Transposon Mutagenesis to Identify Trans-Acting Regulatory Factors of the nif Genes in Anabaena sp. Strain PCC 7120

Abstract

When grown in the absence of combined nitrogen, the photosynthetic cyanobacterium Anabaena sp. strain PCC 7120 fixes nitrogen inside terminally differentiated cells called heterocysts. Differentiation is a complex process involving genome rearrangements to yield functional nifHDK and nifB-fdxN-nifSU operons. The regulation of expression of the nifHDK operon has been studied extensively in other nitrogen-fixing bacteria, but nifHDK regulation in Anabaena is not yet understood. This study used transposon mutagenesis to identify possible trans-acting factors which regulate nifHDK transcription in Anabaena 7120. We analyzed mutants to identify colonies which produced normal looking heterocysts but could not fix nitrogen. Northern blot analysis followed to determine if the nifH promoter was active. DNA was isolated from mutants in which nifH expression was down or off and direct cloning was used to isolate the transposon. Other mutants in heterocyst formation were also subjected to direct cloning.