Identification of lthB, a Gene Encoding a Putative Glycosyltransferase Family 8 Protein Required for Leptothrix Sheath Formation
Identification of lthB, a Gene Encoding a Putative Glycosyltransferase Family 8 Protein Required for Leptothrix Sheath Formation
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DOI：
10.1128/aem.01919-22
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发表时间：
2023-03-23
影响因子：
4.4
通讯作者：
Nomura，Nobuhiko
中科院分区：
文献类型：
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作者：
Kunoh，Tatsuki;Yamamoto，Tatsuya;Nomura，Nobuhiko
细菌Leptothrix cholodnii产生包裹在鞘中的细胞链，鞘由编织的纳米原纤维组成。纳米纤维主要由糖缀合物重复序列组成，并且其生物合成需要几种糖基转移酶（GT）。然而，到目前为止，只有一种GT（LthA）被确定。在这项研究中，我们筛选自发变异的L。cholodniiSP6来寻找那些形成光滑菌落的，这是无鞘变体的特征之一。基因组DNA测序的一个分离的变体揭示了插入locusLcho_0972，它编码一个推定的GT家族8蛋白。因此，我们将这种蛋白质命名为LthB，并使用缺失突变体和抗体对其进行表征。LthB定位于邻近细胞包膜。Δ lthB细胞链不含纳米原纤维，因此无鞘，表明LthB参与纳米原纤维生物合成。与Δ lthA突变体和野生型菌株不同，它们通常产生增殖细胞，大多数Δ lthB生物体在静态条件下呈长细胞链，导致膜形成不足，这需要运动的增殖细胞。这些结果表明，鞘是不需要的细胞链的伸长。最后，由于鞘损失导致细胞链断裂的钙耗尽，废除了LthA的表达，但不是LthB，这表明这些GT在不同的信号控制下协同参与糖缀合物的生物合成。通过细胞外信号调节丝状细菌的细胞链延长作为防止水分配系统堵塞的潜在策略已经引起了注意，丝状菌膨胀的活性污泥在工业设置。然而，对丝状细菌生态学的基本理解仍然难以捉摸。由于鞘的形成与大多数这些细菌中的细胞链伸长相关，因此必须阐明纳米原纤维鞘形成的分子机制，包括响应于细胞外刺激的细胞内信号级联。在这里，我们分离出了L的一个无鞘变体。cholodniiSP6，从而鉴定了一种新的糖基转移酶LthB。虽然缺失了编码另一个GT的lthA和lthB的突变体都不能形成纳米纤维，但它们表现出不同的细胞链延伸和表膜形成表型。此外，LthA的表达，而不是LthB的表达，受到细胞外钙的影响，这是已知的影响纳米纤维的形成，表明LthA和LthB的功能性双功能。这种分子见解对于更好地理解丝状细菌的生态学至关重要，而丝状细菌的生态学又可用于改进控制工业设施中丝状细菌的策略。
The bacterium Leptothrix cholodnii generates cell chains encased in sheaths that are composed of woven nanofibrils. The nanofibrils are mainly composed of glycoconjugate repeats, and several glycosyltransferases (GTs) are required for its biosynthesis. However, only one GT (LthA) has been identified to date. In this study, we screened spontaneous variants ofL. cholodniiSP6 to find those that form smooth colonies, which is one of the characteristics of sheathless variants. Genomic DNA sequencing of an isolated variant revealed an insertion in the locusLcho_0972, which encodes a putative GT family 8 protein. We thus designated this protein LthB and characterized it using deletion mutants and antibodies. LthB localized adjacent to the cell envelope. ΔlthBcell chains were nanofibril free and thus sheathless, indicating that LthB is involved in nanofibril biosynthesis. Unlike the ΔlthAmutant and the wild-type strain, which often generate planktonic cells, most ΔlthBorganisms presented as long cell chains under static conditions, resulting in deficient pellicle formation, which requires motile planktonic cells. These results imply that sheaths are not required for elongation of cell chains. Finally, calcium depletion, which induces cell chain breakage due to sheath loss, abrogated the expression of LthA, but not LthB, suggesting that these GTs cooperatively participate in glycoconjugate biosynthesis under different signaling controls.IMPORTANCEIn recent years, the regulation of cell chain elongation of filamentous bacteria via extracellular signals has attracted attention as a potential strategy to prevent clogging of water distribution systems and filamentous bulking of activated sludge in industrial settings. However, a fundamental understanding of the ecology of filamentous bacteria remains elusive. Since sheath formation is associated with cell chain elongation in most of these bacteria, the molecular mechanisms underlying nanofibril sheath formation, including the intracellular signaling cascade in response to extracellular stimuli, must be elucidated. Here, we isolated a sheathless variant ofL. cholodniiSP6 and thus identified a novel glycosyltransferase, LthB. Although mutants with deletions oflthA, encoding another GT, andlthBwere both defective for nanofibril formation, they exhibited different phenotypes of cell chain elongation and pellicle formation. Moreover, LthA expression, but not LthB expression, was influenced by extracellular calcium, which is known to affect nanofibril formation, indicating the functional diversities of LthA and LthB. Such molecular insights are critical for a better understanding of ecology of filamentous bacteria, which, in turn, can be used to improve strategies to control filamentous bacteria in industrial facilities.