An immunological approach to detect phosphate stress in populations and single cells of photosynthetic picoplankton.

In the marine cyanobacterium Synechococcus sp. strain WH7803, PstS is a 32-kDa cell wall-associated phosphate-binding protein specifically synthesized under conditions of restricted inorganic phosphate (P1) availability (D. J. Scanlan, N. H. Mann, and N. G. Carr, Mol. Microbiol. 10:181-191, 1993). We have assessed its use as a potential diagnostic marker for the P status of photosynthetic picoplankton. Expression of PstS in Synechococcus sp. strain WH7803 was observed when the P1 concentration fell below 50 nM, demonstrating that the protein is induced at concentrations of P1 typical of oligotrophic conditions. PstS expression could be specifically detected by use of standard Western blotting (immunoblotting) techniques in natural mesocosm samples under conditions in which the N/P ratio was artificially manipulated to force P depletion. In addition, we have developed an immunofluorescence assay that can detect PstS expression in single Synechococcus cells both in laboratory cultures and natural samples. We show that antibodies raised against PstS cross-react with P-depleted Prochlorococcus cells, extending the use of these antibodies to both major groups of prokaryotic photosynthetic picoplankton. Furthermore, DNA sequencing of a Prochlorococcus pstS homolog demonstrated high amino acid sequence identity (77%) with the marine Synechococcus sp. strain WH7803 protein, including those residues in Escherichia coli PstS known to be directly involved in phosphate binding.

Identification of iron superoxide dismutase and a copper/zinc superoxide dismutase enzyme activity within the marine cyanobacterium Synechococcus sp. WH 7803.

Three constitutive forms of superoxide dismutase activity have been demonstrated in the cyanobacterial marine picoplankter Synechococcus sp. WH 7803 using polyacrylamide gel activity staining techniques. A protein which gave a positive non-haem iron stain on native polyacrylamide gels exhibited N-terminal similarity to both the iron superoxide dismutase and the manganese superoxide dismutase of Escherichia coli. The metal prosthetic group of each of the three activity bands was characterised by analysing their differential sensitivities to 5 mM H2O2, 2 mM cyanide and 2 mM of the copper chelator diethyldithiocarbamate. Three distinct superoxide dismutase activities were observed, an iron superoxide dismutase, a copper/zinc superoxide dismutase and a third form which has not been identified. Growth of Synechococcus cells in ASW medium containing no added iron resulted in no alteration in the activity of the iron superoxide dismutase. Growth of cultures in the absence of copper or zinc resulted in differential changes in the activities of the copper/zinc superoxide dismutase and the unidentified superoxide dismutase.

ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803.

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.

Characterization of a zwf mutant of Synechococcus sp. strain PCC 7942.

A mutant of the cyanobacterium Synechococcus sp. strain PCC 7942 carrying a disrupted gene encoding glucose-6-phosphate dehydrogenase (zwf) produced no detectable glucose-6-phosphate dehydrogenase as assessed by enzyme assay and Western blot (immunoblot) analysis. This mutant exhibited significantly impaired dark viability.

Organization and transcription of the class I phycoerythrin genes of the marine cyanobacterium Synechococcus sp. WH7803.

The nucleotide sequences of the class I phycoerythrin (PE) alpha- and beta-subunit genes (cpeA and cpeB) from the marine cyanobacterium Synechococcus sp. WH7803 are reported. The cpeB gene is located upstream of cpeA with a separation of 56 nucleotides and the two genes are co-transcribed as a transcript of 1.3 kb, with the transcription startpoint being localized to 110-111 bp upstream of cpeB. The sequence of the promoter region bears no similarity to promoters reported for other cyanobacterial PE genes. Pentanucleotide repeats found upstream of some PE operons, particularly in the case of cyanobacterial strains capable of chromatic adaption, are not found in Synechococcus sp. WH7803; instead the sequence 5'-CGGTT-3' is repeated three times in the promoter region.

Isolation and Molecular Characterization of Five Marine Cyanophages Propagated on Synechococcus sp. Strain WH7803.

Five marine cyanophages propagated on Synechococcus sp. strain WH7803 were isolated from three different oceanographic provinces during the months of August and September 1992: coastal water from the Sargasso Sea, Bermuda; Woods Hole harbor, Woods Hole, Mass.; and coastal water from the English Channel, off Plymouth Sound, United Kingdom. The five cyanophage isolates were found to belong to two families, Myoviridae and Styloviridae, on the basis of their morphology observed in the transmission electron microscope. DNA purified from each of the cyanophage isolates was restricted with a selection of restriction endonucleases, and three distinguishably different patterns were observed. DNA isolated from Myoviridae isolates from Bermuda and the English Channel had highly related restriction patterns, as did DNA isolated from Styloviridae isolates from Bermuda and the English Channel. DNA isolated from the Myoviridae isolate from Woods Hole had a unique restriction pattern. The genome size for each of the Myoviridae isolates was ca. 80 to 85 kb, and it was ca. 90 to 100 kb for each of the Styloviridae isolates. Southern blotting analysis revealed that there was a limited degree of homology among all cyanophage DNAs probed, but clear differences were observed between cyanophage DNA from the Myoviridae and that from the Styloviridae isolates. Polypeptide analysis revealed a clear difference between Myoviridae and Styloviridae polypeptide profiles, although the major, presumably structural, protein in each case was ca. 53 to 54 kDa.

The response of the picoplanktonic marine cyanobacterium Synechococcus species WH7803 to phosphate starvation involves a protein homologous to the periplasmic phosphate-binding protein of Escherichia coli.

During phosphate-limited growth the marine phycoerythrin-containing picoplanktonic cyanobacterium Synechococcus sp. WH7803 synthesizes novel polypeptides, including two abundant species of 100 kDa and 32 kDa. The 32 kDa polypeptide was localized to the cell wall, although in a related strain, Synechococcus sp. WH8103, it could be detected in both the cell wall fraction and the periplasm. The gene (designated pstS) encoding this polypeptide was cloned and shown to be present in a single copy. The deduced amino acid sequence indicated a polypeptide consisting of 326 amino acids with a calculated M(r) of 33,763. Comparison of this sequence with that obtained by microsequencing the N-terminus of the 32 kDa polypeptide showed that the mature protein was synthesized as a precursor, the first 24 amino acid residues being cleaved between two alanine residues at positions 24 and 25. The amino acid sequence of the mature polypeptide showed 35% identity and 52% similarity to the periplasmic phosphate-binding protein (PstS) from Escherichia coli, including three regions of much stronger homology which, by comparison with E. coli PstS, are directly involved in phosphate binding. Northern blot analysis revealed a pstS transcript of 1.2 kb in RNA extracted from cells grown in Pi-replete conditions and one of 1.4 kb in considerably increased abundance under Pi-depleted conditions. Homologues of the pstS gene were detected in other marine phycoerythrin-containing Synechococcus strains, but not in freshwater or halotolerant species.

Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus PCC 7942.

The glucose-6-phosphate dehydrogenase (EC 1.1.1.49) gene (zwf) of the cyanobacterium Synechococcus PCC 7942 was cloned on a 2.8 kb Hind III fragment. Sequence analysis revealed an ORF of 1572 nucleotides encoding a polypeptide of 524 amino acids which exhibited 41% identity with the glucose-6-phosphate dehydrogenase of Escherichia coli.

Bicarbonate Concentration by Synechocystis PCC6803 : Modulation of Protein Phosphorylation and Inorganic Carbon Transport by Glucose.

The ability of the cyanobacterium Synechocystis PCC6803 to transport inorganic carbon in the form of bicarbonate rapidly decreased following a shift from bicarbonate-limited growth to either excess bicarbonate supply or to photoheterotrophic growth on glucose. Nonmetabolizable analogs of glucose did not exert this effect. The rate at which the bicarbonate uptake rate declined was too rapid to be accounted for by dilution of the activity by culture growth and suggested that posttranslational modification may be involved. Several proteins that were unphosphorylated during bicarbonate-limited growth became phosphorylated during the shifts to high CO(2) conditions and to photoheterotrophic growth. A similar alteration in the profile of phosphopolypeptides was observed following a shift into the dark. The changes in protein phosphorylation were not blocked by chloramphenicol or rifampicin.

Construction of lacZ promoter probe vectors for use in Synechococcus: application to the identification of CO2-regulated promoters.

It was shown that the Escherichia coli lacZ gene could be expressed in the cyanobacterium Synechococcus R2 PCC7942 both as a plasmid-borne form and also integrated into the chromosome. A promoterless form of the lacZ gene was constructed and used as a reporter gene to make transcriptional fusions with cyanobacterial promoters using a shuttle vector system and also via a process of integration by homologous recombination. Synechococcus R2 promoter-lacZ gene fusions were then used to identify CO2-regulated promoters, by quantitatively assessing beta-galactosidase activity under high and low CO2 conditions using a fluorescence assay. Several promoters induced under low CO2 conditions were detected.

Novel Role for Phycoerythrin in a Marine Cyanobacterium, Synechococcus Strain DC2.

Cyanobacterial picoplankton contribute substantially to oceanic primary productivity. The colored protein phycoerythrin is the major component of their light-harvesting apparatus. It was found that in Synechococcus strain DC2 a variable proportion of the light energy absorbed by phycoerythrin is lost as autofluorescence and therefore is not passed to a photoreaction center. Phycoerythrin may serve two functionally distinct roles in this organism: as a nitrogen reserve and as a collector of quanta for photosynthesis.

Compensation, radiographic changes, and survival in applicants for asbestosis compensation.

The survival of 354 claimants for compensation for pulmonary asbestosis among former workers of the Wittenoom crocidolite mine and mill in Western Australia has been examined. There were 118 deaths up to December 1982. The median time between start of work and claim for compensation was 17 years. The standardised mortality ratio (SMR) for deaths from all causes was 2.65 (p less than 0.0001). The SMR for pneumoconiosis was 177.2 (p less than 0.0001), bronchitis and emphysema 2.6 (p = 0.04), tuberculosis 44.6 (p less than 0.0001), respiratory cancer (including five deaths from malignant pleural mesothelioma) 6.4 (p less than 0.0001), gastrointestinal cancer 1.6 (p = 0.22), all other cancers 1.6 (p = 0.17), heart disease 1.4 (p = 0.07), and all other causes 2.18 (p = 0.004). Plain chest radiographs taken within two years of claiming compensation were found for 238 subjects and were categorised independently by two observers according to the International Labour Organisation criteria without knowledge of exposure or compensation details. Profusion of radiographic opacities, age at claiming compensation, work in the Wittenoom mill, and degree of disability awarded by the pneumoconiosis medical board were significant predictors of survival, but total estimated exposure to asbestos was not. Radiographic profusion and degree of disability were, however, predictable by total exposure. The median survival from claim for compensation was 17 years in subjects with ILO category 1 pneumoconiosis, 12 years in category 2, and three years in category 3.

Resistance of DNA from filamentous and unicellular cyanobacteria to restriction endonuclease cleavage.

Chromosomal DNA from nine species of filamentous cyanobacteria as diverse as Nostoc, Gloeotrichia and Plectonema is suggested to be extensively modified (methylated) by its resistance to cleavage by a number of restriction endonucleases. A remarkably similar pattern of DNA modification in these species contrasts with the known heterogeneity of their type II restriction endonuclease content. In particular, Nostoc PCC 73102, which lacks detectable sequence-specific endonucleases, is shown to possess extensive DNA modification. The use of isoschizomers demonstrates the presence of a methylase in the filamentous strains analogous to the dam enzyme of Escherichia coli. As a preliminary to assessing the significance of the DNA modification, a study of susceptibility to restriction endonuclease cleavage of the genomes of five unicellular cyanobacteria revealed considerable variation between the different strains. The significance of the DNA modification patterns elucidated is discussed in terms of the restriction endonuclease content and cellular differentiation of the relevant cyanobacterial strains.

Polyadenylated RNA in two filamentous cyanobacteria.

Polyadenylated RNA was detected in the cyanobacteria Nostoc sp. strain MAC and Anabaena variabilis by oligodeoxythymidylic acid-cellulose chromatography and by hybridization to [3H]polyuridylic acid. Polyadenylate tracts from A. variabilis were located at the 3' end of RNA chains and had an estimated length of 15 to 22 nucleotides.

Isolation of polysomes from Nostoc sp. MAC and translation of messenger RNA in a heterologous cell-free system.

A method has been established which isolated polysomes from the lysozyme/EDTA-shocked cyanobacterium, Nostoc sp. MAC. In a typical preparation the total recovery of RNA as polysomes was 83%, in which 77% of the polysome fraction was present at sizes greater than 5-mers and 23% as 2-4-mers. Messenger RNA isolated from such a preparation of polysomes produced a 10-fold stimulation in the incorporation of [35S]methionine into polypeptides by a cell-free system of Escherichia coli. The in vitro-synthesized polypeptides were analysed on an SDS-polyacrylamide gradient gel together with in vivo-labelled proteins of Nostoc sp. MAC: seven polypeptides co-migrated with the in vivo-synthesized products. This is the first report of the expression of cyanobacterial messenger RNA in a heterologous cell-free system from E. coli; the efficiency of the system is discussed.

Detection of glutamate synthase in heterocysts of Anabaena sp. strain 7120.

The formation of [(14)C]glutamate from [(14)C]glutamine in the presence of alpha-oxoglutarate was observed in extracts of heterocysts of Anabaena sp. strain 7120 under conditions that indicate the operation of glutamate synthase.

Heterocyst differentiation and cell division in the cyanobacterium Anabaena cylindrica: effect of high light intensity.

Heterocyst differentiation in the cyanobacterium Anabaena cylindrica is initiated by the removal of fixed nitrogen from the medium. These specialized cells occur singly at regular intervals within filaments of vegetative cells. Incubation of cultures for periods of up to 12 h immediately prior to or following removal of fixed nitrogen, at a light intensity (500 mi Einsteins cm-2 s-1) approximately 10-fold higher than that required for optimum growth, resulted in the differentiation of pairs of adjacent (double) heterocysts. The frequency of double heterocysts was proportional to the length of the period of high light intensity. During growth at normal light intensity approximately 5% of cell divisions were symmetrical, but this increased more than 3-fold during 10-h incubation at high light intensity. The frequency of dividing cells remained constant during this period, but increased rapidly on return to normal light. The frequency of double heterocysts was reduced if a period of incubation at normal light intensity was interposed between the 12-h period at high light intensity and transfer to nitrogen-free medium. A period of 8 h normal light was required to reduce the frequency of double heterocysts to control values, and this corresponded to the length of time needed for the frequency of symmetrical divisions to return to control levels following 12 h at high light intensity. We confirm that cell division in Anabaena cylindrica is asymmetrical and conclude that the presence of double heterocysts results from an increase in the symmetry of cell division during incubation at high light intensity. The results also support the finding of previous workers that a cell is only susceptible to differentiation during a short period following its formation. During the period of high light the rate of doubling of the absorbance of the culture at 750 mn increased from 24 h to approximately 10 h and decreased to more than 100 h on return to normal light. The very high rate could be explained by increases in the volume and granular content of cells during incubation at high light intensity and did not represent an equivalent increase in the rate of cell division.