The CagRS Two-Component System Regulates Clavulanic Acid Metabolism via Multiple Pathways in Streptomyces clavuligerus F613-1

Abstract

Streptomyces clavuligerus F613-1 produces a clinically important β-lactamase inhibitor, clavulanic acid (CA). Although the biosynthesis pathway of CA has essentially been elucidated, the global regulatory mechanisms of CA biosynthesis remain unclear. The paired genes cagS and cagR , which are annotated, respectively, as orf22 and orf23 in S. clavuligerus ATCC 27064, encode a bacterial two-component regulatory system (TCS) and were found next to the CA biosynthetic gene cluster of S. clavuligerus F613-1. To further elucidate the regulatory mechanism of CA biosynthesis, the CagRS TCS was deleted from S. clavuligerus F613-1. Deletion of cagRS resulted in decreased production of CA, but the strain phenotype was not otherwise affected. Both transcriptome and ChIP-seq data revealed that, in addition to CA biosynthesis, the CagRS TCS mainly regulates genes involved in primary metabolism, such as glyceraldehyde 3-phosphate (G3P) metabolism and arginine biosynthesis. Notably, both G3P and arginine are precursors of CA. Electrophoretic mobility shift assays demonstrated that the response regulator CagR could bind to the intergenic regions of argG, argC, oat1, oat2, ceaS1 , and claR in vitro , suggesting that CagR can directly regulate genes involved in arginine and CA biosynthesis. This study indicated that CagRS is a pleiotropic regulator that can directly affect the biosynthesis of CA and indirectly affect CA production by regulating the metabolism of arginine and G3P. Our findings provide new insights into the regulation of CA biosynthetic pathways and provide an innovative approach for future metabolic engineering efforts for CA production in S. clavuligerus .

The two-component system CepRS regulates the cephamycin C biosynthesis in Streptomyces clavuligerus F613-1

“…All the strains and plasmids used in this study are listed in Additional file 1: Table S1. Culturing of Escherichia coli , S. clavuligerus and its derivative strains were performed as described previously (Fu et al 2019; Qin et al 2017). In this study, for phenotypic analysis, S. clavuligerus F613-1 (CGMCC NO.…”

“…The genome of S. clavuligerus F613-1 encodes 47 paired two-component systems (TCSs), amongst which, CagRS has been characterized as a global regulator of both primary and secondary metabolism (Fu et al 2019). To further study the regulation mechanism of TCSs on the secondary metabolism, we constructed a series of single-gene and double-gene knockout strains.…”

Tools to map target genes of bacterial two‐component system response regulators

“…Chromatin Immunoprecipitation (ChIP) combined with sequencing has been used to determine the in vivo binding sites for a large number of TCS RRs. (Wang et al ., ; Pelliciari et al ., ; Fishman et al ., ; Fu et al ., ). In contrast to transcriptomics analysis, which indirectly hypothesizes targets of transcriptionally active RRs, ChIP‐seq directly detects the DNA‐binding targets through protein–DNA interactions (Fig.…”

“…Bioinformatic analysis of the ChIP‐seq output can then be used to identify the binding site motif that determines the specific target of protein–DNA interaction. Electrophoretic mobility shift assays and/or DNase I footprinting assays are often carried out to validate the binding site motif, and qRT‐PCR assays may be used to verify changes in gene expression (Pelliciari et al ., ; Fu et al ., ).…”

“…Many recent studies carry out both RNA‐seq and ChIP‐seq analyses and compare the results (Minch et al ., ; Fishman et al ., ; Fu et al ., ). These combination studies help to reveal if the RR is activating or repressing the genes at the detected binding sites.…”