Characterization of staphylococcal plasmids hybridizing with the fosfomycin resistance gene fosB

The distribution of the fosB gene, coding for fosfomycin resistance, in 105 fosfomycin-resistant isolates of Staphylococcus from various geographical areas, was studied by Southern blot hybridization. Nucleotide sequences related to fosB were detected in 36 strains belonging to five species. Plasmids bearing fosB were often of a size similar to that of pIP1842 (2.54 kb) in S. epidermidis, most often small (2.4 to 4.1 kb) in other species including S. aureus where a 2.7-kb plasmid was found in 16 out of the 18 strains studied. The fosB gene was geographically dispersed since it was present in six different locations in France and also in Japan. The weak hybridization observed with plasmid DNA of certain strains of S. aureus, S. epidermidis, S. haemolyticus, S. saprophyticus, and S. warneri may indicate gene heterogeneity for fosfomycin resistance in Staphylococcus spp.     

The Fosfomycin Resistance Gene fosB3 Is Located on a Transferable,Extrachromosomal Circular Intermediate in Clinical Enterococcus faecium Isolates

Some VanM-type vancomycin-resistant Enterococcus faecium isolates from China are also resistant to fosfomycin. To investigate the mechanism of fosfomycin resistance in these clinical isolates, antimicrobial susceptibility testing, filter-mating, Illumina/Solexa sequencing, inverse PCR and fosfomycin resistance gene cloning were performed. Three E. faecium clinical isolates were highly resistant to fosfomycin and vancomycin with minimal inhibitory concentrations (MICs) >1024 µg/ml and >256 µg/ml, respectively. The fosfomycin and vancomycin resistance of these strains could be co-transferred by conjugation. They carried a fosfomycin resistance gene fosB encoding a protein differing by one or two amino acids from FosB, which is encoded on staphylococcal plasmids. Accordingly, the gene was designated fosB3. The fosB3 gene was cloned into pMD19-T, and transformed into E. coli DH5α. The fosfomycin MIC for transformants with fosB3 was 750-fold higher than transformants without fosB3. The fosB3 gene could be transferred by an extrachromosomal circular intermediate. The results indicate that the fosB3 gene is transferable, can mediate high level fosfomycin resistance in both Gram-positive and Gram-negative bacteria, and can be located on a circular intermediate.     

FosB, a cysteine-dependent fosfomycin resistance protein under the control of sigma(W), an extracytoplasmic-function sigma factor in Bacillus subtilis

We demonstrate that the Bacillus subtilis fosB(yndN) gene encodes a fosfomycin resistance protein. Expression of fosB requires sigma(W), and both fosB and sigW mutants are fosfomycin sensitive. FosB is a metallothiol transferase related to the FosA class of Mn(2+)-dependent glutathione transferases but with a preference for Mg(2+) and L-cysteine as cofactors.     

The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation

For the study of DNA-membrane interaction and the regulation of replication initiation we have determined the total nucleotide sequence of pUB110. As previously reported, this plasmid replicates in B. subtilis at a copy number of 30-50 per cell, with a majority of plasmids (60-80%) bound to the membrane (type-I binding). The type-I membrane binding is apparently necessary for pUB110 initiation of replication in vivo, but the membrane binding site is not known. Furthermore, four areas of the plasmid specifically bind to Bacillus subtilis membrane in an in vitro binding reaction (type II binding). These two types of membrane binding of pUB110 are different in that the in vivo binding (type-I) requires one (dnaBI) of the host initiation genes and is high-salt resistant, whereas the in vitro binding (type-II) does not require the dnaBI gene product and is high-salt sensitive. 7-mer double-strand sequence, TCAGCAA/AGTCGTT, or one-base derivatives of this sequence are frequently (17 of 23 of the 7-mer sequences) found in or close to the type-II binding areas. One of them is found at a restriction enzyme recognition site of a binding area that destroys the type-II membrane binding. These sequences may or may not have significance in type-II membrane binding. In addition to the neomycin resistance gene, the sequence data indicate two sizable open reading frames, ORF alpha and ORF beta, and two small ORF, gamma, and delta. All of these reading frames are in the same direction, which coincides with the direction of the replication. The open reading frame alpha (ORF alpha) corresponding to 334 amino acids close to the replication origin may be essential for the initiation of replication of PUB110. The putative protein alpha corresponding to this open reading frame contains a consensus sequence of the DNA binding sites which are found in a number of known DNA-binding proteins. The consensus DNA binding site of protein alpha is flanked by two hydrophobic areas. These two observations suggest that the corresponding protein may have both an affinity to a specific site in pUB110, and an affinity to the membrane.     

蓝藻Synechococcus sp.PCC7942穿梭表达质粒的构建及胸腺素α1基因的表达

利用本实验室构建的含蓝藻Plectonema boryanum内源小质粒的穿梭质粒pPRS-1,改建成含热诱导启动子、泛素融合的胸腺素α1（UB-Tα1）目的基因、卡那霉素抗性选择标记、rbcS终止子的新的穿梭表达重组质粒pPREUT。将这种重组质粒转化蓝藻Synechococcus sp.PCC7942,通过抗性筛选获得了具卡那霉素抗性的转化藻株。经Southern-blot杂交证实,穿梭表达质粒已转入蓝藻Synechococcus sp.PCC7942细胞中,在42℃热诱导30min后,目的基因UB－Tα1得到较高水平表达,表达量约占总蛋白量的7．5％。     

Tn292l, a transposon encoding fosfomycin resistance.

The determinant of resistance to fosfomycin of the Serratia marcescens plasmid pOU900 was transposed into the plasmid ColE1 and into the plasmid RP4 in the absence of the RecA function of the host. In each case, the acquisition of fosfomycin resistance was correlated with the presence of a discrete piece of DNA, uniform in size and in restriction pattern, This new, 7.5-megadalton transposable element encoding resistance to fosfomycin has been called Tn2921. A preliminary map of the transposon is presented.     

Amplification of the bacA gene confers bacitracin resistance to Escherichia coli.

An Escherichia coli genomic library was constructed in order to facilitate selection for genes which confer bacitracin resistance through amplification. One of the plasmids from the library, plasmid pXV62, provided a high level of bacitracin resistance for E. coli. Deletion and nucleotide sequence analyses of bacitracin resistance plasmid pXV62 revealed that a single open reading frame, designated the bacA gene, was sufficient for antibiotic resistance. The bacA gene mapped to approximately 67 min on the E. coli chromosome by proximity to a previously mapped locus. The deduced amino acid sequence of the bacA-encoded protein suggests an extremely hydrophobic protein of 151 amino acids, approximately 65% of which were nonpolar amino acids. E. coli cells containing plasmid pXV62 have increased isoprenol kinase activity. The physical characteristics of the deduced protein and enhanced lipid kinase activity suggest that the bacA gene may confer resistance to bacitracin by phosphorylation of undecaprenol.     

Identification of a novel fosfomycin-resistant UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from a soil metagenome

A soil metagenomic library was constructed and screened for clones that conferred fosfomycin resistance. A novel protein with 46 % identity to UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from Desulfuromonas acetoxidans DSM 684 (GenBank accession number: ZP_01311756) was identified. Multiple sequence alignment revealed that the novel protein was a natural MurA, in which an aspartic acid instead of a cysteine was located in the active site. An Asp120Cys mutant of Escherichia coli was constructed from the subclone through site-specific mutagenesis, and minimum inhibitory concentration of fosfomycin for the resistant subclone and its mutant were determined. These results showed that fosfomycin resistance was a result of the aspartic acid in the active site. Analysis of all existing MurA sequences revealed that MurAs with an active site aspartic acid that can confer fosfomycin resistance occur in ~14 % of bacteria.     

Identification of a novel UDP-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine enolpyruvyl transferase (MurA) from <Emphasis Type="Italic">Vibrio fischeri</Emphasis> that confers high fosfomycin resistance in <Emphasis Type="Italic">Escherichia coli</Emphasis>

MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 μg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.     

Molecular cloning of the penicillin G acylase gene from Arthrobacter viscosus

Penicillin G acylase was purified from the cultured filtrate of Arthrobacter viscosus 8895GU and was found to consist of two distinct subunits with apparent molecular weights of 24,000 (alpha) and 60,000 (beta). The partial N-terminal amino acid sequences of the alpha and beta subunits were determined with a protein gas phase sequencer, and a 29-base oligonucleotide corresponding to the partial amino acid sequence of the alpha subunit was synthesized. An Escherichia coli transformant having the penicillin G acylase gene was isolated from an A. viscosus gene library by hybridization with the 29-base probe. The resulting positive clone was further screened by the Serratia marcescens overlay technique. E. coli carrying a plasmid designated pHYM-1 was found to produce penicillin G acylase in the cells. This plasmid had an 8.0-kilobase pair DNA fragment inserted in the EcoRI site of pACYC184.     

Plasmid-mediated resistance to fosfomycin in Staphylococcus epidermidis

Staphylococcus epidermidis strain BM2641, isolated from a patient, was resistant to penicillin G, methicillin, aminoglycosides, chloramphenicol, macrolide, lincosamide and streptogramin B-type (MLS) antibiotics, and to high levels of fosmycin. Resistance to forsfomycin and/or to MLS was lost at low frequencies either spontaneously or after curing with novobiocin. The plasmid DNA from BM2641 and its cured derivatives was purified, analyzed by agarose gel electrophoresis and transferred to a nitrocellulose sheet. Comparative analysis of the resistance phenotypes with the plasmid content of the strains indicated that fosfomycin and MLS resistance were encoded by plasmids pIP1842 (2.5 kb) and pIP1843 (2.6 kb), respectively. Southern hybridization with a probe specific for gene fosA of Serratia marcescens showed that the fosfomycin resistance determinant in Staphylococcus is not homologous to that of Gram-negative bacteria.     

Molecular cloning of the penicillin G acylase gene from Arthrobacter viscosus.

Penicillin G acylase was purified from the cultured filtrate of Arthrobacter viscosus 8895GU and was found to consist of two distinct subunits with apparent molecular weights of 24,000 (alpha) and 60,000 (beta). The partial N-terminal amino acid sequences of the alpha and beta subunits were determined with a protein gas phase sequencer, and a 29-base oligonucleotide corresponding to the partial amino acid sequence of the alpha subunit was synthesized. An Escherichia coli transformant having the penicillin G acylase gene was isolated from an A. viscosus gene library by hybridization with the 29-base probe. The resulting positive clone was further screened by the Serratia marcescens overlay technique. E. coli carrying a plasmid designated pHYM-1 was found to produce penicillin G acylase in the cells. This plasmid had an 8.0-kilobase pair DNA fragment inserted in the EcoRI site of pACYC184.     

Characterization of the pTZ2162 encoding multidrug efflux gene qacB from Staphylococcus aureus

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.     

Characterization of Fosfomycin Resistant Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates from Human and Pig in Taiwan

To investigate the efficacy of fosfomycin against extended-spectrum β-lactamases (ESBL) producing Escherichia coli in Taiwan and the resistance mechanisms and characterization of human and pig isolates, we analyzed 145 ESBL-producing isolates collected from two hospitals (n = 123) and five farms (n = 22) in Taiwan from February to May, 2013. Antimicrobial susceptibilities were determined. Clonal relatedness was determined by PFGE and multi-locus sequence typing. ESBLs, ampC, and fosfomycin resistant genes were detected by PCR, and their flanking regions were determined by PCR mapping and sequencing. The fosfomycin resistant mechanisms, including modification of the antibiotic target (MurA), functionless transporters (GlpT and UhpT) and their regulating genes such as uhpA, cyaA, and ptsI, and antibiotic inactivation by enzymes (FosA and FosC), were examined. The size and replicon type of plasmids carrying fosfomycin resistant genes were analyzed. Our results revealed the susceptibility rates of fosfomycin were 94% for human ESBL-producing E. coli isolates and 77% for pig isolates. The PFGE analysis revealed 79 pulsotypes. No pulsotype was found existing in both human and pig isolates. Three pulsotypes were distributed among isolates from two hospitals. ISEcp1 carrying bla _{CTX-M-group 9} was the predominant transposable elements of the ESBL genes. Among the thirteen fosfomycin resistant isolates, functionless transporters were identified in 9 isolates. Three isolates contained novel amino acid substitutions (Asn67Ile, Phe151Ser and Trp164Ser, Val146Ala and His159Tyr, respectively) in MurA (the target of fosfomycin). Four isolates had fosfomycin modified enzyme (fosA3) in their plasmids. The fosA3 gene was harboured in an IncN-type plasmid (101 kbp) in the three pig isolates and an IncB/O-type plasmid (113 kbp) in the human isolate. In conclusion, we identified that 6% and 23% of the ESBL-producing E. coli from human and pigs were resistant to fosfomycin, respectively, in Taiwan. No clonal spread was found between human and pig isolates. Functionless transporters were the major cause of fosfomycin resistance, and the fosA3-transferring plasmid between isolates warrants further monitoring.     

Polypeptide chain elongation factor 1 alpha (EF-1 alpha) from yeast: nucleotide sequence of one of the two genes for EF-1 alpha from Saccharomyces cerevisiae.

Messenger RNA for yeast cytosolic polypeptide chain elongation factor 1 alpha (EF-1 alpha) was partially purified from Saccharomyces cerevisiae. Double-stranded complementary DNA (cDNA) was synthesized and cloned in Escherichia coli with pBR327 as a vector. Recombinant plasmid carrying yEF-1 alpha cDNA was identified by cross-hybridization with the E. coli tufB gene and the yeast mitochondrial EF-Tu gene (tufM) under non-stringent conditions. A yeast gene library was then screened with the EF-1 alpha cDNA and several clones containing the chromosomal gene for EF-1 alpha were isolated. Restriction analysis of DNA fragments of these clones as well as the Southern hybridization of yeast genomic DNA with labelled EF-1 alpha cDNA indicated that there are two EF-1 alpha genes in S. cerevisiae. The nucleotide sequence of one of the two EF-1 alpha genes (designated as EF1 alpha A) was established together with its 5'- and 3'-flanking sequences. The sequence contained 1374 nucleotides coding for a protein of 458 amino acids with a calculated mol. wt. of 50 300. The derived amino acid sequence showed homologies of 31% and 32% with yeast mitochondrial EF-Tu and E. coli EF-Tu, respectively.     

Construction and properties of a cell line constitutively expressing the herpes simplex virus glycoprotein B dependent on functional alpha 4 protein synthesis.

We report the construction of a cell line constitutively expressing the glycoprotein B (gB) of herpes simplex virus (HSV) 1. The cell line was constructed in two steps. In the first, a baby hamster kidney cell line was transfected with the DNA of a plasmid containing the neomycin phosphotransferase gene that confers resistance to the antibiotic G418 and the gene specifying a temperature-sensitive (ts-) alpha 4 protein of HSV-1, the major viral regulatory protein. A clonal cell line, alpha 4/c113, selected for resistance to the antibiotic G418, expressed high levels of alpha 4 protein constitutively. Superinfection of these cells with HSV-2 resulted in twofold induction of the resident HSV-1 alpha 4 gene. In the second step, alpha 4/c113 cells were transfected with the DNA of a plasmid carrying the gB gene and the mouse methotrexate resistance dihydrofolate reductase gene. A clonal cell line, alpha 4/c113/gB, selected for methotrexate resistance expressed gB constitutively. Expression of both gB and alpha 4 continued unabated for at least 32 serial passages. Cells passaged serially in medium containing both methotrexate and G418 after passage 10 contained a higher copy number of the alpha 4 gene and produced larger amounts of both gB and alpha 4 proteins than did cells maintained in medium containing methotrexate alone. Expression of gB was dependent on the presence of functional alpha 4 protein inasmuch as expression of gB ceased on shift up to nonpermissive temperatures, when shifted to permissive temperatures, the cell line reinitiated expression of gB after a delay commensurate with the length of incubation at the nonpermissive temperature, and the cell-resident HSV-1 gB gene was expressed at the nonpermissive temperature in cells infected with a recombinant expressing a ts+ alpha 4 protein and an HSV-2 gB. The properties of the alpha 4/c113 cell line suggest that it may express other viral genes induced by alpha 4 protein constitutively, provided that the product is not toxic to the cells.     

AG alpha 1 is the structural gene for the Saccharomyces cerevisiae alpha-agglutinin, a cell surface glycoprotein involved in cell-cell interactions during mating.

We have cloned the alpha-agglutinin structural gene, AG alpha 1, by the isolation of alpha-specific agglutination-defective mutants, followed by isolation of a complementing plasmid. Independently isolated alpha-specific agglutination-defective mutations were in a single complementation group, consistent with biochemical results indicating that the alpha-agglutinin is composed of a single polypeptide. Mapping results suggested that the complementation group identified by these mutants is allelic to the ag alpha 1 mutation identified previously. Expression of AG alpha 1 RNA was alpha specific and inducible by a-factor. Sequences similar to the consensus sequences for positive control by MAT alpha 1 and pheromone induction were found upstream of the AG alpha 1 initiation codon. The AG alpha 1 gene could encode a 650-amino-acid protein with a putative signal sequence, 12 possible N-glycosylation sites, and a high proportion of serine and threonine residues, all of which are features expected for the alpha-agglutinin sequence. Disruption of the AG alpha 1 gene resulted in failure to express alpha-agglutinin and loss of cellular agglutinability in alpha cells. An Escherichia coli fusion protein containing 229 amino acids of the AG alpha 1 sequence was recognized by an anti-alpha-agglutinin antibody. In addition, the ability of this antibody to inhibit agglutination was prevented by this fusion protein. These results indicate that AG alpha 1 encodes alpha-agglutinin. Features of the AG alpha 1 gene product suggest that the amino-terminal half of the protein contains the a-agglutinin binding domain and that the carboxy-terminal half contains a cell surface localization domain, possibly including a glycosyl phosphatidylinositol anchor.