EXTRACELLULAR LYSIS OF THE BLUEGREEN ALGA PHORMIDIUM LURIDUM BY BDELLOVIBRIO BACTERIOVORUS1

When either cells of the bacterium, Bdellovibrio bacteriovorus (Stolp & Starr), strain 15143, or a heat-resistant lytic factor derived from these cells is added to viable cultures of Phormidium luridum var. olivacea Boresch all the algal cells underwent gradual lysis. This effect was obtained with a mean initial bdellovibrio:algal cell ratio of 7.5:1. When P. luridum was mixed with the bdellovibrio cultures the algal chlorophyll content showed an 8-fold decrease. Concomitantly, this interspecies interaction caused, a 75% inhibition of algal photosynthesis after-4 h. Heat, treatment of the B. bacteriovorus culture supernatant fluid increased its ability to inhibit photosynthesis approximately 14%. Light, microscopy showed pale granules and intracellular spaces to form in the P. luridum within 16 h after adding the bdellovibrio lytic factor. Subsequent morphological changes included the development of large intracellular spaces, intercellular spaces, spheroplast formation and finally Complete lysis of the algal cells.     

THE EFFECT OF PHOSPHATE STATUS ON THE KINETICS OF CYANOPHAGE INFECTION IN THE OCEANIC CYANOBACTERIUM SYNECHOCOCCUS SP. WH78031

Phycoerythrin-containing Synechococcus species are considered to be major primary producers in nutrient-limited gyres of subtropical and tropical oceanic provinces, and the cyanophages that infect them are thought to influence marine biogeochemical cycles. This study begins an examination of the effects of nutrient limitation on the dynamics of cyanophage/Synechococcus interactions in oligotrophic environments by analyzing the infection kinetics of cyanophage strain S-PM2 (Cyanomyoviridae isolated from coastal water off Plymouth, UK) propagated on Synechococcus sp. WH7803 grown in either phosphate-deplete or phosphate-replete conditions. When the growth of Synechococcus sp. WH7803 in phosphate-deplete medium was followed after infection with cyanophage, an 18-h delay in cell lysis was observed when compared to a phosphate-replete control. Synechococcus sp. WH7803 cultures grown at two different rates (in the same nutritional conditions) both lysed 24 h postinfection, ruling out growth rate itself as a factor in the delay of cell lysis. One-step growth kinetics of S-PM2 propagated on host Synechococcus sp. WH7803, grown in phosphate-deplete and-replete media, revealed an apparent 80% decrease in burst size in phosphate-deplete growth conditions, but phage adsorption kinetics ofS-PM2 under these conditions showed no differences. These results suggested that the cyanophages established lysogeny in response to phosphate-deplete growth of host cells. This suggestion was supported by comparison of the proportion of infected cells that lysed under phosphate-replete and-deplete conditions, which revealed that only 9.3% of phosphate-deplete infected cells lysed in contrast to 100% of infected phosphate-replete cells. Further studies with two independent cyanophage strains also revealed that only approximately 10% of infected phosphate-deplete host cells released progeny cyanophages. These data strongly support the concept that the phosphate status of the Synechococcus cell will have a profound effect on the eventual outcome of phage-host interactions and will therefore exert a similarly extensive effect on the dynamics of carbon flow in the marine environment.     

The heat shock response and acquired thermotolerance in three strains of cyanobacteria

The heat shock response of three cyanobacterial strains,Anabaena sp. Strain PCC (paris Culture Collection) 7120,Plectonema boryanum Strain PCC 6306, andSynechococcus sp. Strain PCC 7942, was characterized by polyacrylamide gel electrophoresis.Anabaena produced 33 heat shock proteins,P. boryanum 35 proteins, andSynechoccus 19 proteins. The rapid response to heat shock was consistent for all three strains, although the number of time-dependent proteins varied. All strains developed thermotolerance when first pretreated with a sublethal heat shock and then challenged with a previously lethal temperature. A 30-min 30°C incubation was required between the heat shock and challenge forSynechococcus, but not forAnabaena andP. boryanum. Synechococcus cells required a higher challenge temperature (51° vs. 49°C) than the other two strains to destroy control cells that were not pretreated with a heat shock.     

Estimation of Organic Carbon Content of the Cyanobacterium Synechococcus sp. by Soft X-ray Microscopy

A method for the accurate evaluation of the organic carbon (OC) content of phytoplankton by soft X-ray microscopy (XM) was developed and applied to the picophytoplankton Synechococcus sp., whose cells are covered by extracellular polysaccharides (EPS). Based on the X-ray absorption coefficients and gray levels of the XM images, the OC content of EPS-covered cells of Synechococcus sp. (0.39–0.47 pg/cell) was found to be 2.0–2.4 times larger than that of EPS-removed cells (0.20 pg/cell). These findings suggest that soft XM could be a useful tool for evaluating the OC content of picophytoplankton and EPS without pretreatment steps.     

Spectral Light Quality Affects Protein Profile of Synechococcus sp. PCC 7942: A Comparative 2-Dimensional Gel Electrophoresis (2-DGE) Analysis

We report here a comparative analysis of the effect of blue (450 nm), red (660 nm), and white light (400–700 nm) on the protein profile of cyanobacteria Synechococcus sp. PCC 7942. In vivo labeling of cells with [³⁵S] methionine and their subsequent analysis by two-dimensional gel electrophoresis (2-DGE) showed that eight polypeptides were unique to dark adapted cells, ten were blue light specific, and four were specifically induced in red light. The results show that Synechococcus sp. respond to various light treatments rapidly and synthesize new polypeptides in dark and blue/red light. Received: 12 October 1999 / Accepted: 16 November 1999     

Synechococcus sp. (PTCC 6021) cultivation under different light irradiances—Modeling of growth rate-light response

Synechococcus sp. (PTCC 6021), a cyanobacterium species, was cultivated in an internally illuminated photobioreactor. The reactor was designed to achieve a monoseptic cultivation of the species. The goal was to study the growth–irradiance behavior of Synechococcus sp. (PTCC 6021). To accomplish this, different initial light irradiances were implemented inside the photobioreactor and the growth of the cells was monitored. It was observed that cell growth increased with higher light intensity until the photoinhibition occurrence at light irradiance higher than 250?μE?m^?2?s^?1. The maximum OD₆₀₀, maximum growth rate, and biomass productivity increased, and hence the extinction coefficient decreased, with the increase in light irradiance before photoinhibition. The maximum optical density (OD₆₀₀) of 5.91 was obtained with irradiance below 250?μE?m^?2?s^?1 during a growth period of 80 days. The modified Monod function could model the growth–irradiance of cells with satisfactory agreement with the experimental data. The comparison of growth–irradiance of the studied species with other photosynthetic organisms showed the same trend as for cyanobacteria with photoinhibition.     

PHOSPHORUS BIOAVAILABILITY MONITORING BY A BIOLUMINESCENT CYANOBACTERIAL SENSOR STRAIN 1

Phosphorus (P) is widely considered to be the main nutrient limiting the productivity of freshwater phytoplankton, but an assessment of its bioavailability in natural samples is highly complex. In an attempt to provide a novel tool for this purpose, the promoter of the alkaline phosphatase gene, phoA, from Synechococcus sp. PCC 7942 was fused to the luxAB luciferase genes of the bioluminescent bacterium Vibrio harveyi. The resulting construct was introduced into a neutral site on the Synechococcus sp. PCC 7942 genome to yield strain APL, which emitted light when inorganic P concentrations fell below 2.3 μM. Light emission of P‐deprived cells decreased rapidly upon inorganic P readdition. The reporter was demonstrated to be a sensitive tool for monitoring the bioavailability of both inorganic and organic P sources. In water samples taken from a natural freshwater environment (Lake Kinneret, Israel), the luminescence measured correlated with total dissolved phosphate concentrations.     

Selective production of glutamate by an immobilized marine blue-green alga,Synechococcus sp.

Summary Among 200 strains of marine bluegreen algae isolated from the coastal areas of Japan, the marine blue-green alga Synechococcus sp. NKBG 040607 excreted glutamate at the highest rate, 82.6% of total amino acids production being glutamate. Synechococcus sp. NKBG 40607 was immobilized in calcium alginate gel. Glutamate production by immobilized cells was double that of native cells. Maximal glutamate production (25 g/cm³ gel per day) of the immobilized cells was observed under a light intensity of 144 Einstein/m² per second at a cell concentration of 7.5 mg dry cells/cm³ gel. Immobilized cells of Synechococcus sp. can use nitrate as a nitrogen source. Immobilized marine Synechococcus sp. produced 0265 mg/cm³ gel of glutamate for 7 days in the presence of chloramphenicol.     

Attempts to grow bdellovibrios micurgically-injected into animal cells

Incubation in buffer of Bdellovibrio bacteriovorus 109J, B. stolpii UKi2, or B. starrii A3.12 with washed eucaryotic animal cells (mouse liver, hamster kidney, or bovine mammary gland) resulted in neither attachment nor growth of the bdellovibrios. When cells of these bdellovibrio strains were incubated with erythrocyte suspensions (bovine or rabbit) a very low level of bdellovibrio attachment and penetration occurred, but no growth could be detected. Using micurgical procedures, bdellovibrios were injected into the perivetelline space or the cytoplasm of rabbit ova. After 18–24h incubation, neither a significant loss nor increase of injected, intracellular bdellovibrios was observed. Limited axenic growth of bdellovibrios (109J or UKi2) occurred in media containing rabbit ova extracts and dilute nutrient broth. It is concluded that eucaryotic rabbit ova do not provide a suitable environment for intracellular bdellovibrio growth.     

One-step plasmid construction for generation of knock-out mutants in cyanobacteria: studies of glycogen metabolism in <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002

Genome sequences of microorganisms typically contain hundreds of genes with vaguely defined functions. Targeted gene inactivation and phenotypic characterization of the resulting mutant strains is a powerful strategy to investigate the function of these genes. We have adapted the recently reported uracil-specific excision reagent (USER) cloning method for targeted gene inactivation in cyanobacteria and used it to inactivate genes in glycogen metabolism in Synechococcus sp. PCC 7002. Knock-out plasmid constructs were made in a single cloning step, where transformation of E. coli yielded about 90% colonies with the correct construct. The two homologous regions were chosen independently of each other and of restriction sites in the target genome. Mutagenesis of Synechococcus sp. PCC 7002 was tested with four antibiotic resistance selection markers (spectinomycin, erythromycin, kanamycin, and gentamicin), and both single-locus and double-loci mutants were prepared. We found that Synechococcus sp. PCC 7002 contains two glycogen phosphorylases (A0481/glgP and A2139/agpA) and that both need to be genetically inactivated to eliminate glycogen phosphorylase activity in the cells.     

Characterization of two Synechococcus sp. PCC7002‐related cyanobacterial strains in relation to 16S rDNA,crtR gene,lipids and pigments

Two Synechococcus strains from the Culture Collection of the Institute for Marine Sciences of Andalusia (Cádiz, Spain), namely Syn01 and Syn02, were found to be closely related to the model strain Synechococcus sp. PCC7002 according to 16S rDNA (99% identity). Pigment and lipid profiles and crtR genes of these strains were ascertained and compared. The sequences of the crtR genes of these strains were constituted by 888 bp, and showed 99% identity between Syn01 and Syn02, and 94% identity of Syn01 and Syn02 to Synechococcus sp. PCC7002. There was coincidence in photosynthetic pigments between the three strains apart from the pigment synechoxanthin, which could be only observed in Synechococcus sp. PCC7002. Species of sulfoquinovosyl‐diacyl‐glycerol (SQDG), phosphatidyl‐glycerol (PG), mono‐ and di‐galactosyl‐diacyl‐glycerol (MGDG and DGDG) were detected by high performance liquid chromatography‐mass spectrometry analysis of lipid extracts. The most abundant species within each lipid class were those containing C18:3 together with C16:0 fatty acyl substituents in the glycerol backbone of the same molecule. From these results it is concluded that these cyanobacterial strains belong to group 2 of the lipid classification of cyanobacteria.     

Isolation and purification of siderophores produced by cyanobacteria,Synechococcus sp. PCC 7942 andAnabaena variabilis ATCC 29413

New siderophores were isolated and purified from the spent growth medium of the cyanobacteriaSynechococcus sp. PCC 7942 (Anacystis nidulans R2) andAnabaena variabilis ATCC 29413 by solvent extraction and thin-layer chromatography. For each species the siderophore was released into the medium when the cells were grown at low iron concentrations and was not found in the medium of cells grown in iron-sufficient medium. Through a series of biological and chemical tests, combined with spectral analysis, the dihydroxamate nature of each siderophore was confirmed. The siderophores produced bySynechococcus sp. PCC 7942 andA. variabilis had distinct relative molecular masses of 310–313 Da and 520–525 Da, respectively. Neither of the two strains produced Arnow-positive extracellular organics, which indicate the excretion of extracellular catechol-type siderophores.     

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance ¹³C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 ± 1.5 to 62.3 ± 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His•bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and ¹H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.     

The outer cyst wall ofBdellovibrio bdellocysts is made de novo and not from preformed units from the prey wall

Bdellovibrio sp. strain W bdellocysts were produced inEscherichia coli using three sources of³H-diaminopimelic acid (DAP) for incorporation into the cyst wall peptidoglycan: (a) labeledE. coli peptidoglycan, (b) labeledBdellovibrio peptidoglycan, and (c) exogenous³H-DAP in the encystment medium. After cysts were produced, they were either sonicated to remove the prey cell wall, or germinated to solubilize the cyst wall. The results show that label was incorporated into the cyst wall preferentially from the exogenous DAP in the medium, and not from the bdellovibrio or bdelloplast peptidoglycan. The encysting bdellovibrio does not therefore incorporate existing peptidoglycan units from the bdelloplast for synthesis of the cyst wall.     

Growth and H2 production by Synechococcus spp. using organic/inorganic electron donors

Growth with simultaneous photoproduction of H₂ was obtained on various organic and inorganic compounds using axenic cultures of the oxygenic phototrophic bacteria Synechococcus sp. OU 103 and S. cedrorum. Highest H₂ production occurred with resting cells of S. cedrorum on malate (11.8 mmol H₂/vessel), whereas Synechococcus sp. OU 103 preferred sulphide (10.3 mmol H₂/vessel) as electron donor.The authors are with the Microbial Biotechnology Lab. Department of botany, Osmania University, Hyderabad 500 007, India.     

Diel Division Cycle and Growth Rates of Synechococcus in Lakes Huron and Michigan1

A clone of Synechococcus isolated from Lake Huron and natural populations of Synechococcus from lakes Huron and Michigan were studied in 1989 to examine the diel division cycle and to provide estimates of the in situ growth rate based on the frequency of dividing cells (FDC) method. Cultured populations of Synechococcus exhibited a consistent diel division pattern with a midday/afternoon (1100–1800 h) peak in the percent of dividing cells. The maximum percent of dividing cells varied among cultures (8-27%) and was related to the growth rate. A small fraction of dividing cells (3-5%) remained throughout the dark period, suggesting that some cells were arrested in the doublet stage prior to division. The duration of division (t_d) ranged from 2.6-4.9 h, with a 3.7 h mean for cultures with growth rates ≥0.34 d⁻¹ but increased to 8 h at a lower growth rate of 0.20 d⁻¹. The diel division pattern for natural populations was very similar to the laboratory clone; an afternoon peak (1400-2100 h) in dividing cells and a small fraction of dividing cells (2-5%) remained during the dark period. The maximum percent of dividing cells for natural populations ranged from 6-10%. In situ growth rates, determined from the FDC and assuming a constant t_d of 3.7 h, ranged from 0.30-0.42 d⁻¹. The FDC method may provide accurate estimates of in situ growth, particularly in environments where the growth rate is >0.34 d⁻¹, but in lakes Huron and Michigan where growth rates can be lower and t_d values may increase, FDC-growth rates must be viewed with caution.     

Effects of nitrogen on interspecific competition between two cell-size cyanobacteria: Microcystis aeruginosa and Synechococcus sp.

Micro-cyanobacteria and pico-cyanobacteria coexist in many lakes throughout the world. Their distinct cell sizes and nutrient utilization strategies may lead to dominance of one over the other at varying nutrient levels. In this study, Microcystis aeruginosa and Synechococcus sp. were chosen as representative organisms of micro- and pico-cyanobacteria, respectively. A series of nitrate and ammonia conditions (0.02, 0.1, 0.5, and 2.5 mg N L⁻¹) were designed in mono- or co-cultured systems, respectively. Growth rates of the two species were calculated and fitted by the Monod and Logistic equations. Furthermore, the interspecific competition was analyzed using the Lotka–Volterra model. In mono-cultures, the two cyanobacteria displayed faster growth rates in ammonia than in nitrate. Meanwhile, Synechococcus sp. showed faster growth rates compared to M. aeruginosa in lower N groups (≤ 0.5 mg N L⁻¹). However, in the highest nitrate treatment (2.5 mg N L⁻¹), M. aeruginosa achieved much higher biomass and faster growth rates than Synechococcus sp.. In co-cultures, Synechococcus sp. dominated in the lowest N treatment (0.02 mg N L⁻¹), but M. aeruginosa dominated under the highest nitrate condition (2.5 mg N L⁻¹). Based on the analysis of Raman spectra of living cells in mono-cultures, nitrate (2.5 mg N L⁻¹) upgraded the pigmentary contents of M. aeruginosa better than ammonia (2.5 mg N L⁻¹), but nitrogen in different forms showed little effects on the pigments of Synechococcus sp.. Findings from this study can provide valuable information to predict cyanobacterial community succession and aquatic ecosystem stability.     

Diel patterns of grazing by pigmented nanoflagellates on <Emphasis Type="Italic">Synechococcus</Emphasis> spp. in the coastal ecosystem of subtropical western Pacific

Natural populations of planktonic Synechococcus spp. exhibit diel variations in abundance and frequency of dividing cells (FDC) during the warm seasons (>25°C) in coastal water of the western subtropical Pacific ocean. We hypothesized that differences in grazing rates during the day/dark cycle were a major cause of the observed diel variations in Synechococcus spp. abundance. We used fluorescently labeled particles (FLP) as tracers of feeding on Synechococcus spp. in June and August 2007. Our results showed that FDC of Synechococcus spp. were highest at dusk (50%) and lowest (<10%) between midnight and early morning. Synechococcus spp. had three abundance phases, accrual (I), peak (II), and diminished (III). Moreover, FDC values gradually increased in phase I, declined to the lowest values during phase II, and remained at low levels throughout phase III. Our results strongly indicated that pigmented nanoflagellate (PNF) grazing was the underlying biological factor regulating diel variations in Synechococcus spp. abundance. And, the rate of PNF ingestion peaked during the night on smaller non-dividing cells following a peak of Synechococcus spp. abundance in phase II. These findings provide evidence that diel variations in ingestion rates are affected by non-dividing cells of Synechococcus spp. and imply that the impacts of PNF grazing on Synechococcus spp. is based on food selectivity by size.