Bioinformatic Prediction And Experimental Verification Of The Ely Genes In P. Aeruginosa Bacteriophages

Research background and objectives:The human genome project (HGP) has greatly promoted the development of thousands of organism genome projects since the year of 1990. Up to now, there are thousands of organism genomes have been submitted to GenBank, and there still hundreds of genome sequencing projects were under way. However, the functional genomics research has lagged far behind the genome sequences research and there only few genes were functionally verified by experiment. In addition, there are still lots of organism genomes sequences were totally not annotated, and needed to be verified. The main interests of the biologists have been transferred from the structural genomics to the functional genomics which focus on elucidating the function of genes.Previously three new bacteriophages (phages) of pseudomonas aeruginosa were isolated from hospital sewage in our laboratory and named as PaP1, PaP2 and PaP3 (PaP: pseudomonas aeruginosa phage) respectively. Then the whole genome of the temperate phage PaP2 and PaP3 were sequenced and submitted to GenBank. The partial genome of PaP1, a virulent phage, was also sequenced and kept in our lab. Since most predicated ORFs (open reading frames) of the three phages are functionally unknown, it is necessary for us to analyze and identify these unknown genes of the three phages.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. The bacterial cell wall is of great significance to the survival of bacteria, so if a kind of enzyme which has peptidoglycan lytic activity, was found it would be potentially used as a useful antibacterial agent in the future. In this research, we firstly and foremostly focused our work on the bacteriophage enzymes which can cause the lysis of the host cell wall. To the best of our knowledge, phages are the bacteria viruses which elaborate Endolysins to degrade the cell wall of their host. Endolysins (or lysins) are double-stranded DNA bacteriophage encoded peptidoglycan enzymes and they were produced in phage-infected bacterial cells at the end of the life cycle. Endolysins are also capable of degrading peptidoglycan when applied to the bacterial cell wall, and cause a rapid lysis of the bacterial cell. Because of their unique ability to cleave peptidoglycan in a generally species-specific manner, Endolysins represent a novel class of antibacterial agents. In general, Endolysins have no secretary signal sequence. They were unable to across the cytoplasmic membrane directly and thus accumulate in the cytoplasm, so another protein which was named as Holin is needed for the release of Endolysin. For the reason that bacteriophage Endolysins can be potentially used as the antibacterial agents, a deep insight into their enzymic function is of much helpful not only for investigating the interaction between phages and their host, but also for revealing the diversity of phages, and exploiting their antibacterial application for phage therapy in the future.Materials and Methods:Firstly, the P. aeruginosa bacteriophage genome sequences, which include all the ds-DNA phages that had been submitted to GenBank and all the genomes that had been sequenced by our lab, were collected. Secondly, the genome composition, sequences similarity of these phages was analyzed. In order to find all the endolysin and holin genes, open reading frame (ORF) predication, similarity alignments of the predicated gene sequences were then performed by bio-tools. Thirdly the predicted endolysin and holin genes were analyzed by the methods such as sequence similarity, phylogeny group classification tertiary structure predication and so on. Lastly, one predicated endolysin gene of bacteriophage PaP1 was cloned, expressed and purified. The purified recombinant protein was then verified subsequently.Results and discussion1. Analysis of the whole genome sequences of P. aeruginosa bacteriophages. There are total 20 P. aeruginosa ds-DNA bacteriophage genome sequences were collected. Of the 20 phages, genome sequences of 10 phages have been analyzed and annotated. The sequences of other 10 phages were unannotated till now. In this research, the genome composition, sequences similarity of the 20 phages was performed. Results reveal that with three exceptions, the P. aeruginosa phages show little sequence relatedness to each other. These three exceptions are PA7 and PA16 (more than 99% identity), 119X and PaP2 (93% identity), and SD1-M and phiKZ (more than 99% identity). We predicated that the evolution of these phages is evolved not only by gradual evolution, by which phages change their genomes by single or few base pairs gradually, but also by explosive evolution, by which phages change their genomes by large segments. The explosive evolution can also be explained by the theory of modular evolution, and the results of this evolution method make the genome of phages more chimeric like (or mosaic like), and there are more mutations of the terminal region than of the middle region in the phage genomes.2. Enzymatic function predication of the P. aeruginosa bacteriophage Endolysins. There are 12 endolysin genes had been reported or nominated in GenBank. Based on the ORF predication and homology alignments of the 20 phage genome sequences, 8 endolysin genes were newly found from 6 phages. These 8 endolysin genes are the ORF37273～37890 of bacteriophage F10, the ORF24127～26709 and ORF34978～35637F8 of bacteriophage F8, the ORF13111～13932 of bacteriophage M6, the ORF21764～22279 of bacteriophage Pa11, the ORF147382~148161 and ORF190416~197138 of bacteriophage SD1-M, the ORF25043～25603 of bacteriophage PaP1.3 classifications of the P. aeruginosa bacteriophage Endolysins: Results show that all the Endolysins of these phages can be divided into four categories which include muramidases, glucosaminidases, transglycosylases and unknown categories. Of the 8 newly found Endolysins, the ORF21764～22279 of bacteriophage pa11 is belong to muramidase; the ORF34978～35637 of phage F8 and the ORF37273～37890 of phage F10 are belong to glucosaminidase; the ORF13111～13932 of phage M6, the ORF24127~26706 of phage F8 and the two Endolysin (ORF147382~148161 and ORF190416~197138) of phage SD1-M are belong to transglycosylase; the ORF25043～25603 of phage PaP1 is belong to the unknown categories due to its strange structures and unknown enzymatic functions.4. Predication of holin genes of the P. aeruginosa bacteriophages. There are 3 holin genes had been reported or nominated in GenBank. Based on ORF predication and homology alignments of these phage genome sequences, 2 holin genes were newly found from 2 phages. The 2 newly found holins are the ORF36944~37276 of Bacteriophage F10 and the ORF12887~13219 of bacteriophage M6. All the Holin of P. aeruginosa bacteriophages have the common character of other bacteriophage Holin's such as there are at least two putative transmembrane domains separated by short linker sequences; holin genes are usually located upstream of the endolysin genes of the phage genome; the encoded amino acid sequences length is between 71 and 111 and there are gene overlap by several base pairs between the holin gene and the endolysin gene. Although the holin genes from most other phages were not find in this research, but there are some genes which also have one or more transmembrane domains of those phage genomes and whether or not these function unknown genes are the holin gene candidates is needed to be further studied.5. Molecular cloning, expression and purification of the bacteriophage PaP1 ely gene: The whole PaP1 genome DNA was used as template to amplify the ely gene. The amplification products were finally constructed into the expression vector pQE31 and transformed into the competent cells of E. coli M15 (pREP4) and induced by IPTG. The recombinant ELY-6H can be expressed at high level after 6 hours treated with 0.5mmol/L IPTG, and most of the fusion protein was of a soluble form. The soluble fractions of the six-His-tagged fusion proteins was purified by nickel-affinity chromatography using gravity-flow, and then repurified on a cation exchange resin and desalted on a molecular-sieve chromatography.6. Enzymatic activity assay of the ELY-6H. The peptidoglycan substrate of P. aeruginosa purification was obtained by boiling P. aeruginosa cells in SDS (4%, w/v). Enzymatic activity was detected by zymography assay, gel diffusion assay and turbidimetry assay respectively. The results of enzymatic assay show that both ELY-6H and hen egg white lysozyme can cause the lysis of the purified P. aeruginosa peptidoglycan. The results of gel diffusion assay show that the digest zone of the ELY-6H to the peptidoglycan substrate is more clearly against the background than that of hen egg white lysozyme, and the results of turbidimentry assay show that the absorption of the peptidoglycan solutions which treated with ELY-6H changes greatly than that treated with hen egg white lysozyme.7.Antibacterial activity assay of the ELY-6H. The inhibition activity assay of ELY-6H was applied exogenously to the cultures of P. aeruginosa, S. aureaus (ATCC 25923), Micrococcus lysoleikticus and E. coli JM109, respectively. Results show that the bacterial lawn growth on the agar of Gram-positive bacteria S. aureaus (ATCC 25923) was inhibited by both ELY-6H and hen egg white lysozyme. The inhibition of hen egg white lysozyme to the growth of S. aureaus (ATCC 25923) was stronger than that of ELY-6H. The inhabitation of ELY-6H to the lawn growth of the other bacteria (such as P. aeruginosa and E. coli BL21) was unobvious.Conclusions:8 new endolysin genes and 2 holin genes were found by the analysis of the 20 P. aeruginosa bacteriophage genomes. All the Endolysins of these phages can be divided into four categories including transglycosylases, muramidases, glucosaminidases and the unknown categories. The PaP1 endolysin gene was cloned and expressed in E. coli M15 (pREP4). The fusion protein ELY-6H was purified and its enzymatic activity was detected. Results show that the cell wall of P. aeruginosa can be degraded by the purified recombinant fusion protein ELY-6H. The antibacterial activity to its host was not found of the purified recombinant protein in this research, but it can inhibit the growth of Staphylococcus aureaus. All the results of this research will bring light to the identification the bacteriophage Endolysin and make it to be used as the potential antibacterial materials in the future.