Construction Of Small Non-Coding RNA McaS Deletion Mutant Of Escherichia Coli O157:H7and Related Function

Escherichia coli0157:H7belonging to Enteroheamorrhagic E coli, EHEC, can cause Hemorrhagic Colitis, sometimes concurrently Hemolytic Urinary Syndrome. Over the years, because it has broken out for many times in the United States, Japan, Canada, Europe and other countries and regions, causing serious harm, EHEC0157:H7infection has become a global threat to human health public security problem and attracted worldwide attention.SRNA in bacteria has been shown to be important components of many control circuits.Bacteria under suitable growth conditions after entering into the host, can access to relevant information from the surroundings, which affects the expressions of bacterial virulence genes through certain pathways, and cause damage to the host. SRNA plays a very important role in the information transmission process. Studying the mechanism of action of bacteria sRNA and its effects on bacterial virulence and pathogenesis mechanism is a hot issue in today’s scientific community.This experiment is devoted to researching the effects of small RNA McaS on the hemorrhagic Escherichia coli0157:H7virulence.1The construction of mcaS gene mutant and its complemented strainBased on the original sequences of mcaS gene of MG511, we constructed a mcaS gene mutant using Red recombination system, and then we constructed its complemented strain using the mutant strain with the recombinant vector pACYC\84-mcaS. First, the chloramphenicol resistant (cat) gene flanked by homology extensions of mcaS gene was amplified by PCR. The PCR products were introduced into the wild type strain by electroporation, with the help of λ Red-mediated recombination system, leading to replacing the target gene with homology extensions connected with cat gene. The strains expressing chloramphenicol resistant gene were selected by chloramphenicol agar, and the chloramphenicol resistance gene was then eliminated by using a helper plasmid pCP20which can encode the Flp recombinase. Using this system, mcaS gene in chromosome of O157:H7was deleted successfully. O157:H7ΔmcaS mutant was obtained and further confirmed by PCR amplification and sequencing. Then we amplified the complemented gene using a template of wild type O157:H7, with the introduction of restriction sites BamHI and SalI, and integrated complemented gene into pACYC184which was then transformed into deletion mutant. The mutant and its complemented will be the base to study the function of sRNA McaS gene.2The research on the effects of its upstream and downstream genes expressions after missing gene mcaS using qRT-PCRThe research is to study the effects of its upstream and downstream genes expressions after missing gene mcaS using qRT-PCR. First, we acquired the RNA of wild type O157:H7, the mutant and its complemented by Trnzol method. With the help of reverse transcription Kit PrimeScript RT reagent Kit with gDNA Eraser, the genome DNA was eliminated from the RNA extraction in order to reversed transcript to cDNA.After PCR identification, reverse transcription product was used in the TaKaRa company SYBR Premix Ex Taqll fluorescence quantitative PCR kit for qRT-PCR reaction.According to the result, the gene RNA expressions of its upstream and downstream genes are not significantly changed after the mcaS mutant, which eliminates interference and improves the experimental rigor for subsequent experiments to explore the function of sRNA McaS.3The regulatory function of sRNA McaS in Escherichia coli O157:H7This test part is mainly to explore the regulatory function of McaS in Escherichia coli0157:H7from Bacterial adhesion process, motility process, quorum sensing process by comparing the differences in the wild type, mutant and its complemented.Meanwhile, we did the McaS small RNA homology analysis between different bacteria.(1) The influence of the motility process. We found that the movement ring of mutant was smaller than the wild strains by motility assays and the expression of the main control transcription factors of flhD in the mutant fell to56.2%compared with the wild type. As a result, we summarize that the small RNA McaS can affect the synthesis of the flagellum through increasing the expression of flagella flhD main regulation of transcription factor, thus affect bacteria0157:H7motility ability.(2) The influence of the adhesion process. Using Caco-2cells in vitro adhesion test, we found that the adhesion ability of mutant decreased to70.8%, while its complemented recovers the adhesive ability of wild strains. The expression of pgaA, a porin required for the export of the polysaccharide poly β-1,6-N-acetyl-D-glucosamine, decreased to70.4%by qRT-PCR. As a result, we conclude that McaS can affect the bacteria0157:H7adhesion ability by increasing the expression of gene pgaA.(3) The influence of the quorum sensing process. AI-2molecules can induce bioluminescence reaction in report strain BB170, thus researchers employ this method to measure expression level of AI-2. We used this method to detect whether O157:H7, the mutant and its complemented supernatant could induce bioluminescence. And after comparing with wild type, we found that the reaction of the mutant fell to59.9%and that the RNA expression of luxS fell to82.1%compared to the wild type. Therefore, small RNA McaS increasing luxS gene expression by RNA level, lead to decrease in the ability of influencing the quorum sensing ability after deleted.(4) Small RNA homology analysis between McaS in different bacteria.We randomly selected strains of E. coli K88ac, CE129,3030-2,1194,0157: H7and Muscovy ducks of E. coli meningitis, cloned mcaS gene sequences, and found that except four point mutations, the other area has high homology. Results showed that the McaS in pig source (K88ac,3030-2,1194), anthropogenic (0157:H7), and poultry (Muscovy ducks meningitis, CE129) E. coli had high homology, and were relatively conservative. Through a series of experiments, we summarize that the small RNA McaS in E. coli0157:H7plays an important role in regulating quorum sensing process,bacterial adhesion and motilty process. Therefore, McaS plays an important role in bacteria and host contact, adhesion, and the process of releasing the toxins through quorum sensing; Study of its function has great significance in both Pathogenic Escherichia coli0157:H7and other subtypes of E. coli for its conservative property and high homology. This test has provided a reference for the subsequent explore on small RNA McaS regulation function of the whole Escherichia coli.