Complete genome analysis of Bacillus velezensis TS5 and its potential as a probiotic strain in mice

IntroductionIn recent years, a large number of studies have shown that Bacillus velezensis has the potential as an animal feed additive, and its potential probiotic properties have been gradually explored.MethodsIn this study, Illumina NovaSeq PE150 and Oxford Nanopore ONT sequencing platforms were used to sequence the genome of Bacillus velezensis TS5, a fiber-degrading strain isolated from Tibetan sheep. To further investigate the potential of B. velezensis TS5 as a probiotic strain, in vivo experiments were conducted using 40 five-week-old male specific pathogen-free C57BL/6J mice. The mice were randomly divided into four groups: high fiber diet control group (H group), high fiber diet probiotics group (HT group), low fiber diet control group (L group), and low fiber diet probiotics group (LT group). The H and HT groups were fed high-fiber diet (30%), while the L and LT groups were fed low-fiber diet (5%). The total bacteria amount in the vegetative forms of B. velezensis TS5 per mouse in the HT and LT groups was 1 × 109 CFU per day, mice in the H and L groups were given the same volume of sterile physiological saline daily by gavage, and the experiment period lasted for 8 weeks.ResultsThe complete genome sequencing results of B. velezensis TS5 showed that it contained 3,929,788 nucleotides with a GC content of 46.50%. The strain encoded 3,873 genes that partially related to stress resistance, adhesion, and antioxidants, as well as the production of secondary metabolites, digestive enzymes, and other beneficial nutrients. The genes of this bacterium were mainly involved in carbohydrate metabolism, amino acid metabolism, vitamin and cofactor metabolism, biological process, and molecular function, as revealed by KEGG and GO databases. The results of mouse tests showed that B. velezensis TS5 could improve intestinal digestive enzyme activity, liver antioxidant capacity, small intestine morphology, and cecum microbiota structure in mice.ConclusionThese findings confirmed the probiotic effects of B. velezensis TS5 isolated from Tibetan sheep feces and provided the theoretical basis for the clinical application and development of new feed additives

DHAV-1 2A1 Peptide – A Newly Discovered Co-expression Tool That Mediates the Ribosomal “Skipping” Function

Duck hepatitis A virus 1 (DHAV-1) belongs to the genus Avihepatovirus in the family Picornaviridae. Little research has been carried out on the non-structural proteins of this virus. This study reports that 2A1 protein, the first non-structural protein on the DHAV-1 genome, has a ribosomal “skipping” function mediated by a “-GxExNPGP-” motif. In addition, we prove that when the sequence is extended 10aa to VP1 from the N-terminal of 2A1, the ribosome “skips” completely. However, as the N-terminus of 2A is shortened, the efficiency of ribosomal “skipping” reduces. When 2A1 is shortened to 10aa, it does not function. In addition, we demonstrate that N18, P19 G20, and P21 have vital roles in this function. We find that the expression of upstream and downstream proteins linked by 2A1 is different, and the expression of the upstream protein is much greater than that of the downstream protein. In addition, we demonstrate that it is the nature of 2A1 that is responsible for the expression imbalance. We also shows that the protein “cleavage” is not due to RNA “cleavage” or RNA transcription abnormalities, and the expressed protein level is independent of RNA transcriptional level. This study provides a systematic analysis of the activity of the DHAV-1 2A1 sequence and, therefore, adds to the “tool-box” that can be deployed for the co-expression applications. It provides a reference for how to apply 2A1 as a co-expression tool

Surface display of OmpC of Salmonella serovar Pullorum on Bacillus subtilis spores.

Salmonellosis is a major public health problem throughout the world. Thus, there is a huge need for diversified control strategies for Salmonella infections. In this work, we have assessed the potential use of Bacillus subtilis (B. subtilis) spores for the expression of a major protective antigen of Salmonella serovar Pullorum, OmpC. The expression of OmpC on the surface of spores was determined by immunofluorescence microscopy. Mice immunized with recombinant spores expressing the OmpC antigen presented significant levels of OmpC-specific serum IgG and mucosal SIgA antibodies than in mice immunized with non-recombinant spores (p<0.01). In addition, oral immunization with recombinant spores was able to induce a significant level of protection in mice against lethal challenge with Salmonella serovar Typhimurium. These results suggest that B. subtilis spores have promising potential in the development of mucosal vaccines against Salmonella infections

Schematic representation of the construction of the recombinant spores.

<p>The <i>cotC</i>::<i>ompC</i> gene fusion and <i>cat</i> (chloramphenicol-resistance gene) gene carried by plasmid pDG364-<i>cotC</i>-<i>ompC</i> were integrated into the <i>amyE</i> gene locus of <i>B</i>. <i>subtilis</i> 168 chromosome by double cross-over recombination events. Arrows indicate direction of transcription.</p

Oligonucleotides list.

<p>Oligonucleotides list.</p

Amylase activity analysis.

<p>Recombinant and nonrecombinant strains grew on the starch-containing LB plate before (A) and after (B) being stained by iodine. The integration of <i>cotC</i>::<i>ompC</i> gene fusion disrupts <i>amyE</i> gene and made the strain amylase deficient, transparent halo was produced around the <i>B</i>. <i>subtilis</i> 168, but in the recombinant <i>B</i>. <i>subtilis</i> SE2 clones, no transparent halo was produced.</p

PCR analysis with different primer pairs.

<p>Site-directed PCR analysis of <i>cotC</i>::<i>ompC</i> gene fusion integrated into the chromosome of <i>B</i>. <i>subtilis</i> 168. lane WT, <i>B</i>. <i>subtilis</i> 168; lane RT, recombinant <i>B</i>. <i>subtilis</i> SE2; primer pairs used in PCR are labeled above.</p

Immunofluorescence microscopy analysis.

<p>Sporulation of <i>B</i>. <i>subtilis</i> strains was induced by the exhaustion method, and spores were collected in DSM after 48h. Spores were labeled with OmpC antiserum followed by Cy3-labeled goat anti-chicken IgG. Spores were visualized by phase-contrast (PC) and immunofluorescence (IF) microscopy.</p

Serum IgG and intestinal mucosal SIgA antibodies responses.

<p>PI, pre-immune samples. Samples obtained from the mice immunized with recombinant spores (A and C) and wild-type spores (B and D); E, samples obtained from a naïve, untreated control group. ** indicates <i>p</i><0.01.</p

Protection of mice against challenge with <i>S</i>. Typhimurium strain SL1344.

<p>Protection of mice against challenge with <i>S</i>. Typhimurium strain SL1344.</p