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.     

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.     

Structural basis of selection and thermostability of laboratory evolved Bacillus subtilis lipase

Variation in gene sequences generated by directed evolution approaches often does not assure a minimalist design for obtaining a desired property in proteins. While screening for enhanced thermostability, structural information was utilized in selecting mutations that are generated by error-prone PCR. By this approach we have increased the half-life of denaturation by 300-fold compared to the wild-type Bacillus subtilis lipase through three point mutations generated by only two cycles of error-prone PCR. At lower temperatures the activity parameters of the thermostable mutants are unaltered. High-resolution crystal structures of the mutants show subtle changes, which include stacking of tyrosine residues, peptide plane flipping and a better anchoring of the terminus, that challenge rational design and explain the structural basis for enhanced thermostability. The approach may offer an efficient and minimalist solution for the enhancement of a desired property of a protein.     

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.     

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.     

Structure of a photosystem I-ferredoxin complex from a marine cyanobacterium provides insights into far-red light photoacclimation

Far-red light photoacclimation exhibited by some cyanobacteria allows these organisms to use the far-red region of the solar spectrum (700–800 nm) for photosynthesis. Part of this process includes the replacement of six photosystem I (PSI) subunits with isoforms that confer the binding of chlorophyll (Chl) f molecules that absorb far-red light (FRL). However, the exact sites at which Chl f molecules are bound are still challenging to determine. To aid in the identification of Chl f-binding sites, we solved the cryo-EM structure of PSI from far-red light-acclimated cells of the cyanobacterium Synechococcus sp. PCC 7335. We identified six sites that bind Chl f with high specificity and three additional sites that are likely to bind Chl f at lower specificity. All of these binding sites are in the core-antenna regions of PSI, and Chl f was not observed among the electron transfer cofactors. This structural analysis also reveals both conserved and nonconserved Chl f-binding sites, the latter of which exemplify the diversity in FRL-PSI among species. We found that the FRL–PSI structure also contains a bound soluble ferredoxin, PetF1, at low occupancy, which suggests that ferredoxin binds less transiently than expected according to the canonical view of ferredoxin-binding to facilitate electron transfer. We suggest that this may result from structural changes in FRL-PSI that occur specifically during FRL photoacclimation.     

Thermal properties of NADP:ferredoxin oxidoreductase and ferredoxin isolated from a thermophilic blue-green alga

NADP:ferredoxin oxidoreductase (EC. 1.6.7.1.) isolated from a thermophilic blue-green alga, Synechococcus sp., was stable at temperatures up to 65°C. The diaphorase and cytochrome c reductase activities of the enzyme were low at 25°C but increased with elevated temperature to reach a maximum at about 60°C. The pH-profile of the diaphorase activity showed a peak at pH 9.0 at 55°C, whereas the activity was largely independent of pH at 25°C. High concentrations of NaCl suppressed activity at both high and low temperatures. In the cytochrome c reductase activity catalyzed by the enzyme, ferredoxin served as an electron carrier in a temperature-insensitive manner over a wide range of temperature. The results support the view that the optimum and the upper limiting temperatures for photosynthesis in this alga are related to thermal properties of proteins.     

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.     

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.     

Participation of ferredoxin in oxygen reduction by the photosynthetic electron transport chain

Oxygen reduction by isolated pea thylakoids was studied in the presence of ferredoxin (Fd), Fd + NADP, and cytochrome c. At Fd concentrations optimal for NADP reduction, it contributed 30–50% of the reducing equivalents (as deduced by comparing the rates of oxygen reduction and light oxidation of reduced Fd). The oxygen reduction rate in the presence of Fd + NADP was 3–4 times lower than with Fd alone, and comparable to that with cyt c. It is supposed that the process involves a photosystem I component whose reaction with oxygen depends on the rate of electron efflux from the PS I terminal acceptors, and that this component is phylloquinone.     

Isolation and biliprotein characterization of phycobilisomes from the thermophilic cyanobacterium Mastigocladus laminosus Cohn

A method for the effective isolation of functionally intact phycobilisomes from the thermophilic cyanobacterium M. laminosus is presented, using an unconventional high buffer molarity for stabilizing the aggregates and introducing a DNAse treatment of the disrupted cells to obtain sharp banding of the phycobilisomes in the linear sucrose density gradients.The structural integrity of the isolated phycobilisomes is demonstrated by a fluorescence emission maximum at 673 nm of aggregated allophycocyanin and by electron microscopy.Besides C-phycocyanin and allophycocyanin, phycoerythrocyanin is a constituent pigment of the phycobilisomes. These pigments indicated in the absorption spectrum of phycobilisomes with a maximum at 610 nm and two shoulders at 650 and 580 nm, respectively, were characterized by spectral data and isoelectric points.     

Refined three-dimensional structure of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus at 2.7 A

The structure of the phycobiliprotein phycoerythrocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 2.7 A resolution by X-ray diffraction methods on the basis of the molecular model of C-phycocyanin from the same organism. Hexagonal phycoerythrocyanin crystals of space group P6(3) with cell constants a = b = 156.86 A, c = 40.39 A, alpha = beta = 90 degrees, gamma = 120 degrees are almost isomorphous to C-phycocyanin crystals. The crystal structure has been refined by energy-restrained crystallographic refinement and model building. The conventional crystallographic R-factor of the final model was 19.2% with data to 2.7 A resolution. In phycoerythrocyanin, the three (alpha beta)-subunits are arranged around a 3-fold symmetry axis, as in C-phycocyanin. The two structures are very similar. After superposition, the 162 C alpha atoms of the alpha-subunit have a mean difference of 0.71 A and the 171 C alpha atoms of the beta-subunit differ by 0.51 A. The stereochemistry of the chiral atoms in the phycobiliviolin chromophore A84 is C(31)-R, C(4)-S. The configuration of the chromophore is C(10)-Z, C(15)-Z and the conformation C(5)-anti, C(9)-syn and C(14)-anti like the phycocyanobilin chromophores in phycoerythrocyanin and C-phycocyanin.     

Isolation and localization of N4-methylasparagine in phycobiliproteins from the cyanobacterium Mastigocladus laminosus

The occurrence of post-translationally methylated asparagine residues in beta AP from Anabaena variabilis, Synechococcus PCC 6301 and Porphyridium cruentum has recently been reported (Klotz, A.V., Leary, J.A. & Glazer, A.N. (1986) J. Biol. Chem. 261, 15891-15894). We reinvestigated the amino-acid compositions of all phycobiliproteins from Mastigocladus laminosus. During total hydrolysis of beta AP, beta 16.2 and beta PC one mol methylamine per mol protein was released. These proteins were chemically and enzymatically fragmented and the sequences of the fragments containing the modified asparagine residue were determined by automated Edman degradation. Residues beta AP72, beta 16.2 72 and beta PC 72 were identified as N4-methylasparagine. This derivative of asparagine was also found at a homologous position in beta PE of Calothrix. In the x-ray structure model of C-phycocyanin (PC) the residue beta PC 72 points towards the chromophore beta 84, presumably having an effect on the spectroscopic characteristics of this light harvesting pigment protein 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.     

Crystallization of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus and preliminary characterization of two crystal forms

The light-harvesting protein phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus Cohn has been crystallized in two different crystal forms by vapour diffusion. In 5% (w/v) polyethylene glycol at pH 8.5, hexagonal crystals of space group P63 with cell constants a = b = 158 A, c = 40.6 A were obtained, which turned out to be almost isomorphous with the hexagonal crystals of C-phycocyanin from the same organism. Consequently, the conformation of both phycobiliproteins must be very similar. From 1.5 M-ammonium sulfate (pH 8.5), orthorhombic crystals of space group P2221 with cell constants a = 60.5 A, b = 105 A, c = 188 A could be grown. Density measurements of these crystals indicate that the unit cell contains 18 (alpha beta)-units. A detailed packing scheme is proposed that is consistent with the observed pseudo-hexagonal X-ray intensity pattern and with the known size and shape of (alpha beta)3-trimers of C-phycocyanin. Accordingly, disc-like (alpha beta)3-trimers are associated face-to-face and stacked one upon another in rods with a period of 60.5 A, corresponding to the cell dimension a.     

Genes encoding both subunits of phycoerythrocyanin, a light-harvesting biliprotein from the cyanobacterium Mastigocladus laminosus

Phycocyanin (PC) and phycoerythrocyanin (PEC) are light-harvesting components of the phycobilisome (PbS) from the cyanobacterium Mastigocladus laminosus. These two biliproteins are closely related, and show a particularly high degree of sequence homology in the C-terminal part of their beta-subunits. A 198-bp gene fragment encoding this region of PC from M. laminosus was therefore used as a heterologous hybridization probe to identify the genes coding for PEC from the same organism. A 1.7-kb HindIII fragment was cloned and its sequence determined. Three open reading frames (ORFs) were found on this fragment. The gene coding for the beta-subunit of PEC (pecB) was followed downstream by the alpha-subunit encoding gene (pecA). This gene arrangement had also been found in the PC-encoding (cpc) gene pair from M. laminosus, and is conserved in cpc genes from other organisms. This finding is compatible with a model of evolution of the cpc and pec gene pairs as the product of gene duplication of an ancestral beta- and alpha-subunit-encoding pair. A third ORF starts downstream from pecA. It codes for the 34.5-kDa linker protein, which forms complexes with PEC with a 1:6 stoichiometry in the PbS. Biliprotein- and linker protein-encoding genes are frequently clustered, and this provides mechanisms for the production of the different stoichiometric amounts of these gene products required in the PbS and for coregulation by environmental factors.     

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     

Linker polypeptides of the phycobilisome from the cyanobacterium Mastigocladus laminosus: amino-acid sequences and relationships

Three linker polypeptides of the phycobilisome from the cyanobacterium Mastigocladus laminosus were isolated: A 8.9-kDa polypeptide, L8.9R(C), which is probably associated with C-phycocyanin, a 34.5-kDa polypeptide, L34.5,PCR, which forms a complex with C-phycocyanin, and a 34.5-kDa polypeptide, L34.5,PECR, which is linked to phycoerythrocyanin. The complete amino-acid sequence (80 residues) of the L8.9R(C) polypeptide was determined as well as the N-terminal 44 residues of both L34.5R polypeptides and the 114 C-terminal residues of L34.5,PECR. L8.9R(C) is homologous to L8.9C (Füglistaller et al. (1984) Hoppe-Seyler's Z. Physiol. Chem. 365, 1085-1096) and to the C-terminal sequence of L34.5,PECR. The N-terminal sequences of L34.5,PECR and L34.5,PCR exhibit 34% homology. The 44 N-terminal residues of L34.5,PECR are related to the beta-subunit of phycoerythrocyanin (23% homology), while the C-terminal sequence of L34.5,PECR is more related to alpha PEC (21% homology within 60 residues). This suggests that the 30-kDa-linker polypeptide family originates from a fusion of the alpha- and beta-subunit genes and the corresponding intercistronic DNA sequence, as might have arisen through mutation in the stop-codon of the beta-subunit gene. Hence, all polypeptides of the phycobilisome (including perhaps the anchor polypeptide) may be derived from an early ancestor phycobiliprotein subunit, which itself is also related to myoglobin (Schirmer et al. (1985) J. Mol. Biol. 184, 251-277).     

The complete amino acid sequence of both subunits of C-phycocyanin from the cyanobacterium Mastigocladus laminosus.

The amino acid sequences of both subunits of the C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus have been determined. The alpha-chain consists of 162 amino acid residues and has a molecular weight of 18000, whereas the beta-chain consists of 172 residues and has a molecular weight of 19400. For the first three quarters of their length the polypeptide chains are 31% homologous, whereas there is no significant homology in the final quarter up to the C-terminus. This could mean that the introduction of an additional chromophore binding site in the last quarter of the beta-chain during evolution was achieved via a large number of point mutations or by exchange of the whole C-terminal part in an ancestral gene.     

Isolation and characterization of phycoerythrocyanin and chromatic adaptation of the thermophilic cyanobacterium Mastigocladus laminosus

The thermophilic cyanobacterium Mastigocladus laminosus exhibits chromatic adaptation: In green light the production of the phycobiliprotein phycoerythrocyanin is enhanced drastically.Phycoerythrocyanin was characterised with respect to its molecular weight, isoelectric point, absorption spectra and size of its aggregates. The two subunits of the protein were separated and characterised according to these criteria. Their chromophore contents, amino acid compositions and N-terminal amino acid sequences were also determined. The sequences were compared with those of allophycocyanin and C-phycocyanin from Mastigocladus laminosus.Abbreviations PEC phycoerythrocyanin - -PEC -subunit of PEC - -PEC -subunit of PEC - PE phycoerythrin - PC phycocyanin - APC allophycocyanin - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - chl a chlorophyll a