Purification of HlyX, a potential regulator of haemolysin synthesis, and properties of HlyX:FNR hybrids.
The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae is homologous to FNR, an anaerobic transcriptional regulator of Escherichia coli. It endows a haemolytic phenotype upon E. coli, and will complement the anaerobic respiratory deficiencies of fnr mutants of E. coli. The coding region of the hlyX gene was expressed in E. coli and the HlyX protein was purified by using an assay based on its immunological cross-reactivity with anti-FNR antibodies. The HlyX protein had the predicted N-terminal sequence, and resembled the isolated FNR protein in size (Mr 29,000) and monomeric organization. It has no detectable haemolysin activity per se, and is therefore presumed to confer a haemolytic phenotype by activating a latent haemolysin gene in E. coli. Studies with gene fusions showed that HlyX, like FNR, can function as an anaerobic activator and repressor of FNR-regulated genes in vivo. Plasmids that express hybrid HlyX:FNR proteins in which the 189/190-residue N-terminal segments and the remaining 50/60-residue C-terminal segments are exchanged, retained their FNR-specific functions but failed to confer a haemolytic phenotype. This suggests that the specificity for activating the haemolytic response requires the participation of unique features in both the N- and C-terminal segments of HlyX.
Green
J
J
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, U.K.
Sharrocks
A D
AD
MacInnes
J I
JI
Guest
J R
JR
D016977:Actinobacillus pleuropneumoniae / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000235:genetics; D006824:Hybridization, Genetic; D007506:Iron-Sulfur Proteins; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D055503:Protein Multimerization; D014157:Transcription Factors / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D006460:Hemolysin Proteins; D007506:Iron-Sulfur Proteins; D009838:Oligodeoxyribonucleotides; D014157:Transcription Factors