RESULTS:
1 - 9 of 9 for "Neil Hunter"
Carboxypeptidase activity common to viridans group streptococci cleaves angiotensin I to angiotensin II: an activity homologous to angiotensin-converting enzyme (ACE)
We have found that Streptococcus gordonii FSS2 an infective endocarditis (IE) isolate expresses a dipeptidyl-carboxypeptidase with activity homologous to angiotensin-converting enzyme (ACE). The carboxypeptidase activity was purified to homogeneity as a complex/aggregate from a bacterial surface extract and was also active as a 165 kDa monomer. The specific activity for the carboxypeptidase activity was eightfold higher than that for recombinant human ACE. Selected ACE inhibitors captopril lisinopril and enalapril did not inhibit the ACE activity. The carboxypeptidase also hydrolysed the Aα and Bβ-chains of human fibrinogen which resulted in impaired fibrin formation by thrombin. The gene encoding ACE carboxypeptidase activity was sequenced and the inferred polypeptide product showed 99 % amino acid homology to SGO_0566 sgc ‘challisin’ of S. gordonii CL1 Challis and had no significant amino acid sequence homology to human ACE. Homologues of challisin ACE activity were commonly detected among the viridans group streptococci most often associated with IE.
Verification of a topology model of PorT as an integral outer-membrane protein in Porphyromonas gingivalis
PorT is a membrane-associated protein shown to be essential for the maturation and secretion of a class of cysteine proteinases the gingipains from the periodontal pathogen Porphyromonas gingivalis. It was previously reported that PorT is located on the periplasmic surface of the inner membrane to function as a chaperone for the maturing proteinases. Our modelling suggested it to be an integral outer-membrane protein with eight anti-parallel membrane-traversing β-strands. In this report the outer-membrane localization model was confirmed by the structural and functional tolerance of PorT to hexahistidine (6×His) tag insertions at selected locations within the protein using site-directed mutagenesis. Interestingly those PorT mutations adversely affecting gingipain secretion enhanced expression of the porT gene but at the same time suppressed the transcription of the gingipain rgpB gene. Further PorT mutants deficient in gingipain activities produced significantly more di- and triaminopeptidase activities. PorT homologues have been found in restricted members of the Bacteroidetes phylum where there is potential for PorT to participate in the maturation and secretion of proteins with characteristic C-terminal domains (CTDs). Knowledge of the cellular localization of PorT will enable analysis of the role of this protein in a new secretory pathway for the export of gingipains and other CTD-class proteins.
Veillonella denticariosi sp. nov., isolated from human carious dentine
Selective culture of human carious dentine for Veillonella strains resulted in the isolation of two strains of a Gram-negative coccus-shaped bacterium that has not been described previously. Comparative 16S rRNA and dnaK gene sequence analysis indicated that the two strains were homogeneous and comprised a distinct lineage within the genus Veillonella phylogenetically most closely related to Veillonella rodentium. This was supported by DNA–DNA hybridization which showed clearly that the two strains were similar and distinct from other Veillonella species and the production of major cellular fatty acids (C13 : 0 and C17 : 1 ω8) which is consistent with other members of the genus Veillonella. Based on these observations strains RBV81 and RBV106T represent a novel species for which the name Veillonella denticariosi sp. nov. is proposed with the type strain RBV106T (=CIP 109448T =CCUG 54362T =DSM 19009T).
Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set
The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergey’s Manual of Determinative Bacteriology. However the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number since rapid growth could result in multiple replication forks and hence in effect more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods demonstrating the utility of real-time PCR in the analysis of this environment.
Characterization and Complementation of a Mutant of Rhodobacter sphaeroides with a Chromosomal Deletion in the Light-harvesting (LH2) Genes
An LH2− strain of Rhodobacter sphaeroides DBC1 has been constructed by deleting the puc operon which encodes the LH2 α and β polypeptides from the chromosome and replacing it with a kanamycin resistance gene. Southern blot analysis indicates that the 950 bp BamHI restriction fragment which contains the puc operon has been lost and has been replaced by the 1·25 kb KmR cassette derived from Tn903. Strain DBC1 lacked the LH2 complex as shown by loss of the characteristic absorbance bands at 800 and 850 nm. The LH2 polypeptides were also found to be absent after SDS-PAGE. The wild-type phenotype was restored to DBC1 by the transfer of a 3·8 kb BscI fragment containing the puc operon in plasmid pMA81. Transconjugants possessed a wild-type absorbance spectrum and LH2 polypeptides.
Transposon Tn5 Mutagenesis of Genes Encoding Reaction Centre and Light-harvesting LH1 Polypeptides of Rhodobacter sphaeroides
The puf operon of the photosynthetic bacterium Rhodobacter sphaeroides encodes reaction centre and B875 (LH1) antenna polypeptides of the photosynthetic apparatus. This region of the genome was used to establish the applicability of random transposon Tn5 mutagenesis in this bacterium. Four Tn5 insertions have been mapped and one of the mutants characterized using a variety of techniques in order to establish that the fluorescence properties and polypeptide composition were consistent with the absence of reaction centre polypeptides. Following the ‘rescue’ of the transposon along with flanking regions of the puf operon re-introduction of this construction into wild-type Rb. sphaeroides yielded the original mutation. This demonstrates that following homologous recombination localized mutagenesis can direct Tn5 into a predetermined region of the Rb. sphaeroides chromosome. Accordingly puf genes borne on plasmid pSRC2 were mutagenized with Tn5 in Escherichia coli and the sites of insertion mapped physically. pSRC2 derivatives containing Tn5 were transferred to wild-type Rb. sphaeroides. puf genes have been mapped by correlating the photosynthetic properties of resulting strains with the sites of Tn5 insertion into pSRC2.
Transfer of Genes Coding for Apoproteins of Reaction Centre and Light-harvesting LH1 Complexes to Rhodobacter sphaeroides
Several methods of introducing mobilizable cloning vectors by conjugation into the photosynthetic bacterium Rhodobacter sphaeroides have been examined. The efficiency of transfer was sufficiently high to enable a bank of Rb. sphaeroides genes in Escherichia coli to complement non-photosynthetic mutants of Rb. sphaeroides thus providing a generally applicable method of isolating Rb. sphaeroides genes. With a mutant incapable of synthesizing reaction centres and light-harvesting LH1 complexes as a recipient the transfer of puf genes encoding reaction centre and light-harvesting LH1 polypeptides was examined in some detail. The spectroscopic and electrophoretic properties of this mutant and the newly photosynthetic transconjugant strain were consistent with the efficient transfer and expression of puf genes.
Cloning and Oxygen-regulated Expression of the Bacteriochlorophyll Biosynthesis Genes bch E, B, A and C of Rhodobacter sphaeroides
Four mutants of the photosynthetic bacterium Rhodobacter sphaeroides were isolated which were incapable of photosynthetic growth due to inability to synthesize bacteriochlorophyll. A Rb. sphaeroides gene bank was constructed in the mobilizable vector pSUP202 and was transferred into these mutants using the helper plasmid pRK2073. Three clones that produced photosynthetic transconjugants from one or more of the bch mutants were isolated and characterized. These clones were used as probes to estimate levels of specific transcripts in cells undergoing a 100-fold increase in bacteriochlorophyll content. The maximum level of transcripts was observed at an early stage of photosynthetic membrane synthesis when only 7% of the eventual level of pigment had been synthesized.
Characteristics of a High Molecular Weight Extracellular Protein of Streptococcus mutans
A high molecular weight protein antigen designated P1 has been isolated from the culture fluid of chemostat-grown Streptococcus mutans strain Ingbritt and shown to be free of other antigens including glucosyltransferase. Antiserum against the protein was used in rocket immunoelectro-phoresis to confirm and extend the previous observation that there were major differences in the amount of the protein produced under different growth conditions. Physico-chemical and serological studies indicated that protein P1 was indistinguishable from antigens B I II and IF isolated in other laboratories. Mammalian tissue cross-reactivity of protein P1 was demonstrated by binding of antiserum to P1 to sections of normal rabbit tissues particularly heart. There was also a statistically significant increase in the number of mononuclear leucocytes in heart tissue of rabbits which had been injected with protein P1 when compared with the levels in control uninjected rabbits; injection with whole cells of S. mutans Ingbritt did not produce this effect.