Abstract
Biosynthesis of secondary metabolites in actinobacteria is regulated by complex regulatory mechanisms on responding environmental changes. In this study, we have identified a two-component system (TCS) designated as RimA1A2 in the genome of Streptomyces rimosus M4018, with high homology to the TCS RapA1A2 from Streptomyces coelicolor, known for its positive regulatory function towards actinorhodin (ACT) biosynthesis. Using RT-PCR analysis, we demonstrate that rimA1 encodes response regulator (RR) and rimA2 encoding histidine kinase (HK) from S. rimosus that are co-transcribed as a single-polycistronic mRNA. When S. rimosus was cultivated on standard media, no significant difference in culture growth or morphological properties was observed between the rimA1-disrupted mutant and parent strain M4018. However, the rimA1-disrupted strain displayed significant increase in oxytetracycline (OTC) titer when cultivated in minimal medium (MM) containing glycine as sole nitrogen source, and the transcription of selected genes involved in OTC biosynthesis was increased, supporting the hypothesis that RimA1A2 has a negative regulatory role in OTC biosynthesis via global regulation. We observed an increased resistance of the rimA1-disrupted mutant strain to selected antibiotics. Interestingly, in the rimA1-disrupted strain, OTC biosynthesis was affected under different environmental stress conditions such as osmotic and oxidative stress. Accordingly, this phenotype was observed in a medium-dependent manner. Considering complexity of regulatory networks in antibiotic-producing organisms, this study demonstrates the importance of cultivation conditions, which is often neglected.
Highlights
Streptomyces rimosus is a Gram-positive, aerobic, and filamentous actinobacterium, which produces the medically important broad-spectrum antibiotic oxytetracycline (OTC) (Finlay et al 1950)
After thorough evaluation of phenotype of the S. rimosus rimA1-disrupted mutant strain culture under diverse cultivation conditions, and under influence of different environmental stress conditions, we demonstrate that RimA1A2 plays a significant role in regulation of OTC biosynthesis only under specific environmental stress conditions, which are, not comparable to the optimal conditions used for industrial OTC production
The most RRs identified in the genome of M4018 strain belong to the OmpR and NarL families (Fabret et al 1999; Hutchings et al 2004) (Fig. 1). 50 pairs of typical two-component system (TCS) comprising a histidine kinase and a cognate response regulator protein were screened, considering that HK and RR of a typical TCS are adjacent to each other on the chromosome, and they are generally transcribed in the same orientation
Summary
Streptomyces rimosus is a Gram-positive, aerobic, and filamentous actinobacterium, which produces the medically important broad-spectrum antibiotic oxytetracycline (OTC) (Finlay et al 1950). The pathway-specific regulators and the global regulators play important roles in the regulation of secondary metabolism of antibiotic production. These are capable of repressing or triggering the expression of genes involved in biosynthesis of antibiotic. The Streptomyces antibiotic regulatory proteins (SARPs) are the best-characterized pathway-specific regulators, which possess an OmpR-like winged helix–turn–helix (HTH) DNA-binding motif near the N-terminus and an adjacent bacterial transcriptional activation domain (BTAD) (Wietzorrek and Bibb 1997). An SARP positive regulatory protein OtcR was located in
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