A Two-Component regulatory system with opposite effects on glycopeptide antibiotic biosynthesis and resistance

Abstract

The glycopeptide A40926, produced by the actinomycete Nonomuraea gerenzanensis, is the precursor of dalbavancin, a second-generation glycopeptide antibiotic approved for clinical use in the USA and Europe in 2014 and 2015, respectively. The final product of the biosynthetic pathway is an O-acetylated form of A40926 (acA40926). Glycopeptide biosynthesis in N. gerenzanensis is dependent upon the dbv gene cluster that encodes, in addition to the two essential positive regulators Dbv3 and Dbv4, the putative members of a two-component signal transduction system, specifically the response regulator Dbv6 and the sensor kinase Dbv22. The aim of this work was to assign a role to these two genes. Our results demonstrate that deletion of dbv22 leads to an increased antibiotic production with a concomitant reduction in glycopeptide resistance. Deletion of dbv6 results in a similar phenotype, although the effects are not as strong as in the Δdbv22 mutant. Consistently, quantitative RT-PCR analysis showed that Dbv6 and Dbv22 negatively regulate the regulatory genes (dbv3 and dbv4), as well as some dbv biosynthetic genes (dbv23 and dbv24), whereas Dbv6 and Dbv22 positively regulate transcription of the single, cluster-associated resistance gene. Finally, we demonstrate that exogenously added acA40926 and its precursor A40926 can modulate transcription of dbv genes but with an opposite extent: A40926 strongly stimulates transcription of the Dbv6/Dbv22 target genes while acA40926 has a neutral or negative effect on transcription of those genes. We propose a model in which glycopeptide biosynthesis in N. gerenzanensis is modulated through a positive feedback by the biosynthetic precursor A40926 and a negative feedback by the final product acA40926. In addition to previously reported control systems, this sophisticated control loop might help the producing strain cope with the toxicity of its own product. This work, besides leading to improved glycopeptide producing strains, enlarges our knowledge on the regulation of glycopeptide biosynthesis in actinomycetes, setting N. gerenzanensis and its two-component system Dbv6-Dbv22 apart from other glycopeptide producers.

Figure 1

Chemical structures of O-acetyl A40926 (A) and of dalbavancin (B) and genetic organization of the dbv cluster (C). (A,B). Only the component B0 (the major congener in the A40926 complex) is shown for simplicity. The acetyl group of A40926 and the chemical modification present in dalbavancin are indicated in red circle and pink, respectively. (C) The thin black arrows indicate experimentally determined operons. Red and green arrows represent the transcriptional units controlled by Dbv4 and Dbv3, respectively. The dbv genes are grouped by functional category as indicated. Asterisks indicate the genes analyzed by qRT-PCR analysis in the present work.

Figure 2

Growth curves and antibiotic production by N. gerenzanensis wild type, Δdbv6 and Δdbv22 strains in RARE3 medium. (A) The dry weight of the wild type, Δdbv6 and Δdbv22 strains are shown with green, red and blue lines, respectively. Standard deviation was calculated as average of three technical and two biological replicates. (B) Bioassays of 50 µL of the culture broth of the wild type, Δdbv6 and Δdbv22 strains using K. rhizophila as test strain. (+) and (−) indicate the positive (culture broth collected from the parental strain after 120 h) and negative (only growth medium) control, respectively.

Figure 3

AcA40926 production and growth in V40P medium of the wild type, Δdbv6 and Δdbv22 strains. Squares indicate acA40926 concentrations, while triangles represent %PMV. Brown, red and violet symbols correspond to the wild type, Δdbv6 and Δdbv22 strains, respectively.

Figure 4

qRT-PCR of dbv genes in the wild type and Δdbv6 and Δdbv22 mutant strains. For each gene, data are normalized to the parental strain (blue bars). Red and green bars represent relative RNA levels seen in the Δdbv6 and Δdbv22 strains, respectively. Error bars are calculated from three independent determinations of mRNA abundance in each sample. RNAs were extracted after 54 h of growth in RARE3 medium.

Figure 5

Resistance assay of the wild type and Δdbv6 and Δdbv22 mutant strains. Plating efficiency of the wild type, Δdbv6 and Δdbv22 strains on different concentrations of A40926 (solid lines) or of acA40926 (dashed lines). Reported data are the average of two independent counts. The limit of detection were 10⁵ CFU/mL for the wild type, Δdbv6, and Δdbv22 strains.

Figure 6

qRT-PCR analysis of the gene expression of dbv3, dbv4, dbv6, dbv7, dbv23 and dbv24 in the wild type strain. RNA was isolated from the wild type strain incubated in V40P medium for 2 and 5 h in the presence of 0.5 µg/mL A40926 (black bars) or 0.5 µg/mL acA40926 (red bars). Data are expressed as relative values to the no-addition controls. Within each sample, mRNA levels were normalized to hrdB, arbitrarily setting the ratio for each sample to 1. Standard deviations are calculated from three independent determinations of mRNA abundance in each sample.

Figure 7

Gel mobility shift assays of DNA regions upstream of dbv3, dbv4, dbv7, dbv23, dbv24 and dbv6. Lanes labeled with an asterisk contained the probe only (updbv3, updbv4, updbv7, updbv23, updbv24 and updbv6). All lanes contained 50 ng of target DNA. Increasing concentrations of His-Dbv6 (0.5, 1, 1.5, 2 mM) were incubated in the presence of probes as indicated.

Figure 8

Phylogenetic analysis of 62 response regulators (left panel) and 65 sensory kinases (right panel). Sequences associated with established gene clusters are identified by the compound name followed by the genus of the corresponding strain; characterized S. coelicolor proteins are identified by the protein name followed by the SCO code; and N. gerenzanensis sequences are denoted by the SBO or SBP codes. The branches associated with the Van-and Dbv-related TCSs are identified by green and orange boxes, respectively. Green and red arrows indicate the adjacent members of two different, uncharacterized N. gerenzanensis TCSs closely associated with the Dbv branches. Sequences associated with N. gerenzanensis biosynthetic gene clusters, as identified by AntiSMASH 5.0, have a yellow highlight.