No abstract

dStreptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci.

The crystallization and melting behavior of erythritol in and around two-dimensional hexagonal mesoporous silica SBA-15 with a pore diameter range of 7.5-9.2 nm were investigated.Erythritol inside the mesopores did not crystallize but became amorphous when the temperature decreased from 423 K, a value above the fusion point in the bulk, at 1 K min -1 . Subsequently, as the temperature increased at 1 K min -1 , the amorphous cold-crystallized and then fused around 320-360 K. If the temperature decreased from around the fusion point in the mesopores at the same cooling rate, erythritol inside the mesopores become a crystal. The XRD pattern of the erythritol inside the mesopores exhibited the crystal structure at 353 K, suggesting that the crystal grew around the nuclei of crystal. The heterogeneity of mesopores induces partial crystallization or partial melting, resulting in smooth phase transition. However, because the relaxation time of dynamic properties of erythritol confined in mesopores becomes longer due to strong interactions with pore walls, glass transition occurs easily instead of crystallization. The phase transition of erythritol on the outer surface of mesoporous silica particles was also clarified.

We report a novel bioluminescent protein-protein interaction (PPI) assay, which is based on the functional complementation of two mutant firefly luciferases (Fluc). The chemical reaction catalyzed by Fluc is divided into two half reactions of ATP-driven luciferin adenylation and subsequent oxidative reactions. In the former adenylation half-reaction, a luciferyl-adenylate (LH2-AMP) intermediate is produced from LH2 and ATP. With this intermediate, the latter oxidative reactions produce oxyluciferin via proton abstraction at the C4 carbon of LH2-AMP. We created and optimized two Fluc mutants; one is named "Donor", which virtually lacks oxidative activity, while the other, named "Acceptor", is almost defective in the adenylation activity. Then, the two mutants are fused to interacting partners, and prepared as pure proteins. When the interaction between the partners is induced, higher efficiency of LH2-AMP transfer between the Donor and Acceptor enzymes resulted in increased luminescence. The assay was found to work both in vitro and in cultured cells with strong signals. This would be the first example of reconstituting two divided reactions of one enzyme to detect PPI, which will not only be utilized as a robust PPI assay, but also open a way to control the activity of similar enzymes in acyl/adenylate-forming enzyme superfamily.

Streptococcus constellatus is a member of the Anginosus group streptococci (AGS) and primarily inhabits the human oral cavity. S. constellatus is composed of three subspecies: S. constellatus subsp. constellatus (SCC), S. constellatus subsp. pharyngis and the newly described subspecies S. constellatus subsp. viborgensis. Although previous studies have established that SCC contains b-haemolytic strains, the factor(s) responsible for b-haemolysis in b-haemolytic SCC (b-SCC) has yet to be clarified. Recently, we discovered that a streptolysin S (SLS) homologue is the b-haemolytic factor of b-haemolytic Streptococcus anginosus subsp. anginosus (b-SAA), another member of the AGS. Furthermore, because previous studies have suggested that other AGS species, except for Streptococcus intermedius, do not possess a haemolysin(s) belonging to the family of cholesterol-dependent cytolysins, we hypothesized that, as with b-SAA, the SLS homologue is the b-haemolytic factor of b-SCC, and therefore aimed to investigate and characterize the haemolytic factor of b-SCC in the present study. PCR amplification revealed that all of the tested b-SCC strains were positive for the sagA homologue of SCC (sagA SCC ). Further investigations using b-SCC strain W277 were conducted to elucidate the relationship between sagA SCC and b-haemolysis by constructing sagA SCC deletion mutants, which completely lost bhaemolytic activity. This loss of b-haemolytic activity was restored by trans-complementation of sagA SCC . Furthermore, a co-cultivation assay established that the cytotoxicity of b-SCC was clearly dependent on the presence of sagA SCC . These results demonstrate that sagA SCC is the factor responsible for b-SCC b-haemolysis and cytotoxicity. INTRODUCTIONAnginosus group streptococci (AGS) are commensal bacteria in the human oral cavity. Current classification assigns AGS to three species, including five subspecies (Jensen et al., 2013): Streptococcus anginosus subsp. anginosus (SAA), S. anginosus subsp. whileyi (SAW), Streptococcus constellatus Abbreviations: AGS, Anginosus group streptococci; CSP, competence-stimulating peptide; erm, erythromycin resistance gene cassette; GAS, group A streptococci; GBS, group B streptococci; PGS, Pyogenic group streptococci; SAA, Streptococcus anginosus subsp. anginosus; SAW, Streptococcus anginosus subsp. whileyi; SCC, Streptococcus constellatus subsp. constellatus; SCP, Streptococcus constellatus subsp. pharyngis; SCV, Streptococcus constellatus subsp. viborgensis; SLS, streptolysin S.The GenBank/EMBL/DDBJ accession numbers for the sag operon homologue from b-haemolytic SCC strain W277, the sagA homologues from bhaemolytic SCC strains HW74 and 1340, and the sag operon homologues from the SCP type strain MM9889a T (5SK1060 T ), the SAW strain MAS624 and the SCV type strain SK1359 T are AB389362, AB389363, AB389364, AB389365, AB389366 and AB860419, respectively. (2014), 160, 980-991 Microbiology

DNA alkylation damage can be repaired by nucleotide excision repair (NER), base excision repair (BER) or by direct removal of alkyl groups from modified bases by O(6)-alkylguanine DNA alkyltransferase (AGT; E.C. 2.1.1.63). DNA mismatch repair (MMR) is also likely involved in this repair. We have investigated alkylation-induced mutagenesis in a series of NER- or AGT-deficient Escherichia coli strains, alone or in combination with defects in the MutS, MutL or MutH components of MMR. All strains used contained the F'prolac from strain CC102 (F'CC102) episome capable of detecting specifically lac GC to AT reverse mutations resulting from O(6)-alkylguanine. The results showed the repair of O(6)-methylguanine to be performed by AGT ≫ MMR > NER in order of importance, whereas the repair of O(6)-ethylguanine followed the order NER > AGT > MMR. Studies with double mutants showed that in the absence of AGT or NER repair pathways, the lack of MutS protein generally increased mutant frequencies for both methylating and ethylating agents, suggesting a repair or mutation avoidance role for this protein. However, lack of MutL or MutH protein did not increase alkylation-induced mutagenesis under these conditions and, in fact, reduced mutagenesis by the N-alkyl-N-nitrosoureas MNU and ENU. The combined results suggest that little or no alkylation damage is actually corrected by the mutHLS MMR system; instead, an as yet unspecified interaction of MutS protein with alkylated DNA may promote the involvement of a repair system other than MMR to avoid a mutagenic outcome. Furthermore, both mutagenic and antimutagenic effects of MMR were detected, revealing a dual function of the MMR system in alkylation-exposed cells.

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This paper presents some modifications of the gamma-based model developed by K.M. Shyue (J. Comput. Phys., 1998, 142, 208-242). In deriving process of the original model, pressure equilibrium and an isentropic condition are not involved explicitly. Therefore, to adopt these physical conditions, we improved evolution equations for problem-dependent material quantities (e.g. ratio of specific heat). As a result, a non-conservative term associated with the pressure relaxation was added to the equations. The modifications have no effect on an oscillation-free property of the original model. Furthermore, because division by a volume fraction is not included in the present model, an inherent property of mixture-type models is maintained. To capture contact discontinuity sharply, we utilized an approximate Riemann solver HLLC with a third-order MUSCL interpolation. Finally, we carried out one-dimensional numerical tests in order to evaluate the influence on accuracy. Numerical results show that the modified model has good capability for simulating low Mach number flow problems.