Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Biology, Molecular Biology & Biotechnology

Date of Defense

5-12-2011

Graduate Advisor

Teresa Thiel

Committee

Wendy Olivas

Lisa Schechter

Bethany Zolman

Abstract

Little is known about the regulation of nitrogenase genes in cyanobacteria. Transcription of the nifH1 and vnfH genes, encoding dinitrogenase reductases for the heterocyst-specific Mo-nitrogenase and the alternative V-nitrogenase, respectively, was studied using a lacZ reporter. Two promoters were found to be responsible for expression of nifHDK; a weak promoter within nifU1 and a strong promoter upstream of nifB (the nifB1 promoter). The region immediately upstream of vnfH did not drive expression of lacZ, however the region that included the promoter for the upstream gene, ava4055 did. Characterization of the previously reported nifH1 and vnfH transcriptional start sites by 5' RACE revealed that these 5' ends resulted from processing of larger transcripts rather than by de-novo transcription initiation. Anabaena variabilis grows heterotrophically using fructose while the close relative Anabaena sp. strain PCC 7120 does not. Introduction of a cluster of genes encoding a putative ABC-transporter, herein named frtRABC, into Anabaena sp. strain PCC 7120 on a replicating plasmid allowed that strain to grow in the dark using fructose, indicating that these genes were necessary and sufficient for heterotrophic growth. FrtR, a putative LacI-like regulatory protein, was essential for heterotrophic growth of both cyanobacterial strains. Transcriptional analysis revealed that the transport system was induced by fructose and that in the absence of FrtR, frtA was very highly expressed with or without fructose. In the frtR mutant, high-level expression of the fructose transporter resulted in cells that were extremely sensitive to fructose. A. variabilis grew better with increasing concentrations of fructose up to 50 mM, showing increased cell size and heterocyst frequency. Anabaena sp. strain PCC 7120 did not show any of these changes when growing with fructose. Thus, although Anabaena sp. strain PCC 7120 could take up fructose and use it in the dark, fructose did not improve growth in the light.

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