Phosphate-controlled regulator for the biosynthesis of the dalbavancin precursor A40926

Abstract

The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of the novel antibiotic dalbavancin. Previous studies have shown that phosphate limitation results in enhanced A40926 production. The A40926 biosynthetic gene (dbv) cluster, which consists of 37 genes, encodes two putative regulators, Dbv3 and Dbv4, as well as the response regulator (Dbv6) and the sensor-kinase (Dbv22) of a putative two-component system. Reverse transcription-PCR (RT-PCR) and real-time RT-PCR analysis revealed that the dbv14-dbv8 and the dbv30-dbv35 operons, as well as dbv4, were negatively influenced by phosphate. Dbv4 shows a putative helix-turn-helix DNA-binding motif and shares sequence similarity with StrR, the transcriptional activator of streptomycin biosynthesis in Streptomyces griseus. Dbv4 was expressed in Escherichia coli as an N-terminal His(6)-tagged protein. The purified protein bound the dbv14 and dbv30 upstream regions but not the region preceding dbv4. Bbr, a Dbv4 ortholog from the gene cluster for the synthesis of the glycopeptide balhimycin, also bound to the dbv14 and dbv30 upstream regions, while Dbv4 bound appropriate regions from the balhimycin cluster. Our results provide new insights into the regulation of glycopeptide antibiotics, indicating that the phosphate-controlled regulator Dbv4 governs two key steps in A40926 biosynthesis: the biosynthesis of the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine and critical tailoring reactions on the heptapeptide backbone.

FIG. 1.

Organization of the 71-kb dbv cluster and transcriptional map. (A) Genomic organization of the 71-kb dbv cluster. The thin arrows represent the experimentally determined transcriptional units; the thick arrows indicate the Dbv4-controlled dbv14-dbv8 and dbv30-dbv35 operons; triangles denote DNA fragments used in gel retardation experiments; asterisks and the symbol “Q” indicate genes targeted by RT-PCR and quantitative RT-PCR, respectively. dbv genes are grouped by category as indicated (33, 35). (B) RT-PCR analysis of intergenic regions. Total RNA, extracted after 47 h of growth under LowP conditions, was used as a template in the presence (+) or in the absence (−) of reverse transcriptase. Lanes D and C represent positive (DNA) and negative (water) controls, respectively.

FIG. 2.

A40926 production in batch fermentation. Growth (squares), phosphate concentration (circles), and A40926 production (triangles) were monitored with initial phosphate concentrations of 2 mM (LowP; closed symbols) and 4.2 mM (HighP; open symbols).

FIG. 3.

Transcriptional analysis of selected dbv genes under LowP and HighP conditions. RNA samples, extracted from mycelium after 28, 35, 42, 47, 60, and 71 h were analyzed by RT-PCR using primers specific for each dbv gene and for hrdB (see Table S1 in the supplemental material). Asterisks indicate negative controls (no reverse transcriptase).

FIG. 4.

Real-time RT-PCR analysis of dbv3, dbv4, dbv6, dbv8, dbv9, dbv10, dbv11, dbv12, dbv13, and dbv14 (A) and dbv15, dbv16, dbv22, dbv29, dbv30, dbv31, dbv32, dbv33, dbv34, and dbv35 (B) under LowP and HighP conditions. mRNA levels after 42 and 60 h are expressed as relative values to hrdB, arbitrarily setting the ratio values for the 60-h, HighP sample to 1. Error bars are calculated from three independent determinations of mRNA abundance in each sample (see Materials and Methods).

FIG. 5.

Identification of the Dbv4 binding site. (A) dbv14 promoter region. The open arrow represents the dbv14 gene, the bent arrow represents the most probable transcription start site, and the boxes indicate the deduced −10 and −35 regions. The black arrows indicate the putative Dbv4 binding site. The fragments (14A to 14D) used as electrophoretic mobility shift assay probes are indicated below. (B) Alignment of the StrR-like binding sites. Nucleotides conserved in the four sequences are in boldface. The inverted repeats are indicated by arrows.

FIG. 6.

Purification of His₆-tagged Dbv4 and gel mobility shift assays. (A) SDS-PAGE analysis of His₆-tagged Dbv4 purification. Lane 1, cell extract (25 μg of proteins) from an IPTG-induced culture of E. coli BL21(DE3)pLysS containing pRSET-Dbv4. Lanes 2, 3, and 4, pooled Ni-NTA fractions were eluted with 50, 150, and 250 mM imidazole, respectively. The arrow indicates the His₆-tagged Dbv4 protein. Molecular mass standards are indicated on the left. (B to F) Gel mobility shift assays of DNA regions upstream of dbv4 (B), dbv14 (C), dbv30 (D), bbr (E), and oxyA (F) with Dbv4, Bbr, or Nonomuraea total proteins (CE). Lanes labeled with an asterisk contained the probe only. All lanes contained 0.4 ng of ³²P-end-labeled target DNA. The binding reactions reported in panel C were carried out in the presence of a 200-fold molar excess of unlabeled probe (lane 1) and a 200-fold molar excess of unlabeled competitor DNA (lane 2).

FIG. 7.

Gel mobility shift assays of ³²P-end-labeled fragments 14A (A), 14B (B), 14C (C), and 14D (D) with Dbv4, Bbr, or Nonomuraea total proteins (CE). All samples contained 0.4 ng of labeled target DNA. The asterisks indicate the lane containing the probe only. Lanes: 1, binding reaction; 2, same as in lane 1 but with a 200-fold molar excess of unlabeled probe; 3, same as in lane 1 but with a 200-fold molar excess of unlabeled aspecific competitor DNA added. Arrows denote bands observed with fragments A and D and total Nonomuraea proteins.