Studies On The Lactobacillus Bacteriophage And Its Lysis Mechanism

Lactic acid bacteria (LAB) are industrially important microorganisms. Considering the GRAS status (generaly regarded as safe), LAB has a long history of application and consumption in the yoghourt and cheese. They cause rapid acidification of the raw material through the production of lactic acid. Also, their production of aroma compounds, sweeteners, vitamin, exopolysaccharides and bactcriocins contribute to the microbial safety or offer organoleptic, nutritional or health advantages. However, in the non-sterile environment of raw or heat-treated milk, the added LAB cells will come into contact with bacteriophages found in milk. The breakout of a specific LAB bacteriophage will delay or even halt the milk fermentation process leading to low-quality products and huge economic losses. Alternatively, bacteriophage is also demonstrated a favorable side. The lytic proteins produced by bacteriophages could degrade pcptidoglycan rapidly with the cell wall hydrolase activities. Thus, bacteriophage endolysin has received renewed attention as a novel class of antimicrobial agents for biocontrol of pathogenic antibiotic resistant bacteria. Futhermore, the cell lysis is effective to facilitate the release of bacterial intracellular flavor generating enzymes and aroma forming compounds during cheese ripening. Therefore, the in-depth study of genetic information and lysis mechanisms will promote the control as well as usage of bacteriophage lysis capacity. In this thesis, a survey is carried out on the presence of prophages in Laclohacillus stains, followed by whole-genome sequencing of two Lactohacillus delhrueckii subsp. hulguricus bacteriophages and construction of a novel antibacterial agent, as well as the lysis mechanism of Lb. fermentum temperate bacteriophage φpPYB5. The detailed contents and results are listed as follows: 1. Widely distributed lysogeny in Lactobacillus strainsLactobacillus is an essential starter strain in dairy fermentation, in which the lysogeny phenomenon is widely distributed. Prophages are not silent units within bacterial chromosomes. In fact, they may be induced by diverse agents, and frequently present insertions, deletions and rearrangements. Moreover, DNA of defective prophages is able to recombine and generate virulent phages infective for the host strain. Because of the potential risks, a set of20commercial and traditional dairy isolated Lactobacillus strains were used to investigate the occurrence of lysogeny on the genomes. All the strains were induced with mitomycin C, allowing direct recovering of prophages in6cases which was evidenced for60%of the strains analyzed. All of four traditional strains as well as2out of6commercial strains studied contained prophages, indicating the potential risks of their use at industrial scale. According to BamHI-restriction profiles of phage DNA, two of them,φ2008and φ2028, were the same kind of prophage, which was confirmed by SDS-PAGE profiles. Lysogenic strains were also induced with other agents susceptible of acting as an inducer in a dairy plant, including UV light, hydrogen peroxide, NaCl, lactate and nisin. The result showed that UV light was able to induce prophage release for all the strains tested, while no prophage induction was perceived on any of the assayed strains by the other five agents.2. Genome sequence of Lactobacillus delbrueckii phagesDue to their economical importance, LAB bacteriophages become the most thoroughly studied phage group in the database. At present, about100complete genomic sequences of LAB phages are publicly available, but only four of them represent Lb. delbrueckii phages. More information is clearly needed to extend the knowledge of Lb. delbrueckii phages. In this study, the genome sequences of two Lb. delbrueckii phages were analyzed in detail, and the evolution, genetic diversity as well as horizontal gene transfer were explored. The double-stranded DNA genomes of Lb. delbrueckii virulent phage phiLdb and temperate phage phiJB were36,677bp and36.969bp, with41.9%and47.7%G+C content, respectively. The phages phiLdb and LL-Ku, c5showed a minimum nucleotide sequence identity of90%. A total of52possible open reading frames (ORFs) were identified in the phiLdb genome, of which37ORFs had putative functions. The phiJB genome consisted of46putative ORFs;52%showed siginaficant identity with phage LL-H, while the others showed similarities to proteins from Gram-positive bacteria and phages. According to the comparative genomic analysis, phage phiLdb and phiJB genomes showed functionally related genes clustered together in a genome structure composed of modules for DNA replication, DNA packaging, head and tail morphogenesis, and lysis. In phiJB genome, the DNA replication module had littel similarities with other Lb. delbrueckii phages. Together with its higher G+C content, it could be deduced that the phage phiJB was ongoing fast evolution and the diversification of Lb. delbrueckii phages was mainly due to deletions, insertions and recombination.3. A novel antibacterial using the endolysin of phage phiLdbThe endolysin of phage phiLdb was used to develop a novel antibacterial for the biocontrol of Staphylococcus aureus and Listeria monocytogenes. After the endolysin Lysdb from Lb. delbrueckii virulent phage was cloned and expressed in Eseherichia coli, the recombinant protein was purified and its wide lytic spectrum was analyzed, when Lysdb was added externally for10h, the lysis efficiency of S. aureus ATCC33591, S. aureus ATCC27217, L. monocytogenes ATCC19114and L. monocytogenes ATCC19115were42.5%,40.2%,69.9%and68.5%, respectively. In addition, a chimeric endolysin consisting of the Usp45signal peptide fused to Lysdb was produced to develop a recombinant strain with endolysin secreted. The results showed that in the co-culture system, the Lysdb which was produced and translocated to the medium by the recombinant strain Lb. casei/pBLysdb could effectively reduce the number of the aforesaid pathogens, while the number of Lb. casei was not affected. 4. Functional analysis of the N-terminal region of endolysin Lyb5Lactobacillus fermentum temperate bacteriophage φPYB5utilizes a typical holin-endolysin system to achieve cell lysis. Previous result showed that sole expression of endolysin Lyb5caused Escherichia coli lysis slowly, and therefore we supposed Lyb5passed cytoplasmic membrane to peptidoglycan in a holin-independent way. In this work, the signal peptide (SP) motif at the N-terminal region of Lyb5was identified as a signal-anchor-release (SAR) domain both in Gram-negative and Gram-positive bacteria. In E. coli, experimental evidences indicated that the SPLyb5motif was not cleaved as a typical SP and delivered Lyb5to peptidoglycan through host Sec machinery. In addition, the SPLyb5motif was necessary for the transmembrane movement of Lyb5, as SPLyb5deletion converted Lyb5into a nontoxic endolysin in cytoplasm. Subcellular localization also confirmed the presence of Lyb5in cytoplasm, cytoplasmic membrane and periplasm with the same molecular mass. In Lactococcus lactis, we observed that Lyb5expression was not able to lyse the host cells, which resulted from50%of Lyb5protein restrained in cytoplasmic membrane and less than5%secreted into extracellular medium. The NucleaseB fusion confirmed that the SPLyb5motif was sufficient to drive protein to extracytoplasm in E. coli as well as L. lactis. These results strongly demonstrated the SPLyb5motif shared obvious characteristics with SAR domain, which was firstly identified in lactic acid bacteria bacteriophage endolysins.5. Topological structure of Hyb5and interaction model of Hyb5-Lyb5Analysis of the amino acid sequence of phage φpPYB5holin Hyb5showed that the presence of a single transmembrane domain (TMD) from residues10to29in the N terminus with a highly charged and hydrophilic C-terminal domain, structural characteristics of class Ⅲ holins, and a predicated N-out, C-in topology. The potential orfs of hyb5were coexpressed with hyb5or hyb5/lyb5to identify the antiholin. The results showed that the residues157to465of Hyb5(named ORF2) could delay the lysis timing, which was predicted to act as antiholin for Hyb5. The pull-down assay directly confirmed the interaction between Hyb5and ORF2. When the R, GFP and β-Gal were selected as reporters to size the holin lesion, we found that Hyb5formed pin holes at the cytoplasmic membrane, not the typical large membrane holes. As a result, Hyb5was designated as a pinholin, as opposed to large-hole-forming holins. Additionally, cross-linking expreiments showed that Hyb5oligomerized to form tetramer in the membrane. According to the function of Lyb5and Hyb5, it could be concluded that the pin holes formed by Hyb5depolarize the membrane, leading to the release of membrane arrested Lyb5to the periplasmic.6. Fine tuning of the metabolites production through promoter engineering in Lactococcus lactisLactococcus lactis is a well-studied bacterium widely used in dairy fermentation. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H2O-forming NADH oxidase gene in L. lactis. The library consisted of30promoters covering a wide range of activities from7,000to380,000relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H2O-forming NADII oxidase gene in L. lactis, and the NADH oxidase activity increased from9.43to58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from21.15±0.08mM to9.94±0.07mM, and the corresponding diacetyl production increased from1.07±0.03mM to4.16±0.06mM with the intracellular NADU/NAD+ratios varying from0.711±0.005to0.383±0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD+ratios. Interestingly, the increased H2O-forming NADH oxidase activity led to76.95%lower H2O2concentration in the recombinant strain than that of the wild-type strain after24h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within28days of storage at4℃.