Isolation And Characterization Of Lactobacillus Phages

Lactobacillus generally recognized as safe is widely used in food industry,pharmaceutical engineering and animal husbandry,and Lactobacillus plantarum(L.plantarum),Lactobacillus casei(L.casei)and Lactobacillus pentosus(L.pentosus)were more commonly used.However,the use of L.plantarum and L.pentosus can be affected by phage outbreaks,which can result in depreciated products even fermentation failure.Phage infection is considered to be the main reason for substandard dairy fermentation processes,which result in economic losses.Two novel virulent phages of Lactobacillus were isolated from Chinese sauerkraut samples in this study,designated as LpeD and Lpa804 respectively.The results showed that both of two belong to the Siphoviridae family based on their morphology with an icosahedral capsid and a noncontractile tail.LpeD infected both Lactobacillus pentosus and Lactobacillus plantarum and Lpa804 infected Lactobacillus plantarum.One-step growth curves showed,LpeD and Lpa804 had a latent period of 15 min and 30 min,a burst period of about 90 min and 105 min,and the burst size of 17 and 25 PFU per infected cell,respectively.Both phage LpeD and phage Lpa804 were sensitive to temperature,no LpeD particles were detected after incubation at 56? for 2 min,and Lpa804 lost entire activity after incubation at 63 ? for only 2 min.Phage LpeD and Lpa804 were tolerance with pH because of their stability in the environment of pH 4-12.Phage LpeD and Lpa804 could not be completely inactivated under UV irradiation,there were still part of both two phage particle survival after 20 min in the ultraviolet irradiation.Phage LpeD and Lpa804 were sensitive to biocides,LpeD was inactivated completely when incubated with 35%ethanol for 10 min,and Lpa804 was inactivated completely when incubated with 25%ethanol for 10 min.Lpa804 was more sensitive than LpeD,and the effect of isopropanol was similar to ethanol.Furthermore,temperature,magnesium ions and pH all had an influence on the adsorption of phages LpeD and Lpa804,as well as viable and nonviable host cells.Divalent Mg2+ was of some use to adsorped on cells rapidly for LpeD and Lpa804.The adsorption rates of Lpa804 and LpeD had no change in the range of pH from 4 to 8,but the adsorption rates of Lpa804 decreased 15%at pH 9 and 10,and the adsorption rates of LpeD decreased 20%.Compared with viable host cells,the adsorptions of LpeD on the nonviable host cells decreased 50%,and Lpa804 decreased 30%.The adsorption rates of LpeD on two kinds of host cells were not significently affected by temperature.But the maximum adsorption rate(more than 98%)were obtained at 25 ?.The adsorption rates of Lpa804 were influenced obviously by temperature.With the increase of temperature,the adsorption rates of Lpa804 decreased.The maximum adsorption rate of Lpa804 was achieved at 4?.The cell wall of host cells was treated by chemical and enzyme method,then the properties of phage receptor can be identified.The adsorption rate of phage LpeD on the cell wall treated by TCA decreased significantly,and the adsorption rate of phage Lpa804 on the cell wall treated by Lysozyme reduced significantly.These suggest that the adsorbent receptor of Lpa804 may be peptidoglycan,and the adsorbent receptor of LpeD may be cell wall polymer linked to the peptide polycarbohydrates.In this study,the whole genome sequencing and sequence analysis of phage LpeD and Lpa804 were carried out.LpeD has the largest genome of 145,162 bp reported up to now for L.plantarum phages with the lowest GC content of 34.11%and 182 open reading frames(ORFs).LpeD was the only one L.pentosus phage that has been completely sequencing.The genome size of Lpa804 is 142,567 bp with the GC content of 36.62%,and 185 ORFs were predicted.These two new phage genomes,LpeD and Lpa804,are organized into four functional modules:Morphogenesis,Lysogeny,Lysis,DNA replication and regulation.By phylogenetic relationship analysis,we found that the relationship of phage LpeD,Lpa804 and Lactobacillus plantarum bacteriophage LP65 was close.The result of collinear comparison analysis showed that the similarity between LpeD and Lpa804 was 79.68%.In conclusion,phage LpeD and Lpa804 were new Lactobacillus phage,and the homology of the two phages was higher.The characterizations of phage LpeD and phage Lpa804 are necessary to take measures to control phage in industrial production.The analysis of genome of phage LpeD and phage Lpa804 indicated that they are different from other L.plantarum phages reported.Above all,thermo and biocides could be used to control phages contamination,and the increasing knowledge of L.plantarum and L.pentosus phages genomes help us understand the diversity of Lactobacillus phages.