The expression in Staphylococcus aureus of cloned DNA encoding methicillin resistance

A 4 kb fragment of chromosomal DNA was cloned from a clinical strain of methicillin-resistant Staphylococcus aureus. It comprises part of a section of the chromosome that was lost when the strain was cured of resistance to methicillin and to other antimicrobial agents. The fragment mediates an increased level of methicillin resistance when inserted into a shuttle vector and transformed back into the sensitive strain generated when the original DNA was deleted.     

Cloning and sequence determination of six Staphylococcus aureus beta-lactamases and their expression in Escherichia coli and Staphylococcus aureus

The plasmid-encoded beta-lactamase genes of six strains of Staphylococcus aureus were cloned and shown to be expressed in Escherichia coli. The cloned genes were re-introduced into S. aureus via a shuttle vector, and expressed beta-lactamase. However, clones containing only the small amount of DNA found necessary for expression of ampicillin resistance in E. coli did not express beta-lactamase in S. aureus. Much larger pieces of DNA from the original plasmid were necessary to obtain expression in S. aureus. Some of the six strains of S. aureus synthesized beta-lactamase constitutively and some released only a small proportion of the enzyme into the medium. Both these characteristics were maintained in the clones so it is concluded that they are features either of the gene itself or of the surrounding DNA. The cloned genes were sequenced and the putative amino acid sequences of the beta-lactamases were compared. There are several differences between the sequences and in particular one change in the N-terminal region, at a position believed to be especially important for export of proteins from the cell, is thought to have a key effect on whether or not the enzyme is found in the medium.     

Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus.

A novel penicillin-binding protein, PBP-2' (Mr about 75,000), is known to be induced in excessively large amount by most beta-lactam compounds in cells of a clinically isolated strain of Staphylococcus aureus, TK784, that is highly resistant to beta-lactams and also most other antibiotics. This protein has very low affinities to most beta-lactam compounds and has been supposed to be the cause of the resistance of the cells to beta-lactams. A 14-kilobase DNA fragment was isolated from the cells that carried the gene encoding this penicillin-binding protein and also a genetically linked marker that is responsible for the resistance to tobramycin. This DNA was cloned on plasmid pACYC184 and was shown to cause both production of PBP-2' and resistance to tobramycin in Escherichia coli cells. However, the formation of PBP-2' in E. coli was only moderate and was independent of normal inducer beta-lactams. The PBP-2' formed in the E. coli cells showed slow kinetics of binding to beta-lactams similar to that of PBP-2' formed in the original S. aureus cells and gave a similar pattern of peptides to the latter when digested with the proteolytic V8 enzyme of S. aureus.     

Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones.

The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The 2.7-kb DNA fragment containing the norA gene had a long open reading frame coding for 388 amino acid residues with a molecular weight of 42,265, which was consistent with the experimental value of about 49,000 obtained on DNA-directed translation. The deduced NorA polypeptide has 12 hydrophobic membrane-spanning regions and is partly homologous to tetracycline resistance protein and sugar transport proteins. The uptake of a hydrophilic quinolone, enoxacin, by S. aureus harboring a plasmid carrying the norA gene was about 50% that by the parent strain lacking the plasmid, but it increased to almost the same level as that by the latter strain with carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, the uptake of a hydrophobic quinolone, sparfloxacin, was similar in the two strains. These results suggest that the NorA polypeptide may constitute a membrane-associated active efflux pump of hydrophilic quinolones.     

Additional DNA in methicillin-resistant Staphylococcus aureus and molecular cloning of mec-specific DNA.

Additional DNA was shown to be present in methicillin-resistant Staphylococcus aureus by one- and two-dimensional restriction endonuclease analyses of the chromosomal DNA. A 3.5-kilobase Bg/II fragment, which was present in methicillin-resistant strains but not in the isogenic methicillin-sensitive parental strain, was cloned into newly constructed plasmid pWDB1 in Escherichia coli. Hybridization of this 3.5-kilobase Bg/II fragment with different methicillin-sensitive and methicillin-resistant S. aureus clinical isolates indicated that the fragment represents part of the methicillin resistance determinant (mec). In addition, the fragment carries a sequence that is present in some large staphylococcal plasmids, as well as in penicillinase plasmid pI524.     

The Streptomyces peucetius drrC gene encodes a UvrA-like protein involved in daunorubicin resistance and production.

The drrC gene, cloned from the daunorubicin (DNR)- and doxorubicin-producing strain of Streptomyces peucetius ATCC 29050, encodes a 764-amino-acid protein with a strong sequence similarity to the Escherichia coli and Micrococcus luteus UvrA proteins involved in excision repair of DNA. Expression of drrC was correlated with the timing of DNR production in the growth medium tested and was not dependent on the presence of DNR. Since introduction of drrC into Streptomyces lividans imparted a DNR resistance phenotype, this gene is believed to be a DNR resistance gene. The drrC gene could be disrupted in the non-DNR-producing S. peucetius dnrJ mutant but not in the wild-type strain, and the resulting dnrJ drrC double mutant was significantly more sensitive to DNR in efficiency-of-plating experiments. Expression of drrC in an E. coli uvrA strain conferred significant DNR resistance to this highly DNR-sensitive mutant. However, the DrrC protein did not complement the uvrA mutation to protect the mutant from the lethal effects of UV or mitomycin even though it enhanced the UV resistance of a uvrA+ strain. We speculate that the DrrC protein mediates a novel type of DNR resistance, possibly different from the mechanism of DNR resistance governed by the S. peucetius drrAB genes, which are believed to encode a DNR antiporter.     

金黄色葡萄球菌甲氧西林耐药辅助基因femB的克隆和原核表达

金黄色葡萄球菌femB基因与甲氧西林高水平耐药密切相关,可能成为开发抗MRSA药物的新靶位.以金葡菌基因组DNA为模板,PCR扩增femB全长基因,所得片段与pGM-T载体连接并转化感受态大肠杆菌DH5α,阳性克隆以PCR、双酶切及测序鉴定.将鉴定正确的目的片段定向克隆到pGEX-4T-1表达载体中,转化至大肠杆菌BL21后经IPTG诱导表达GST/FemB融合蛋白;采用SDS-PAGE及Western blot对融合蛋白进行鉴定.结果显示,重组质粒在宿主菌中获得了高效表达,融合蛋白相对分子质量为75 kD,该融合蛋白可与抗GST-tag抗体特异结合;表明femB基因的原核表达系统构建成功,为进一步研究其生物学功能奠定了基础.     

Analysis of the beta-lactamase plasmid of borderline methicillin-susceptible Staphylococcus aureus: focus on bla complex genes and cadmium resistance determinants cadD and cadX

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.     

A staphylococcal plasmid that replicates and expresses ampicillin, gentamicin and amikacin resistance in Escherichia coli

Plasmid pPG1 from Staphylococcus aureus coding for ampicillin (Apr), gentamicin (Gmr) and amikacin (Akr) resistance was transformed into Escherichia coli. Transformation efficiency was about 2 x 10(3) transformants/micrograms of plasmid DNA. The plasmids present in the E. coli transformants were identical to pPG1 according to their restriction patterns. The copy number of pPG1 was estimated to be at least 20-times less in E. coli than in S. aureus. The minimal inhibitory concentrations (MICs) for Ap and Gm were lower in E. coli than in S. aureus. However, the MIC for Ak was higher in E. coli transformants than in S. aureus. pPG1 was maintained in the E. coli transformants for at least 80 generations at 37 degrees C without antibiotic selection pressure.     

Molecular cloning and analysis of Staphylococcus aureus chromosomal aminoglycoside resistance genes

Most of the aminoglycoside resistant Staphylococcus aureus strains isolated in France are resistant to all the antibiotics belonging to this family. Two aminoglycoside-modifying enzymes were detected in the wild-type strains studied: an APH3'III and an AAC6'-APH2". These strains also carry two types of streptomycin resistance: high-level resistance due to chromosomal mutation(s) affecting ribosome affinity and low-level resistance, the mechanism of which was not characterized. All the aminoglycoside resistance genes were located on the chromosome. DNA fragments of 1.5 and 1.95 kb carrying the aphA and aacA genes, respectively, were isolated, by cloning, from the cellular DNA of a clinical isolate. When these genes were introduced into Escherichia coli and Bacillus subtilis strains, the enzymes synthesized were indistinguishable from those produced by the S. aureus strains. When the cellular DNAs of wild-type and resistant strains were hybridized with the cloned fragments, sequences homologous to the fragment carrying the aphA gene were found to be located at the same chromosomal site, while those hybridizing with the fragment carrying the aacA gene were at different chromosomal sites.     

Physical and biochemical characterization of the qacA gene encoding antiseptic and disinfectant resistance in Staphylococcus aureus

We have previously cloned a 3.5 kb fragment from the Staphylococcus aureus multiresistance plasmid pSK1 which carries the qacA determinant responsible for linked resistance to acriflavine (Acr), ethidium bromide (Ebr), quaternary ammonium compounds (Qar), propamidine isethionate (Pir), and diamidinodiphenylamine dihydrochloride (Ddr). This report presents a biochemical and physical analysis of qacA and shows the widespread carriage of this gene on S. aureus resistance plasmids. Tn5 insertion mutagenesis defined the extent of qacA to within 2.40 kb of pSK1 DNA. Examination of the expression of insertion and deletion mutants of the cloned qacA sequences in both maxicells and minicells led to the association of a 50 kDa protein, designated QacA, with the AcrEbrQarPirDdr phenotype. Based on fluorimetric and isotopic assays used to determine the extent of accumulation of ethidium bromide by S. aureus strains harbouring pSK1, we propose that the basis of AcrEbrQarPirDdr in S. aureus is a qacA-mediated efflux system.     

Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection

Abstract Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE ) gene of Escherichia coli , a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB -like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium .     

Characterization and expression of a cloned tetracycline resistance determinant from the chromosome of Streptococcus mutans

A chromosomal tetracycline resistance (Tcr) determinant previously cloned from Streptococcus mutans into Streptococcus sanguis (Tobian and Macrina, J. Bacteriol. 152:215-222, 1982) was characterized by using restriction endonuclease mapping, deletion analysis, and Southern blot hybridization. Deletion analysis allowed localization of the Tcr determinant to a 2.8-kilobase region of the originally cloned 10.4-kilobase sequence. This cloned determinant hybridized to a representative of the tetM class of streptococcal Tcr determinants but not to representatives of the tetL and tetN classes and, like other tetM determinants, mediated high-level resistance to tetracycline and low-level resistance to minocycline. A portion (approximately 3 kilobases) of the isolated streptococcal fragment was subcloned into Escherichia coli, where it conferred resistance to tetracycline and minocycline. Two proteins with apparent molecular weights of 33,000 and 35,000, encoded by the S. mutans DNA, were synthesized in E. coli minicells. Insertion of DNA into a unique SstI site of the cloned S. mutans fragment resulted in inactivation of Tcr expression in E. coli and S. sanguis, as well as loss of production of both the 33,000- and 35,000-dalton proteins in E. coli minicells. Incubation of minicells in subinhibitory concentrations of tetracycline did not result in changes in the levels of synthesis of either protein. Our data suggest that at least one of these proteins is involved in the expression of Tcr.     

Spectinomycin resistance and associated DNA amplification in Streptomyces achromogenes subsp. rubradiris.

Streptomyces achromogenes subsp. rubradiris plated at low density on 1,000 micrograms of spectinomycin per ml initially produces slow-growing, bald colonies from which arise, in a spatially and temporally random fashion, foci of rapidly growing aerial mycelium-forming cells whose DNA contains an approximately 200- to 300-fold amplification of an 8-kilobase (kb) sequence. This sequence was cloned in Escherichia coli on pBR322 and physically characterized. It was separately cloned also in Streptomyces lividans as a BglII fragment and shown to impart high-level resistance to spectinomycin in an orientation-independent manner when present in either the high-copy-number vector pIJ702 or the unit-copy-number vector pIJ943. A spectinomycin resistance determinant was shown to reside on a 1.7-kb SphI-BglII subfragment. Analysis of Southern blots of restriction enzyme digests of wild-type S. achromogenes DNA probed with the labeled 8-kb DNA sequence resulted in the identification and subsequent cloning in S. lividans of a 10.4-kb BamHI fragment which probably includes the complete 8.8-kb amplifiable unit of DNA. This unit is present in wild-type S. achromogenes and in the initially slow-growing, bald colonies arising on 1,000 micrograms of spectinomycin per ml as a single copy. It carries two 0.8-kb direct repeats at its termini as well as the spectinomycin resistance determinant close to one of these termini. About 5% of protoplast regenerants from wild-type S. achromogenes and 77% of protoplast regenerants from the rapidly growing strains lost both the ability to grow on spectinomycin at 10 micrograms/ml and the sequences that hybridize with the 8-kb probe DNA. The 1.7-kb Bg/II-SphI resistance fragment, when introduced via the vector pIJ702 into an S. achromogenes strain sensitive to 10 microgram of spectinomycin per ml, permitted its vigorous growth on 1,000 micrograms of the antibiotic per ml.     

Cloning a DNA marker associated to wheat scab resistance

Wheat head blight caused mainly by Fusarium graminearum, is an important wheat disease, causing yield and quality losses. The breeding of resistant varieties is the key measure to control this disease, but the conventional breeding method is of low efficiency. The marker-assisted selection (MAS) can significantly improve the breeding efficiency. In this study, four RAPD (randomly amplified polymorphic DNA) markers linked to FHB resistance were obtained and one was cloned and sequenced. F7 recombinant inbred lines (RILs) were derived from the F1 of the cross Ning894037 (resistant)/Alondra (susceptible) by the single-seed descent method. Scab resistance of F7 RILs was evaluated in the greenhouse by injecting conidiospores into a central floret. Scab symptoms were evaluated on the 21st day after inoculation. Disease severity was assessed as the percentage of infected spikelets/spike. The F7 RIL population displayed a normal distribution, transgressive segregation and significant variation for FHB severity. DNA from resistant and susceptible parents was analyzed with 520 RAPD primers. Four markers (S1384-640,S1360-600, S1319-350,S1319-820) linked to FHB resistance were obtained. DNA of S1384-640 was recovered, subjected to re-amplification by using S1384 primer and the same protocol as for RAPD analysis and identified the rightness. The PCR product of S1384-640 was ligated into the pUCm-T vector, and cloned into fresh competent cells of Escherichia coli strain DH5alpha RAPD analysis showed that the inserts of the recombinant plasmids were DNA of S1384-640. The sequencing result showed that the cloned fragment was 648 bp.     

Plasmid-determined bleomycin resistance in Staphylococcus aureus

A 1580-bp fragment of Staphylococcus aureus plasmid pUB110 encoding resistance to the DNA synthesis inhibitor bleomycin has been cloned and sequenced. A DNA sequence containing an open reading frame of 405 bp was subcloned into several expression vectors and bleomycin resistance was expressed at high level in Escherichia coli under the control of lambda PL promoter. On induction, a ca. 14,000-Da protein was detected by gel electrophoresis. The bleomycin resistance determinant of the gram-positive plasmid pUB110 was compared to that of the enterobacterial transposon Tn5; limited regions of close relatedness could be identified.     

DNA cloning and organization of the Staphylococcus aureus gyrA and gyrB genes: close homology among gyrase proteins and implications for 4-quinolone action and resistance.

Staphylococcus aureus gyrA and gyrB genes, which encode the DNA gyrase A and B proteins, have been isolated and found to map contiguously. DNA sequence analysis revealed close homology between the S. aureus gyrase subunits and their counterparts in Bacillus subtilis and Escherichia coli, including several conserved amino acid residues whose substitution in E. coli confers resistance to 4-quinolones. These results are discussed in regard to quinolone resistance mechanisms in S. aureus.