Identification in methicillin-susceptible Staphylococcus hominis of an active primordial mobile genetic element for the staphylococcal cassette chromosome mec of methicillin-resistant Staphylococcus aureus

We previously reported that the methicillin resistance gene mecA is carried by a novel type of mobile genetic element, SCCmec (staphylococcal cassette chromosome mec), in the chromosome of methicillin-resistant Staphylococcus aureus (MRSA). These elements are precisely excised from the chromosome and integrated into a specific site on the recipient chromosome by a pair of recombinase proteins encoded by the cassette chromosome recombinase genes ccrA and ccrB. In the present work, we detected homologues of the ccr genes in Staphylococcus hominis type strain GIFU12263 (equivalent to ATCC 27844), which is susceptible to methicillin. Sequence determination revealed that the ccr homologues in S. hominis were type 1 ccr genes (ccrA1 and ccrB1) that were localized on a genetic element structurally very similar to SCCmec except for the absence of the methicillin-resistance gene, mecA. This genetic element had mosaic-like patterns of homology with extant SCCmec elements, and we designated it SCC(12263) and considered it a type I staphylococcal cassette chromosome (SCC). The ccrB1 gene identified in the S. hominis strain is the first type 1 ccrB gene discovered to retain its function through the excision process as judged by two criteria: (i) SCC(12263) was spontaneously excised during cultivation of the strain and (ii) introduction of the S. hominis ccrB1 into an MRSA strain carrying a type I SCCmec whose ccrB1 gene is inactive generated SCCmec excisants at a high frequency. The existence of an SCC without a mec determinant is indicative of a staphylococcal site-specific mobile genetic element that serves as a vehicle of transfer for various genetic markers between staphylococcal species.     

The recombination-deficient mutant RPA (rfa1-t11) is displaced slowly from single-stranded DNA by Rad51 protein

Replication protein-A (RPA) is involved in many processes of DNA metabolism, including DNA replication, repair, and recombination. Cells carrying a mutation in the largest subunit of RPA (rfa1-t11: K45E) have defects in meiotic recombination, mating-type switching, and survival after DNA damage caused by UV and methyl methanesulfonate, as well as increased genome instability; however, this mutant has no significant defect in DNA replication. We purified the RPA heterotrimer containing the rfa1-t11 substitution (RPA(rfa1-t11)). This mutant RPA binds single-stranded DNA (ssDNA) with the same site size, and the RPA(rfa1-t11).ssDNA complex shows a similar sensitivity to disruption by salt as the wild-type RPA.ssDNA complex. RPA(rfa1-t11) stimulates DNA strand exchange, provided that the Rad51 protein.ssDNA nucleoprotein complex is assembled prior to introduction of the mutant RPA. However, RPA(rfa1-t11) is displaced from ssDNA by Rad51 protein more slowly than wild-type RPA and, as a consequence, Rad51 protein-mediated DNA strand exchange is inhibited when the ssDNA is in a complex with RPA(rfa1-t11). Rad52 protein can stimulate displacement of RPA(rfa1-t11) from ssDNA by Rad51 protein, but the rate of displacement remains slow compared with wild-type RPA. These in vitro results suggest that, in vivo, RPA is bound to ssDNA prior to Rad51 protein and that RPA displacement by Rad51 protein is a critical step in homologous recombination, which is impaired in the rfa1-t11 mutation.     

Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

A recombinant molt-inhibiting hormone of the kuruma prawn has a similar secondary structure to a native hormone: determination of disulfide bond arrangement and measurements of circular dichroism spectra

In crustaceans, molt-inhibiting hormone (MIH) is presumed to regulate molting through suppressing synthesis and/or secretion of ecdysteroids by the Y-organ. Recently, a recombinant MIH of the kuruma prawn Penaeus japonicus was produced in E. coli. To approximate the secondary structure of native and recombinant MIH of P. japonicus containing six cysteine residues, the arrangements of disulfide bridges in both MIHs were determined by characterizing their enzymatic digests, and their circular dichroism spectra were measured. The arrangements of disulfide bonds in both MIHs were determined to be identical, and they were linked between Cys7 and Cys44, Cys24 and Cys40, and Cys27 and Cys53. The circular dichroism spectra of both MIHs were very close, and demonstrated that they were rich in a-helix. a-Helix contents in native and recombinant MIHs were calculated to be 49.3% and 46.0%, respectively. All these results strongly suggested that the recombinant MIH was folded in the same manner as the native MIH.     

The calcium-binding loops of the tandem C2 domains of synaptotagmin VII cooperatively mediate calcium-dependent oligomerization

Synaptotagmin VII (Syt VII), a proposed regulator for Ca2+-dependent exocytosis, showed a robust Ca2+-dependent oligomerization property via its two C2 domains (Fukuda, M., and Mikoshiba, K. (2001) J. Biol. Chem. 276, 27670-27676), but little is known about its structure or the critical residues directly involved in the oligomerization interface. In this study, site-directed mutagenesis and chimeric analysis between Syt I and Syt VII showed that three Asp residues in Ca2+-binding loop 1 or 3 (Asp-172, Asp-303, and Asp-357) are crucial to robust Ca(2+)-dependent oligomerization. Unlike Syt I, however, the polybasic sequence in the beta4 strands of the C2 structures (so-called "C2 effector domain") is not involved in the Ca2+-dependent oligomerization of Syt VII. The results also showed that the Ca2+-binding loops of the two C2 domains cooperatively mediate Syt VII oligomerization (i.e. the presence of redundant Ca2+-binding site(s)) as well as the importance of Ca2+-dependent oligomerization of Syt VII in Ca2+-regulated secretion. Expression of wild-type tandem C2 domains of Syt VII in PC12 cells inhibited Ca2+-dependent neuropeptide Y release, whereas mutant fragments lacking Ca2+-dependent oligomerization activity had no effect. Finally, rotary-shadowing electron microscopy showed that the Ca2+-dependent oligomer of Syt VII is "a large linear structure," not an irregular aggregate. By contrast, in the absence of Ca2+ Syt VII molecules were observed to form a globular structure. Based on these results, we suggest that the linear Ca2+-dependent oligomer may be aligned at the fusion site between vesicles and plasma membrane and modulate Ca2+-regulated exocytosis by opening or dilating fusion pores.     

Structure and electrochemistry of the bridging-ligand mono-substituted diruthenium compound, [Ru2(II,III)(O2CCH3)3(admpym)(Cl)(MeOH)] (Hadmpym=2-amino-4,6-dimethylpyrimidine)

The ligand substitution reaction of Ru₂(O₂CCH₃)₄Cl with 2-amino-4,6-dimethylpyrimidine (Hadmpym) under gentle refluxing conditions in methanol led to the formation of a bridging-ligand mono-substituted compound, [Ru₂(O₂CCH₃)₃(admpym)(Cl)(MeOH)] (1). Compound 1 crystallized in monoclinic space group P2₁/n (no. 14) with a=8.3074(8) Å, b=12.3722(8) Å, c=18.913(1) Å, β=95.559(3)°, V=1934.8(3) ų, and Z=4. Temperature dependence of the magnetic susceptibility of 1 revealed it to be in a spin ground state S=3/2 arising from the electronic configuration of σ²π⁴δ²(δ*π*)³. Compound 1 undergoes three metal-centered redox reactions in electrochemistry: E_1/2 ^(ox)=+0.72 V (I_a/I_c<1, ΔE_p=0.17 V); E_1/2 ^(1,red)=−0.65 V (I_a/I_c≈1, ΔE_p=0.10 V); and E_1/2 ^(2,red)=−1.80 V (I_a/I_c?1, ΔE_p=0.16 V). Then, the redox species produced by electrolysis were characterized by spectroscopic studies.