Nucleotide sequencing of the Proteus mirabilis calcium-independent hemolysin genes (hpmA and hpmB) reveals sequence similarity with the Serratia marcescens hemolysin genes (shlA and shlB).
We cloned a 13.5-kilobase EcoRI fragment containing the calcium-independent hemolysin determinant (pWPM110) from a clinical isolate of Proteus mirabilis (477-12). The DNA sequence of a 7,191-base-pair region of pWPM110 was determined. Two polypeptides are encoded in this region, HpmB and HpmA (in that transcriptional order), with predicted molecular masses of 63,204 and 165,868 daltons, respectively. A putative Fur-binding site was identified upstream of hpmB overlapping the -35 region of the proposed hpm promoter. In vitro transcription-translation of pWPM110 DNA and other subclones confirmed the assignment of molecular masses for the predicted polypeptides. These polypeptides are predicted to have NH2-terminal leader peptides of 17 and 29 amino acids, respectively. NH2-terminal amino acid sequence analysis of purified extracellular hemolysin (HpmA) confirmed the cleavage of the 29-amino-acid leader peptide in the secreted form of HpmA. Hemolysis assays and immunoblot analysis of Escherichia coli containing subclones expressing hpmA, hpmB, or both indicated that HpmB is necessary for the extracellular secretion and activation of HpmA. Significant nucleotide identity (52.1%) was seen between hpm and the shl hemolysin gene sequences of Serratia marcescens despite differences in the G+C contents of these genes (hpm, 38%; shl, 65%). The predicted amino acid sequences of HpmB and HpmA are also similar to those of ShlB and ShlA, the respective sequence identities being 55.4 and 46.7%. Predicted cysteine residues and major hydrophobic and amphipathic domains have been strongly conserved in both proteins. Thus, we have identified a new hemolysin gene family among gram-negative opportunistic pathogens.
Uphoff
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TS
Department of Medical Microbiology, University of Wisconsin Medical School, Madison 53706.
Welch
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RA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000187:drug effects; D006460:Hemolysin Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011513:Proteus mirabilis / Q000235:genetics*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D012706:Serratia marcescens / Q000235:genetics*
D006460:Hemolysin Proteins; D002118:Calcium