Distribution of Synechococcus spp. determined by immunofluorescent assay

By using two polyclonal antisera against WH 7803 strain (Synechococcus sp.) and WH 5701 strain (Synechococcus bacillaris) it is possible to detect and to enumerate cells of the two cyanobacterial serogroups. The immunofluorescence technique was used to study the distribution of the two serogroups in the estuarine, coastal and upwelling waters of the Mediterranean Sea surrounding Messina. In the estuarine waters of the Alcantara River (Ionian Sea), the WH 7803 serogroup was present at a concentration in the order of 10² cells ml⁻¹ and the WH 5701 serogroup at a concentration of 5·5 × 10² cellsml⁻¹. In the coastal waters of Messina, where urban and industrial wastes are usuallydumped, the concentration of total phycoerythrin- Synechococcus ranged from 1·3 × 10² to 4·1 × 10³ cells ml⁻¹; the WH 7803 serogroup accounted for 50–94% of the totalpopulation in Ionian stations, whereas the WH 5701 serogroup ranged from1·4 × 10¹ to6·7 × 102cells ml⁻¹. In the upwelling area (Straits of Messina) bothserogroups were found. Vertical distribution of two Synechococcus strains had anopposite trend and their concentrations were of the order of 10¹–10²cells ml⁻¹. Theuse of the Scan laser system allows both autofluorescent and labelled organismsto be distinguished in a preparation for optical microscopy. It also allows false-positivecells to be distinguished.     

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

Abstract 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.     

A suppression subtractive hybridization approach reveals niche-specific genes that may be involved in predator avoidance in marine Synechococcus isolates

Picocyanobacteria of the genus Synechococcus are important contributors to marine primary production and are ubiquitous in the world's oceans. This genus is genetically diverse, and at least 10 discrete lineages or clades have been identified phylogenetically. However, little if anything is known about the genetic attributes which characterize particular lineages or are unique to specific strains. Here, we used a suppression subtractive hybridization (SSH) approach to identify strain- and clade-specific genes in two well-characterized laboratory strains, Synechococcus sp. strain WH8103 (clade III) and Synechococcus sp. strain WH7803 (clade V). Among the genes that were identified as potentially unique to each strain were genes encoding proteins that may be involved in specific predator avoidance, including a glycosyltransferase in strain WH8103 and a permease component of an ABC-type polysaccharide/polyol phosphate export system in WH7803. During this work the genome of one of these strains, WH7803, became available. This allowed assessment of the number of false-positive sequences (i.e., sequences present in the tester genome) present among the SSH-enriched sequences. We found that approximately 9% of the WH8103 sequences were potential false-positive sequences, which demonstrated that caution should be used when this technology is used to assess genomic differences in genetically similar bacterial strains.     

Ferric iron-reducing Shewanella putrefaciens and N2-fixing Bradyrhizobium japonicum with uptake hydrogenase are unable to oxidize atmospheric H2

Abstract Bradyrhizobium japonicum and Shewanella putrefaciens were unable to oxidize hydrogen at atmospheric concentrations (0.55 ppmv), neither in suspension nor when added to sterile soil. The K _m-value of S. putrefaciens for H₂ (39 ppmv in gas phase, 0.22 μM in aqueous phase), using Fe(III) as electron acceptor, showed a 4–5-fold higher affinity for H₂ than that of B. japonicum (1200 ppmv; 0.84 μM) or other hydrogen-oxidizing bacteria. However, the V _max (4.54 fmol H₂ h⁻¹ cell ⁻¹) and threshold (> 0.5 ppmv; 0.35 nM) of S. putrefaciens and the V _max (7.19 fmol H₂ h⁻¹ cell⁻¹) and threshold (> 0.5 ppmv; 0.35 nM) of B. japonicum were in the same order of magnitude as data for Knallgas bacteria from relevant literature. To enable hydrogen oxidation in soil the soil-samples with S. putrefaciens even had to be supplemented with Fe(III). Fresh soil, on the other hand, oxidized hydrogen very efficiently below atmospheric mixing ratios, demonstrating that there must be other oxidation activities in soil.     

Oxygen control prevents denitrifiers and barley plant roots from directly competing for nitrate

Abstract Two denitrifying bacteria ( Pseudomonas chlororaphis and P. aureofaciens ) and a plant (barley, Hordeum vulgare ) were used to study the effect of O₂ concentration on denitrification and NO₃⁻ uptake by roots under well-defined aeration conditions. Bacterial cells in the early stationary phase were kept in a chemostat vessel with vigorous stirring and thus a uniform O₂ concentration in the solution. Both Pseudomonads lacked N₂O reductase and so total denitrification could be directly measured as N₂O production.
Denitrification decreased to 6–13% of the anaerobic rate at 0.01% O₂ saturation (0.14 μM O₂) and was totally inhibited at 0.04% O₂ saturation (0.56 μM O₂). In this well-mixed system denitrification was 10-times more oxygen sensitive than stated in earlier reports. Uptake of nitrate by plants was measured in the same system under light. The NO₃⁻ uptake rate decreased gradually from a maximum in 21% O₂-saturated medium (air saturated) to zero at 1.6% O₂ saturation (22.4 μM O₂). Owing to the very different non-overlapping oxygen requirements of the two processes, direct competition for nitrate between plant roots and denitrifying bacteria cannot occur.     

Characteristics of ammonium absorption by excised root and leaf tissues of maize

Absorption of ammonium from solutions of ammonium chloride by maize ( Zea mays L. cv. GS-2) tissue was studied. In contrast to an initial rapid phase of absorption in root tissue, a one hour lag period was recorded in leaf tissue. The maximum rate of uptake was observed at 5–10 m M NH₄Cl in both tissues. Roots had a K_m value of 1.0 m M and V_max of 24.3 μmol ammonium (g fresh weight)⁻¹ h⁻¹, whereas the leaf tissue had a higher K_m (4.1 m M ) and a lower V_max (8.7 μmol). There was a concentration dependent increase in ethanol soluble and insoluble fractions of organic nitrogen during ammonium supply. The optimum pH for ammonium absorption for both tissues was 7.4. The optimal concentration of CaCl₂ for ammonium absorption was 5 m M whereas that of KCl was only 1 m M . In both tissues, the absorption was inhibited substantially by DCMU, DNP, cycloheximide, lincomycin, sodium tungstate, sodium arsenate and to some extent also by the anions nitrate and sulfate. It is suggested that a carrier is involved in an active uptake of ammonium in the leaf tissues.     

Tracing red-pigmented marine cyanobacteria using in vivo absorption maxima

Abstract The relative abundance of small coccoid cyanobacteria in the Banda Sea (Indonesia) was examined during the first cruise of the "Pelagic Systems" Theme 3 of the Snellius II expedition in August 1984. The survey study embraced 34 stations that were located 50 miles apart. The peak-height of the distinct maximum at 545 nm in the in vivo absorption spectra of continuous cultures of Synechococcus WH7803 (DC2) appeared to be an excellent marker for fast tracing of small red-pigmented Synechococcus -type cyanobacteria. These picocyanobacteria were found at 75% of the Banda Sea Stations, ranging from 10⁴ to 10⁵ cells ml⁻¹ and with a preference for the deeper layers where light intensities were low. Their low surface abundance seemed to show a diurnal pattern (i.e., decreased during the course of the day), correlating with surface irradiance. These observations suggest that red-pigmented picocyanobacteria are well adapted to grow and photosynthesis at relatively low irradiances. This is consistent with laboratory results.     

Swimming marine Synechococcus strains with widely different photosynthetic pigment ratios form a monophyletic group

Unicellular marine cyanobacteria are ubiquitous in both coastal and oligotrophic regimes. The contribution of these organisms to primary production and nutrient cycling is substantial on a global scale. Natural populations of marine Synechococcus strains include multiple genetic lineages, but the link, if any, between unique phenotypic traits and specific genetic groups is still not understood. We studied the genetic diversity (as determined by the DNA-dependent RNA polymerase rpoC1 gene sequence) of a set of marine Synechococcus isolates that are able to swim. Our results show that these isolates form a monophyletic group. This finding represents the first example of correspondence between a physiological trait and a phylogenetic group in marine Synechococcus. In contrast, the phycourobilin (PUB)/phycoerythrobilin (PEB) pigment ratios of members of the motile clade varied considerably. An isolate obtained from the California Current (strain CC9703) displayed a pigment signature identical to that of nonmotile strain WH7803, which is considered a model for low-PUB/PEB-ratio strains, whereas several motile strains had higher PUB/PEB ratios than strain WH8103, which is considered a model for high-PUB/PEB-ratio strains. These findings indicate that the PUB/PEB pigment ratio is not a useful characteristic for defining phylogenetic groups of marine Synechococcus strains.     

DIFFERENCES IN GROWTH AND PHYSIOLOGY OF MARINE SYNECHOCOCCUS (CYANOBACTERIA) ON NITRATE VERSUS AMMONIUM ARE NOT DETERMINED SOLELY BY NITROGEN SOURCE REDOX STATE1

The preference of phytoplankton for ammonium over nitrate has traditionally been explained by the greater metabolic cost of reducing oxidized forms of nitrogen. This “metabolic cost hypothesis” implies that there should be a growth disadvantage on nitrate compared to ammonium or other forms of reduced nitrogen such as urea, especially when light limits growth, but in a variety of phytoplankton taxa, this predicted difference has not been observed. Our experiments with three strains of marine Synechococcus (WH7803, WH7805, and WH8112) did not reveal consistently faster growth (cell division) on ammonium or urea as compared to nitrate. Urease and glutamine synthetase (GS) activities varied with nitrogen source in a manner consistent with regulation by cellular nitrogen status via NtcA (rather than by external availability of nitrogen) in all three strains and indicated that each strain experienced some degree of nitrogen insufficiency during growth on nitrate. At light intensities that strongly limited growth, the composition (carbon, nitrogen, and pigment quotas) of WH7805 cells using nitrate was indistinguishable from that of cells using ammonium, but at saturating light intensities, cellular carbon, nitrogen, and pigment quotas were significantly lower in cells using nitrate than ammonium. These and similar results from other phytoplankton taxa suggest that a limitation in some step of nitrate uptake or assimilation, rather than the extra cost of reducing nitrate per se, may be the cause of differences in growth and physiology between cells using nitrate and ammonium.     

Nitrification in vitro by a range of filamentous fungi and yeasts

A wide range of fungi including yeasts, growing on Czapek Dox medium, nitrified added ammonium and the ammonium released by urea hydrolysis. Phanerochaete chrysosporium and Hymenoscyphus ericiae were the only fungi tested which failed to nitrify. A soil yeast (isolate 1) was the most active nitrifier of ammonium in vitro , forming 0·80 μg nitrate mg^-1 biomass over the 7 d incubation period.     

Influence of bicarbonate and metal ions on the development of root Fe(III) reducing capacity by Fe-deficient cucumber (Cucumis sativus) plants

The effect of bicarbonate and selected metal ions on the development of enhanced root Fe(III) reducing capacity (a response to Fe deficiency of dicotyledons) was studied in young plants of cucumber ( Cucumis sativus L. cv. Ashley) grown in nutrient solution. Pretreatment of 11-day-old Fe-deficient cucumber plants with 20 m M NaHCO₃, for at least 23 h prior to determination of root Fe(III) reducing capacity, markedly inhibited this response. The inhibitory effect of bicarbonate could be partly reversed by a 4- to 8-h treatment with either 10 μ M MnSO₄, 10 μ M FeEDDHA, 2 μ M ZnSO₄, 0.5 μ M NiCl₂, or 0.25 μ M , or CoSO₄ (final concentrations), added to the nutrient solution. By contrast, the addition of other salts of metal ions, like CuSO₄ and Cd(NO₃)₂, at 0.25, 0.5 or 1 μ M , or MgSO₄, at 0.5, 1 or 2 m M (final concentrations), had no beneficial effect. The results suggest that bicarbonate may inhibit the development of root Fe(III) reducing capacity by diminishing the availability of certain metal ions required for this response.     

The marine picoplankter Synechococcus sp. WH 7803 exhibits an adaptive response to restricted iron availability

Abstract: Laboratory cultures of marine Synechococcus sp. WH 7803 were grown under conditions of restricted iron availability. The culture medium was adjusted to restrict iron availability: (i) by adding the iron chelator EDDA; (ii) by omitting iron; and (iii) by omitting both iron and EDTA. An adaptive response was observed to these iron-restricted conditions, including a decrease in cellular phycoerythrin and synthesis of a 36 kDa polypeptide in [³⁵S]methionine radiolabelled whole cell lysates separated by SDS-PAGE. The polypeptide was synthesized within 48 h of transferring exponential phase cells to the iron-restricted medium. The protein was localized to the cell membranes and not the cytoplasmic fraction.     

Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea

Phylogenetic relationships among members of the marine Synechococcus genus were determined following sequencing of the 16S ribosomal DNA (rDNA) from 31 novel cultured isolates from the Red Sea and several other oceanic environments. This revealed a large genetic diversity within the marine Synechococcus cluster consistent with earlier work but also identified three novel clades not previously recognized. Phylogenetic analyses showed one clade, containing halotolerant isolates lacking phycoerythrin (PE) and including strains capable, or not, of utilizing nitrate as the sole N source, which clustered within the MC-A (Synechococcus subcluster 5.1) lineage. Two copies of the 16S rRNA gene are present in marine Synechococcus genomes, and cloning and sequencing of these copies from Synechococcus sp. strain WH 7803 and genomic information from Synechococcus sp. strain WH 8102 reveal these to be identical. Based on the 16S rDNA sequence information, clade-specific oligonucleotides for the marine Synechococcus genus were designed and their specificity was optimized. Using dot blot hybridization technology, these probes were used to determine the in situ community structure of marine Synechococcus populations in the Red Sea at the time of a Synechococcus maximum during April 1999. A predominance of genotypes representative of a single clade was found, and these genotypes were common among strains isolated into culture. Conversely, strains lacking PE, which were also relatively easily isolated into culture, represented only a minor component of the Synechococcus population. Genotypes corresponding to well-studied laboratory strains also appeared to be poorly represented in this stratified water column in the Red Sea.     

Effect of algal deposition on acetate and methane concentrations in the profundal sediment of a deep lake (Lake Constance)

Abstract In the profundal sediment ot Lake Constance (143 m depth) the temperature is constant at 4 °C. Despite the constant temperature, CH₄ concentrations changed with season between about 120 μM in winter and about 750 μM in summer, measured down to 30 cm depth. The acetate concentration profiles also varied between seasons. In summer, acetate concentration reached a maximum at about 100 μM in 2 or 4 cm depth. In winter, maximal concentrations of about 5 μM were observed over the entire depth. Input of organic material in late spring may be the reason for the seasonal change of both compounds. To simulate such a sedimentation event, intact sediment cores were covered with suspensions of Porphyridium aerugenium or Synechococcus sp. The addition of the phytoplankton material resulted in a drastic increase of acetate concentrations with a maximum at 2 cm depth, similar to in situ acetate concentrations measured in summer. The same applies for CH₄ for which increased concentrations were observed down to 6 cm depth. H₂ concentrations, on the other hand, showed no distinct increase. Treatment of intact sediment cores with ¹⁴C-labeled Synechococcus cells resulted in the formation of ¹⁴C-acetate, ¹⁴CH₄ and ¹⁴CO₂. Maximum concentrations of ¹⁴CH₄ were found at 4 cm depth, i.e. just above the depth to which ¹⁴C-acetate penetrated. The results show that phytoplankton blooms may cause a seasonal variation of acetate and CH₄ in profundal sediments of deep lakes despite the constant low temperature. They also indicate that acetate is the dominant substrate for methanogenic bacteria in the profundal sediments of Lake Constance.     

Role of nitrate and nitrite for production and consumption of nitric oxide during denitrification in soil

Abstract Anaerobic production and consumption of NO was measured in a calcic cambisol (KBE; pH 7.3) and a forest luvisol (PBE; pH 4.4) which were incubated at 80% water-holding capacity and continuously flushed with N₂. Both NO production and NO consumption were negligibly low when nitrate and nitrite concentrations in the soil were exhausted. Addition of glucose alone had no effect, but addition of nitrate ± glucose greatly stimulated both NO production and NO consumption. NO consumption followed an apparent first-order reaction at low NO mixing ratios (1–3 ppmv), but a higher NO mixing ratios it followed Michaelis-Menten kinetics. In PBE the apparent K _m was 980 ppbv NO (1.92 nM in soil water). During reduction of nitrate, nitrite intermediately accumulated and simultaneously, production rates of NO and N₂O were at the maximum. Production rates of NO plus N₂O amounted to 20% and 34% of the nitrate reduction rate in KBE and PBE, respectively. NO production was hyperbolically related to the nitrite concentration, indicating an apparent K_m of 1.6 μg nitrite-N g⁻¹ d.w. soil (equivalent to 172 μM nitrite in soil solution) for the reduction of nitrite to NO in KBE. Under nitrate and nitrite-limiting conditions, 62–76% and 93–97% of the consumed NO-N were recovered as N₂O-N in KBE and PBE, respectively. Gassing of nitrate plus nitrite-depretsu KBE with increasing mixing ratios of NO₂ resulted in increasing rates of NO₂ uptake and presumably in the formation of low concentrations of nitrite and nitrate. This NO₂ uptake resulted in increasing rates of both NO production and NO consumption indicating that nitrite or nitrate was limiting for both reactions.     

The effect of nitrogen source on photosynthesis of carob at high CO2 concentrations

Carob seedlings ( Ceratonia siliqua L. cv. Mulata), fed with nitrate or ammonium, were grown in growth chambers containing two levels of CO₂ (360 or 800 μl l⁻¹), three root temperatures (15, 20 or 25°C), and the same shoot temperature (20/24°C, night/day temperature). The response of the plants to CO₂ enrichment was affected by environmental factors such as the type of inorganic nitrogen in the medium and root temperature. Increasing root temperature enhanced photosynthesis rate more in the presence of nitrate than in the presence of ammonium. Differences in photosynthetic products were also observed between nitrate- and ammonium-fed carob seedlings. Nitrate-grown plants showed an enhanced content of sucrose, while ammonium led to enhanced storage of starch. Increase in root temperature caused an increase in dry mass of the plants of similar proportions in both nitrogen sources. The enhancement of the rates of photosynthesis by CO₂ enrichment was proportionally much larger than the resulting increases in dry mass production when nitrate was the nitrogen source. Ammonium was the preferred nitrogen source for carob at both ambient and high CO₂ concentrations. The level of photosynthesis of a plant is limited not only by atmospheric CO₂ concentration but also by the nutritional and environmental conditions of the root.     

Photosynthesis and respiration of a diatom biofilm cultured in a new gradient growth chamber

Abstract A diatom biofilm was grown in a chamber developed for culture of biofilms in chemical gradients. The diatoms grew on a polycarbonate membrane filter which separated a sterile reservoir, with added phosphate, from a reservoir without phosphate. Within 3 weeks of inoculation, a thick biofilm developed on the surface of the filter. The biofilms were homogeneous and therefore suitable for calculations of O₂ diffusion fluxes from concentration profiles of O₂. Profiles of O₂, pH, and gross photosynthesis at different light intensities and liquid medium concentrations of dissolved inorganic carbon and O₂ were measured with microelectrodes. Respiratory activity in a layer of the biofilm was determined as the difference between gross photosynthesis and outflux of O₂ from that layer. The photosynthetic activity in a well-developed biofilm grown at 360 μEinst m⁻² s⁻¹ and 2.4 mM HCO₃⁻ was limited by the supply of inorganic carbon. Exposure to light above 360 μEinst m⁻² s⁻¹ stimulated gross photosynthesis as well as respiratory processes without affecting net outflux of O₂. Higher concentrations of inorganic carbon, on the other hand, enhanced gross photosynthesis without concurrent increase in respiratory rate, resulting in an increased outflux of O₂. High concentrations of O₂ in the liquid medium decreased the net outflux of O₂ with little effect on the gross photosynthesis. The effects of inorganic carbon and O₂ on the metabolic activities of the biofilm were consistent with the presence of photorespiratory activity.     

GROWTH RATE RELATIONSHIPS TO PHYSIOLOGICAL INDICES OF NUTRIENT STATUS IN MARINE DIATOMS

The growth of two species of marine diatom, Thalassiosira weissflogii (Grunow) and Thalassiosira pseudonana (Hustedt), was followed in batch cultures at four concentrations of dissolved inorganic carbon from N- and C-replete lag phase into N- and/or C-deplete stationary phase. Results describe the relationship between carbon-specific growth rate (μ_C) and chl a :carbon (chl a :C) and glutamine:glutamate (gln:glu) ratios with changes in the cells' nutritional status (N:C), during the utilization of either NO₃ ⁻ or NH₄ ⁺ . The use of the gln:glu ratio as an index of N:C requires further clarification. For both species and N sources, N stress resulted in a decrease in μ_C, chl a :C, and N:C relative to μ_Cmax values, whereas C stress resulted in a decrease in μ_C and an increase in chl a :C and N:C relative to μ_Cmax values. Both species attained a chl a :C ratio of approximately 15 μg·g ^{− 1} at μ_Cmax using either N source. However, this value was not necessarily an indicator of maximal growth rate. NC colimitation resulted in decreased μ_C to values less than 20% of μ_Cmax with only minor changes in chl a :C and N:C relative to μ_Cmax values. Chl a :C results suggest a similarity between the light stress and C stress responses of marine diatoms. The potential for C stress in the marine environment needs to be addressed.