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

Abstract

A cAMP receptor protein (CRPVH2) 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. CRPVH2 shares a sequence identity of 79% with GlxR, a well-studied global regulator of Corynebacterium glutamicum. Furthermore, CRPVH2 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 CRPVH2 confirmed the existence as a homodimer with a native molecular weight of 44.1 kDa in the presence of cAMP. CRPVH2 bound to the TGTGAN6TCACT motif within the 131-bp intergenic region of divergently oriented lcp1VH2 and lcpRVH2, encoding a latex clearing protein and its putative repressor, respectively. DNase I footprinting assays revealed the exact operator size of CRPVH2 in the intergenic region (25 bp), which partly overlapped with the proposed promoters of lcpRVH2 and lcp1VH2. Our findings indicate that CRPVH2 represses the expression of lcpRVH2 while simultaneously directly or indirectly activating the expression of lcp1VH2 by binding the competing promoter regions. Furthermore, binding of CRPVH2 to upstream regions of additional putative enzymes of poly(cis-1,4-isoprene) degradation was verified in vitro. In silico analyses predicted 206 CRPVH2 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 CRPVH2 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 (CRPVH2) 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.

Microbial Degradation of Rubber: Actinobacteria

“…Recently, a novel key regulator in poly( cis -1,4-isoprene) degradation, cAMP receptor protein (CRPVH2), was detected for Gordonia polyisoprenivorans VH2, having 16 bp TGTGAN 6 TCACT motif [ 87 ]. CRP is also known as catabolite activator protein, CAP.…”

“…CRP is also known as catabolite activator protein, CAP. CRPVH2 and G1xR has 79% sequence identity, both having a common oligomerization state, similar binding motifs and thus most 26 likely similar functions as global regulator [ 87 ]. G1xR is a well -studied global regulator of Corynebacterium glutamicum .…”

Structural and functional diversity of bacterial cyclic nucleotide perception by CRP proteins

“…In Actinobacteria of industrial importance, CRP proteins were found to play a role in regulating biotechnologically important functions, which instigated their molecular characterization. Gordonia polyisoprenivorans from the order Corynebacteriales is a rubber-degrading bacterium in which the CRP protein CRPVH2 (Table 1 ) was proposed as a key regulator of poly(cis-1,4-isoprene) degradation (de Witt et al 2020 ). CAP-like motifs were found in promoter regions of rubber degradation genes.…”

Corynebacterium glutamicum regulation beyond transcription: Organizing principles and reconstruction of an extended regulatory network incorporating regulations mediated by small RNA and protein-protein interactions

“…Also, the inclusion of post-transcriptional regulations mediated by sRNAs into the global regulatory networks has been performed in other organisms (e.g., [14] in E. coli as a undirected network). Previous versions of the C. glutamicum transcriptional regulatory network have been used for the transfer of regulatory interactions to other corynebacterial strains hosted in the CoryneRegNet database [15], the construction of a model for the inference of the number of interactions once the regulatory networks are complete [6], an assessment of the NDA robustness to random remotion of nodes and interactions [13], as gold standard for the benchmarking of a network inference approach based on sequence data (unpublished results), and as reference for the identification of global regulators [16].…”