RfaH enhances elongation of Escherichia coli hlyCABD mRNA.
Escherichia coli hlyCABD operons encode the polypeptide component (Hly A) of an extracellular cytolytic toxin, as well as proteins required for its acylation (HlyC) and sec-independent secretion (HlyBD). Previous reports suggested that the E. coli protein RfaH is required for wild-type hemolysin expression, either by positively activating hly transcript initiation (M. J. A. Bailey, V. Koronakis, T. Schmoll, and C. Hughes, Mol. Microbiol. 6:1003-1012, 1992) or by promoting proper insertion of hemolysin export machinery in the E. coli outer membrane (C. Wandersman and S. Letoffe, Mol. Microbiol. 7:141-150, 1993). RfaH is also required for wild-type levels of mRNA transcribed from promoter-distal genes in the rfaQ-K, traY-Z, and rplK-rpoC gene clusters, suggesting that RfaH is a transcriptional antiterminator. We tested these models by analyzing the effects of rfaH mutations on hlyCABD mRNA synthesis and decay, HlyA protein levels, and hemolytic activity. The model system included a uropathogenic strain of E. coli harboring hlyCABD on the chromosome and E. coli K-12 transformed with the hlyCABD operon on a recombinant plasmid. Our results suggest that RfaH enhances hlyCABD transcript elongation, consistent with the model of RfaH involvement in transcriptional antitermination in E. coli. We also demonstrated that RfaH increases toxin efficacy. Modulation of hemolysin activity may be an indirect effect of RfaH-dependent E. coli outer membrane chemotype, which is consistent with the model of lipopolysaccharide involvement in hemolytic activity.
Leeds
J A
JA
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison 53706, USA.
Welch
R A
RA
D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D006460:Hemolysin Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D012333:RNA, Messenger