Balhimycin, a vancomycin-type glycopeptide, is a lipid II targeting antibiotic produced by Amycolatopsis balhimycina. A. balhimycina has developed a self-resistance mechanism based on the synergistic action of different enzymes resulting in modified peptidoglycan. The canonical resistance mechanism against glycopeptides is the synthesis of peptidoglycan precursors ending with acyl-d-alanyl-d-lactate (d-Ala-d-Lac) rather than acyl-d-alanyl-d-alanine (d-Ala-d-Ala). This reprogramming is the result of the enzymes VanH, VanA, and VanX. VanH and VanA are required to produce d-Ala-d-Lac; VanX cleaves cytosolic pools of d-Ala-d-Ala, thereby ensuring that peptidoglycan is enriched in d-Ala-d-Lac. In A. balhimycina, the ΔvanHAXAb mutant showed a reduced glycopeptide resistance in comparison to the wild type. Nevertheless, ΔvanHAXAb was paradoxically still able to produce d-Ala-d-Lac containing resistant cell wall precursors suggesting the presence of a novel alternative glycopeptide resistance mechanism. In silico analysis, inactivation studies, and biochemical assays led to the characterization of an enzyme, Ddl1Ab, as a paraloguous chromosomal d-Ala-d-Lac ligase able to complement the function of VanAAb in the ΔvanHAXAb mutant. Furthermore, A. balhimycina harbors a vanYAb gene encoding a d,d-carboxypeptidase. Transcriptional analysis revealed an upregulated expression of vanYAb in the ΔvanHAXAb mutant. VanYAb cleaves the endstanding d-Ala from the pentapeptide precursors, reducing the quantity of sensitive cell wall precursors in the absence of VanXAb. These findings represent an unprecedented coordinated layer of resistance mechanisms in a glycopeptide antibiotic producing bacterium.

Induction of staphylococcal penicillinase by benzylpenicillin: effect of pH, concentration of ferrous ion and inducer, and duration of exposure of cells to inducer. J. Bacteriol. 86:717-727. 1963.-The kinetics of induction of penicillinase by benzylpenicillin in exponentially multiplying Staphylococcus aureus strain 55-C-1 were shown to vary with the pH. At pH 7.3 in the absence of free inducer, the rate of increase of penicillinase activity rapidly declined and came to a halt. At pH 5.4 to 5.5 and in the presence of optimal concentrations of Fe++, the penicillinase activity of the induced culture increased linearly with time for 2.5 or more generations, but the rate of increase usually declined eventually. Evidence was advanced to support the concept that the acidic pH and optimal Fe++ concentration maintain the induced formation of enzyme. The induced increase in penicillinase activity appeared 3 to 4 min after the addition of benzylpenicillin. The degree of induction of penicillinase varied with the duration of exposure of the staphylococci to the inducer and with the concentration of inducer. Maximal induction under our conditions was attained by exposure for 15 min to benzylpenicillin at an initial concentration between 0.6 and 2 units/ml. Although the inducibility of staphylococcal penicillinase has been known for some time (Geronimus and Cohen, 1957; Steinman, 1961; Crompton et al., 1962), more information on the characteristics of this system, comparable with that developed for classical systems such as penicillinase in Bacillus cereus and f-galactosidase in Escherichia coli, has been lacking. In this paper, we present results of studies of the kinetics of induction of staphylococcal penicillinase. We have found that the kinetics of induction of this enzyme in exponentially multiplying cultures of Staphylococcus aureus vary markedly with pH and Fe concentration. At an acidic pH in the range from 5.4 to 5.5 and a favorable Fe concentration, penicillinase activity increases linearly with time in the absence of free inducer, in a fashion kinetically similar to but usually not so prolonged as that described by Pollock (1950) for B. cereus. Other aspects of induction in this system, including the effects of time of exposure of cells to inducer, are also presented. MATERIALS AND METHODS Organisms. S. aureus strain 55-C-1 (Geronimus and Cohen, 1957) was the routine test organism. Drops of overnight broth cultures taken up on porcelain beads and then desiccated over silica gel served as routine inocula. The dried beads, stored at 4 C, were used for periods up to 6 months. A few experiments were repeated with several other penicillinase-producing strains of S. aureus to test the generality of the observations. These strains were stored and inoculated from nutrient agar slants. Media. Cultures were grown in tryptic digest broth (TD), lot no. 003607 (BBL), unless otherwise stated. Several other lots failed to sustain growth at pH 5.5. It was eventually found that acetic acid added to the tryptic meat digest during t...