Michael L. Summers

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.

Akinetes: Dormant Cells of Cyanobacteria

Cyanobacteria are an ancient and morphologically diverse group of photosynthetic prokaryotes, which were the first to evolve oxygenic photosynthesis. Cyanobacteria are widely distributed in diversed environments. In the case of members of the orders Nostocales and Stigonematales, their persistence and success were attributed to their ability to form specialized cells: heterocysts, capable of fixing atmospheric nitrogen and spore-like cells, the akinetes. This review focuses on akinetes of Nostocales, emphasizing environmental triggers and cellular responses involved in differentiation, maturation, dormancy, and germination of these resting cells. Morphological and structural changes, variation in akinete composition, and metabolism are summarized. Special attention is given to the genetic regulation of the differentiation process in an attempt to close gaps in our understanding of the dormancy phenomenon in cyanobacteria and to identify open questions for future research.

Expression and regulation of phosphate stress inducible genes in Sinorhizobium meliloti

Sinorhizobium meliloti 104A14 was mutated with transposon Tn5B22, which creates lacZ transcriptional fusions when inserted in the correct orientation relative to the promoter. This promoter reporter allowed us to identify six phosphate stress inducible (psi) genes in S. meliloti that are up-regulated in response to inorganic phosphate (Pi) starvation. The transposon and flanking DNA were cloned from each psi::Tn5B22 reporter mutant and the junction DNA sequenced. High identity/similarity of the inferred peptides with those in major data bases allowed identification of the following genes: dnaK, expC, pssB, ackA, vipC, and prkA. The prkA homolog was also found to be up-regulated in response to carbon starvation and when nitrate replaced ammonium as the nitrogen source. Through allele replacement techniques, PhoB- mutants were generated for the expC, ackA, vipC, and pssB reporter strains. Loss of a functional PhoB resulted in the absence of Pi-sensitive induction in all four genes. These experiments suggest the Pho regulon in S. meliloti includes genes that presumably are not directly linked to Pi acquisition or assimilation. The psi strains were tested for their symbiotic properties under growth conditions that were Pi-limiting or Pi-nonlimiting for the host plant. All were Nod+ and Fix+ except the reporter strain of dnaK transcription, which was less effective than the wild-type strain under both P treatments, indicating DnaK is required for optimum symbiotic function.

Heat- and acid-tolerance of a grass commonly found in geothermal areas within Yellowstone National Park

Surveys of geothermally-heated environments in Yellowstone National Park have revealed an exceptionally heat-resistant grass Dichanthelium lanuginosum. Individuals of this species were able to withstand rhizosphere temperatures ranging from 40 to 57°C. Long-term (July and August, 1996) rhizosphere temperature measurements at three sites confirmed that geothermal heat maintained high soil temperatures during the night. Plants grown in the lab from field-collected seed display significantly higher shoot fresh weight when grown at soil temperatures of 35–41°C vs. 23–27°C. Though there is no difference in root fresh weight of plants grown at these two temperature regimes, the roots from the warmer soils are significantly shorter and more highly branched compared with plants grown in the cooler soils. This species also displays acid tolerance both in the field, with rhizosphere pH<3, measured at several sites, and when grown in the lab. In response to increased temperature, individual D. lanuginosum plants, either grown in the lab or collected in the field, expressed a low molecular weight protein that cross-reacted with heat shock protein antibodies.

Composition and occurrence of lipid droplets in the cyanobacterium Nostoc punctiforme

Abstract Inclusions of neutral lipids termed lipid droplets (LDs) located throughout the cell were identified in the cyanobacterium Nostoc punctiforme by staining with lipophylic fluorescent dyes. LDs increased in number upon entry into stationary phase and addition of exogenous fructose indicating a role for carbon storage, whereas high-light stress did not increase LD numbers. LD accumulation increased when nitrate was used as the nitrogen source during exponential growth as compared to added ammonia or nitrogen-fixing conditions. Analysis of isolated LDs revealed enrichment of triacylglycerol (TAG), α-tocopherol, and C17 alkanes. LD TAG from exponential phase growth contained mainly saturated C16 and C18 fatty acids, whereas stationary phase LD TAG had additional unsaturated fatty acids characteristic of whole cells. This is the first characterization of cyanobacterial LD composition and conditions leading to their production. Based upon their abnormally large size and atypical location, these structures represent a novel sub-organelle in cyanobacteria.

Characterization of a model system for the study of Nostoc punctiforme akinetes

Nostoc punctiforme is a filamentous cyanobacterium that is capable of dark heterotrophy and cellular differentiation into nitrogen-fixing heterocysts, motile hormogonia, or spore-like akinetes. The study of akinete differentiation at the molecular level has been limited by the asynchronous development and limited number of akinetes formed within a filament. A system in which to study the development and genetic regulation of akinetes was investigated using a zwf mutant lacking glucose-6-phosphate dehydrogenase, the initial enzyme of the oxidative pentose phosphate pathway. Upon dark incubation in the presence of fructose, the zwf− strain ceased growth and differentiated into akinete-like cells, whereas the wild-type strain exhibited heterotrophic growth. Dark-induced zwf akinetes exhibited periodic acid–Schiff staining characteristics identical to that observed for wild-type akinetes, and synchronous induction of akinetes occurred in treated cultures. Dark-induced zwf akinetes exhibited increased resistance to the environmental stresses of desiccation, cold, or treatment with lysozyme relative to vegetative cells of both strains. Transcription of the avaK akinete marker gene was strongly induced in developing zwf akinetes as shown by Northern blotting and green fluorescent protein transcriptional reporter fusions. ATP levels did not vary significantly between dark incubated strains, indicating that a signal other than energy level may trigger akinete formation. This phenotypic and genetic evidence showing near-synchronous induction of dark-induced zwf akinetes indicates that this system will provide a valuable tool for the molecular genetic study of akinete development in N. punctiforme.

Identification of Nostoc punctiforme akinete‐expressed genes using differential display

Akinetes are spore‐like resting cells formed by certain filamentous cyanobacteria that have increased resistance to environmental stress. They can be found at low frequencies in dense cultures experiencing low light or phosphate limitation, but also form at high frequencies in a zwf mutant strain of Nostoc punctiforme following dark incubation in the presence of fructose. The wild‐type strain is capable of facultative heterotrophic growth under these conditions and does not form akinetes. To identify genes associated with akinete development, differential display was used to amplify and compare cDNA from a wild‐type and zwf mutant strain of N. punctiforme following a switch to dark heterotrophic conditions. Screening of candidate genes by reverse transcriptase real‐time quantitative PCR and subsequent testing for akinete‐specific expression using GFP transcriptional reporter plasmids lead to the identification of three novel akinete‐expressed genes. The genes identified from the screening encoded for proteins homologous to an aminopeptidase (aapN), a zinc protease (hap) and an ATP‐binding cassette (ABC)‐type transporter (aet). Expression of hap was also increased in developing hormogonia, a transient type of differentiated filament capable of gliding motility. Transcriptional start sites for akinete‐expressed genes were determined using random amplification of cDNA ends (RACE), and promoter regions were compared with orthologues in other filamentous cyanobacteria to identify putative regulatory sequences.

The Sinorhizobium meliloti Lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa.

While screening for Sinorhizobium meliloti Pho regulatory mutants, a transposon mutant was isolated that constitutively expressed higher levels of acid and alkaline phosphatase enzymes. This mutant was also found to form pseudonodules on alfalfa that were delayed in appearance relative to those formed by the wild-type strain, it contained few bacteroids, and it did not fix nitrogen. Sequence analysis of the transposon insertion site revealed the affected gene to have high homology to Lon proteases from a number of organisms. In minimal succinate medium, the mutant strain was found to grow more slowly, reach lower maximal optical density, and produce more extracellular polysaccharide (EPS) than the wild-type strain. The mutant fluoresced brightly on minimal succinate agar containing calcofluor (which binds to EPSI, a constitutively expressed succinoglycan), and gas chromotographic analysis of purified total EPS showed that the glucose-to-galactose ratio in the lon mutant total EPS was 5.0 +/- 0.2 (mean +/- standard error), whereas the glucose-to-galactose ratio in the wild-type strain was 7.1 +/- 0.5. These data suggested that in addition to EPSI, the lon mutant also constitutively synthesized EPSII, a galactoglucan which is the second major EPS known to be produced by S. meliloti, but typically is expressed only under conditions of phosphate limitation. (13)C nuclear magnetic resonance analysis showed no major differences between EPS purified from the mutant and wild-type strains. Normal growth, EPS production, and the symbiotic phenotype were restored in the mutant strain when the wild-type lon gene was present in trans. The results of this study suggest that the S. meliloti Lon protease is important for controlling turnover of a constitutively expressed protein(s) that, when unregulated, disrupts normal nodule formation and normal growth.

Cloning and characterization of a Rhizobium meliloti nonspecific acid phosphatase.

Abstract Nodulated legumes require high levels of phosphorus for optimal symbiotic performance. However, the basis for this elevated phosphorus requirement is poorly understood, and very little information regarding bacteroid phosphorus metabolism is available. To develop an understanding of the relative importance of organic and inorganic phosphorus sources for bacteroids, we investigated phosphatase activity in Rhizobium meliloti. An R. meliloti plasmid library clone that complemented an Escherichia coli phosphatase mutant was isolated, and the clone was sequenced. The complementing fragment contained a 337-amino-acid open reading frame that has a potential leader sequence and processing sites characteristic of periplasmic proteins. The phosphatase activity was located in the periplasm of R. meliloti and of E. coli containing the cloned gene. The subunit molecular mass of the cloned phosphatase was 33 kDa, and gel filtration indicated the active enzyme was a 66-kDa homodimer. Lack of substrate specificity suggests the cloned gene, napD, encodes a nonspecific acid phosphatase with a pH optimum of approximately 6.5. An R. melilotinapD transposon-insertion mutant was constructed, and its symbiotic phenotype was determined to be Fix+ regardless of the level of phosphorus provided to the host plant.