Departamento de Microbiología y Genética and CIALE, Universidad de Salamanca, Salamanca, Spain.
Microb Cell Fact. 2012 Sep 12;11:125. doi: 10.1186/1475-2859-11-125.
The synthesis of cellulose is among the most important but poorly understood biochemical processes, especially in bacteria, due to its complexity and high degree of regulation. In this study, we analyzed both the production of cellulose by all known members of the Rhizobiaceae and the diversity of Rhizobium celABC operon predicted to be involved in cellulose biosynthesis. We also investigated the involvement in cellulose production and biofilm formation of celC gene encoding an endoglucanase (CelC2) that is required for canonical symbiotic root hair infection by Rhizobium leguminosarum bv. trifolii.
ANU843 celC mutants lacking (ANU843ΔC2) or overproducing cellulase (ANU843C2+) produced greatly increased or reduced amounts of external cellulose micro fibrils, respectively. Calcofluor-stained cellulose micro fibrils were considerably longer when formed by ANU843ΔC2 bacteria rather than by the wild-type strain, in correlation with a significant increase in their flocculation in batch culture. In contrast, neither calcofluor-stained extracellular micro fibrils nor flocculation was detectable in ANU843C2+ cells. To clarify the role of cellulose synthesis in Rhizobium cell aggregation and attachment, we analyzed the ability of these mutants to produce biofilms on different surfaces. Alteration of wild-type CelC2 levels resulted in a reduced ability of bacteria to form biofilms both in abiotic surfaces and in planta.
Our results support a key role of the CelC2 cellulase in cellulose biosynthesis by modulating the length of the cellulose fibrils that mediate firm adhesion among Rhizobium bacteria leading to biofilm formation. Rhizobium cellulose is an essential component of the biofilm polysaccharidic matrix architecture and either an excess or a defect of this "building material" seem to collapse the biofilm structure. These results position cellulose hydrolytic enzymes as excellent anti-biofilm candidates.
纤维素的合成是最重要但了解甚少的生化过程之一，尤其是在细菌中，因为其复杂性和高度调控性。在这项研究中，我们分析了根瘤菌科所有已知成员的纤维素生产以及预测参与纤维素生物合成的 Rhizobium celABC 操纵子的多样性。我们还研究了纤维素生产和生物膜形成的参与，其中 celC 基因编码一种内切葡聚糖酶（CelC2），它是 Rhizobium leguminosarum bv. trifolii 典型共生根毛感染所必需的。
缺乏 celC 基因（ANU843ΔC2）或过度表达纤维素酶（ANU843C2+）的 ANU843 celC 突变体分别产生了大量增加或减少的外部纤维素微纤维。与野生型菌株相比，形成的 Calcofluor 染色纤维素微纤维更长，与它们在批式培养中的絮凝显著增加相关。相比之下，在 ANU843C2+细胞中既检测不到 Calcofluor 染色的细胞外微纤维，也检测不到絮凝。为了阐明纤维素合成在 Rhizobium 细胞聚集和附着中的作用，我们分析了这些突变体在不同表面上形成生物膜的能力。改变野生型 CelC2 水平会导致细菌在非生物表面和植物体内形成生物膜的能力降低。
我们的结果支持 CelC2 纤维素酶在纤维素生物合成中的关键作用，通过调节介导 Rhizobium 细菌之间牢固粘附的纤维素纤维的长度来调节纤维素生物合成，从而导致生物膜形成。根瘤菌纤维素是生物膜多糖基质结构的重要组成部分，这种“建筑材料”的过剩或缺陷似乎会破坏生物膜结构。这些结果将纤维素水解酶定位为优秀的抗生物膜候选物。
