Streptococcus uberis, an environmental organism also associated with dairy animals, is a common and persistent cause of bovine mastitis. New approaches to control these infections need to be identified. One such strategy may be the application of bacteriocins; proteinaceous antimicrobials elaborated by bacteria that typically inhibit the growth of strains closely related to the producer organism. The well-characterized lactococcal bacteriocin nisin is the active ingredient in two commercial products currently in use for the prevention of mastitis. However, reports of resistance development have prompted the investigation of alternative bacteriocins to be used in conjunction with nisin in 'bacteriocin cocktails' designed to have more comprehensive inhibitory activity against mastitis pathogens. The bacteriocins of gram-positive bacteria have been divided into four distinct classes: (I) lantibiotics, (II) non-lantibiotic peptides, (III) large proteins, and (IV) circular peptides.
Although it has been known for more than twenty years that S. uberis commonly produce bacteriocin-like inhibitory substances (BLIS), none had been characterised prior to the present study. The first step in the current investigation was a survey of the BLIS activities of a set of fifteen S. uberis and S. bovis strains against a set of standard indicators as well as common gram-positive mastitis pathogens. Additional tests using a deferred antagonism agar plate-based assay showed that some of the BLIS activities were heat-sensitive and their production was influenced by the presence of either blood or a fermentable carbohydrate source in the test medium. On the basis of the results obtained from these tests it became apparent that S. uberis and S. bovis may commonly produce more than a single inhibitory agent.
S. uberis 42 became the focus of this study because (a) it had broad inhibitory activity against mastitis-associated bacteria, (b) it did not display cross-resistance to nisin, and (c) from the preliminary screening results it appeared to produce both heat-stable and heat-labile inhibitory agents. Acid extracts of S. uberis 42 cells yielded inhibitory activity that, when fractionated by reversed-phase HPLC, yielded a peptide of 3029 Da. Although this peptide was blocked to Edman degradation at position 2, following propanethiol-modification a 20-amino acid sequence was obtained. Degenerate primers to lantibiotic biosynthesis gene homologs were used to initiate inverse PCR and primer walking, ultimately yielding a 15-kb contiguous sequence encompassing 11 genes typical of those involved in lantibiotic synthesis, regulation and immunity. Due to the close similarities to nisin of the S. uberis 42 lantibiotic precursor (78%), and the organisation and composition of the locus, this inhibitor was named nisin U. Nucleotide sequences homologous to insertion sequences were detected in the vicinity of the nisin U locus, and indicate a possible mechanism of acquisition of this locus by S. uberis. The locus was detected in ten other S. uberis, and also in two S. agalactiae and two S. thoraltensis strains, and in one S. porcinus and one S. pluranimalium strain. The amino acid sequences of some of these differed in one or two amino acids, and these variants were named nisin U2 and nisin U3 accordingly. Nisin U, the two nisin U variants, and nisin A exhibited cross-immunity (i.e. all of the producer strains were insensitive to each form of nisin) and cross-inducibility (i.e. all of the producer strains displayed enhanced production when exposed to each form of nisin).
Nisin U did not contribute to the entire spectrum of inhibitory activity of S. uberis 42. Freeze thaw extracts of S. uberis 42 agar cultures yielded heat-labile inhibitory activity that was inhibitory to L. lactis A5, a producer of nisin Z. Subsequent purification by cation-exchange chromatography, gel filtration, and reversed-phase HPLC yielded a peptide of mass 7048 Da, which was resistant to Edman degradation. Digestion with chymotrypsin released an 819 Da peptide fragment of sequence NHâ‚‚-KAQAVIW-COOH. Tn916 mutagenesis of S. uberis 42 enabled the identification of the genetic locus of the inhibitor, comprising six genes potentially involved in its biosynthesis and immunity. The detection of a pair of flanking 159-bp direct repeats indicates possible acquisition of the locus by 'long target duplication'. The inhibitor was inferred to be a circular peptide, on the basis of its behaviour to Edman degradation, and by comparison of its locus with that of other circular bacteriocins. On the basis that the purified peptide appears to induce lysis in sensitive bacteria, although by an as-yet unidentified mechanism, the inhibitor was named uberolysin. The uberolysin structural gene was detected in eight other strains of S. uberis, however not all of these appeared to be producing active inhibitor.
No bacteriocins closely resembling the two reported in this thesis have been demonstrated previously to be produced by members of the genus Streptococcus. The remarkable diversity in the structures, activity spectra and basic modes of action of these two bacteriocins produced by a single strain of S. uberis, combined with the observation of apparent greater heterogeneity in properties of a preliminary sampling of BLIS-producing strains, indicates that these bacteria may be an important source of novel antimicrobials of potential value for the treatment of mixed bacterial infections and for minimising potential resistance development.
| Identifer | oai:union.ndltd.org:ADTP/217488 |
| Date | January 2007 |
| Creators | Wirawan, Ruth E., n/a |
| Publisher | University of Otago. Department of Microbiology & Immunology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Ruth E. Wirawan |
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