Thermal stabilization of an endoglucanase by cyclization.

An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-ß-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins.

Changes in abundance of Lactobacillus spp. and Streptococcus suis in the stomach, jejunum and ileum of piglets after weaning.

This present study investigated the changes in bacterial community composition, with an emphasis on Lactobacillus spp. and Streptococcus suis populations as potentially beneficial and harmful groups, in the stomach, jejunum and ileum of piglets after weaning (21 days postpartum) by 16S rRNA gene-based methods. Denaturing gradient gel electrophoresis analysis showed that, after weaning, predominant bands related to Lactobacillus spp. disappeared and were replaced by potential pathogenic species, such as Peptostreptococcus anaerobius, Moraxella cuniculi, S. suis and Porphyromonas catoniae. Real-time PCR revealed that the abundances of lactobacilli and Lactobacillus sobrius as a proportion of total bacterial abundance were significantly lower in the stomach, jejunum and ileum of weaned piglets than in 21-day-old piglets. A specific and sensitive real-time PCR assay was developed for quantification of the important pathogen S. suis within gastrointestinal microbiota. The assay showed that S. suis predominated in the stomach samples of weaned piglets with population levels up to 10(7) copies g(-1) digesta, while it was not detected in the stomach before weaning. Streptococcus suis was not dominant in the jejunum and ileum digesta before weaning, but became dominant after weaning, with population levels up to 10(7) copies g(-1) digesta. The results demonstrated for the first time the postweaning dominance of the potentially harmful S. suis in piglet intestine. The results also suggest that the defensive barrier of the stomach can be impaired as S. suis became dominant while the proportion of Lactobacillus populations decreased after weaning, which may further result in an increase of S. suis abundance in the intestine.

16S ribosomal RNA-based methods to monitor changes in the hindgut bacterial community of piglets after oral administration of Lactobacillus sobrius S1.

16S ribosomal RNA (rRNA) gene based PCR/denaturing gradient gel electrophoresis (DGGE) and real-time PCR were used to monitor the changes in the composition of microbiota in the hindgut of piglets after oral administration of Lactobacillus sobrius S1. Six litters of neonatal piglets were divided randomly into control group and treatment group. At 7, 9, and 11 days of age, piglets in the treatment group orally received a preparation of L. sobrius S1. At 7, 14, 21(weaning), 24, and 35 days of age, one piglet from each litter was sacrificed and digesta samples of hindgut were collected. DGGE analysis of 16S rRNA gene V6-V8 region for all bacteria showed that several populations present in the hindgut of piglets, represented by far-migrating bands, disappeared after weaning. Most of these bands corresponded to Lactobacillus spp. as revealed by sequence analysis. Quantitative real-time PCR specific for lactobacilli further demonstrated that the number of lactobacilli population tended to decrease after the piglets were weaned. Drastic changes of L. amylovorus and L. sobrius in total Lactobacillus populations were also observed in the colon of piglets around weaning, as monitored by 16S rRNA gene V2-V3 region based Lactobacillus-specific PCR-DGGE. Species-specific real-time PCR also revealed that the population of L. sobrius declined apparently in the colon of piglets after weaning. No remarkable changes in the overall microbial community in the hindgut were found between control and treatment groups. However, comparison of DGGE profiles between the two groups revealed a specific band related to Clostridium disporicum that was found in treatment group on day 14. On day 35, a specific band appeared only in the control group, representing a population most closely related to Streptococcus suis (99%). Real-time PCR showed that L. sobrius 16S rRNA gene copies in treatment group were relatively higher than in the control group (10(8.45) vs. 10(6.83)) on day 35, but no significant difference was observed between the two groups.