Highly Efficient Staphylococcus carnosus Mutant Selection System Based on Suicidal Bacteriocin Activation

Abstract

Strains from various staphylococcal species produce bacteriocin peptides, which are thought to play important roles in bacterial competition and offer interesting biotechnological avenues. Many bacteriocins are secreted as inactive prepeptides with subsequent activation by specific proteolytic cleavage. By deletion of the protease gene gdmP in Staphylococcus gallinarum Tü3928, which produces the highly active lanthionine-containing bacteriocin gallidermin (lantibiotic), a strain was created producing inactive pregallidermin. On this basis, a new suicidal mutant selection system in the food-grade bacterium Staphylococcus carnosus was developed. Whereas pregallidermin was inactive against S. carnosus, it exerted potent bactericidal activity toward GdmP-secreting S. carnosus strains. To take advantage of this effect, gdmP was cloned in plasmid vectors used for random transposon mutagenesis or targeted allelic replacement of chromosomal genes. Both mutagenesis strategies rely on rare recombination events, and it has remained difficult and laborious to identify mutants among a vast majority of bacterial clones that still contain the delivery vectors. The gdmP-expressing plasmids pGS1 and pGS2 enabled very fast, easy, and reliable identification of transposon and gene replacement mutants, respectively. Mutant selection in the presence of pregallidermin caused suicidal inactivation of all clones that had retained the plasmids and allowed growth of only plasmid-cured mutants. Efficiency of mutant identification was several magnitudes higher than standard screening for the absence of plasmid-encoded antibiotic resistance markers and reached 100% specificity. Thus, the new pregallidermin-based mutant selection system represents a substantial improvement of staphylococcal mutagenesis methodology.

An update on the molecular genetics toolbox for staphylococci

“…Among them, the temperature-sensitive S. aureus/E. coli shuttle vector pBT2 (Brückner, 1997) has been frequently applied for gene replacement in S. aureus , S. xylosus , S. carnosus and S. epidermidis as well as in the human pathogen S. lugdunensis and the lantibiotic producer S. gallinarum , as reported recently (Krismer et al , 2012; Marlinghaus et al , 2012). The pBT2 vector backbone, which carries a cat gene and the temperature sensitive pE194ts replicon, permits stable inheritance in staphylococci at 30 °C.…”

“…Wild-type sacB is a widely applied marker for counterselection in several bacteria, but for S. aureus the use of a variant with a defective secretion signal sequence appears to be critical. A system for allelic replacement in S. carnosus (Krismer et al , 2012) employs the lantibiotic precursor pregallidermin, naturally produced by S. gallinarum (Kellner et al , 1988) and the cognate protease GdmP which converts pregallidermin to the toxic form by removal of a leader peptide. By exploiting a pBT2 derivative bearing gdmP , plasmid-free mutants were achieved in pregallidermin spiked medium.…”

The Peptidoglycan Pattern of Staphylococcus carnosus TM300—Detailed Analysis and Variations Due to Genetic and Metabolic Influences

“…For overexpression of HlyD, FtsE and FtsX, the genes SCA_1997, SCA_1995-1996 and SCA_1995-1997 were cloned into the xylose inducible plasmid pPTX [52] and cut with the same enzymes. SCA_0214 (putative ld -CP): For deletion, the knockout vector pGS1 was used [59]. The overexpression plasmid was constructed in the xylose inducible plasmid pPTX [52] and cut with the same enzymes.…”

“…The overexpression plasmid was constructed in the xylose inducible plasmid pPTX [52] and cut with the same enzymes. SCA_1342 ( ccpA ): For deletion, knockout vector pGS1was used [59]. For constitutive complementation, SCA_1342 was ligated into pRAB11-EF-TU, cut with the same enzymes.…”