Transcriptional organization of the Escherichia coli hemolysin genes

Abstract

The transcriptional organization of the Escherichia coli hemolysin genes (hlyCABD) encoded by pSF4000 was examined. The use of different hemolysin gene-specific radiolabeled probes in blots containing isolated in vivo RNA revealed 4.0-kilobase hlyCA and 8.0-kilobase hlyCABD transcripts. The treatment of cells with rifampin just before RNA isolation showed the half-lives of these mRNAs to be 10.2 and 4.4 min, respectively. The 5' ends of the hly transcripts were 462 and 464 nucleotides from the putative initiation codon of hlyC based on a primer extension method of RNA mapping. Deletion analysis of pSF4000 combined with quantification of the hemolysin structural protein HlyA by immunoblotting confirmed that major control of HlyA expression occurs within a 168-base-pair PstI fragment located 433 base pairs upstream of the start of hlyC. A second recombinant plasmid, pANN202-312, encoding an E. coli hemolysin of different origin expressed 6-fold less total HlyA and 50-fold less extracellular HlyA than pSF4000 in identical cell backgrounds. The pANN202-312 recombinant had a different hly promoter, with the hly mRNA beginning 264 nucleotides upstream from the start of hlyC. We showed by RNA blotting that cells harboring pANN202-312 compared with pSF4000 have similar steady-state levels of the hlyCA transcript but they lack a consistently detectable hlyCABD transcript. We propose that one reason for the disparate levels of extracellular hemolysin produced by hemolytic W. coli is dissimilar levels of mRNA encoding in part the transport genes hlyB and hlyD.

hlyM, a transcriptional silencer downstream of the promoter in the hly operon of Escherichia coli

“…Transcription of the hly operon produces a major 4-kb hlyCA transcript and a minor 8-kb hlyCABD transcript (31). The two transcripts initiate at the same promoter, located upstream of the hlyC gene (12,31).…”

“…Transcription of the hly operon produces a major 4-kb hlyCA transcript and a minor 8-kb hlyCABD transcript (31). The two transcripts initiate at the same promoter, located upstream of the hlyC gene (12,31). The minor transcript is thought to be produced by readthrough, or antitermination, at a Rho-independent transcriptional terminator located between hlyA and hlyB (15).…”

Bacterial hemolysins as virulence factors

“…On the contrary, little sequence homology was observed between several E.coli determinants in the upstream and downstream regions flanking the four structural hly genes. A region (upstream of hlyC) of more than 400 bp was defined as a regulatory site in a chromosomal hly determinant which apparently contain two promoters from which a polycistronic m-RNA is transcribed (11) . A terminator-like structure was observed in all h!Y determinants in the intercistronic region between hlyA and hlyB and evidence was presented that most transcripts terminate at this point (11).…”

In Vivo Proteolytic Degradation of the Escherichia coli Acyltransferase HlyC

“…Moreover, since hlyA is transcribed after hlyC to generate hlyCA or hlyCABD mRNA (24,25), detection of HlyA is an indirect proof that hlyC is indeed transcribed. Fully acylated HlyA was detected in culture supernatants of cells carrying hlyA and hlyC in trans or in cis (26), demonstrating that HlyC must have been produced.…”

“…Recently, a novel type of fatty acyltransferase activity, that catalyzes the formation of an internal protein amide bond has been described for HlyC (22,23), despite the fact that HlyC does not show any significant similarity to known acyl transferases (13,19), the protein seems to acylate HlyA in an equimolar fashion, and it appears to be functionally consumed during the reaction (16,22). In this respect, it is also notable that HlyC and HlyA are encoded in the same mRNA and therefore synthesized at nearly equimolar amounts (24,25). The unique acylation reaction catalyzed by HlyC does not involve covalent attachment of fatty acid molecules to the acyltransferase but a direct interaction between acylACP-HlyC and pro-HlyA through a non-covalent ternary complex (22).…”