Core substructure in Mastigocladus laminosus phycobilisomes

A native high molecular complex (M_r 850000) containing about 50% of the allphycocyanin of the phycobilisome but lacking allophycocyanin B was separated from isolated phycobilisomes by gel electrophoresis. It was designated AP_CM since the large linker polypeptide L_CM was exclusively localized in this complex. The complex exhibited a ?196°C fluorescence emission maximum at 673 nm (671 nm at 25°C). In addition, a core complex (designated AP_C, M_r≥1000000) consisting of both AP_CM and AP 680 was isolated by combined gel filtration and linear gradient centrifugation. At 25°C this complex showed dual emission peaks at 670 and 680 nm demonstrating functional independence of the terminal emitters. A complex similar to AP_CM can be isolated from phycobilisomes of Anabaena variabilis. This is evidence that AP_CM is the constitutive center of the tricylindrical core of hemidiscoidal cyanobacterial phycobilisomes. Two models summarizing the structural and functional consequences of the results are presented in the discussion.     

Core substructure in Mastigocladus laminosus phycobilisomes

Three allophycocyanin complexes were separated by gel electrophoresis, isoelectric focusing and ion exchange chromatography from a low molecular fraction (M_r 100–150000) of partially dissociated phycobilisomes of Mastigocladus laminosus: A. (^APAP); B. (^*AP₂ ^AP₂ ^AP*AP) · L _C ¹⁰ ; and C. (^*APAPBAP₂ ^AP*AP) · L _C ¹⁰ . According to their fluorescence emission maximum at room temperature the complexes A., B. and C. are designated AP 660, AP 664 and AP 680. The different subunits of the AP complexes have apparent molecular weights of M_r 18500 ^*AP, 18200 ^APB, 18000 ^AP, 17000 ^AP and 16500 ^*AP. This hitherto unrecognized microheterogeneity within the AP subunits of complexes B. and C. of Mastigocladus laminosus phycobilisomes could also be demonstrated and confirmed with the two phycocyanin complexes PC 642 and PC 646. PC 642 is characterized by a L _R ¹¹ linker polypeptide.Abbreviations AP allophycocyanin - PC phycocyanin - PEC phycoerythrocyanin - PE phycoerythrin - PAGE polyacrylamide gel electrophoresis - IEF isoelectric focusing - pI isoelectric point - M_r apparent molecular weight - TMED tetramethylethylenediamine - APS ammonium persulphate - SDS sodium dodecylsulphate - O.D. optical density A preliminary account of this work has been presented at the Embo Workshop on Oxygenic and Anoxygenic Electron Transport Systems in Cyanobacteria (Blue-green Algae) in Cape Sounion, Greece, 20–25 September 1987     

Phylogeography of the thermophilic cyanobacterium Mastigocladus laminosus

We have taken a phylogeographic approach to investigate the demographic and evolutionary processes that have shaped the geographic patterns of genetic diversity for a sample of isolates of the cosmopolitan thermophilic cyanobacterial Mastigocladus laminosus morphotype collected from throughout most of its range. Although M. laminosus is found in thermal areas throughout the world, our observation that populations are typically genetically differentiated on local geographic scales suggests the existence of dispersal barriers, a conclusion corroborated by evidence for genetic isolation by distance. Genealogies inferred using nitrogen metabolism gene sequence data suggest that a significant amount of the extant global diversity of M. laminosus can be traced back to a common ancestor associated with the western North American hot spot currently located below Yellowstone National Park. Estimated intragenic recombination rates are comparable to those of pathogenic bacteria known for their capacity to exchange DNA, indicating that genetic exchange has played an important role in generating novel variation during M. laminosus diversification. Selection has constrained protein changes at loci involved in the assimilation of both dinitrogen and nitrate, suggesting the historic use of both nitrogen sources in this heterocystous cyanobacterium. Lineage-specific differences in thermal performance were also observed.     

Phylogeography of the Thermophilic Cyanobacterium Mastigocladus laminosus

We have taken a phylogeographic approach to investigate the demographic and evolutionary processes that have shaped the geographic patterns of genetic diversity for a sample of isolates of the cosmopolitan thermophilic cyanobacterial Mastigocladus laminosus morphotype collected from throughout most of its range. Although M. laminosus is found in thermal areas throughout the world, our observation that populations are typically genetically differentiated on local geographic scales suggests the existence of dispersal barriers, a conclusion corroborated by evidence for genetic isolation by distance. Genealogies inferred using nitrogen metabolism gene sequence data suggest that a significant amount of the extant global diversity of M. laminosus can be traced back to a common ancestor associated with the western North American hot spot currently located below Yellowstone National Park. Estimated intragenic recombination rates are comparable to those of pathogenic bacteria known for their capacity to exchange DNA, indicating that genetic exchange has played an important role in generating novel variation during M. laminosus diversification. Selection has constrained protein changes at loci involved in the assimilation of both dinitrogen and nitrate, suggesting the historic use of both nitrogen sources in this heterocystous cyanobacterium. Lineage-specific differences in thermal performance were also observed.     

Ultrastructure of the cyanobacterium,Mastigocladus laminosus

The morphology and ultrastructure of the thermophilic cyanobacteriumMastigocladus laminosus were examined by scanning and transmission electron microscopy. Mature cultures consisted of relatively old, wide filaments that branched frequently to form younger, thinner filaments. The cells of the younger filaments had a consistently cylindrical morphology, while those of older filaments were rounded and pleomorphic. The internal ultrastructure of the cells depended somewhat on their age. As young cells became larger and wider, their thylakoids underwent slight rearrangement and spread out toward the center of the cytoplasm. Polyphosphate bodies, carboxysomes (polyhedral bodies), and lipid-body-like structures increased in number as the cells aged, but ribosomes and cyanophycin granules were depleted. Cell division involved septum formation followed by ingrowth of the outer membrane and sheath. Cells in older filaments were separated from each other by a complete layer of sheath material. Septum formation in older cells was also seen to occur parallel to the long axis of the filament, thereby confirming that true branching took place.     

Heterocyst differentiation in the cyanobacterium Mastigocladus laminosus.

The morphological and ultrastructural aspects of heterocyst differentiation in the branching, filamentous cyanobacterium Mastigocladus laminosus were examined with light and electron microscopy. The earliest differentiation stages involved cytoplasmic changes, including (i) rapid degradation of carboxysomes, (ii) degradation of polysaccharide granules, and (iii) accumulation of electron-dense ribosomal or protein material (or both). Intermediate differentiation stages involved synthesis of a homogeneous extra wall layer, development of necks leading to adjacent cells, and elaboration of a complex system of intracytoplasmic membranes. Late differentiation stages included further development of necks and continued elaboration of membranes. Mature heterocysts possessed a uniformly electron-dense cytoplasm that contained large numbers of closely packed membranes, some of which were arranged in lamellar stacks. Mature heterocysts lacked all of the inclusion bodies present in undifferentiated vegetative cells, but contained a number of unusual spherical inclusions of variable electron density. Cells in both narrow and wide filaments were capable of differentiating. No regular heterocyst spacing pattern was observed in the narrow filaments; the number of vegetative cells between consecutive heterocysts of any given filament varied by a factor of 10. Heterocysts developed at a variety of locations in the wide, branching filaments, although the majority of them were situated adjacent to branch points. M. laminosus displayed a marked tendency to produce sets of adjacent heterocysts or proheterocysts (or both) that were not separated from each other by vegetative cells. Groups of four or more adjacent heterocysts or proheterocysts occurred frequently in wide filaments, and in some of these filaments virtually all of the cells appeared to be capable of differentiating into heterocysts.     

Chromophore assignment in phycoerythrocyanin from Mastigocladus laminosus

The component spectra (maxima of absorption, circular and linear dichroism) of individual chromophores have been assigned for phycoerythrocyanin (PEC) trimer, monomer(s), and its subunits (-PEC and -PEC) by titration with p-chloromercury-benzene-sulfonate (PCMS), linear dichroism and photochemical transformations, as well as by deconvolution using a bilin line-shape spectrum based on the -84 phycoviolobilin-chromophore in the -subunit. The level ordering PVB--84 PCB--155 PCB--84 is the same irrespective of aggregation. Two different monomers () were observed. In 4 M urea, the spectra are appropriately weighted sums of the subunit spectra, whereas in the monomer obtained in 1 M KSCN, both -chromophores are red-shifted by 4–5 nm. Formation of trimer ()_{_3}gives considerable spectral changes: (1) the absorption is narrowed, which has been rationalized by excitonic coupling between neighbouring monomers, (2) the short wavelength part in the CD spectrum is missing and (3) a fourth band (+) at 528 in the LD spectrum appears. A deconvolution of the trimeric aggregation state using only the bilin line-shape model is not possible.     

Isolation of hydrogenase from the thermophilic cyanobacterium Mastigocladus laminosus

Summary A soluble, cytoplasmic hydrogenase was detected and partially purified from Mastigocladus laminosus. It was found to be unstable but could be stabilized to some extent by Mg⁺⁺; 50% of the activity remained after one week in air at 4 °C.     

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.     

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.     

Characterization of the motile hormogonia of Mastigocladus laminosus.

The cyanobacterium Mastigocladus laminosus produces motile hormogonia which move by gliding motility. These hormogonia were characterized in terms of their morphology, state of differentiation of the cells, optimal temperature for production and motility, minimal nutritional requirements to sustain motility, liberation of the hormogonium from its parental trichome, average surface velocity, and maximal concentration of agar through which the hormogonium may move. We found that an average hormogonium consisted of 13.6 cells of only the narrow-cell-type morphology. Gliding motility and the production of hormogonia were maximal at 45 degrees C. Agarose plus 0.20 mM Ca2+ was sufficient to sustain gliding motility. Hormogonia were liberated from the parental trichome by formation and lysis of a necridium. The average surface velocity of a hormogonium was 1.7 micron/s with a maximal velocity of 3 micron/s. Hormogonia were motile through 7% agar. Motile hormogonia leave a record of their passage in the form of easily visible tracks on the surface of solid media. Three types of tracks were observed: straight, sinusoidal, and circular. Normal, forward-directed motion involves screwlike rotation that describes a right-handed helix. However, observations are presented which suggest that rotational motion is not a prerequisite for gliding motility in this cyanobacterium.     

Significance of braided trichomes in the cyanobacterium Mastigocladus laminosus.

Loops and braids in filaments of the cyanobacterium Mastigocladus laminosus were observed. Braided filaments were generally in the form of a right-handed helix (87%) but were occasionally observed as left-handed helices (13%). It was demonstrated by time-lapse photomicrography that braids continued to grow as braids and that loops coiled into braids as growth proceeded. Measurements of the distance between grooves in 74 braids yielded an average distance of 13 +/- 3 micron, a result which suggests that braid formation is not random. We propose that the braids arise as a consequence of the helical growth of cells that make up the filaments of M. laminosus.     

Hydrogen evolution by a thermophilic blue-green alga Mastigocladus laminosus

The thermophilic blue-green alga (cyanobacterium), Mastigocladuslaminosus isolated from a hot spring, evolved hydrogen gas undernitrogen-starved conditions in light when algal cells were grownin a nitrate-free medium. Cells grown in a nitrate-medium evolvedno detectable hydrogen gas in light. The optimal temperatureand pH for hydrogen evolution were 44–49?C and 7.0–7.5.High activity of hydrogen evolution. 1.6 ml H₂/mg chl.hr, wasinduced when algal cells grown in the nitrate medium were activelyforming heterocysts in the nitrate-free medium in air. Hydrogenevolution in light was depressed by nitrogen gas and inhibitedby salicylaldoxime or DNP. This hydrogen evolution by M. laminosusis attributed to the action of nitrogenase. (Received June 20, 1979; )     

Structural basis for the thermostability of ferredoxin from the cyanobacterium Mastigocladus laminosus

Plant-type ferredoxins (Fds) carry a single [2Fe-2S] cluster and serve as electron acceptors of photosystem I (PSI). The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus displays optimal activity at 65 degrees C. In order to reveal the molecular factors that confer thermostability, the crystal structure of M.laminosus Fd (mFd) was determined to 1.25 A resolution and subsequently analyzed in comparison with four similar plant-type mesophilic ferredoxins. The topologies of the plant-type ferredoxins are similar, yet two structural determinants were identified that may account for differences in thermostability, a salt bridge network in the C-terminal region, and the flexible L1,2 loop that increases hydrophobic accessible surface area. These conclusions were verified by three mutations, i.e. substitution of L1,2 into a rigid beta-turn ((Delta)L1,2) and two point mutations (E90S and E96S) that disrupt the salt bridge network at the C-terminal region. All three mutants have shown reduced electron transfer (ET) capabilities and [2Fe-2S] stability at high temperatures in comparison to the wild-type mFd. The results have also provided new insights into the involvement of the L1,2 loop in the Fd interactions with its electron donor, the PSI complex.     

Morphology and ultrastructure of the cyanobacterium Mastigocladus laminosus growing under nitrogen-fixing conditions

The morphological and ultrastructural characteristics of the cyanobacterium Mastigocladus laminosus growing under N₂-fixing conditions were examined with light and electron microscopy. Vegetative cells in narrow filaments contained randomly arranged segments of thylakoid membrane, centrally located carboxysomes (polyhedral bodies), peripherally located lipid bodies, and large numbers of polysaccharide granules in addition to nuclear material and ribosomes. The ultrastructural characteristics of cells in wide filaments were similar, except for increased numbers of carboxysomes and lipid bodies. Heterocytes and proheterocysts developed at a variety of locations in narrow filaments, wide filaments, and the lateral branches off of wide filaments. Akinetes were not observed in any of the filaments. The morphological characteristics of heterocysts and proheterocysts were variable and depended on those of the vegative cells from which the heterocysts and proheterocysts developed. Mature M. laminosus heterocysts were somewhat similar to those formed in other cyanobacterial genera, but they possessed a number of distinct and unique ultrastructural characteristics, including (i) the absence of a fibrous and, possibly, a laminated wall layer, (ii) the presence many closely packed membranes throughout most of the cytoplasm, and (iii) the presence of unidentified, spherical inclusion bodies of variable electron density.     

Ecological Specialization in a Spatially Structured Population of the Thermophilic Cyanobacterium Mastigocladus laminosus

Laboratory evolution experiments suggest the potential for microbial populations to contribute significant ecological variation to ecosystems, yet the functional importance of genetic diversity within natural populations of microorganisms is largely unknown. Here, we investigated the distribution of genetic and phenotypic variation for a population of the cyanobacterium Mastigocladus laminosus distributed along the temperature gradient of White Creek, Yellowstone NP. A total of 153 laboratory strains were directly isolated from five sites with mean annual temperatures ranging between 39 and 54°C. Genetic characterization at four nitrogen metabolism genes identified 15 closely related lineages in the population sample. These lineages were distributed nonrandomly along White Creek, but the observed geographic structure could not be explained by limited dispersal capabilities. Temperature performance experiments with six M. laminosus lineages that maximized their respective relative abundances at different positions along the gradient provided evidence for niche differentiation within the population. Niche differentiation included a tradeoff in performance at high and low temperatures, respectively. The physiological variation of these lineages in laboratory culture was generally well matched to the prevailing temperature conditions experienced by these organisms in situ. These results suggest that sympatric diversification along an ecological selection gradient can be a potent source of evolutionary innovation in microbial populations.     

Photosynthetic activities of a membrane preparation of the thermophilic cyanobacterium Mastigocladus laminosus

Photosynthetically active membranes have been prepared from the thermophilic cyanobacterium Mastigogladus laminosus by treatment with lysozyme. The membranes were active in electron transport through photosystem I and II as well as in photophosphorylation and proton uptake. Cells were grown at 40°, 45° and 55°C respectively. The temperature optimum of oxygen evolution of whole cells was about 10°C higher than the growth temperature. In isolated membranes the temperature optimum for cyclic photophosphorylation was identical to the growth temperature of the cells whereas the optimum for photosystem II electron transport never exceeded 40°C. Photophosphorylation was inhibited by N, N-dicyclohexyl carbodiimide (DCCD), carbonyl-cyanide-m-chlorophenylhydrazone and NH₄Cl, whereas proton uptake was enhanced by DCCD. Electron transport was slightly inhibited by these treatments. The membranes could be stored for several weeks at-20°C in 50% glycerol without any loss in the activities.Abbreviations DPIP 2,6-dichlorophenolindophenol - CCCP Carbonyl-cyanide-m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - PMS N-methylphenazonium methosulfate - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - TMP 20 mM Tris-HCl buffer pH 7.8, 10 mM MgCl₂, 5 mM phosphate buffer pH 7.8     

Cloning of the nifHDK genes and their organisation in the heterocystous cyanobacterium Mastigocladus laminosus

Abstract The heterocystous, nitrogen-fixing cyanobacterium Mastigocladus laminosus UTEX 1931 has an adjacent arrangement of the nifH, nifD and nifK genes, apparently similar to Fischerella sp. 27929, but unlike Anabaena 7120. In addition, unlike Fischerella sp. 27929, M. laminosus UTEX 1931 contains an additional nifH -like sequence located approximately 10 kb from the nifHDK gene cluster.