Screening Pseudomonas Aeruginosa Genes Required For Phage Infection And Genomic Analysis Of Phage C11

Phage infection can be affected by host bacteria gene expression level.The study on host generelated to phage infection will help to understand the interaction between phage and host bacteria,and revealthe corresponding molecular mechanism,providing a basis for the use of phage application.To construct the transposon Tn5G insertion mutant library,7 Pseudomonas aeruginosa phages O1,O2,K1,K2,K3,C10,C11 respectively were coculturedwith Pseudomonas aeruginosa strain PAK as a host.A total of 30 mutants were obtained in 7 transposon libraries.The corresponding phage adsorption rate of 25 mutantswas significantly reduced,displayingmutant genesmay be associated with the synthesis pathway of phage receptor;phage C11 adsorption rate of 5 mutantswas 100%,and the mutant genesmay be involved in other processes of phage infection independent of adsorption process.The mutant genes were found by the inverse PCR technique.For 25 mutants with the reduced adsorption rate,11 different genes were respectively blocked by transposon Tn5G,of which eight genes are involved in lipopolysaccharide LPS synthesis.Gene wbpO and wbpVparticipate inbiosynthetic pathways for sugar nucleotide precursors UDP-GalNAcA and UDP-QuiNAc used for synthesis of polysaccharide molecules;gene wbpR,wbpT and wbpLall encodea glycosyltransferase;gene PALES₁6971 encodes a protein with homology to E.coli polymerase O-antigen,and the degree of homology is 96%;gene PA5001 and PA5004both encode a glycosyltransferase,responsible for LPS core oligosaccharide assembly.Gene PA4367 encodes a protein BifA,exercisingthe hydrolysis of cyclic diguanylate.Involved in folate synthesis,gene PA0583 encodes 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase.Gene PA5181encodes molybdenum oxidoreductase.The results of complement function experiments showed that sixteencould make correspondingmutants restore the sensitivity to phage.For five mutants with constant adsorption rate,five genes were blocked.GeneBN889₀5221encodes a 75 aasmall protein;the proteinencodedby gene PA3808could providesulfurfor iron sulfur cluster assembly;gene PA1993 encodesa major facilitator superfamily?MFS?transporter;gene PA0243encodesa TeR regulator factor;gene PA1115 encodesa sulfatase.Phage C11 were respectively co-cultured with five mutants of constant adsorption rate.Mutant growth rate gradually declined withthe increasing of the concentration of phage.The results suggested that the phage genome DNA may be injected into the host cell,delayingthe growth of the host bacteria.Further by real-time quantitative PCR technology,it was found phage genome could be replicated within five mutants.The results showed five mutant genes were associated with the non-adsorbed phase during phage infection.Assembled genome size of C11 is 93010 bp.It was found that the genome 5 'end and 3' end have the same direct repeats,a length of 1173 bp.The revised genome size is 94109 bp,encoding 12 tRNA and 172 ORFs.Comparative genomic analysis showedthe genome sequence of phage C11 had a homology of 92.41%with phage PaP1,and amino acidof major capsid protein has a homology of 100%.Phage C11 is classified as a new member of PaP1 family.