Abstract
Escherichia coli hemolysin (HlyA) is the prototype toxin of a major family of exoproteins produced by Gram-negative bacteria known as "repeats in toxins." Only fatty acid-acylated HlyA molecules at residues Lys564 and Lys690 are able to damage the target cell membrane. Fatty acylation of pro-HlyA is dependent on the co-synthesized acyltransferase HlyC and the acylated form of acyl-carrier protein. By using a collection of hlyA and hlyC mutant strains, the processing of HlyC was investigated. HlyC was not detected by Western blot in an E. coli strain encoding hlyC and hlyA, but it was present in a strain encoding only hlyC. The hlyC mRNA pattern, however, was similar in both strains indicating that the turnover of HlyC does not occur at the transcriptional level. HlyC was detected in Western blots of cell lysates from an E. coli strain encoding HlyC and a HlyA derivative where both acylation sites were substituted. Similar results were obtained when HlyC was expressed in a hlyA mutant strain lacking part of a putative HlyC binding domain, indicating that this particular HlyA region affects HlyC stability. We did not detect HlyC in cell lysates from hlyC mutants with different abilities to acylate pro-HlyA, suggesting that the degradation of HlyC is not related to the HlyA acylation process. The protease systems ClpAP, ClpXP, and FtsH were found to be responsible for the HlyA-dependent processing of HlyC.
Highlights
Escherichia coli hemolysin (HlyA)1 is the major representative of a family of proteins secreted by proteobacteria known as “repeats in toxin” (RTX)
E. coli hemolysin (HlyA) is synthesized as an inactive precursor, which is activated through fatty acid acylation of residues Lys564 and Lys690 [13, 14] when both the HlyC protein and the acylated form of acyl-carrier protein are present [13, 15, 16]
Only HlyC from E. coli and CyaC from B. pertussis have been shown to be involved in the fatty acylation of their corresponding RTX toxin [13, 21]
Summary
HlyA, Escherichia coli hemolysin; RTX, repeats in toxins; GST, glutathione S-transferase; PCR, polymerase chain reaction; IPTG, isopropyl -D-thiogalactopyranoside; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; MALDI, matrix-assisted laser desorption/ionization; HlyC, Escherichia coli HlyC acyltransferase. 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]. We present evidence that HlyC is degraded in vivo by different protease systems when HlyA is present
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