Cloning and sequencing of sarA of Staphylococcus aureus, a gene required for the expression of agr.

To evaluate the effect of a sar mutation on the agr locus, Northern (RNA) blotting was performed to determine the levels of RNAIII, the agr regulatory molecule, in two isogenic pairs of Staphylococcus aureus strains. Our results demonstrated that RNAIII was either significantly diminished or absent in both sar mutants compared with the parents. The RNAIII level was partially restored in sar mutants complemented with an intact sar gene (designated sarA). Additionally, we were able to complement selected sar phenotypes with a plasmid carrying RNAIII (pRN6735). These studies suggest that the sarA gene is necessary for the optimal expression of agr. The sarA gene of strain RN450 was subsequently cloned and sequenced. Sequence analysis revealed an open reading frame of 372 bp with a predicted molecular size of 14,718 Da and a deduced pI of 8.52. The deduced protein sequence has a predominance of charged residues (33%) and shares sequence similarity with the virF gene of Shigella flexneri.     

Cloning of genes affecting capsule expression in Staphylococcus aureus strain M

Staphylococcus aureus strain M produces large amounts of capsular polysaccharide. It produces a non-encapsulated variant at a frequency of 0.01% at 37 degrees C. At high temperature (43 degrees C), the frequency of capsule loss was shown to be 1-38%. A 19 kb plasmid and a prophage were found to be carried by the M strain, but curing of these elements did not affect capsular production. To clone the capsular (cap) genes, a plasmid library of S. aureus M was constructed directly in S. aureus RN4200. The library was then infected with phage 80 alpha. After transduction of the phage lysates to a Cap- mutant derived from M strain, a recombinant plasmid was obtained which complemented the mutant to a Cap+ phenotype. Chromosomal walking experiments were used to clone additional nearby cap genes. Complementation tests using a collection of Cap- mutants showed that most of the mutants were complemented by a 19.4 kb DNA fragment, suggesting that the majority of the cap genes affecting capsule production are clustered together.     

Cloning and expression of the exfoliative toxin B gene from Staphylococcus aureus.

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.     

Cloning, sequencing, expression and characterization of DNA photolyase from Salmonella typhimurium.

We have cloned the phr gene that encodes DNA photolyase from Salmonella typhimurium by in vivo complementation of Escherichia coli phr gene defect. The S.typhimurium phr gene is 1419 base pairs long and the deduced amino acid sequence has 80% identity with that of E. coli photolyase. We expressed the S.typhimurium phr gene in E.coli by ligating the E.coli trc promoter 5' to the gene, and purified the enzyme to near homogeneity. The apparent molecular weight of S.typhimurium photolyase is 54,000 dalton as determined by SDS-polyacrylamide gel electrophoresis, which is consistent with the calculated molecular weight of 53,932 dalton from the deduced phr gene product. S.typhimurium photolyase is purple-blue in color with near UV-visible absorption peaks at 384, 480, 580, and 625 nm and a fluorescence peak at 470 nm. From the characteristic absorption and fluorescence spectra and reconstitution experiments, S.typhimurium photolyase appears to contain flavin and methenyltetrahydrofolate as chromophore-cofactors as do the E.coli and yeast photolyases. Thus, S.typhimurium protein is the third folate class photolyase to be cloned and characterized to date. The binding constant of S.typhimurium photolyase to thymine dimer in DNA is kD = 1.6 x 10(-9) M, and the quantum yield of photorepair at 384 nm is 0.5.     

Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus.

The gene encoding the fibronectin-binding protein (FNBP) from Staphylococcus aureus strain 8325-4 was isolated from a gene bank in pBR322. The original clone, containing a 6.5-kb insert, gave a functional product present in the periplasm of Escherichia coli. Analysis of polypeptides isolated after affinity chromatography on fibronectin-Sepharose followed by ion-exchange chromatography revealed two gene products, 87 and 165 kd in mol. wt. The amino acid compositions of these two polypeptides and a native FNBP from S. aureus strain Newman were very similar. Antibodies raised against the native FNBP from strain Newman precipitated the 125I-labelled 165-kd polypeptide, and unlabeled 165- and 87-kd polypeptides as well as native FNBP inhibited the immunoprecipitation reactions. The region of the fnbp-gene encoding the fibronectin-binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene. The resulting product in E. coli is an extracellular fusion protein consisting of two IgG-binding domains of protein A followed by a fibronectin-binding region. The fusion protein binds to fibronectin and completely inhibits the binding of fibronectin to intact cells of S. aureus.     

Cloning,sequencing and expression of the nhaA and nhaR genes from Salmonella entiritidis

Na⁺/H⁺ antiporter activity is wide-spread and plays essential physiological roles. We found that several Enterobacteriaceae share conserved sequences with nhaA, the gene coding for an E. coli antiporter. A nhaA strain which is sensitive to Na⁺ and Li⁺, was used to clone by complementation a DNA fragment from Salmonella enteritidis which confers resistance to the ions. The cloned fragment increased Na⁺/H⁺ antiport activity in membranes isolated from strains carrying the respective hybrid plasmid. DNA sequence analysis of the insert revealed two open reading frames. Both encode putative polypeptides which are closely homologous to the nhaA and nhaR gene products from Escherichia coli. The antiporter activity displays properties very similar to that of the E. coli NhaA, namely, it is activiated by alkaline pH and recognizes Li⁺ with high affinity.Abbreviations _H ⁺ Proton electrochemical potential - pH transmembrane pH gradient - _Na ⁺ Sodium electrochemical potential - SDS Sodium dodecyl sulfate - CIP Calf intestine alkaline phosphates - ORF open reading frame     

Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase A and B proteins.

We have determined the nucleotide sequence of a 5.3-kb segment of the Staphylococcus aureus chromosome that includes the gyrA and gyrB genes coding for both subunits of DNA gyrase, the enzyme that catalyzes ATP-dependent DNA supercoiling. The gene order at this locus, dnaA-dnaN-recF-gyrB-gyrA, is similar to that found in the Bacillus subtilis replication origin region. S. aureus recF, gyrB, and gyrA genes are closely spaced, occupy the same reading frame, and may be coordinately expressed. The S. aureus gyrB and gyrA genes encode 640- and 889-residue proteins, respectively, that share strong homology with other bacterial gyrase subunits, notably those from B. subtilis. These results are discussed in regard to the mechanism of DNA gyrase and its role as a target for the 4-quinolones and other antistaphylococcal agents.     

Cloning, expression and sequencing of Helicobacter felis urease genes

Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid cione led to the construction of plLL205 (9.5 kb) which conferred a urease activity of 1.2±0.5 μmole urea min^-1 mg^-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on plLL205 which were necessary for the expression of an urease-positive phenotype in E. coii clones. To localize the putative structural genes of H. felis on plLL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant cione did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had caicuiated moiecuiar masses of 26 074 and 61 663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.     

Identification of LytSR-regulated genes from Staphylococcus aureus.

In this report, the characterization of a Staphylococcus aureus operon containing two LytSR-regulated genes, lrgA and lrgB, is described. Sequence and mutagenesis studies of these genes suggest that lrgA encodes a murein hydrolase exporter similar to bacteriophage holin proteins while lrgB may encode a protein having murein hydrolase activity.     

Cloning, sequencing, and expression of the Pseudomonas putida protocatechuate 3,4-dioxygenase genes.

The genes that encode the alpha and beta subunits of protocatechuate 3,4-dioxygenase (3,4-PCD [EC 1.13.11.3]) were cloned from a Pseudomonas putida (formerly P. aeruginosa) (ATCC 23975) genomic library prepared in lambda phage. Plaques were screened by hybridization with degenerate oligonucleotides designed using known amino acid sequences. A 1.5-kb SmaI fragment from a 15-kb primary clone was subcloned, sequenced, and shown to contain two successive open reading frames, designated pcaH and pcaG, corresponding to the beta and alpha subunits, respectively, of 3,4-PCD. The amino acid sequences deduced from pcaHG matched the chemically determined sequence of 3,4-PCD in all except three positions. Cloning of pcaHG into broad-host-range expression vector pKMY319 allowed high levels of expression in P. putida strains, as well as in Proteus mirabilis after specific induction of the plasmid-encoded nahG promoter with salicylate. The recombinant enzyme was purified and crystallized from P. mirabilis, which lacks an endogenous 3,4-PCD. The physical, spectroscopic, and kinetic properties of the recombinant enzyme were indistinguishable from those of the wild-type enzyme. Moreover, the same transient enzyme intermediates were formed during the catalytic cycle. These studies establish the methodology which will allow mechanistic investigations to be pursued through site-directed mutagenesis of P. putida 3,4-PCD, the only aromatic ring-cleaving dioxygenase for which the three-dimensional structure is known.     

Cloning, characterization, and sequencing of an accessory gene regulator (agr) in Staphylococcus aureus.

We have previously identified a gene in Staphylococcus aureus, agr, whose activity is required for high-level post-exponential-phase expression of a series of secreted proteins. In this paper, we describe the cloning of this gene in Escherichia coli by using an inserted transposon (Tn551) as a cloning probe. The cloned gene, consisting of a 241-codon open reading frame containing the site of the transposon insertion, was recloned to an S. aureus vector, pSK265, and shown to be functional in S. aureus. Activity was evaluated by determinations of alpha-hemolysin, beta-hemolysin, and toxic shock syndrome toxin-1 production in early-stationary-phase cultures. The cloned gene showed considerable variation with respect to different exoproteins and different host strains compared with the chromosomal agr determinant; this variation could not be attributed to the higher copy number of the cloned gene and probably reflects inapparent subtleties of the regulatory system.     

Cloning and sequencing of the gene, fmtC, which affects oxacillin resistance in methicillin-resistant Staphylococcus aureus

Two Tn551 insertional mutants with reduced methicillin resistance were isolated from methicillin-resistant Staphylococcus aureus KSA8. These two mutants showed increased susceptibility to beta-lactam antibiotics and bacitracin, but not to fosfomycin and vancomycin. Tn551 in these mutants was inserted into the same gene, termed fmtC. The fmtC gene has an open reading frame of 840 amino acid residues with an estimated molecular mass of 96.9 kDa. The N-terminal half of the deduced FmtC protein is very hydrophobic, implying that this protein is a membrane-associated protein.     

Cloning of lysozyme and lysostaphin genes of Staphylococcus aureus and their expression in Bacillus subtilis cells

The gene of microbial lysozyme (lyz) of S. aureus 118 and the gene of lysostaphin (lzf) of S. aureus RN 3239 were cloned and their expression in B. subtilis cells was shown. Lysozyme production in B. subtilis recombinant clone pLF14-Lyz, obtained as the result of cloning, was 2.5-fold greater than lysozyme production in S. aureus wild strain 118. Lysostaphin production in B. subtilis recombinant strain pLF14-Lzf which had inherited the cloned genes was approximately equal to lysostaphin production observed in S. aureus initial strain RN 3239. The production of lysozyme and lysostaphin in the cells of B. subtilis recombinant strains was observed at 30 degrees C and pH 5.5, while in S. aureus initial strains 118 and RN 3239 bacteria produced lysozyme and lysostaphin at 37 degrees C and pH 7.5 respectively.     

Rapid isolation of DNA from Staphylococcus aureus.

We describe a Staphylococcus aureus bulk DNA isolation procedure which uses detergent and guanidine hydrochloride to free the nucleic acid from contaminants. The procedure is rapid and yields high-molecular-weight DNA suitable for molecular biological procedures.     

Cloning and expression of the Staphylococcus aureus glucosaminidase in Escherichia coli

Abstract A fragment of Staphylococcus aureus DNA encoding the glucosaminidase determinant was cloned in Escherichia coli by inserting the Sau 3A genomic fragments in the Bam HI site of the plasmid vector pBR322. One clone selected on the basis of its lytic activity was shown to contain a hybrid plasmid (pEU213) carrying a 4.7 kb insert of S. aureus DNA. Lytic activity was tested using different assays, and the enzyme production was confirmed by immunological reactions. An appreciable reduction of lytic activity was noted after few subcultures. The E. coli carrying pEU213 had a slower growth rate and increased autolytic activity compared to the parental strain. The possible reasons for this behavior are discussed.     

An improved expression plasmid for affinity purification of Staphylococcus aureus gyrase A subunit

Of the bacterial topoisomerases, the gyrase A subunit (GyrA) of Staphylococcus aureus is particularly difficult to purify because of its tendency to form inclusion bodies. Previous attempts at purification yielded low concentrations of protein with reduced specific activity. To overcome this problem, we modified the commercially available plasmid expression vector, pBAD/Thio-TOPO, via the addition of DNA sequences encoding a hexahistidine tag upstream and a cleavage site for tobacco etch virus protease downstream of the gene encoding thioredoxin. The resulting expression system consisting of the modified plasmid, pSAGA7, and the recommended host strain, Escherichia coli TOP 10, facilitated high level expression of soluble GyrA and its affinity purification to over 95% homogeneity. Purified GyrA had high biological activity as evidenced by a specific activity of 4.3x10(5)U/mg. The pSAGA7/TOP10 expression system also facilitated the expression and purification of a subunit of S. aureus topoisomerase IV, ParE, and a recently discovered protein unrelated to topoisomerases, QnrB, two "hard to purify" proteins. We conclude that pSAGA7 might be useful for high-level soluble expression and purification of diverse microbial proteins.