Pristinamycin IIA

Date fruits extract was assessed as substrate for pristinamycin production in shake flasks usingStreptomyces pristinaespiralisDSMZ 40338 as production organism. A process of microfiltration, concentration, and solvent extraction was used to recover the antibiotic from the fermentation broth and its antimicrobial activity was tested using a well assay method. During a fermentation period of 120 h, the bacterium consumed approximately 28.4 g/l sugars, grew from 0.4 g/l to 4.0 g/l dry weight, and produced 51.0 mg/L pristinamycin. The microfiltration, concentration, and ethyl acetate solvent extraction applied resulted in 71% pristinamycin recovery. The antibiotic showed inhibition capacity comparable to that of the pristinamycins IA and IIA standards. The inhibited bacteria wereStaphylococcus aureus,Escherichia coli, andEnterococcus faecium. Clear inhibition zones of 13.3-19.6 mm diameter were formed.

A wild strain of Staphylococcus aureus which inactivates a wide variety of antibiotics has been found to inactivate pristinamycin IIA, an antistaphylococcal antibiotic. This phenomenon has been demonstrated to be plasmid mediated. The plasmid directs the biosynthesis of an acetyltransferase which is able to O-acetylate the drug. We propose to call the new enzyme PAC (IIA): Pristinamycin acetyltransferase.

In Gram-positive bacteria, a large subfamily of dual ATP-binding cassette proteins confers acquired or intrinsic resistance to macrolide, lincosamide, and streptogramin antibiotics by a far from well understood mechanism. Here, we report the first biochemical characterization of one such protein, Vga(A), which is involved in streptogramin A (SgA) resistance among staphylococci. Vga(A) is composed of two nucleotide-binding domains (NBDs), separated by a charged linker, with a C-terminal extension and without identified transmembrane domains. Highly purified Vga(A) displays a strong ATPase activity (K(m) = 78 mum, V(m) = 6.8 min(-1)) that was hardly inhibited by orthovanadate. Using mutants of the conserved catalytic glutamate residues, the two NBDs of Vga(A) were shown to contribute unequally to the total ATPase activity, the mutation at NBD2 being more detrimental than the other. ATPase activity of both catalytic sites was essential for Vga(A) biological function because each single Glu mutant was unable to confer SgA resistance in the staphylococcal host. Of great interest, Vga(A) ATPase was specifically inhibited in a non-competitive manner by the SgA substrate, pristinamycin IIA (PIIA). A deletion of the last 18 amino acids of Vga(A) slightly affected the ATPase activity without modifying the PIIA inhibition values. In contrast, this deletion reduced 4-fold the levels of SgA resistance. Altogether, our results suggest a role for the C terminus in regulation of the SgA antibiotic resistance mechanism conferred by Vga(A) and demonstrate that this dual ATP-binding cassette protein interacts directly and specifically with PIIA, its cognate substrate.

A Streptomyces pristinaespiralis strain, which produces a streptogramin antibiotic pristinamycin II (PII) as a mixture of two biologically active molecules PIIB and PIIA, was genetically engineered to produce exclusively PIIA. The snaA,B genes, which encode a PIIA synthase that performs oxidation of the precursor (PIIB) to the final product (PIIA), were integrated in the chromosome of S. pristinaespiralis using an integrative derivative of the pSAM2 genetic element from Streptomyces ambofaciens. Integration was due to site-specific recombination at the attB site of S. pristinaespiralis, and no homologous recombination at the snaA,B locus was observed. The attB site of S. pristinaespiralis was sequenced and found to overlap the 3' end of a pro-tRNA gene. The integrants were stable in industrial conditions of pristinamycin production and showed no decrease in PII biosynthesis. Western blot analysis showed a constant production of the PIIA synthase in the overall fermentation process due to expression of the cloned snaA,B genes from the constitutive ermE promoter. This allows the complete conversion of the PIIB form into PIIA.