Shimkets L J
Department of Microbiology, University of Georgia, Athens 30602.
J Bacteriol. 1990 Jan;172(1):24-30. doi: 10.1128/jb.172.1.24-30.1990.
The fprA gene is immediately adjacent to the csgA gene (formerly known as spoC) of Myxococcus xanthus. Whereas the csgA gene has an essential role in cell interactions during the developmental cycle, the function of the fprA gene is unknown. Gene disruption was used to determine what affect a null mutation in this gene has on the phenotype of the cell. A csgA-fprA deletion and an fprA frameshift mutation were constructed in vitro in a cloned copy of this locus and then inserted into the M. xanthus chromosome to create a merodiploid with the wild-type and mutant alleles in tandem. The merodiploid was then allowed to segregate one of the two alleles along with the vector sequences in an effort to replace the wild-type allele with the mutant allele. All of the segregants had the wild-type allele, suggesting that a functional fprA gene is essential for vegetative growth. The fprA gene was placed under control of the lacZ transcriptional and translational signals and overexpressed in Escherichia coli, and the new host was examined for any phenotypic changes. A 27-kilodalton protein was observed in sodium dodecyl sulfate-polyacrylamide gels of total-cell protein as predicted from the DNA sequence of this gene. Overexpression of FprA caused the accumulation of a yellow pigment with spectral and redox properties similar to that of the flavins. The pigment cochromatographed with flavin mononucleotide by Silica Gel G thin-layer chromatography. Approximately two-thirds of the total cellular flavin was associated with soluble protein. The major soluble flavin-associated protein was purified on DEAE-Bio-Gel A and Phenyl-Sepharose CL-4B and by polyacrylamide gel electrophoresis. The amino acid composition of the purified protein was similar to that predicted from the DNA sequence of the FprA fusion protein. Apparently, overproduction of FprA (for flavin-associated protein A) in E. coli resulted in a large increase in flavin biosynthesis. Together, these results suggest that the fprA gene encodes a protein that is associated with flavin mononucleotide and has an essential function in M. xanthus.
fprA基因紧邻黄色粘球菌的csgA基因（以前称为spoC）。虽然csgA基因在发育周期中的细胞相互作用中起重要作用，但fprA基因的功能尚不清楚。基因破坏被用于确定该基因的无效突变对细胞表型有何影响。在该基因座的克隆拷贝中体外构建了csgA-fprA缺失和fprA移码突变，然后插入到黄色粘球菌染色体中，以创建一个带有串联的野生型和突变等位基因的部分二倍体。然后让部分二倍体分离两个等位基因之一以及载体序列，试图用突变等位基因取代野生型等位基因。所有分离株都具有野生型等位基因，这表明功能性fprA基因对于营养生长至关重要。fprA基因置于lacZ转录和翻译信号的控制之下，并在大肠杆菌中过表达，然后检查新宿主是否有任何表型变化。如该基因的DNA序列所预测的那样，在全细胞蛋白的十二烷基硫酸钠-聚丙烯酰胺凝胶中观察到一种27千道尔顿的蛋白质。FprA的过表达导致一种黄色色素的积累，其光谱和氧化还原特性与黄素相似。该色素通过硅胶G薄层色谱与黄素单核苷酸共色谱。大约三分之二的总细胞黄素与可溶性蛋白相关。主要的可溶性黄素相关蛋白在DEAE-生物凝胶A和苯基-琼脂糖CL-4B上纯化，并通过聚丙烯酰胺凝胶电泳进行纯化。纯化蛋白的氨基酸组成与FprA融合蛋白的DNA序列所预测的相似。显然，大肠杆菌中FprA（黄素相关蛋白A）的过量产生导致黄素生物合成大幅增加。总之，这些结果表明fprA基因编码一种与黄素单核苷酸相关的蛋白，并且在黄色粘球菌中具有重要功能。
