Biomedical subjects
Publications and source records attributed to Nicholas C K Heng.
The production of streptocins STH(1) and STH(2) by Streptococcus gordonii DL1 (Challis) is directly controlled by the competence regulon, which requires intact comR and comAB loci. The streptocin (sth) locus comprises two functional genes, sthA and sthB. Whereas STH(1) activity requires sthA alone, STH(2) activity depends on both genes.
Streptococcus salivarius strains commonly produce bacteriocins as putative anti-competitor or signalling molecules. Here we report that bacteriocin production by the oral probiotic strain S. salivarius K12 is encoded by a large (ca. 190 kb) plasmid. Oral cavity transmission of the plasmid from strain K12 to a plasmid-negative variant of this bacterium was demonstrated in two subjects. Tests of additional S. salivarius strains showed large (up to ca. 220 kb) plasmids present in bacteriocin-producing isolates. Various combinations (up to 3 per plasmid) of loci encoding the known streptococcal lantibiotics salivaricin A, salivaricin B, streptin and SA-FF22 were localised to these plasmids. Since all bacteriocin-producing strains of S. salivarius tested to date appear to harbour plasmids, it appears that they may function as mobile repositories for bacteriocin loci, especially those of the lantibiotic class.
Dysgalacticin is a novel bacteriocin produced by Streptococcus dysgalactiae subsp. equisimilis strain W2580 that has a narrow spectrum of antimicrobial activity directed primarily against the principal human streptococcal pathogen Streptococcus pyogenes. Unlike many previously described bacteriocins of Gram-positive bacteria, dysgalacticin is a heat-labile 21.5 kDa anionic protein that kills its target without inducing lysis. The N-terminal amino acid sequence of dysgalacticin [Asn-Glu-Thr-Asn-Asn-Phe-Ala-Glu-Thr-Gln-Lys-Glu-Ile-Thr-Thr-Asn-(Asn)-Glu-Ala] has no known homologue in publicly available sequence databases. The dysgalacticin structural gene, dysA, is located on the indigenous plasmid pW2580 of strain W2580 and encodes a 220 aa preprotein which is probably exported via a Sec-dependent transport system. Natural dysA variants containing conservative amino acid substitutions were also detected by sequence analyses of dysA elements from S. dysgalactiae strains displaying W2580-like inhibitory profiles. Production of recombinant dysgalacticin by Escherichia coli confirmed that this protein is solely responsible for the inhibitory activity exhibited by strain W2580. A combination of in silico secondary structure prediction and reductive alkylation was employed to demonstrate that dysgalacticin has a novel structure containing a disulphide bond essential for its biological activity. Moreover, dysgalacticin displays similarity in predicted secondary structure (but not primary amino acid sequence or inhibitory spectrum) with another plasmid-encoded streptococcal bacteriocin, streptococcin A-M57 from S. pyogenes, indicating that dysgalacticin represents a prototype of a new class of antimicrobial proteins.
In naturally-competent streptococci such as Streptococcus pneumoniae, expression of the late competence operons is regulated by ComX (sigma(X)), the competence-specific alternative sigma factor. In this study, duplicate genes (comR1 and comR2) encoding the putative ComX homologue of the oral bacterium Streptococcus gordonii were identified. Like the identical twin comX loci of S. pneumoniae, both comR determinants are independently functional as well as responsive to the ComDE signal transduction system activated by competence-stimulating peptide. However, in contrast to the comX system, nucleotide sequence analyses in combination with in trans complementation studies with a comR null mutant demonstrate that the identical 83 bp tracts (Region I) located immediately upstream of the comR structural genes are insufficient to confer wild-type competence levels. Wild-type transformation levels required additional distal nonhomologous DNA segments (Region II). Our findings suggest that alternative regulatory elements, under overall control of the ComDE pathway, may influence expression of the comR loci.
Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by the nlm locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).
Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic bacteriocin (mutacin) activity. In this study, we have combined bioinformatic and mutational analyses to identify the ABC transporter designated NlmTE, which is required for mutacin biogenesis in strain UA159 as well as in another mutacin producer, S. mutans N.
Production of the novel bacteriocin streptococcin A-M57 (SA-M57) by Streptococcus pyogenes strains of M-protein type 57 is plasmid-associated. Plasmid pDN571 (3351bp) harbored by S. pyogenes 71-724, the prototype M-type 57 strain, has been completely sequenced and contains three putative open reading frames (repA, scnM57 and ORF3). In addition, the double-strand and single-strand (SSO) origins of replication were identified. Analysis of the replication-associated genetic elements places pDN571 in the ubiquitous pC194/pUB110 family of rolling-circle plasmids. The SSO of pDN571 is of the ssoA type. SA-M57 (encoded by scnM57) is synthesized as a secreted 179-amino acid polypeptide with a 27-residue secretion signal peptide and has no homology to proteins of known function.
Lactobacilli are common inhabitants of the gastrointestinal tracts of mammals and have received considerable attention due to their putative health-promoting properties. Little is known about the traits that enhance the ability of these bacteria to inhabit the gastrointestinal tract. In this paper we describe the development and application of a strategy based on in vivo expression technology (IVET) that enables detection of Lactobacillus reuteri genes specifically induced in the murine gut. A plasmid-based system was constructed containing 'ermGT (which confers lincomycin resistance) as the primary reporter gene for selection of promoters active in the gastrointestinal tract of mice treated with lincomycin. A second reporter gene, 'bglM (beta-glucanase), allowed differentiation between constitutive and in vivo inducible promoters. The system was successfully tested in vitro and in vivo by using a constitutive promoter. Application of the IVET system with chromosomal DNA of L. reuteri 100-23 and reconstituted lactobacillus-free mice revealed three genes induced specifically during colonization. Two of the sequences showed homology to genes encoding xylose isomerase (xylA) and peptide methionine sulfoxide reductase (msrB), which are involved in nutrient acquisition and stress responses, respectively. The third locus showed homology to the gene encoding a protein whose function is not known. Our IVET system has the potential to identify genes of lactobacilli that have not previously been functionally characterized but which may be essential for growth of these bacteria in the gastrointestinal ecosystem.