Complete Genome Sequence And Annotation Of Bacteriophage MmP1, With Biological Characterization Of This Phage

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.Bacteriophages are viruses of bacteria. They are ubiquitous in nature, and are now recognized to be the most numerous entities in the biosphere. When the sensitive bacterium was infected by a phage, there will be two reactions, one is lytic reaction, the other is lysogenic reaction. The lysogenic bacteriophage can alter the biologic features of the host bacterium by its inheritance material, meanwhile, they can change host's inheritance material constituent in the process of the DNA recombination, integration, transposition and excision in host genome, which result in the microbial genetic diversity. Lytic phages can lyse and kill the host bacteria, and lysogenic phages also have a lytic period. So that phages are in the hope to be developed as anti-bacteria pharmaceutics. Deep-looking insight into the genetic background of phage is very important for no matter investigating the interaction of phage and its host bacteria, and revealing the biological diversity mechanism resulting from gene horizontal transfer caused by phages, or engineering remodeling of phage and phage-therapy. Now the genomic era has re-focussed researchers'attention onto the importance of bacteriophages. To date, there are totally 484 completed bacteriophage genomes available in public databases. In this dissertation, we are focused on the whole genome sequence and annotation of Morganella morganii phage 1 ( MmP1 ). The results are as follows:1. The biological characterization of MmP1. The phage MmP1 is a temperate phage of Morganella morganii. Electron microscopy revealed that the phage has an isometric head (about 55 nm in diameter) and a short tail, and it belongs to the Podoviridae family. The optimal multiplicity of infection (MOI) of MmP1 is about 0.1, and hosts infected by the phages with an MOI of 0.1 will produce highest phage titer at 3.5h after infection. One-step growth kinetics of the phage showed that the latent period is about 10 min, the rise period is about 40 min, and the average burst size is about 105 phage particles per an infected cell.2. The general characterization of MmP1 genome. The complete nucleotide sequence of phage MmP1 is 38,233 bp, and includes terminal repeats of 61bp, which is highly similar to T7-like phage. The G+C content of the MmP1 genome averages 46.54%. There are two regions with significantly higher G+C content than the average, among which two genes, gene 35 and gene 42, having the highest G+C content, may be acquired genes by horizontal transfer. Genes are all transcribed from the same DNA strand and occupy almost 90% of the nucleotide sequence, the same value found for T7. This efficiency is presumably the result of evolutionary packaging of the maximum amount of useful information into a DNA molecule whose length is limited by a virion of fixed size.3. The bioinformatics analysis of phage genome. 152 ORFs longer than 100bp are found out by ORF finder software at NCBI website. Predictions using the GeneMark and Softberry programs revealed that the phage genome has 49 predicted genes. No potential tRNA coding regions were found in the phage genome. The predicted genes are tightly organized with little space between them, and there are 10 overlapping gene regions with total 91 bp, ranging from 1 to 32 bp. At the DNA level, 12 of the phage-encoded putative proteins show homology to known proteins in the database using BLASTn program, and at the protein level, 28 of the phage-encoded putative proteins show homology to known proteins in the database using BLASTp program. The function of these 35 genes show homology to phage T7 and phage phiA1122. Thus , MmP1 is thought to be a member of the T7 group. Similar to the T3 and T7 genomes, MmP1 genes could be divided into three classes: I (early), II (middle) and III(late). Multiple sequence alignment was conducted among those homologous proteins using software CLUSTALX, and then the phylogenetic trees were drawn by phylip software.4. The phage capsid proteins and their coding genes. Phage particles were purified with CsCl gradient centrifugation, then the total phage capsid proteins were separated at least 7 bands by SDS-PAGE, respectively with molecular weight 150, 95, 82, 69, 62, 44 and 38kD. After transferred to a PVDF filter, Most bands were excised from the membrane and were subjected to N-terminal sequence with Edman degradation method. Only 38kD proteins could be sequenced and the N-terminal sequences were figured out. By comparative analysis of the N-terminal amino acid sequences of the proteins to the products coded by all the predicted genes, we got to know that gene 35 coded the 38kD proteins.5. The non-coding sequences of the genome. There are 37 intergenic regions ranging from 1 to 1321 bps. There are non-coding sequences with length of 140 bps and 204 bps respectively on the 5'-and 3'-terminus of the phage genome. 17 inverted repeats (palindrome sequence) and 1 long tandem repeats(61bp) are found in the genome. These repeat sequences are related to the transcription, recombination of DNA and genomic evolution. 5 possible terminators are found in the intergenic regions, and 5 possible promoter was predicted at the genomic positive strand.The genome data of phage MmP1 has been deposited in GenBank under the accession No. EU652770.