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

Abstract

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.

The VanRS Homologous Two-Component System VnlRSAbof the Glycopeptide ProducerAmycolatopsis balhimycinaActivates Transcription of thevanHAXScGenes inStreptomyces coelicolor, but not inA. balhimycina

“… 47 Investigations of the PG of A. balhimycina revealed the presence of 3–3 cross-linked tetra- and tripeptides. 40 , 45 Furthermore, we could identify at least three ldt genes in the genome of A. balhimycina . 45 We therefore speculate that by activating the expression of vanY Ab , VnlR Ab is involved in regulating an alternative, VanHAX Ab -independent glycopeptide resistance mechanism in A. balhimycina .…”

“…However, in contrast to other glycopeptide-resistant bacteria, the vanHAX Ab genes in A. balhimycina are located 2 Mb apart from the vnlRS Ab genes, which are part of the balhimycin biosynthetic gene cluster. 40 …”

“…Resistance test, reverse transcriptase polymerase chain reaction (RT-PCR) analyses, extraction of PG precursors, PG isolation, and the high-performance liquid chromatography–mass spectrometry (HPLC-MS) analyses were performed as described. 27 , 40 …”

“…The balhimycin concentration in 1 ml culture was quantified using HPLC with a photodiode array detector (HPLC-DAD) as described. 40 The balhimycin concentration was calculated to 100 μg/ml total DNA.…”

Genetics and Genomics of the Genus Amycolatopsis

“…Within actinobacteria, members of genus Streptomyces contribute to almost 70-80 % of secondary metabolites while a small percentage is contributed by Amycolatopsis, Actinoplanes, Micromonospora and Saccharopolyspora [1]. Among these genera, the genus Amycolatopsis is of special importance for its capacity to produce several commercially and medicinally important antibiotics as balhimycin, vancomycin and rifamycin [2][3][4], other secondary metabolites as immuno-suppressants, anti-cancer agents etc. [5] and other applications [6][7][8].…”