Lipopolysaccharide in the outer membrane of the filamentous prochlorophyte Prochlorothrix hollandica

Abstract A lipopolysaccharide (LPS) fraction was isolated from Prochlorothrix hollandica by hot phenol/water extraction. Negatively stained preparations of an aqueous LPS dispersion showed the triple-layered appearance of the LPS aggregates. Glucose (main sugar), rhamnose, fucose, galactose, mannose, xylose, and 3- O -methyl-xylose were found as the constituents of the polysaccharide moiety. Glucosamine and the 3-hydroxy fatty acids, 3-OH-16:0, 3-OH-14:0, and the rarely detected iso-3-OH-15:0, constitute the lipid A of the LPS. l -glycero- d -manno-heptose and 3-deoxy- d -manno-2-octulosonic acid (dOclA), typical components of inner core oligosaccharides from enterobacterial LPS, were lacking in the isolated LPS fraction from Prochlorothrix hollandica .     

Ultrastructural studies on the membrane systems and cell inclusions of the filamentous prochlorophyte Prochlorothrix hollandica

The outer membrane of Prochlorothrix hollandica is covered with a network of fine fibrils on its surface and separated from the cytoplasmic membrane by an electrondense peptidoglycan layer (8 to 20 nm thick). The thylakoid membranes are arranged in stacked and unstacked regions which present four characteristic fracture faces with different numbers and sizes of intramembrane particles. Cell inclusions such as polyhedral bodies (carboxysomes), ribosomes, and polyphosphate granules were found in Prochlorothrix hollandica. Another type of cell inclusions was identified by its characteristic shape (a cylindre with conical caps) and a regular striation as gas vesicles. It is concluded that the organism is in its morphological structure similar to the cyanobacteria.Abbreviations C carboxysome - CM cytoplasmic membrane - EF_s, EF_u exoplasmic fracture face of stacked and unstacked membrane area, respectively - ES exoplasmic surface - PF_s, PF_u plasmic fracture face of stacked and unstacked membrane area, respectively - PG peptidoglycan layer - TM thylakoid membrane Dedicated to Prof. Dr. D. Peters, Hamburg, on the occasion of his 75th birthday     

Light-Dependent Tyrosine Phosphorylation in the Cyanobacterium Prochlorothrix hollandica

A light-dependent tyrosine kinase activity is present in soluble extracts from the cyanobacterium Prochlorothrix hollandica. The substrate of this tyrosine kinase activity is a soluble 88-kD protein that is phosphorylated when cultures of P. hollandica are adapted to high-light conditions. This phosphoprotein was identified by probing western blots of 32P-labeled soluble proteins from P. hollandica with an antibody specific for phosphotyrosine. This specificity was confirmed by competition experiments in which the antibody binding was abolished completely in the presence of excess phosphotyrosine but not phosphoserine and phosphothreonine. The kinetics of phosphorylation in vivo were determined by probing western blots with this antibody. Within 1 h following a switch from extended darkness to high light (200 [mu]mol photons m-2 s-1), the 88-kD protein was detectable upon India ink staining of western blots. After 3 h, the antibody recognized the phosphorylated form of this polypeptide. Within 6 h of a downshift from high to low light, the 88-kD protein was dephosphorylated. In vitro phosphorylation studies also showed that cell extracts can phosphorylate a tyrosine-containing artificial substrate; acid hydrolysis of both the artificial substrate and the 88-kD protein showed that phosphorylation occurred exclusively on tyrosine residues. Finally, experiments with high-light-adapted Synechococcus sp. PCC7942 suggest that a similar tyrosine phosphorylation event occurs in a phycobilisome-containing cyanobacterium.     

Photosynthesis of Prochlorothrix hollandica under Sulfide-Rich Anoxic Conditions

The photosynthetic activity and photosystem II fluorescence of Prochlorothrix hollandica were studied under anoxic, sulfide-rich conditions. Oxygenic photosynthetic activity with water as the electron donor was highly resistant to inhibition by sulfide. Cells still retained 50% of their oxygenic photosynthetic activity at >1 mM sulfide. In the presence of DCMU [N-(3,4-dichlorophenyl)-N(prm1)-dimethylurea], an inhibitor of photosystem II activity, P. hollandica cells exhibited a low but significant anoxygenic photosynthetic activity when sulfide was present. This activity increased with higher sulfide concentrations and reached maximal rates at concentrations exceeding 1 mM sulfide. The effects of hydroxylamine on both oxygen evolution and fluorescence induction kinetics were similar to those observed for sulfide. It was concluded that the oxidizing site of photosystem II was the site of sulfide action leading to reduced or even fully inhibited electron donation to photosystem II. These observations bear similarity to the situation in some cyanobacteria in which both hydroxylamine and sulfide inhibit electron donation from H(inf2)O to P(inf680). The high resistance of photosystem II to sulfide is related to the hydrophobic nature of the manganese-stabilizing protein in P. hollandica (T. S. Mor, A. F. Post, and I. Ohad, Biochim. Biophys. Acta 1141:206-212, 1993). The observed sulfide tolerance of P. hollandica may confer a competitive advantage in its natural environment, where it forms a dominant fraction of phytoplankton in waters in which sulfide presence is a recurring phenomenon.     

Purification and characterization of a surface-associated carotenoid-binding complex from the photosynthetic prokaryote,Prochlorothrix hollandica

We have identified a water-soluble surface-associated complex from Prochlorothrix hollandica, composed of two polypeptides of 56 and 58 kilodaltons (kDa), zeaxanthin, and lipopolysaccharide. The complex was purified by preparative isoelectric focusing (pI=3.0). The outer membrane lipopolysaccharide co-purified with the complex. Immunocytochemisty employing a polyclonal antibody to the apoproteins exclusively labeled the cell surface. Both zeaxanthin and the protein accumulated under high light intensities, thus we propose that the complex may play a role in photoprotection.     

Major outer membrane proteins of Prochlorothrix hollandica (Prochloraceae)

Abstract Rhizobium sp. isolated from Lablab purpureus utilized catechol as sole carbon and energy source, a property which is plasmid encoded. The heat curable (39–41°C) plasmid, designated as pAMG1, was transferred to cat⁻ strains of Rhizobium sp. with a transfer frequency of 2.6 × 10⁻⁶ transconjugants/donor cell.     

Computational simulation of the docking of Prochlorothrix hollandica plastocyanin to potosystem I: modeling the electron transfer complex

We have used several docking algorithms (GRAMM, FTDOCK, DOT, AUTODOCK) to examine protein-protein interactions between plastocyanin (Pc)/photosystem I (PSI) in the electron transfer reaction. Because of the large size and complexity of this system, it is faster and easier to use computer simulations than conduct x-ray crystallography or nuclear magnetic resonance experiments. The main criterion for complex selection was the distance between the copper ion of Pc and the P700 chlorophyll special pair. Additionally, the unique tyrosine residue (Tyr(12)) of the hydrophobic docking surface of Prochlorothrix hollandica Pc yields a specific interaction with the lumenal surface of PSI, thus providing the second constraint for the complex. The structure that corresponded best to our criteria was obtained by the GRAMM algorithm. In this structure, the solvent-exposed histidine that coordinates copper in Pc is at the van der Waals distance from the pair of stacked tryptophans that separate the chlorophylls from the solvent, yielding the shortest possible metal-to-metal distance. The unique tyrosine on the surface of the Prochlorothrix Pc hydrophobic patch also participates in a hydrogen bond with the conserved Asn(633) of the PSI PsaB polypeptide (numbering from the Synechococcus elongatus crystal structure). Free energy calculations for complex formation with wild-type Pc, as well as the hydrophobic patch Tyr(12)Gly and Pro(14)Leu Pc mutants, were carried out using a molecular mechanics Poisson-Boltzman, surface area approach (MM/PBSA). The results are in reasonable agreement with our experimental studies, suggesting that the obtained structure can serve as an adequate model for P. hollandica Pc-PSI complex that can be extended for the study of other cyanobacterial Pc/PSI reaction pairs.     

NMR solution structure of plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica

The solution structure of a divergent plastocyanin (PC) from the photosynthetic prokaryote Prochlorothrix hollandica was determined by homonuclear 1H NMR spectroscopy. Nineteen structures were calculated from 1222 distance restraints, yielding a family of structures having an average rmsd of 0.42 +/- 0.08 A for backbone atoms and 0.71 +/- 0.07 A for heavy atoms to the mean structure. No distance constraint was violated by more than 0.26 A in the structure family. Despite the low number of conserved residues shared with other PC homologues, the overall folding pattern of P. hollandica PC is similar to other PCs, in that the protein forms a two-sheet beta-barrel tertiary structure. The greatest variability among the backbone structures is seen in the loop region from residues 47-60. The differences seen in the P. hollandica PC homologue likely arise due to a small deletion of 2-4 residues compared to the PC consensus; this yields a less extended loop containing a short alpha-helix from residues Ala52-Leu55. Additionally, the protein has an altered hydrophobic patch thought to be important in binding reaction partners. Whereas the backbone structure is very similar within the loops of the hydrophobic region, the presence of two unique residues (Tyr12 and Pro14) yields a structurally different hydrophobic surface likely important in binding P. hollandica Photosystem I.     

Salinity Tolerance of the Chlorophyll b-synthesizing Cyanobacterium Prochlorothrix hollandica Strain SAG 10.89

Ecophysiological investigations on the salinity acclimation of the cyanobacterium Prochlorothrix hollandica SAG 10.89 led to significantly revised salinity tolerance limits. Besides potential effects of cultivation techniques, clear ion composition effects mainly explain formerly described hypersensitivity to NaCl-mediated salinity and lack of osmolyte detection. An extraordinarily broad plasticity of cellular chlorophyll a/b ratios occurred with variations of NaCl-induced salinity. Photosynthesis characteristics, pigment regulation, respiration, and biomass yield in growth medium with field-like ion composition indicated generally reduced acclimation pressure. A simultaneously significant increase in osmolyte (sucrose) accumulation indicated more efficient osmotic acclimation. Minor growth inhibition up to salinities of 10 practical salinity units enlarged the potential habitat of P. hollandica but at the most to about 300,000 km² in the Baltic Sea. This supports probable observations of Prochlorothrix sp. in phytoplankton assemblages of open waters in Baltic Sea-monitoring studies. Brackish habitats differ from so far known habitats of Prochlorothrix spp. in turbidity, productivity, and plankton composition. Adjusted physiological features dispel fundamental doubts on the establishment of filamentous prochlorophytes in brackish waters.     

The unique proline of the Prochlorothrix hollandica plastocyanin hydrophobic patch impairs electron transfer to photosystem I.

A number of surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in amino acids located in the amino-terminal hydrophobic patch of the copper protein, which presents a variant structure as compared with other plastocyanins. The single mutants Y12G, Y12F, Y12W, P14L, and double mutant Y12G/P14L have been produced. Their reactivity toward photosystem I has been analyzed by laser flash absorption spectroscopy. Plots of the observed rate constant with all mutants versus plastocyanin concentration show a saturation profile similar to that with wild-type plastocyanin, thus suggesting the formation of a plastocyanin-photosystem I transient complex. The mutations do not induce relevant changes in the equilibrium constant for complex formation but induce significant variations in the electron transfer rate constant, mainly with the two mutants at proline 14. Additionally, molecular dynamics calculations indicate that mutations at position 14 yield small changes in the geometry of the copper center. The comparative kinetic analysis of the reactivity of plastocyanin mutants toward photosystem I from different organisms (plants and cyanobacteria) reveals that reversion of the unique proline of Prochlorothrix plastocyanin to the conserved leucine of all other plastocyanins at this position enhances the reactivity of the Prochlorothrix protein.     

Immunological characterization of the Prochlorothrix hollandica and Prochloron sp. chlorophyll a/b antenna proteins

Polyclonal antibodies were prepared against the major antenna chlorophyll (Chl) a/b-binding protein from the prokaryote Prochlorothrix hollandica (Burger-Wiersma et al. (1986) Nature (Lond.) 320, 262-264). Immunoblotting experiments on Triton X-114 phase-partitioned P. hollandica thylakoids revealed that the antibody recognizes intrinsic membrane polypeptides of 33 and 30 kDa, and immunocytochemistry of P. hollandica thin sections showed that the antibody preferentially decorates the thylakoid. The antibody was immunopurified against a LacZ fusion protein produced in Escherichia coli by an immunopositive phage clone retrieved from a lambda ZAP expression library. This purified antibody crossreacted to both the 33 and 30 kDa polypeptides, indicating that these proteins are either structurally related products of different genes, or modified forms of the same gene product. Whereas immunological crossreactivity of Prochlorothrix antibody to the major LHC-II Chl a/b antenna of maize could not be detected, the immunopurified antibody reacted strongly to the major 34 kDa Chl a/b antenna protein from the prokaryote Prochloron sp. (Lewin (1975) Phycologia 14, 153-160). These data confirm the structural similarity of the prochlorophyte photosynthetic antenna systems.     

Chlorophyll-protein composition of the thylakoid membrane from Prochlorothrix hollandica, a prokaryote containing chlorophyll b

The chlorophyll-protein complexes of the thylakoid membrane from Prochlorothrix hollandica were identified following electrophoresis under nondenaturing conditions. Five complexes, CP1-CP5, were resolved and these green bands were analyzed by spectroscopic and immunological methods. CP1 contains the photosystem I (PSI) reaction center, as this complex quenched fluorescence at room temperature, and had a 77 K fluorescence emission peak at 717 nm. CP4 contains the major chlorophyll-a-binding proteins of the photosystem II (PSII) core, because this complex contained polypeptides which cross-reacted to antibodies raised against Chlamydomonas PSII proteins 5 and 6. Furthermore, fluorescence excitation studies at 77 K indicated that only a Chl a is bound to CP4. Complexes CP2, CP3 and CP5 contained functionally bound Chl a and b as judged by absorption spectroscopy at 20 degrees C and fluorescence excitation spectra at 77 K. CP2, CP3 and CP5 all contain polypeptides of 30-33 kDa which are immunologically distinct from the LHC-II complex of higher plant thylakoids.     

Association of chlorophyll a/b-binding Pcb proteins with photosystems I and II in Prochlorothrix hollandica

Action spectra for photosystem II (PSII)-driven oxygen evolution and of photosystem I (PSI)-mediated H(2) photoproduction and photoinhibition of respiration were used to determine the participation of chlorophyll (Chl) a/b-binding Pcb proteins in the functions of pigment apparatus of Prochlorothrix hollandica. Comparison of the in situ action spectra with absorption spectra of PSII and PSI complexes isolated from the cyanobacterium Synechocystis 6803 revealed a shoulder at 650 nm that indicated presence of Chl b in the both photosystems of P. hollandica. Fitting of two action spectra to absorption spectrum of the cells showed a chlorophyll ratio of 4:1 in favor of PSI. Effective antenna sizes estimated from photochemical cross-sections of the relevant photoreactions were found to be 192+/-28 and 139+/-15 chlorophyll molecules for the competent PSI and PSII reaction centers, respectively. The value for PSI is in a quite good agreement with previous electron microscopy data for isolated Pcb-PSI supercomplexes from P. hollandica that show a trimeric PSI core surrounded by a ring of 18 Pcb subunits. The antenna size of PSII implies that the PSII core dimers are associated with approximately 14 Pcb light-harvesting proteins, and form the largest known Pcb-PSII supercomplexes.     

Functional Analysis of the Photosynthetic Apparatus of Prochlorothrix hollandica (Prochlorales), a Chlorophyll b Containing Procaryote

Light-shade adaptation of the chlorophyll a/b containing procaryote Prochlorothrix hollandica was studied in semicontinuous cultures adapted to 8, 80 and 200 μmole quanta per square meter per second. Chlorophyll a contents based on dry weight differed by a factor of 6 and chlorophyll b by a factor of 2.5 between the two extreme light conditions. Light utilization efficiencies determined from photosynthesis response curves were found to decrease in low light grown cultures due to lower light harvesting efficiencies; quantum requirements were constant at limiting and saturating irradiances for growth. At saturating growth irradiances, changes in light saturated oxygen evolution rate originated from changes in chlorophyll a antenna relative to the number of reaction centers II. At light-limiting conditions both the number of reaction centers II and the antenna size changed. The amount of chlorophyll b relative to reaction center II remained constant. As in cyanobacteria, the ratio of reaction center I to reaction center II was modulated during light-shade adaptation. On the other hand, time constants for photosynthetic electron transport (4 milliseconds) were low as observed in green algae and diatoms. The occurrence of state one to two and state two to one transitions is reported here. Another feature linking photosynthetic electron transport in P. hollandica to that in the eucaryotic photosynthetic apparatus was blockage of the state one to two transition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Although chlorophyll b was reported in association with photosystem I, the 630 nanometer light effect does not exclude that chlorophyll b is the photoreceptor for the state one to two transition.     

Feeding in Euchlanis dilatata lucksiana Hauer on filamentous cyanobacteria and a prochlorophyte

Ingestion and assimilation rates of Euchlanis dilatata lucksiana Hauer, isolated from Lake Loosdrecht (The Netherlands) and cultured on lake water (seston < 33 µm), were measured in the laboratory using the ¹⁴C-tracer technique. The five taxa used as tracer foods, together with 6–7 mg C l^–1 of lake seston in each case, included four species of filamentous cyanobacteria (Oscillatoria redekei, O. limnetica, Aphanizomenon flos-aquae, Anabaena PCC 7120) and a prochlorophyte (Prochlorothrix hollandica). Except Anabaena, they are all commonly encountered in eutrophic Loosdrecht lakes, including Lake Loosdrecht, and their dimensions ranged between 150 and 250 µm in length and 2 and 3.5 µm in width. The small and large Euchlanis used as experimental animals had mean lengths of 217–223 µm and 276–305 µm, respectively. Euchlanis fed on all the taxa offered as food. Clearance rates ranged from 51 to 99 µl ind^–1 d^–1 for the large animals and from 22 to 41 µl ind^–1 d^–1 for the small animals. The highest ingestion rate observed, 1.7 µg ind^–1 d^–1, was for the large animals feeding on Aphanizomenon. The daily ration for both size classes far exceeded 100% of body weight, reaching up to 690% for the small animals feeding on Aphanizomenon. The small animals generally appeared to assimilate the ingested food more efficiently (assimilation efficiencies: 37–100%) than the large animals (34–77%). Compared with an earlier study in which only lake seston (<33 µm) was used as food, the specific clearance rates of Euchlanis on the cyanobacteria and Prochlorothrix were generally somewhat lower.     

The incorporation of mannoproteins in the cell wall of S. cerevisiae and filamentous Ascomycetes

In yeast, glucanase extractable cell wall proteins are anchored to the plasma membrane at an intermediate stage in their biogenesis via a glycosylphosphatidylinositol (GPI) moiety before they become anchored to the wall glucan via a 1,6-glucan linkage. The mechanism of the membrane processing step of cell wall proteins is not known. Here, we report that Ascomycete filamentous fungi involved in food spoilage such as Aspergillus, Paecilomyces and Penicillium, also contain GPI membrane-anchored proteins some of which are processed by an endogenous phospholipase C activity. Furthermore, similar to the situation in yeast, their cell walls contain mannoproteins which are linked to the glucan backbone through a 1,6-glucan linkage. Interestingly, one mould which contains a significant amount of non covalently linked 1,6-glucosylated cell wall proteins, is much more sensitive towards 1,3-glucanases and membrane perturbing peptides than the others.     

The 38 kDa chlorophyll a/b protein of the prokaryote Prochlorothrix hollandica is encoded by a divergent pcb gene

The chlorophyll (Chl) a/b proteins of the photosynthetic prokaryotes appear to have evolved by gene duplication and divergence of the core Chl a antenna family, which also includes CP43 and CP47 and the iron-stress induced Chl a-binding IsiA proteins. We show here that Prochlorothrix hollandica has a cluster of three pcb (prochlorophyte chlorophyll b) genes which are co-transcribed. The major antenna polypeptides of 32 and 38 kDa are encoded by pcbA and pcbC respectively. The pcbC gene is significantly divergent from the other two and may have originated by a gene duplication independent of the one that led to isiA and the other prochlorophyte pcb genes. The distant relatedness of the three prochlorophyte genera implies that not only the ability to make Chl b and use it for light-harvesting arose independently in the three lineages, but also that the pcb genes may have arisen as the result of independent gene duplications in each lineage.