Cbl upregulates cysH for hydrogen sulfide production in Aeromonas veronii

Bacterial strains, plasmids and culture conditions

The bacterial strains and plasmids used in this study were shown in Table S1. The smpB deletion strain of Aeromonas veronii C4 was constructed previously (Liu et al., 2015). The derivative Aeromonas veronii C4 strains were grown in LB/M9 medium supplemented with 50 mg/mL ampicillin at 30 °C, and E. coli strains were grown in LB medium supplemented with 50 mg/mL kanamycin and 25 mg/mL chloramphenicol at 37 °C. And all plasmids were sequenced for verification. LB medium contained 10% tryptone, 5% yeast extract and 10% NaCl. M9 medium contained 20% 5×M9 salts, 0.2% 1 M MgSO₄, 0.01% 1 M CaCl₂ and 0.4% Glucose, of which 5×M9 salts included 6.4% Na₂PO₄·7H₂O, 1.5% KH₂PO₄, 0.25% NaCl and 0.5% NH₄Cl.

Complemented strain construction

The DNA fragment including both cbl gene and its promoter was amplified by PCR (Wang et al., 2019). PCR reaction was performed as follow: 98 °C for 2 min, stepped by 98 °C for 30 s, 55 °C for 30 s and 72 °C for one kb/min in 30 cycles. The purified PCR product was inserted into pBBR plasmid for creating pBBR-Cbl expression plasmid. The cbl deletion strains were complemented by the conjugation of recipient WM3064 strains carrying pBBR-Cbl.

H₂S detection

The Pb(Ac)₂ detection (Shatalin et al., 2011) method and WSP5 fluorescent H₂S probe (Peng et al., 2014) were performed for monitoring H₂S production in gas and liquid phases, respectively. Bacteria were grown in M9 at 30 °C for 48 h with soaked Pb(Ac)₂ paper strips hanging from the mouth of the conical bottle, and five mg/L Na₂SO₃ was added as a source of sulfur. Pb(Ac)₂-soaked paper strips showed a PbS brown stain as a result of the reaction with H₂S. The color length of one mm represented 12 μg/L of H₂S production. After 10 μM WSP5 was added to liquid bacterial culture, the samples were incubated at 37 °C for 30 min and then washed in PBS buffer to remove excess probe. Synergy H1 (BioTek) was used to take fluorescent readings at excitation 500 nm and emission 533 nm. Each reaction was performed at least in triplicate.

RNA extraction and qRT-PCR

The qPCR reaction was conducted with ABI Prism^® 7300 (ABI, New York, NY, USA) for fluorescent detection utilizing SYBRR^® Green real time PCR Master Mix (Toyonbo, Shanghai, China). The cDNA was synthesized by RNA reverse transcription reaction and was used as the template for real-time PCR. The primers used to monitor expression of the objective genes were summarized in Table S2. Each reaction was performed at least in triplicate, and wild type (WT) was chosen as the control. And the data was analyzed by the comparative CT method (Schmittgen & Livak, 2008).

Fluorescence analysis

The promoter of cysJIH was fused with eGFP and inserted into pUC19 plasmid. The cbl gene was cloned into pTRG plasmid simultaneously. Both the above plasmids were co-transformed into E. coli Reporter strain. Meanwhile, the recombinant pUC19 plasmid and the empty pTRG plasmid were co-transformed as the negative control. After bacteria were grown in LB at 37 °C, the total amount of 1 × 10⁸ cells were harvested in 1.5 mL eppendorf tube at interval time. The samples were washed with PBS twice, and placed on Synergy H1 (Biotek) for the fluorescent readings at excitation 425 nm and emission 525 nm. Each reaction was performed at least in triplicate.

Bacterial one-hybrid analysis

To identify whether the transcription factor Cbl interacted with the promoter of cysJIH, Cbl was inserted into pTRG, and the promoter of cysJIH was ligated with pBXcmT, following with both the recombinant plasmids were cotransformed into E. coli Reporter strain. The transformants were placed on a selective NM medium plate containing five mM 3-amino-1, 2, 4-triazole (3-AT) and 12.5 mg/mL streptomycin for incubation at 37 °C for 48 h. The pTRG and pBXcmT plasmids were co-transformed as the negative control, and pTRG-GAL and pBT-LGF2 were co-transformed as the positive control.

Protein expression and purification

The cbl gene was inserted into pET28a plasmid and transformed into E. coli BL21 strain. The expression and purification were performed according to previous procedure (Bykowski et al., 2002). Cbl protein was purified from E. coli BL21 harboring the pET28a-Cbl plasmid. The recombinant bacteria were grown in LB at 37 °C until the logarithmic phase, followed by the addition of 0.1 mM IPTG to induce protein expression at 15 °C for 14 h. The cells were harvested in Tris-HCl and lysed by sonication. The supernatant was collected after centrifugation and loaded onto a Ni-NTA column. The sample was eluted prior to the dialysis, and SDS-PAGE was used to assess protein purity.

Electrophoretic mobility shift assay (EMSA)

Double stranded DNA probes were radiolabeled with Fluorophore 6-carboxy-fluorescein (FAM) and purified by FastPure Gel DNA Extraction Mini Kit (Vazyme). For the EMSA, DNA probe was incubated with Cbl protein samples in reaction buffer (10 mM Tris–HCl, one mM MgCl₂, one mM DTT, 40 mM KCl, 0.1 mg/mL BSA, 5% (w/v) glycerol) at 37 °C for 30 min. After the samples were separated using a 6% native acrylamide gel (Zhang et al., 2020), the gel was then exposed to a phosphorscreen and visualized on Typhoon FLA 9500.

Transcriptome analysis

To perform the whole-transcriptome analysis, the wild type and smpB deletion of Aeromonas veronii C4 were grown in M9 at 30 °C for 20 h, and 2 OD₆₀₀ of cells were collected. Illumina HiSeq-X ten based on the service of RNA-Seq Quantification library at BGI-Shenzhen (China) was used to obtain the transcriptome sequencings. And the RNA-seq raw data was assembled and analyzed by comparing with the translational region of the annotated DNA sequence in reference genome (GCA_001593245.1) using HISAT (Kim, Langmead & Salzberg, 2015). The DESeq. 2 package in R was used for the estimation of fold changes and other analysis (Love, Huber & Anders, 2014).

Statistical analyses

Statistical significance was determined by t test (two-tailed distribution with two-sample, equal variance) when directly comparing two conditions or a one-way analysis of variance (ANOVA) and Tukey post-test by pairwise comparisons.

Transcriptomic analysis

Based on the transcriptomic analysis, the deletion of SmpB mainly caused the changes in 20 biological pathways, including two-component system, sulfur metabolism, plant pathogenic bacteria interaction, and phenylalanine metabolism. Sulfur metabolism was the most influential on metabolic pathways (Fig. 1A).

In Aeromonas veronii C4, the H₂S synthesis pathway included the sulfate assimilation pathway, the organic pathway, and the 3-MST pathway. But compared with others, Aeromonas veronii C4 lacked cystathionine β-synthase (CBS) in the transsulfuration pathway and cysteine aminotranferase (CAT) in the 3MST pathway. The deletion of SmpB mainly up-regulated the transcription levels of cysN, cysD, cysC, cysH, cysJ, cysI and cbl (Fig. 1B). Also, these genes were mainly involved in sulfate assimilation pathway (Fig. 1C). The transcription of cysB did not change. Therefore, we speculated that SmpB deficiency was able to increase H₂S synthesis.

The production of H₂S was increased in the absence of SmpB under nutritionally deficient conditions

To figure out how sulfur metabolism was affected by SmpB deficiency, H₂S production was measured in rich and deficient nutrition conditions by Pb(Ac)₂ detection test. There is no difference between wild type (WT) and smpB deletion in a rich medium (LB medium) (Fig. 2A). Under the condition of nutritional deficiency (M9 medium), the smpB deletion produced less amount of H₂S in the early stage of growth, but it enhanced to synthesize H₂S in the stationary stage (Fig. 2B). The final H₂S yield of smpB deletion was significantly higher than that of WT. This suggested that the production of H₂S was increased in the absence of SmpB during auxotrophic conditions, especially predominant during the stationary phase of bacterial growth.

Cbl affects the generation of H₂S

Using both the classic Pb(Ac)₂ detection test and a fluorescent-based probe WSP5 (Zhang et al., 2020), we confirmed that, the production of H₂S in cbl deletion strain was significantly lower than that of WT in M9 medium (Figs. 3A and 3B). And the difference was offset when Cbl protein was complemented (Figs. 3A and 3B). All the results were consistent with the transcriptome data, implying that Cbl had a positive regulatory effect on the synthesis of H₂S under nutritional deficiency.

Cbl promotes the transcription of cysH

The amino acid sequence of cbl gene was highly homologous to the cysB family, and CysB was proved to binding with the promoter of sulfur reductase (CysJIH) as a transcription factor for regulation. Therefore, it was speculated that cbl regulated the transcription of genes such as cysH, cysJ and cysI. The relative expression of cysH decreased significantly compared WT with cbl deletion by RT-qPCR, while those of cysI, cysJ revealed no differences (Fig. 3C).

Furthermore, the fusion of the promoter cysJIH (PcysJIH) and eGFP was constructed as the indicator plasmid for the fluorescent measurement. When co-expressed with Cbl, the fluorescence value was extremely significantly higher than that of the strain containing only PcysJIH (Fig. 3D). Collectively, Cbl had a positive regulation on PcysJIH.

Cbl regulates downstream cysH by binding to the P cysJIH

To confirm whether Cbl bound to PcysJIH, the PcysJIH promoter sequence and Cbl coding sequence were cloned into pBXcmT and pTGR plasmids respectively, and then co-transformed into E. coli XL 1-Blue MRF’ reporter strain for bacterial one-hybrid experiment. Only the co-expressed strain and the positive control grew on the minimum medium supplemented with six mM 3-AT and streptomycin (Fig. 4A), suggesting that the strong interaction between PcysJIH and Cbl.

Next, PcysJIH was labelled with Fluorophore 6-carboxy-fluorescein (6-FAM) for electrophoretic mobility shift assay (EMSA). The Cbl protein reduced the mobility of the 6-FAM-PcysJIH DNA probe corresponding to the increased Cbl concentration with the enhanced Cbl–DNA complex (Fig. 4B). So Cbl protein was able to bind with PcysJIH following with the regulation of H₂S production.

Determination of the binding region of P cysJIH with Cbl protein

To confirm the binding region of PcysJIH with Cbl protein, we truncated the full length of PcysJIH to 150 bp and 50 bp upstream of transcriptional initiation which were named as PcysJIH¹⁵⁰ and PcysJIH⁵⁰. PcysJIH¹⁵⁰ was able to form a complex with Cbl protein, while PcysJIH⁵⁰ lost the binding ability (Fig. 4C). The result suggested that the regions between 50 bp and 150 bp upstream of transcriptional initiation in PcysJIH were responsible for the binding of Cbl.