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1.
We cloned genes, designated smdAB, that encode a multidrug efflux pump from the chromosomal DNA of clinically isolated Serratia marcescens NUSM8906. For cells of the drug-hypersensitive strain Escherichia coli KAM32 harboring a recombinant plasmid carrying smdAB, structurally unrelated antimicrobial agents such as norfloxacin, tetracycline, 4′,6-diamidino-2-phenylindole (DAPI), and Hoechst 33342 showed elevated MICs. The deduced amino acid sequences of both SmdA and SmdB exhibited similarities to the sequences of ATP-binding cassette (ABC)-type multidrug efflux pumps. The efflux of DAPI and Hoechst 33342 from E. coli cells expressing SmdAB was observed, and the efflux activities were inhibited by sodium o-vanadate, which is a well-known ATPase inhibitor. The introduction of smdA or smdB alone into E. coli KAM32 did not elevate the MIC of DAPI; thus, both SmdA and SmdB were required for function. These results indicate that SmdAB is probably a heterodimeric multidrug efflux pump of the ABC family in S. marcescens.  相似文献   

2.
In gram-negative bacteria, transporters belonging to the RND family are the transporters most relevant for resistance to antimicrobial compounds. In Pseudomonas aeruginosa, a clinically important pathogen, the RND-type pump MexAB-OprM has been recognized as one of the major multidrug efflux systems. Here, homologues of MexAB-OprM in the plant pathogens Pseudomonas syringae pv. phaseolicola 1448A, P. syringae pv. syringae B728a, and P. syringae pv. tomato DC3000 were identified, and mexAB-oprM-deficient mutants were generated. Determination of MICs revealed that mutation of MexAB-OprM dramatically reduced the tolerance to a broad range of antimicrobials. Moreover, the ability of the mexAB-oprM-deficient mutants to multiply in planta was reduced. RNA dot blot hybridization revealed growth-dependent regulation of the mexAB-oprM operon in P. syringae; the expression of this operon was maximal in early exponential phase and decreased gradually during further growth.  相似文献   

3.
Transport of K+ and H+ in the anaeronically and aerobically grown bacterium Serratia marcescens has been studied. The volumes of one cell of the anaerobically and aerobically grown bacterium were 3.7 X 10(-13) cm3 and 2.4 X 10(-13) cm3, respectively. Irrespective of the growth conditions the bacteria manifested the same respiration rate. However, the values of membrane potential for the anaerobically and aerobically grown bacterium were different and equal to -130 mV and -175 mV (interior negative), respectively, in the absence of an exogenic energy source. KCN + DCCD decreases delta psi down to almost zero in both species. DCCD alone decreases delta psi partially in anaerobes and increases delta psi in aerobes, whereas KCN alone reduces delta psi partially in both species. The introduction of glucose into the medium containing K+ reduces the absolute value of delta psi to [-160] mV in aerobes and to [-20] mV in anaerobes. The effect is not observed without external K+. In the presence of arsenate a delta psi is not reduced after the addition of glucose. At pH 7.5-7.8 the ATP level in aerobes grows notably faster than in anaerobes. The H+ extrusion becomes intensified when K+ uptake is activated by the increase in external osmotic pressure. Apparent Km and Vmax for K+ accumulation are 1.2 mM and 0.4 mM.min-1.g-1. The decrease of delta psi by glucose or KCN + DCCD have no effect on the K+ uptake whereas CCCP inhibits potassium accumulation. At the same time, arsenate stabilizes the delta psi value, but blocks K+ uptake. The accumulation of K+ correlates with the potassium equilibrium potential of -200 mV calculated according to the Nernst equation, whereas the delta psi measured was not more than [-25] mV. The calculated H+/ATP stoichiometry was 3.3 for aerobes. It was assumed that a constitutive K+ pump having a K+/ATP ratio equal to 2 or 3 operates in S. marcescens membranes.  相似文献   

4.
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.  相似文献   

5.
The Escherichia coli AcrB multidrug efflux pump is a membrane protein that recognizes many structurally dissimilar toxic compounds. We previously reported the X-ray structures of four AcrB-ligand complexes in which the ligands were bound to the wall of the extremely large central cavity in the transmembrane domain of the pump. Genetic studies, however, suggested that discrimination between the substrates occurs mainly in the periplasmic domain rather than the transmembrane domain of the pump. We here describe the crystal structures of the AcrB mutant in which Asn109 was replaced by Ala, with five structurally diverse ligands, ethidium, rhodamine 6G, ciprofloxacin, nafcillin, and Phe-Arg-beta-naphthylamide. The ligands bind not only to the wall of central cavity but also to a new periplasmic site within the deep external depression formed by the C-terminal periplasmic loop. This depression also includes residues identified earlier as being important in the specificity. We show here that conversion into alanine of the Phe664, Phe666, or Glu673 residue in the periplasmic binding site produced significant decreases in the MIC of most agents in the N109A background. Furthermore, decreased MICs were also observed when these residues were mutated in the wild-type AcrB background, although the effects were more modest. The MIC data were also confirmed by assays of ethidium influx rates in intact cells, and our results suggest that the periplasmic binding site plays a role in the physiological process of drug efflux.  相似文献   

6.
7.
Characterization of Serratia marcescens surviving in disinfecting footbaths   总被引:1,自引:0,他引:1  
AIM: To determine if disinfecting footbaths in the food industry were contaminated with bacteria and to characterize some of the bacteria present. METHODS AND RESULTS: Bacterial strains were isolated from disinfecting footbaths containing TEGO 103G (amphoteric disinfectant) or TP-99 (alkyl amino acetate-based disinfectant) in five of six dairy factories. Fourteen strains identified as Cedecea spp. by their fatty acid composition were further characterized. Results from Rapid ID 32 E API analysis and 16S-rDNA-sequencing showed that all strains were Serratia marcescens. Unlike S. marcescens ATCC 13880, the isolates from disinfecting footbaths were not killed (<5 log10 reduction) by the recommended in-use concentration of TEGO 103G, TEGO 51 or benzalkonium chloride. Survival and multiplication in tap water with an in-use concentration of TEGO 103G was demonstrated for one of the strains. All strains were killed by the in-use concentrations of commercial disinfectants based on peracetic acid, hypochlorite, quaternary ammonium compounds and alkyl amino acetate (TP-99). There were no indications of cross-resistance between disinfectants and antibiotics. CONCLUSION: Serratia marcescens may survive and multiply in disinfecting footbaths containing TEGO 103G or alkyl amino acetate because of disinfectant resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Disinfecting footbaths may act as contamination sources in food factories and should not be used without regular hygienic monitoring.  相似文献   

8.
The plasmid-borne multidrug efflux gene qacB is widely distributed in methicillin-resistant Staphylococcus aureus (MRSA). We analyzed the complete nucleotide sequence of the plasmid pTZ2162 (35.4 kb) encoding qacB. The plasmid pTZ2162 contains 47 ORFs and four copies of IS257 (designated IS257A to D). The 24.7-kb region of pTZ2162, which excluding the region flanked by IS257A and IS257D, is 99.9% identical to pN315 carried by MRSA N315. However, the repA-like region of pTZ2162 was divided into two ORFs, ORF46 and ORF47. Functional analysis with the pUC19-based vector pTZN03 showed that both ORF46 and ORF47 were essential for the replication of pTZ2162 and ORF1 is required for the stable maintenance of pTZ2162 in S. aureus. When pTZ2162 was searched for evidence of mobile elements, an 8-bp duplicated sequence (GATAAAGA) was existed at the left boundary of IS257A and the right boundary of IS257D. Therefore, the 10.7-kb region between IS257A and IS257D in pTZ2162 has the potential to act as a transposon. In addition to qacB, the pTZ2162 transposon-like element contains a novel fosfomycin resistance determinant fosD and an aminoglycoside resistance determinant aacA-aphD. This transposon-like element appears to have translocated into the beta-lactamase gene blaZ. Our data suggest that qacB is transferred between MRSA as a multiple antibiotic resistance transposon.  相似文献   

9.
10.
The gene coding for the mannose-sensitive hemagglutinating fimbriae in Serratia marcescens US5 was cloned into Escherichia coli K4 with a cosmid vector system. One of the transformants, US5-1, expressed two morphologically distinct fimbriae, one that was 5-nm wide and one that was 3-nm wide. The latter fimbria was morphologically and serologically indistinguishable from that of strain US5. Genetic analysis of transformant US5-1 showed that the gene responsible for the 5-nm-wide fimbriae was located more than 10 kilobases away from the gene responsible for the 3-nm-wide fimbriae. The molecular sizes of the subunits of these two fimbriae, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 19 kilodaltons for the 3-nm-wide fimbriae and 20 kilodaltons for the 5-nm-wide fimbriae. Serologically, the 5-nm-wide fimbriae did not cross-react with monoclonal or polyclonal antibodies raised against the mannose-sensitive hemagglutinating fimbriae of strain US5. Strain EL101, which expressed only the 5-nm-wide fimbriae, did not agglutinate chicken or human erythrocytes. These experimental results suggest that the gene for the 5-nm-wide fimbriae is cryptic in strain US5 and is expressed in E. coli K4 only after it is moved by transformation.  相似文献   

11.
The 2,3-butanediol (2,3-BD) dehydrogenase gene budC of Serratia marcescens G12 was disrupted to construct the acetoin (AC) producing strain G12M. In shake-flask cultures, AC production was enhanced by increased concentrations of glucose or sodium acetate in G12M. In fed-batch fermentation, G12M produced 47.5 g/L AC along with 9.8 g/L 2,3-BD. The expression of the key enzymes for AC synthesis was further investigated. Alpha-acetolactate synthase gene budB decreased its expression significantly in G12M compared with G12. This probably explained the moderate AC production in G12M cultures. Additionally, overexpression of budB gene and α-acetolactate decarboxylase gene budA was conducted in G12M and no significant increase of AC was observed. The results suggested that intracellular AC accumulation might inhibit the expression of budB and budA gene and induce budC gene expression in G12M. Our analyses offered the bases for further genetic manipulations in improving AC production in microbial fermentations.  相似文献   

12.
A tolC-like gene (hasF) was identified upon scanning the incomplete database of the S. marcescens genome. This gene was amplified using PCR and cloned in the pUC18 vector to yield pUCHF. Sequencing of the S. marcescens tolC-like hasF gene and subsequent amino acid sequence prediction revealed approximately 80% amino acid homology with the Escherichia coli TolC. A tolC-deficient strain of E. coli (BL923) containing pUCHF/hasF was analyzed for susceptibility to fluoroquinolones (ciprofloxacin, norfloxacin, and ofloxacin), chloramphenicol, sodium dodecyl sulfate (SDS), and ethidium bromide. Antibiotic susceptibility assays of the E. coli tolC-deficient mutant BL923 demonstrated a 64-fold increase in resistance to SDS and ethidium bromide upon introduction of the S. marcescens tolC-like hasF gene. No change was observed for susceptibility to fluoroquinolones and chloramphenicol. Ethidium bromide accumulation assays performed using E. coli BL923:pUCHF established the role of the S. marcescens hasF gene product in proton gradient-dependent efflux.  相似文献   

13.
Summary Utilizing the DNA sequence of the metalloprotease fromSerratia strain E-15, we isolated and sequenced the homologous gene fromSerratia strain SM6. These two genes are similar at both the DNA and protein sequence level. Expression of the protease gene inEscherichia coli was achieved by use of thelac promoter. This resulted in the production and excretion of an immunologically detectable but inactive protein of slightly higher molecular weight than that fromSerratia. We introduced the cloned gene into previously described protease mutants. The observed pattern of protease expression suggested that these mutations fall into three classes.  相似文献   

14.
Imipenem is often used to treat intensive care unit patients infected by Enterobacter aerogenes, but it is leading to an increasing number of antibiotic resistant strains. Clinical isolates and imipenem resistant variants presented a high level of resistance to beta-lactam antibiotic group and to chemically unrelated drugs. We report here that imipenem selects strains which contain active efflux pumps ejecting various unrelated antibiotics including quinolones, tetracycline, and chloramphenicol. An increase of AcrA, an efflux pump component, was observed in the imipenem resistant variants. The overexpression of marA, involved in the genetic control of membrane permeability via porin and efflux pump expression, indicated the activation of the resistance genetic cascade in imipenem resistant variants.  相似文献   

15.
Chloramphenicol has been reported to act as an inducer of the multidrug resistance in Escherichia coli. A resistant variant able to grow on plates containing 64 microg/ml chloramphenicol was obtained from the Enterobacter aerogenes ATCC 13048-type strain. Chloramphenicol resistance was due to an active efflux of this antibiotic and it was associated with resistance to fluoroquinolones and tetracycline, but not to aminoglycoside or beta-lactam antibiotics. MDR in the chloramphenicol-resistant variant is linked to the overexpression of the major AcrAB-TolC efflux system. This overexpression seems unrelated to the global Mar and the local AcrR regulatory pathways.  相似文献   

16.
Summary To overproduce Serratia marcescens metalloprotease(SMP), various recombinant plasmids encoding SMP gene were constructed and the SMP productivities from the recombinant S. marcescens strains were examined. The recombinant S. marcescens strains indispensably required proteinaceous substrates such as casein for the extracellular production of SMP. We obtained maximum 9,100U/ml of SMP from the culture supernatant of S. marcescens ATCC27117 containing a regulatory plasmid pTSP2 encoding SMP gene fused with a strong trc99a promoter and its repressor gene lacIq, which is about 23 times higher than that of wild type strain. SMP produced from the recombinant S. marcescens(pTSP2) was 88.3% of total extracellular proteins.  相似文献   

17.
粘质沙雷氏菌脂肪酶基因的克隆表达和酶学性质的研究   总被引:1,自引:0,他引:1  
目的:克隆粘质沙雷氏菌脂肪酶基因(lipA)使其在大肠杆菌B121(DE3)中实现高效表达,并对重组酶进行酶学性质研究.方法:以产脂肪酶粘质沙雷氏菌总DNA为模板,PCR扩增脂肪酶基因lipA,构建重组表达载体pET-lipA,并将其导入大肠杆菌进行诱导表达,对表达产物进行SDS-PAGE和酶学性质的测定.结果:经过优化培养条件,脂肪酶活力最高能达到104U/mL.重组脂肪酶的最适反应温度为40~45℃,最适pH为7.0~7.5,在50℃保温1h下仍能保持80%的酶活力,Ca2+、Sr2+、Mn2+和Mg2+对脂肪酶酶活有较强的激活作用,尤其是Ca2+使脂肪酶酶活提高了1倍多,而Ni2+、Fe2+、Fe3+、Cu2+、Zn2+和Al3+对酶活具有较强的抑制作用,尤其是Zn2+和Al3+使酶活力几乎完全丧失.该酶对一些有机溶剂有较好的耐受性,与50%甲醇混合24h,仍能保持84%的酶活力.结论:该脂肪酶具有较好的热稳定性和甲醇耐受力,作为生产生物柴油的催化剂具有很大的应用价值,为基因工程酶法生产生物柴油打下良好的基础.  相似文献   

18.
Abstract The nucleotide sequence of the chiA gene from Serratia marcescens strain BJL200 was determined. The gene was found to encode a protein of 563 amino acid residues, with a typical N-terminal signal peptide of 23 residues, that is cleaved off during export. The gene exhibited striking differences with two previously characterized chiA genes of S. marcescens in the region corresponding to amino acid residues 410–467 of the gene product. Periplasmic fractions of an Escherichia coli strain harbouring the cloned gene were used as starting material for the development of a fast, one-step purification protocol for the chitinase that is based on hydrophobic interaction chromatography.  相似文献   

19.
20.
Multidrug resistance (MDR) in bacteria has been associated with efflux pumps that export structurally unrelated compounds and decrease cytoplasmic drug accumulation. To investigate MDR in mycobacteria, we studied the Mycobacterium smegmatis mutant mc(2)11, which is resistant to doxorubicin, tetracycline, rhodamine, ethidium bromide and the hydrophilic fluoroquinolones. A genomic library constructed from this mutant was used to select clones conferring resistance to doxorubicin. Surprisingly, the clone selected encodes the efflux pump LfrA, which has been reported to confer resistance to hydrophilic fluoroquinolones, ethidium bromide, rhodamine, and acriflavine. To define the contribution of LfrA to the innate mycobacterial drug resistance and to the MDR phenotype in mc(2)11, the lfrA gene was disrupted in both the mc(2)11 mutant and the mc(2)155 wild-type parent. LfrA disruption of the wild-type strain decreased resistance to ethidium bromide and acriflavine, and increased accumulation of ethidium bromide. However, disruption of lfrA gene results only in a 2-fold decrease in minimal inhibitory concentrations (MICs) for ciprofloxacin, doxorubicin, rhodamine, and accumulation of [(14)C]ciprofloxacin was unchanged. LfrA disruption of the MDR strain mc(2)11 produced a similar phenotype. Thus, LfrA contributes significantly to the intrinsic MICs of M. smegmatis for ethidium bromide and acriflavine, but not for ciprofloxacin, doxorubicin or rhodamine.  相似文献   

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