Cloning, characterisation and phylogenetic analysis of the sigA σ70factor gene sequence from the actinomycete Frankia

Abstract

The σ70 family of transcriptional factors is essential for bacterial life. Members of this family have been duplicated through evolution and have evolved and specialized for all sorts of physiological responses. Here, we have performed the first analysis of this gene family in Frankia. PCR was used to amplify Frankia σ70 DNA regions well-conserved among primary, stationary phase and alternative actinomycetal sigma factors. DNA sequence analyses confirmed the identity of the obtained PCR fragment, which was then used to screen a Frankia ACN14a gene library. A hybridizing library clone was obtained and characterized. A 2045 bp-long DNA sequence harboring the probed region was sequenced and analysed. A single complete orf was detected. This orf has 82% identities with Streptomyces coelicolor hrdA gene sequence and its deduced protein has 59% identities and 74% similarities with the HrdA protein. A phylogenetic analysis shows the deduced protein sequence to belong to a well-resolved phylogenetic radiation containing primary σ70 factors (essential for growth), and was thus named SigA. Early duplication events in this radiation led to the presence of several paralogs of primary-like σ70 factors among the actinomycetes. These paralogous lineages could be divided into at least three well-resolved clusters: (A) a HrdA lineage found among Streptomyces; (B) a MysB-SigB lineage found among Mycobacterium and Brevibacterium, and (C) a MysA-HrdB lineage widespread among the actinomycetes. Frankia SigA represents a distinct lineage in this radiation. Blot analysis of digested Frankia ACN14a total DNA showed sigA to be in a single copy.