Kazuhisa Sekimizu

Whole genome sequencing of meticillin-resistant Staphylococcus aureus

BACKGROUNDnStaphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism.nnnMETHODSnWhole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction.nnnFINDINGSnThe Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors.nnnINTERPRETATIONnThe remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

The Initiator Function of DnaA Protein Is Negatively Regulated by the Sliding Clamp of the E. coli Chromosomal Replicase

The beta subunit of DNA polymerase III is essential for negative regulation of the initiator protein, DnaA. DnaA inactivation occurs through accelerated hydrolysis of ATP bound to DnaA; the resulting ADP-DnaA fails to initiate replication. The ability of beta subunit to promote DnaA inactivation depends on its assembly as a sliding clamp on DNA and must be accompanied by a partially purified factor, IdaB protein. DnaA inactivation in the presence of IdaB and DNA polymerase III is further stimulated by DNA synthesis, indicating close linkage between initiator inactivation and replication. In vivo, DnaA predominantly takes on the ADP form in a beta subunit-dependent manner. Thus, the initiator is negatively regulated by action of the replicase, a mechanism that may be key to effective control of the replication cycle.

Replication cycle-coordinated change of the adenine nucleotide-bound forms of DnaA protein in Escherichia coli

The ATP‐bound but not the ADP‐bound form of DnaA protein is active for replication initiation at the Escherichia coli chromosomal origin. The hydrolysis of ATP bound to DnaA is accelerated by the sliding clamp of DNA polymerase III loaded on DNA. Using a culture of randomly dividing cells, we now have evidence that the cellular level of ATP–DnaA is repressed to only ∼20% of the total DnaA molecules, in a manner depending on DNA replication. In a synchronized culture, the ATP–DnaA level showed oscillation that has a temporal increase around the time of initiation, and decreases rapidly after initiation. Production of ATP–DnaA depended on concomitant protein synthesis, but not on SOS response, Dam or SeqA. Regeneration of ATP–DnaA from ADP–DnaA was also observed. These results indicate that the nucleotide form shifts of DnaA are tightly linked with an epistatic cell cycle event and with the chromosomal replication system.

Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus.

Abstract. We isolated and characterized temperature-sensitive mutants for two genes, dnaE and polC, that are essential for DNA replication in Staphylococcus aureus. DNA replication in these mutants had a slow-stop phenotype when the temperature was shifted to a non-permissive level. The dnaE gene encodes a homolog of the α-subunit of the DNA polymerase III holoenzyme, the replicase essential for chromosomal DNA replication in Escherichia coli. The polC gene encodes PolC, another catalytic subunit of DNA polymerase, which is specifically found in gram-positive bacteria. The wild-type dnaE or polC gene complemented the temperature-sensitive phenotypes of cell growth and DNA replication in the corresponding mutant. Single mutations resulting in amino-acid exchanges were identified in the dnaE and polC genes of the temperature-sensitive mutants. The results indicate that these genes encode two distinct DNA polymerases which are both essential for chromosomal DNA replication in S. aureus. The number of viable mutant cells decreased at non-permissive temperature, suggesting that inactivation of DnaE and PolC has a bactericidal effect and that these enzymes are potential targets of antibiotics.

Negative control of DNA replication by hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli

DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by ATP. ATP bound to DnaA protein is slowly hydrolyzed to ADP, but the physiological role of ATP hydrolysis is unclear. We constructed, by site‐directed mutagenesis, mutated DnaA protein with lower ATPase activity, and we examined its function in vitro and in vivo. The ATPase activity of purified mutated DnaA protein (Glu204→Gln) decreased to one‐third that of the wild‐type DnaA protein. The mutation did not significantly affect the affinity of DnaA protein for ATP or ADP. The mutant dnaA gene showed lethality in wild‐type cells but not in cells growing independently of the function of oriC. Induction of the mutated DnaA protein in wild‐type cells caused an overinitiation of DNA replication. Our results lead to the thesis that the intrinsic ATPase activity of DnaA protein negatively regulates chromosomal DNA replication in E.coli cells.

Inactivation of Escherichia coli DnaA protein by DNA polymerase III and negative regulations for initiation of chromosomal replication

Genetic and biochemical evidence indicates that initiation of chromosomal replication in Escherichia coli occurs in a nucleoprotein complex at the replication origin (oriC) formed with DnaA protein. The frequency of initiation at oriC is tightly regulated to only once per chromosome per cell cycle. To prevent untimely, extra initiations, negative control for initiation is indispensable. Recently, we found that the function of the initiator protein, DnaA, is controlled by DNA polymerase III holoenzyme, the replicase of the chromosome. The ATP-bound form of DnaA protein, an active form for initiation, is efficiently converted to the ADP bound form, an inactive form, since a subunit of the polymerase loaded on DNA (beta subunit sliding clamp) stimulates hydrolysis of ATP bound to DnaA protein. Comparison of this system, RIDA (regulatory inactivation of DnaA), with other systems for negative regulation of initiation is included in this review, and the roles of these systems for concerted control for initiation during the cell cycle are discussed.

Site-directed Mutational Analysis for the ATP Binding of DnaA Protein FUNCTIONS OF TWO CONSERVED AMINO ACIDS (LYS-178 AND ASP-235) LOCATED IN THE ATP-BINDING DOMAIN OF DnaA PROTEIN IN VITRO AND IN VIVO

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, is activated by binding to ATP in vitro. We introduced site-directed mutations into two amino acids of the protein conserved among various ATP-binding proteins and examined functions of the mutated DnaA proteins, in vitro and in vivo. Both mutated DnaA proteins (Lys-178 → Ile or Asp-235 → Asn) lost the affinity for both ATP and ADP but did maintain binding activity for oriC. Specific activities in an oriC DNA replication system in vitro were less than one-tenth those of the wild-type protein. Assay of the generation of oriC sites sensitive to P1 nuclease, using the mutated DnaA proteins, revealed a defect in induction of the duplex opening at oriC. On the other hand, expression of each mutated DnaA protein in the temperature-sensitivednaA46 mutant did not complement the temperature sensitivity. We suggest that Lys-178 and Asp-235 of DnaA protein are essential for the activity needed to initiate oriC DNA replication in vitro and in vivo and that ATP binding to DnaA protein is required for DNA replication-related functions.

Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the replicase and in DnaA

Abstract. In Escherichia coli, an interaction between the replication initiator DnaA and the sliding clamp protein, the β subunit (DnaN) of DNA polymerase III, is required to regulate the chromosomal replication cycle. We report here that colony formation by, and cell division of, the temperature (42°C)-sensitive dnaN59 mutant are inhibited at 34–35°C when DnaA is moderately (4-to 8-fold ) overexpressed, although chromosomal replication and the β subunit-dependent regulation of DnaA activity are not significantly inhibited. Immunoblotting analysis revealed that the β subunit is abundant (present at a level of about 5000 dimers per cell) at 34°C, and its concentration per unit cell volume was practically unaffected in the dnaN59 mutant by the overexpression of DnaA. The dnaN mutant cells that overexpress DnaA become filamentous at 34°C via an sfiA-independent pathway, different from that activated by the SOS response. This filamentation is accompanied by inhibition of nucleoid partition and FtsZ ring formation. In the dnaN59 mutant, oversupply of DnaA may disturb the coordinated action of cell cycle-regulating molecules, thus leading to the inhibition of these events.

Inhibition of thymidine transport in dnaA mutants of Escherichia coli

DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. We report here our evidence that thymidine transport across cytoplasmic membranes in temperature-sensitive dnaA mutants is greatly decreased at a permissive temperature for growth of the mutants. Complementation analysis with a plasmid containing the wild type dnaA gene and P1 phage-mediated transduction confirmed that mutations in thednaA gene were responsible for the phenotype. A low level of nucleoside transport in the dnaA mutant was specific for thymidine; transport activities for other nucleosides were much the same as those in wild type cells. Membrane vesicles prepared from thednaA mutant showed much the same activity of thymidine transport as did those from the wild type cells. No significant difference in the activity of thymidine kinase, which is known to facilitate thymidine transport, was seen between the mutant and the wild type cells. An increase in the pool of dTTP, a negative regulator for thymidine kinase, was observed in the dnaA mutant. Based on these observations, we suggest that inhibition of thymidine transport in dnaA mutants is caused by increases in the dTTP pool.

Amounts of proteins altered by mutations in the dnaA gene of Escherichia coli

© 1997 Federation of European Biochemical Societies.