Prediction, Cloning, Expression And Activity Determination Of Bacteriophage Mmp1 Endolysin In Morganella Morganii

Morganella morganii is a gram-negative rod commonly found in the environment and in the intestinal tracts of humans, mammals, and reptiles as normal flora. The genus Morganella belongs to the family Enterobacteriaceae. Despite its wide distribution, it is an uncommon cause of community-acquired infection and is most often encountered in postoperative and other nosocomial settings.As we know, Phages are the bacteria viruses which can produce endolysins to degrade the cell wall of their host. During their infection from outside or offspring release from inside of the host cells, phages must elaborate enzymes to destroy the cell wall of their bacterial hosts. Endolysins are double-stranded DNA bacteriophage-encoded peptidoglycan hydrolases produced in phage-infected bacterial cells toward the end of the lytic cycle, and Endolysins are also have the capable of degrading peptidoglycan when applied to the bacterial cell wall, cause a rapid lysis of the bacterial cell. Because of this ability, Endolysins represent a novel class of antibacterial agents.The ORF12 within the genome of Morganella morganii bacteriophage Mmp1 was predicted to encode endolysin. So structure analysis of the amino acid sequence of endolysin was first carried out. Subsequently we constructed its recombinational plasmid, expressed and purified the recombinant protein, finally we examined its bacteriostasis activity in vitro. The content and results of our study include the following aspects:1. Sequence and structure analysis of Morganella morganii phage MmP1 endolysin gene: The molecular phylogeny analysis of MmP1 endolysin and its homologous protein showed that endolysins of MmP1 and bacteriophage K11 were probably originated from the same ancestor sequence; the tertiary structure prediction showed that MmP1 endolysin possess active center consisting of His17,Tyr46,His122,Lys128及Cys130 which is similar to T7 lysozyme, the result indicated that MmP1 endolysin may be a N-acetylmuramyl-L-alanine amidases. 2. The cloning of full sequence of Morganella morganii phage MmP1 endolysin gene: The whole MmP1 genome DNA was used as template to amplify the full sequence of endolysin gene. We inserted the full sequence of phage MmP1 endolysin gene into prokaryotic expressing vector pQE-31,and succeeded in constructing the recombinant plasmid of phage MmP1 endolysin (MmP1-pQE31),which was confirmed by BamHⅠand sal I double-enzyme incision and DNA sequencing, then we know that its sequence and reading frame are correct.3. The expression and purification of full sequence of Morganella morganii phage MmP1 endolysin gene: The recombinant plasid was transformed into JM109 in order to obtain stably expressed clone. The recombinant protein is to be secretory proteins,and exist in supernatant.The recombinant protein in supernatant was purified by nickel-affinity chromatography using gravity-flow, and desalted on a molecular-sieve chromatography.4. Enzymatic activity assay of the recombinant protein: The peptidoglycan substrate of Morganella morganii purification was obtained by boiling Morganella morganii cells in SDS(4%,w/v).Enzymatic acivity was detected by zymographu assay and gel diffusion assay respectively. The results of enzymatic assay show both recombinant protein and hen egg white lysozyme can cause the lysis of the Morganella morganii peptidoglycan. The results of gel diffusion assay show that both of recombinant protein and hen egg white lysozyme have the degradation activity to the peptidoglycan substrate.5. Antibacterial activity assay of the recombinant protein: The inhibition activity assay of recombinant protein was applied exogenously to the cultures of S. aureaus, P. aeruginosa and Morganella morganii, respectively. Results show that the bacterial lawn growth on the agar of Gram-positive bacteria S. aureaus was inhibited by both recombinant protein and hen egg white lysozyme. The inhabitation of recombinant protein to the lawn growth of other bacteria(such as P. aeruginosa and Morganella morganii) was unobvious. The synergistic effect of MmP1 endolysin and antibiotics was investigated by determination of MIC, the result indicated that MmP1 endolysin could notably enhance the bacteriostasis activity of antibiotics against S. aureaus.To sum up, Sequence and structure analysis indicated that MmP1 endolysin may be a N-acetylmuramyl-L-alanine amidases. Subsequently the gene of Mmp1 endolysin was amplified, expressed and purified as a fusion protein to acquire its biologically active form. The results of enzymatic assay show the recombinant endolysion have the activity of degrading the peptidoglycan substrate;and the exogenous antibacterial activity assay showed that the recombinant endolysion can inhibit the growth of Gram-positive bacteria and enhance the bacteriostasis activity of antibiotics, which provides the very foundation for its further study and possible clinical application to the treatment of infectious diseases.