Entsch B, Nan Y, Weaich K, Scott K F
Department of Biochemistry, Microbiology and Nutrition, University of New England, Armidale, N.S.W., Australia.
Gene. 1988 Nov 30;71(2):279-91. doi: 10.1016/0378-1119(88)90044-3.
The only recognized gene for the metabolism of p-hydroxybenzoate in Pseudomonads (pobA) has been isolated from Pseudomonas aeruginosa to provide the DNA for mutagenesis studies of the protein product, p-hydroxybenzoate hydroxylase. Since pobA is induced by p-hydroxybenzoate to produce large amounts of enzyme, its regulation in P. aeruginosa is significant. The nucleotide sequence of pobA is presented with the derived amino acid (aa) sequence, which has only two substitutions compared to the amino acid sequence obtained from the enzyme from P. fluorescens. The derived amino acid sequence predicts that the enzyme is a single polypeptide of 394 aa residues and contains one molecule of FAD. The complete structure of the protein from P. aeruginosa can be derived by analogy from the published structure of the protein from P. fluorescens. Transcription mapping was used to determine that there is one site for the initiation of mRNA synthesis in P. aeruginosa. The presence of a putative operator in the sequence suggests primary regulation by a repressor protein which binds p-hydroxybenzoate. The ribosome-binding site permits translation of the gene in Escherichia coli at levels comparable to its production in P. aeruginosa, but it produces no detectable product in E. coli under the influence of its own promoter sequence. The promoter does not conform to the common consensus sequence of E. coli promoters. The results have identified an apparent novel promoter for P. aeruginosa, which may reflect the presence of a sigma factor required for pobA induction. Repression of expression by glucose suggests a binding site in the sequence for catabolite repression.
假单胞菌中唯一已知的对羟基苯甲酸代谢基因（pobA）已从铜绿假单胞菌中分离出来，以提供用于对其蛋白质产物对羟基苯甲酸羟化酶进行诱变研究的DNA。由于pobA由对羟基苯甲酸诱导产生大量酶，因此其在铜绿假单胞菌中的调控具有重要意义。本文给出了pobA的核苷酸序列及其推导的氨基酸序列，与从荧光假单胞菌中获得的该酶的氨基酸序列相比，仅有两个替换。推导的氨基酸序列预测该酶是一个由394个氨基酸残基组成的单链多肽，并且含有一分子FAD。铜绿假单胞菌中该蛋白质的完整结构可通过类比已发表的荧光假单胞菌中该蛋白质的结构推导得出。转录图谱分析用于确定铜绿假单胞菌中mRNA合成起始的一个位点。序列中存在一个假定的操纵基因，提示主要由一种结合对羟基苯甲酸的阻遏蛋白进行调控。核糖体结合位点使得该基因在大肠杆菌中能够以与其在铜绿假单胞菌中产生的水平相当的水平进行翻译，但在其自身启动子序列的影响下，在大肠杆菌中不产生可检测到的产物。该启动子不符合大肠杆菌启动子的常见共有序列。这些结果确定了一个明显的铜绿假单胞菌新启动子，这可能反映了pobA诱导所需的一种σ因子的存在。葡萄糖对表达的抑制表明该序列中存在一个分解代谢物阻遏的结合位点。
