A Study Of MicC Promoter In Escherichia Coli

ObjectivemicC is one of sRNA-coding genes in many gram-negative bacteria. InEschericchia coli （E.coli). micC is located between ybdK and ompN on thegenome, which is transcribed inversely to ybdk and ompN genes in direction. Thepromoter of micC is a fragment of227bp between micC and ompN, about whichmany detailed issues remained unclear, such as, whether it could transcribe both ofompN and micC in double directions. There is still great space for studying micCgene regulation expression. This study focused on the basic study of bidirectionaltranscription of micC promoter and the micC expression under various stimuli.Method(1) DH5ɑ, one of E.coli K-12derivative strains, was used in this study. Its genomeDNA was extracted and purified by TianGen Bacteria Genome Extracting Kit.Two DNA fragments were amplified by PCR with primers, and then inserteddirectly and inversely upstream of GUS (β-glucuronidase) reporter gene ofpJRS462plasmid. Finally, the GUS activity of DH5ɑwith different transformedrecombined vectors was tested.(2) On the base of positive results of experiment (1) above, the promoters linkedwith GUS gene was recombined into DH5ɑ genome by Red homologousrecombination system, and then the GUS activities of recombinants were tested toidentify the bidirectional transcription in advance.(3) We also tested the GUS activity of mutated DH5ɑ strains living in different stressenvironments, such as antibiotics present, high or low temperatures, oxidativestress， different osmotic pressures， acid and alkaline stimuli and so on.Results(1) The two amplified DNA fragments both could express gus gene in two directionson plasmid, and the GUS activities of test groups were more than two-fold thannegative control. (2) The GUS activity was greatly improved after the recombinants with only one copyof micC promoter linked gus gene stepped into stationary growth phase fromexponential phase.(3) The GUS activities were different when the recombined DH5ɑ strains lived indifferent adverse environments. The promoter is highly activated whenenvironmental temperature is raised and inactivated when the temperature islowered. The gus gene is impressed at high osmotic pressure. However, it is veryweak for pH to change the levels of gus expression.Conclusions1. The DNA fragment between ompN and micC was a bidirectional promoter.2. There was no great difference between the transcriptions of the same promoter inboth directions.3. The expression of micC gene was greatly improved when E.coli stepped intostationary growth phase from exponential phase.4. Temperature and osmotic pressure are the mainly stimuli in micC expression, micCpromoter was induced and impressed in both directions when the growth temperaturewas elevated and lowered，respectively.5. The micC promoter was impressed in both directions at high osmotic pressure.6. pH stimulus was ineffective to regulate micC expression. There might be otherstress-responsive pathways in response to pH stimulus.