Measuring the force production of the hormogonia of Mastigocladus laminosus

The cyanobacterium Mastigocladus laminosus forms hormogonia, which glide slowly away from the parent colony by extruding slime out of nozzles. Using video microscopy, we observed hormogonia embedded in and moving through 1-4% agar solutions with an average velocity of 0.5 microm/s. Agar is non-Newtonian and is subject to shear-thinning so that its viscosity greatly increases at low shear rates. We measured the viscosity of these agar solutions at the very low shear rates appropriate for gliding hormogonia and found it to vary from 1 to 52 million centipoise. Then, by applying a Newtonian drag coefficient for a 100-microm-long, cigar-shaped hormogonium, we found that it produced a force of several million pN. A typical hormogonium has 10-100 thousand 9-nm-wide slime extrusion nozzles. Wolgemuth et al. have proposed hydration-driven swelling of the polyelectrolyte slime ejected from these nozzles as the force production mechanism, and our experiment found a large nozzle force that was consistent with this hypothesis. Average single nozzle force depended on viscosity, being large when the viscosity was high: 71 pN in 3% and 126 pN in 4% agar.     

Symbiosis between the cyanobacterium Nostoc and the liverwort Blasia requires a CheR-type MCP methyltransferase

In response to environmental change, the cyanobacterium Nostoc punctiforme ATCC 29133 produces highly adapted filaments known as hormogonia that have gliding motility and serve as the agents of infection in symbioses with plants. Hormogonia sense and respond to unidentified plant-derived chemical signals that attract and guide them towards the symbiotic tissues of the host. There is increasing evidence to suggest that their interaction with host plants is regulated by chemotaxis-related signal transduction systems. The genome of N. punctiforme contains multiple sets of chemotaxis (che)-like genes. In this study we characterize the large che5 locus of N. punctiforme. Disruption of NpR0248, which encodes a putative CheR methyltransferase, results in loss of motility and significantly impairs symbiotic competency with the liverwort Blasia pusilla when compared with the parent strain. Our results suggest that chemotaxis-like elements regulate hormogonia function and hence symbiotic competency in this system.     

Molecular analysis of genes in Nostoc punctiforme involved in pilus biogenesis and plant infection

Hormogonia are the infective agents in many cyanobacterium-plant symbioses. Pilus-like appendages are expressed on the hormogonium surface, and mutations in pil-like genes altered surface piliation and reduced symbiotic competency. This is the first molecular evidence that pilus biogenesis in a filamentous cyanobacterium requires a type IV pilus system.     

Sacrificial cell death and trichome breakage in an oscillatoriacean blue-green alga: the role of murein

Summary Trichomes of Microcoleus vaginatus, a motile blue-green alga of the family Oscillatoriaceae, were studied by light and electron microscopy in an effort to determine the sites of trichome breakage during production of hormogonia.According to the evidence presented herein, transcellular breakage of trichomes is the only mechanism of hormogonium production in M. vaginatus. Tearing of the murein sacculus appears to be necessary and sufficient for transcellular breakage to ensue. As Fuhs and earlier investigators have correctly claimed, this process always involves the death of the cell whose wall is torn.When trichomes of M. vaginatus break across cells to produce hormogonia, the murein sacculus usually tears along a circumferential set of junctional pores. This particular mechanism of trichome breakage is not universal among members of the family Oscillatoriaceae.This report is based on a thesis submitted in partial fulfillment of the requirements for a Ph. D. degree in Biology at Harvard University.     

Development of motility in cultures of the cyanobacterium Mastigocladus laminosus

Abstract The time course for the development of motility in cultures of the cyanobacterium Mastigocladus laminosus was established quantitatively using a slicer tool as described here. The slicer tool produces samples of trichomes from centrifuged pellets that, under identical conditions, shed comparable numbers of hormogonia. The number of hormogonia formed in liquid culture rises steeply between 24 and 31 h of incubation, returning to essentially zero in the next 24 h. The initial lag may be devoted to the cell divisions needed to form the cells of the hormogonium. The drop in motility could be due to one or more heat-stable substance(s) accumulated in the medium, since used media inhibited motility and the effect resisted autoclaving. The fact that the inoculum needed to be ground in order for motility to occur suggests that the structure of the clump inhibits the shedding of hormogonia. Some ecological implications are proposed, assuming that the clump structure interferes with light and mass transfer.     

Hormogonia in two nostocalean cyanophytes (cyanobacteria) from the genera Hapalosiphon and Fischerella

The formation of hormogonia in the nostocalean cyanophytes/cyanobacteria Hapalosiphon fontinalis (C. Agardh) Bornet and Fischerella sp. was studied in natural populations collected from the Klin peatbog, northern Slovakia. Hormogonia were produced terminally in lateral branches of filaments (both species), or also directly on the main branches (Fischerella sp.). In contrast to vegetative filaments, hormogonia were not ramified, lacked heterocytes, were embedded in mucilaginous envelopes, were able to move, and their cells contained aerotopes. They were released by gliding through an opening in the sheath at the end of lateral branches of filaments. Released hormogonia of H. fontinalis were solitary or agglomerated into common fascicles morphologically resembling planktic colonies of Aphanizomenon flos-aquae (L.) Ralfs ex Bornet et Flahault or Dolichospermum affine (Lemmermann) Wacklin, Hoffmann et Komárek (syn. Anabaena affinis Lemmermann). Occasionally, lateral or sessile Nostochopsis-like heterocytes and apical spherical monocytes were formed on the main filaments. Hormogonia of Fischerella sp. were formed not only in apical part of lateral trichomes, but also directly on the main trichomes. Their cells were markedly larger than the vegetative cells and possessed well-developed aerotopes. Released hormogonia remained solitary, and were not agglomerated into fascicles. Apical hormogonia were released by gliding through an opening in the sheath at the end of lateral branches of filaments, and basal hormogonia were released by breaking off the main axis. In contrast to filaments of H. fontinalis which were very common and represented the dominant species of the cyanophyte communities in the locality, filaments of Fischerella sp. were observed only in one sample and for a limited period. This is the first record of a representative of the genus Fischerella in Slovakia.     

丝状体蓝藻藻殖段的分化及其调节机制

本文介绍了丝状体蓝藻(亦称蓝细菌) 的藻殖段的分化及其调节机制。藻殖段与正常藻丝体的区别在于细胞形状、细胞内存有气囊和可移动的短而直的藻丝链等。本文对许多环境因子包括光和营养因素等促进或抑制藻殖段的分化进行了讨论;还介绍了念珠藻(Nostoc) ,单歧藻(Tolypothrix) 和眉藻(Calothrix)所具有复杂的细胞发育过程,即具气囊又可移动的藻殖段分化,异形胞分化以及营养细胞的补偿性色适应。这三种细胞类型的适应形成取决于两种不同的光受体系统。藻殖段和异形胞两者的分化可能取决于光合电子传递链;而营养细胞的补偿性色适应则受光敏色素的调节。此外,谷酰胺合成酶合成和活性调节的PII蛋白,在协同藻殖段分化、异形胞分化及营养细胞的补偿色适应中起重要作用。由于蓝藻藻殖段分化及其调节机制是一个新的研究领域,关于它的知识尚不完整,亟待人们加强研究。     

Electron Transport Regulates Cellular Differentiation in the Filamentous Cyanobacterium Calothrix

Differentiation of the filamentous cyanobacteria Calothrix sp strains PCC 7601 and PCC 7504 is regulated by light spectral quality. Vegetative filaments differentiate motile, gas-vacuolated hormogonia after transfer to fresh medium and incubation under red light. Hormogonia are transient and give rise to vegetative filaments, or to heterocystous filaments if fixed nitrogen is lacking. If incubated under green light after transfer to fresh medium, vegetative filaments do not differentiate hormogonia but may produce heterocysts directly, even in the presence of combined nitrogen. We used inhibitors of thylakoid electron transport (3-[3,4-dichlorophenyl]-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) to show that the opposing effects of red and green light on cell differentiation arise through differential excitations of photosystems I and II. Red light excitation of photosystem I oxidizes the plastoquinone pool, stimulating differentiation of hormogonia and inhibiting heterocyst differentiation. Conversely, net reduction of plastoquinone by green light excitation of photosystem II inhibits differentiation of hormogonia and stimulates heterocyst differentiation. This photoperception mechanism is distinct from the light regulation of complementary chromatic adaptation of phycobilisome constituents. Although complementary chromatic adaptation operates independently of the photocontrol of cellular differentiation, these two regulatory processes are linked, because the general expression of phycobiliprotein genes is transiently repressed during hormogonium differentiation. In addition, absorbance by phycobilisomes largely determines the light wavelengths that excite photosystem II, and thus the wavelengths that can imbalance electron transport.     

PHOTOSYNTHETIC ACTIVITY OF TWO DEVELOPMENTAL STAGES OF A NOSTOC STRAIN (CYANOBACTERIA) ISOLATED FROM GEOSIPHON PYRIFORME (MYCOTA)1

Sessile colonies and motile hormogonia, the two main developmental stages in the life cycle of a Nostoc strain isolated from the endocytobiosis with Geosiphon pyriforme (Kützing) F. v. Wettstein, were investigated for their photosynthetic competence. Large-scale fractionation of the two stages is presented. Photosynthetic parameters were assessed by measurement of chlorophyll fluorescence and oxygen evolution. Hormogonia were as photosynthetically competent as the colonial stage. In addition, hormogonia showed an enhanced capability for nonradiative dissipation of absorbed light energy, a feature that might be important for their function as propagula. Data for the quantum yield of photosystem II of the isolated Nostoc strain were compared to the values determined in situ in G. pyriforme and indicated the possibility of a higher photosynthetic capacity of the endosymbiotic as compared to the isolated cyanobacterium.     

Identification of a hormogonium polysaccharide-specific gene set conserved in filamentous cyanobacteria

Cyanobacteria comprise a phylum defined by the capacity for oxygenic photosynthesis. Members of this phylum are frequently motile as well. Strains that display gliding or twitching motility across semisolid surfaces are powered by a conserved type IV pilus system (T4P). Among the filamentous, heterocyst-forming cyanobacteria, motility is usually confined to specialized filaments known as hormogonia, and requires the deposition of an associated hormogonium polysaccharide (HPS). The genes involved in assembly and export of HPS are largely undefined, and it has been hypothesized that HPS exits the outer membrane via an atypical T4P-driven mechanism. Here, several novel hps loci, primarily encoding glycosyl transferases, are identified. Mutational analysis demonstrates that the majority of these genes are essential for both motility and production of HPS. Notably, most mutant strains accumulate wild-type cellular levels of the major pilin PilA, but not extracellular PilA, indicating dysregulation of the T4P motors, and, therefore, a regulatory interaction between HPS assembly and T4P activity. A co-occurrence analysis of Hps orthologs among cyanobacteria identified an extended set of putative Hps proteins comprising most components of a Wzx/Wzy-type polysaccharide synthesis and export system. This implies that HPS may be secreted through a more canonical pathway, rather than a T4P-mediated mechanism.     

Characterization of gliding motility in Flexibacter polymorphus

Motility of the marine gliding bacterium Flexibacter polymorphus was studied by using microcinematographic techniques. Following adhesion to a glass surface, multicellular filaments and individual cells usually began to glide within a few seconds at a speed of approximately 12 micron per second (at 23 degrees C). Adhesion to the glass surface was evidently mediated by multitudes of extremely fine extracellular fibrils. Gliding velocity was independent of filament length but directly related to electron-transport activity and substratum temperature in the range 3-35 degrees C. The rate of gliding was inversely related to medium viscosity, suggesting that the locomotor apparatus functions at constant torque. Forward motion was occasionally interrupted by direction reversals, somersaults (observed primarily in single cells of short filaments), or spinning of filaments tethered by one pole. The frequency of direction reversal was found to be an inverse function of filament length. Translational motility was invariably accompanied by sinistral revolution about the longitudinal axis of a filament. The sense and pitch of revolution were constant among filaments of different length. Polystyrene microspheres or India ink particles adsorbed to gliding cells were actively displaced in either direction, their movement tracing either a regular zigzag or helical path along the filament surface. Because microspheres were also observed to move on nonmotile filaments, particle translocation was evidently not obligatorily linked to gliding locomotion. Multiple particles adsorbed to a single filament often moved independently. The data are consistent with a motility mechanism involving limited motion in numerous mechanically independent (yet functionally coordinated) domains on the cell surface.     

Characteristics of Hormogonia Formation by Symbiotic Nostoc spp. in Response to the Presence of Anthoceros punctatus or Its Extracellular Products

Nostocacean cyanobacteria typically produce gliding filaments termed hormogonia at a low frequency as part of their life cycle. We report here that all Nostoc spp. competent in establishing a symbiotic association with the hornwort Anthoceros punctatus formed hormogonial filaments at a high frequency in the presence of A. punctatus. The hormogonia-inducing activity was produced by A. punctatus under nitrogen-limited culture conditions. The hormogonia of the symbiotically competent Nostoc spp. were characterized as motile (gliding) filaments lacking heterocysts and with distinctly smaller cells than those of vegetative filaments; the small cells resulted from a continuation of cell division uncoupled from biomass increase. An essentially complete conversion of vegetative filaments to hormogonia occurred within 12 h of exposure of Nostoc sp. strain 7801 to A. punctatus growth-conditioned medium. Hormogonia formation was accompanied by loss of nitrogen fixation (acetylene reduction) and by decreases in photosynthetic CO₂ fixation and in vivo NH₄⁺ assimilation of 30% and approximately 40%, respectively. The rates of acetylene reduction and CO₂ fixation returned to approximately the control rates within 72 to 96 h after hormogonia induction, as the cultures of Nostoc sp. strain 7801 differentiated heterocysts and reverted to the vegetative growth state. The relationship between hormogonia formation and symbiotic competence is discussed.     

Comparison of Nostocean hormogonium induction and its motility on solid plates between agar and gellan gum at varying gel matrix concentrations

To establish a sensitive bioassay for Nostocean hormogonium induction, we compared the effectiveness of the morpho-differentiation induction on two gelled plates, agar and gellan gum, for anacardic acid C15:1-Δ⁸ decyl ester (1) (100 nmol/disc). On BG-11₀ (nitrogen-free) medium-based 0.6 and 0.8% agar plates, Nostoc sp. strain Yaku-1 isolated from a coralloid root of Cycas revoluta in Yakushima Island showed clear morpho-differentiation from filamentous aggregates into hormogonia, and the induced hormogonia dispersed within 24 h; however, similar hormogonium formation was not observed at agar concentrations of 1.0% or higher. Conversely, hormogonium induction was considerably more pronounced on gellan gum plates than those on agar plates through concentrations ranging from 0.6 to 1.6% even after 12 h of incubation, particularly active on the 0.8–1.0% gellan gum plates. Thus, gellan gum plates can achieve clear results within 12 h and are thus highly useful for primary screening for hormogonium-inducing factors (HIFs).     

丝状体蓝藻藻殖段的分化及其调节机制

本文介绍了丝状体蓝藻（亦称蓝细菌）的藻殖段的分化及其调节机制。藻殖段与正常藻丝体的区别在于细胞开状、细胞内存有气囊和可移动的短而真的藻丝链等。本文对许多环境因子包括光和营养因素等促进或抑制藻殖段的分化进行一讨论;还介绍了含球藻（Ｎｏｓｔｏｃ）,单歧藻（Ｔｏｌｙｐｏｔｈｒｉｘ）和眉藻（Ｃａｌｏｔｈｒｉｘ）所具有复杂的细胞发育过程,即具气囊又可移动的藻殖段分化,异形胞分化以及营养细胞的被偿性色适应。这     

VULNERABILITY OF NOSTOC MUSCORUM AGARDH (CYANOPHYCEAE) MOTILE HORMOGONIA TO CILIATE GRAZING1

Experiments were carried out to investigate if the stage of life cycle of Nostoc muscorum Agardh alters vulnerability to grazing by Pseudomicrothorax dubius Maupas. When the percentage of motile hormogonia of all counted trichomes exceeded 10%, most of the grazers (80%–100%) became satiated within 2 h. In most cases (90%) grazers successfully attacked motile hormogonia. Attacks on nonmotile trichomes were much rarer (8%) and mainly unsuccessful. Direct observations revealed that hormogonia could be ingested by the ciliates as long as they remained motile. Hormogonia already adhered to the bottom were still recognized by ciliates as potential food but were not ingested. We did not observe attacks on old vegetative colonies. This is apparently the first report on the motile stage of Nostoc being susceptible to ciliate grazing. Experiments with other grazers, Nassula tumida Maskell and two different clones of Furgassonia blochmanni Faure‐Fremiet, showed that only one clone of F. blochmanni was able to feed on motile hormogonia, whereas the second clone and N. tumida showed no interest in them.     

STEROLS AS BIOMARKERS IN GYMNODINIUM BREVE: DISTRIBUTION IN DINOFLAGELLATES

A poorly understood feature of nostocacean growth and development is the formation of ordered macroscopic structures from microscopic cells, trichomes, and filaments. Using macro-photography, time-lapse micro-cinematography, light and electron microscopy of Nostoc species in pure culture, it has been possible to demonstrate how motility, adhesion and aggregation of photo-induced hormogonia result in macro-morphogenesis of dendroid forms. Red-light induced hormogonia from synchronized cultures aggregate rapidly on agar as tight flowing streams, in patterns responsive to the direction and quality of incident light. Unlike the even textured cell surfaces of heterocystous filaments, the cell walls of swarming hormogonia are covered with a striate mucoid layer containing pili attached to cells of adjacent hormogonia. During differentiation to an aseriate phase, cell wall fusions occur and a gelatinous matrix forms around the enlarging sub-globose cells. Liquid suspensions of hormogonia aggregate in a solid mass following the net centripetal movement of interlaced loops of curved hormogonia attached by adhesive pili. In darkness or dim white light, compressed hormogonial aggregates form erect tree-like (dendroid) macro-structures by photo-tactic reversal of streaming motility. Hormogonia within the aggregates re-organize into streams that push upward into the light, forming structured, positively phototropic protuberances, several millimeters in length. Under weak illumination, the structures become branched with crowns of waving hormogonia. The dendroid morphology is stabilized by deposit of gelatinous material derived from successive cycles of cell-filament development, liberation of heterocysts and formation of dormant cells and trichomes.