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 highly active as a 5' --> 3' helicase and had a weaker 3' --> 5' helicase activity. The RepC initiator protein encoded by pT181 nicks at the origin of replication and becomes covalently attached to the 5' end of the DNA. The 3' OH end at the nick then serves as a primer for displacement synthesis. PcrA helicase showed an origin-specific unwinding activity with supercoiled plasmid pT181 DNA that had been nicked at the origin by RepC. We also provide direct evidence for a protein-protein interaction between PcrA and RepC proteins. Our results are consistent with a model in which the PcrA helicase is targeted to the pT181 origin through a protein-protein interaction with RepC and facilitates the movement of the replisome by initiating unwinding from the RepC-generated nick.     

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 plasmid pT181 replication. Suppressor mutations in the repC initiator gene have been isolated that allow pT181 replication in the pcrA3 mutant. One such suppressor mutant contains a D57Y change in the RepC protein. To identify the nature of the defect in PcrA3, we have purified this mutant protein and studied its biochemical activities. Our results show that while PcrA3 retains its DNA binding activity, it is defective in its helicase and RepC-dependent pT181 DNA unwinding activities. We have also purified the RepC D57Y mutant and shown that it is similar in its biochemical activities to wild-type RepC. RepC D57Y supported plasmid pT181 replication in cell-free extracts made from wild-type S. aureus but not from the pcrA3 mutant. We also demonstrate that both wild-type RepC and its D57Y mutant are capable of a direct physical interaction with both wild-type PcrA and the PcrA3 mutant. Our results suggest that the inability of PcrA3 to support pT181 replication is unlikely to be due to its inability to interact with RepC. Rather, it is likely that a defect in its helicase activity is responsible for its inability to replicate the pT181 plasmid.     

Plasmid replication initiator protein RepD increases the processivity of PcrA DNA helicase

The replication initiator protein RepD encoded by the Staphylococcus chloramphenicol resistance plasmid pC221 stimulates the helicase activity of the Bacillus stearothermophilus PcrA DNA helicase in vitro. This stimulatory effect seems to be specific for PcrA and differs from the stimulatory effect of the Escherichia coli ribosomal protein L3. Whereas L3 stimulates the PcrA helicase activity by promoting co-operative PcrA binding onto its DNA substrate, RepD stimulates the PcrA helicase activity by increasing the processivity of the enzyme and enables PcrA to displace DNA from a nicked substrate. The implication of these results is that PcrA is the helicase recruited into the replisome by RepD during rolling circle replication of plasmids of the pT181 family.     

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 fork 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° on the chromosome and belongs to a putative operon together with a ligase gene ( lig ) and two unknown genes named pcrB and yerH . As PcrA was essential for cell viability, conditional mutants were constructed. In such mutants, chromosomal DNA synthesis was slightly decreased upon PcrA depletion, and rolling-circle replication of the plasmid pT181 was inhibited. Analysis of the replication intermediates showed that leading-strand synthesis of pT181 was prevented upon PcrA depletion. To compare PcrA with Rep and UvrD directly, the protein was produced in rep and uvrD mutants of E. coli . PcrA suppressed the UV sensitivity defect of a uvrD mutant but not its mutator phenotype. Furthermore, it conferred a Rep⁻ phenotype on E. coli . Altogether, these results show that PcrA is an helicase used for plasmid rolling-circle replication and suggest that it is also involved in UV repair.     

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 plasmids containing two pT181 origins. In vitro replication experiments showed that 18- and 24-bp synthetic oligonucleotides containing the RepC nick site were active in the termination of replication. However, initiation of replication required a larger region which also includes the RepC binding site. Plasmids containing the 18- and 24-bp region were also found to be nicked by the RepC protein. Our results demonstrate that sequence requirements for initiation and termination of pT181 replication overlap, but while the RepC binding site is required for initiation, it is dispensable for termination.     

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 lo the PT181 replication origin rather than on the absolute amount of RepC in the cell. In a wild-type host, the increase in RepC/plasmid copy also inhibited chromosome replication and cell division. The changes in host physiology did not play any role in the decrease in pT181 replication caused by RepC overexpression since pT181 replication responded in the same way in a host mutant insensitive to the effects of RepC induction. These results suggest that pT181, the prototype of an entire class of plasmids from Gram-positive bacteria, responds to overexpression of its replication initiator by a decrease in plasmid replication.     

The beta-propeller protein YxaL increases the processivity of the PcrA helicase

The DNA helicase PcrA is found in gram-positive bacteria and belongs to the superfamily 1 (SF1) of helicases, together with Rep and UvrD helicases from Escherichia coli. These helicases have been extensively studied in vitro and their mode of unwinding are well characterised. However, little is known about the putative cellular partners of such helicases. To identify PcrA-interacting factors, PcrA was used as a bait in a genome-wide yeast two-hybrid screen of a Bacillus subtilis library. Three proteins with unknown functions - YxaL, YwhK and YerB - were found to interact specifically with PcrA. The yxaL gene was cloned, the product was overexpressed and purified, and its effect on the PcrA activity was investigated in vitro. YxaL enhanced the processivity of the PcrA helicase. A comparison of the amino acid sequence of YxaL with other proteins from data banks suggests that YxaL belongs to a family of proteins with a repeated domain, which adopt a typical three-dimensional structure designated as a "beta-propeller". This raises the possibility that YxaL acts as a connector protein between PcrA and another cellular component.     

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 circle mechanism. Two other small, multicopy plasmids pC221 and pS194 belong to the pT181 family and have common structural organization and replication properties. The replication proteins and replication origins of these plasmids have extensive sequence homologies, although they belong to different incompatibility groups. In spite of this homology, the replication proteins and replication origins of these three plasmids do not show any cross-reactivity in vivo. We have carried out a series of in vitro experiments to determine the specificity of pT181-encoded initiator protein, RepC. DNA binding experiments showed that although the binding of RepC to the pT181 origin was very efficient, little or no binding was seen with pC221 and pS194 origins. The nicking-closing activity of RepC was found to be equally efficient with the pC221 and pS194 plasmids. The plasmids pC221 and pS194 replicated efficiently in a RepC-dependent in vitro system. However, replication of these plasmids was greatly reduced in the presence of a competing pT181 origin. The results presented here suggest that nicking-closing by RepC at the origin is not sufficient for maximal replication and that tight binding of RepC to the origin plays an important role in the initiation of DNA replication.     

Plasmids of the pT181 family show replication-specific initiator protein modification.

The rolling circle plasmids of Staphylococcus aureus regulate their replication by controlling initiator (Rep) protein synthesis. It was demonstrated recently that the pT181 initiator protein RepC is inactivated during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC* (A. Rasooly and R. P. Novick, Science, 262:1048-1050). We establish here that this initiator modification occurs with four other members of the pT181 family and that it occurs in Bacillus subtilis as well as S. aureus. These results suggest that Rep conversion to Rep* is probably universal among plasmids of the pT181 family and is not host dependent.     

Plasmid rolling-circle replication: highlights of two decades of research

This review provides a historical perspective of the major findings that contributed to our current understanding of plasmid rolling-circle (RC) replication. Rolling-circle-replicating (RCR) plasmids were discovered approximately 20 years ago. The first of the RCR plasmids to be identified were native to Gram-positive bacteria, but later such plasmids were also identified in Gram-negative bacteria and in archaea. Further studies revealed mechanistic similarities in the replication of RCR plasmids and the single-stranded DNA bacteriophages of Escherichia coli, although there were important differences as well. Three important elements, a gene encoding the initiator protein, the double strand origin, and the single strand origin, are contained in all RCR plasmids. The initiator proteins typically contain a domain involved in their sequence-specific binding to the double strand origin and a domain that nicks within the double strand origin and generates the primer for DNA replication. The double strand origins include the start-site of leading strand synthesis and contain sequences that are bound and nicked by the initiator proteins. The single strand origins are required for synthesis of the lagging strand of RCR plasmids. The single strand origins are non-coding regions that are strand-specific, and contain extensive secondary structures. This minireview will highlight the major findings in the study of plasmid RC replication over the past twenty years. Regulation of replication of RCR plasmids will not be included since it is the subject of another review.     

Genetic relationship among Bacillus licheniformis rolling-circle-replicating plasmids and complete nucleotide sequence of pBL63.1, an atypical replicon

The degree of biodiversity among Bacillus licheniformis plasmids and their relation to other Bacillus subtilis group plasmids has been evaluated. To attain this goal we surveyed the diversity and linkage of replication modules in a collection of 21 naturally occurring plasmids of B. licheniformis strains, isolated from different geographical areas. On the basis of rep gene sequence analysis it was possible to group the B. licheniformis plasmids rep genes in two main cluster. Comparison with known rep genes from Bacillus rolling-circle-replicating (RCR) plasmids revealed the presence in B. licheniformis plasmids of replication genes with a DNA sequence peculiar to B. licheniformis species together with rep genes with a very high sequence similarity to B. subtilis plasmids. Furthermore, the molecular organization of an atypical replicon, pBL63.1, was shown. This plasmid did not display any significant similarity with known Bacillus RCR plasmids. The complete nucleotide sequence evidenced a replication module with an unexpected similarity with Rep proteins from RCR plasmids of bacterial species phylogenetically distantly related to Bacillus. pBL63.1 represents an exception to the low-level diversity hypothesis among Bacillus RC replicons.     

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 located in the loop of a hairpin region (IRII) within the pT181 origin of replication. By mutational analysis, we have found that several nucleotides within the stem of IRII which are critical for the initiation activity are dispensable for termination of replication. We also demonstrate that nucleotides in the right arm of IRII, but not the left arm, are absolutely required for termination of RC replication. We have also identified specific nucleotides in IRII that are critical for its termination activity. The sequence of the right arm of the hairpin must be located downstream of the initiator nick site for termination, suggesting that termination requires a specific orientation of the initiator protein at the origin.     

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, acting in trans only at the cognate replication origin (ori) of the encoding plasmid. Previous studies in vitro of the RepC protein of pT181 demonstrated replication initiator, topoisomerase-like, and DNA binding activities, which appeared to be specific for the origin (oriC) of pT181 when compared with unrelated staphylococcal plasmids. Although RepD, specified by the inc4 chloramphenicol resistance plasmid pC221, has a range of activities similar to those noted previously for RepC, manipulation of in vitro conditions has revealed discrete steps in the overall reaction of RepD with oriD. In addition, factors have been identified which are necessary not only for sequence-dependent discrimination in vitro by Rep proteins for all pT181-like plasmids but also for the absolute specificity of RepD for its cognate pC221 replication origin (oriD), the latter occurring in vivo and a function of the topological state of the ori-containing target DNA. Here we also demonstrate the presence of a covalent phosphoryl-tyrosine linkage between the RepD protein of plasmid pC221 and an oligonucleotide substrate corresponding to its replication origin (oriD). The reactive tyrosine (Tyr-188) was identified from amino acid sequences of 32P-labeled peptide-oligonucleotide fragments. Substitution of Tyr-188 with phenylalanine confirms the importance of the tyrosyl hydroxyl group since the Y188F protein retains the sequence-specific DNA-binding capabilities of wild-type RepD but is unable to attach covalently to the replication origin or participate in the nicking-closing reaction in vitro.     

Kinetic mechanism of initiation by RepD as a part of asymmetric, rolling circle plasmid unwinding

Some bacterial plasmids carry antibiotic resistance genes and replicate by an asymmetric, rolling circle mechanism, in which replication of the two strands is not concurrent. Initiation of this replication occurs via an initiator protein that nicks one DNA strand at the double-stranded origin of replication. In this work, RepD protein from the staphylococcal plasmid pC221 carries this function and allows PcrA helicase to bind and begin unwinding the plasmid DNA. This work uses whole plasmid constructs as well as oligonucleotide-based mimics of parts of the origin to examine the initiation reaction. It investigates the phenomenon that nicking, although required to open a single-stranded region at the origin and so allow PcrA to bind, is not required for another function of RepD, namely to increase the processivity of PcrA, allowing it to unwind plasmid lengths of DNA. A kinetic mechanism of RepD initiation is presented, showing rapid binding of the origin DNA. The rate of nicking varies with the structure of the DNA but can occur with a rate constant of >25 s(-1) at 30 °C. The equilibrium constant of the nicking reaction, which involves a transesterification to form a phosphotyrosine bond within the RepD active site, is close to unity.     

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.     

Purification and characterization of the PcrA helicase of Bacillus anthracis

PcrA is an essential helicase in gram-positive bacteria, and a gene encoding this helicase has been identified in all such organisms whose genomes have been sequenced so far. The precise role of PcrA that makes it essential for cell growth is not known; however, PcrA does not appear to be necessary for chromosome replication. The pcrA gene was identified in the genome of Bacillus anthracis on the basis of its sequence homology to the corresponding genes of Bacillus subtilis and Staphylococcus aureus, with which it shares 76 and 72% similarity, respectively. The pcrA gene of B. anthracis was isolated by PCR amplification and cloning into Escherichia coli. The PcrA protein was overexpressed with a His6 fusion at its amino-terminal end. The purified His-PcrA protein showed ATPase activity that was stimulated in the presence of single-stranded (ss) DNA (ssDNA). Interestingly, PcrA showed robust 3'-->5' as well as 5'-->3' helicase activities, with substrates containing a duplex region and a 3' or 5' ss poly(dT) tail. PcrA also efficiently unwound oligonucleotides containing a duplex region and a 5' or 3' ss tail with the potential to form a secondary structure. DNA binding experiments showed that PcrA bound much more efficiently to oligonucleotides containing a duplex region and a 5' or 3' ss tail with a potential to form a secondary structure than to those with ssDNAs or duplex DNAs with ss poly(dT) tails. Our results suggest that specialized DNA structures and/or sequences represent natural substrates of PcrA in biochemical processes that are essential for the growth and survival of gram-positive organisms, including B. anthracis.     

The patchwork nature of rolling-circle plasmids: comparison of six plasmids from two distinct Bacillus thuringiensis serotypes

Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200kb. Apart from the genes coding for the biopesticide delta-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B. thuringiensis strains. The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared. Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified and analyzed. Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3). On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer (oriT)) were identified. Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly-X-Y triplets). These genes were termed bcol for Bacillus-collagen-like genes.