Pruneda-Paz José Luis, Linares Mauricio, Cabrera Julio E, Genti-Raimondi Susana
Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.
J Steroid Biochem Mol Biol. 2004 Jan;88(1):91-100. doi: 10.1016/j.jsbmb.2003.10.010.
Comamonas testosteroni is a soil bacterium, which can use a variety of steroids as carbon and energy source. Even if it can be estimated that the complete degradation of the steroid nucleus requires more than 20 enzymatic reactions, the complete molecular characterization of the genes encoding these steroid degradative enzymes as well as the genetic organization of them remain to be elucidated. We have previously reported the cloning and nucleotide sequence of two steroid-inducible genes, beta hsd and stdC encoding 3 beta-17 beta-hydroxysteroid dehydrogenase and a hypothetical protein respectively, located in both ends of a 3.2kb HindIII fragment. Herein, we report the cloning and characterization of another steroid-inducible gene, called sip48 (steroid inducible protein), located between these two genes. The analysis of Sip48 amino acid sequence predicts a protein of 438 amino acids with a molecular mass of 48.5 kDa. This protein bears high homology with conserved hypothetical proteins of unknown function described in Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas putida, Burkholderia fungorum, Shewanella oneidensis, Pseudomonas fluorescens and Thauera aromatica. The predicted protein shows a typical structure of a leader peptide at its N-terminus. A 48.5 kDa protein encoded by the recombinant plasmid was detected by SDS-PAGE analysis of in vitro [35S]-methionine labeled polypeptides. Analysis of gene expression indicates that Sip48 is tightly controlled at the transcriptional level by several steroid compounds. In addition, transcriptional analysis of sip48 and beta hsd in a sip48 mutant strain, indicates that both genes are transcribed as a polycistronic mRNA. lacZ transcriptional fusions integrated into the chromosome of C. testosteroni demonstrate that a steroid-inducible promoter located upstream of sip48 regulates the expression of both genes.
睾丸酮丛毛单胞菌是一种土壤细菌，它能够利用多种类固醇作为碳源和能源。即便据估计类固醇核的完全降解需要20多种酶促反应，但编码这些类固醇降解酶的基因的完整分子特征及其基因组织仍有待阐明。我们之前报道了两个类固醇诱导基因β hsd和stdC的克隆及核苷酸序列，它们分别编码3β - 17β - 羟基类固醇脱氢酶和一种假定蛋白，位于一个3.2kb HindIII片段的两端。在此，我们报道了另一个位于这两个基因之间的类固醇诱导基因sip48（类固醇诱导蛋白）的克隆及特征。对Sip48氨基酸序列的分析预测其为一个由438个氨基酸组成、分子量为48.5 kDa的蛋白质。该蛋白与铜绿假单胞菌、丁香假单胞菌、恶臭假单胞菌、真菌伯克霍尔德菌、希瓦氏菌、荧光假单胞菌和芳香陶厄氏菌中描述的功能未知的保守假定蛋白具有高度同源性。预测的蛋白在其N端显示出典型的前导肽结构。通过对体外[35S] - 甲硫氨酸标记的多肽进行SDS - PAGE分析，检测到了由重组质粒编码的48.5 kDa蛋白。基因表达分析表明，Sip48在转录水平受到几种类固醇化合物的严格调控。此外，对sip48突变株中sip48和β hsd的转录分析表明，这两个基因都作为多顺反子mRNA进行转录。整合到睾丸酮丛毛单胞菌染色体中的lacZ转录融合实验表明，位于sip48上游的一个类固醇诱导型启动子调控这两个基因的表达。
