No abstract

YdiV is a negative regulator of cell motility. It interacts with FlhD4C2 complex, a product of flagellar master operon, which works as the transcription activator of all other flagellar operons. Here, we report the crystal structures of YdiV and YdiV2–FlhD2 complex at 1.9 Å and 2.9 Å resolutions, respectively. Interestingly, YdiV formed multiple types of complexes with FlhD4C2. YdiV1–FlhD4C2 and YdiV2–FlhD4C2 still bound to DNA, while YdiV3–FlhD4C2 and YdiV4–FlhD4C2 did not. DNA bound FlhD4C2 through wrapping around the FlhC subunit rather than the FlhD subunit. Structural analysis showed that only two peripheral FlhD subunits were accessible for YdiV binding, forming the YdiV2–FlhD4C2 complex without affecting the integrity of ring-like structure. YdiV2–FlhD2 structure and the negative staining electron microscopy reconstruction of YdiV4–FlhD4C2 suggested that the third and fourth YdiV molecule bound to the FlhD4C2 complex through squeezing into the ring-like structure of FlhD4C2 between the two internal D subunits. Consequently, the ring-like structure opened up, and the complex lost DNA-binding ability. Thus, YdiV inhibits FlhD4C2 only at relatively high concentrations.

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Daptomycin is a potent cyclic lipopeptide antibiotic. It is widely used against various Gram-positive bacterial pathogens. Historically, a poor understanding of the transcriptional regulation of daptomycin biosynthesis has limited the options for targeted genetic engineering toward titer improvement. Here, we isolated a TetR family transcriptional regulator, DepR1, from the industrial producer Streptomyces roseosporus SW0702 using a biotinylated dptE promoter (dptEp) as a probe. The direct interaction between DepR1 and dptEp then was confirmed by electrophoretic mobility shift assays and DNase I footprinting assays. The deletion of depR1 led to a reduction in dptEp activity and the cessation of daptomycin production. The ⌬depR1 mutant produced less red pigment and failed to sporulate on R5 medium. This suggests that DepR1 plays a positive role in the control of morphological differentiation. Moreover, DepR1 was positively autoregulated by directly binding to its own promoter. This might account for the positive feedback regulation of daptomycin production. Based on these positive effects, genetic engineering by overexpression of depR1 raised daptomycin production and shortened the fermentation period both in flask and in fermentor. Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus via nonribosomal peptide synthases (1). It contains 13 amino acids and a straight-chain decanoic acid attached to the terminal amino group of Trp (2). In the presence of Ca 2ϩ , the lipid tail can insert into the bilayer membrane of pathogens. This results in potassium efflux, membrane depolarization, and eventual cell death (3). Daptomycin has been approved for the clinical treatment of diseases caused by drug-resistant Gram-positive bacterial pathogens. Such pathogens include vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, and others (4-7).Due to its importance in clinical medicine, several approaches have been employed for the production of daptomycin derivatives. These include semisynthetic modification (8, 9), chemoenzymatic synthesis (10, 11), and combinatorial biosynthesis (12)(13)(14), with the anticipation of altered pharmaceutical spectra and/or increased antimicrobial activities. In addition, many efforts have been made to increase daptomycin production. These have included random mutagenesis (15), in silico-based metabolic engineering (16,17), and attempts to increase precursor supply (18). However, up to the present, the poor understanding of the transcriptional regulation of daptomycin biosynthesis has limited options for targeted genetic engineering toward titer improvement.The gene cluster for daptomycin biosynthesis originally was identified in Streptomyces roseosporus NRRL 11379. Three putative cluster-situated transcriptional regulators, DptR1, DptR2, and DptR3 (1), also have been identified. We previously reported that the industrial strain S. roseosporus SW0702 also contained a similar daptomycin gene clus...