Contribution of sul to sulfonamide resistance
MIC tests confirmed that the sulfonamide resistance gene was generated successfully. The sulfonamide resistance gene expressed normally and donated sulfisoxazole resistance to the cells. E. coli BL21: pET23a-sul1, E. coli BL21: pET23a-sul2 and E. coli BL21: pET23a-sul3 had MIC values of 4 mg/mL for sulfisoxazole.
The fitness cost of sulfonamide resistance genes in Escherichia coli
The plasmid stability of four genetically engineered strains was analyzed under free selection pressure and sulfisoxazole inhibition concentration. The results showed no plasmid loss in strains E. coli BL21: pET23a-sul1, E. coli BL21: pET23a-sul2 and control strains E. coli BL21: pET23a, but the plasmid loss was 76% ± 4% after 10 days of subculture. No plasmid loss was observed in the control strain E. coli BL21: PET23a at the subinhibitory concentration (5 % MIC, 12.5 μg/ mL).
The growth curve of 4 genetically engineered strains was drawn under adequate nutrition and in the absence of drug selection pressure and other influencing factors. E. coli BL21: pET23a-Sul3 showed a reduction in growth ability; however, the other two strains showed no significant difference from the control strain (P 0.05). In the same way, E. coli BL21: PET23a-sul3 had the lowest microbiota diameter and its motor ability was decreased compared with the other three strains (P<0.05). E. coli BL21: PET23a-sul1, E. coli BL21: PET23a-sul2 and the control strain E. coli BL21: PET23a showed no significant difference in colony circle diameter (P 0.05). E. coli BL21: PET23A was used as the control, and three pairs of competitive tests were carried out using the method of combining resistance plate screening and PCR verification. An inevitable decrease was observed in adaptability by combining the above experimental results and comparing E. coli BL21: PET23a-sul3 with E. coli BL21: PET23a-sul1 and E. coli BL21: PET23a-sul2.
Quality control analysis of proteome
A total of 2446 proteins were identified by uniprot_Escherichia_coli_1124415_20180910 database. The number of proteins identified in 12 samples of 4 groups is shown in the table 1. The 2446 proteins identified by LC-MS/MS were compared quantitatively by MaxQuant software LFQ algorithm.
Bioinformatics analysis of differentially expressed proteins
Gene Ontology (GO) functional annotation analysis
In terms of cell composition, the protein changes were mainly located in the cell membrane and cell-matrix among all six groups. These changes are reflected primarily in catalytic activity and binding functions, which involved cells' metabolic process.
KEGG pathway analysis
In the paired comparison between the engineered strains carrying sul1, sul2 and control strains, proteins involved in the pathways were relatively concentrated. Apart from the glucose and nucleotide metabolisms, they were also involved in the ABC transporter and the two-component systems. However, in the paired comparison between E. coli BL21: PET23a-sul3 strains in the experimental and control groups, the metabolic pathways were relatively dispersed, mainly focused on glucose and nucleotide metabolism pathways.
Analysis of the variation trend of differentially expressed proteins
In E. coli BL21: pET23a-sul1 and E. coli BL21: pET23a-sul2 strains, proteins fliA, fliC and their upstream transcriptional activator fliZ in the two-component system were significantly upregulated. However, lrhA, the transcriptional inhibitor expressed by type I pili, was significantly downregulated.
The protein SUL1, which is involved in folic acid metabolism, was significantly upregulated in E. coli BL21: pET23a-sul1 strain. Besides, molybtrexate adenosine transferase, molybtrexate synthase catalyzed subunit and cyclopyranxate monophosphate synthase were upregulated by 1.7 and 1.6 times, respectively. 2-amino-4-hydroxy-6-hydroxymethyl dihydroteropterine diphosphate kinase was changed from zero to zero, and 4-amino-4-deoxy branched-acid lythase was altered from existing to non-existing. The protein expression of QueE (7-carboxy-7-deazaguanine synthase), which is involved in the pathway of folic acid metabolism, was downregulated 1.8 times in E. coli BL21: pET23a-sul2 strain. E. coli BL21: pET23a-sul3 strain significantly reduced the number of differential proteins involved in the pathway of folic acid metabolism, and significantly upregulated the expression of dihydrospteric acid synthase SUL3
In this study, obvious differences were observed in the ABC transporter system pathway by KEGG pathway analysis. The expressions of ATP-binding proteins ugpC, rbsA and gsiA, which are closely related to energy supply, were significantly increased in E. coli BL21: pET23a-sul1 or E. coli BL21: pET23a-sul2, but there was no significant change in E. coli BL21: pET23a-sul3.
Analysis of individual differentially expressed proteins showed that the differential expression ratio of the outer membrane pore protein ompD was the highest among all the proteins. In E. coli BL21: pET23a-sul3, the quantitative strength of ompD LFQ protein was 1.7 × 109, which was increased by 29.93 times compared with the control strain E. coli BL21: pET23a.
Transcriptional level analysis of differentially expressed proteins
Genes fliZ, fliA, fliC, lrhA (bacteria movement-related), sul1, sul2, sul3 (folic acid metabolic pathways related), moaB (molybdenum with poison adenosine transferase), moaE (molybdenum with poison synthetase catalytic subunit), moaC (pyran ring poison monosodium phosphate synthetase), APT94_19870 (dihydrogen pteridine diphosphate kinase), C5P43_33360, ugpC, rbsA, gsiA (ATP binding protein related) and ompD (outer membrane channel proteins), and other 16 genes were chosen for Real-time PCR analysis. Based on 16s rDNA internal genes, 2-ΔΔCt relative quantitative method was used for comparative quantitative analysis of target genes.
The comparative quantitative analysis results showed that the variation trend of target gene mRNA level expression obtained by real-time PCR analysis was the same as that obtained by Label-free proteomic analysis (Figure 6).