The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression

IRIS, Chiron S.r.l., Via Fiorentina 1, 53100 Siena, Italy Correspondence Cesira L. Galeotti cesira_galeotti{at}chiron.it The 60 nucleotides encoding the signal peptide of the Neisseria meningitidis membrane-bound lytic transglycosylase (MltA) homologue GNA33 were found to exert a negative regulatory effect on expression of GNA33 from either a T7- or a P lac -driven system in Escherichia coli . Down-regulation was observed to occur at the transcriptional/post-transcriptional level and could possibly be ascribed to the formation of a stem–loop secondary structure within the signal peptide sequence. Slowing down the transcription rate through inhibition/titration of the RNA polymerase resulted in a considerable increase in mRNA accumulation, suggesting that a better coupling of translation to transcription would impede the formation of the putative secondary structure. Screening of synonymous mutations in the signal peptide sequence that showed high-level expression of an in-frame fusion to a reporter resulted in the isolation of several deletion mutants lacking most of the sequence participating in the putative secondary structure. Interestingly, the increase in the steady-state mRNA level observed in deletion mutants was higher, reaching a 300-fold increment, than that found in substitution mutants. Our results support the hypothesis that the rate of transcription controls the formation of a secondary structure in the region of the GNA33 transcript corresponding to the signal peptide sequence and this, when formed, negatively regulates expression. Abbreviations: p NPP, p -nitrophenyl phosphate; RNAP, RNA polymerase; TPMG, Tryptose phosphate/phenolphthalein/methyl green