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a vector for recombinant dna in staphylococcus aureus.staphylococcal plasmids ps194 and psc194 which confer streptomycin and streptomycin-chloramphenicol resistance respectively have been used as vectors for construction of recombinant dna, since they each carry one single recipient site for endonuclease ecori. hybrid dna does not express streptomycin resistance, a marker which is present in both vectors, presumably because the marker gene is cleaved by ecori. a chloramphenicol marker present in psc194 was used for positive hybrid selection. hybrid ...1978658667
improved method for electroporation of staphylococcus aureus.we have developed a significantly improved method for the electroporation of plasmid dna into staphylococcus aureus. the highest transformation efficiency achieved with this procedure was 4.0 x 10(8) transformants per microgram of plasmid psk265 dna. this represents a 530-fold improvement over the previously reported optimum efficiency of 7.5 x 10(5) transformants per microgram of plasmid dna after electroporation of s. aureus cells [9]. identical results were obtained when electrocompetent cell ...19921521761
nucleotide sequence of beta-lactamase regulatory genes from staphylococcal plasmid pi258. 19911861992
isolation of transposon tn551 insertions near chromosomal markers of interest in staphylococcus aureus.a procedure was developed to isolate insertions of transposon tn551 near other markers of interest on the chromosome of staphylococcus aureus nctc 8325. when an inoculum of strain 8325-4 carrying a thermosensitive mutant of plasmid pi258 (on which tn551 resides) was inoculated into brain heart infusion agar plus erythromycin and grown to saturation at 43 degrees c, the transforming dna extracted from this population of cells contained a random collection of different chromosomal insertions of tn ...19846090397
terminal nucleotide sequences of tn551, a transposon specifying erythromycin resistance in staphylococcus aureus: homology with tn3.the erythromycin resistance determinant of staphylococcus aureus plasmid pi258 resides on a 5.3 kb transposon, tn551. we have determined dna sequences surrounding the junctions between the transposon and the flanking dna in the wild-type plasmid, in an insertion into a second plasmid, and in two transposon-related deletions. the ends of the transposon consist of an inverted repeat of 40 base pairs flanked by a direct repeat of 5, thus placing the transposon in the same class as tn3, is2, tn501, ...19806100928
exfoliative toxin plasmids of bacteriophage group 2 staphylococcus aureus: sequence homology.the plasmid contents of seven exfoliative toxin-producing strains of phage group 2 staphylococcus aureus were analyzed by agarose gel electrophoresis and deoxyribonucleic acid-deoxyribonucleic acid hybridization. all strains were found to contain a large plasmid with a molecular weight of 27 x 10(6) except for strain rw1005. a comparison of the restriction endonuclease cleavage products by agarose gel electrophoresis showed that the number and size distribution of the fragments of all these tox ...19806254889
a functional chimeric membrane subunit of an ion-translocating atpase.a chimeric transport protein was made by expression of a fusion of the arsb genes from escherichia coli plasmid r773 and staphylococcus aureus plasmid pi258. the two genes were fused to encode a functional protein with first eight membrane spanning alpha-helices of the s. aureus and the last four helices of the e. coli protein. the hybrid protein provided arsenite resistance and transport. when an arsa gene was expressed in trans with the arsb proteins encoded by the r773, pi258 and fusion genes ...19947832592
arsenate reductase of staphylococcus aureus plasmid pi258.arsenate reductase encoded by staphylococcus aureus arsenic-resistance plasmid pi258 was overproduced in escherichia coli and purified. the purified enzyme reduced radioactive arsenate to arsenite when coupled to thioredoxin, thioredoxin reductase, and nadph. nadph oxidation coupled to arsenate reduction also required thioredoxin and thioredoxin reductase. glutaredoxin and reduced glutathione did not stimulate arsenate reduction. nadph oxidation showed michaelis-menten kinetics with a km of 1 mi ...19948003493
detection of genes regulating beta-lactamase production in enterococcus faecalis and staphylococcus aureus.four beta-lactamase-producing clinical isolates (wh245, wh257, ch570, and del) of enterococcus faecalis were examined for the presence of the staphylococcal beta-lactamase regulatory genes (blar1 and blai) by pcr using six primer pairs. all isolates produced small amounts of beta-lactamase constitutively. in wh245, ch570, and del, the corresponding regions of the regulatory genes have lost sequences of various lengths. however, the regulatory genes in wh257 appeared to be the same as those in st ...19968913462
cadc, the transcriptional regulatory protein of the cadmium resistance system of staphylococcus aureus plasmid pi258.the cadc protein from the cada cadmium resistance operon of staphylococcus aureus plasmid pi258 regulates transcription of this system in vitro. the cadc protein was overproduced in escherichia coli cells and partially purified. gel shift assays of the proposed cada operator/promoter region dna showed specific association with the cadc protein. control arsenic resistance operator/promoter dna from the same plasmid was not shifted by the cadc protein. cd2+, bi3+, and pb2+ caused the release of ca ...19957543476
inducible plasmid-determined resistance to arsenate, arsenite, and antimony (iii) in escherichia coli and staphylococcus aureus.plasmids in both escherichia coli and staphylococcus aureus contain an "operon" that confers resistance to arsenate, arsenite, and antimony(iii) salts. the systems were always inducible. all three salts, arsenate, arsenite, and antimony(iii), were inducers. mutants and a cloned deoxyribonucleic acid fragment from plasmid pi258 in s. aureus have lost arsenate resistance but retained resistances to arsenite and antimony, demonstrating that separate genes are involved. arsenate-resistant arsenite-s ...19817016838
dna homology between the arsenate resistance plasmid psx267 from staphylococcus xylosus and the penicillinase plasmid pi258 from staphylococcus aureus.a 29.5-kb plasmid, psx267, from staphylococcus xylosus dsm 20267 was found to code for arsenate, arsenite, and antimony (iii) resistance. the isolated plasmid was transformed into s. aureus, where the same resistances were expressed. it was of special interest to see whether psx267 showed any dna sequence homology with the well-studied penicillinase plasmid from s. aureus pi258, also conferring arsenate, arsenite, and antimony iii resistance. by the use of the southern blotting technique, it was ...19836602348
resistance to mercury and to cadmium in chromosomally resistant staphylococcus aureus.apparently chromosomally located mercury resistance determinants in five methicillin-resistant staphylococcus aureus strains of different geographical origin were structurally homologous to plasmid-located mercury resistance determinants in s. aureus. these were all located on a 6.3-kilobase (kb) bg/ii fragment, as evident from southern hybridization experiments with the 6.3-kb bg/ii fragment of plasmid pi258 as the probe. these methicillin-resistant s. aureus strains exhibited similar phage sus ...19863635384
nucleotide sequence and expression of the beta-lactamase gene from staphylococcus aureus plasmid pi258 in escherichia coli, bacillus subtilis, and staphylococcus aureus.the structural gene for beta-lactamase in the staphylococcus aureus plasmid pi258 was cloned into a staphylococcus aureus-bacillus subtilis-escherichia coli shuttle vector, pwn101, and the nucleotide sequence of the gene was determined. pwn101 was structurally stable and the beta-lactamase gene was expressed efficiently from its native promoter and ribosome-binding site in all three hosts.19873104315
nucleotide sequence and expression of the mercurial-resistance operon from staphylococcus aureus plasmid pi258.the mercurial-resistance determinant from staphylococcus aureus plasmid pi258 is located on a 6.4-kilobase-pair bgl ii fragment. the determinant was cloned into both bacillus subtilis and escherichia coli. mercury resistance was found only in b. subtilis. the 6404-base-pair dna sequence of the bgl ii fragment was determined. the mer dna sequence includes seven open reading frames, two of which have been identified by homology with the mera (mercuric reductase) and merb (organomercurial lyase) ge ...19873037534
nucleotide sequence of a chromosomal mercury resistance determinant from a bacillus sp. with broad-spectrum mercury resistance.a 13.5-kilobase hindiii fragment, bearing an intact mercury resistance (mer) operon, was isolated from chromosomal dna of broad-spectrum mercury-resistant bacillus sp. strain rc607 by using as a probe a clone containing the mercury reductase (mera) gene. the new clone, pyw33, expressed broad-spectrum mercury resistance both in escherichia coli and in bacillus subtilis, but only in b. subtilis was the mercuric reductase activity inducible. sequencing of a 1.8-kilobase mercury hypersensitivity-pro ...19892536669
cadmium resistance from staphylococcus aureus plasmid pi258 cada gene results from a cadmium-efflux atpase.cadmium resistance specified by the cada determinant of staphylococcus aureus plasmid pi258 results from the functioning of a cadmium-efflux system. in the nucleotide sequence of the dna fragment containing the cada determinant, two open reading frames were identified. the larger one, corresponding to a predicted polypeptide of 727 amino acid residues, is necessary and sufficient for expression of cadmium resistance. comparison of the cada amino acid sequence with known protein sequences suggest ...19892524829
tn552, a novel transposable element from staphylococcus aureus.tn552, one of several closely related beta-lactamase-encoding transposons from staphylococcus aureus, has a novel set of putative transposition functions. each is homologous with a well-characterized function from a different type of mobile genetic element. thus, tn552 encodes: (i) resl-binl, a co-integrate resolution system homologous with those of tn3 family elements; (ii) p480, a potential transposase significantly homologous with the dna integrases of eukaryotic retroviruses and retrotranspo ...19902170815
deletion mutant analysis of the staphylococcus aureus plasmid pi258 mercury-resistance determinant.deletion mutant analysis of the mercury-resistant determinant (mer operon) from the staphylococcus aureus plasmid pi258 was used to verify the location of the mera and merb genes and to show the existence of mercuric ion transport gene(s). orf5 was confirmed to be a transport gene and has an amino acid product sequence homologous to the mert gene products from several gram-negative bacteria and a bacillus species. deletion analysis established that inactivation of mera on a broad-spectrum mer re ...19911954576
kinetics and active site dynamics of staphylococcus aureus arsenate reductase.arsenate reductase (arsc) encoded by staphylococcus aureus arsenic-resistance plasmid pi258 reduces intracellular arsenate(v) to the more toxic arsenite(iii), which is subsequently extruded from the cell. it couples to thioredoxin, thioredoxin reductase and nadph to be enzymatically active. arsc is extremely sensitive to oxidative inactivation, has a very dynamic character hampering resonance assignments in nmr and produces peculiar biphasic michaelis-menten curves with two v(max) plateaus. in t ...200211862551
regulation of the cada cadmium resistance determinant of staphylococcus aureus plasmid pi258.regulation of the cada cadmium and zinc resistance determinant of staphylococcus aureus plasmid pi258 was demonstrated by using gene fusions and direct measurements of transcription. in growth experiments, cells harboring the intact cada operon were induced with different cations and challenged by an inhibitory concentration of zncl2, a substrate of the cada resistance system. uninduced cells did not grow for 8 h after zn2+ addition, whereas induced cells grew in the presence zn2+. cd2+ was a st ...19911938960
a second gene in the staphylococcus aureus cada cadmium resistance determinant of plasmid pi258.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 trunca ...19911938959
transcriptional analysis of the staphylococcus aureus plasmid pi258 mercury resistance determinant.northern blot dna-rna hybridization analysis of staphylococcus aureus cells bearing pi258 showed that upon induction the amount of mer operon transcript present increased 49-fold over that observed in uninduced cells. maximum induction occurred after 45 min in the presence of 5 microm hgcl2. two transcripts, 5.0 and 5.8 kb long, were observed. both transcripts encoded merr through merb (inclusive). primer extension analysis determined that the 5' end of at least one transcript (and presumably of ...19911860831
regulation and expression of the arsenic resistance operon from staphylococcus aureus plasmid pi258.the arsenic resistance operon from staphylococcus aureus plasmid pi258 was cloned and sequenced. the dna sequence contains three genes in the order arsr, arsb, and arsc. the predicted amino acid sequences of the gene products are homologous with those of the products of the ars operons of plasmids psx267 from staphylococcus xylosus and r773 from escherichia coli. the cloned staphylococcal ars operon confers resistances to arsenate, arsenite, and antimonite in s. aureus and bacillus subtilis. the ...19921534328
atp-dependent cadmium transport by the cada cadmium resistance determinant in everted membrane vesicles of bacillus subtilis.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. 109 ...19921530844
reduction of arsenate to arsenite by the arsc protein of the arsenic resistance operon of staphylococcus aureus plasmid pi258.the arsenic resistance operon of staphylococcus aureus plasmid pi258 consists of three genes, arsr (encoding the repressor regulatory protein), arsb (the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cells), and arsc (the small gene whose protein product is required for arsenate resistance only, not for arsenite resistance). arsc has now been shown to be an arsenate reductase, converting intracellular arsenate [as(v)] to arsenite [as(iii)], which ...19921409657
regulation of the staphylococcus aureus plasmid pi258 mercury resistance operon.experiments involving fusion between the staphylococcus aureus plasmid pi258-encoded mer operon and the reporter gene beta-lactamase, mutational analysis, and trans-complementation studies have shown that the merr gene of pi258, which shows dna sequence similarity with known merr genes from other bacteria, regulates the expression of the mer operon in vivo. the merr gene product is a trans-acting protein that activates mer operon transcription in the presence of the inducers hg2+ and cd2+. a glu ...19921400255
the cadc gene product of alkaliphilic bacillus firmus of4 partially restores na+ resistance to an escherichia coli strain lacking an na+/h+ antiporter (nhaa).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 ...19921321115
characterization and construction of molecular cloning vehicles within staphylococcus aureus.four chloramphenicol resistance (cm) and four tetracycline resistance (tc) plasmids from staphylococcus aureus were characterized by restriction endonuclease mapping. all four tc plasmids had molecular masses of 2.9 megadaltons (mdaltons) and indistinguishable responses to seven different restriction endonucleases. the four cm plasmids (pcw6, pcw7, pcw8, and pc221) had molecular masses of 2.6, 2.8, 1.9, and 2.9 mdaltons, respectively. the four cm plasmids also differed both in the level of resis ...1978711670
genetic translocation in staphylococcus aureus.a 5.2-kilobase pair transposon, tn551, has been found in staphylococcus aureus, a gram-positive bacterium. initially detected on plasmid pi258, it undergoes rec-independent transposition to multiple chromosomal and plasmid sites, sometimes causing insertional inactivation. unlike most other transposons, tn551 undergoes apparently precise excision as a rule. the initial observation of tn551 transition involved uv inactivation of the carrier plasmid; this would appear to be a general means of dete ...1979284355
deoxyribonucleic acid sequence common to staphylococcal and streptococcal plasmids which specify erythromycin resistance.plasmids from erythromycin-resistant staphylococcus aureus, streptococcus sanguis, and streptococcus faecalis show deoxyribonucleic acid sequence homology. the homologous sequences can be localized to specific restriction endonuclease fragments, which in the case of s. aureus plasmid pi258 involves a single fragment from either ecori or hindiii digest known to contain the erythromycin resistance determinant. complementary ribonucleic acid probes prepared from s. aureus plasmid pi258 and s. sangu ...1979110774
recombinant luminescent bacteria for measuring bioavailable arsenite and antimonite.luminescent bacterial strains for the measurement of bioavailable arsenite and antimony were constructed. the expression of firefly luciferase was controlled by the regulatory unit of the ars operon of staphylococcus aureus plasmid pi258 in recombinant plasmid ptoo21, with s. aureus rn4220, bacillus subtilis br151, and escherichia coli mc1061 as host strains. strain rn4220(ptoo21) was found to be the most sensitive for metal detection responding to arsenite, antimonite, and cadmium, the lowest d ...19979361432
arsenic efflux governed by the arsenic resistance determinant of staphylococcus aureus plasmid pi258.the arsenic resistance operon of staphylococcus aureus plasmid pi258 determined lowered net cellular uptake of 73as by an active efflux mechanism. arsenite was exported from the cells; intracellular arsenate was first reduced to arsenite and then transported out of the cells. resistant cells showed lower accumulation of 73as originating from both arsenate and arsenite. active efflux from cells loaded with arsenite required the presence of the plasmid-determined arsb gene. efflux of arsenic origi ...19938501052
luxab gene fusions with the arsenic and cadmium resistance operons of staphylococcus aureus plasmid pi258.pc101, a novel shuttle vector between escherichia coli and staphylococcus aureus carrying the lux genes encoding luciferase from vibrio harveyi, selectable ampicillin and chloramphenicol markers and origins of replication for gram-negative and gram-positive bacteria has been constructed. the inducibility of the arsenic and cadmium operon from s. aureus plasmid pi258 to different ions has been tested in e. coli and in s. aureus with two fusions in pc101: an arsb-luxab and a cada-luxab transcripti ...19938349095
plasmid-borne cadmium resistance genes in listeria monocytogenes are similar to cada and cadc of staphylococcus aureus and are induced by cadmium.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 ar ...19948188605
the ars operon in the skin element of bacillus subtilis confers resistance to arsenate and arsenite.the bacillus subtilis skin element confers resistance to arsenate and arsenite. the ars operon in the skin element contains four genes in the order arsr, orf2, arsb, and arsc. three of these genes are homologous to the arsr, arsb, and arsc genes from the staphylococcal plasmid pi258, while no homologs of orf2 have been found. inactivation of arsr, arsb, or arsc results in either constitutive expression of ars, an arsenite- and arsenate-sensitive phenotype, or an arsenate-sensitive phenotype, res ...19989537360
pb(ii)-translocating p-type atpases.the cad operon of staphylococcus aureus plasmid pi258, which confers cadmium resistance, encodes a transcriptional regulator, cadc, and cada, an atp-coupled cd(ii) pump that is a member of the superfamily of cation-translocating p-type atpases. the escherichia coli homologue of cada, termed znta, is a zn(ii)/cd(ii) pump. the results described in this paper support the hypothesis that znta and cada are pb(ii) pumps. first, cadc is a metal-responsive repressor that responds to soft metals in the o ...19989830000
luminescent bacterial sensor for cadmium and lead.a sensor plasmid was constructed by inserting the regulation unit from the cada determinant of plasmid pi258 to control the expression of firefly luciferase. the resulting sensor plasmid ptoo24 is capable of replicating in gram-positive and gram-negative bacteria. the expression of the reporter gene as a function of added extracellular heavy metals was studied in staphylococcus aureus strain rn4220 and bacillus subtilis strain br151. strain rn4220(ptoo24) mainly responded to cadmium, lead and an ...19989839381
chromosome-determined zinc-responsible operon czr in staphylococcus aureus strain 912.a novel operon, czrab (zinc-responsible genes), was identified in the chromosome of staphylococcus aureus. the operon consists of two genes, czra and czrb. the czra gene, coding for an 11.5 kda protein, was homologous to cadc, arsr of s. aureus plasmid pi258 and smtb of synechococcus pcc7942. the czrb, coding for a 36 kda membrane spanning protein, was homologous to the czcd gene, cobalt, zinc and the cadmium-resistant factor of bacillus subtilis and alcaligenes eutrophus. in the presence of zin ...199910229265
the essential catalytic redox couple in arsenate reductase from staphylococcus aureus.arsenate reductase (arsc) encoded by staphylococcus aureus arsenic-resistance plasmid pi258 reduces intracellular as(v) (arsenate) to the more toxic as(iii) (arsenite), which is subsequently extruded from the cell. arsc couples to thioredoxin, thioredoxin reductase, and nadph to be enzymatically active. a novel purification method leads to high production levels of highly pure enzyme. a reverse phase method was introduced to systematically analyze and control the oxidation status of the enzyme. ...199910606519
development of a downstream process for the isolation of staphylococcus aureus arsenate reductase overproduced in escherichia coli.arsenate reductase (arsc) encoded by staphylococcus aureus arsenic-resistance plasmid pi258 reduces intracellular as(v) (arsenate) to the more toxic as(iii) (arsenite). in order to study the structure of arsc and to unravel biochemical and physical properties of this redox enzyme, wild type enzyme and a number of cysteine mutants were overproduced soluble in escherichia coli. in this paper we describe a novel purification method to obtain high production levels of highly pure enzyme. a reversed- ...200010681053
a cadmium resistance plasmid, pxu5, in staphylococcus aureus, strain atcc25923.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.200010913869
role of cysteinyl residues in sensing pb(ii), cd(ii), and zn(ii) by the plasmid pi258 cadc repressor.the cadca operon of staphylococcus aureus plasmid pi258 confers resistance to salts of the soft metals lead, cadmium, and zinc. the operon is regulated by cadc, a member of the arsr family of metal-responsive transcriptional repressors. in this study the role of the five cysteine residues of cadc in soft metal ion sensing was investigated. cys-7, cys-11, cys-52, cys-58, and cys-60 were changed individually to glycine or serine residues. the effect of the cadc mutations was examined in escherichi ...200111278706
arsenate reductase from s. aureus plasmid pi258 is a phosphatase drafted for redox duty.arsenate reductase (arsc) from staphylococcus aureus plasmid pi258 plays a role in bacterial heavy metal resistance and catalyzes the reduction of arsenate to arsenite. the structures of the oxidized and reduced forms of arsc were solved. arsc has the ptpase i fold typical for low molecular weight tyrosine phosphatases (lmw ptpases). remarkably, kinetic experiments show that pi258 arsc also catalyzes the tyrosine phosphatase reaction in addition to arsenate reduction. these results provide evide ...200111573087
investigation of the yvgw bacillus subtilis chromosomal gene involved in cd(2+) ion resistance.analysis of the complete genome sequence of bacillus subtilis has identified the gene yvgw encoding a protein of 703 amino acids with sequence similarity to the cadmium resistance determinant cada from the staphylococcus aureus plasmid pi258. deletion of yvgw (designated cada) resulted in increased sensitivity of the strain to cadmium. the cada gene is expressed from its own promoter, and its expression is induced by cadmium. northern hybridization analysis showed that cadmium induces the synthe ...200211934502
elucidation of primary (alpha(3)n) and vestigial (alpha(5)) heavy metal-binding sites in staphylococcus aureus pi258 cadc: evolutionary implications for metal ion selectivity of arsr/smtb metal sensor proteins.despite a common evolutionary origin, individual members of the arsr/smtb family of bacterial metal-responsive transcriptional repressors sense a wide range of heavy-metal ions. the molecular basis for this metal ion selectivity is unclear. here, we establish that staphylococcus aureus plasmid pi258 cadc, a cd(ii)/pb(ii)/bi(iii)/zn(ii) sensor, contains two distinct metal-binding sites: a thiolate-rich alpha(3)n site comprised exclusively of cysteine ligands that preferentially binds larger, soft ...200212054863
both metal binding sites in the homodimer are required for metalloregulation by the cadc repressor.the cadca operon of plasmid pi258, which confers resistance to the soft metals cd(ii), pb(ii) and zn(ii), is regulated by cadc, a metal-responsive transcriptional repressor. cadc is a 27.6 kda homodimer composed of two 122-residue monomers. three cysteine residues, cys-7, cys-58 and cys-60, have been shown to be required for sensing soft metals. thus, the repressor has two potential inducer binding sites, one on each monomer. however, it is not known whether both binding sites are required for d ...200212068812
the soft metal ion binding sites in the staphylococcus aureus pi258 cadc cd(ii)/pb(ii)/zn(ii)-responsive repressor are formed between subunits of the homodimer.the staphylococcus aureus plasmid pi258 cadc is a homodimeric repressor that binds cd(ii), pb(ii), and zn(ii) and regulates expression of the cadac operon. cadc binds two cd(ii) ions per dimer, with a tetrathiolate binding site composed of residues cys(7), cys(11), cys(58), and cys(60). it is not known whether each site consists of residues from a single monomer or from residues contributed by both subunits. to examine whether cys(7) and cys(11) are spatially proximate to cys(58) and cys(60) of ...200212176999
specific potassium binding stabilizes pi258 arsenate reductase from staphylococcus aureus.arsenate reductase (arsc) from staphylococcus aureus plasmid pi258 catalyzes the reduction of arsenate to arsenite and plays a role in bacterial heavy metal resistance. the high resolution x-ray structure of arsc reveals the atomic details of the k+ binding site situated next to the catalytic p-loop structural motif of this redox enzyme. a full thermodynamic study of the binding characteristics of a series of monovalent cations (li+, na+, k+, rb+, and cs+) and their influence on the thermal stab ...200312682056
ratiometric pulsed alkylation mass spectrometry as a probe of thiolate reactivity in different metalloderivatives of staphylococcus aureus pi258 cadc.the coordination structure and reactivities of metal ligands in metal-sensing metalloregulatory coordination complexes may well dictate their biological properties. here, we use the technique of ratiometric pulsed alkylation mass spectrometry (rpa-ms) to probe the structure and reactivities of metal coordination complexes formed by different metalloderivatives of staphylococcus aureus plasmid pi258-encoded cadc, the metal-regulated transcriptional repressor of the cad operon. the cad operon prov ...200415049689
crystal structure of the staphylococcus aureus pi258 cadc cd(ii)/pb(ii)/zn(ii)-responsive repressor.the staphylococcus aureus plasmid pi258 cadca operon encodes a p-type atpase, cada, that confers resistance to the heavy metals cd(ii), zn(ii), and pb(ii). expression of this heavy-metal efflux pump is regulated by cadc, a homodimeric repressor that dissociates from the cad operator/promoter upon binding of cd(ii), pb(ii), or zn(ii). cadc is a member of the arsr/smtb family of metalloregulatory proteins. here we report the x-ray crystal structure of cadc at 1.9 angstroms resolution. the dimensio ...200515937183
structural and functional characterization of mycobacterium tuberculosis cmtr, a pbii/cdii-sensing smtb/arsr metalloregulatory repressor.the smtb/arsr family of prokaryotic metalloregulators are winged-helix transcriptional repressors that collectively provide resistance to a wide range of both biologically required and toxic heavy-metal ions. cmtr is a recently described cd(ii)/pb(ii) regulator expressed in mycobacterium tuberculosis that is structurally distinct from the well-characterized smtb/arsr cd(ii)/pb(ii) sensor, staphylococcus aureus plasmid pi258-encoded cadc. from functional analyses and a multiple sequence alignment ...200515966722
development and testing of a green fluorescent protein-based bacterial biosensor for measuring bioavailable arsenic in contaminated groundwater samples.a green fluorescent protein (gfp)-based bacterial biosensor for the detection of bioavailable as(iii), as(v), and sb(iii) was developed and characterized. the biosensor strain escherichia coli dh5alpha (pvlas1) was developed based on the expression of gfp under the control of the ars promoter and the arsr gene of staphylococcus aureus plasmid pi258. strain dh5alpha (pvlas1) responded mainly to as(iii), as(v), and sb(iii), with the lowest detectable concentrations being 0.4, 1, and 0.75 microm, r ...200516050578
assessment of heavy metal bioavailability in contaminated sediments and soils using green fluorescent protein-based bacterial biosensors.a green fluorescent protein (gfp)-based bacterial biosensor escherichia coli dh5alpha (pvlcd1) was developed based on the expression of gfp under the control of the cad promoter and the cadc gene of staphylococcus aureus plasmid pi258. dh5alpha (pvlcd1) mainly responded to cd(ii), pb(ii), and sb(iii), the lowest detectable concentrations being 0.1 nmol l(-1), 10 nmol l(-1), and 0.1 nmol l(-1), respectively, with 2h exposure. the biosensor was field-tested to measure the relative bioavailability ...200616298031
interplay between ion binding and catalysis in the thioredoxin-coupled arsenate reductase family.in the thioredoxin (trx)-coupled arsenate reductase family, arsenate reductase from staphylococcus aureus plasmid pi258 (sa_arsc) and from bacillus subtilis (bs_arsc) are structurally related detoxification enzymes. catalysis of the reduction of arsenate to arsenite involves a p-loop (cys10thr11gly12asn13ser14cys15arg16) structural motif and a disulphide cascade between three conserved cysteine residues (cys10, cys82 and cys89). for its activity, sa_arsc benefits from the binding of tetrahedral ...200616797027
role of bound zn(ii) in the cadc cd(ii)/pb(ii)/zn(ii)-responsive repressor.the staphylococcus aureus plasmid pi258 cadca operon encodes a p-type atpase, cada, that confers resistance to cd(ii)/pb(ii)/zn(ii). expression is regulated by cadc, a homodimeric repressor that dissociates from the cad operator/promoter upon binding of cd(ii), pb(ii), or zn(ii). cadc is a member of the arsr/smtb family of metalloregulatory proteins. the crystal structure of cadc shows two types of metal binding sites, termed site 1 and site 2, and the homodimer has two of each. site 1 is the ph ...200919286656
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