Response from Nizet: CylI and group B streptococcal hemolysis

I appreciate the comments of Dr. Spellerberg who has played a principal role in the discovery and elucidation of the genetic locus for GBS β-hemolysin/cytolysin (β-h/c) activity. Dr. Spellerberg correctly identifies a sequencing error that was made in our original 1999 GeneBank submissions (AF157015) of genes downstream of the putative GBS β-h/c structural gene cylE. This error led to inadvertent division of a single open reading frame (ORF) into two apparent ORFs that we named cylH and cylI, an error similarly misreported in our manuscript [1xGenetic basis for the beta-haemolytic/cytolytic activity of group B Streptococcus. Pritzlaff, C.A. et al. Mol. Microbiol. 2001; 39: 236–247Crossref | PubMed | Scopus (93)See all References[1]. Dr. Spellerberg's publication [2xThe cyl genes of Streptococcus agalactiae are involved in the production of pigment. Spellerberg, B. et al. FEMS Microbiol. Lett. 2000; 188: 125–128Crossref | PubMedSee all References[2] correctly identifies the single ORF with strong homology to 3-oxoacyl-ACP-synthases and names it cylI, a designation appropriately adopted by the two recently completed GBS genome projects [3xGenome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease. Glaser, P. et al. Mol. Microbiol. 2002; 45: 1499–1513Crossref | PubMed | Scopus (297)See all References, 4xComplete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae. Tettelin, H. et al. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12391–12396Crossref | PubMed | Scopus (319)See all References]. In Figure 2a of the Trends in Microbiology review article [5xStreptococcal beta-hemolysins: genetics and role in disease pathogenesis. Nizet, V. Trends Microbiol. 2002; 10: 575–580Abstract | Full Text | Full Text PDF | PubMed | Scopus (75)See all References[5], I joined the two ORFs together but mislabeled them as cylH instead of cylI.Dr. Spellerberg also points out that a nonhemolytic, nonpigmented GBS mutant identified from a random ISS1 transposition mutagenesis library mapped to the cylI gene [2xThe cyl genes of Streptococcus agalactiae are involved in the production of pigment. Spellerberg, B. et al. FEMS Microbiol. Lett. 2000; 188: 125–128Crossref | PubMedSee all References[2]. Although suggestive of a role in hemolysin production, I have from personal experience been cautious to ascribe a specific function to a single ORF solely on the basis of transposon or other random insertional mutagenesis strategies. The possibility of spontaneous mutations or polar effects should be ruled out by targeted mutagenesis and single gene complementation analysis. For example, we found targeted disruption of the ORFs harboring the transposon insertions in previously published nonhemolytic GBS tranposon mutants COH31.cl5 and COH1-20 does not affect hemolysin activity of the parent strain [6xGroup B streptococcal beta-hemolysin induces mortality and liver injury in experimental sepsis. Ring, A. et al. J. Infect. Dis. 2002; 185: 1745–1753Crossref | PubMed | Scopus (35)See all References[6]. Although I do not discount for a moment the possibility that other genes besides cylE could contribute to GBS β-h/c production, we have demonstrated that targeted mutagenesis of the cylI gene (then called cylH) does not affect the hemolysin or pigment phenotype of GBS serotype III strain COH1 [1xGenetic basis for the beta-haemolytic/cytolytic activity of group B Streptococcus. Pritzlaff, C.A. et al. Mol. Microbiol. 2001; 39: 236–247Crossref | PubMed | Scopus (93)See all References[1]. Moreover, the cylE gene alone is sufficient to confer β-hemolysis when cloned and expressed in Escherichia coli [1xGenetic basis for the beta-haemolytic/cytolytic activity of group B Streptococcus. Pritzlaff, C.A. et al. Mol. Microbiol. 2001; 39: 236–247Crossref | PubMed | Scopus (93)See all References[1].