Characterization of a new operon, as-48EFGH, from the as-48 gene cluster involved in immunity to enterocin AS-48
Characterization of a new operon, as-48EFGH, from the as-48 gene cluster involved in immunity to enterocin AS-48
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DOI：
10.1128/aem.69.2.1229-1236.2003
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发表时间：
2003-02-01
影响因子：
4.4
通讯作者：
Maqueda, M
中科院分区：
文献类型：
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作者：
Diaz, M;Valdivia, E;Maqueda, M
肠菌素AS-48是由粪肠球菌S-48产生的环肽，其遗传决定簇已在接合质粒pMB 2中鉴定。先前已在pAM 401（pAM 401 -52）中克隆并测序了一个7.8 kb的区域，该区域携带AS-48产生和抗AS-48免疫所需的最低信息。在该区域，已鉴定出as-48 A结构基因、as-48 B、as-48 C、as-48 C、as-48 D和as-48 D基因以及开放阅读框6（ORF 6）和ORF 7。在本工作中，对从最后一个鉴定的ORF（ORF 7）下游克隆的pMB 2的BglII B片段（6.6-kb）进行的序列分析揭示了存在两个新的ORF，as-48 G和as-48 H，这是AS-48完全表达所必需的。因此，用pAM 401 -81质粒获得的JH 2 -2转化体成为生产者，并在野生型水平上具有抗性。在最后一个基因as-48 EFGH中的Tn 5破坏实验不能再现这些表达水平，证实了这些基因的表达对于获得野生型pMB 2质粒赋予的表型是必需的。as-48 EFGH操纵子编码一种新的ABC转运蛋白，可能参与生产者的自我保护。根据所观察到的相似性，As-48 G将是ATP结合结构域，As-48 E和As-48-H的推导的氨基酸序列可以被指定为跨膜亚基，并且As-48 F，具有N-末端跨膜段和卷曲螺旋结构域，强烈地类似于一些已知的ABC转运蛋白辅助蛋白的结构，其在细胞中的定位进行了讨论。该基因簇由JH 2 -2（pAM 401 -81）中的两个多顺反子mRNA T-2和T-3协同表达。我们的结果还表明，T1的表达是可以调节的，因为在JH 2 -2（pAM 401（EH））转化体中，没有检测到T3，这表明这些基因本身并不赋予免疫力，这与AS-48免疫所需的AS-48 D（1）基因一致。
Enterocin AS-48 is a cyclic peptide produced by Enterococcus faecalis S-48 whose genetic determinants have been identified in the conjugative plasmid pMB2. A region of 7.8 kb, carrying the minimum information required for production of and immunity against AS-48, had been previously cloned and sequenced in pAM401 (pAM401-52). In this region, the as-48A structural gene and as-48B, as-48C, as-48C,, as-48D, and as-48D, genes and open reading frame 6 (ORF6) and ORF7 had been identified. The sequence analysis carried out in this work in the BglII B fragment (6.6-kb) from pMB2 cloned downstream from the last ORF identified (ORF7) revealed the existence of two new ORFs, as-48G and as-48H, necessary for full AS-48 expression. Thus, JH2-2 transformants obtained with the pAM401-81 plasmid became producers and resistant at the wild-type level. Tn5 disruption experiments in the last genes, as-48EFGH, were not able to reproduce these expression levels, confirming that expression of these genes is necessary to get the phenotype conferred by the wild-type pMB2 plasmid. The as-48EFGH operon encodes a new ABC transporter that could be involved in producer self-protection. On the basis of the observed similarities, As-48G would be the ATP-binding domain, the deduced amino acid sequences of As-48E and As48-H could be assigned as transmembrane subunits, and As-48F, with an N-terminal transmembrane segment and a coiled-coil domain, strongly resembles the structure of some known ABC transporter accessory proteins whose localization in the cell is discussed. This cluster of genes is expressed by two polycistronic mRNAs, T-2 and T-3 in JH2-2(pAM401-81) in coordinate expression. Our results also suggest that expression of T, could be regulated, because in JH2-2(pAM401(EH)) transformants, T-3 was not detected, suggesting that these genes do not by themselves confer immunity, in accordance with the requirement for the as-48D(1) gene for immunity against AS-48.