| the effect of plasmid copy number mutations on pt181 replication initiator protein expression. | previous studies have shown that plasmid pt181 controls its replication by countertranscript-mediated regulation of the rate of synthesis of the pt181 initiator, repc. in this study, the relation has been studied between plasmid copy number and repc synthesis for a series of pt181 copy number mutants. for each mutant plasmid, the repc coding sequence along with its 5' regulatory region was translationally fused to the beta-lactamase structural gene on a vector plasmid unrelated to pt181. by mean ... | 1991 | 1924557 |
| mechanism of plasmid pt181 dna replication. | the origin of replication of plasmid pt181 is nicked by the plasmid-encoded repc protein. this nick presumably serves as the start-site of pt181 replication by extension synthesis. in vitro replication of pt181 was found to generate single-stranded dna in addition to the supercoiled, double-stranded dna. the single-stranded dna was circular and corresponded to the pt181 leading strand. in vitro replication of a recombinant plasmid carrying two pt181 origins in direct orientation was shown to gen ... | 1988 | 3061471 |
| the nucleotide sequence of staphylococcus aureus plasmid pt48 conferring inducible macrolide-lincosamide-streptogramin b resistance and comparison with similar plasmids expressing constitutive resistance. | the complete nucleotide sequence of a naturally occurring staphylococcus aureus plasmid, pt48 (from s. aureus strain t48), has been determined. the 2475 bp plasmid confers inducible resistance to macrolide-lincosamide-streptogramin b (mls) type antibiotics. it is similar to the constitutive mls resistance plasmid, pne131, from staphylococcus epidermidis and shows homology with s. aureus plasmids psn2 and pe194. it contains a pala structure homologous to that on s. aureus plasmid pt181. the open ... | 1988 | 3141573 |
| cleavage of single-stranded dna by plasmid pt181-encoded repc protein. | repc protein encoded by plasmid pt181 has single-stranded endonuclease and topoisomerase-like activities. these activities may be involved in the initiation (and termination) of pt181 replication by a rolling circle mechanism. repc protein cleaves the bottom strand of dna within the origin of replication at a single, specific site when the dna is in the supercoiled or linear (double or single-stranded) form. we have found that repc protein will also cleave single-stranded dna at sites other than ... | 1987 | 3588285 |
| the replication initiator protein of plasmid pt181 has sequence-specific endonuclease and topoisomerase-like activities. | initiation of pt181 dna replication specifically requires the plasmid-encoded repc protein. here we demonstrate that highly purified repc protein has sequence-specific endonuclease and topoisomerase-like activities. a maximum sequence of 127 base pairs containing the pt181 origin of replication is required for nicking-closing by repc protein. repc introduces a single strand break within the pt181 origin. the nick site has been shown by dna sequencing to lie between nucleotides 70 and 71 in the b ... | 1985 | 2995991 |
| purification of pt181-encoded repc protein required for the initiation of plasmid replication. | the plasmid pt181 of staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. initiation of pt181 replication specifically requires the plasmid-encoded repc protein. an in vitro system has been shown to carry out semiconservative replication of pt181 and its derivative plasmids (khan, s a., carleton, s. m., and novick, r. p. (1981) proc. natl. acad. sci. u. s. a. 78, 4902-4906). we have used this replication assay to isolate repc protein, which was purified to ne ... | 1985 | 2989292 |
| functional organization of the plasmid pt181 replication origin. | replication of the staphylococcal plasmid pt181 is initiated at the origin (ori) with the introduction of a site-specific nick by the plasmid-encoded initiator protein repc. deletion analysis showed that a sequence of about 70 base-pairs is required for full ori function, including the ability to compete with a co-resident wild-type origin for the trans-acting repc protein. a shorter sequence of 43 base-pairs is sufficient for origin function in the absence of competition. single and double poin ... | 1989 | 2926812 |
| synthesis of single-stranded plasmid pt181 dna in vitro. initiation and termination of dna replication. | the origin of replication of plasmid pt181 is nicked by the plasmid-encoded repc protein. the free 3'-hydroxyl end at the nick is presumably used as primer for leading strand dna synthesis. in vitro replication of pt181 was found to generate single-stranded dna in addition to the supercoiled, double-stranded dna. the single-stranded dna was circular and corresponded to the pt181 leading strand. recombinant plasmids were constructed that contain two pt181 origins of replication in either direct o ... | 1989 | 2910844 |
| staphylococcus aureus chromosomal mutation plac1 amplifies plasmid pt181 by depressing synthesis of its negative-effector countertranscripts. | a staphylococcus aureus chromosomal mutation, plac1, which leads specifically to the amplification of plasmid pt181 has previously been described (s. iordanescu, plasmid 10:130-137, 1983). the mechanism by which plac1 amplifies plasmid pt181 has been approached in two ways: determination of the plasmid region required for the specific response to the plac1 mutation and evaluation of different parameters of pt181 replication control by using transcriptional and translational fusions with the beta ... | 1989 | 2788645 |
| specificity of the interactions between the rep proteins and the origins of replication of staphylococcus aureus plasmids pt181 and pc221. | pt181 and pc221 are closely related staphylococcus aureus plasmids with the same genome organization, which is characterized by the overlapping of the origin of replication with the sequence encoding a protein, rep, essential for plasmid replication. former results have shown the lack of in vivo cross-complementation between these two plasmids, while in vitro studies have revealed the ability of both rep proteins to act on either origin. one possible explanation for this difference was based on ... | 1989 | 2770700 |
| plasmid pt181 replication is regulated by two countertranscripts. | a transcription map of the replication control region of the staphylococcus aureus plasmid pt181 has been constructed. two major leftward transcripts, rna iii and rna iv, start at positions 339 and 413, respectively. these two rnas can serve as mrnas for a plasmid-specific replication protein repc. two short rightward transcripts, rna i and rna ii, approximately 85 and 150 nucleotides long, respectively, start at position 246. these rightward transcripts (referred to as countertranscripts) do no ... | 1985 | 2579377 |
| specificity of repc protein in plasmid pt181 dna replication. | the plasmid pt181 of staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. initiation of pt181 dna replication specifically requires the plasmid-encoded initiator protein, repc. the initiator protein binds specifically to a 32-base pair sequence within the pt181 origin of replication. repc protein also has a nicking-closing activity that is specific for the pt181 origin. replication of pt181 initiates by covalent extension of the nick and proceeds by a rolling ... | 1990 | 2303456 |
| initiation of rolling-circle replication in pt181 plasmid: initiator protein enhances cruciform extrusion at the origin. | plasmid pt181 dna secondary structures have been analyzed in vitro by nuclease s1 digestion and in vivo by bromoacetaldehyde treatment. a cruciform structure occurring at the pt181 replication origin in vitro is greatly enhanced by the binding of the plasmid-encoded initiator protein repc. in vivo a dna secondary structure also existed in the replication origin. its frequency of formation was correlated with efficiency of repc utilization. these data suggest that cruciform extrusion at the origi ... | 1990 | 2236066 |
| in vitro studies of the initiation of staphylococcal plasmid replication. specificity of repd for its origin (orid) and characterization of the rep-ori tyrosyl ester intermediate. | several staphylococcal plasmids from different incompatibility (inc) groups which replicate by a rolling circle mechanism each specify a replication initiator protein (rep) which is homologous with that of the inc3 tetracycline resistance plasmid pt181. the rep gene sequences of six pt181-like plasmids are known, each encoding proteins of molecular mass 38 kda with 62% overall amino acid sequence identity. the initiation of replication in vivo by each of the rep proteins is plasmid specific, act ... | 1990 | 2156820 |
| the staphylococcus aureus mutation pcra3 leads to the accumulation of pt181 replication initiation complexes. | in staphylococcus aureus cells carrying the pcra3 chromosomal mutation, plasmid pt181 and its derivatives were maintained at a reduced copy number. a significant proportion of their dna migrated during agarose gel electrophoresis as nicked dna. the results obtained in the characterization of this plasmid dna species show that it represents replication initiation complexes. such complexes could not be detected in a wild-type host. the replication initiation complexes present in pcra3 cells could ... | 1991 | 1942047 |
| the staphylococcus aureus chromosomal gene plac, identified by mutations amplifying plasmid pt181, encodes a sigma factor. | the staphylococcus aureus chromosomal gene plac, identified by mutations such as plac1 that lead to the amplification of plasmid pt181, has been cloned and sequenced. the plac gene encodes a protein with high similarity (79% identity) with the vegetative sigma factor of bacillus subtilis, siga, suggesting that it acts as an rna polymerase sigma factor in s.aureus. the plac1 mutation was found to be a c to t transition leading to a proline to serine substitution at amino acid residue 209 of the p ... | 1991 | 1923759 |
| molecular structure of the replication origin of a bacillus subtilis (natto) plasmid, puh1. | the structure of a 2.0-kb bsteii dna sequence necessary and sufficient for the replication of a 5.7-kb natto plasmid, puh1, which is responsible for gamma-polyglutamate production by bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from b. subtilis chromosomal dna as a selective marker. the 2.0-kb dna sequence contains an open reading frame, rep, stretching for 999 bp; a promoter region for rep expression; and a possible replication origin for the ... | 1991 | 1840479 |
| replication of single-stranded plasmid pt181 dna in vitro. | plasmid pt181 is a 4437-base-pair, multicopy plasmid of staphylococcus aureus that encodes tetracycline resistance. the replication of the leading strand of pt181 dna initiates by covalent extension of a site-specific nick generated by the initiator protein at the origin of replication and proceeds by an asymmetric rolling circle mechanism. the origin of the leading strand synthesis also serves as the site for termination of replication. replication of pt181 dna in vivo and in vitro has been sho ... | 1992 | 1729700 |
| mutational and physiological analyses of plasmid pt181 functions expressing incompatibility. | plasmid pt181 is a small multicopy plasmid from staphylococcus aureus that belongs to incompatibility group 3 and expresses two distinct types of incompatibility, inc3a and inc3b. inc3a incompatibility is expressed by the primary replication control determinant, copa, which specifies two small transcripts, rna i and rna ii, that jointly inhibit the synthesis of the rate-limiting initiator protein, repc. inc3b incompatibility is expressed by the leading strand replication origin and is due to com ... | 1990 | 1693440 |
| sequence analysis of replication origin of plasmid pls11 of bacillus subtilis ifo 3022. | the structure of a 1.6-kb sphi-hindiii dna sequence necessary and sufficient for the replication of a 8.6-kb plasmid pls11 of bacillus subtilis ifo 3022, which is responsible for gamma-polyglutamate production, has been characterized by using a trimethoprim (tmp)-resistance gene derived form b. subtilis ttk24 chromosomal dna as a selective marker. the 1.6-kb dna sequence contains a rep gene encoding the protein (333 amino acids) essential for initiation of replication and a possible origin of re ... | 1992 | 1368298 |
| an 18-base-pair sequence is sufficient for termination of rolling-circle replication of plasmid pt181. | pt181 and related plasmids of gram-positive bacteria replicate by a rolling-circle mechanism. the replication initiator protein of pt181, repc, has origin-specific nicking-closing activities. replication of the plasmid pt181 leading strand initiates by covalent extension of the repc-generated nick, and the origin of replication contains signals for both initiation and termination of dna replication. we have investigated the sequence requirements for the initiation and termination steps by using ... | 1996 | 8752341 |
| the use of synthetic oligonucleotides with universal templates for rapid dna sequencing: results with staphylococcal replicon pc221. | a rapid sequencing strategy has been devised and applied to determine the complete nucleotide sequence (4555 bp) of staphylococcus aureus plasmid pc221. the entire replicon was cloned into phage m13mp8 in both orientations to provide 'universal templates' for primed dna synthesis from internally-sited oligonucleotide primers. the latter were synthesized by a modification of a recently described paper disc method which employs phosphotriester chemistry. less than 4 weeks was required for the synt ... | 1985 | 3860383 |
| static and initiator protein-enhanced bending of dna at a replication origin. | dna bending has been suggested to play a role in the regulation of gene expression, initiation of dna replication, dna packaging, and the recognition of specific dna sequences by proteins. it has recently been demonstrated that dna bending can be sequence-directed. bent dna has also been observed as a consequence of sequence-specific binding of proteins to dna. in this report dna of plasmid pt181 is shown to contain a bend at the replication origin. furthermore, this bend is enhanced by the bind ... | 1986 | 3749879 |
| the cloning of chromosomal dna associated with methicillin and other resistances in staphylococcus aureus. | competitive hybridization was used to detect the deletion of chromosomal dna accompanying the loss of resistance to methicillin (and concomitantly, to cadmium, mercury and tetracycline) from a clinical strain of methicillin-resistant staphylococcus aureus (mrsa). the method was also used to screen a partial plasmid library of chromosomal hindiii fragments from the mrsa strain. eight recombinant plasmid clones were identified as containing dna included in the deletion. these clones were used as p ... | 1987 | 3668502 |
| effect of the deletion of a fragment dispensable for the autonomous maintenance of plasmid pt181 on the competition between incompatible plasmids. | the deletion of the 560-bp hindiii c fragment from pt181 derivatives does not change the stability or copy number of the plasmid but affects its ability to compete with undeleted, incompatible plasmids for maintenance in the host cell. the disadvantage of the deleted plasmids seems to be manifested at the level of replication. it results that for plasmid pt181 a sequence dispensable for autonomous maintenance and replication control could affect the outcome of the competition between autonomous, ... | 1986 | 3520616 |
| the inc3b determinant of plasmid pt181. a mutational analysis. | a region encompassing the origin of replication of staphylococcal plasmid pt181 has previously been shown to express an incompatibility effect denoted inc3b, when cloned into another replicon (novick et al. 1984). in an attempt to understand the mechanism of this incompatibility effect, and its relationship with the function of the replication origin, mutants deficient in this property were isolated and characterized. the results obtained suggest that the inc3b effect is due to the competition f ... | 1987 | 3474496 |
| sequence-specific interaction between the replication initiator protein of plasmid pt181 and its origin of replication. | the replication of the pt181 plasmid is dependent on the plasmid-encoded initiator protein repc. we have previously shown that repc protein has sequence-specific endonuclease and topoisomerase-like activities. in this paper we demonstrate that this initiator protein has sequence-specific dna-binding properties. based on filter binding of plasmid restriction fragments, repc protein specifically recognizes only the pt181 origin region. using dnase i and neocarzinostatin "footprinting" techniques, ... | 1986 | 3461445 |
| intermediates in plasmid pt181 dna replication. | staphylococcus aureus plasmid pt181 is thought to replicate via an asymmetric rolling-circle mechanism. by studying pulse labeled replicative intermediates, here we report that pt181 replication involves: (1) a post-replicative hypersupercoiled monomer and (2) a partially replicated intermediate which lacks superhelicity but is unlike a typical rolling-circle intermediate in that only nascent strands of less than unit length are released by alkali denaturation. a model for pt181 replication is p ... | 1988 | 3368310 |
| an enhancer of dna replication. | cmp, a nucleotide sequence element in the plasmid pt181 of staphylococcus aureus, acts as an enhancer of dna replication. when cmp is present on an unrelated vector along with the pt181 origin of replication, it increases the ability of the linked pt181 origin to compete with a coresident pt181 plasmid for the initiator protein repc. cmp is contained within a 156-base-pair segment, and its deletion from pt181 reduces by twofold the frequency of plasmid replication under derepressed conditions. t ... | 1988 | 3192513 |
| characterization of the tetracycline resistance gene of plasmid pt181 of staphylococcus aureus. | some genetic and biochemical properties of the tetracycline resistance element of the staphylococcus aureus plasmid pt181 have been studied. resequencing of a portion of the tetracycline resistance gene (tet) showed the presence of a single open reading frame of 1,299 nucleotides capable of encoding a polypeptide of 433 amino acids. analysis of bal 31 nuclease-generated deletion mutants of the tet gene showed the presence of two complementation groups within this region. northern blot hybridizat ... | 1988 | 3142848 |
| sequence analysis of a cryptic plasmid from flavobacterium sp. kp1, a psychrophilic bacterium. | a cryptic plasmid found at high copy number was isolated from flavobacterium sp. kp1, a psychrophilic gram-negative bacterium, cloned, and sequenced. the sequence will appear in the ddbj/embl/genbank databases under the accession number ab007196. the pfl1 plasmid is 2311 nucleotides in length with 32.7% gc content, and shows a distinctive nucleotide sequence without homology to other plasmids of similar length. the plasmid contains two open reading frames of significant length, orfi and orfii. o ... | 1999 | 9919674 |
| control of pt181 replication ii. mutational analysis. | we describe the isolation and analysis of mutations affecting the regulation of staphylococcus aureus plasmid pt181 replication. previous results suggested that regulation is achieved by control of the synthesis of repc, a plasmid-coded replication protein and that the primary negative control element is copa rna, which consists of two transcripts that are complementary to the 5' region of the repc mrna leader. copa inhibition probably involves a base pairing interaction with the complementary r ... | 1984 | 6437809 |
| reciprocal intrapool variation in plasmid copy numbers: a characteristic of segregational incompatibility. | an experimental analysis of the concept that incompatible plasmids occupy a common intracellular pool from which copies are drawn at random for replication and assortment is presented. intrapool variations in an incompatible heteroplasmid strain are inevitable and it is shown that these variations can be exploited by differential selection to amplify one plasmid at the expense of the other. constant overall copy number is demonstrated for isogenic wild-type replicons and also for isogenic copy m ... | 1984 | 6494316 |
| staphylococcus aureus chromosomal mutation specifically affecting the copy number of inc3 plasmids. | a chromosomal mutation leading to an important increase in the copy number of plasmid pt181 and its derivatives has been isolated from staphylococcus aureus strain 8325. the amplification effect in the mutant strain sa1350 was found to be specific for plasmids of the inc3 group, to which belongs pt181. there are some other differences in the behavior of inc3 plasmids between sa1350 and 8325, including stable maintenance in sa1350 at high copy number of temperature-sensitive replication mutants a ... | 1983 | 6635012 |
| replication of plasmid pt181 dna in vitro: requirement for a plasmid-encoded product. | pt181 is a naturally occurring 4.5-kilobase staphylococcus aureus plasmid encoding resistance to tetracycline. the plasmid has a copy number of about 20 per cell; a mutant, cop-608, that has a copy number of 800-1000 has been isolated. a cell-free extract has been developed that carries out complete replication of this plasmid. extracts made from a strain containing the mutant have much greater replication activity than do extracts of strains containing pt181. in an extract from which endogenous ... | 1981 | 6946436 |
| control of replication of the lactobacillus pentosus plasmid p353-2: evidence for a mechanism involving transcriptional attenuation of the gene coding for the replication protein. | the synthesis of plasmid dna and of rna encoded by the replication protein gene (rep) of plasmid p353-2 of lactobacillus pentosus was studied for the wild-type plasmid and for a mutant plasmid with a deletion in the 5' untranslated region of the rep gene. plasmid p353-2 codes for two countertranscript rnas (ct-rna) of approximately 75 and 250 nucleotides transcribed from the 5' untranslated region of the rep gene, in opposite directions. in a mutant plasmid with a deletion of the promoter and pa ... | 1994 | 7510019 |
| plasmid pt181 replication is decreased at high levels of repc per plasmid copy. | the replication of staphylococcal plasmid pt181 is indirectly controlled at the level of the synthesis of its replication initiator, repc. as a result, high levels of repc synthesis per plasmid copy were expected to lead to autocatalytic plasmid replication, which secondarily would affect host physiology. surprisingly, repc overexpression was found to lead to a rapid decrease in pt181 copy number and replication rate. these effects depended on the ratio of repc to the pt181 replication origin ra ... | 1995 | 7565108 |
| a bacillus anthracis-based in vitro system supports replication of plasmid pxo2 as well as rolling-circle-replicating plasmids. | capsule-encoding virulence plasmid pxo2 of bacillus anthracis is predicted to replicate by a unidirectional theta-type mechanism. to gain a better understanding of the mechanism of replication of pxo2 and other plasmids in b. anthracis and related organisms, we have developed a cell-free system based on b. anthracis that can faithfully replicate plasmid dna in vitro. the newly developed system was shown to support the in vitro replication of plasmid pt181, which replicates by the rolling-circle ... | 2007 | 17575005 |
| replication-specific conversion of the staphylococcus aureus pt181 initiator protein from an active homodimer to an inactive heterodimer. | the staphylococcus aureus rolling circle plasmid pt181 regulates its replication by controlling the synthesis of its initiator protein repc. repc is inactivated during pt181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, repc*. we analyzed repc and repc* in four classes of mutants: plasmid copy number mutants, two classes of repc mutants affecting different portions of the protein and oric (origin) mutants. we have found that in the cell with wild-type repc th ... | 1994 | 7957090 |
| is257 and small plasmid insertions in the mec region of the chromosome of staphylococcus aureus. | four copies of the insertion sequence is257 are found in the mec region of the chromosome of the australian methicillin resistant staphylococcus aureus (mrsa) strain ans46, two flanking a meramerb sequence (encoding resistance to mercurial compounds), the other two flanking an integrated copy of the plasmid pt181 (tetracycline resistance). the termini of the integrated copy of the plasmid pt181 carry a direct repeat of 8 bp of plasmid sequence, but otherwise there are no similarities in the 8 bp ... | 1994 | 8171122 |
| tetracycline resistance genes in staphylococci from the skin of pigs. | forty-seven tetracycline-resistant staphylococci from the skin of pigs were examined for genes mediating this resistance. seventeen isolates were also resistant to minocycline and all hybridized with the tet(m) gene; 23 carried the tet(k) gene and 10 the tet(l) gene. three carried more than one gene and two did not hybridize with any of the three probes tested. maps were constructed for two plasmids carrying the tet(k) gene, all were very similar in size (4.35-4.7 kb) and structure and closely r ... | 1994 | 8200858 |
| characterization of the staphylococcus aureus chromosomal gene pcra, identified by mutations affecting plasmid pt181 replication. | the staphylococcus aureus chromosomal gene pcra, identified by mutations, such as pcra3, that affect plasmid pt181 replication, has been cloned and sequenced. the pcra gene encodes a protein with significant similarity (40% identity) to two escherichia coli helicases: the helicase ii encoded by the uvrd gene and the rep helicase. the pcra3 mutation was found to be a c to t transition leading to a threonine to isoleucine substitution at amino acid residue 61 of the protein. the pcra gene seems to ... | 1993 | 8232203 |
| the tet(k) gene of plasmid pt181 of staphylococcus aureus encodes an efflux protein that contains 14 transmembrane helices. | the corrected dna sequence of the tet(k) structural gene, encoding the tetracycline efflux protein from staphylococcus aureus, is reported. the tet(k) protein is homologous to related tetracycline efflux proteins throughout the protein, including at the c-terminal end. hydropathy plotting now clearly indicates that the tet(k) protein contains 14 transmembrane alpha-helices. | 1993 | 8234490 |
| replication-specific inactivation of the pt181 plasmid initiator protein. | replication of the staphylococcus aureus plasmid pt181, which occurs by the rolling circle mechanism, is accompanied by the covalent attachment of a approximately 12-residue oligodeoxy-nucleotide to one subunit of the dimeric plasmid-coded initiator protein, repc. this oligonucleotide represents the plasmid sequence immediately 3' to the initiating nick site. the resulting heterodimeric protein lacks the topoisomerase and replication activities of unmodified repc, suggesting that the regulation ... | 1993 | 8235621 |
| sequence and structure of cmp, the replication enhancer of the staphylococcus aureus plasmid pt181. | the staphylococcus aureus plasmid pt181 possesses a dna replication enhancer element, called cmp, that is required in cis for optimal utilization of the initiator protein by the origin of replication. the minimal nucleotide sequence required for cmp activity was defined by testing progressively smaller dna fragments for their ability to restore cmp activity in a plasmid mutant deleted for cmp. these experiments indicate that cmp is a sequence of 100 base pairs (bp) characterized by a loosely rep ... | 1993 | 8244037 |
| is257-mediated cointegration in the evolution of a family of staphylococcal trimethoprim resistance plasmids. | analyses of the staphylococcus epidermidis multiresistance plasmids psk697 and psk818 have revealed them to be closely related to the trimethoprim resistance plasmid psk639, also isolated from s. epidermidis. psk697 and psk818 were found to contain a cointegrated copy of a second plasmid related to the s. epidermidis multidrug antiseptic and disinfectant resistance plasmid psk108 and the s. aureus tetracycline resistance plasmid pt181, respectively. in contrast to psk639, both plasmids were foun ... | 1996 | 8830710 |
| integration of pt181-like tetracycline resistance plasmids into large staphylococcal plasmids involves is257. | four large staphylococcal plasmids ranging in size from 31 to 82 kbp have been shown to mediate tetracycline resistance via an integrated copy of the tet(k)-encoding plasmid pt181 which was flanked by copies of the insertion element is257. in two cases, is257 elements interrupted the repc reading frame of pt181 and an 8-bp sequence from within the repc gene was duplicated at the interrupted site. in the third plasmid, the is257 elements interrupted the pt181 dna immediately upstream of the repc ... | 1996 | 8913460 |
| the inactive pt181 initiator heterodimer, repc/c, binds but fails to induce melting of the plasmid replication origin. | staphylococcus aureus plasmid pt181 replicates via a rolling circle mechanism. the synthesis of the pt181 initiator protein (repc) is regulated by antisense rnas, and repc is inactivated after usage by the attachment of an oligonucleotide to one of its subunits. the inactivated heterodimeric repc/c* has been shown be unable to initiate replication in vitro (rasooly, a., and novick, r. p. (1993) science 262, 1048-1050). the inactive repc/c* has been found to be very stable and constitute about 90 ... | 1996 | 8940104 |
| binding of a novel host factor to the pt181 plasmid replication enhancer. | replication enhancers are cis-acting genetic elements that stimulate the activity of origins of dna replication. the enhancer found in plasmid pt181 of staphylococcus aureus, called cmp, functions at a distance of 1 kb from the origin of dna replication to stimulate the interaction between the replication initiation protein and the origin. dna encoding cmp-binding activity was isolated by screening an expression library of s. aureus dna in escherichia coli, and a novel gene, designated cbf1, was ... | 1997 | 9006021 |
| the inactivated plasmid inititator protein repc/repc* may have a regulatory role. | during replication of the plasmid pt181, the initiator protein repc is modified by the addition of an oligodeoxynucleotide, giving rise to a new form, repc*. here we show that during in vitro replication, repc* is radioactively labeled, suggesting that the source of the repc* oligodeoxynucleotide is the newly synthesized pt181 dna. the repc/repc* heterodimer retains its ability to bind the pt181 double-strand origin and, therefore, it may act as a competitive inhibitor of the repc homodimer duri ... | 1997 | 9023233 |
| sequence requirements for the termination of rolling-circle replication of plasmid pt181. | most small multicopy plasmids of gram-positive bacteria and many in gram-negative bacteria replicate by a rolling-circle (rc) mechanism. the replication initiator proteins encoded by the rc plasmids and single-stranded bacteriophages of escherichia coli have origin-specific nicking-closing activities that are required for the initiation and termination of rc replication. we have investigated the sequence requirements for termination of rc replication of plasmid pt181. the initiator nick site is ... | 1997 | 9179847 |
| double-stranded origin nicking and replication initiation are coupled in the replication of a rolling circle plasmid, pt181. | the staphylococcus aureus rolling circle plasmid pt181 initiator repc is modified by the addition of an oligodeoxynucleotide, giving rise to a new form, repc*. repc/repc* heterodimer is an inhibitor of replication. however, in order to act effectively, the initiator/inhibitor protein must be stable. we show here that repc is stable for at least 90 min, which enables it to function effectively as an inhibitor of replication. this finding also allowed us to carry out the two stages in pt181 replic ... | 1997 | 9228752 |
| pcra is an essential dna helicase of bacillus subtilis fulfilling functions both in repair and rolling-circle replication. | the only dna helicase essential for escherichia coli viability is dnab, the chromosome replication for helicase. in contrast, in bacillus subtilis, in addition to the dnab counterpart called dnac, we have found a second essential dna helicase, called pcra. it is 40% identical to the rep and uvrd dna helicases of e. coli and 61% identical to the pcra helicase of staphylococcus aureus. this gene is located at 55 degree on the chromosome and belongs to a putative operon together with a ligase gene ... | 1998 | 9701819 |
| antisense rna-mediated transcriptional attenuation: an in vitro study of plasmid pt181. | antisense rnas regulate plasmid replication by several different mechanisms. one of these mechanisms, transcriptional attenuation, was first described for the staphylococcal plasmid pt181, and later for the streptococcal plasmids pip501 and pambeta1. previously, we performed detailed in vitro and in vivo analyses of the pip501 system. here, we present an in vitro analysis of the antisense system of plasmid pt181. the secondary structures of antisense and sense rna species of different lengths we ... | 2000 | 10760147 |
| biochemical characterization of the staphylococcus aureus pcra helicase and its role in plasmid rolling circle replication. | previous genetic studies have suggested that a putative chromosome-encoded helicase, pcra, is required for the rolling circle replication of plasmid pt181 in staphylococcus aureus. we have overexpressed and purified the staphylococcal pcra protein and studied its biochemical properties in vitro. purified pcra helicase supported the in vitro replication of plasmid pt181. it had atpase activity that was stimulated in the presence of single-stranded dna. unlike many replicative helicases, pcra was ... | 2002 | 12244110 |
| dna-protein interactions during the initiation and termination of plasmid pt181 rolling-circle replication. | initiation of dna replication requires the generation of a primer at the origin of replication that can be utilized by a dna polymerase for dna synthesis. this can be accomplished by several means, including the synthesis of an rna primer by a dna primase or rna polymerase, by nicking of one strand of the dna to generate a free 3'-oh end that can be used as a primer, and by the utilization of the oh group present in an amino acid such as serine within an initiation protein as a primer. furthermo ... | 2003 | 14604011 |
| bacillus anthracis and bacillus cereus pcra helicases can support dna unwinding and in vitro rolling-circle replication of plasmid pt181 of staphylococcus aureus. | replication of rolling-circle replicating (rcr) plasmids in gram-positive bacteria requires the unwinding of initiator protein-nicked plasmid dna by the pcra helicase. in this report, we demonstrate that heterologous pcra helicases from bacillus anthracis and bacillus cereus are capable of unwinding staphylococcus aureus plasmid pt181 from the initiator-generated nick and promoting in vitro replication of the plasmid. these helicases also physically interact with the repc initiator protein of pt ... | 2004 | 15028705 |
| the pcra3 mutant binds dna and interacts with the repc initiator protein of plasmid pt181 but is defective in its dna helicase and unwinding activities. | plasmid rolling-circle replication initiates by covalent extension of a nick generated at the plasmid double-strand origin (dso) by the initiator protein. the repc initiator protein binds to the plasmid pt181 dso in a sequence-specific manner and recruits the pcra helicase through a protein-protein interaction. subsequently, pcra unwinds dna at the nick site followed by replication by dna polymerase iii. the pcra3 mutant of staphylococcus aureus has previously been shown to be defective in plasm ... | 2005 | 16122559 |
| genetic and biochemical characterization of the streptococcus pneumoniae pcra helicase and its role in plasmid rolling circle replication. | pcra is a chromosomally encoded dna helicase of gram-positive bacteria involved in replication of rolling circle replicating plasmids. efficient interaction between pcra and the plasmid-encoded replication initiator (rep) protein is considered a requirement for the plasmid to replicate in a given host, and thus, the ability of a rep protein to interact with heterologous pcra helicases has been invoked as a determinant of plasmid promiscuity. we characterized transcription of the streptococcus pn ... | 2006 | 16936036 |
| a novel type-iii staphylococcal cassette chromosome mec (sccmec) variant among indian isolates of methicillin-resistant staphylococcus aureus. | we identified a novel type-iii staphylococcal cassette chromosome mec (sccmec) element carried by eight methicillin-resistant staphylococcus aureus (mrsa) strains from different wards and patients in an indian hospital. although the pulsed-field gel electrophoresis pattern and spa types of eight strains were identical and clonally related to other nosocomial indian isolates that belonged to sequence type (st) 239 and spa type t037, the minimum inhibitory concentration (mic) of these eight varian ... | 2009 | 19187210 |
| versatile rna-sensing transcriptional regulators for engineering genetic networks. | the widespread natural ability of rna to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. previous work in rna synthetic biology has engineered rna mechanisms that independently regulate multiple targets and integrate regulatory signals. however, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. in this work, we ... | 2011 | 21555549 |
| plasmid pt181-linked suppressors of the staphylococcus aureus pcra3 chromosomal mutation. | plasmid pt181 replication is affected in hosts carrying the chromosomal pcra3 mutation, resulting in significantly lower plasmid copy numbers. mutations suppressing this effect have been isolated and characterized. the suppressor mutations were found to map in the plasmid repc gene and manifested pcra allele specificity, suggesting the existence of a direct repc-pcra interaction. | 1993 | 8509346 |
| genetic evidence for replication enhancement from a distance. | rolling circle replication of the staphylococcus aureus plasmid pt181 requires interaction of the repc initiator protein with the origin of replication (the ori site). a second site named cmp, which is distant from ori, is thought to stimulate replication, since a mutant pt181 plasmid lacking cmp cannot coexist with a cmp+ wild-type plasmid. second-site mutations compensating for the loss of cmp were shown to map in repc. the compensatory mutations produced repc proteins that, unlike the wild-ty ... | 1993 | 8516296 |
| countertranscript-driven attenuation system of the pam beta 1 repe gene. | the plasmid-encoded repe protein is absolutely essential and rate-limiting for replication of the promiscuous plasmid pam beta 1 originating from enterococcus faecalis. we previously showed that the rep gene is transcribed from a promoter that is negatively regulated (approximately 10-fold reduction) by the copf repressor. in this report, we show that this transcription is decreased a further approximately 10-times by a countertranscript-driven transcriptional attenuation system. extensive mutag ... | 1996 | 8809762 |