Beta‐lactamase topology probe analysis of the OutO NMePhe peptidase, and six other Out protein components of the Erwinia carotovora general secretion pathway apparatus
Beta‐lactamase topology probe analysis of the OutO NMePhe peptidase, and six other Out protein components of the Erwinia carotovora general secretion pathway apparatus
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OutO NMePhe 肽酶和胡萝卜软腐欧文氏菌一般分泌途径装置的其他六种 Out 蛋白成分的 β-内酰胺酶拓扑探针分析
DOI：
10.1111/j.1365-2958.1994.tb01033.x
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发表时间：
1994
影响因子：
3.6
通讯作者：
G. Salmond
中科院分区：
文献类型：
--
作者：
P. Reeves;Paul Douglas;G. Salmond
欧文氏菌的外基因簇。编码果胶酶和纤维素酶分泌所需的一般分泌途径（GSP）装置的蛋白质。我们已经使用胡萝卜软腐欧文氏菌胡萝卜软腐欧文氏菌胡萝卜胡萝卜软腐菌（Ecc）out基因和拓扑探针blaM，以评估Out蛋白区域在大肠杆菌和Ecc中跨细胞质膜输出BlaM的能力。对于outO基因产物（NMePhe肽酶），已经鉴定了7个跨膜区，并预测了另外一个跨膜区。具有最高亲水性水平的OutO区域可能作为位于两个疏水结构域之间的大细胞质环存在，并且位于蛋白质的N末端。当BlaM融合在OutO的最后一个疏水段的C末端侧时，所得的杂合蛋白将BlaM部分转移到周质，同时保留OutO活性。去除一部分的这种疏水拉伸导致的损失的OutO活性，这表明有严格的限制拓扑结构的完整性OutO保持催化功能。当outG、-H、-I、-J、-K和-N与blaM融合时，所得表型表明每种蛋白质的大部分靶向周质。我们的结果表明，这六个Out蛋白，当由E。coli或Ecc中的每一种都至少暂时地在细胞质膜中采用II型双位构象。对于OutG、-H、-I和-J，这可能表示在Ecc中由OutO处理之前的膜拓扑。当从T7基因10启动子构建体体内产生时，outG产物在Ecc中加工，而outO突变体RJP 249不能加工前OutG。位于前OutG、-H、-I和-J疏水段C端的BlaM融合体由野生型Ecc处理，但不由RJP 249或E处理。因此，这些蛋白质的周质结构域在肽酶切割反应中不起作用。使用OutG-BlaM融合构建体来证明NMePhe肽酶在其他细菌菌株中的活性，包括E.胡萝卜软腐菌亚种carotovora（ATCC 39048）、胡萝卜软腐肠杆菌E.胡萝卜软腐菌亚种atroseptica（SCR 11043）和欧文氏菌（Erwiniachenii）（3937）。
The out gene cluster of Erwinia spp. encodes the proteins of the general secretory pathway (GSP) apparatus that is required for pectinase and cellulase secretion. We have used fusions between Erwinia carotovora subsp. carotovora (Ecc) out genes and the topology probe blaM to assess the ability of Out protein regions to export BlaM across the cytoplasmic membrane in Escherichia coli and Ecc. For the outO gene product (an NMePhe peptidase), seven transmembrane regions have been identified and one more is predicted. The region of OutO with the highest level of hydrophilicity is likely to exist as a large cytoplasmic loop, located between two hydrophobic domains, and is positioned towards the N‐terminus of the protein. When BlaM was fused on the C‐terminal side of the last hydrophobic stretch of OutO, the resulting hybrid protein transferred the BlaM moiety to the periplasm whilst retaining OutO activity. Removal of a portion of this hydrophobic stretch resulted in the loss of OutO activity, suggesting that there are tight constraints on the topological integrity of OutO for maintaining catalytic function. When outG, ‐H, ‐I, ‐J, ‐K and ‐N were fused to blaM, the resulting phenotype suggested that the majority of each protein was targeted to the periplasm. Our results indicate that these six Out proteins, when produced by E. coli or Ecc, each adopt, at least temporarily, a type II bitopic conformation in the cytoplasmic membrane. For OutG, ‐H, ‐I and ‐J this probably represents the membrane topology prior to processing by OutO in Ecc. When produced in vivo from a T7 gene 10 promoter construct, the outG product was processed in Ecc whereas the outO mutant RJP249 failed to process pre‐OutG. BlaM fusions positioned on the C‐termtnal side of the hydrophobic stretches of pre‐OutG, ‐H, ‐I, and ‐J were processed by wild‐type Ecc but not RJP249 or E. coli DHL Thus the periplasmic domains of these proteins play no role in the peptidase cleavage reaction. An OutG‐BlaM fusion construct was used to demonstrate NMePhe peptidase activity in other bacterial strains including E. carotovora subsp. carotovora (ATCC39048), E. carotovora subsp. atroseptica (SCR11043) and Erwinia chrysanthemi (3937).
DOI：
10.1073/pnas.82.4.1074
发表时间：
1985-01-01
影响因子：
11.1
作者：
TABOR, S;RICHARDSON, CC
通讯作者：
RICHARDSON, CC