Molekularbiologische und genetische Untersuchungen zur Regulation des zentralen Regulators der Stickstofffixierung NifA in Rhizobium leguminosarum bv. viciae VF39

Abstract

Expression of nif and fix genes in rhizobia is strictly regulated. The dominating regulator within the regulation of nif and fix gene expression is NifA. This work presents, that two promotors are involved in nifA gene expression in Rhizobium leguminosarum bv. viciae VF39, namely P1, located at the downstream end of fixX and PfixA, the promotor of the fixA gene, located 3600bp upstream of P1. P2, a promotor identified by transcriptional mapping was shown to be pointless in earlier works and could be identified as an artefact caused by several hypothetical secondary structures located downstream the fixX gene, within the fixX nifA intergenic region. P2, identified within this region could be deleted without having negative effects on nifA expression. In fact, expression of nifA was boosted when deleting P2. It was shown that this region has a general effect on upstream promotor activity, which is downregulated by 70 percent. This could result in some sort of fine tuning mechanism, regulating upstream P1 and PfixA promotor activity. Within nifA expression, P1 and PfixA show clear interactions. During symbiosis, NifA independent P1 promotor activity results in a ground level expression of nifA. Since oxygen sensibly NifA is stable under these conditions, nifA expression will result in activation of NifA dependent promotors, such as the fixA promotor, located upstream P1. NifA is directed by a well conserved nifA box (UAS) located upstream the fixA promotor and activates transcription of the s 54 dependent fixA promotor, which transcribes the fixABCX genes as well as nifA, resulting in even more NifA. In turn, NifA will amplify its own expression via PfixA. This feedback loop explains the autoregulatory character of nifA expression and why nifA mutants show no symbiotic activation of nifa expression. Factors involved in symbiotic P1 activation and regulation could not yet be clearly identified. Most seemingly, FixL as well as FnrN are involved in the regulation of P1 activity, as P1 is clearly FixL dependent, FnrN is essential for nifA Expression and FnrN itself needs FixL for microaerobic activation. During experiments to identify possible regulators involved in regulation of P1 activity and nifA expression, a gene could be identified by TN5 mutagenesis, showing strong similarities to a transcriptional regulator. This regulator seems to be involved in oxygen dependent regulation of nifA, as mutations within this region lead to nifA overexpression under aerobic conditions. Furthermore these effects could neither be demonstrated under microaerobic conditions nor during symbiosis with Pisum sativum outlining the regulatory effect ouf this gene