Environmental Microbiology

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

Jan de Witt, Sylvia Oetermann, Mariana Parise, Doglas Parise, Jan Baumbach, Alexander Steinbüchel
Shuang-Jiang Liu, Editor
DOI: 10.1128/AEM.00774-20

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.

INTRODUCTION

Increasing amounts of rubber waste materials, predominantly originating from car tires, lead to huge challenges in waste management (1). A variety of bacteria could contribute to the development of ecofriendly and economically expedient recycling processes by their ability to degrade rubber (2, 3). In order to use such biological recycling processes, it is important to investigate the molecular mechanisms of microbial rubber degradation. Rubber oxygenases were identified as key enzymes involved in the degradation of poly(cis-1,4-isoprene), the main component of natural rubber (4–7). These extracellular enzymes cleave the polymer by introducing molecular oxygen at the double bond, creating oligoisoprenes with terminal aldehyde and ketone groups. Subsequently, the cleavage products are imported into the cells and metabolized via β-oxidation (5, 6, 8). In total, three types of rubber oxygenases have been discovered so far. While RoxAs (9) and RoxBs (10, 11) were only found in Gram-negative bacteria, latex clearing proteins (Lcps) were thought to be only present in Gram-positive bacteria (7, 12–15). However, the recently identified LcpHR-BB of Solimonas fluminis refuted this theory, since it was the first lcp ortholog identified in a Gram-negative species (16). All Lcps characterized so far, including LcpHR-BB, harbor heme-b as cofactor (7, 16–18). Furthermore, cleavage of poly(cis-1,4-isoprene) by Lcps gives a spectrum of oligoisoprenes as a result of an endocleavage mechanism (6, 19).

Transcription analyses with Gordonia polyisoprenivorans VH2, Nocardia farcinica NVL3, and Streptomyces sp. strain K30 revealed a strong induction of lcp genes (lcp1VH2, lcpNVL3, and lcpK30) when the respective cells were grown with poly(cis-1,4-isoprene) as the sole carbon source (13, 15, 20). However, when cells were grown in the absence of poly(cis-1,4-isoprene), no expression of lcp1VH2 and lcpNVL3, and only a low basal expression of lcpK30, was observed. These results indicate a regulatory mechanism ensuring the production of Lcps only when poly(cis-1,4-isoprene) is present as the sole carbon source.

G. polyisoprenivorans serves as one of the model organisms to study degradation of rubber. It possesses two Lcps (Lcp1VH2 and Lcp2VH2). Recently, LcpRVH2 was identified as the first known lcp regulator (21). LcpRVH2 belongs to the family of TetR regulators and is located 131 bp upstream of lcp1VH2 in G. polyisoprenivorans VH2. Electrophoretic mobility shift assays (EMSAs) revealed the binding of LcpRVH2 upstream of both lcp1VH2 and lcp2VH2, indicating that both lcp genes are regulated by LcpRVH2. Furthermore, an alignment of 17 lcpR-lcp intergenic regions of different actinomycetes revealed a conserved motif, similar to the detected binding site of LcpRVH2 (21). Binding of LcpRVH2 to the obtained consensus sequence strongly indicated a widespread regulatory mechanism of lcp genes by LcpRs in Actinobacteria. It was suggested that LcpRVH2 acts as a repressor of lcp1VH2 and lcp2VH2, when poly(cis-1,4-isoprene) is absent (21). A short time later, a second type of lcp regulator named LcpRBA3(2) was discovered downstream of lcpA3(2) in Streptomyces coelicolor A3(2) (22). Although LcpRBA3(2) is also a member of the TetR family, it shows no homology to LcpRVH2. Moreover, LcpRBA3(2) and LcpRVH2 exhibit different properties, including their oligomerization states, molecular weights, and operator sites. LcpRBA3(2) occurs as a monomer in its native state with a molecular weight of 44 kDa, whereas LcpRVH2 was identified as a homodimer with a native molecular weight of 52.7 kDa (21, 22). It was shown that LcpRBA3(2) does not recognize the binding motif of LcpRVH2 and vice versa (21, 22). Some actinomycetes, such as Actinosynnema mirum 101T and Streptomyces sp. strain CFMR 7, harbor coding sequences for both LcpR and LcpRB regulators. Furthermore, they both have three open reading frames coding for Lcps with binding sites for either LcpRs or LcpRBs (22). These findings indicate that there are at least two different regulatory mechanisms for the expression of lcp genes in actinomycetes.

Since Lcps are the key enzymes of poly(cis-1,4-isoprene) degradation, a more complex regulation network is likely. Often, a master or global regulator senses a lack of carbon source so that a second, local regulator then regulates the expression of a specific gene, as is known from the well-studied lac operon (23, 24). The most important and characterized global regulators are cAMP receptor proteins (CRPs, also known as catabolite activator proteins [CAPs]) that belong to the widespread CRP/FNR protein family. CRPEc of Escherichia coli and GlxR of Corynebacterium glutamicum both regulate hundreds of genes associated with several functional categories, including carbon metabolism, fatty acid biosynthesis, cellular stress response, etc. (25–29). Moreover, regulating the expression of local and master regulators, CRPs are involved in complex regulation networks. Following the attachment of cAMP, CRPs bind to the consensus sequence TGTGAN6TCACA and regulate the activity of nearby promoters (27, 29). Furthermore, CRPs are dual regulators, as they can act as (co)activator or (co)repressor (30). The regulation of divergently oriented genes by CRPs is especially complex, as single or multiple CRP molecules can simultaneously (co)activate and (co)repress each of the divergent genes (30, 31).

To date, nothing was known about a global regulator involved in the regulation of poly(cis-1,4-isoprene) degradation. Furthermore, the recent identification of two distinct local lcp regulators that could be regulated by a global regulator attracted our interest. In addition, the discovery of LcpR and LcpRB could not explain the increased abundance of several proteins under poly(cis-1,4-isoprene)-degrading conditions (32). Thus, we aimed at identifying a global regulator that could be involved in the (in)direct regulation of poly(cis-1,4-isoprene) degradation in G. polyisoprenivorans VH2. Moreover, our aim was to extend the knowledge of poly(cis-1,4-isoprene) degradation and its regulation in other lcp-harboring actinomycetes by connecting a putative involvement of a global regulator with the regulation by LcpRs or LcpRBs.

RESULTS

Discovery and properties of CRPVH2.An alignment of 15 lcpR-lcp intergenic regions of actinomycetes unraveled a conserved motif with unknown function (Fig. 1a). The motif was identified as TTGTGN8CACN3GTGNC and showed no similarity to the previously discovered LcpR-binding site in the lcpR-lcp intergenic regions. In silico analyses revealed sequence similarities (E value 1.15e−12 and overlap of 10) to the consensus binding site of GlxR, a well-characterized global regulator of C. glutamicum. Thus, we screened the genome of G. polyisoprenivorans VH2 for a GlxR homolog that might bind to the identified motif in the lcpRVH2-lcp1VH2 intergenic region. Using BLAST we identified a single homolog that shared a sequence identity of 79% toward GlxR (GenBank accession number AFA71800.1). Since GlxR is a cAMP receptor protein, we designated the newly discovered protein CRPVH2. CRPVH2 is encoded by a 675-bp gene (GPOL_RS03655) that is located on the chromosome and shows no proximity to the intergenic region of lcpRVH2 and lcp1VH2. Comparing the amino acid sequence of CRPVH2 with GlxR, we detected identical cAMP-binding motifs (Fig. 2a and b). Previous crystallization and 3D structure modeling revealed the essential amino acids and their positions for cAMP binding in GlxR (PDB 4CYD) as follows: Gly-82, Glu-83, Ser-85, Arg-92, Thr-93, Leu-134, Thr-137, and Asn-138 (Fig. 2a) (33). CRPVH2 possesses the identical cAMP-binding site, shifted by three amino acids to the N terminus as follows: Gly-79, Glu-80, Ser-82, Arg-89, Thr-90, Leu-131, Thr-134, and Asn-135 (Fig. 2b). Furthermore, homology structure modeling predicted that CRPVH2 is a homodimer binding cAMP to form an active complex (Fig. 2c). In addition, two C-terminal DNA-binding helices were predicted for CRPVH2 that are also characteristic for CRP regulators (30).

FIG 1
FIG 1

Alignment and sequence logo of putative CRP-binding sites within lcpR-lcp intergenic regions. (a) A multiple alignment was performed in which bases with a conservation of at least 70% are highlighted. Strains in boldface are known to utilize rubber as a carbon source (8, 55–57). Genome sequences of all strains are available in the prescribed order under the following RefSeq IDs (NCBI): NC_016906.1, NZ_CP017717.1, NC_013595.1, NZ_CP006850.1, NC_013510.1, NZ_CP022580.1, NZ_CP018063.1, NZ_CP016594.1, NZ_CP008953.1, NC_018581.1, NC_013093.1, NZ_CP012752.1, NZ_CP016353.1, NZ_CP023405.1, NZ_CP011522.1. (b) The resulting sequence logo was constructed to highlight the frequency of the bases in the alignment.

FIG 2
FIG 2

Comparison of the GlxR and CRPVH2 cAMP-binding sites and the predicted structure of CRPVH2. (a) cAMP-binding site of GlxR. Essential amino acids for cAMP binding are highlighted in red and displayed with their position (33). Binding of cAMP is shown. (b) Putative cAMP-binding site of CRPVH2 with essential amino acids highlighted in red. (c) Predicted structure of homodimeric CRPVH2. Monomers are shown in green and blue, respectively. Essential amino acids for cAMP binding are highlighted in red. Yellow arrows indicate the C-terminal DNA-binding helices.

Purification and oligomerization state of CRPVH2.To characterize CRPVH2, it was purified by Ni-nitrilotriacetic acid (Ni-NTA) affinity chromatography using an N-terminal His6 tag as was performed for GlxR (34). The calculated molecular weight of CRPVH2 carrying the His6 tag was 25.7 kDa. CRPVH2 was present in the soluble fraction and eluted in the fraction containing 100 mM imidazole with the previously calculated molecular weight (Fig. 3a). Since CRP regulators are only active in their dimeric state when bound to cAMP, size exclusion chromatography (SEC) was performed to determine the native molecular weight of CRPVH2. A clear peak at 217 ml was detected, which corresponded to a protein of 44.1 kDa (Fig. 3b). As a dimer, CRPVH2 should have a molecular weight of 51.4 kDa, but for a monomer it was clearly too large. GlxR of C. glutamicum exhibited a native molecular weight of 44 kDa (24 kDa for the monomer) during SEC, which is also smaller than the calculated molecular weight (28). Hence, we assume that CRPVH2 occurs as a dimer, and the detection of a lower molecular weight of the protein might indicate a compact form of the CRPVH2 complex.

FIG 3
FIG 3

Purification and size exclusion chromatography of CRPVH2. (a) SDS-gel showing the purification steps of CRPVH2 (25.7 kDa). Protein (10 μg) was applied from crude extract (CE), soluble fraction (SF), and flow through (FT). Protein (5 μg) was applied from elution steps with 100 mM imidazole (E1), 200 mM (E2), and purified and desalted CRPVH2 for EMSA studies (P). PageRuler prestained protein ladder (10 to 180 kDa) (Thermo Fisher Scientific, Waltham, MA, USA) was used as marker mixture (M). Gels were stained with Coomassie blue. (b) Chromatogram of the size exclusion chromatography. One milliliter containing 5 mg of purified CRPVH2 was applied to a Superdex 200 prep grade XK 26/60 column (GE Healthcare, Little Chalfont, UK) for molecular weight determination.

EMSAs revealed binding of CRPVH2 to the lcpRVH2-lcp1VH2 intergenic region.To verify whether CRPVH2 recognizes the discovered motif in the lcpRVH2-lcp1VH2 intergenic region, electrophoretic mobility shift assays (EMSAs) were performed. For that, a 40-bp oligonucleotide consisting of the proposed 16-bp recognition site (TGTGAN6TCACT) and 12 bp of the flanking sequences on each site were hybridized and used for the assay. A shift became visible when the samples contained CRPVH2 and were incubated with cAMP, verifying the binding of CRPVH2 to the tested oligonucleotide and thus to the intergenic region (Fig. 4a). Without the addition of cAMP, no binding of CRPVH2 was detected, resulting in a lower fluorescence and hence the intensity of the original band was decreased (Fig. 4a). Thus, CRPVH2 requires cAMP for the formation of an active homodimer to bind specifically to the DNA. In control samples, CRPVH2 did not bind to the LcpRVH2 binding site and vice versa, revealing specific binding of CRPVH2 to the tested binding motif (Fig. 4a). In order to confirm the proposed recognition site, the 12-bp flanking regions were randomly exchanged, while the 16-bp motif was unaltered (Fig. 4b). Indeed, CRPVH2 still bound to the altered 40-mer. However, exchanging position 1 (T) or 16 (T) of the proposed recognition site with an A or C impaired the binding of CRPVH2 to the oligonucleotide (Fig. 4c). Furthermore, variations of the N6 spacer were tested. When the six spacing nucleotides were randomly exchanged but a spacer length of six nucleotides was maintained, CRPVH2 still bound to the tested oligonucleotide (Fig. 4d). However, when the spacer length was extended or shortened by a single base pair, CRPVH2 did not recognize the motif (Fig. 4e). Thus, a spacer length of six nucleotides was detected to be mandatory for CRPVH2 binding, irrespective of its sequence. These findings confirm the 16-bp sequence TGTGAN6TCACT as recognition site of CRPVH2 within the intergenic region of lcpRVH2 and lcp1VH2. Thus, gene expression regulation of one or both genes, and hence an involvement of CRPVH2 in the regulation network of rubber degradation, is likely.

FIG 4
FIG 4

Properties of the CRPVH2 recognition site in the lcpRVH2–lcp1VH2 intergenic region. A set of 40-bp FAM (6-carboxyfluorescein)-labeled DNA segments with indicated variations (red) were used for binding studies. If indicated, samples were incubated with 19.5 μM CRPVH2 or LcpRVH2 as a control and 0.4 mM cAMP. (a) Native 40-bp sequence of the intergenic region of lcpRVH2 and lcp1VH2, including the 16-bp motif TGTGAN6TCACT with 12-bp flanking regions on each site. LcpRVH2 was used as a control for random protein-DNA interactions. Asterisks (*) indicate the use of the 40-bp LcpRVH2 consensus binding sequence as an additional control. (b) The 12-bp flanking regions on each site were randomly exchanged, while the 16-bp motif was unaltered. (c) Position 1 (T) or position 16 (T) of the 16-bp motif was exchanged with an A or C, respectively. (d) The six nucleotides of the N6 spacer were randomly exchanged while the spacer length remained unchanged. (e) The spacer length was shortened or extended by deleting or adding a T at position 18. (f) A 40-bp sequence containing the GlxR-binding site upstream of gntP (TGTGG N6 TCTCA).

In addition, CRPVH2 bound to a GlxR recognition site (TGTGGN6TCTCA) (Fig. 4f). This indicates that CRPVH2 tolerates certain alterations of the core sequence. Furthermore, the 167-bp upstream region of crpVH2 was screened for putative CRPVH2 recognition sites. However, only sequences with a spacer length of seven and eight base pairs were identified and were not bound by CRPVH2 (data not shown). Thus, an autoregulation of CRPVH2, as has been described for other CRP-type regulators, could not be confirmed (30, 35). In addition, CRPVH2 did not bind to the upstream region of lcp2VH2, which is the second lcp gene in G. polyisoprenivorans VH2 (data not shown).

Determination of the CRPVH2 operator site.After the recognition site of CRPVH2 in the lcpRVH2-lcp1VH2 intergenic region was confirmed, DNase I footprinting assays were performed to identify the exact CRPVH2 operator site. During digestion of FAM (6-carboxyfluorescein)- and NED (ATTO 550)-labeled intergenic regions, the CRPVH2 operator site was protected against digestion by DNase I when CRPVH2 was present. After that, the oligonucleotides were analyzed by capillary electrophoresis to show a gap that corresponds to the protected area. For both fluorescent signals, an identical 25-bp gap was detected (5′-AATCTGTTGTGATGCCAATCACTGT-3′) when the samples contained CRPVH2. Thus, the detected 25-bp sequence, harboring the 16-bp recognition site (TGTGAN6TCACT), was covered through binding by CRPVH2 (Fig. 5). The CRPVH2 operator site in the intergenic region is located 84 bp to 108 bp upstream of lcp1VH2 or 24 bp to 48 bp upstream of lcpRVH2. The GC base pair located 100 bp upstream of the lcp1VH2 translation start was exposed to DNase I digestion, although being located within the CRPVH2 binding site. This might be explained by alteration of the DNA topology upon binding of CRPVH2, leading to exposure to DNase I.

FIG 5
FIG 5

DNase I footprinting assay. Electropherogram of the DNase I footprinting assay of FAM-labeled (a) and NED-labeled (b) lcpRVH2-lcp1VH2 intergenic region containing 200 μM CRPVH2 and 0.4 mM cAMP (blue line). The control reaction was performed without CRPVH2 (green line). Sequence numbering is relative to the translation start of lcp1VH2. The electropherogram of the sequencing reaction is shown together with an enlargement of the operator site of CRPVH2, in which the recognition site (TGTGAN6TCACT) is colored red.

Proposed promoter regions of lcp1VH2 and lcpRVH2.Since lcp1VH2 and lcpRVH2 are divergently oriented and located in close proximity, it is conceivable that binding of CRPVH2 to the intergenic region might regulate the expression of both genes. As the mode of transcription regulation (activation or repression) by CRPVH2 depends on the location of its operator site relative to the promoter regions (36, 37), identification of the −35 and −10 regions would lead to a first prediction of the CRPVH2 regulatory role(s).

Capping rapid amplification of cDNA ends (RACE) was performed to identify the 5′ untranslated mRNA regions of lcpRVH2 and lcp1VH2, thus identifying their putative −35 and −10 regions. A capping-RACE product for lcp1VH2 could only be obtained when the cells were grown with poly(cis-1,4-isoprene) as the sole carbon source. Sequencing of the capping RACE product revealed a size of 344 bp, of which 66 bp were identified as the 5′ untranslated mRNA region of lcp1VH2. The proposed −35 and −10 regions matched in silico predicted promoter regions of lcp1VH2, so that the promoter was designated lcp1VH2p. The −35 region of lcp1VH2p was identified as CTGTTG spaced by 15 bp to the −10 region, which exhibited the TATGCT sequence (Fig. 6). It was discovered that the entire −35 region and the first invariant T of the −10 region of lcp1VH2p are located within the CRPVH2 operator site. This strongly indicates a regulation of lcp1VH2p activity by CRPVH2.

FIG 6
FIG 6

Location of the CRPVH2-binding site in relation to lcpRVH2p and lcp1VH2p. CRPVH2 and LcpRVH2 binding sites are highlighted as blue and red boxes, respectively. Transcription start sites of lcpRVH2 and lcp1VH2 are indicated with +1. The proposed −35 and −10 promoter regions of lcpRVH2p and lcp1VH2p are underlined.

For lcpRVH2, a capping-RACE product was detected when propionate was used as the carbon source. Sequencing of the capping RACE product revealed a size of 326 bp, of which 36 bp were identified as 5′ untranslated region. Again, the proposed −35 and −10 regions agreed with in silico predicted promoter regions. The −35 region of lcpRVH2p (TTGTAA) was spaced by 14 bp to the −10 region (TACAGT) (Fig. 6). It was discovered that the −10 region of lcpRVH2p was almost completely located within the CRPVH2 operator site. Hence, we assume that CRPVH2 might regulate the activity of both lcpRVH2p and lcp1VH2p. Furthermore, lcpRVH2p and lcp1VH2p overlap, so there might be a competition for RNA polymerase binding and transcription of lcpRVH2 and lcp1VH2 regulated by CRPVH2.

Promoter reporter gene assays using lacZ fusions.As both lcpRVH2p and lcp1VH2p overlap the CRPVH2 operator site, promoter reporter gene assays were performed to investigate the regulatory role of CRPVH2 in the promoter activities. For that, the lcpRVH2-lcp1VH2 intergenic region was fused to lacZ in lcpRVH2 and lcp1VH2 orientation, respectively. Two control constructs were designed in which the P1 promoter of lacZ (lacZp1) or no promoter region was fused upstream to the reporter gene. Unfortunately, lacZ was not expressed in G. polyisoprenivorans VH2 after testing various conditions. Because of this, promoter reporter gene assays were conducted in E. coli K-12. E. coli was chosen because CRPEc recognizes motifs similar to the recognition motif of CRPVH2. In order to compare the promoter activity in the presence and absence of CRPEc, the strains BW25113 and the crpEc deletion mutant JW5702-4 were transformed with the reporter gene plasmids (Table 1).

View this table:
TABLE 1

Bacterial strains, plasmids, and oligonucleotides used in this studya

As proof of concept, the activation of lacZp1 by CRPEc was verified. A LacZ activity of 172 Miller units (MU) was measured in the presence of CRPEc, whereas in the absence of CRPEc a LacZ activity of only 3.7 MU was measured (Fig. 7a). When no promoter region was located upstream of lacZ, a low basal LacZ activity of only 0.2 MU was obtained, irrespective of the presence or absence of CRPEc (Fig. 7b). Likewise, a similar low LacZ activity was obtained in the presence or absence of CRPEc when lcp1VH2p was fused to lacZ (Fig. 7c). Thus, lcp1VH2p is not recognized by strains BW25113 and JW5704-2, and a regulatory effect of CRPEc on the activity of lcp1VH2p could not be investigated. Interestingly, we perceived an interspecies regulatory mechanism in which CRPEc of E. coli repressed lcpRVH2p of G. polyisoprenivorans VH2. In the presence of CRPEc, a LacZ activity of 3.6 MU was detected, whereas the LacZ activity increased to 79 MU in the absence of CRPEc (Fig. 7d). Hence, the activity of lcpRVH2p is repressed by the foreign CRPEc.

FIG 7
FIG 7

Analysis of promoter regulation by CRPEc using lacZ fusions. The promoter-dependent expression of lacZ was determined in the E. coli K-12 strains BW25113 (wild type [Wt]) and JW5702-4 (ΔcrpEc) by the Miller assay. Miller units (MU) are displayed on the y-axis. lacZp1 (a), which is known to be activated by CRPEc, and no promoter region (b) were used as references. No activity of lcp1VH2p was observed in the presence or absence of CRPEc (c). Activity of lcpRVH2p (d) increased in the absence of CRPEc, revealing repression of lcpRVH2p by CRPEc. All experiments were performed in biological triplicates, each consisting of three technical replicates, with standard deviations shown.

Further CRPVH2 binding sites related to poly(cis-1,4-isoprene) degradation.Previous studies identified several proteins of G. polyisoprenivorans VH2 that are more abundant during poly(cis-1,4-isoprene)-degrading conditions (32). These mainly include enzymes involved in β-oxidation of the oligo(cis-1,4-isoprene) cleavage products. We screened the upstream regions of these genes for putative CRPVH2-binding sites to identify further genes associated with poly(cis-1,4-isoprene) degradation whose expression might be regulated by CRPVH2. In total, CRPVH2 bound to the upstream regions of three genes, encoding an aldehyde dehydrogenase (GPOL_RS01280), a long-chain fatty acid-CoA ligase (GPOL_RS12170), and a putative trehalose importer operon (GPOL_RS08770-08785), all of which were upregulated during growth with poly(cis-1,4-isoprene) (Fig. 8a to c). Furthermore, binding to the upstream region of a gene encoding an α-methylacyl-CoA racemase (GPOL_RS18275) was verified (Fig. 8d). All four upstream regions harbor different CRPVH2-recognition motifs, confirming that CRPVH2 tolerates certain alterations to the TGTGAN6TCACA core motif of CRPs. The recognition motifs were identified as TGTGCN6TCACA, TGTGAN6ACACT, TGTGAN6TCACA, and TGTGTN6ACACG for the aldehyde dehydrogenase, the long-chain fatty acid-CoA ligase, the putative trehalose importer operon, and the α-methylacyl-CoA racemase, respectively. Previous studies already discovered a participation of these enzymes in the degradation of poly(cis-1,4-isoprene) (32, 38). Hence, we identified additional CRPVH2 regulation targets related to the degradation of the polymer in G. polyisoprenivorans VH2. The verification of several CRPVH2-recognition sites supports our hypothesis of a function as a global regulator that might be involved in more than regulation of poly(cis-1,4-isoprene) degradation.

FIG 8
FIG 8

CRPVH2-binding sites in upstream regions of genes related to poly(cis-1,4-isoprene) degradation. A set of 40-bp FAM (6-carboxyfluorescein)-labeled DNA was used for binding studies. If indicated, samples were incubated with 19.5 μM CRPVH2 and 0.4 mM cAMP. Negative controls were performed without CRPVH2 or cAMP, respectively. The corresponding gene products and CRPVH2 recognition sites are indicated. Binding of CRPVH2 was shown for upstream regions of GPOL_RS01280 (aldehyde-dehydrogenase) (a), GPOL_RS12170 (long-chain fatty acid-CoA ligase) (b), GPOL_RS08785 (putative trehalose importer operon) (c), and GPOL_RS18275 (α-methylacyl-CoA racemase) (d).

In silico analyses identified CRPVH2 as a global regulator.Since CRPVH2 shows a sequence identity of 79% to the global regulator GlxR of C. glutamicum, a similar function for CRPVH2 as a global regulator in G. polyisoprenivorans VH2 is assumed. According to Schröder and Tauch, global regulators regulate the expression of >20 transcription units in ≥5 functional categories, including the gene expression regulation of master and local regulators to create hierarchic regulatory networks (29). To verify our hypothesis, we performed in silico analyses and screened the genome of G. polyisoprenivorans VH2 for in vitro-verified and similar CRPVH2-binding sites. In total, 206 CRPVH2-binding sites were predicted, comprising 244 genes under putative regulation of CRPVH2. The functions of the encoded proteins were analyzed and classified into functional categories. About 17% of all predicted CRPVH2-regulated genes code for hypothetical proteins (Fig. 9a). About one quarter (24.3%) of the predicted CRPVH2-regulated genes encode proteins that are associated with carbon metabolism. These proteins are involved in the main pathways of direct carbon metabolism, such as the pentose phosphate pathway (e.g., gluconokinase GPOL_RS03405), the glyoxylate cycle (e.g., isocitrate lyase GPOL_RS04695), or the citric acid cycle (e.g., succinate dehydrogenase GPOL_RS08320). Furthermore, expression of several sugar transporters was predicted to be under regulation of CRPVH2. Of the predicted CRPVH2 regulation targets, 15% encode proteins that are related to peptide metabolism, such as amino acid transporters (e.g., amino acid permease GPOL_RS22850). Moreover, enzymes of direct amino acid catabolism (e.g., asparaginase GPOL_RS13845) were predicted to be regulated by CRPVH2. In addition, genes encoding several enzymes related to β-oxidation (11.7%) were predicted to be regulated by CRPVH2, including aldehyde-dehydrogenases (GPOL_RS20865, GPOL_RS01460 etc.), acyl-CoA dehydrogenases (GPOL_RS09505, GPOL_RS00670 etc.), acyltransferases (GPOL_RS23210), and many more. Furthermore, CRPVH2 was predicted to regulate the expression of genes that are associated with DNA replication processes (5.8%), such as transposases (GPOL_RS12530), DNA-ligases (GPOL_RS23700), and DNA-topoisomerases (GPOL_RS00030). The same number of the identified target genes were identified as encoding oxidoreductases (5.8%), and 4.4% were related to stress response, such as a universal stress protein (GPOL_RS09155) or a cold-shock protein (GPOL_RS19925). Moreover, CRPVH2 was predicted to regulate the gene expression of 33 transcriptional regulators (16%), comprising several regulator families such as TetR-, LysR-, LytR-, MarR-, LuxR-, GntR-, or Fis-family regulators (Table 2). Furthermore, gene expression of GPOL_RS08005, coding for a σ70-factor, was predicted to be regulated by CRPVH2. As these regulators and the housekeeping σ70-factor regulate the expression of further genes in different functional categories, a complex hierarchic CRPVH2 regulon is indicated by this in silico analysis.

FIG 9
FIG 9

In silico prediction of CRPVH2 regulation targets and their functions. (a) Binding sites (206) were predicted, comprising 244 genes under putative regulation of CRPVH2. The functions of the 244 encoded proteins were investigated and classified into eight groups, whose relative distribution is shown. (b) CRPVH2 consensus binding sequence logo based on the 206 identified binding sites.

View this table:
TABLE 2

In silico prediction of CRPVH2 regulated genes encoding transcriptional regulators

DISCUSSION

Since degradation of poly(cis-1,4-isoprene) requires great effort for cells, the existence of regulatory mechanisms for the expression of Lcps and other related enzymes to occur only when rubber is available seems highly likely. Transcription analyses performed with G. polyisoprenivorans VH2, N. farcinica NVL3, and Streptomyces sp. K30 already demonstrated the induction of lcp genes in the presence of poly(cis-1,4-isoprene) (13, 15, 20). Furthermore, two different regulatory proteins regulating the expression of lcp genes were recently identified, namely LcpRs and LcpRBs (21, 22). However, no superior regulator was known that could regulate the expression of local LcpR or LcpRB regulators. In addition, the discovery of LcpR and LcpRB could not explain the increased abundance of several proteins that are related to poly(cis-1,4-isoprene) degradation, as lcp genes were identified as their sole regulation targets (21, 22, 32).

Our studies led to the discovery of a conserved CRP binding site in the lcpR-lcp intergenic regions of rubber-degrading actinomycetes, indicating an involvement of CRPs in the regulation of poly(cis-1,4-isoprene) utilization. Furthermore, we discovered CRPVH2 of G. polyisoprenivorans VH2 and characterized its recognition site in the lcpRVH2-lcp1VH2 intergenic region. As is typical for CRP regulators, the 16-bp TGTGAN6TCACT motif was verified as the CRPVH2 recognition site. However, the presence of an additional conserved GTGNT motif at a distance of three base pairs from the recognition motif was not a requirement for CRPVH2 binding. Identification of the promoter lcp1VH2p revealed that the conserved Ts of the short motif belong to the −10 region of lcp1VH2p (TATGCT). Thus, it is conceivable that the GTGNT motif is conserved due to the corresponding −10 regions of lcp promoters located in proximity to the CRP-binding sites. Hence, CRPs might regulate the activity of the proximate lcp promoters. Since the −10 region of lcpRVH2p and the −35 region of lcp1VH2p overlap the CRPVH2-binding site, we suggest a dual regulation for CRPVH2 by simultaneously repressing lcpRVH2p and activating lcp1VH2p. Promoter reporter gene assays support our hypothesis, as CRPEc repressed the activity of lcpRVH2p. Since CRPEc and CRPVH2 are homologs, this strongly indicates repression of lcpRVH2p by CRPVH2. When the expression of lcpRVH2 was repressed, expression of lcp1VH2 and lcp2VH2 was indirectly activated by CRPVH2, as LcpRVH2 is the putative lcp repressor. Our results are in agreement with the recently published PredCRP ruleset (37). According to repression rule 2, CRPVH2 acts as a repressor of lcpRVH2p since its binding site is located within the region ranging from 30 bp upstream to 22 bp downstream of the promoter, harboring neither a TTAC sequence nor a GAGC sequence in its binding motif. Hence, the binding site of CRPVH2 strongly overlaps with the RNA polymerase binding site that might block the transcription process of lcpRVH2 or prevent its initiation. However, it is unknown if repression of lcpRVH2p might be sufficient for activating lcp1VH2p, or if simultaneously the direct activation of lcp1VH2p by CRPVH2 is required for lcp1VH2 expression. As the CRPVH2 binding site overlaps with the −35 region of lcp1VH2p, class II CRP-dependent activation of lcp1VH2p by CRPVH2 is conceivable (36). However, lcp1VH2p was not active in E. coli K-12; therefore, future studies must show whether the proposed activation of the promoter by CRPVH2 also occurs here. Due to the conserved CRP-binding sites in various lcpR-lcp intergenic regions, we assume that the expression regulation of lcpR genes and thus of lcp genes by CRPs is a widespread mechanism in rubber-degrading actinomycetes. This hypothesis is supported by the discovery of CRPVH2 homologs in the investigated strains. However, the question arises how CRPs and LcpRs respond to the presence of poly(cis-1,4-isoprene). Based on our results and literature review, we predict that CRPs sense a lack of carbon sources when intracellular cAMP levels increase due to starvation (39). For LcpRs, which are TetR family regulators (TFRs), no ligand could yet be identified. Nevertheless, TFRs are known to interact with a diverse set of ligands, including carbon metabolites (40). Hence, we assume that oligo(cis-1,4-isoprene) molecules or further degradation products, originating from a low basal lcp expression, are the ligands of LcpRs, as already postulated in Oetermann et al. (21). Thus, both stimuli, i.e., the lack of carbon sources signaled by cAMP-CRPs and the extracellular presence of poly(cis-1,4-isoprene) signaled by metabolite bound LcpRs, might be required for full induction of lcp expression.

No CRP-binding sites could be identified in the upstream regions of lcpRB genes in A. mirum 101T, Streptomyces sp. strain CFMR 7, or S. coelicolor A3(2), all of which harbor both LcpR and LcpRB. Thus, regulation of lcp genes by LcpRs could differ from regulation by LcpRBs, as CRPs might only regulate expression of lcpR genes. Because of that, regulation of lcpRB expression might follow a different, yet unknown mechanism.

In addition to lcpR genes and lcp genes, CRPs might regulate the expression of additional enzymes that are also directly and indirectly related to poly(cis-1,4-isoprene) degradation, as was shown for CRPVH2. A regulation at different stages of the degradation pathway by CRPVH2 homologs in other rubber-degrading actinomycetes is likely. In addition to the β-oxidation pathway, CRPVH2 might also be involved in regulating the expression of an operon putatively encoding a trehalose importer. Previous studies discovered an increased abundance of the putative trehalose importer under poly(cis-1,4-isoprene)-degrading conditions (32). As trehalose is linked to mycolic acids, enabling adhesive growth on the polymer, CRPVH2 might also promote adhesive growth of G. polyisoprenivorans VH2 by increasing trehalose recycling (32). Because of that, we assume that CRPs regulate the degradation of poly(cis-1,4-isoprene) at different stages of the proposed pathway in various rubber-degrading actinomycetes.

Since CRPVH2 shares a sequence identity of 79% to GlxR, a well-studied global regulator of C. glutamicum, we assume a similar function as global regulator for CRPVH2 in G. polyisoprenivorans VH2. In addition, several CRPVH2-binding sites harboring different recognition sites were discovered in vitro that show substantial similarity to the CRPVH2 recognition site in the lcpRVH2-lcp1VH2 intergenic region. These results strengthen our hypothesis of various CRPVH2 regulation targets in the organism. In silico analyses predicted 206 CRPVH2 binding sites in the genome of G. polyisoprenivorans VH2, comprising 244 genes under predicted regulation of CRPVH2. As is typical for global CRP regulators such as GlxR and CRPEc, CRPVH2 was predicted to regulate the expression of genes encoding proteins of several functional categories, such as carbon and peptide metabolism, subordinated transcriptional regulators, or stress response. Because of that, we predict that many genes are indirectly regulated by CRPVH2. Furthermore, we assume a complex regulon with several regulatory hubs in which CRPVH2 acts as a superior key regulator, as has been described for GlxR and CRPEc (25–27, 41). Moreover, various approaches to generate a crpVH2 knockout mutant of G. polyisoprenivorans VH2 were not successful (data not shown). Also, in C. glutamicum, several attempts to construct the ΔglxR mutant failed but finally resulted in a mutant with severe growth defects (28, 42). Hence, we assume that CRPVH2 has an important function in the viability of G. polyisoprenivorans VH2 and that disruption of crpVH2 results in a lethal mutant. Future studies could use high-throughput methods to verify the indicated complex CRPVH2 regulon if a ΔcrpVH2 mutant can be constructed.

Our studies identified CRPVH2 as a novel key regulator participating in the regulation of the poly(cis-1,4-isoprene) degradation network. We assume that the recently discovered regulation of lcp expression by LcpRs is much more complex, since CRPs might regulate the lcpR expression in many other rubber-degrading actinomycetes. Furthermore, the identification of several CRPVH2 targets that are involved in poly(cis-1,4-isoprene) degradation at different stages of the pathway gives novel insights into its complex regulation. Because of the conserved CRP-binding sites in various lcp-lcpR intergenic regions, we assume a widespread involvement of CRPs in the networks of poly(cis-1,4-isoprene) degradation in several rubber-degrading actinomycetes that can be investigated in future studies.

MATERIALS AND METHODS

Bacterial strains, plasmids, oligonucleotides, and growth conditions.A list of all strains, plasmids, and oligonucleotides used and constructed in this study is displayed in Table 1. Strains of E. coli were grown at 37°C in lysogeny broth (LB) medium containing 100 μg ml−1 ampicillin if it was necessary for plasmid stability. G. polyisoprenivorans VH2 was cultivated at 30°C, either in standard I (St-I) medium or in mineral salts medium (MSM), supplemented with 0.2% (wt/vol) of the indicated carbon source. Oligonucleotides were obtained from Eurofins Genomics GmbH (Ebersberg, Germany).

Bioinformatic analyses.Using the Multiple Em for Motif Elicitation (MEME) suite, the putative CRP-binding motifs in the lcpR-lcp intergenic regions were discovered (43). Subsequently, Tomtom was used to align the discovered motif with a database of known motifs to reveal its putative function (44). Similarity searches of amino acid sequences were performed with the basic local alignment search tool (BLAST) of the NCBI website (45). Alignments were performed using ClustalW with default parameters (46). SWISS-MODEL was used to predict the 3D structure of CRPVH2 by homology structure modeling (47). PromoterHunter of the phiSITE database was used to predict the promoter regions of lcpRVH2 and lcp1VH2 with default parameters (48).

We screened the genome of G. polyisoprenivorans VH2, including the chromosome (RefSeq: NC_016906.1) and the plasmid (RefSeq: NC_016907.1), to predict further CRPVH2 transcription factor binding sites (TFBSs). For that, we used TFBSs from CRPVH2 identified in this study and TFBSs from the global regulator GlxR of C. glutamicum (49) to build two profile HMMs, one with only CRPVH2 TFBSs and the other with CRPVH2 plus GlxR TFBSs. Profile HMMs were built with hmmer-build from HMMER package (50). We scanned upstream regions (−20,480) of the chromosome and the plasmid with each of the profile HMMs by using nhmmer with default parameters (51). Then, we calculated Hamming distance by comparing the hmmer hits with the experimental CRPVH2 TFBSs and removed the hits that matched the negative-verified TFBSs. Hmmer hits with hamming distance of ≤2 were selected as highly probable true CRPVH2-binding sites.

Purification of CRPVH2.Genomic DNA of G. polyisoprenivorans VH2 was extracted with the NucleoSpin Tissue kit purchased from Macherey-Nagel (Düren, Germany). Using Phusion High-Fidelity DNA polymerase (New England BioLabs, Ipswich, MA, USA) and genomic DNA as the template, crpVH2 was amplified. The primers (CRP-fw and CRP-rv-his) harbored NdeI and BamHI restriction sites and encoded an N-terminal His6 tag, analogously to GlxR (34). The resulting fragment was purified from a 1% (wt/vol) agarose gel and ligated into the cloning vector pJET1.2/blunt (Thermo Fisher Scientific, Waltham, MA, USA), and E. coli Mach1 T1 was transformed with the plasmid. The plasmid was isolated and digested by NdeI and BamHI, and the 705-bp insert was ligated into the expression vector pET23a(+). Expression took place in E. coli C41(DE3) that harbored pET23a(+)::HiscrpVH2. The cells were cultivated at 37°C in LB containing 100 μg ml−1 ampicillin. At an optical density at 600 nm (OD600) of 0.4, expression was induced by the addition of 0.2 mM IPTG (isopropyl-β-D-thiogalactopyranoside) and the temperature was reduced to 20°C. Cells were harvested after 20 h and disrupted with a French press, and the pellet was resuspended in 50 mM Tris-HCl buffer (pH 7.4) containing 500 mM NaCl and 40 mM imidazole (resuspension buffer). CRPVH2 was purified using His SpinTrap purification columns (GE Healthcare, Little Chalfont, UK). For this, the samples were washed with resuspension buffer and were eluted in 50 mM Tris-HCl buffer (pH 7.4) containing 500 mM NaCl and 100 mM or 200 mM imidazole, respectively. An additional chromatographic step was performed to remove salt and imidazole. For this a HiPrep 26/10 desalting column (GE Healthcare, Little Chalfont, UK) was chosen and TG buffer (30 mM Tris-HCl pH 7.4 and 5% [vol/vol] glycerol) was used for desalting. If necessary, the samples were concentrated using a VivaSpin 6 ml centrifugal concentrator tube (Sartorius, Stonehouse, UK) with 10,000 Da as the molecular weight cutoff. The success of purification was verified by SDS-PAGE and protein concentration was measured spectrophotometrically using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Size exclusion chromatography.To ascertain the native molecular weight of CRPVH2 and its oligomerization state, size exclusion chromatography was performed. A Superdex 200 prep grade XK 26/60 column (GE Healthcare, Little Chalfont, UK) was equilibrated with TE buffer (30 mM Tris-HCl pH 7.5, 1 mM EDTA) and 1 ml purified CRPVH2 (5 mg ml−1) was applied. During the run, TE buffer was used with a flow rate of 1 ml min−1. The system was calibrated applying the Gel Filtration LMW Calibration kit according to the manufacturer’s instructions (GE Healthcare, Little Chalfont, UK).

Electrophoretic mobility shift assays.Binding of CRPVH2 to the tested sequences was investigated by electrophoretic mobility shift assays (EMSAs). FAM (6-carboxyfluorescein)-labeled DNA was prepared by hybridization of 40-bp oligonucleotides. Equimolar amounts of each oligonucleotide were heated to 95°C for 2 min. Over a period of 45 min, the mixture was cooled to 25°C. Reaction mixtures contained 30 nM DNA, 19.5 μM purified CRPVH2 and binding buffer (10 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM EDTA, 6% [vol/vol] glycerol) in a total volume of 20 μl. When cAMP was added to the samples, a concentration of 0.4 mM was used. Controls contained purified LcpRVH2 instead of CRPVH2 or the LcpRVH2 consensus binding sequence instead of the tested CRPVH2 binding sequence. Samples were incubated for 20 min at room temperature in the dark. Separate native polyacrylamide gels (8%, wt/vol) were prepared with either only TE running buffer (15 mM Tris-HCl pH 8.0, 1 mM EDTA), or additional 0.4 mM cAMP, added prior to the polymerization. Gels were equilibrated by a prerun for 15 min at 160 V and 4°C. After that, the samples were loaded into the gels with loading dye (20 mM Tris/HCl pH 8.0, 25% [wt/vol] sucrose, 0.05% [wt/vol] bromophenol blue). Gels were run at 160 V and 4°C for approximately 2 h. The fragments were visualized with a ChemiDoc MP Imaging System choosing Alexa488 as the excitation and emission method.

DNase I footprinting assays.DNase I footprinting assays were performed to determine the operator site of CRPVH2 in the intergenic region of lcpRVH2 and lcp1VH2. The intergenic region was amplified from pJET1.2::FP_lcp1VH2 with the fluorescently labeled primers pJET_fw_FAM and pJET_rv_NED. Thus, the fragment used for the analysis was labeled on both strands with either FAM (6-carboxyfluorescein) or NED (ATTO 550). The footprinting assay itself was performed as described previously (52). For this, 9.3 nM of the labeled fragment was incubated with 150 μM CRPVH2 for 20 min at 30°C in reaction buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 6% [wt/vol] glycerol, 0.4 mM MgCl2, 1.0 mM dithiothreitol [DTT] and 0.4 mM cAMP) in a total volume of 100 μl. DNase I digestion (0.2 U ml−1) was carried out for 30 s at 30°C in a final volume of 100 μl and stopped by the addition of 2 mM EDTA. The DNA was extracted with phenol, precipitated with ethanol, resuspended in Hi-Di formamide, and mixed with the size standard 500 ROX (Applied Biosystems, Foster City, CA, USA). An ABI 3730 XL DNA analyzer was used for analysis. Genescan results were evaluated with PeakScanner 2 software (Applied Biosystems, Foster City, CA, USA).

RNA isolation and capping RACE.To determine the 5′ untranslated mRNA regions of lcp1VH2 and lcpRVH2, G. polyisoprenivorans VH2 was grown in MSM containing 0.2% (wt/vol) poly(cis-1,4-isoprene) or propionate until the mid-exponential phase. Total RNA was extracted using the innuPREP RNA minikit 2.0 (Analytik Jena AG, Jena, Germany). For that, 109 cells were harvested and resuspended in 100 μl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 15 mg ml−1 lysozyme. After 2 h of incubation at 37°C, total RNA was purified according to the manufacturer’s instructions. Up to 10 μg of RNA was subjected to DNase I digestion (Roche, Mannheim, Germany) performed for 30 min at 25°C to remove residual DNA. RNA was purified using RNA Clean & Concentrator-5 (Zymo Research, Freiburg, Germany). The RNA concentration was measured spectrophotometrically using NanoDrop, whereas the integrity of isolated RNA was verified on a 1% (wt/vol) agarose gel containing 1% (vol/vol) NaClO. Capping rapid amplification of cDNA ends (RACE) was performed as described previously (53). For that, 10 μg of purified RNA was heated at 70°C for 2 min and subsequently incubated with 0.5 mM GTP, 0.1 mM SAM, and 1.5 U ml−1 vaccinia capping enzyme (NEB, Ipswich, MA, USA) for 30 min at 37°C to add a 7-methylguanylate cap structure at the 5′ end. Capped RNA was purified again using RNA Clean & Concentrator-5 and was used for RACE. For that, 3 μg of capped RNA was incubated for 5 min at 60°C with deoxynucleoside triphosphates (dNTPs) and gene-specific primers for lcpRVH2 (Gsp1-lcpR) and lcp1VH2 (Gsp1-lcp1), respectively. cDNA extension was performed for 60 min at 50°C using SuperScript III reverse transcriptase (Thermo Fischer Scientific). After that, the template switching oligonucleotide (TSO-5G) was added and template switching was performed for 90 min at 42°C. RNA of DNA-RNA hybrids was degraded by the addition of 2 U RNase H (NEB, Ipswich, MA, USA) for 20 min at 37°C. Subsequent nest-PCR was performed using the primer set of the inner TSO and Gsp2-lcpR and Gsp2-lcp1, respectively. Capping RACE products were visualized on a 2% (wt/vol) agarose gel and were sequenced.

Promoter reporter gene assays.To investigate the regulation of promoter activity by CRP regulators, promoter-lacZ fusions were constructed. For that, the lcpRVH2-lcp1VH2 intergenic region was fused to lacZ in lcpRVH2 or lcp1VH2 orientation via fusion PCR. Thus, the corresponding promoter regions (lcpRVH2p and lcp1VH2p) were located upstream of the reporter gene. Two control constructs were designed in which the P1 lacZ promoter (lacZp1) or no promoter was fused to lacZ. All four fusion constructs were digested with BamHI and SalI and ligated into pJET1.2/blunt. E. coli K-12 strains BW25113 and the crpEc deletion mutant JW5702-4 (Δcrp-765::kan) were purchased from Horizon Discovery and transformed with the reporter plasmids. Construction of the plasmids was confirmed by DNA sequencing. Overnight cultures in LB medium were diluted 1:1,000 into fresh LB medium and cells were grown to an OD600 of 0.5 to 0.6. LacZ activity was quantified according to the assay of Miller (54). Experiments were performed in biological triplicates, each comprising three technical replicates.

ACKNOWLEDGMENTS

We thank Bodo Philipp for the use of the ChemiDoc MP Imaging System.

We declare no conflicts of interest.

No experimental work with humans or animals was performed.

No funding was received for this study.

FOOTNOTES

    • Received 31 March 2020.
    • Accepted 17 May 2020.
    • Accepted manuscript posted online 22 May 2020.

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KEYWORDS

cAMP receptor protein
CRP
global regulator
Gordonia
latex clearing protein
poly(cis-1,4-isoprene) degradation

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