H-077. HetR Transcription Control in the Developmental Regulation of Anabaena sp. PCC 7120

R. Rajagopalan, S. M. Callahan;
Univ. of Hawaii, Honolulu, HI.

The photosynthetic cyanobacterium Anabaena PCC 7120 fixes atmospheric nitrogen in specially differentiated cells called heterocysts in response to nitrogen-starvation. Approximately every tenth cell in a filament of vegetative cells differentiates, and this one-dimensional pattern is maintained as the filament elongates. HetR, PatS, HetN and PatA are regulatory proteins involved in differentiation and patterning. HetR is necessary and sufficient for differentiation. It is a DNA-binding protein, and positively upregulates its own transcription. Four transcription start points (tsp) have been mapped at nucleotides -728, -696, -271 and -184 with respect to the translational start site. The transcript originating at -271 is dependant on HetR and has been implicated in auto-regulation. The goal of this project is to understand how transcription control of HetR facilitates developmental regulation. To see how certain negative (PatS and HetN) and positive (PatA) heterocyst regulators affect hetR transcription, a RACE (Rapid Amplification of cDNA ends) procedure and northern blot were done on RNA extracted from the wildtype and strains overexpressing either PatS or HetN, or with patA deleted. HetN overexpression prevented transcription from -271, and down-regulated transcription from -728 and -696, while PatS down-regulated transcription from -184 as well. The -271 transcript appeared weak in the PatA mutant. To see the hetR transcription timeline, RACE was done on RNA extracted from the wildtype 0, .5, 1, 2, 3, 4, 5 and 6 hours post-induction. The -728, -696 and -184 transcripts were present at all time-points, while the -271 transcript was present from 2 hours on. To elucidate the pattern of transcription from the tsps, tsp-green fluorescent protein fusions were introduced into the wildtype and examined by fluorescence microscopy. The -728, -696 and -184 tsps were transcribed in all cells, while -271 was transcribed specifically in heterocysts and developing heterocysts. Our results indicate that the -271 tsp is the only tsp regulated in a cell-type specific and switch-like manner.