Alternative Pathway to a Glycopeptide-Resistant Cell Wall in the Balhimycin Producer Amycolatopsis balhimycina

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 Delta vanHAX(Ab) mutant showed a reduced glycopeptide resistance in comparison to the wild type. Nevertheless, Delta vanHAX(Ab) 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, Ddl1(Ab), as a paraloguous chromosomal D-Ala-D-Lac ligase able to complement the function of VanA(Ab) in the Delta vanHAX(Ab) mutant. Furthermore, A balhimycina harbors a vanY(Ab) gene encoding a D,D-carboxypeptidase. Transcriptional analysis revealed an upregulated expression of vanY(Ab) in the Delta vanHAX(Ab) mutant. VanY(Ab) cleaves the endstanding D-Ala from the pentapeptide precursors, reducing the quantity of sensitive cell wall precursors in the absence of VanX(Ab). These findings represent an unprecedented coordinated layer of resistance mechanisms in a glycopeptide antibiotic producing bacterium.