Global Regulator of Rubber Degradation in Gordonia polyisoprenivorans VH2: Identification and Involvement in the Regulation Network

ABSTRACT

A cAMP receptor protein (CRP_VH2) was detected as a global regulator in Gordonia polyisoprenivorans VH2 and was proposed to participate in the network regulating poly(cis-1,4-isoprene) degradation as a novel key regulator. CRP_VH2 shares a sequence identity of 79% with GlxR, a well-studied global regulator of Corynebacterium glutamicum. Furthermore, CRP_VH2 and GlxR have a common oligomerization state and similar binding motifs, and thus most likely have similar functions as global regulators. Size exclusion chromatography of purified CRP_VH2 confirmed the existence as a homodimer with a native molecular weight of 44.1 kDa in the presence of cAMP. CRP_VH2 bound to the TGTGAN₆TCACT motif within the 131-bp intergenic region of divergently oriented lcp1_VH2 and lcpR_VH2, encoding a latex clearing protein and its putative repressor, respectively. DNase I footprinting assays revealed the exact operator size of CRP_VH2 in the intergenic region (25 bp), which partly overlapped with the proposed promoters of lcpR_VH2 and lcp1_VH2. Our findings indicate that CRP_VH2 represses the expression of lcpR_VH2 while simultaneously directly or indirectly activating the expression of lcp1_VH2 by binding the competing promoter regions. Furthermore, binding of CRP_VH2 to upstream regions of additional putative enzymes of poly(cis-1,4-isoprene) degradation was verified in vitro. In silico analyses predicted 206 CRP_VH2 binding sites comprising 244 genes associated with several functional categories, including carbon and peptide metabolism, stress response, etc. The gene expression regulation of several subordinated regulators substantiated the function of CRP_VH2 as a global regulator. Moreover, we anticipate that the novel lcpR regulation mechanism by CRPs is widespread in other rubber-degrading actinomycetes.

IMPORTANCE In order to develop efficient microbial recycling strategies for rubber waste materials, it is required that we understand the degradation pathway of the polymer and how it is regulated. However, only little is known about the transcriptional regulation of the rubber degradation pathway, which seems to be upregulated in the presence of the polymer. We identified a novel key regulator of rubber degradation (CRP_VH2) that regulates several parts of the pathway in the potent rubber-degrader G. polyisoprenivorans VH2. Furthermore, we provide evidence for a widespread involvement of CRP regulators in the degradation of rubber in various other rubber-degrading actinomycetes. Thus, these novel insights into the regulation of rubber degradation are essential for developing efficient microbial degradation strategies for rubber waste materials by this group of actinomycetes.