Glycoside hydrolase family 32 enzymes from Bombella spp. catalyze the formation of high-molecular weight fructans from sucrose.

AIMS: Acetic acid bacteria of the genus Bombella have not been reported to produce exopolysaccharides (EPS). In this study, the formation of fructans by B. apis TMW 2.1884 and B. mellum TMW 2.1889 was investigated. METHODS AND RESULTS: Out of eight strains from four different Bombella species, only B. apis TMW 2.1884 and B. mellum TMW 2.1889 showed EPS formation with 50 g l-1 sucrose as substrate. Both EPS were identified as high-molecular weight (HMW) polymers (106-107 Da) by asymmetric flow field-flow fractionation coupled to multi angle laser light scattering and UV detecors (AF4-MALLS/UV) and high performance size exclusion chromatography coupled to MALLS and refractive index detectors (HPSEC-MALLS/RI) analyses. Monosaccharide analysis via trifluoroacetic acid hydrolysis showed that both EPS are fructans. Determination of glycosidic linkages by methylation analysis revealed mainly 2,6-linked fructofuranose (Fruf) units with additional 2,1-linked Fruf units (10%) and 2,1,6-Fruf branched units (7%). No glycoside hydrolase (GH) 68 family genes that are typically associated with the formation of HMW fructans in bacteria could be identified in the genomes. Through heterologous expression in Escherichia coli Top10, an enzyme of the GH32 family could be assigned to the catalysis of fructan formation. The identified fructosyltransferases could be clearly differentiated phylogenetically and structurally from other previously described bacterial fructosyltransferases. CONCLUSIONS: The formation of HMW fructans by individual strains of the genus Bombella is catalyzed by enzymes of the GH32 family. Analysis of the fructans revealed an atypical structure consisting of 2,6-linked Fruf units as well as 2,1-linked Fruf units and 2,1,6-Fruf units.

Characterization of a novel inulosucrase from Lactiplantibacillus plantarum.

Fructansucrases produce fructans by polymerizing the fructose moiety released from sucrose. Here, we describe the recombinant expression and characterization of a unique fructansucrase from Lactiplantibacillus plantarum DKL3 that showed low sequence similarity with previously characterized fructansucrases. The optimum pH and temperature of fructansucrase were found to be 4.0 and 35 °C, respectively. Enzyme activity increased in presence of Ca2+ and distinctly in presence of Mn2+. The enzyme was characterized as an inulosucrase (LpInu), based on the production of an inulin-type fructan as assessed byNMR spectroscopy and methylation analysis. In addition to ß-2,1-linkages, the inulin contained a few ß-2,1,6-linked branchpoints. High-performance size exclusion chromatography with refractive index detection (HPSEC-RI) revealed the production of inulin with a lower molecular weight compared to other characterized bacterial inulin. LpInu and its inulin product represent novel candidates to be explored for possible food and biomedical applications.

Detailed structural characterization of five water-insoluble α-glucans produced by glucansucrases from Streptococcus spp.

Water-insoluble α-glucans synthesized from sucrose by glucansucrases from Streptococcus spp. are essential in dental plaque and caries formation. Because limited information is available on the fine structure of these biopolymers, we analyzed the structures of unmodified glucans produced by five recombinant Streptococcus (S.) mutans DSM 20523 and S. salivarius DSM 20560 glucansucrases in detail. A combination of methylation analysis, endo-dextranase and endo-mutanase hydrolyses, and HPSEC-RI was used. Furthermore, crystal-like regions were analyzed by using XRD and 13C MAS NMR spectroscopy. Our results showed that the glucan structures were highly diverse: Two glucans with 1,3- and 1,6-linkages were characterized in detail besides an almost exclusively 1,3-linked and a linear 1,6-linked glucan. Furthermore, one glucan contained 1,3-, 1,4-, and 1,6-linkages and thus had an unusual, not yet described structure. It was demonstrated that the glucans had a varying structural architecture by using partial enzymatic hydrolyses. Furthermore, crystal-like regions formed by 1,3-glucopyranose units were observed for the two 1,3- and 1,6-linked glucans and the linear 1,3-linked glucan. 1,6-linked regions were mobile and not involved in the crystal-like areas. Altogether, our results broaden the knowledge of the structure of water-insoluble α-glucans from Streptococcus spp.

Influence of ultrasonication and hydrolysis conditions in methylation analysis of bacterial homoexopolysaccharides.

Homoexopolysaccharides (HoEPS) such as α-glucans and ß-fructans are synthesized by lactic and acetic acid bacteria. Methylation analysis is an important and well-established tool for the structural analysis of these polysaccharides, however, multiple steps are required for polysaccharide derivatization. Because ultrasonication during methylation and the conditions during acid hydrolysis may influence the results, we investigated their role in the analysis of selected bacterial HoEPS. The results reveal that ultrasonication is crucial for water insoluble α-glucan to swell/disperse and deprotonate prior to methylation whereas it is not necessary for water soluble HoEPS (dextran and levan). Complete hydrolysis of permethylated α-glucans requires 2 M trifluoroacetic acid (TFA) for 60/90 min at 121 °C while levan is hydrolyzed in 1 M TFA for 30 min at 70 °C. Nevertheless, levan was also detectable after hydrolysis in 2 M TFA at 121 °C. Thus, these conditions can be used to analyze a levan/dextran mixture. However, size exclusion chromatography of permethylated and hydrolyzed levan showed degradation and condensation reactions at harsher hydrolysis conditions. Application of reductive hydrolysis with 4-methylmorpholine-borane and TFA did not lead to improved results. Overall, our results demonstrate that conditions used for methylation analysis have to be adjusted for the analysis of different bacterial HoEPS.

Analysis of locomotor behavior in the German Mouse Clinic.

BACKGROUND: Generation and phenotyping of mutant mouse models continues to increase along with the search for the most efficient phenotyping tests. Here we asked if a combination of different locomotor tests is necessary for comprehensive locomotor phenotyping, or if a large data set from an automated gait analysis with the CatWalk system would suffice. NEW METHOD: First we endeavored to meaningfully reduce the large CatWalk data set by Principal Component Analysis (PCA) to decide on the most relevant parameters. We analyzed the influence of sex, body weight, genetic background and age. Then a combination of different locomotor tests was analyzed to investigate the possibility of redundancy between tests. RESULT: The extracted 10 components describe 80% of the total variance in the CatWalk, characterizing different aspects of gait. With these, effects of CatWalk version, sex, body weight, age and genetic background were detected. In addition, the PCA on a combination of locomotor tests suggests that these are independent without significant redundancy in their locomotor measures. COMPARISON WITH EXISTING METHODS: The PCA has permitted the refinement of the highly dimensional CatWalk (and other tests) data set for the extraction of individual component scores and subsequent analysis. CONCLUSION: The outcome of the PCA suggests the possibility to focus on measures of the front and hind paws, and one measure of coordination in future experiments to detect phenotypic differences. Furthermore, although the CatWalk is sensitive for detecting locomotor phenotypes pertaining to gait, it is necessary to include other tests for comprehensive locomotor phenotyping.

Expression analysis of Lrrk1, Lrrk2 and Lrrk2 splice variants in mice.

Missense mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are linked to autosomal dominant forms of Parkinson's disease (PD). In order to get insights into the physiological role of Lrrk2, we examined the distribution of Lrrk2 mRNA and different splice variants in the developing murine embryo and the adult brain of Mus musculus. To analyse if the Lrrk2-paralog, Lrrk1, may have redundant functions in PD-development, we also compared Lrrk1 and Lrrk2 expression in the same tissues. Using radioactive in situ hybridization, we found ubiquitous expression of both genes at low level from embryonic stage E9.5 onward, which progressively increased up until birth. The developing central nervous system (CNS) displayed no prominent Lrrk2 mRNA signals at these time-points. However, in the entire postnatal brain Lrrk2 became detectable, showing strongest level in the striatum and the cortex of adult mice; Lrrk1 was only detectable in the mitral cell layer of the olfactory bulb. Thus, due to the non-overlapping expression patterns, a redundant function of Lrrk2 and Lrrk1 in the pathogenesis of PD seems to be unlikely. Quantification of Lrrk2 mRNA and protein level in several brain regions by real-time PCR and Western blot verified the striatum and cortex as hotspots of postnatal Lrrk2 expression. Strong expression of Lrrk2 is mainly found in neurons, specifically in the dopamine receptor 1 (DRD1a) and 2 (DRD2)-positive subpopulations of the striatal medium spiny neurons. Finally, we identified 2 new splice-variants of Lrrk2 in RNA-samples from various adult brain regions and organs: a variant with a skipped exon 5 and a truncated variant terminating in an alternative exon 42a. In order to identify the origin of these two splice variants, we also analysed primary neural cultures independently and found cell-specific expression patterns for these variants in microglia and astrocytes.