Characteristics Of Meiothermus Phage MMP7' Genomics And Its Lysin MMPpgh

The lysin is a type of cell wall hydrolase that is expressed and released in the late stage of phage-infected host,which lyses bacteria by hydrolyzing cell wall peptidoglycan and releases progeny phage.Phage lysin efficiently lyse bacteria,making them promising to replace traditional antibiotic treatments,especially for multi-drug resistant pathogens.In this paper,a strain of Miothermus Myoviridae phage 7 was isolated from Yunnan Eryuan Hot Spring.The morphology was observed by transmission electron microscopy,and the genome was sequenced and analyzed.The MMPpgh gene was amplified,ligated and transformed by PCR to obtain the expression strain pET28a-MMPpgh/Rosetta.After induction and purification,the recombinant protein MMPpgh was obtained and its enzymatic properties were studied.Thereafter,the killing and cleavage effects of the lysin MMPpgh on part of Escherichia coli,Salmonella,Staphylococcus aureus,multidrug-resistant K.pneumoniae and probiotic strains were studied by double-layer plate and coating counting method;Simultaneous scanning electron microscopy?SEM?was used to observe the cleavage effect of MMPpgh on Escherichia coli,Salmonella,Staphylococcus aureus and Miothermus TG07.Finally,a mouse model of traumatic infection was established to study the role of MMPpgh in the treatment of multi-drug resistant Staphylococcus aureus in traumatic infection.Transmission electron microscopy showed that phage MMP7 is a typical Myoviridae phage.Genomic sequencing showed that the MMP7 genome is a double-stranded DNA virus with genome size of 32864 bp and GC content of 63.99%,with total of 53 ORFs.ORF28 is the cleavage MMPpgh gene,the size is 630 bp,the GC content is 60.32%.Homologous alignment analysis indicated that MMPpgh has high homology with peptidoglycan hydrolase from the Thermus phage,and the conserved domain indicates that MMPpgh belongs to the M23 peptidase family.After cleavage of the MMPpgh gene,a recombinant protein having a molecular weight of 24.53 KDa was obtained.The MMPpgh has an optimum enzyme activity temperature of 60?and an enzyme activity from 30 to 68?,which is higher than the optimum temperature of most of the reported phage lysin;It has the strongest activity at pH 7-8;the presence of metal ions Ca²⁺and Fe²⁺leads to a decrease in MMPpgh enzyme activity,while Zn²⁺and Mg²⁺activate the enzyme.At the concentration of enzyme concentration of 50?g/mL,MMPpgh has obvious antibacterial activity against Meiothermus TG07 compared with Kan⁺penicillin,and the relative inhibition rate is as high as 91.67%.The mortality rate of MMPpgh against Salmonella CMCC?B?50094 and Escherichia coli CMCC?B?44102 was90%,and the mortality rate of multi-drug resistant Klebsiella pneumoniae was 87%;The SEM observation showed that MMPpgh achieved its bactericidal function by cleavage of Escherichia coli,Salmonella,Staphylococcus aureus ATCC6538 and Meiothermus TG07.In the mouse traumatic infection model,the MMPpgh has a 50%killing effect on multi-drug resistant Staphylococcus aureus,and has a good healing effect on the wound.In summary,the lysin MMPpgh has high temperature catalytic activity and stability.At 37?,the growth of Gram-positive and Gram-negative bacteria can be inhibited.MMPpgh has a relatively broad cleavage spectrum and has obvious killing effect on multi-drug resistant Klebsiella pneumoniae and Staphylococcus aureus,and it is expected to become a new type of antibacterial substance.This study provides a new idea for the alternative technology of antibiotics.