Screening Of The Lactobacillus Brevis M8and Cloning And Expression Analysis Of Its Surface Layer Gene SlpM

S-layer proteins (Slps) are crystalline surface protein arrays which are composed of a single protein or glycoprotein species. Diverse functions have been attributed to the Slps, including cell's protection from external environment invasion; metabolism regulation and nutrition transportation; as well as adhesion and surface recognition ability. Slps exist in dozens of microorganisms; with few of them belong to Lactobacillus genus.Lactobacillus strains carrying Slps are quite scarce. Up to present, researches of Lactobacillus Slps in domestic and abroad countries focused their attention on several type strains of Lactobacillus. However, the structures and functions of Lactobacillus Slps have not been studied thoroughly. Thus, Lactobacillus strains from six rich in Lactic Acid Bacteria (LAB) samples were selected by using Slps screening index. The biological characteristic, protein molecular structure, immunogenicity, cloning, expression and prediction analysis of S-layer gene slpM of Lactobacillus brevis M8strain were studied. Several results are as follows:1. Screenig and identification of Slps carrying Lactobacillus strainsEight Slps carrying Lactobacillus strains were isolated from six different matrixes with SL medium. Five strains were identified and belonged to three different species:Lactobacillus reuteri, Lactobacillus plantarum and Lactobacillus brevis. These five strains were named L. reuteri Y4, L. reuteri Y6, L. plantarum P4, L. brevis C7and L. brevis M8. The morphological characteristics, SDS-PAGE result and sequence alignment showed that strains Y4and Y6expressed various kinds of Slps, while P4, C7and M8strains abundantly secreted one kind of Slps.However, Slps of M8strain were more stable after20times generation; therefore further study was conducted on M8strain.2. Study of biological characteristics of L. brevis M8strain and its Slps M8strain and its Slps showed good digestive enzymes tolerance and adhesion characteristics during in vivo simulation of host gastrointestinal microenvironment. Though artificial gastric juice treatment decreased the viable number from log8.18to log7.01, the survival rate was still60%. Subsequently, the number of viable cells proliferated from6.01to7.30after4hours artificial intestinal juice treatment.The intact cells of M8strain were treated by single use of pepsin and trypsin, but SDS-PAGE analysis showed that a large number of the Slps were not digested. It could be concluded that Slps, distributed in the outermost layer of cell walls, was able to effectively resist the digestive enzymes invasion.The adhesion ability of M8strain was analyzed with Caco-2cells model. The cells of M8strain was self-aggregated and attached to Caco-2cells. However, when Slps were removed or the active sites were blotted by Slps antibody, the adhesion ability was significantly disturbed, showing that Slps were closely related to the adhesion of this strain.3. Slps purification from M8strainThe purified Slps (PAGE grade) of M8strain were obtained by gel filtration column chromatography. It had the same general characteristics of Slps from other microorganisms.Maximum absorption peak was appeared at52minute at the experiment condition of gel filtration chromatography. The molecular weight size of this fraction was coincided to the target Slps and the purification efficiency reached82.22%. The amino acid composition analysis of Slps from M8strain showed no methionine (Met) and cystine (Cys). The content of both hydrophilic and hydrophobic amino acids was nearly30%. M8strain's Slps had a rich content of positively charged amino acids, similar to other microorganisms'Slps.4. Detection surface morphology and immunogenicity of SlpsThe supra-molecular structure of Slps and the immunogenicity of Slps were studied. Two-dimensional morphology of supra-molecular structure assembled by Slps monomer was detected by atomic force microscopy (AFM). AFM tapping mode scan test found that the surface layers of M8strain were unevenly wrinkled, having a number of small granular morphology. The supra-molecular structure assembled by Slps monomer in Tris-HCl buffer or super purified water was a "middle concave nearly circular cluster" and had a nanometer scale crystal structure. These cluster structures could be further aggregated into a200-500nm size "flower-like" crystal. The rough state of Slps were self-aggregated, gave it a huge surface area and a good adhering ability.ELISA detection of the rabbit anti-Slps polyclonal antiserum titer was very high, about18-fold higher compared to control, while western blotting analysis showed a strong positive specificity of Slps-polyclonal antibody. Those results proved a good immunogenicity of Slps from M8strain.5."SlpM" gene cloning and expressionThe slpM gene (GenBank accession number: JQ063472) was obtained through twice PCR amplification by using a pair of universal primers (P1&P2) and a pair of designed primers (1f&1416r) successively."SlpM-pQE30" expression vector was constructed and heterologous expression in E. coli JM109successfully. The recombined Slps was named "SlpM".6. Prediction of structures and functions of "SlpM"A series of bioinformatics softwares gave some further informations of SlpM. Its theoretical molecular mass was49.96kD. The content of polar and non-polar amino acids was almost equal. Disulfide bond might not be existed since it contained no Cys and low Met. Basic amino acids such as Lys caused a high pI value9.5.Many β-sheets, random coils and a few a-helices constituted the secondary structures of SlpM. The first30amino acid residues constituted the signal peptide (SP) sequence that might play an important part in going across the membrane, which was the reason why SlpM was existed in the outermost of cell wall. The3D-structure of SlpM was matched with the invasion protein InlB GW domains of Listeria monocytogenes. Both of them were surface proteins; bound to cell's surface with non-covalent bond; and could adhere to the epithelium of host cells. Thus, the GW domain might become the rudiment model of SlpM and its homogeneous proteins. This study replenished the resource of Slps carrying Lactobacillus and provided a theoretical basis for using and developing M8strain and its Slps.