Coding
Part:BBa_K3519000
Designed by: Omkar Mohapatra Group: iGEM20_IISER-Tirupati_India (2020-10-07)
Revision as of 06:15, 24 October 2020 by Omkarmohapatra123 (Talk | contribs)
sulX
This part codes for the gene sulX, sulfonamide monooxygenase. The genes sulX and sulR are a two-component flavin-dependent monooxygenase system present in Microbacterium sp. CJ77 and were first described by Kim et al., 2019. The expression of these genes in E. coli has been shown to degrade sulfonamides in the media.
Usage and Biology
1.1 Isolation and characterization:
The microbacterium sp. CJ77, a bacterial strain capable of degrading sulfonamide was isolated from a sulfonamide contaminated site. Isolation was carried out using sulfathiazole (100 μg/mL) as a sole carbon source in the minimal medium at 30 °C for four weeks. Isolate identification was done by carrying out PCR amplification of the 16S rRNA gene followed by sequencing[1]. The 16S rRNA gene was aligned with the nearest sequences obtained from the database of the EzBioCloud server.
It was seen that the isolate was able to utilize various types of sulfonamides as a carbon source for its growth. When the expression levels of proteins from cells grown on glucose (control) and sulfonamides were compared by proteome analysis, several genes in a gene cluster were highly up-regulated in cultures containing each sulfonamide as a carbon source (Fig 1). The gene cluster was found to contain homologs of sulfonamide monooxygenase (SadA) and flavin reductase (SadC) which were previously identified to be responsible for the initial cleavage of sulfonamides in Microbacterium sp. BR1[2].
Fig. 1. Genetic organization and expression profiles of a gene cluster for sulfonamide degradation. The locations of transposases and integrases are shown as red bars in the genome of Microbacterium sp. CJ77. Expression levels are displayed by normalized spectral counts below the genetic map of the cluster. GLU, SNM, SMX, and SMZ indicate glucose, sulfanilamide, sulfamethoxazole, and sulfamethazine, respectively, used as a sole carbon source.
It was seen that the isolate was able to utilize various types of sulfonamides as a carbon source for its growth. When the expression levels of proteins from cells grown on glucose (control) and sulfonamides were compared by proteome analysis, several genes in a gene cluster were highly up-regulated in cultures containing each sulfonamide as a carbon source (Fig 1). The gene cluster was found to contain homologs of sulfonamide monooxygenase (SadA) and flavin reductase (SadC) which were previously identified to be responsible for the initial cleavage of sulfonamides in Microbacterium sp. BR1[2].
Fig. 1. Genetic organization and expression profiles of a gene cluster for sulfonamide degradation. The locations of transposases and integrases are shown as red bars in the genome of Microbacterium sp. CJ77. Expression levels are displayed by normalized spectral counts below the genetic map of the cluster. GLU, SNM, SMX, and SMZ indicate glucose, sulfanilamide, sulfamethoxazole, and sulfamethazine, respectively, used as a sole carbon source.
1.2. Sulfonamide degradation assay:
To examine antibiotic degradation, the culture was set in 50ml minimal media at 30 °C and cell-free cultures were used as control. To monitor adsorption of sulfonamides heat-killed cells were used. The culture was centrifuged at 13000g for 20min and the supernatant obtained was separated and analyzed using HPLC.
Crude extract assay
Sulfonamide-grown cells were disrupted by sonication in 50 mM Tris-HCl buffer (pH 7.5). Cell debris was removed by centrifugation at 13,000 ×g at 4 °C for 1 h and filtered (0.2 μm) to obtain cell-free protein extracts. The reaction mixture contained 250 μg of protein, 200 μM sulfonamide, 1 mM NADH, and 5 μM FMN in 1 mL of 50 mM Tris-HCl buffer (pH 7.5) and incubated at 30 °C. The reaction was stopped by adding 12% phosphoric acid. Samples taken from the reaction mixture were analyzed by HPLC. To detect metabolites in the reaction, the reaction mixture was extracted with an equal volume of ethyl acetate three times. The ethyl acetate extract was evaporated to dryness under nitrogen gas. The residue was dissolved in methanol for HPLC analysis.
Activity assay for recombinant strains
E. coli BL21(DE3) harboring appropriate plasmid constructs were grown at 37 °C overnight in 5 mL of LB medium supplemented with 100 mg/L of ampicillin. The overnight culture was transferred into 50 mL of fresh LB medium containing ampicillin and incubated at 30 °C. Isopropyl-β-D- thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM when the cells reached an OD600 of 0.4–0.6 and the culture was induced for 3 h. Sulfamethoxazole was added to the culture at a final concentration of 0.5 mM and the culture was further incubated for 16 h. The culture supernatant was taken at intervals for HPLC analysis.
Cloning, expression, and purification of monooxygenase and flavin reductases
Cloning and expression of genes were conducted using pET28-(a) vector for single gene expression and pETDuet-1 vector for co-expression of two genes in E. coli BL21 (DE3). Using appropriate primers Monooxygenase and flavin reductase genes were PCR amplified.. E. coli BL21(DE3) harboring appropriate plasmid constructs were cultivated at 37 °C. At an OD600 of 0.5, cultures were induced with IPTG at a final concentration of 0.1 mM, and then further incubated at 37 °C for 3 h or at 20 °C for 12 h as required. Cells were harvested and re-suspended in 20 mM Tris-HCl buffer (pH 7.5). Cell-free protein extracts were obtained as described previously. The recombinant His- tagged proteins were purified using His GraviTrap column.
Enzyme assay for kinetic studies
Sulfonamide degradation activity by purified enzymes was determined by HPLC analysis as described above. The reaction mixture contained 50 μM sulfamethazine, 0.75 μM sulfonamide monooxygenase, 0.5 μM, flavin reductase, 1.0 μM FMN and 400 μM NADH in 50mM Tris-HCl buffer (pH 7.5) and was incubated at 25 °C. The reaction was stopped by adding 12% phosphoric acid and sulfamethazine and its metabolites were quantified at every 1 min for 5 min. ( Fig 2). Steady state kinetic parameters were obtained by fitting initial velocity data to the standard Michaelis-Menten equation. The initial velocities for various concentrations of sulfonamides were obtained with sulfonamide monooxygenase (0.5, 5.0 and 2.5 μM), the equivalent amounts of flavin reductase and FMN, and 200 μM NADH at 25 °C for 1 min.(Fig 3).
In the presence of NADH and flavin cofactor (FMN or FAD), the heterologously expressed and purified sulfonamide monooxygenase and flavin reductase of strain CJ77 resulted in the rapid degradation of sulfonamides with concomitant production of the dead-end products and 4-aminophenol in stoichiometric manners.
Fig. 2. UV–visible spectrum during the sulfamethazine degradation by purified proteins. The reaction mixture contained 50 μM sulfamethazine, 2 μM sulfonamide monooxygenase, 0.1 μM flavin reductase,2.0 μM FMN, and 200 μM NADH in 50mM Tris-HCl buffer (pH 7.5) and was incubated at 25 °C. Scan was taken at every 30 s for 5 min[3].
Fig. 3. Kinetics of the sulfonamide degradation reaction. The reaction mixture contained 50 μM sulfamethazine, 0.75 μM sulfonamide monooxygenase, 0.5 μM flavin reductase, 1.0 μM FMN, and 400 μM NADH in 50mM Tris-HCl buffer (pH 7.5) and incubated at 25 °C. Sulfamethazine (closed circle), 2‑amino‑4,6‑dimethylpyrimidine (open square) and 4‑aminophenol (open triangle) were analyzed over time[3].
Crude extract assay
Sulfonamide-grown cells were disrupted by sonication in 50 mM Tris-HCl buffer (pH 7.5). Cell debris was removed by centrifugation at 13,000 ×g at 4 °C for 1 h and filtered (0.2 μm) to obtain cell-free protein extracts. The reaction mixture contained 250 μg of protein, 200 μM sulfonamide, 1 mM NADH, and 5 μM FMN in 1 mL of 50 mM Tris-HCl buffer (pH 7.5) and incubated at 30 °C. The reaction was stopped by adding 12% phosphoric acid. Samples taken from the reaction mixture were analyzed by HPLC. To detect metabolites in the reaction, the reaction mixture was extracted with an equal volume of ethyl acetate three times. The ethyl acetate extract was evaporated to dryness under nitrogen gas. The residue was dissolved in methanol for HPLC analysis.
Activity assay for recombinant strains
E. coli BL21(DE3) harboring appropriate plasmid constructs were grown at 37 °C overnight in 5 mL of LB medium supplemented with 100 mg/L of ampicillin. The overnight culture was transferred into 50 mL of fresh LB medium containing ampicillin and incubated at 30 °C. Isopropyl-β-D- thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM when the cells reached an OD600 of 0.4–0.6 and the culture was induced for 3 h. Sulfamethoxazole was added to the culture at a final concentration of 0.5 mM and the culture was further incubated for 16 h. The culture supernatant was taken at intervals for HPLC analysis.
Cloning, expression, and purification of monooxygenase and flavin reductases
Cloning and expression of genes were conducted using pET28-(a) vector for single gene expression and pETDuet-1 vector for co-expression of two genes in E. coli BL21 (DE3). Using appropriate primers Monooxygenase and flavin reductase genes were PCR amplified.. E. coli BL21(DE3) harboring appropriate plasmid constructs were cultivated at 37 °C. At an OD600 of 0.5, cultures were induced with IPTG at a final concentration of 0.1 mM, and then further incubated at 37 °C for 3 h or at 20 °C for 12 h as required. Cells were harvested and re-suspended in 20 mM Tris-HCl buffer (pH 7.5). Cell-free protein extracts were obtained as described previously. The recombinant His- tagged proteins were purified using His GraviTrap column.
Enzyme assay for kinetic studies
Sulfonamide degradation activity by purified enzymes was determined by HPLC analysis as described above. The reaction mixture contained 50 μM sulfamethazine, 0.75 μM sulfonamide monooxygenase, 0.5 μM, flavin reductase, 1.0 μM FMN and 400 μM NADH in 50mM Tris-HCl buffer (pH 7.5) and was incubated at 25 °C. The reaction was stopped by adding 12% phosphoric acid and sulfamethazine and its metabolites were quantified at every 1 min for 5 min. ( Fig 2). Steady state kinetic parameters were obtained by fitting initial velocity data to the standard Michaelis-Menten equation. The initial velocities for various concentrations of sulfonamides were obtained with sulfonamide monooxygenase (0.5, 5.0 and 2.5 μM), the equivalent amounts of flavin reductase and FMN, and 200 μM NADH at 25 °C for 1 min.(Fig 3).
In the presence of NADH and flavin cofactor (FMN or FAD), the heterologously expressed and purified sulfonamide monooxygenase and flavin reductase of strain CJ77 resulted in the rapid degradation of sulfonamides with concomitant production of the dead-end products and 4-aminophenol in stoichiometric manners.
Fig. 2. UV–visible spectrum during the sulfamethazine degradation by purified proteins. The reaction mixture contained 50 μM sulfamethazine, 2 μM sulfonamide monooxygenase, 0.1 μM flavin reductase,2.0 μM FMN, and 200 μM NADH in 50mM Tris-HCl buffer (pH 7.5) and was incubated at 25 °C. Scan was taken at every 30 s for 5 min[3].
Fig. 3. Kinetics of the sulfonamide degradation reaction. The reaction mixture contained 50 μM sulfamethazine, 0.75 μM sulfonamide monooxygenase, 0.5 μM flavin reductase, 1.0 μM FMN, and 400 μM NADH in 50mM Tris-HCl buffer (pH 7.5) and incubated at 25 °C. Sulfamethazine (closed circle), 2‑amino‑4,6‑dimethylpyrimidine (open square) and 4‑aminophenol (open triangle) were analyzed over time[3].
1.3. Antibiotic susceptibility test:
The minimum inhibitory concentration (MIC) was determined by the broth microdilution method. LB broth medium containing 0.1 mM IPTG was used for susceptibility testing. The susceptibility of E. coli BL21(DE3) harboring appropriate plasmid constructs was tested against sulfamethoxazole. For disk diffusion assay, E. coli BL21(DE3) harboring appropriate plasmid constructs was grown at 37 °C overnight in 5 mL of LB medium supplemented with 100mg/L of ampicillin. The overnight culture was transferred into fresh medium containing ampicillin and incubated at 37 °C up to an OD600 = 0.4. The bacterial suspension was spread on LB agar supplemented with ampicillin and IPTG (0.2 mM final concentration). Filter paper disks with sulfamethoxazole (20 μg) were overlaid onto the E. coli lawn and plates incubated at 30 °C overnight.
Fig. 4. Sulfonamide-cleavage activity associated with resistance of E. coli cells where sulfonamide monooxygenase and flavin reductase were heterologously expressed. The activity was assayed using cells of E. coli strains harboring the plasmid pET-Duet derivatives. Susceptibility of E. coli cells against sulfamethoxazole was tested by broth dilution assay and disk-diffusion assay[3].
Genes encoding sulfonamide monooxygenase and flavin reductase were introduced into a sulfamethoxazole-susceptible E. coli strain to examine the role of these genes. When antibiotic susceptibility was tested, the following observations were made (Fig 4).
In conclusion, the two-component system consisting of sulfonamide monooxygenase and flavin reductase is required for both sulfonamide degradation activity and novel resistance mechanism via drug inactivation. (This monooxygenase responsible for sulfonamide resistance was named SulX in analogy to TetX by the researchers, to distinguish from the previously known sulfonamide resistance genes)
Fig. 4. Sulfonamide-cleavage activity associated with resistance of E. coli cells where sulfonamide monooxygenase and flavin reductase were heterologously expressed. The activity was assayed using cells of E. coli strains harboring the plasmid pET-Duet derivatives. Susceptibility of E. coli cells against sulfamethoxazole was tested by broth dilution assay and disk-diffusion assay[3].
Genes encoding sulfonamide monooxygenase and flavin reductase were introduced into a sulfamethoxazole-susceptible E. coli strain to examine the role of these genes. When antibiotic susceptibility was tested, the following observations were made (Fig 4).
- Compared to control cells E. coli cells harboring both of two-component genes showed a significant increase in resistance.
- E. coli cells harboring only the monooxygenase gene also displayed a lower level of resistance, suggesting that indigenous flavin reductases present in E. coli contribute to the slight increase in resistance. But both genes were required for the acquisition of resistance to the drugs.
In conclusion, the two-component system consisting of sulfonamide monooxygenase and flavin reductase is required for both sulfonamide degradation activity and novel resistance mechanism via drug inactivation. (This monooxygenase responsible for sulfonamide resistance was named SulX in analogy to TetX by the researchers, to distinguish from the previously known sulfonamide resistance genes)
1.3. Future experiments:
Due to the imposed lockdowns in relation to the COVID-19 only preliminary experiments could be conducted for our project. For designing these preliminary experiments and their protocols the above mentioned data was taken as reference. In one of the experiments we conducted, excreta samples were analysed using HPLC-MS to detect sulfonamides. For this an approach similar to the above mentioned was used. Also, for designing future experiments of our project all the above collected information from literature was used as the basis.
References:
References:
- Yoon, S. H., Ha, S. M., Kwon, S., Lim, J., Kim, Y., Seo, H., & Chun, J. (2017). Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. International journal of systematic and evolutionary microbiology, 67(5), 1613–1617. https://doi.org/10.1099/ijsem.0.001755
- Ricken, B., Kolvenbach, B.A., Bergesch, C. et al. (2017). FMNH2-dependent monooxygenases initiate catabolism of sulfonamides in Microbacterium sp. strain BR1 subsisting on sulfonamide antibiotics. Sci Rep 7, 15783 https://doi.org/10.1038/s41598-017-16132-8
- Kim, D. W., Thawng, C. N., Lee, K., Wellington, E., & Cha, C. J. (2019). A novel sulfonamide resistance mechanism by two-component flavin-dependent monooxygenase system in sulfonamide-degrading actinobacteria. Environment international, 127, 206–215. https://doi.org/10.1016/j.envint.2019.03.046
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 23
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 448
Illegal AgeI site found at 576 - 1000COMPATIBLE WITH RFC[1000]
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Categories
Parameters
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