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.

Fig 1

Construction of an S. gallinarum Tü3928 gdmP mutant. (A) Schematic representation of the biosynthetic and regulatory genes of the gallidermin operon. The genes responsible for transport and producer self-immunity, which are located upstream, are not shown. Flanking regions (FR1 and FR2) were amplified by PCR. (B) The majority of gdmP was replaced by an erythromycin resistance cassette (ermB) located in the same orientation as gdmP, while the ribosomal binding site of the neighboring gdmQ was not affected. The chromosomal integration was achieved by allelic replacement via FR1 and FR2 (indicated by dotted lines).

Fig 2

Identification of pregallidermin in the culture supernatant of the S. gallinarum Tü3928 ΔgdmP::erm mutant and in vitro conversion into active gallidermin by culture filtrate of S. carnosus TM300 pT182-gdmP. (A) After cultivation of the S. gallinarum Tü3928 ΔgdmP::erm mutant in gallidermin production medium YE4, a peak can be detected after 6.1 min of retention. (B) One hour of incubation of the filter-sterilized culture supernatant from panel A with supernatant of the GdmP-producing S. carnosus TM300 pT182-gdmP in the ratio 1:1 at 37°C results in the reduction of the pregallidermin peak at 6.1 min and the appearance of a peak at 6.46 min corresponding to active gallidermin. (C) UV/Vis absorption spectra of the HPLC peak eluting at 6.1 min and of purified gallidermin, showing overall spectrum similarity and the characteristic absorption at 270 nm, supporting the assumption that the newly appearing peak is pregallidermin. (D) HPLC chromatogram showing the different retention times of purified gallidermin and pregallidermin (1 mg/ml gallidermin; 0.5 mg/ml pregallidermin). The detection wavelength in Fig. 2A, B, and D was 210 nm.

Fig 3

Suicidal conversion of inactive pregallidermin into active gallidermin by a GdmP-secreting S. carnosus TM300 strain. The gallidermin producer S. gallinarum Tü3928 (A) produces active gallidermin, thereby inhibiting S. carnosus pT182 (upper plate) and pT182-gdmP (lower plate). The S. gallinarum ΔgdmP::erm mutant (B) produces pregallidermin, which has no effect on S. carnosus pT182 (upper plate) but is converted into active gallidermin by S. carnosus pT182-gdmP (lower plate), leading to suicidal growth inhibition of the protease producer.

Fig 4

Determination of the selective pregallidermin (PG) concentration on the GdmP-expressing S. carnosus TM300 (pT182-gdmP). The pictures show agar plates with increasing concentrations of pregallidermin (A) or filter-sterilized culture supernatant of the S. gallinarum ΔgdmP::erm mutant (B). (A) Whereas 0.01 μg/ml pregallidermin has no significant influence on CFU numbers and colony morphology, 0.05 μg/ml pregallidermin led to a slight decrease in colony size but not in CFU numbers. A total of 0.1 μg/ml pregallidermin results in colony formation of about 3 to 5% of plated cells, whereas 0.5 μg/ml pregallidermin results in complete inhibition of GdmP-expressing S. carnosus cells. The control S. carnosus pT182 is not affected by 0.5 μg/ml pregallidermin. (B) Filter-sterilized culture supernatant of the S. gallinarum ΔgdmP::erm mutant has only little growth inhibitory impact on S. carnosus pT182 at a dilution rate of 1:100 and an inhibitory effect at a 1:50 dilution. In contrast, the 1:1,000 dilution has already a strong growth inhibitory effect on S. carnosus pT182-gdmP.

Fig 5

Schematic presentation of the newly constructed pregallidermin selection vectors pGS1 and pGS2. The shuttle vector pGS1 is a derivative of the allelic replacement vector pBT2 (9), sharing the same multiple cloning site (MCS). For counterselection on pregallidermin, gdmP was inserted. In contrast, pGS2 is no shuttle vector and is replicating only in Gram-positive bacteria. It was generated by the insertion of gdmP into the transposon delivery vector pTV1ts (53). Both pGS1 and pGS2 share a large portion of identity. The location of the transposon Tn917 is indicated by the double arrow. bla, β-lactamase resistance gene; gdmP, gallidermin protease gene; cat, chloramphenicol resistance gene; repY, replication protein gene; erm, erythromycin resistance gene; tnpR, resolvase gene; tnpA, transposase gene.

Fig 6

Schematic presentation of the sequencing of Tn917 mutants with impaired β-galactosidase activity (A) or absent acid production from mannitol (B). A total of 1,300 Tn917 mutants obtained on erythromycin- and pregallidermin-containing agar plates were screened by replica plating on X-Gal agar plates for defects in β-galactosidase activity and mannitol-containing Morse agar plates for defects in mannitol utilization, respectively. Three independent mutants were obtained for each screening. (A) Chromosomal sequencing showed the Tn917 insertions in the genes for the lactose transporter lacP, the lactose operon regulator lacR, or the odhA gene encoding a hypothetical protein with homology to the 2-oxoglutarate-decarboxylase sucA. (B) The three mutants in mannitol utilization all had the Tn917 insertion in different positions of the mtlA transcript, encoding the (EII) component of the mannitol-specific PTS system.