Purification and Characterization of Streptin, a Type A1 Lantibiotic Produced by Streptococcus pyogenes

ABSTRACT Approximately 10% of Streptococcus pyogenes strains inhibit the growth of all nine indicators in a standardized streptococcal bacteriocin typing scheme. The present study has shown that this inhibitory profile, referred to as bacteriocin producer (P)-type 777 activity, is due to the type A1 lantibiotic streptin. Two major forms of streptin were purified to homogeneity from 95% acidified (pH 2) methanol extracts of S. pyogenes M25 cells by using a series of reversed-phase chromatographic separations. The fully processed form of streptin (streptin 1) is a 23-amino-acid peptide with a mass of 2,424 Da. The 2,821-M r form of the peptide (streptin 2) has three additional amino acids (TPY) at the N terminus. Strain M25 extracts also contained small quantities of the streptin 1 and streptin 2 peptides in various stages of dehydration. Streptin 1 and streptin 2 were each capable of specifically inducing streptin production when added to strain M25 cultures. The streptin gene cluster resembled that of other type A1 lantibiotics but appeared to lack a streptin-specific proteinase gene. Although the streptin structural gene (srtA) was widespread within S. pyogenes, being detected in 40 of 58 strains, each representing a different M serotype, only 10 of these srtA-positive strains produced active streptin. The failure of some strains to express streptin was attributed to an ∼4.5-kb deletion in their streptin loci, encompassing genes putatively encoding proteins involved in streptin processing (srtB and srtC) and transport (srtT). In other strains, srtA transcription appeared to be defective. No direct association could be detected between the production of streptin and the production of the lantibiotic-like hemolysin streptolysin S in strain M25.

An Introduction to the Hows and Whys of Molecular Typing†

Until recently, the relatedness of bacterial isolates has been determined solely by testing for one or several phenotypic markers, using methods such as serotyping, phage typing, biotyping, antibiotic susceptibility testing, and bacteriocin typing. However, there are problems in the use of many of these phenotype-based methods. For example, phage and bacteriocin typing systems are not available for all bacterial species and serotyping can be labor-intensive and costly. In addition, phenotypic markers may not be stably expressed under certain environmental or culture conditions. In contrast, some of the newer molecular typing methods involving the analysis of DNA offer many advantages over traditional techniques. One of the more important advantages is that since DNA can always be extracted from bacteria, all bacteria should be typeable. Another is that the discriminatory power of DNA-based methods is greater than that of phenotypic procedures. This review focuses on the basics of molecular typing along with the advantages and disadvantages of several of the newer genotypic typing techniques. This includes methods such as plasmid typing, pulsed-field gel electrophoresis, ribotyping and its variations, and polymerase chain reaction-based methods such as random amplified polymorphic DNA analysis. Molecular typing of microorganisms has made great strides in the last decade, and many food microbiology laboratories have become more knowledgeable and better equipped to carry out these new molecular techniques. Molecular typing procedures can be broadly defined as methods used to differentiate bacteria, based on the composition of biological molecules such as proteins, fatty acids, carbohydrates, etc., or nucleic acids. The latter can also be more specifically defined as genotyping, and is the subject of this review.

Comparison of serotype, biotype and bacteriocin type with rDNA RFLP patterns for the type identification ofSerratia marcescens

SummaryVariations in rDNA gene loci in DNA digests of 209 clinical isolates ofSerratia marcescenswere determined with anEscherichia colirRNA probe. Forty-one restriction fragment length polymorphism patterns (ribotypes) were identified, based on the size of 4–14 (mean 7·5) hybridization bands. The patterns differed by more than a single band in 98% of pair-wise comparisons. On a subset of 76 isolates, ribotyping proved to be marginally more discriminating than biotyping (discrimination index 0·92v. 0·89) followed by serotyping (0·87) and bacteriocin typing (0·74). About one-third of isolates belonged to unique ribotypes and only two ribotypes exceeded 5% in frequency (23·0 and 6·4% respectively). A combination of serotype or biotype with ribotyping defined a similar number of strains, although none of the methods alone was sufficiently discriminatory to identify strains. We conclude that due to the accessibility of biotyping and the lack of commercially available antisera forS. marcescens, the biotype and ribotype together provide reliable markers of strain identity.