Benzopyrene is a high molecular weight polycyclic aromatic hydrocarbon with highly recalcitrant and develops carcinogenic effects. CsrA is a conserved regulatory protein, which controls the translation and stability of its target transcripts, having negative or positive effects depending on the target mRNAs. It is known that Bacillus licheniformis M2-7 has the ability to grow and survive in concentrations of hydrocarbons as benzopyrene, which prompted in part by CsrA, as it occurs in the presence of gasoline. However, there are a few studies that reveal the genes involved in that process. In order to know the involved genes in the Bacillus licheniformis M2-7 degradation pathway, the plasmid pCAT-sp containing a mutation in the catEgen was constructed and used to transform B. licheniformis M2-7 and generate the CAT1 strain. We determined the capacity of the mutant B. licheniformis(CAT1) to grow in the presence of glucose and benzopyrene as a carbon source. We observed that CAT1 strain presented an increase in growth in the presence of glucose, but a statistically considerable decrease in the presence of benzopyrene with respect to the wild strain M2-7. Also we demonstrated that the Csr system regulates its expression positively since it was observed that the expression of the gene in the mutant strain LYA12 (M2-7 csrA:: Sp, SpR) decreased considerably with respect to the wild strain. This allowed to characterize the metabolic pathway that Bacillus licheniformis M2-7 implements in the presence of benzopyrene.
Research Article
The catE gene of Bacillus licheniformis M2-7 is essential for growth in Benzopyrene and its expression is regulated by the Csr system
https://doi.org/10.21203/rs.3.rs-2507976/v1
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Journal Publication
published 28 Apr, 2023
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Csr system
post-transcriptional regulation
Benzopyrene
Microbial growth
Benzopyrene (BaP) is a high molecular weight Polycyclic Aromatic Hydrocarbon (PAH) composed of five highly recalcitrant benzene rings (Habe and Omori, 2003, Fanali et al. 2018). When BaP is released into the environment, it is incorporated into the particles present in sediments, water, air and soil (Guevara-Luna et al. 2018, Qin et al. 2018). The degradation and/or biotransformation of PAHs depends on the pH, temperature, oxygen, microbial population, degree of acclimatization, accessibility of nutrients, chemical structure of the compound, properties of cell transport and chemical division in the growth medium (Huisman et al. 1996). The genes that encode the enzymes involved in the different metabolic steps of the degradation pathways of PAHs have been described in a wide variety of Gram-negative and some Gram-positive bacterial strains (Cébron et al. 2008). Functional PAH-RHD genes (encoding ring hydroxylating dioxygenases) were examined to understand BaP degradation mechanisms, as the nah, pah, arh, and phn genes in Gram-negative bacteria and the nid, nir, phd, and nar genes evolutionarily correlated in Gram-positive bacteria, which are responsible for the first step of hydroxylation of PAHs under aerobic conditions (Song et al. 2015). In the degradation of BaP by Mycobacterium vanbaalenii PYR-1, the dioxygenase of the K region and the nidA and nidB genes that encode the α and β subunits of terminal dioxygenase have been identified. It has been observed that the degradation of HAPs in Mycobacterium spp. isolated from sites contaminated with these compounds possess the genes nidA and nidB similar to those of M. vanbaalenii PYR-1 (Moody et al. 2004). Similarly, that in M. vanbaalenii PYR-1 strain, using the genome sequence and previously published genetic data for PYR-1, the genes / ORFs were assigned to each enzymatic step required for the pathway (Kim et al. 2007). In B. licheniformis M2-7, the genes catE, pobA and fabHB were proposed to be involved in the biotransformation of BaP (Rojas-Aparicio et al. 2018). The catE gene encodes the enzyme catechol 2,3-dioxygenase (EC 1.13.11.2, 2,3-CTDs, catechol: oxygen 2,3-oxidoreductase [decyclization]), wich catalyzes the incorporation of dioxygen into catechol and cleavage of the ring extradiol to form semialdehyde 2-hydroxuconate, having a key role in the bacterial degradation of aromatic molecules present in the environment (Ishida et al. 2002; Kita et al. 1999). Guevara-Luna et al. (2018) evaluated the enzymatic activity of the protein catechol 2, 3-dioxygenase in Bacillus licheniformis M2-7, and proposed that this enzyme incorporates molecular oxygen at carbons 9 and 10 of benzo [a] pyrene cis- 9, 10-dihydrodiol and is subsequently metabolized by the meta route until the generation of phthalic acid during the first 24 h of culture at 50 °C. The Csr (Carbon Storage Regulator) or Rsm (Repressor of Stationary phase Metabolites) system is among the most widely studied bacterial RNA regulatory systems (Romeo and Babitzke, 2018). The csrA gene and its rsmA ortholog, have been characterized in a wide variety of bacterial species (LeGrand et al. 2015), and encode CsrA / RsmA proteins, which are widely distributed regulators of bacterial gene expression that bind to target mRNAs near or overlapping the ribosome binding site (RBS) (Altegoer et al. 2016). Different biochemical and genetic approaches revealed that CsrA / RsmA interacts more frequently with highly conserved GGA motifs, altering their stability and translation (Wang et al. 2017). CsrA and RsmA mediate post-transcriptional regulation of a wide variety of processes, such as central carbon metabolism, secondary metabolites, motility, biofilm formation, regulates virulence genes of pathogens associated with plant diseases, invasion of mammalian cells, and quorum sensing (Romeo and Babitzke, 2018). Serrano-Ángel et al. (2020) constructed and observed that B. licheniformis carrying the mutant csrA gene showed ability to grow in the presence of gasoline than the wild strain, suggesting that the Csr system plays a fundamental plays a key role in the hydrocarbon degradation, however, it is nuclear how CsrA regulates that process in B. licheniformis M2-7 (Rojas-Aparicio et al. 2018; Serrano-Angel et al. 2020). Therefore, the objective of this work was to determine if the catE gene is essential for the growth of Bacillus licheniformis M2-7 in the presence of Benzoapiren and if the Csr system is involved in the regulation of gene expression, in order to describe the regulatory networks that activate this metabolic capacity.
Bacterial strains and growth conditions.
The strains, plasmids and oligonucleotides used in this study are listed in Table 1. The B. licheniformis M2-7 and E. coli DH5α strains were grown in Luria Bertani medium (LB) with a composition in g/L: peptone of casein 10, sodium chloride 10, yeast extract 5 for liquid medium and supplemented with 15 g/L of agar for solid medium) at 37°C. The concentration of spectinomycin (μg/mL) used for B. licheniformis CAT1, B. licheniformis LYA12 and E. coli DH5α was 200 and 100, respectively. Competent E. coli DH5a were prepared as in Sambrook protocol (Sambrook and Green 2012), whereas Bacillus licheniformis M2-7 were prepared according to the Hoffman’s methodology (Hoffman et al. 2010). For growth assays, minimum saline medium (MSM) supplemented with 2% glucose (MMS+GLUC) or 1mg/ml of benzoapyrene (MMS+BaP) was used (Guevara-Luna et al. 2018).
Construction of the plasmid pCAT/sp and the mutant strain of Bacillus licheniformis CAT1.
To inactivate the catE gene of Bacillus licheniformis M2-7, the plasmid pCAT/sp was constructed (Table 1), which was generated from the insertion of the spectinomycin cassette, (Obtained by cutting the plasmid pHP45-omega2 with the enzyme SmaI) in the pCAT8 plasmid linearized with the restriction enzyme EcoRV and ligated with the T4 DNA ligase (Thermoscientific ®). The generation of the pCAT/sp plasmid was confirmed by PCR using plasmid DNA and the oligonucleotides FSp19 and RSp19 and by cutting with the EcoRV enzyme. Once the plasmid pCAT/sp was generated, competent Bacillus licheniformis M2-7 cells were transformed and the transformant cells resistant to spectinomycin were selected. The deletion of the catE gene by double recombination was confirmed by PCR using genomic DNA of the recombinant strain with the oligonucleotides FSp19 and RSp19 and FC2-3d and RC2-3d. Amplification control was carried out using genomic DNA from the wild strain Bacillus licheniformis M2-7 and CAT1 and the oligonucleotides FphoA1 and RphobA1 for the amplification of the pobA gene.
Evaluation of the growth of B. licheniformis M2-7 and CAT1 in glucose and benzopyrene as a carbon source.
The growth kinetics of the M2-7 and CAT1 strains were performed in minimal saline medium supplemented with glucose 2% (MMS + GLUC) or minimal saline medium supplemented with benzoapyren 1 µg / mL (MMS + BaP). For this, 1 mL of a 12-hour growth culture was inoculated in LB broth (50 mL) at 37 °C / 200 rpm, the cell pellet was washed 3 times with sterile saline (0.9%) and resuspended in 10 mL of MMS to determine DO 600nm, adjusted to a final OD 600nm of 0.1 in 50 mL of medium. Finally, the volume to be inoculated was adjusted by the following equation:
The inoculated flasks were incubated at 37 ° C / 36 h / 200 rpm and the kinetic characterization was evaluated by taking 1 mL of the culture at 0, 3, 6, 12, 24 and 36 h. The quantification of total protein was carried out using the Bradford protocol, 1976. The growth kinetics were carried out in triplicate.
Expression of the catE gene by Real Time PCR.
To quantify the level of expression of the catE gene, the extraction and purification of the total RNA of the strain of Bacillus licheniformis M2-7 and LYA12 was carried out, following the protocol described by Damm et al., (2015) with some modifications. The contaminating DNA was treated with DNase I (Thermo Scientific®) and the RNA was stored at -70°C. For complementary DNA (cDNA) synthesis by reverse transcription, the RevertAid kit (Thermo Scientific®) was used. The reaction was performed as follows: 1 μg of total RNA, 20 pmol/μL of the reverse oligonucleotides (Table 1), 2 μL of dNTP's (10mM), 4 μL of 5X reaction buffer, 1 μL of RNase inhibitor (40 U) and 0.5 μL of reverse transcriptase. The reaction was incubated at 42°C for 1 h, inactivated at 70°C for 10 minutes and the cDNA was stored at -20°C. The obtained cDNA was used as a template for the qRT- PCR reaction, the oligonucleotides used are listed in Table 1. The rpoB gene was used as an endogenous control. The qRT-PCR was performed on a CFX96 Touch™. Each reaction contained: 1 μL of cDNA, 10 μL of Master Mix 2x (LightCycler® 480 SYBR Green I, from Roche) and 2.5 mM of oligonucleotide mixture (Table 1). Each reaction was performed in triplicate. The amplification conditions were performed as follows: denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 10 seconds and 60 °C for 60 seconds. The analysis of the relative expression was carried out using the 2-ΔΔCT method (Schmittgen and Livak 2008).
Statistical analysis.
All experiments were performed in triplicate at different times. The GraphPad Prism5 software was used to create graphics. For the Statistical Analysis ANOVA was performed, for independent samples (P≤0.05), and the Student t test with a value of p ≤0.05 was considered significant.
CatE is required to the growth of Bacillus licheniformis M2-7 in the presence of glucose and benzopyrene
The role of the catE gene in the degradation of BaP was evaluated during 36 h through growth kinetics. Figure 1 shows the comparison of the growth of the wild strain M2-7 and mutant CAT1(csrA-) in MMNS + GLU (20%) or MMS + BaP (1 µg / mL). Both strains showed a similar growth pattern during the first 3 h in the presence of glucose, however, from 6 to 36 h the wild strain M2-7 showed greater growth in comparison to the mutant strain CAT1 (Figure 1a). The growth of the strain M2-7 in the presence of BaP was significantly greater from 3 to 12 h of growth with respect to the mutant strain CAT1, which presented less growth from the first hours and declined at 6 hours of growth (Fig. 1b). These results corroborated that the CAT1 strain has a greater sensitivity to stress caused by BaP, compared to the wild strain, due to the inability to metabolize BaP, confirming that the catE gene has a fundamental role in the BaP degradation pathway.
CsrA regulates the expression catE gene in the presence of hydrocarbons with respect to the glucose carbon source in Bacillus licheniformis M2-7
The relative level of mRNA of the catE gene, of B. licheniformis strains M2-7 and LYA12 at times 6, 12 and 24 h of culture was evaluated by RT-qPCR, using the rpoB gene as internal control. It was observed that the expression of the catE gene in the LYA12 strain decreased with respect to the wild strain (Fig. 2). These results suggest that the role of CsrA in the growth of B. licheniformis M2-7 in the presence of BaP is through regulating the catE gene, thus participating in its positive regulation.
BaP is a highly recalcitrant PAH with a high molecular weight, which is incorporated into the particles present in water, air and soil (Fanali et al. 2018; Qin et al. 2018; Guevara-Luna et al. 2018). Up to this date, there are few studies that address the genes and regulators involved in the degradation of BaP, as well as the enzymatic and physiological responses that cause changes in the metabolic pathways that are crucial for using BaP as the sole source of carbon and energy (Sowada et al. 2014; Eskandari et al. 2017). It has been reported that the enzymatic activity of catechol 2,3-dioxygenase, product of the catE gene, is related to the catabolism of hydrocarbons in B. subtilis, and is essential in the growth and viability of this bacterium in the presence of catechol (Tam et al. 2006). In Bacillus licheniformis M2-7, a BaP transformation pathway was proposed, in accordance with the results recently reported by our group on the BaP biotransformation intermediary (Guevara-Luna et al. 2018) and the expression analysis of the catE, pobA and fabHB genes (Rojas-Aparicio et al. 2018). The reactions began with the incorporation of molecular oxygen at carbons 9 and 10 of BaP through the enzyme catechol 2, 3-dioxygenase, generating the benzo [a] pyrene cis-9,10-dihydrodiol (Guevara-Luna et al. 2018), which undergoes another dioxygenation through catechol 2,3-dioxygenase producing cis-4- (hydroxypyrene-8-yl) -2-oxobut-3-enoic acid via meta (Cerniglia 1992; Schneider et al. 1996). This is follow by the formation of 7,8-pyrene-dihydro-7-carboxylic acid (Schneider et al. 1996), through the degradation pathway of high molecular weight PAHs described by Mahaffey and collaborators (Mahaffey et al. 1988), which involves various enzymes and intermediate metabolites. There would be a second stage, where the enzymes 4-hydroxybenzoate 3-monooxygenase and ketoacyl-ACP synthase III, products of the pobA and fabHB genes, respectively, would participate (Rojas-Aparicio et al. 2018) in the formation of protocatecuate under the β-ketoadipate pathway to finally generate phthalic acid, until the intermediates can enter the tricarboxylic acid cycle (Sim et al. 2013). Given this background, the study was designed to determine the role of the catE gene, which codes for catechol 2,3-dioxygenase, one of the first enzymes involved in the BaP degradation pathway in B. licheniformis M2-7. We observed that inactivation of the catE gene diminished the growth of B. licheniformis strain CAT1 in BaP similarly to the mutant of B. subtilis 168 (yfiE) constructed by Tam et al. (2006) in the presence of catechol. This decrease in the growth of the strain, in comparison with the wild strain may be due to the fact that, as in B. subtilis, the enzyme catechol 2,3-dioxygen de B. licheniformis is a key enzyme in the meta cleavage pathway, in not being present, cell lysis occurs (Pi and Helmann 2018). Therefore, as proposed by Rojas-Aparicio et al. (2018), BaP is subject to degradation by catechol 2,3 dioxygenase and enters the target pathway, so that its participation would be involved in the first degradation phases of this compound (Bhatt et al. 2018), becoming a key enzyme in the BaP degradation pathway, leading it to the formation of phthalic acid as suggested by Guevara-Luna et al. (2018) and their subsequent entry into the β-ketoadipate pathway (Rojas-Aparicio. 2018). Among the proteins related to bacterial growth, mobility, virulence and response to stress is the highly conserved CsrA protein that affects the translation or stability of its target transcripts, which has been widely studied in the γ proteobacteria subfamily and very little in the genus Bacillus (Mukherjee et al. 2011, 2013). Its activity is mediated by sRNAs that have highly repeated sequences similar to those of the binding site in their transcripts, sequestering it and preventing binding to their target RNAs (Mukherjee, et al. 2013; Romeo and Babitzke 2018). It is interesting to observe that when comparing the expression levels of the catE gene in the LYA12 and M2-7 strains of Bacillus licheniformis, in the presence of BaP as a carbon source, the mutant strain (LYA12) presented an extremely low relative expression (Fig. 2), this is indicative that the post-transcriptional regulator CsrA exerts a positive regulation on the gene, when the strain is in the presence of PAHs. However, in B. subtilis the catDE operon is under negative regulation CatR, YodB and Fur, which would suggest that they are involved in the regulation of its homologue in the strain of B. licheniformis M2-7 and that CsrA would be putatively having a upstream interaction with these regulators, inhibiting them and causing overexpression in the catE gene (Porwal et al. 2009; Pi and Helmann 2018). However, additional studies are needed to corroborate these hypotheses. All of the above suggests that in addition to regulating the hag gene, it regulates genes necessary for PAH degradation pathways, directly influencing the metabolism of these compounds, adaptability and growth of the strain under adverse conditions (Diomandé et al. 2015; Mukherjee et al. 2013).
Possible model of regulation of CsrA on catE in Bacillus licheniformis M2-7
The results obtained in the present work allow proposing a possible model for the regulation produced by CsrA on the catE gene in the strain of B. licheniformis M2-7, where the CsrA protein binds to a different site to the Shine-Delgarno sequence of the Bicistronic mRNA of the catDE operon, generating a greater stability and increasing its translation. Other posibility is that CsrA could be regulate the mRNAs of the yodB, catR and fur genes, preventing their binding to DNA, allowing their transcription and subsequent translation. Likewise, CsrA would bind directly with the mRNA of these proteins, preventing their translation and thereby avoiding binding to the DNA of the catDE operon.
Acknowledgment
This work was supported by grants 18-PFA-IIDTTT-000089-L000-DF from SAGARPA. Yamilet Morales Blancas and José Daniel Reyna Teránthanks CONACyT for M.Sc. scholarships.
Statements and Declarations
The authors have no relevant financial or non-financial interests to disclose.
Author Contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Alejandro Bolaños Dircio], [José Alberto Hernandez Eligio], [Erubiel Toledo Hernández] and [Carlos Ortuño pineda]. The first draft of the manuscript was written by [Yanet Rmero Ramírez], [Jeiry Toribio Jiménez] and [Miguel Ángel Rodríguez Barrera] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.”
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Table 1. Strains, plasmids, enzymes and oligonucleotides used in this work.
|
|
Description |
Reference |
|
Strain |
|
|
|
Bacillus licheniformis M2-7 |
Filamentous colony, flat, beige, with a Ø of 0.7 mm, chemoorganotroph, oxidase (+), VP (+) and nitrate (+), Sps. |
Guevara-Luna et al., (2018) |
|
CAT1 |
Derived from strain M2-7 with inactivation in the catE gene by insertion of the fusion catE::Sp, SpR |
This work |
|
LYA12 |
Derived of the M2-7 with a blockage in the csrA gene by insertion of a spectinomicyn resistance cassette, (csrA::Sp, SpR) |
Serrano-Angel et al., (2020) |
|
Escherichia coli XL1-Blue |
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´ proAB lacIq Z∆M15 Tn10 (Tetr)] |
Stratagene |
|
Plasmids |
|
|
|
pJET1.2/blunt |
Plasmid of clone PCR products, ApR |
Thermo Fisher Scientific (2015) |
|
pCAT8 |
Plasmid derived of pJET1.2/blunt containing fragment of catE gene from B. licheniformis M2-7, ApR |
This work |
|
pHP45-omega2 |
Plasmid with the cassette de resistance to spectinomycin, SpR |
Fellay et al., (1987) |
|
pCAT/sp |
Plasmid derived of plasmid pCAT, containing the fusion catE::Sp, SpR, |
This work |
|
Enzymes |
|
|
|
EcoRV |
5 ´GAT ↓ ATC ´3 3 ´CTA ↑ TAG ´5 |
Thermo Fisher Scientific |
|
BamHI |
5 ´G ↓ GATCC ´3 3 ´CCTAG ↑ G ´5 |
Thermo Fisher Scientific |
|
Oligonucleotides |
(5´-------´3) |
|
|
FC2-3d |
GCT CGG ATA TGT CAA GC |
Rojas-Aparicio et al., (2018) |
|
RC2-3d |
GGT CTT TAA CAA ACC ATG C |
|
|
FSp19 |
GGA CAA ATT CTT CCA ACT G |
Serrano-Ángel et al., (2020) |
|
RSp19 |
GCA ATT TGG AGA ATG GCA |
|
|
FphoA1 |
AAG AGA CCG TCC GTG CAG |
This work |
|
RphobA1 |
GCG GAG GCA AGA CTC AGA |
|
|
HBMo Fw |
AGA AGC GCC TGT CAT CAC TT |
Rojas-Aparicio et al., (2018) |
|
HBMo Rev |
TCC TTC GGA ATG CTT TGT CT |
Rojas-Aparicio et al., (2018) |
|
rpoB Fw |
GGT TTT ACC GCG ACT GTC AT |
Rojas-Aparicio et al., (2018) |
|
rpoB Rev |
GAA CCG TAA CCG GCA ACT TA |
Rojas-Aparicio et al., (2018) |
|
ApR: resistant ampicillin, SpR: resistant spectinomycin, SpS::sensitive spectinomycin, oxidase (+): oxidase positive, VP (+): Voges Proskauer positive, nitrate (+): nitrate positive, Ø: diameter, recA1: mutation in the recombination, endA1: DNA specific endonuclease I mutation, gyrA96: DNA gyrase mutation, thi-1: mutation in thiamine metabolism, hsdR17:It allows cloning without cleavage of transformed DNA by endogenous restriction endonucleases. DNA prepared from this strain can be used to transform strain rK + E. coli, supE44: suppressor mutations, relA1: mutation in ppGpp synthase I, lac [F´ proAB lacIq Z∆M15 Tn10 (Tetr)]: mutations in proline metabolism, overproduction of the lac repressor, partial deletion of the β-d-galactosidase gene, resistance to tetracycline. |
||
No competing interests reported.