Physical and genetic characterization of an outer-membrane protein (OmpM1) containing an N-terminal S-layer-like homology domain from the phylogenetically Gram-positive gut anaerobe Mitsuokella multacida
Physical and genetic characterization of an outer-membrane protein (OmpM1) containing an N-terminal S-layer-like homology domain from the phylogenetically Gram-positive gut anaerobe Mitsuokella multacida
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DOI：
10.1016/j.anaerobe.2009.01.001
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发表时间：
2009-06-01
期刊：
影响因子：
2.3
通讯作者：
Selinger, L. B.
中科院分区：
文献类型：
--
作者：
Kalmokoff, M. L.;Austin, J. W.;Selinger, L. B.
绵羊瘤胃分离株 Mitsuokella multacida 菌株 46/5(2) 的薄切片和冷冻断裂蚀刻揭示了革兰氏阴性包膜超微结构，由外膜覆盖的肽聚糖壁组成。对全细胞、细胞包膜和 Triton X-100 提取的包膜进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SIDS-PAGE) 分析，并结合薄片和 N 端序列分析表明，外膜含有两种共享相同 N 端的主要蛋白质（45 和 43 kDa）（A-A-N-P-F-S-D-V-P-A-D-H-W-A-Y-D）。克隆了编码蛋白质的基因，该蛋白质的预测 N 末端与 43 和 45 kDa 外膜蛋白质的 N 末端相同。 1290 bp 开放阅读框编码 430 个氨基酸的多肽，预计分子量为 47,492 Da。预测的 23 个氨基酸前导序列的切割将产生分子量为 45,232 Da 的蛋白质。质谱分析证实克隆的基因 (ompM1) 编码 45 kDa 的外膜蛋白。成熟 OmpM1 蛋白的 N 末端（残基 24-70）与各种规则结构表面层（S 层）中发现的表面层同源 (SLH) 结构域具有同源性。然而，外膜区域、对 SDS 和高温变性的抵抗力以及 C 端残基是苯丙氨酸的发现表明 ompM1 编码孔蛋白。线程分析与跨膜结构域的鉴定相结合表明，OmpM1 的 C 端区域（残基 250-430）可能形成 16 链 β 桶，并且似乎与先前在蓝藻聚球藻 PCC6301 中描述的不寻常的 N 端 SLH 结构域含有 β 桶孔蛋白有关。 (C) 2009 Elsevier Ltd. 保留所有权利。
Thin sectioning and freeze-fracture-etch of the ovine ruminal isolate Mitsuokella multacida strain 46/5(2) revealed a Gram-negative envelope ultra-structure consisting of a peptidoglycan wall overlaid by an outer membrane. Sodium-dodecyl-sulfate-polyacrylamide gel electrophoretic (SIDS-PAGE) analysis of whole cells, cell envelopes and Triton X-100 extracted envelopes in combination with thin-section and N-terminal sequence analyses demonstrated that the outer membrane contained two major proteins (45 and 43 kDa) sharing identical N-termini (A-A-N-P-F-S-D-V-P-A-D-H-W-A-Y-D). A gene encoding a protein with a predicted N-terminus identical to those of the 43 and 45 kDa outer-membrane proteins was cloned. The 1290 bp open reading frame encoded a 430 amino acid polypeptide with a predicted molecular mass of 47,492 Da. Cleavage of a predicted 23 amino acid leader sequence would yield a protein with a molecular mass of 45,232 Da. Mass spectroscopic analysis confirmed that the cloned gene (ompM1) encoded the 45 kDa outer-membrane protein. The N-terminus of the mature OmpM1 protein (residues 24-70) shared homology with surface-layer homology (SLH) domains found in a wide variety of regularly structured surface-layers (S-layers). However, the outer-membrane locale, resistance to denaturation by SDS and high temperatures and the finding that the C-terminal residue was a phenylalanine suggested that ompM1 encoded a porin. Threading analysis in combination with the identification of membrane spanning domains indicated that the C-terminal region of OmpM1 (residues 250-430) likely forms a 16-strand beta-barrel and appears to be related to the unusual N-terminal SLH-domain-containing beta-barrel-porins previously described in the cyanobacterium Synechococcus PCC6301. (C) 2009 Elsevier Ltd. All rights reserved.