The Myxococcus xanthus FprA protein causes increased flavin biosynthesis in Escherichia coli

Abstract

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.