All strains and plasmids used in this study were listed in Table 1. n-Dodecane, n-tetradecane, n-hexadecane, n-octadecane, n-pentadecane, n-eicosane, n-docosane, n-tetracosane, n-triacontane, n-hexane and their derivates (of HPLC grade, >99.0%) were purchased from Sigma-Aldrich (St. Louis, MO, United States). All other reagents were of analytical reagent grade. Luria-Bertani (LB) medium (10.0 g/L tryptone, 5.0 g/L yeast extract, 10.0 g/L NaCl, pH 7.0) and the minimal medium (BSM) [4.5 g/L KH₂PO₄, 13.75 g/L K₂HPO₄⋅3H₂O, 2.0 g/L (NH₄)₂SO₄, 0.16 g/L MgSO₄⋅7H₂O, 5.0 μg/L FeSO₄, 1.0 μg/L CaCl₂⋅2H₂O, 2.0 μg/L MnCl₂⋅4H₂O, pH 7.4] were used in this study. The alkane-hexane solutions were prepared by dissolving the n-alkanes into the n-hexane (v/v or g/v) to 500 mg/mL and were supplied into the minimal medium to obtain various concentrations. The n-hexane was neither toxic nor utilized by strain P. aeruginosa SJTD-1 (Liu et al., 2014). All the enzymes for DNA-operation (DNA polymerases, T4 DNA ligases, restriction endonucleases and etc.) were purchased from TaKaRa Co. (Dalian, China) and the DNase I was the product of Thermo Fisher Scientific Inc. (Waltham, MA, United States). Oligonucleotides used for gene amplification, plasmid construction, reverse transcription (RT), quantitative PCR (q-PCR), homologous recombination, and electrophoretic mobility shift assay (EMSA) in this work were all synthesized by Invitrogen Ltd. (Shanghai, China) and listed in Table 2.

The PCR procedure was performed at 95°C for 5 min, 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min, and 72°C for 5 min. All the plasmids were constructed by ligating the restriction-enzyme-treated PCR fragments and plasmid bones with T4 DNA ligase. Chemical transformation and the electroporation-mediated transformation were used for the plasmids transformation into E. coli strains and P. aeruginosa strains, respectively. All constructed plasmids were confirmed by DNA sequencing in Invitrogen Ltd. (Shanghai, China). The recovery of PCR fragment, extraction of plasmid DNA, and isolation of genome DNA were achieved by respective kits from TIANGEN Co. (Beijing, China) and following the corresponding protocols. RT and q-PCR were performed using the PrimeScript Reverse Transcriptase kit and Probe qPCR kit (TaKaRa, Dalian, China). Other general techniques for the agarose gel electrophoresis, native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) were carried out with standard protocols.

Pseudomonas aeruginosa SJTD-1 was cultured in 1 L BSM medium with 500 mg/L n-octadecane as the carbon source at 37°C to OD₆₀₀ about 1.0. Cells were harvested by centrifugation at 8,000 rpm for 10 min at 4°C and re-suspended in 40 mL ice-cold lysis buffer [20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, 5 mM β-Mercaptoethanol (β-ME), 1 mM phenylmethanesulfonyl fluoride (PMSF), 10% glycerol, pH 7.9]. Crude extracts was obtained by sonication on ice and centrifugation at 12,000 rpm for 30 min at 4°C. Then the clarified solution was mixed into the streptavidin-coated magnetic particles with the immobilized DNA fragment, the 500 bp upstream fragment of alkB2 gene. Proteins that bound to the target DNA in magnetic particles were isolated and detected by mass spectrometry. Mass spectral data were collected in +ESI mode in separate runs on a Waters HDMS-QTOF synapt GI mass spectrometer operated in a scan mode of 50–500 m/z. The predicted composition was calculated with MASCOT software.

The multiple sequences alignments (MSA) of DNAs and proteins were performed using DNAMAN software. The phylogenetic tree of CrgA homologues was constructed based on their gene sequences using MEGA 7.0 software in the neighbor-joining method with 1,000 replications. The structure-homology modeling of the CrgA protein (from P. aeruginosa SJTD-1) was performed in SWISS-MODEL with the CrgA protein (3HHG) of Neisseria meningitidis as a template.

The recombinant CrgA protein was expressed in E. coli BL21 (DE3) strain and purified by affinity chromatography. Briefly, E. coli BL21 (DE3) cells containing plasmid pET28a-crgA was inoculated in 10 mL LB with 50 μg/mL kanamycin and cultured at 37°C. The overnight cultures were transferred into fresh LB with 50 μg/mL kanamycin and cultured to about 0.5 OD₆₀₀. 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and 3 h induction at 37°C was performed. Cells were harvested by centrifugation at 8,000 rpm for 10 min at 4°C and re-suspended in 25 mL ice-cold lysis buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, 5 mM β-ME, 1 mM PMSF, 10% glycerol, pH 7.9). Cell disruption was performed with sonication on ice, and the cell lysate was clarified by centrifugation at 12,000 rpm and 4°C for 30 min. The cell supernatants were loaded into the Ni-NTA resin (Bio-Rad, Hercules, CA, United States) at 4°C and washed with washing buffer (20 mM Tris-HCl, 300 mM NaCl, 5 mM β-ME, 10% glycerol, 1 mM PMSF, 20 mM or 50 mM imidazole, pH 7.9). Finally, the recombinant protein was eluted from the column using elution buffer (20 mM Tris-HCl, 300 mM NaCl, 5 mM β-ME, 10% glycerol, 1 mM PMSF, 250 mM imidazole, pH 7.9). All the eluted fractions were analyzed with 15% SDS-PAGE followed by staining with Coomassie Brilliant Blue R250 (Sigma-Aldrich, St. Louis, MO, United States). The eluted recombinant CrgA protein was dialyzed and stored in storage buffer (20 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 50% glycerol, pH 8.0) at -80°C.

The crgA gene was knocked out using the two-step homologous recombination method (Hoang et al., 1998). The pEX-crgA-UD plasmid was constructed by inserting the 500 bp upstream and 500 bp downstream fragments of crgA gene into plasmid pEX18Gm (Hoang et al., 1998). Then plasmid pEX-crgA-UD was transformed into E. coli SM10 strain before the conjugation with P. aeruginosa SJTD-1. Fifty μg/mL gentamycin and 25 μg/mL tetracycline were used for the first-step selection; 10% sucrose was used to induce the second-step recombination and generate the crgA-deleted mutant. The deletion of crgA gene was performed in the wild-type strain SJTD-1, the alkB1-knockout strain S1_ΔalkB1 and the alkB2-knockout strain S1_ΔalkB2 (Liu et al., 2014); the resulting strains were named S1_ΔcrgA, S1_ΔalkB1&crgA, S1_ΔalkB2&crgA respectively (Table 1).

The cell growth of wild-type strain SJTD-1 and all the knockout mutants (S1_ΔalkB1, S1_ΔalkB2, S1_ΔcrgA, S1_ΔalkB1&crgA, S1_ΔalkB2&crgA) were determined with n-alkanes as the sole carbon source (Liu et al., 2014). Single colony was inoculated into 10 mL LB broth and cultured overnight at 37°C. The harvested cells were washed thrice with sterilized water and re-suspended in BSM medium to OD₆₀₀ about 2.0. Ten μL cell pellet was inoculated into the wells of a 10 × 10 multi-well plate, each containing 190 μL BSM medium with different concentrations of n-alkanes (C₁₂–C₂₄). The initial OD₆₀₀ of each well was 0.1. Wells containing cells without n-alkanes and wells containing n-alkanes without cells were used as blank controls. The 10 × 10 multi-well plates were loaded onto an Automatic Growth Curve Analyzer (BioScreen Testing Service, Inc., Torrance, CA, United States), and cultured at 180 r/min for 7 days at 37°C. Cell densities were determined by detecting the OD₆₀₀ every hour. At least five independent experiments (three paralleled samples in each experiment) were conducted and the average values were calculated with standard errors. Statistical analysis were performed with the cell densitities at three time points (96, 120, and 140 h) using SPSS 24 for t-test calculation and the P-value was calculated (P-value < 0.05, significant).

DNA fragments of different length were amplified from the upstream regions of alkB2 gene using different oligonucleotides. The long fragments (>100 bp) were amplified with primers labeled with FAM at 5′-site, and the short fragments (<100 bp) were obtained by annealing two paired oligonucleotides (Table 2). The two oligonucleotides of equimolar concentrations (10 μM) were mixed in the annealing buffer (10 mM Tris-HCl, 50 mM KAc, 1 mM EDTA, pH 8.0), incubated at 95°C for 5 min, and then cooled slowly to room temperature. The EMSA detection was performed by mixing CrgA protein with the labeled DNA fragments at different molecular ratios and incubating in the binding buffer (20 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA, pH 7.5) at 37°C for 30 min. All input DNA amounts were 4 pmol in the 20 μL binding system. The mixture of the binding assay was analyzed with 8% native PAGE and visualization using the BioRad Imaging System (Bio-Rad, Hercules, CA, United States). The EMSA detection of CrgA protein to the upstream fragment of alkB1 gene and the mutant fragments of alkB2 gene (alkB2-b1 to alkB2-b7) were also performed.

Different metabolites of n-hexadecane and n-octadecane (hexadecanol, octadecanol, octadecanoic acid, palmitic acid, sodium palmitate, sodium stearate, hexadecyl coenzyme A, and octadecyl coenzyme A) were used to determine their effects on the binding of CrgA to the upstream region of alkB2 gene. Ethyl acetate and DMSO were used as controls. The binding of CrgA to the three fragments of alkB2 gene (alkB2-b1, alkB2-b2, alkB2-b3) was performed same as above. Then different metabolites of 20, 100, or 500 pmol were added into the 20 μL reaction system, followed with 15-min incubation at 37°C. The mixture was analyzed with 8% native PAGE and visualization by the BioRad Imaging System (Bio-Rad, Hercules, CA, United States).

Four hundred ng DNA fragments (alkB2-U187) amplified from the upstream region of alkB2 gene was bound with CrgA protein at 25°C for 30 min same as above. Then 0.015 U DNase I was added into the binding system with incubation at 25°C for 1 min. The reaction products were extracted with phenol to remove proteins, and ethanol was added to precipitate DNA fragments. The precipitated DNA fragment was dissolved in ultra-pure water and sequenced. The DNA fragment (alkB2-U187) with BSA protein and the DNA fragment without binding ability to CrgA protein (alkB2-U46) were used as controls.

The wild-type strain and the mutant strains were inoculated in BSM medium with glucose or various alkanes (C₁₂–C₂₄) and cultured to the mid-exponential phase. Total RNA was extracted with Total RNA Extraction Reagents (Vazyme, Nanjing, China) according to the protocol. The yield of RNA was estimated using a Nanodrop UV spectrometer (Thermo Fisher Scientific, Waltham, MA, United States). DNase treatment was used to remove the possible genomic DNA contamination. RT was performed with approximately 1 μg RNA and 20 ng random primers using PrimeScript Reverse Transcriptase Kit (TaKaRa, Dalian, China). q-PCR was achieved using Premix Ex Taq (Probe qPCR) (TaKaRa, Dalian, China) and the gene-specific primers in an IQTM 5 Multicolor Real-time PCR Detection System (Bio-Rad, Hercules, CA, United States). The conditions were set at 95°C for 3 min, and 40 cycles of 95°C for 10 s, 57°C for 30 s, and 72°C for 30 s; a final melting analysis was performed by slow heating with 10 s increments of 0.5°C from 57°C to 95°C. The one without reverse transcriptase was used as negative control. The threshold cycle (Cq) value of each sample was determined during the exponential phase of amplification, and the relative fold change in mRNA quantity was calculated using the DDCt method (Livak and Schmittgen, 2001). At least five independent experiments (three paralleled samples in each experiment) were conducted for each sample, and the average values were calculated with standard errors. Statistical analysis were performed with the relative mRNA quantities using SPSS 24 for t-test calculation and the P-value was calculated (P-value < 0.05, significant).

Green fluorescent protein fluorescence assays was performed to detect the promoter activity and expression level of alkB2 gene in different strains. The egfp gene was cloned into pBSPPc-Gm to generate plasmid pBS-eGFP (Table 1). Then the upstream promoter region of alkB2 gene was inserted the upstream of egfp gene to form the plasmid pBS-P_alkB2-eGFP. The two plasmids were transformed into the wild-type strain and the crgA-knockout mutant strains. All the recombinant strains were cultured in BSM medium with 0.4% glucose or 500 mg/L n-alkanes of different lengths. Cells were collected at different time points to measure GFP fluorescence with excitation at 485 nm and emission at 527 nm. At least five independent experiments were performed, and the average values were calculated with standard errors. Statistical analysis were performed with the relative GFP fluorescence using SPSS 24 for t-test calculation and the P value was calculated (P-value < 0.05, significant).

AlkB2 monooxygenase was proved to be the major enzyme hydroxylating the medium-to-long-chain n-alkanes (C₁₄–C₂₀) in strain P. aeruginosa SJTD-1 (Liu et al., 2014). To find its potential regulators, the pull-down assay and mass spectrometry were performed. Results showed that one 34 KD protein was identified because of its specific binding to the upstream region of alkB2 gene, which was predicted to be a CrgA-like protein. This CrgA-like protein (ANI09566) of P. aeruginosa SJTD-1 contained 304 amino acids, encoded by gene crgA (CP015877.1: 3086790-3087704), far from the alkB2 gene (CP015877.1: 3423611-3424744). Secondary structure analysis of this CrgA protein showed that there were 11 α-helixes and 12 β-strands in it; three α-helixes (α1 to α3) and two β-strands (β1 to β2) were in its N-terminal region, forming a conserved winged-helix-turn-helix (w-HTH) structure and functioning as the DNA binding domain. Its C-terminal region containing seven α-helixes (α5 to α11) and ten β-strands (β3 to β12) were highly variable and responsible for the effector binding (Figure 1A). Evolutionary analysis revealed this CrgA-like protein was close to the CrgA protein of Neisseria meningitides (Figure 1B). Tertiary structure prediction showed that their spatial structures were similar; the N-terminal region and C-terminal region were connected through a long α-helix (α4) structure (Figure 1C). Therefore, this identified CrgA-like regulator was a member of the CrgA sub-family of LTTRs.

The recombinant CrgA protein was obtained by heterologous expression and affinity purification, with a yield of 12 mg/L and purity of 98% (Figure 2A). EMSA detection showed that the CrgA protein could bind to the 187 bp upstream fragment of alkB2 gene (alkB2-U187) in a concentration-dependent manner. No dissociation was observed over the 32:1 protein-DNA ratio (Figure 2B). However, very weak binding ability and low specificity was observed when this CrgA protein mixed with the upstream region of alkB1 gene, implying that the regulation preference of this CrgA protein to AlkB2 monooxygenase (Supplementary Figure S1A).

The DNase I foot printing assay showed that CrgA protein bound to the upstream fragment of alkB2 gene at two specific sites, bs1 and bs2 (Figure 2C). No binding was observed when BSA used (Supplementary Figure S2A). Interestingly, the structures of the two binding sites were mirror like, similar to the imperfect mirror repeat (IIR) structure. For the bs1 site, its sequence was GGGA-N3-GGC-N-GG-N-CGG-N3-AGGG, using the middle GG bases for symmetry; for the bs2 site, the central base A was used for symmetry and the sequence was AGC-N5-A-N5-CGA. The IIR structure in bs1 was more stringent than that in bs2 (Figure 2C). Moreover, the bs2 site was close to the predicted -35 region of alkB2 gene; the bs1 site overlapped the -35 region and located between the -35 and -10 region of alkB2 gene (Figure 2D). No obvious mirror structure was found in the upstream region of alkB1 gene (Supplementary Figure S1B). These results indicated CrgA could bind to the specific sites in the promoter region of alkB2 gene and may influence the transcription of AlkB2 monooxygenase.

To study the structure specificity of the CrgA-binding sites, several mutant DNA fragments containing the intact, partial or destroyed mirror structure were constructed and used for EMSA detection. The 46 bp upstream region of alkB2 gene (alkB2-U46), which was confirmed not binding to CrgA protein, was selected as template fragment to insert different binding sequences (Supplementary Figure S2B). The IIR structure was maintained in the mutant fragments (b1/b2), and only half of IIR structure was reserved in the mutant fragments (b4/b5). The destroyed IIR structure was generated by changing the opposite direction of the mirror structure into identical direction (b6/b7) (Figure 3A). EMSA results showed that the binding of CrgA protein to the fragments containing half of the IIR structure (b4/b5) or no IIR structure (b6/b7), were much weaker than that to the fragments with full IIR structure (b1/b2); half of the IIR structure probably acted as the basic binding unit (Figure 3B,C). Therefore, these results demonstrated that the IIR structure and its integrity were crucial for the efficient binding of CrgA to the promoter region of alkB2 gene.

Effects of n-alkanes and their metabolites on the specific binding of CrgA to the promoter region of alkB2 were analyzed. Addition of hexadecyl coenzyme A and octadecyl coenzyme A into the binding system could efficiently release the specific binding of CrgA to the fragments containing the two binding sites (bs1 and bs2), dependent on the chemical’s concentration. No sequence preference was observed and no release was found with acetyl coenzyme A (Figure 4). Although palmitic acid and octadecanoic acid had little influence on the interaction, other derivatives of n-alkanes could not release CrgA binding from the upstream fragments of alkB2 (Supplementary Figure S3). These results indicated that long-chain fatty acyl-CoA (C₁₆–C₁₈) could influence the specific binding of CrgA to the target region of alkB2 gene, probably through a positive feedback of intermediates.

The regulation of CrgA to AlkB2 monooxygenase and n-alkane utilization was analyzed by detecting the cell growth and the transcription and expression of alkB2 gene. For cell growth analysis, at least five independent experiments (three paralleled samples in each experiment) were conducted at each time point, and the average cell densitities at the stationary phase were performed the statistical analysis using SPSS 24 for t-test calculation between every two strains. Results indicated that in the C₁₈-alkane condition, strain S1_ΔalkB1 grew normally, while strain S1_ΔalkB2 grew slower than the wild-type strain, consistent with previous report (Supplementary Figure S4) (Liu et al., 2014). However, at the stationary phase, the growth difference of the three strains (SJTD-1, S1_ΔalkB1 and S1_ΔalkB2) was not statistically significant. Notably, the growth diversity in strains S1_ΔalkB1 and S1_ΔalkB1&crgA was significant at the stationary phase, and the cell densitity of strain S1_ΔalkB1&crgA was higher than that of strain S1_ΔalkB1 (Figure 5A and Supplementary Figure S5). Beside, the growth difference was not statistically significant between the strains S1_ΔalkB2 and S1_ΔalkB2&crgA (Supplementary Figure S4). These results implied that crgA-knockout could not siginificantly influence the cell growth; while the deletion of crgA gene in the alkB1-knockout strain (S1_ΔalkB1&crgA/S1_ΔalkB1) could improve the cell growth, in which alkB2 gene was the major gene for the utilization of medium-to-long chain n-alkane. At the meanwhile, the growth difference was not statistically significant when the crgA gene was knocked out in the alkB2-knockout strain (S1_ΔalkB2&crgA/S1_ΔalkB2). It indicated that CrgA was probably involved in the regulation of AlkB2, not AlkB1. As the growth difference was not so big, it was much likely that other alkane hydroxylases and other transcriptional regulators also participated in utilization of medium-to-long-chain n-alkanes in strain SJTD-1, although the AlkB monooxygenases played important roles (Liu et al., 2014).

Transcriptional levels of alkB2 gene in strains SJTD-1 and S1_ΔcrgA cultured with different carbon sources (glucose and C₁₂–C₂₄ alkanes) were analyzed. When strain SJTD-1 was cultured with C₁₂, C₁₆, and C₁₈ alkanes, the transcription of alkB2 gene was significantly induced (over 10-fold); about 2–4 folds increase in the transcription of alkB2 gene was also observed in the culture with C₂₀ and C₂₄ alkanes (Figure 5B). The transcription of alkB2 gene could be further enhanced after deletion of crgA; in the C₁₆–C₂₀ alkanes culture, significant transcriptional promotion (over 2.4-fold) was observed compared to those in wild-type strain. However, no significant change was observed when strains cultured with C₁₂ and C₂₄ alkanes (Figure 5C). These results demonstrated that the transcription of alkB2 gene was repressed by CrgA and in the medium-to-long-chain alkane conditions, this repression was released to start alkB2 transcription. Furthermore, the expression detection of egfp gene under the promoter of alkB2 gene was performed in strains SJTD-1 and S1_ΔcrgA. Although the GFP fluorescence changes in the two strains were not as significant as the transcription diversity, similar trends were also observed. The GFP fluorescence of strains cultured with C₁₆, C₁₈, and C₂₀ alkanes were higher than those cultured in glucose; when cultured with medium-to-long-chain alkanes, the expression of egfp gene under the promoter of alkB2 gene in strain S1_ΔcrgA was higher than that in strain SJTD-1 (Figure 5D). The inconsistency in the transcription and translation may due to differences in the expression of genome and plasmid or the influences of other regulators. Taken together, these results demonstrated that CrgA could repress the transcription of alkB2 gene and regulate the utilization of medium-to-long-chain n-alkane in strain SJTD-1.