Chair of Microbiology, Technical University of Munich, Gregor-Mendel-Straße 4, 85354 Freising, Germany.
Institute of Chemistry, Division of Food Chemistry, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 2, 06120 Halle (Saale), Germany.
J Appl Microbiol. 2023 Nov 1;134(11). doi: 10.1093/jambio/lxad268.
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.
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.
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.
属于 Bombella 属的醋酸菌尚未报道能产生胞外多糖 (EPS)。在这项研究中，研究了 B.apis TMW 2.1884 和 B. mellum TMW 2.1889 形成果聚糖的情况。
在来自四个不同 Bombella 种的 8 个菌株中，只有 B.apis TMW 2.1884 和 B. mellum TMW 2.1889 用 50 g l-1 蔗糖作为底物显示出 EPS 形成。通过不对称流场流分离与多角度激光散射和 UV 检测器（AF4-MALLS/UV）和高效尺寸排阻色谱与 MALLS 和折射率检测器（HPSEC-MALLS/RI）分析相结合，两种 EPS 均被鉴定为高分子量（HMW）聚合物（106-107 Da）。通过三氟乙酸水解进行的单糖分析表明，两种 EPS 都是果聚糖。通过甲基化分析确定糖苷键揭示了主要的 2,6-连接的果呋喃糖（Fruf）单元，外加 2,1-连接的 Fruf 单元（10%）和 2,1,6-Fruf 支化单元（7%）。在基因组中未鉴定到通常与细菌中 HMW 果聚糖形成相关的糖苷水解酶（GH）68 家族基因。通过在大肠杆菌 Top10 中的异源表达，可以将 GH32 家族的一种酶分配到果聚糖形成的催化作用中。鉴定的果糖基转移酶在系统发育和结构上可与其他先前描述的细菌果糖基转移酶明显区分开来。
Bombella 属的个别菌株形成 HMW 果聚糖是由 GH32 家族的酶催化的。对果聚糖的分析显示出一种由 2,6-连接的 Fruf 单元以及 2,1-连接的 Fruf 单元和 2,1,6-Fruf 单元组成的非典型结构。
