Regulation of the cyclic activities of nitrogen fixation and photosynthesis in the unicellular cyanobacterium, Cyanothece sp. ATCC 51142 and cloning and sequencing of thenifHDK operon

Milagros S Colon-Lopez, Purdue University

Abstract

The unicellular, diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142 fixes N$\sb2$ aerobically. A cyclic pattern of the metabolic activities of N$\sb2$ fixation, photosynthetic O$\sb2$ evolution and respiration have been observed in cultures grown either under continuous light (CL) or in alternating light-dark cycles (LD). During the 12hL-12hD regimen, N$\sb2$ fixation and respiration were restricted to the dark phase, generally peaking 3-6 h into the dark. Photosynthetic activity occurred approximately 12 h out of phase and peaked 4-8 h into the light period. A similar pattern was observed in CL-grown cultures. The temporal separation of N$\sb2$ fixation and photosynthesis is one mechanism used by Cyanothece sp. to protect the nitrogenase enzyme from O$\sb2$-inactivation. This temporal separation has been shown to be transcriptionally and posttranslationally regulated. Transcripts corresponding to the nifHDK operon were maximally detected 1-2 h before the peak of nitrogenase activity, independent of the light regime. In LD, levels of Fe-protein and MoFe-protein increased early in the dark period, but virtually disappeared by the end of this phase. In CL, similar kinetics were observed for the Fe-protein, but significant amounts of the MoFe-protein were immunodetected throughout the 24 h period. Cyclic photosynthetic activity is also regulated transcriptionally and translationally. Comparable patterns of regulation were observed under both light regimes. Transcripts corresponding to psbA, psbDI/psbC, psbDII and psaAB were maximally detected at different times during the light period, coinciding with maximum photosynthesis. Levels of D1 Form II and D2 proteins were higher during the light (subjective light in CL), whereas PsaAB protein was highly abundant during the dark (subjective dark in CL). These results correlated well with biophysical measurements to suggest many changes in the photosynthetic mechanism throughout the day. A 5.4 kb region containing the nifHDK operon have been cloned and sequenced. Cyanothece's Fe-protein and MoFe-protein showed high percentages of identity (up to 80%) when compared with proteins from other diazotrophic bacteria. Two other ORF have been identified: upstream (NifU) and downstream (unknown). Therefore, the gene arrangement on this region is nifSUnifHDK.

Degree

Ph.D.

Advisors

Sherman, Purdue University.

Subject Area

Microbiology|Molecular biology|Botany

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