5S Clavam Biosynthesis Is Controlled by an Atypical Two-Component Regulatory System in Streptomyces clavuligerus

5S Clavam Biosynthesis Is Controlled by an Atypical Two-Component Regulatory System in Streptomyces clavuligerus Thomas Kwong,* Nathan J. Zelyas, Hui Cai,* Kapil Tahlan,* Annie Wong, and Susan E. Jensen Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada C lavulanic acid has seen wide clinical use in recent years because of its potent ␤-lactamase inhibitory properties. It is produced industrially through fermentation, with Streptomyces clavuligerus the only species used for this purpose. In addition to clavulanic acid, S. clavuligerus also produces the conventional ␤-lactam antibiotic cephamycin C as well as four other clavam compounds, 2-formyloxymethylclavam, 2-hydroxymethylclavam (2-HMC), alanylclavam, and clavam-2-carboxylate, which are structurally related to clavulanic acid (6, 27). These four clavam compounds lack ␤-lactamase inhibitory activity and are commonly referred to as the 5S clavams because of their 5S stereochemistry compared to the 5R configuration of clavulanic acid. A better understanding of clavulanic acid production could lead to the design of more-powerful variants as well as to higher levels of production through strain improvement. As a result, the biosynthetic pathway and underlying regulatory mechanisms in S. clavuligerus have been the subject of intensive research (12). It is now known that the pathway for clavulanic acid biosynthesis is shared with the production of the 5S clavams up to the intermediate, clavaminic acid (11). This part of the pathway, referred to as the early steps, has been well characterized (8, 13, 25, 43), but the pathway then diverges to late steps that are much less well understood, leading either to clavulanic acid or the 5S clavams. To date, three unlinked gene clusters have been described that contain genes involved in clavam biosynthesis, the clavulanic acid (40), the paralogue (37), and the clavam (26) gene clusters (24). The clavulanic acid gene cluster includes genes encoding enzymes for each of the early steps of the shared pathway, as well as genes for the late steps specific to clavulanic acid production. It is located on the chromosome in the 4.86-Mba region. The clavam gene cluster is also located on the chromosome in the 3.44-Mba region and contains a second copy of one of the early genes, cas1, as well as cvm1 and cvm5, which are essential for late steps in the production of 5S clavams (36). The paralogue gene cluster (Fig. 1A), September 2012 Volume 56 Number 9 located in the 1.15-Mba region of the giant linear plasmid, pSCL4, is so named because it carries second copies of all of the other early genes except for cas1, as well as additional genes involved in late steps of 5S clavam biosynthesis (36, 42). In particular, orfA, orfB, orfC, and orfD encode biosynthetic enzymes specifically required for alanylclavam production and are located upstream of ceaS1 in one flank of the paralogue gene cluster (42). cvm6p and cvm7p are located in the other flank and are essential for the production of all 5S clavams (36, 42). cvm6p encodes an aminotransferase implicated as the first step in 5S clavam production after clavaminic acid, whereas Cvm7P is a regulatory protein with an N-terminal Streptomyces antibiotic regulatory protein (SARP)-like domain, but also a C-terminal ATPase domain, therefore placing it in a class of antibiotic regulatory proteins similar to PimR from S. natalensis (2). Since cvm7p mutants are blocked specifically in 5S clavam production, Cvm7P is proposed to act as a pathway-specific transcriptional activator for 5S clavam biosynthesis in S. clavuligerus (36). The details of 5S clavam production are beginning to emerge (42), but many steps remain hypothetical, and the regulatory Received 26 May 2012 Returned for modification 18 June 2012 Accepted 22 June 2012 Published ahead of print 2 July 2012 Address correspondence to Susan E. Jensen, . * Present address: Thomas Kwong, Department of Chemistry, The Scripps Research Institute Florida, Jupiter, Florida, USA; Hui Cai, PPD Inc., Middleton, Wisconsin, USA; Kapil Tahlan, Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada. Supplemental material for this article may be found at http://aac.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.01090-12 Antimicrobial Agents and Chemotherapy p. 4845– 4855 aac.asm.org 4845 Downloaded from http://aac.asm.org/ on November 18, 2019 by guest Streptomyces clavuligerus produces a collection of five clavam metabolites, including the clinically important ␤-lactamase inhibitor clavulanic acid, as well as four structurally related metabolites called 5S clavams. The paralogue gene cluster of S. clavuligerus is one of three clusters of genes for the production of these clavam metabolites. A region downstream of the cluster was analyzed, and snk, res1, and res2, encoding elements of an atypical two-component regulatory system, were located. Mutation of any one of the three genes had no effect on clavulanic acid production, but snk and res2 mutants produced no 5S clavams, whereas res1 mutants overproduced 5S clavams. Reverse transcriptase PCR analyses showed that transcription of cvm7p (which encodes a transcriptional activator of 5S clavam biosynthesis) and 5S clavam biosynthetic genes was eliminated in snk and in res2 mutants but that snk and res2 transcription was unaffected in a cvm7p mutant. Both snk and res2 mutants could be complemented by introduction of cvm7p under the control of an independently regulated promoter. In vitro assays showed that Snk can autophosphorylate and transfer its phosphate group to both Res1 and Res2, and Snk-H365, Res1-D52, and Res2-D52 were identified as the phosphorylation sites for the system. Dephosphorylation assays indicated that Res1 stimulates dephosphorylation of Res2⬃P. These results suggest a regulatory cascade in which Snk and Res2 form a two-component system controlling cvm7p transcription, with Res1 serving as a checkpoint to modulate phosphorylation levels. Cvm7P then activates transcription of 5S clavam biosynthetic genes. Kwong et al. mechanisms that govern them are even less clear. In the present study, we examined the region beyond cvm7p of the paralogue gene cluster. Sequence analysis revealed three genes, snk, res1, and res2, encoding a putative sensor kinase and two response regulators, which together form an atypical two-component regulatory system. In vivo and in vitro studies were employed to gain a more complete understanding of how these various regulatory circuits are interconnected. A regulatory cascade is proposed in which this two-component system, along with Cvm7P, regulates 5S clavam biosynthesis. MATERIALS AND METHODS Bacterial strains, plasmids, and culture conditions. The bacterial strains and plasmid and cosmid vectors used in this study are listed in Tables S1 and S2 in the supplemental material. S. clavuligerus str (...truncated)