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    Epistasis Analysis of Four Genes from Anabaena sp. Strain PCC 7120 Suggests a Connection between PatA and PatS in Heterocyst Pattern Formation
    (American Society for Microbiology, 2005-12) Orozco, Christine C. ; Risser, Douglas D. ; Callahan, Sean M.
    The hetR, patA, hetN, and patS genes are part of a regulatory network that regulates the differentiation and patterning of heterocysts in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. In this report, the epistatic interactions of mutant alleles of these four genes have been used to refine our understanding of their relationships to one another. The hetR gene was necessary for differentiation in genetic backgrounds that normally give rise to excessive differentiation, supporting its role as the master regulator of differentiation and indicating that HetR directly regulates factors in addition to hetR and patS genes that regulate differentiation. A functional patS gene was necessary for the delayed multiple-contiguous-heterocyst phenotype observed in hetN mutants as well as for the relative lack of intercalary heterocysts in patA mutants. Epistasis results with mutant alleles of these three genes suggested that PatA attenuates the negative effects of both PatS and HetN on differentiation and promotes differentiation independent of its antagonistic effects on PatS and HetN activity. Cooverxpression of patS and hetR in a synthetic operon indicated that patS acts at a point downstream of hetR transcription in the regulatory network controlling differentiation. A model for the regulation of differentiation that is consistent with these and previous findings is presented.
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    Mutagenesis of hetR Reveals Amino Acids Necessary for HetR Function in the Heterocystous Cyanobacterium Anabaena sp. Strain PCC 7120
    (American Society for Microbiology, 2007-01) Risser, Douglas D. ; Callahan, Sean M.
    HetR is the master regulator of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. Genetic selection was used to identify 33 amino acid substitutions in HetR that reduced the proportion of cells undergoing heterocyst differentiation to less than 2%. Conservative substitutions in the wild-type HetR protein revealed three mutations that dramatically reduced the amount of heterocyst differentiation when the mutant allele was present in place of the wild-type allele on a replicating plasmid in a mutant lacking hetR on the chromosome. An H69Y substitution resulted in heterocyst formation among less than 0.1% of cells, and D17E and G36A substitutions resulted in a Het- phenotype, compared to heterocyst formation among approximately 25% of cells with the wild-type hetR under the same conditions. The D17E substitution prevented DNA binding activity exhibited by wild-type HetR in mobility shift assays, whereas G36A and H69Y substitutions had no affect on DNA binding. D17E, G36A, and H69Y substitutions also resulted in higher levels of the corresponding HetR protein than of the wild-type protein when each was expressed from an inducible promoter in a hetR deletion strain, suggesting an effect on HetR protein turnover. Surprisingly, C48A and S152A substitutions, which were previously reported to result in a Het- phenotype, were found to have no effect on heterocyst differentiation or patterning when the corresponding mutations were introduced into an otherwise wild-type genetic background in Anabaena sp. strain PCC 7120. The clustering of mutations that satisfied the positive selection near the amino terminus suggests an important role for this part of the protein in HetR function.
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    Analysis of LuxR Regulon Gene Expression during Quorum Sensing in Vibrio fischeri
    (American Society for Microbiology, 2007-03) Qin, Nan ; Callahan, Sean M. ; Dunlap, Paul V. ; Stevens, Ann M.
    The regulation of the lux operon (luxICDABEG) of Vibrio fischeri has been intensively studied as a model for quorum sensing in proteobacteria. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl L-homoserine lactone (3-oxo-C6-HSL). To determine if the LuxR-dependent regulation of the genes encoding these proteins was due to direct transcriptional control by LuxR and 3-oxo-C6-HSL or instead was due to indirect control via an unidentified regulatory element, promoters of interest were cloned into a lacZ reporter and tested for their LuxR and 3-oxo-C6-HSL dependence in recombinant Escherichia coli. The promoters for qsrP, acfA, and ribB were found to be directly activated via LuxR-3-oxo-C6-HSL. The sites of transcription initiation were established via primer extension analysis. Based on this information and the position of the lux box-binding site near position -40, all three promoters appear to have a class II-type promoter structure. In order to more fully characterize the LuxR regulon in V. fischeri MJ-100, real-time reverse transcription-PCR was used to study the temporal expression of qsrP, acfA, and ribB during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities of LuxR to the promoters under investigation. Taken together, the results demonstrate that regulation of the production of QsrP, RibB, and AcfA is controlled directly by LuxR at the level of transcription, thereby establishing that there is a LuxR regulon in V. fischeri MJ-100 whose genes are coordinately expressed during mid-exponential growth.
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    HetF and PatA Control Levels of HetR in Anabaena sp. Strain PCC 7120
    (American Society for Microbiology, 2008-10) Risser, Douglas D. ; Callahan, Sean M.
    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that differentiates heterocysts in response to deprivation of combined nitrogen. A hetF deletion strain lacked heterocysts and had aberrant cell morphology. Site-directed mutagenesis of the predicted active-site histidine and cysteine residues of this putative caspasehemoglobinase fold protease abolished HetF function, supporting the hypothesis that HetF is a protease. Deletion of patA, which is necessary for the formation of most intercalary heterocysts, or hetF resulted in an increase in HetR protein, and extra copies of hetF on a plasmid functionally bypassed the deletion of patA. A hetR-gfp translational fusion expressed from an inducible promoter demonstrated that hetF-dependent downregulation of HetR levels occurs rapidly in vegetative cells, as well as developing heterocysts. “Mosaic” filaments in which only one cell of a filament had a copy of hetR or hetF indicated that hetF is required for differentiation only in cells that will become heterocysts. hetF was required for transcription from a hetRdependent transcription start point of the hetR promoter and induction of transcription from the patS promoter. The inverse correlation between the level of HetR protein and transcription from hetR-dependent promoters suggests that the transcriptional activity of HetR is regulated by HetF and PatA.
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    Genetic and cytological evidence that heterocyst patterning is regulated by inhibitor gradients that promote activator decay
    (Proceedings of the National Academy of Sciences of the United States of America, 2009-11) Risser, Douglas D. ; Callahan, Sean M.
    The formation of a pattern of differentiated cells from a group of seemingly equivalent, undifferentiated cells is a central paradigm of developmental biology. Several species of filamentous cyanobacteria differentiate nitrogen-fixing heterocysts at regular intervals along unbranched filaments to form a periodic pattern of two distinct cell types. This patterning has been used to exemplify application of the activator-inhibitor model to periodic patterns in biology. The activator-inhibitor model proposes that activators and inhibitors of differentiation diffuse from source cells to form concentration gradients that in turn mediate patterning, but direct visualization of concentration gradients of activators and inhibitors has been difficult. Here we show that the periodic pattern of heterocysts produced by cyanobacteria relies on two inhibitors of heterocyst differentiation, PatS and HetN, in a manner consistent with the predictions of the activator-inhibitor model. Concentration gradients of the activator, HetR, were observed adjacent to heterocysts, the natural source of PatS and HetN, as well as adjacent to vegetative cells that were manipulated to overexpress a gene encoding either of the inhibitors. Gradients of HetR relied on posttranslational decay of HetR. Deletion of both patS and hetN genes prevented the formation of gradients of HetR, and a derivative of the inhibitors was shown to promote decay of HetR in a concentration-dependent manner. Our results provide strong support for application of the activator-inhibitor model to heterocyst patterning and, more generally, the formation of periodic patterns in biological systems.