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Title: Characterization of genes involved in heterocyst differentiation and pattern formation in the cyanobacterium Anabaena sp. strain PCC 7120 
Author: Borthakur, Pritty B
Date: 2008
Abstract: The goal of this research was to understand regulation of heterocyst differentiation in Anabaena sp. strain PCC 7120 (hereafter Anabaena PCC 7120) by characterizing regulatory genes for heterocyst formation and their mutants. Anabaena is a filamentous cyanobacterium that forms specialized cells for nitrogen fixation. called heterocysts, which differentiate from vegetative cells at intervals of 10 - 12 cells. Two genes, patS and hetN. are known to suppress the differentiation of vegetative cells into heterocysts for establishing and maintaining a pattern of heterocysts along the filament This study has established that PatS and HetN work independently to suppress differentiation. Using a patS-deletion strain with conditional expression of hetN, it was shown in this study that PatS and HetN are members of separate heterocyst suppression pathways. Inactivation of either of these negative regulators enhances heterocyst frequencies to >20%, compared to 9% in the wild type. However, inactivation of both patS and hetN increases heterocyst differentiation to nearly 100%. A mutant, UHM I 00, was created to study the function of both genes by deleting patS and making expression of hetN conditional. In the absence of nitrogen in liquid medium, almost all vegetative cells of UHM100 differentiated to heterocysts, giving rise to a phenotype called 'multiple contiguous heterocysts' (Mch). Interestingly, UHM100 has an Mch phenotype even in the presence of combined nitrogen, which usually suppresses heterocyst differentiation. UHM100 was used to study the time course of heterocyst differentiation. The percentage of cells that differentiated into heterocysts correlated with the time since induction and was independent of cell density in liquid medium lacking a fixed nitrogen source. It was expected that in the patS and hetN double mutant UHM100, hetR will be overexpressed in all cells. However, when UHM100 containing hetR-gfp was grown in nitrogen-free medium, the pattern of fluorescence observed at 48 h after induction showed that hetR was expressed in ~55% cells, suggesting that nitrogen-deprivation did not immediately induce hetR in all cells. Thus, hetR expression in the absence of patS and hetN was asynchronous. The patS and hetN double mutant was also used to determine if the position of heterocysts along the filament was random or not When the positions of heterocysts relative to other vegetative cells was examined using statistical analyses for randomness, the distribution of heterocysts was found to be nonrandom. Time course studies using UHM100 further showed that heterocyst differentiation occurred in clusters of 2-5 cells at 48 h and the size of the clusters increased with time. Heterocyst frequency reached -98% after 144 h in the absence of fixed nitrogen. Clustering of heterocysts in the filaments of UHM100 suggests that besides PatS and HetN, there are other factors that influence pattern formation in Anabaena PCC 7120. A heterocyst-deficient (Her) spontaneous mutant, NSM6, was isolated from UHM100. Complementation of NSM6 by a cosmid clone, pPB6-1, from an Anabaena PCC 7120 genomic library restored the Mch phenotype of this mutant. By sequencing, sub-cloning, and further complementation analyses, a novel gene, alr9018, was identified in a 4.l-kb fragment of pPB6-1. The alr9018 gene is located in the Epsilon plasmid of Anabaena, and it encodes a 148.7-kDa protein. The Alr9018 protein contains an NTPase domain, which is a characteristic of proteins involved in signal transduction. alr9018 is expressed in both vegetative cells and heterocysts. Similar to alr9018, hetR can also restore the Mch phenotype in NSM6, suggesting that the NSM6 mutant can be functionally complemented by multiple copies of either a/r9018 or hetR. When palr9018 was transferred to Anabaena PCC 7120, the transconjugants formed ~15% heterocysts compared to ~10% heterocysts formed by Anabaena PCC 7120. The transconjugants also reduced at least 50% more acetylene than PCC 7120, suggesting that multiple copies of alr9018 enhance heterocyst development. This is the first report showing that the Epsilon plasmid of Anabaena PCC 7120 contains genes involved in heterocyst differentiation. The identification and characterization of alr9018 in the present study further show that the regulation of heterocyst differentiation in Anabaena is complex. Further studies will be required to fully understand the complex interactions between alr9018 and hetR and the role of alr9018 in cell differentiation and pattern formation in Anabaena PCC 7120.
Description: Thesis (Ph.D.)--University of Hawaii at Manoa, 2008. The goal of this research was to understand regulation of heterocyst differentiation in Anabaena sp. strain PCC 7120 by characterizing regulatory genes for heterocyst formation and their mutants. Anabaena is a filamentous cyanobacterium that forms specialized cells for nitrogen fixation, called heterocysts, which differentiate from vegetative cells at intervals of 10--12 cells. Two genes, patS and hetN, are known to suppress the differentiation of vegetative cells into heterocysts for establishing a de novo pattern and maintaining a pattern of heterocysts along the filament. A mutant, UHM100, was created to study the function of both genes by deleting patS and making expression of hetN conditional. This study has established that PatS and HetN are members of two separate heterocyst suppression pathways. In absence of nitrogen, inactivation of both patS and hetN increases heterocyst differentiation to nearly 100%, giving rise to a phenotype called 'multiple contiguous heterocysts' (Mch). UHM100 has an Mch phenotype even in the presence of combined nitrogen, which usually suppresses heterocyst differentiation. In absence of both patS and hetN, the expression of hetR, a master regulator of heterocyst differentiation, was observed in ∼55% cells and was asynchronous. The distribution of heterocysts next to a vegetative cell in UHM 100 was found to be nonrandom. These results suggest that besides PatS and HetN, there are other factors that influence pattern formation in Anabaena PCC 7120. A heterocyst-deficient (Hef) spontaneous mutant, NSM6, was isolated from UHM 100. A novel gene, alr9018, from the Anabaena Epsilon plasmid complemented NSM6 and restored the Mch phenotype of this mutant. Transconjugants of Anabaena PCC 7120 containing the cloned alr9018 gene fixed 50% more N2 than PCC 7120, suggesting that multiple copies of alr9018 enhance heterocyst development. This is the first report showing that the Epsilon plasmid of Anabaena PCC 7120 contains genes involved in heterocyst differentiation. Expression of alr9018 was observed in both vegetative cells and heterocysts. Similar to alr9018, hetR could also restore the Mch phenotype in NSM6, suggesting functional similarity between a1r9018 and hetR. The Alr9018 protein contains an NTPase domain, which is a characteristic of proteins involved in signal transduction. Includes bibliographical references (leaves 89-97). Also available by subscription via World Wide Web 97 leaves, bound 29 cm
ISBN: 9780549600350
URI: http://hdl.handle.net/10125/20730
Rights: All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.

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