Promoter strength influences phase variation of neisserial opa genes

Abstract

The opa multigene family of Neisseria gonorrhoeae encodes 11 related outer-membrane proteins which phase vary in vitro and in vivo. Illegitimate recombination within direct pentameric DNA repeats, encoding the signal-peptide region of pre-Opas, leads to switches in expression states. Despite the conserved nature of the variation mechanism, specific genes are expressed at high frequencies in the transition from Opa- to Opa+. The genes which are expressed at elevated frequencies differ from the rest of the family with respect to promoter structure, based on sequence comparisons between the opa genes of strain MS11mk. We have analysed transcription of the opa gene family of N. gonorrhoeae MS11mk, focussing on the different promoters found among the 11 genes to determine whether increased levels of expression are associated with increased phase-variation rates. Primer extension and Northern blotting was used to assess the levels of transcription of three representative opa genes (opaA, B and C) in 'on' and 'off' states. Full-length opa mRNA was detected primarily in strains expressing the homologous gene. Truncated opa mRNA was constitutively expressed from all opa genes regardless of their expression state. Quantitative comparisons in N. gonorrhoeae were complicated by the simultaneous expression of all 11 genes and the cross-reactivity of mRNA probes. Expression levels from the individual promoters were therefore assessed by creating transcriptional and translational lacZ fusions to each of the representative opa promoters which lacked the DNA repeats responsible for variation. The expression levels were compared to the phase-variation rates of translational opa::phoA fusions containing the same promoters in addition to the corresponding coding repeat regions. A strong correlation was found between expression levels from the different promoters and the variation rates at which 'on' variants appeared from an 'off' population (i.e. opaA > opaB > opaC). These results provide an explanation for the favoured expression of specific Opa proteins and indicate that expression of opa genes may be regulated at the level of transcription.