Elsie L. Campbell

Cellular differentiation in the cyanobacterium Nostoc punctiforme.

Abstract.Nostoc punctiforme is a phenotypically complex, filamentous, nitrogen-fixing cyanobacterium, whose vegetative cells can mature in four developmental directions. The particular developmental direction is determined by environmental signals. The vegetative cell cycle is maintained when nutrients are sufficient. Limitation for combined nitrogen induces the terminal differentiation of heterocysts, cells specialized for nitrogen fixation in an oxic environment. A number of unique regulatory events and genes have been identified and integrated into a working model of heterocyst differentiation. Phosphate limitation induces the transient differentiation of akinetes, spore-like cells resistant to cold and desiccation. A variety of environmental changes, both positive and negative for growth, induce the transient differentiation of hormogonia, motile filaments that function in dispersal. Initiation of the differentiation of heterocysts, akinetes and hormogonia are hypothesized to depart from the vegetative cell cycle, following separate and distinct events. N. punctiforme also forms nitrogen-fixing symbiotic associations; its plant partners influence the differentiation and behavior of hormogonia and heterocysts. N. punctiforme is genetically tractable and its genome sequence is nearly complete. Thus, the regulatory circuits of three cellular differentiation events and symbiotic interactions of N. punctiforme can be experimentally analyzed by functional genomics.

Global Gene Expression Patterns of Nostoc punctiforme in Steady-State Dinitrogen-Grown Heterocyst-Containing Cultures and at Single Time Points during the Differentiation of Akinetes and Hormogonia

The vegetative cells of the filamentous cyanobacterium Nostoc punctiforme can differentiate into three mutually exclusive cell types: nitrogen-fixing heterocysts, spore-like akinetes, and motile hormogomium filaments. A DNA microarray consisting of 6,893 N. punctiforme genes was used to identify the global transcription patterns at single time points in the three developmental states, compared to those in ammonium-grown time zero cultures. Analysis of ammonium-grown cultures yielded a transcriptome of 2,935 genes, which is nearly twice the size of a soluble proteome. The NH(4)(+)-grown transcriptome was enriched in genes encoding core metabolic functions. A steady-state N(2)-grown (heterocyst-containing) culture showed differential transcription of 495 genes, 373 of which were up-regulated. The majority of the up-regulated genes were predicted from studies of heterocyst differentiation and N(2) fixation; other genes are candidates for more detailed genetic analysis. Three days into the developmental process, akinetes showed a similar number of differentially expressed genes (497 genes), which were equally up- and down-regulated. The down-regulated genes were enriched in core metabolic functions, consistent with entry into a nongrowth state. There were relatively few adaptive genes up-regulated in 3-day akinetes, and there was little overlap with putative heterocyst developmental genes. There were 1,827 differentially transcribed genes in 24-h hormogonia, which was nearly fivefold greater than the number in akinete-forming or N(2)-fixing cultures. The majority of the up-regulated adaptive genes were genes encoding proteins for signal transduction and transcriptional regulation, which is characteristic of a motile filament that is poised to sense and respond to the environment. The greatest fraction of the 883 down-regulated genes was involved in core metabolism, also consistent with entry into a nongrowth state. The differentiation of heterocysts (steady state, N(2) grown), akinetes, and hormogonia appears to involve the up-regulation of genes distinct for each state.

Transposon mutagenesis of Nostoc sp. strain ATCC 29133, a filamentous cyanobacterium with multiple cellular differentiation alternatives

Nostoc sp. strain ATCC 29133 (PCC 73102; Nostoc 29133) is a symbiotically-competent, facultatively heterotrophic, diazotrophic cyanobacterium with the capacity to differentiate specialized cells such as heterocysts, akinetes and hormogonial filaments. We have optimized several methods for physiological and molecular genetic analysis of Nostoc 29133. By use of a Tn5 derivative, Tn5-1063 (Km(r)Bm(r)Sm(r)), delivered by conjugation from Escherichia coli, antibiotic-resistant mutants of Nostoc 29133 were generated at a frequency of approximately 1 x 10(-6), 0.4% of which expressed a nitrogen fixation (heterocyst) defective phenotype. Mutant strain UCD 328 was isolated after co-culture of 86 Nostoc 29133::Tn5-1063 clones with the symbiotic plant partner, Anthoceros punctatus; strain UCD 328 expressed a symbiotic phenotype of increased frequency of hormogonia-dependent infection. The transposon and flanking genomic DNA was recovered from strain UCD 328, the mutation and phenotype reconstructed by homologous recombination in Nostoc 29133, and the transposition site identified from a Nostoc 29133 genomic library. Transposon mutagenesis has thus provided the means for isolation and identification of developmental and symbiotic-specific genes of Nostoc 29133.

A polyketide-synthase-like gene is involved in the synthesis of heterocyst glycolipids in Nostoc punctiforme strain ATCC 29133

Abstract A Tn5-1063-derived mutant of Nostoc punctiforme strain ATCC 29133 was unable to fix N2 in air although it reduced acetylene in the absence of O2. Mutant strain UCD 307 formed cells morphologically similar to heterocysts, but it failed to synthesize the characteristic heterocyst glycolipids. Sequence analysis around the site of insertion revealed an ORF of 3,159 base pairs, termed hglE. hglE putatively encodes a 115.4-kDa protein containing two domains with conserved amino acid sequences identified with acyl transferase and the chain length factor variation of β-ketoacyl synthase active sites. These active sites are characteristic of polyketide and fatty acid synthases. The N. punctiforme strain 29133 hglE gene is transcribed only under nitrogen-limiting growth conditions. The hglE gene, or similar sequences, was found in all other heterocyst-forming cyanobacteria surveyed and was absent in unicellular Synechococcus sp. strain PCC 7942. Based on these results, we propose that the synthesis of heterocyst glycolipids follows a pathway characteristic of polyketide synthesis and involves similar large, multienzyme complexes.

DNA Microarray Comparisons of Plant Factor- and Nitrogen Deprivation-Induced Hormogonia Reveal Decision-Making Transcriptional Regulation Patterns in Nostoc punctiforme

Hormogonia are nongrowing filaments, motile by means of a gliding mechanism, that are produced by certain cyanobacteria. Their differentiation is induced by positive and negative factors for growth, such as deprivation of combined nitrogen (nitrogen stress induction [NSI]). In Nostoc punctiforme, they are also induced by the exudate (hormogonium-inducing factor [HIF]) of a symbiotic plant partner. Time course (0.5 to 24 h) transcription profiles were determined by DNA microarray assays for hormogonia of N. punctiforme following induction by HIF and NSI. Clustering analysis revealed both common and distinct transcriptional patterns for the two methods of induction. By 24 h, a common set of 1,328 genes was identified. This 24-h common set of genes arose by the transition of 474 genes from an 819-member common set of genes at 1 h after induction; 405 and 51 genes unique to the HIF and NSI groups at 1 h, respectively; and 398 genes differentially transcribed at later time points. The NSI hormogonia showed a transcriptional checkpoint at 12 h following induction in which up- and downregulated genes were transiently down- or upregulated, respectively. The transient changes in these 1,043 genes appeared to reflect a switch back to a vegetative growth state. Such a checkpoint was not seen in HIF hormogonia. Genes uniquely upregulated in HIF hormogonia included those encoding proteins hypothesized to synthesize a metabolite repressor of hormogonium differentiation. Approximately 34 to 42% of the 6,893 printed genes were differentially transcribed during hormogonium differentiation; about half of those genes were upregulated, and 1,034 genes responded within 0.5 h after induction. These collective results indicate extensive and rapid global changes in the transcription of specific genes during the differentiation of these specialized filaments.

Global Transcription Profiles of the Nitrogen Stress Response Resulting in Heterocyst or Hormogonium Development in Nostoc punctiforme

The filamentous cyanobacterium Nostoc punctiforme differentiates from vegetative cells into three distinct cell types, heterocysts, hormogonia, and akinetes, in response to different stimuli. Cultures growing with ammonium can be induced to form hormogonia or heterocysts upon removal of the combined nitrogen. A DNA microarray consisting of 94% of the open reading frames predicted from the 9.059-Mb N. punctiforme genome was used to generate a global transcription data set consisting of seven time points over a 24-h period of nitrogen deprivation, which results in heterocyst formation. This data set was compared to a similarly generated data set of nitrogen-starved N. punctiforme resulting in hormogonium formation that had previously been published (E. L. Campbell, H. Christman, and J. C. Meeks, J. Bacteriol. 190:7382-7391, 2008). The transition from vegetative cells to either heterocysts or hormogonia resulted in rapid and sustained expression of genes required for utilization of alternate nitrogen sources. Overall, 1,036 and 1,762 genes were found to be differentially transcribed during the heterocyst and hormogonium time courses, respectively, as analyzed with the Bayesian user-friendly software for analyzing time series microarray experiments (BATS). Successive transcription of heterocyst regulatory, structural, and functional genes occurred over the 24 h required to form a functional heterocyst. During hormogonium differentiation, some heterocyst structural and functional genes were upregulated, while the heterocyst master regulator hetR was downregulated. There are commonalities in differential expression between cells bound for differentiation into heterocysts or hormogonia, yet the two paths are distinguished by their developmentally specific transcription profiles.

Genetic Analysis Reveals the Identity of the Photoreceptor for Phototaxis in Hormogonium Filaments of Nostoc punctiforme

In cyanobacterial Nostoc species, substratum-dependent gliding motility is confined to specialized nongrowing filaments called hormogonia, which differentiate from vegetative filaments as part of a conditional life cycle and function as dispersal units. Here we confirm that Nostoc punctiforme hormogonia are positively phototactic to white light over a wide range of intensities. N. punctiforme contains two gene clusters (clusters 2 and 2i), each of which encodes modular cyanobacteriochrome-methyl-accepting chemotaxis proteins (MCPs) and other proteins that putatively constitute a basic chemotaxis-like signal transduction complex. Transcriptional analysis established that all genes in clusters 2 and 2i, plus two additional clusters (clusters 1 and 3) with genes encoding MCPs lacking cyanobacteriochrome sensory domains, are upregulated during the differentiation of hormogonia. Mutational analysis determined that only genes in cluster 2i are essential for positive phototaxis in N. punctiforme hormogonia; here these genes are designated ptx (for phototaxis) genes. The cluster is unusual in containing complete or partial duplicates of genes encoding proteins homologous to the well-described chemotaxis elements CheY, CheW, MCP, and CheA. The cyanobacteriochrome-MCP gene (ptxD) lacks transmembrane domains and has 7 potential binding sites for bilins. The transcriptional start site of the ptx genes does not resemble a sigma 70 consensus recognition sequence; moreover, it is upstream of two genes encoding gas vesicle proteins (gvpA and gvpC), which also are expressed only in the hormogonium filaments of N. punctiforme.

Evidence for plant-mediated regulation of nitrogenase expression in theAnthoceros-Nostoc symbiotic association

Summary: A pleiotropic dinitrogen and nitrate assimilation mutant was obtained by mutagenesis of Nostoc sp. strain ATCC 29133 with N-methyl-N-nitro-N-nitrosoguanidine followed by penicillin counterselection in the presence of NO- 3 and N2. Mutant strain UCD 223 was capable of reducing acetylene in the free-living growth state only under anaerobic conditions, or under atmospheric conditions when in symbiotic association with Anthoceros punctatus. Heterocysts of strain UCD 223 were noticeably lacking the cohesive outer polysaccharide layer of wild-type heterocysts. Oxygen microelectrode profiles of symbiotic Anthoceros-Nostoc tissue revealed an anaerobic environment in the symbiotic cavities containing Nostoc. The acetylene-reducing activities of strain UCD 223, and of its spontaneously-arising Fix+ revertant strain UCD 236, were not repressed by the presence of 10 mm-NO- 3 when in the free-living growth state, in contrast to wild-type Nostoc ATCC 29133. However, in situ activities of acetylene reduction by symbiotically associated Nostoc ATCC 29133 and strains UCD 223 and UCD 236 were repressed by the presence of 10 mm-NO- 3. It appears that the symbiotic cavities of Anthoceros punctatus can physiologically replace the function of the heterocyst outer wall and that the repression of nitrogenase activity in symbiotic Nostoc in response to the presence of NO- 3, and probably NH+ 4, is mediated by Anthoceros.

DNA binding properties of the HrmR protein of Nostoc punctiforme responsible for transcriptional regulation of genes involved in the differentiation of hormogonia

Nostoc punctiforme is an example of a filamentous cyanobacterium that is capable of differentiating non‐growing cells that constitute gliding filaments termed hormogonia. These gliding filaments serve in short distance dispersal and as infective units in establishing a symbiosis with plants, such as the bryophyte Anthoceros punctatus . Mutants of N . punctiforme exist which show elevated levels of initial infection of A . punctatus as a consequence of repeated cycles of hormogonium differentiation. Such mutations occur within the hrmA and hrmU genes. Further characterization of the hrm locus revealed several genes with an organizational and predicted protein sequence similarity to genes of heterotrophic bacteria that are involved in hexuronic acid metabolism. Genes in the N. punctiforme locus are transcribed in response to the presence of a plant extract containing hormogonium‐repressing factors. A predicted transcriptional repressor encoded in the locus, HrmR , was shown herein to be a specific DNA binding protein that regulates the transcription of its own gene and that of hrmE , a nearby gene. The ability of HrmR to bind DNA was abolished upon addition of either galacturonate or lysate from specifically induced N . punctiforme cells, implying that the in vivo HrmR binding activity is modulated via an internal compound, most likely a sugar molecule.

Elements interrupting nitrogen fixation genes in cyanobacteria: presence and absence of a nifD element in clones of Nostoc sp. strain Mac

Nostoc sp. strain Mac is capable of microaerobic, but not aerobic, nitrogen fixation (Fox-). Nostoc Mac grows as long, relatively straight, filaments that are well dispersed in the culture medium. However, spontaneously-arising revertant strains selected for aerobic nitrogen fixation (Fox+) all grow as coiled filaments that associate in macroscopic clumps or balls of varying dimensions. DNA restriction fragment length polymorphism, using nitrogenase (nif) structural genes as probes, established identity between revertants and the parental culture. Mapping of the fragments and lack of hybridization to specific probes indicated the absence of a DNA sequence interrupting the nifD gene in one Fox+ revertant. Such a nifD element is assumed to be present in essentially all heterocyst-forming cyanobacteria. Only one clone out of 223 Fox- and Fox+ Nostoc Mac clones surveyed lacked the nifD element, indicating that loss of the element is a rare event. The nifD element is present in the same location in the genome of Nostoc Mac as it is in all other heterocystforming cyanobacteria analysed. No phenotypic differences could be detected between two Fox+ clones containing or lacking the nifD element, including repression and derepression of nitrogen fixation in response to the presence or absence of combined nitrogen. We suspect that retention of the nifD element in vegetative cells of heterocyst-forming cyanobacteria is a consequence of selective pressure, although such selective conditions in laboratory cultures have not been identified.