Secretion and Transcriptional Regulation of the Latex-Clearing Protein, Lcp, by the Rubber-Degrading Bacterium Streptomyces sp. Strain K30
Secretion and Transcriptional Regulation of the Latex-Clearing Protein, Lcp, by the Rubber-Degrading Bacterium Streptomyces sp. Strain K30
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DOI：
10.1128/aem.01001-08
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发表时间：
2008-07
影响因子：
4.4
通讯作者：
Meral Yikmis;M. Arenskötter;K. Rose;N. Lange;Henrike Wernsmann;Lars Wiefel;A. Steinbüchel
中科院分区：
文献类型：
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作者：
Meral Yikmis;M. Arenskötter;K. Rose;N. Lange;Henrike Wernsmann;Lars Wiefel;A. Steinbüchel
摘要 大约 22,000 个 1-甲基-3-硝基-1-亚硝基胍和紫外线诱导的橡胶降解细菌链霉菌突变体。菌株 K30 的特征在于能够在天然橡胶乳胶覆盖琼脂平板上产生透明区域。 35 个突变体仅在橡胶裂解方面存在缺陷，并且在表型上与野生型 lcp（乳胶清除蛋白）基因互补。 69 个突变体表现出多效性表型，并且在橡胶和木聚糖的利用方面受到损害，表明负责这些聚合物初始裂解的酶通过相同的分泌途径输出（Q. K. Beg、M. Kapoor、L. Mahajan 和 G. S. Hoondal，Appl. Microbiol. Biotechnol. 56:326-3381, 2001; U.K.Laemmli，《自然》227：680-685，1970）。对 lcp 编码的氨基酸序列的分析揭示了双精氨酸基序，表明 Lcp 是双精氨酸易位 (Tat) 途径的底物 (K. Dilks, W. Rose, E. Hartmann, 和 M. Pohlschröder, J. Bacteriol. 185:1478-1483, 2003)。浅青紫链霉菌 10-164 的 tatC 破坏突变体，含有来自链霉菌属的 lcp。菌株 K30 不能在橡胶覆盖琼脂平板上形成透明区域。此外，在上清液中检测到Lcp和增强型绿色荧光蛋白融合蛋白。使用在Lcp上游的信号肽中具有双精氨酸基序的大肠杆菌，获得了分泌Lcp的明确证据。转录分析揭示了 Lcp 在葡萄糖生长的细胞中的基础表达，并且在聚（顺式-1,4-异戊二烯）存在下明显诱导了 Lcp 的转录。相比之下，oxiB 和 oxiA 直接位于 lcp 下游，推测编码一种异多聚醛脱氢酶，氧化 Lcp 从聚（顺式 1,4-异戊二烯）产生的初级裂解产物，仅在聚（顺式 1,4-异戊二烯）存在时才表达。因此，需要低水平的lcp表达来感测介质中的聚合物。然后，橡胶降解产物可能诱导编码酶的基因转录，催化聚（顺式1,4-异戊二烯）降解的后续步骤以及LCP本身的转录。 lcp、oxiB和oxiA似乎构成操纵子，因为检测到包含这三个基因的多顺反子mRNA。 lcp的转录起始位点定位在lcp起始密码子上游400bp处。
ABSTRACT About 22,000 1-methyl-3-nitro-1-nitrosoguanidine- and UV-induced mutants of the rubber-degrading bacterium Streptomyces sp. strain K30 were characterized for the ability to produce clear zones on natural rubber latex overlay agar plates. Thirty-five mutants were defective solely in cleavage of rubber and were phenotypically complemented with the wild-type lcp (latex clearing protein) gene. Sixty-nine mutants exhibited a pleiotropic phenotype and were impaired in utilization of rubber and xylan, indicating that the enzymes responsible for the initial cleavage of these polymers are exported by the same secretion pathway (Q. K. Beg, M. Kapoor, L. Mahajan, and G. S. Hoondal, Appl. Microbiol. Biotechnol. 56:326-3381, 2001; U. K. Laemmli, Nature 227:680-685, 1970). Analysis of the amino acid sequence encoded by lcp revealed a twin-arginine motif, indicating that Lcp is a substrate of the twin-arginine translocation (Tat) pathway (K. Dilks, W. Rose, E. Hartmann, and M. Pohlschröder, J. Bacteriol. 185:1478-1483, 2003). A tatC disruption mutant of Streptomyces lividans 10-164 harboring lcp from Streptomyces sp. strain K30 was not capable of forming clear zones on rubber overlay agar plates. Moreover, Lcp and enhanced green fluorescent protein fusion proteins were detected in the supernatant. Using Escherichia coli having the twin-arginine motif in the signal peptide upstream of Lcp, clear evidence that Lcp is secreted was obtained. Transcriptional analysis revealed basal expression of Lcp in glucose-grown cells and that transcription of lcp is obviously induced in the presence of poly(cis-1,4-isoprene). In contrast, oxiB and oxiA, which are located directly downstream of lcp and putatively encode a heteromultimeric aldehyde dehydrogenase oxidizing the primary cleavage products generated by Lcp from poly(cis-1,4-isoprene), were expressed only in the presence of poly(cis-1,4-isoprene). Expression of lcp at a low level is thus required for sensing the polymer in the medium. Rubber degradation products may then induce the transcription of genes coding for enzymes catalyzing the later steps of poly(cis-1,4-isoprene) degradation and the transcription of lcp itself. lcp, oxiB, and oxiA seem to constitute an operon, as a polycistronic mRNA comprising these three genes was detected. The transcriptional start site of lcp was mapped 400 bp upstream of the lcp start codon.