cadA predominantly mediated resistance to cadmium. Both transporters decreased the cellular content of zinc or cadmium in this host. In the plasmid-free R. metallidurans strain AE104, single gene deletions of cadA or zntA had only a moderate effect on cadmium and zinc resistance, but zinc resistance decreased 6-fold and cadmium resistance decreased 350-fold in double deletion strains. Neither single nor double gene deletions affected zinc resistance in the presence of czcCBA. In contrast, cadmium resistance of the cadA zntA double mutant could be elevated only partially by the presence of CzcCBA. lacZ reporter gene fusions indicated that expression of cadA was induced by cadmium but not by zinc in R. metallidurans strain AE104. In the absence of the zntA gene, expression of cadA occurred at lower cadmium concentrations and zinc now served as an inducer. In contrast, expression of zntA was induced by both zinc and cadmium, and the induction pattern did not change in the presence or absence of CadA. However, expression of both genes, zntA and cadA, was diminished in the presence of CzcCBA. This indicated that CzcCBA efficiently decreased cytoplasmic cadmium and zinc concentrations. It is discussed whether these data favor a model in which the cations are removed either from the cytoplasm or the periplasm by CzcCBA.  相似文献   

16.
A 5.6-kb fragment of alkaliphilic Bacillus firmus OF4 DNA was isolated by screening a library of total genomic DNA constructed in pGEM3Zf(+) for clones that reversed the Na+ sensitivity of Escherichia coli NM81, in which the gene encoding an Na+/H+ antiporter (NhaA) is deleted (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989). The plasmid, designated pJB22, contained two genes that apparently encode transposition functions and two genes that are apparent homologs of the cadA and cadC genes of cadmium resistance-conferring plasmid pI258 of Staphylococcus aureus. E. coli NM81 transformed with pJB22 had enhanced membrane Na+/H+ antiporter activity that was cold labile and that decreased very rapidly following isolation of everted vesicles. Subclones of pJB22 containing cadC as the only intact gene showed identical complementation patterns in vivo and in vitro. The cadC gene product of S. aureus has been proposed to act as an accessory protein for the Cd2+ efflux ATPase (CadA) (K. P. Yoon and S. Silver, J. Bacteriol. 173:7636-7642, 1991); perhaps the alkaliphile CadC also binds Na+ and enhances antiporter activity by delivering a substrate to an integral membrane antiporter. A 6.0-kb fragment overlapping the pJB22 insert was isolated to complete the sequence of the cadA homolog. A partial sequence of a region approximately 2 kb downstream of the cadA locus shares sequence similarity with plasmids from several gram-positive bacteria. These results suggest that the region of alkaliphile DNA containing the cadCA locus is present on a transposon that could reside on a heretofore-undetected endogenous plasmid.  相似文献   

17.
The presence of plasmid gene cadB did not affect Cd2+ accumulation, whereas plasmid gene cadA reduced Cd2+ accumulation by whole cells but not by membrane vesicles. Membrane vesicle studies indicated that Cd2+ uptake occurred via the Mn2+ transport system which was energized by the membrane electrical potential. Mn2+ and Cd2+ were competitive inhibitors of each other's transport, with Km's of 0.95 microM Mn2+ and 0.2 microM Cd2+. The kinetic parameters were nearly identical with vesicles prepared from sensitive and resistant cells, indicating that the cadA-encoded Cd2+ efflux system was inoperative in membrane vesicle preparations. Experiments with energy-inhibited cells indicated that the cadB gene product may bind Cd2+.  相似文献   

18.
Resistance to cadmium conferred by the staphylococcal plasmid pI258 occurs by means of energy-dependent efflux, resulting in decreased intracellular accumulation of cadmium. Recent sequence information suggested that efflux is mediated by a P-type ATPase. The cadA gene was previously expressed in Bacillus subtilis, conferring resistance to cadmium. Everted membrane vesicles were prepared from B. subtilis cells harboring either a plasmid containing the cadA system or the vector plasmid alone. 109Cd2+ transport into the everted membranes was measured in the presence of various energy sources. Cadmium transport was detected only in the presence of ATP as an energy source. The production of an electrochemical proton gradient (delta mu H+) by using NADH or phenazine methosulfate plus ascorbate was not able to drive transport. Reagents which dissipate delta pH abolished calcium transport due to the Ca2+/H+ antiporter but only partially inhibited cadmium transport. Inhibition of transport by the antibiotic bafilomycin A1 occurred at concentrations comparable to those which inhibit P-type ATPases. A band corresponding to the cadA gene product was identified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antibodies to the protein were prepared.  相似文献   

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Sequence of the exfoliative toxin B gene of Staphylococcus aureus.   总被引:3,自引:1,他引:2       下载免费PDF全文
We sequenced the Staphylococcus aureus exfoliative toxin B gene contained on a 1.7-kilobase HindIII fragment of plasmid pRW001. The gene was located by comparison of the amino acid sequences of open reading frames with the amino-terminal sequence of exfoliative toxin B and the total amino acid composition of the protein (A.D. Johnson, L. Spero, J.S. Cades, and B.T. De Cicco, Infect. Immun. 24:679-684, 1979). The primary translation product consists of 274 amino acids and contains a 31-amino-acid N-terminal peptide presumably necessary for transport.  相似文献   

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