Studies On The Isolation, Functional Gene Expression Of Lactobacillus Phages And Mutation Breeding Of Phage-resistance Strains

Lactobacilli are one of the most important strains in dairy fermentation. However, the infection of Lactobacillus phages would result in large damage to dairy industries. In order to control and apply Lactobacillus phages effectively, we studied mainly on characterizations of the Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) virulent phage phiLdb and the Lactobacillus fermentum temperate bacteriophageφPYB5, the results were described as follows:1. Isolation and characterization of a novel virulent phage (phiLdb) of Lactobacillus delbrueckiiL. bulgaricus is an essential starter contributing to fast lactic acid development, and also to flavor and texture modifications in fermented milks. However, this starter activity of L. bulgaricus can be seriously affected when phage infection occurs. Consequently, the fermentation process may be slowed or completely stopped, thereby reducing the quality of the final products. Nevertheless, in comparison with phages of lactococi, the available knowledge about lactobacilli phages is limited and only a few of them have been studied in detail. In this study, A new virulent phage (phiLdb) of L. bulgaricus was isolated from a Chinese yogurt sample showing slow acidification. It belonged to the Siphoviridae family with an icosahedral capsid of 47.7±0.9 nm in diameter and a long tail of 129.8±2 nm. The genome of phage phiLdb was estimated to be approximately 41 kbp, and did not contain cohesive ends. One-step growth kinetics of its lytic development revealed latent and burst periods of 45 min and 75 min, respectively, with a burst size of 56±2 phage particles per infected cell. Phage phiLdb was highly specific for L. bulgaricus. The presence of calcium or magnesium ions was necessary to accelerate cell lysis and improve plaque formation. Phage phiLdb was able to survive in a pH range between 2 and 10, and resist ethanol and isopropanol. However, a treatment of 90℃for 40 min was observed to inactive phage phiLdb thoroughly. Calcium ions, pH as well as temperature did not show significant influence on phage adsorption, and the adsorption kinetics were similar on viable and nonviable cells. The characterization of this novel phage was helpful to establish a basis for adopting the most effective phage control strategies in industrial plants.2. Research on storage methods of phagesPhages are obligate intracellular parasite of bacteria and will easily lose activity without host cells. In order to store phages for a long period, Escherichia coliλphage and Lactobacillus delbrueckii phage phiLdb were kept with a variety of stablizers and temperature for 6 months. And at one month intervals, the phage titers were enumerated and compared to optimize the storage conditions. The results showed that the best storage temperature was 4℃and secondly-80℃; The best stablizer was glycerine, and secondly dimethyl sulfoxide. Therefore, phage storages with glycerine or dimethyl sulfoxide at 4℃,-20℃or-80℃are simple, efficient and recommendable methods.3. Isolation and characterization of phage-resistant derivatives of Lactobacillus delbruekii.The phage problem has promoted the adoption of various strategies. However, phage-resistant starter cultures were traditionally obtained by recombinant DNA technology or conjugal transfer of plasmids confering phage resistance, which holded health potential dangers. Fortunately, the isolation of spontaneous phage-resistant mutants has been considered as a convenient, simple and "natural" strategy to replace phage-sensitive strains.60Co-γray is one of the most common used physical mutagens. And recently,60Co-γray mutagenesis was also used to improve microbe strains. In this study, a total of 60 phage-resistant mutants were isolated from L. bulgaricus ATCC11842 by spontaneous mutaion (30 mutants) and 60Co-γray irradiation (30 mutants). All mutants appeared to be completely refractory to infection with the efficiency of plaquing lower than 10-9, and with high phage-resistant stability. Except for 2 spontaneous mutants, the phage-resistant mechanisms of the last 58 mutants were irrelevant with lysogeny. The obvious decrease of the adsorption rates exhibited by all mutants indicated the presence of adsorption interference during phage infection. And the accessory polysaccharide-peptidoglycan complex was proved to be involved in the phage receptor sites. Proteolytic activities were heterogeneous distributed among the spontaneous mutants. Nevertheless, most of the mutants revealed proteolytic activities and pH values similar to those observed for the parent strain. Two mutants, BIM 10 and y 19 were chosen as model starters for pilot fermentations. Both of them showed favorable technological characteristics regardless of the absence or presence of phage. Therefore, besides spontaneous mutation, the 60Co-γray irradiation was also proved to be a simple and effective method for obtaining phage-resistant mutants from Lactobacillus delbrueckii strains with high technological performance. Moreover, some of the phage-resistant mutants obtained in this work might be used as potential starters when commercial strains become phage-sensitive.4. Construction of a controlled autolysis Lactococcus strainIn the last stage of reproduction cycles, bacteriophages produce a set of enzymes that lyse the host cells to release their progeny particles. The lysis enzymes encoded by all double-stranded DNA phages involve two proteins:a holin and a lysin (also termed endolysin or lysozyme), which constitute a "two-component cell lysis cassette". The temperate bacteriophageφPYB5 was isolated from Lactobacillus fermentum YB5 strain. A putative holin (hyb5) and lysin (lyb5), obtained by sequencing of the genome library ofφPYB5 in E. coli, constituted a two-component cell lysis cassette. To elucidate the characterization of the lysis cassette (Hyb5-Lyb5) encoded by temperate bacteriophageφPYB5 in lactic acid bacteria, we cloned the hyb5 and lyb5 gene into the plasmid pSEC under the control of the nisin-inducible promoter, generating the recombinant plasmid pSEC-hyb5-lyb5. Then, pSEC-hyb5-lyb5 was induced into Lactococcus lactis NZ9000 by electroporation, yielding NZphl. Hyb5 and Lyb5 were expressed successfully under the induction of nisin and induced the lysis of NZphl effectively, resulting in the rapid reduction of OD600 and leakage of abundant intracellular proteins. These results will facilitate the application of autolysis lactic acid bacteria in fermentation to improve the quality of products.5. Clone and expression of the two-component cell lysis cassette encoded by temperate bacteriophageφPYB5 of Lactobacillus fermentum in E. coli.Different to others, Hyb5 was predicted to have a single transmembrane domain and Lyb5 possess an N-terminal signal transmembrane domain. In order to characterize this novel two-component cell lysis cassette encoded byφPYB5, and illustrate the potential application of Lyb5 as therapeutic agents, the hyb5, lyb5 and hyb5-lyb5 cassette were cloned and expressed in E. coli in this paper. The molecular weight of Hyb5 indicated by SDS-PAGE was 19 kDa, and Lyb5 was 45 kDa. Both Hyb5 and Lyb5 protein could induce cell lysis alone, resulting in the leakage ofβ-galactosidase. However, the Hyb5-Lyb5 cassette lysed the host cells more rapidly and extensively. By zymogram analysis, the Lyb5 produced in E. coli exhibited a broad lytic spectrum against Gram positive strains including Staphylococcus aureus as well as Gram negative strains such as Salmonella typhi, suggesting that Lyb5 provides a potential alternative of diagnostic tools and therapeutic agents.6. Lysis mechanism studies on "Hyb5-Lyb5".The lysis mechanism of holin-lysin cassette and the role of holin in the regulation of lysis time were elucidated based on the studies on E. coli phages. In this study, we tried to illuminate the lysis model of Hyb5-Lyb5 cassette and the mechanism of the regulation of Hyb5 in lysis through analyzing the function of N-trminal signal sequence in Lyb5 and determining the activities of different segments in Hyb5. We cloned lyb5 or△SARlyb5 (lyb5 lack of the SAR sequence) into the MCS1 of the pETDuet-1 vector, and inserted hyb5, hyb5p, or hyb5N30 (N-terminal sequence encoding the TMD with 30 amino acids) into the MCS2 of the pETDuet-1 vector, generating a series of recombinant vectors. By determining the lysis of different recombinants, we revealed that the co-expression of△SARlyb5 and Hyb5p resulted in obvious decrease of the culture turbidity in OD600, while the expression of△SARlyb5 accelerated the growth of the host strain, indicating that the SAR was unnecessary to the lysis activity of Lyb5, but was essential to the transmemberane of Lyb5. When hyb5N30 was expressed, it restrained the growth of E. coli, suggesting that the 30 AA could achieve transmemberane alone. However, the co-action of N30hyb5 and Lyb5 or△SARlyb5 did not induce large lysis of E. coli, exhibiting the limination of N30hyb5 in assisting Lyb5 or△SARlyb5 to the periplasmic space. Interestingly, Hyb5 lacking the C-terminal 30 AA could assist the transfer of Lyb5 more effectively than Hyb5, implying that the C-termina might inhibit the activity of Hyb5. However, the cleavability of SAR in the lysis process, the regulation of Hyb5 itself or other factors to the activity of Hyb5 and finally elucidation of the lysis mechanism of the novel Hyb5-Lyb5 cassette as well as the regulation of Hyb5 in the lysis process, all of which require abundant and further investigations.