Identification of the clonal complexes of Staphylococcus aureus strains by determination of the conservation patterns of small genomic islets

Aims:  To investigate the clonality of Staphylococcus aureus isolates, it is important to identify their clonal complexes (CCs) with multilocus sequence typing (MLST). However, it is expensive to carry out MLST analyses for many isolates. The aim of this study, therefore, was to develop a cost-effective method to identify CCs by determining the conservation pattern of 'small genomic islets' (SGIs). SGIs are nonconserved regions between strains and have single or multiple open-reading frames (ORFs).
Methods and Results:  The whole-genome sequences of nine strains were compared in order to select 16 SGIs. The conservation patterns of the 16 SGIs (islet patterns) were investigated in 136 S. aureus isolates, which were classified into 21 CCs. The islet patterns (IPs) exhibited a one-to-one correspondence with the CCs, except for isolates belonging to CC1, CC5 and CC8. The IPs typical of strains belonging to CC1, CC5 and CC8 differed between those of sequence type 1 (ST1) and ST188 (CC1), ST5 and ST6 (CC5) and ST8 and ST239 (CC8).
Significance and Impact of the Study:  The CCs of many isolates can be identified in an easy and inexpensive manner by detecting these 16 SGIs. Emergent clones, particularly methicillin-resistant ones, can be identified by examining numerous islets by IP analysis.     

Purification and some properties of deoxyribonuclease whose synthesis is controlled by gene 49 of bacteriophage T4.

An enzyme which specifically cleaves very-fast-sedimenting DNA of bacteriophage T4 is synthesized after infection of T4, and its synthesis is controlled by gene 49 [1,2]. This enzyme has been proved to be a DNase [2]. We have purified this DNase 3000-fold from extracts of E. coli infected with T4. The purified preparation was practically free from other DNases, and the DNase activity was not detectable in cells infected with a mutant defective in gene 49. The enzyme activity from cells infected with a temperature-sensitive mutant of gene 49 was also temperature-sensitive, suggesting strongly that gene 49 is a structural gene of the DNase. The molecular weight of the wild-type enzyme was estimated to be 50 x 10(3) by gel filtration chromatography. The purified DNase did not cleave native and denatured DNAs of T3 and T4, but cleaved renatured T3 DNA with enzymatically fragmented T3 DNA, indicating that gaps in the DNA duplex are structures susceptible to the DNase. Cleavage of the hybridized T3 DNA occurred when the fragmented DNA was phosphorylated at either the 3' or 5'-strand termini.     

Variation in M protein production among Streptococcus pyogenes strains according to emm genotype

M protein is an important virulence determinant in Streptococcus pyogenes, but the amounts of M protein in various strains of the species remain to be elucidated. To assess the amount of M protein in strains of each emm genotype, dot blot analysis was performed on 141 clinically isolated strains. Among the cell membrane-associated proteins, M protein was present in greater quantities in the emm1, 3, and 6 strains than in the other emm strains. In addition three strains, one each of the emm1, 3, and 6 types, showed prolific M protein production (M protein-high producers). These three emm genotypes are frequently isolated in clinical practice. Sequencing of the csrRS gene, one of the two-component signal transduction systems implicated in virulence, was performed on 25 strains bearing different amounts of M protein. CsrS mutations, in contrast to CsrR protein, were detected in 11 strains. The M protein-high producer strain of emm1 type carried two amino acid substitutions, whereas the other three emm1 strains carried only one substitution each. The M protein-high producer expressed its emm gene more strongly than the corresponding M protein-low producer did according to TaqMan RT-PCR. These observations suggest that the accumulation of amino acid substitutions in CsrS protein may contribute, at least in part, to the large amount of M protein production seen in several emm genotypes.     

Cadmium-copper antagonism in the activation of periplasmic nitrous oxide reductase of copper-deficient cells fromPseudomonas stutzeri

Summary Copper-deficient cells ofPseudomonas stutzeri strain ZoBell synthesize catalytically inactive nitrous oxide (N₂O) reductase which is activated by added Cu(II) in the absence of de novo protein synthesis. The apparentK _m for the activation process is 0.13 M. Activation is temperature-dependent and is inhibited by Cd(II)(K _i 1.27 M) and less strongly by Zn(II), Ni(II), and Co(II). The same metal ions at 20 M have little or no effect on N₂O reduction of intact cells. Apo-N₂O reductase of transposon Tn5-inducednos ^– mutants with defective Cu-chromophore biosynthesis is not reactivated by Cu(II). N₂O reductase of Cu-sufficient and Cu-deficient wild type, and ofnos ^– mutants is localized in the periplasm, the latter providing the likely site of metal incorporation into the apoenzyme.     

A monoclonal antibody against T-cell receptor αβ induces endogenous cytokines and prevents mice from a lethal infection with Listeria monocytogenes

Abstract In vivo induction of cytokines by a monoclonal antibody (mAb) against T-cell receptor (TCR) αβ and the protective effect induced by the mAb on a lethal infection with Listeria monocytogenes were studied. Injection of anti-TCR αβ mAb induced rapid production of endogenous tumour necrosis factor in the spleens, and gamma interferon and interleukin-6 in the bloodstreams and spleens of mice. Administration of anti-CD4 mAb, anti-CD8 mAb, or anti-Thy1.2 mAb resulted in suppression of anti-TCR αβ mAb-induced endogenous cytokine production. Mice were protected against lethal L. monocytogenes infection when treated with anti-TCR αβ mAb. The protective effect was not demonstrated in CD4 + cell- or CD8 + cell-depleted mice. These results suggest that anti-TCR αβ mAb shows a protective effect on a lethal infection with L. monocytogenes in mice and that the mAb-induced endogenous cytokines might be involved in the effect of anti-TCR αβ mAb.     

Purification and characterization of a thermostable carboxypeptidase (carboxypeptidase Taq) from Thermus aquaticus YT-1.

A thermostable carboxypeptidase, which we named carboxypeptidase Taq, was purified from Thermus aquaticus YT-1 and characterized. The molecular weight of the enzyme was estimated to be about 56,000 and 58,000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme has a monomeric structure. The optimum pH of the enzyme was 8.0, and the optimum temperature for the reaction was 80 degrees C. The enzyme activity was dependent on cobalt ion and was inhibited by metal-chelating reagents, indicating that the enzyme is a metalloenzyme. The enzyme had high thermostability independent of cobalt ion; about 90% of its activity remained even after treatment at 80 degrees C for 5 h. The enzyme showed broad substrate specificity, although proline at the C-terminus of peptides was not cleaved. The enzyme released amino acids sequentially from the C-terminus.     

Effect of double mutation on thermostability of lactate oxidase

DNA shuffling was used to make a double mutant lactate oxidase (E160G/V198I LOD) in E. coli was more thermostable than both E160G single-mutant and wild-type LODs. The half-life of this E160G/V198I LOD at 70°C was about 3 times that of E160G LOD, and was about 20 times that of wild-type. In contrast, the thermostability of the V198I single-mutant LOD made by site-directed mutagenesis was almost identical to that of wild-type. This indicates that the V198I mutation alone does not affect LOD thermostability but does affect it when combined with the E160G mutation.