Amycolatopsis balhimycina DSM 5908 (formerly described as Amycolatopsis mediterranei) was first isolated from a soil sample originating from the Himalayas (20). It belongs to the order Actinomycetales and synthesizes the glycopeptide antibiotic balhimycin. Balhimycin differs only in its glycosylation pattern from vancomycin, which is used as an antibiotic of last resort against multidrug-resistant Gram-positive bacteria. Since the activities of balhimycin are comparable to those of vancomycin both in vitro and in vivo and A. balhimycina is accessible to genetic manipulation, this strain has become a model organism for analyzing glycopeptide synthesis and resistance (32). Glycopeptide antibiotics inhibit cell wall biosynthesis in Grampositive bacteria by binding the D-alanyl-D-alanine (D-Ala-DAla) terminus of peptidoglycan precursors on the outside surface of the cytoplasmic membrane (24). However, when the usage of vancomycin steadily increased in the 1980s (14), the first vancomycin-resistant enterococci (VRE) were isolated in hospitals (17). In these pathogens, cell wall biosynthesis was reprogrammed in such a way that the pentapeptide of the peptidoglycan precursor terminated in D-alanyl-D-lactate (DAla-D-Lac) rather than D-Ala-D-Ala, thereby causing an ϳ1,000-fold lower binding affinity of the glycopeptide (5). This alteration of the cell wall precursor requires the following three genes: vanH, coding for a dehydrogenase which converts pyruvate to D-Lac (1); vanA, which codes for a D-Ala-D-Lac ligase (4); and vanX, which codes for a DD-dipeptidase that cleaves the D-Ala-D-Ala dipeptide to ensure that only altered peptidoglycan precursors terminating in D-Ala-D-Lac are built up (25). van-like genes which have similarity to the resistance genes from VRE have been found in several glycopeptide producers, such as various Amycolatopsis spp. (16).We report here the rare case of an antibiotic producer carrying a biosynthetic gene cluster without essential resistance genes. The essential vanHAX resistance genes, located elsewhere in the chromosome, are expressed constitutively and are not controlled by the vanRS-like two component system vnlRS, resulting in constitutive production of peptidoglycan precursors terminating in D-Ala-D-Lac.
MATERIALS AND METHODSBacterial strains and plasmids. The strains and plasmids used for this study are listed in Table 1.Media and culture conditions. Escherichia coli strains were grown in LB (26) at 37°C. Actinomycetes strains were grown in R5 medium (13) at 30°C. Media were supplemented with antibiotics when necessary to maintain plasmids.DNA preparation and manipulation. The methods used for the isolation and manipulation of DNA for E. coli and actinomycetes were described by Kieser et al. (13). PCR fragments were isolated from agarose gels by use of a QIAquick kit (Qiagen). Restriction endonucleases were obtained from various suppliers and were used according to their specifications.