| The adhesion and colonization of lactic acid bacteria in the intestinal tract is a prerequisite for its probiotic effect,and the S-layer protein plays a key role.Lactobacillus crispatus is a typical S-layer-containing strain.However,the composition and proportion of the proteins in the S-layer have not been reported,especially after the S-layer proteins are secreted out of the cell,their assembly process on the cell wall surface is unknown.In view of this,studying the proportion,abundance and assembly method of the proteins in the S-layer of Lactobacillus crispatus will provide theoretical basis and methods for the application of Lactobacillus crispatus in the intervention of livestock and poultry intestinal health and the mining and utilization of the function of the S-layer proteins.This experiment screened out highly adherent Lactobacilli from the intestinal samples of Lueyang black-bone chickens preserved in the laboratory and used them as subsequent test objects to isolate the S-layer proteins and analyze the relationship between the S-layer proteins and tolerance and adhesion.Combined with ultra-high resolution liquid chromatography-mass spectrometry and whole genome sequencing,the protein composition and abundance of Lactobacillus crispatus S layer and the types and proportions of proteins in the soluble supernatant and culture supernatant were analyzed.A detection vector was constructed to analyze the genetic basis of the S layer proteins.After the separated soluble S-layer proteins was lyophilized and quantified,they were assembled on the surface of Bacillus subtilis spores using the self-assembly properties of the S-layer proteins.The experiment yielded the following results:1.Intensively reared Lueyang black-bone chickens had better gut microbial diversity than pure free-range black-bone chickens,but the abundance of lactic acid bacteria in the gut of purely free-range black-bone chickens was higher than that of intensively reared black-bone chickens.Therefore,eight strains of lactic acid bacteria with high S-layer proteins content were screened out from the intestines of free-range black-bone chickens,two strains of L.reuteri,one strain of L.agilis,three strains of L.delbrueckii,one strain of L.crispatus and one strain of E.faecalis.A strain of L.crispatus with the highest adhesion was used as the research object,named L.crispatus LW.This strain is a gram-positive bacterium with a slender rod-like shape,curved and appearing in chains;and L.crispatus strain 5-3-2(KU991813.1)has the highest similarity.2.L.crispatus LW platform-stage bacteria were incubated with 5 M neutral LiCl for 1 h,and the S-layer proteins yield and purity were high.The preliminary determination of the S-layer proteins content was 25%,and the S-layer proteins could not be completely stripped off by 5 M neutral LiCl for 1 h.When the S-layer proteins of L.crispatus LW were removed,the adhesion rate of the strain decreased significantly(P<0.01),and the tolerance to acid-base,mechanical force and lysozyme decreased(P<0.01).3.Whole-genome sequencing results showed that the genome of L.crispatus LW contained 9 S-layer protein-coding genes.Mass spectrometry results of the S-layer proteins showed that only three were expressed under normal conditions,namely orf00226,orf01719,orf00290,of which orf00226 was dominant,and orf00226 was a new gene encoding S-layer protein.In addition to S-layer proteins,there were other secreted proteins and a large number of intracellular proteins outside the cell wall of L.crispatus LW.The results of subcellular localization showed that the proportion of cell wall proteins,membrane proteins,periplasmic proteins and extracellular proteins were 0.46%,16.01 %,62.18 % and 21.35%,respectively.The results of the S-layer mass spectrometry of L.crispatus LW further confirmed the presence of intracellular proteins,compared with the S-layer proteins mass spectrometry,the protein proportion was the same,the overall abundance was decreased,and the expression of orf00226 was the highest.4.The mass spectrometry results of the dialysate supernatant and the cell culture supernatant proved the existence of S-layer proteins,and the proportion of the protein was the same,and the extracellular proteins accounted for the largest proportion of the total proteins.S-layer proteins could be isolated from the surface of spores incubated with soluble S-layer proteins.Research conclusion: The pure free-range Lueyang black-bone chicken is an important source for screening high-adhesive lactic acid bacteria.The S-layer proteins are closely related to the high adhesion of L.crispatus LW,and has a good protective effect on the bacteria.The S-layer of L.crispatus LW is far more diverse and complex than expected.In addition to the typical S-layer proteins,there are also extracellular matrix anchoring proteins and other proteins that play immune regulatory roles.The genes of the S-layer proteins are different from the genes that have been found.Soluble S-layer proteins can be successfully assembled on the surface of spores.It is feasible to use S-layer proteins to assemble on the surface of spores to increase the adhesion of spores. |
