Mustapha A, Hutkins R W, Zirnstein G W
Department of Food Science and Technology, University of Nebraska-Lincoln 68583-0919, USA.
J Dairy Sci. 1995 May;78(5):989-97. doi: 10.3168/jds.S0022-0302(95)76714-5.
The objective of this research was to clone and characterize the galactokinase gene (galK) from Streptococcus thermophilus F410. Partially digested genomic DNA was cloned into pBR322 and transformed into galK Escherichia coli, and a galactose-fermenting transformant was isolated. Restriction analysis revealed that the transformant resulted from a Sau3A-HindIII 4.0-kb fragment. Galactokinase activity in the recombinant was 10 times that of the parent strain. Analysis of the DNA sequence showed the presence of a 1.3-kb open reading frame that had high homology with the galK gene from other organisms. A putative ribosome-binding site, start and stop codons, and -10 and -35 sequences were identified. The predicted protein had a molecular mass of 49 kDa, which corresponded to the estimated size of a band apparent by SDS-PAGE. Amino acid sequence homologies with other galactokinases ranged from 50 to 62% similarity. Northern blots were performed between the galK gene and mRNA from S. thermophilus. No hybridization signals were observed for cells grown in glucose, but cells grown in lactose or galactose gave moderate and strong signals. The results suggest that repression of the galK gene by glucose may be responsible for the galactose-releasing phenotype in these strains.
本研究的目的是克隆和鉴定嗜热链球菌F410的半乳糖激酶基因（galK）。将部分消化的基因组DNA克隆到pBR322中，并转化到galK大肠杆菌中，分离出一株能发酵半乳糖的转化子。限制性分析表明，该转化子是由Sau3A-HindIII 4.0-kb片段产生的。重组体中的半乳糖激酶活性是亲本菌株的10倍。DNA序列分析显示存在一个1.3-kb的开放阅读框，与其他生物体的galK基因具有高度同源性。鉴定出一个假定的核糖体结合位点、起始和终止密码子以及-10和-35序列。预测的蛋白质分子量为4九 kDa，这与SDS-PAGE显示的一条带的估计大小相对应。与其他半乳糖激酶的氨基酸序列同源性在50%至62%之间。对嗜热链球菌的galK基因和mRNA进行了Northern杂交。在葡萄糖中生长的细胞未观察到杂交信号，但在乳糖或半乳糖中生长的细胞给出了中等强度和强信号。结果表明，葡萄糖对galK基因的抑制可能是这些菌株中半乳糖释放表型的原因。
