SsaA, a member of a novel class of transcriptional regulators, controls sansanmycin production in Streptomyces sp. strain SS through a feedback mechanism

Abstract

Sansanmycins, produced by Streptomyces sp. strain SS, are uridyl peptide antibiotics with activities against Pseudomonas aeruginosa and multidrug-resistant Mycobacterium tuberculosis. In this work, the biosynthetic gene cluster of sansanmycins, comprised of 25 open reading frames (ORFs) showing considerable amino acid sequence identity to those of the pacidamycin and napsamycin gene cluster, was identified. SsaA, the archetype of a novel class of transcriptional regulators, was characterized in the sansanmycin gene cluster, with an N-terminal fork head-associated (FHA) domain and a C-terminal LuxR-type helix-turn-helix (HTH) motif. The disruption of ssaA abolished sansanmycin production, as well as the expression of the structural genes for sansanmycin biosynthesis, indicating that SsaA is a pivotal activator for sansanmycin biosynthesis. SsaA was proved to directly bind several putative promoter regions of biosynthetic genes, and comparison of sequences of the binding sites allowed the identification of a consensus SsaA binding sequence, GTMCTGACAN₂TGTCAGKAC. The DNA binding activity of SsaA was inhibited by sansanmycins A and H in a concentration-dependent manner. Furthermore, sansanmycins A and H were found to directly interact with SsaA. These results indicated that SsaA strictly controls the production of sansanmycins at the transcriptional level in a feedback regulatory mechanism by sensing the accumulation of the end products. As the first characterized regulator of uridyl peptide antibiotic biosynthesis, the understanding of this autoregulatory process involved in sansanmycin biosynthesis will likely provide an effective strategy for rational improvements in the yields of these uridyl peptide antibiotics.

Fig 1

Structures of sansanmycins and some pacidamycins and napsamycins. m-Tyr, meta-Tyr; TIC, tetradehydro-3-isoquinoline carboxylic acid; Met^SO, methionine sulfoxide.

Fig 2

Genetic organization of the ssa cluster. Genetic organization of 25 ORFs of the ssa cluster plus 5 ORFs each upstream and downstream is shown. Black lines above the ORFs are DNA fragments retarded in the EMSA experiments, and gray lines are DNA fragments which were not retarded in the EMSA experiments.

Fig 3

Domain structure of SsaA and structure-based alignment of SsaA with its structurally homologous proteins. (A) Predicted domain structure of SsaA. (B) Structure-based alignment of the FHA domain of SsaA with its structurally homologous proteins. Three orthologues of SsaA, PacA (GenBank accession number ADN26237.1), NpsM (ADY76675.1), and SrosN15 (ZP_04694256.1), and the 4 closest FHA structural neighbors of SsaA, EmbR (PDB entry code 2FF4), yeast Rad53 FHA1 (1QU5), yeast Rad53 FHA2 (1QU5), and human Ki67 (1R21), were aligned. The known conserved amino acid residues important for the binding of phosphopeptide are shaded gray. Numbers inserted in the sequences count residues omitted from the nonconserved regions. (C) Structure-based alignment of the DNA binding domain (DBD) of SsaA with its structurally homologous proteins. Three orthologues of SsaA, PacA (GenBank accession number ADN26237.1), NpsM (ADY76675.1), and SrosN15 (ZP_04694256.1), and the 5 closest DBD structural neighbors of SsaA, TraR (PDB entry code 1L3L), QcsR (1P4W), CviR (3QP6), RcsB (3SZT), and ComA (3ULQ), were aligned.

Fig 4

Effects of overexpression of ssaA on sansanmycin production in Streptomyces sp. SS. (A) Antibacterial activities of SS/pL-ssaA and SS/pSET152 at the indicated time points. (B) HPLC analysis of sansanmycins produced by SS/pL-ssaA and SS/pSET152 at 144 h. mAU, milliabsorbance units.

Fig 5

Disruption of ssaA and its effects on sansanmycin production and biosynthetic gene expression. (A) Antibacterial activities of the wild-type strain (1), SS/AKO (2), and SS/AKO/pL-ssaA (3) at the indicated time points. (B) HPLC analysis of sansanmycins produced by the wild-type strain, SS/AKO, and SS/AKO/pL-ssaA at 144 h. (C) Transcriptional analysis of different genes in the wide-type strain, SS/AKO mutant, and its complemented strain, SS/AKO/pL-ssaA. The relative abundance at 48 h of ssaH, ssaN, ssaP, ssaA, ssaX, and ssaC transcripts in mycelia of the wide-type, SS/AKO, and SS/AKO/pL-ssaA strains was determined by quantitative RT-PCR. Data are from three biological samples with one or two PCR determinations of each. The values were normalized to that of hrdB and were represented as means ± standard deviations (SD). The amounts of each particular transcript in the wild-type strain were arbitrarily assigned a value of 1.

Fig 6

EMSA analysis of SsaA with the postulated promoter regions of the ssa cluster. Data represent EMSA analysis of 3′ biotin-11-ddUTP-labeled fragments ssaHp, ssaN-Pp, ssaC-Dp, ssaU-A-1p, ssaU-A-2p, and ssaWp with His₁₀-SsaA. For the ssaHp, ssaN-Pp, ssaU-A-1p, ssaU-A-2p, and ssaC-Dp images, the minus indicates probe only. Lanes under the triangle indicate increasing concentrations of SsaA (0, 0.175, 0.35, 0.7, 1.4, and 1.75 μM) incubated with the probe. C indicates 0.7 μM His₁₀-SsaA incubated with a 200-fold excess of unlabeled specific competitor DNA fragment. In the case of ssaWp, the minus indicates probe only, and a plus indicates probe incubated with 0.175 μM His₁₀-SsaA.

Fig 7

Identification of SsaA binding sites. (A) Identification of SsaA binding site in ssaN-Pp promoter region by DNase I footprinting. The upper electropherogram shows the control reaction, and the lower one shows DNase I footprints of His₁₀-SsaA (2.67 μM) bound to the labeled DNA fragments (50 ng). The protected nucleotide sequences are shaded gray. The nucleotide positions of the protected sequences respective to the ssaP translation start point are shown below the gray shading. (B) Identification of the SsaA consensus binding site by WebLogo. Six sequences were aligned, including 5 sequences detected by the DNase I footprinting experiments and 1 sequence upstream of ssaW that is identical to that of ssaU. The base-pairing nucleotides within the binding site are underlined. (C) Validation of the identified SsaA consensus binding site. The binding of SsaA to the three synthetic duplexes, D1, D2, and D3 (the dashed lines in D3 indicate the deletion of 5 nucleotides), was detected by EMSA analysis. −, probe only; +, probe incubated with 1.75 μM His₁₀-SsaA.

Fig 8

Effect of sansanmycins on the DNA binding activity of SsaA. (A) Effect of different amounts of SS-A on the binding of His₁₀-SsaA (0.175 μM) to ssaC-Dp or ssaU-A-1p. Lane 1, probe only; lane 2, probe incubated with 0.175 μM His₁₀-SsaA; lanes 3 to 10, probe incubated with 0.175 μM His₁₀-SsaA in the presence of increasing concentrations of SS-A (50, 100, 200, 300, 450, 600, 800, and 1,000 μM). (B) Effect of different amounts of SS-H on the binding of His₁₀-SsaA (0.175 μM) to ssaC-Dp or ssaU-A-1p. Lane 1, probe only; lane 2, probe incubated with 0.175 μM His₁₀-SsaA; lanes 3 to 10, probe incubated with 0.175 μM His₁₀-SsaA in the presence of increasing concentrations of SS-H (50, 100, 200, 300, 450, 600, 800, and 1,000 μM). (C) SPR analysis of the effect of SS-A on the DNA binding activity of SsaA. The binding of His₁₀-SsaA (100 nM) to ssaC-Dp-3 fragment immobilized on an SA chip was inhibited by the increasing concentrations of SS-A (50, 100, 200, and 300 μM). (D) SPR analysis of SS-A interaction with immobilized His₁₀-SsaA. Sensorgrams for the increasing concentrations of SS-A (3.125, 6.25 12.5, 25, and 50 μM) show its binding to the immobilized His₁₀-SsaA on a CM5 chip. (E) SPR analysis of SS-H interaction with immobilized His₁₀-SsaA. Sensorgrams for the increasing concentrations of SS-H (12.5, 25, 50, 100, and 200 μM) show its binding to the immobilized His₁₀-SsaA on a CM5 chip.