Eduardo Fernández-Valiente

Microstructural Characterization of Cyanobacterial Mats from the McMurdo Ice Shelf, Antarctica

ABSTRACT The three-dimensional structures of two types of cyanobacterium-dominated microbial mats from meltwater ponds on the McMurdo Ice Shelf were as determined by using a broad suite of complementary techniques, including optical and fluorescence microscopy, confocal scanning laser microscopy, scanning electron microscopy with back-scattered electron-imaging mode, low-temperature scanning electron microscopy, and microanalyitical X-ray energy dispersive spectroscopy. By using a combination of the different in situ microscopic techniques, the Antarctic microbial mats were found to be structures with vertical stratification of groups of cyanobacteria and mineral sediments, high contents of extracellular polymeric substances, and large void spaces occupied by water. In cyanobacterium-rich layers, heterocystous nostocalean and nonheterocystous oscillatorialean taxa were the most abundant taxa and appeared to be intermixed with fine-size deposits of epicellular silica and calcium carbonate. Most of the cyanobacterial filaments had similar orientations in zones without sediment particles, but thin filaments were tangled among thicker filaments. The combination of the microscopic techniques used showed the relative positions of biological and mineral entities within the microbial mats and enabled some speculation about their interactions.

N2-Fixation in Cyanobacterial Mats from Ponds on the McMurdo Ice Shelf, Antarctica

We have investigated the ecological importance of N2-fixation in cyanobacterial mats, dominated by oscillatorean species, in ponds of the Bratina Island area of the McMurdo Ice Shelf, Antarctica (78°S, 166°E). Nitrogenase activity, estimated as acetylene reducing activity (ARA), was found in all the mats investigated (n = 16). The average ARA was 75.9 mmol ethylene m-2 h-1, ranging from 6 to 201 mmol ethylene m-2 h-1. Nitrogenase activity was positively correlated with dissolved reactive phosphorus concentration in pondwater and the C/N ratio of the mat, and was negatively correlated with pondwater NH4+-N concentrations and natural abundance of 15N in the mats. ARA was restricted to the upper, oxic layer of the mats. Experiments conducted to ascribe ARA to different groups of prokaryotes suggested that ARA was mainly conducted by heterocystous cyanobacteria, since no activity was found in the dark and the activity was inhibited by the photosystem II inhibitor DCMU (3-[3,4-dichlorophenyl]-1,1-dimethyl urea). In spite of 24 h of daylight, nitrogenase activity showed a diel cycle with maximum activity at midday (10-18 h) and minimal activity at early morning (6-10 h) when pond temperatures were at their minima. Light dependency of nitrogenase activity for three cyanobacterial communities showed that the irradiance required for saturating ARA was low, in every case lower than 100 mmol photon m-2s-1. Irradiance rarely fell below 100 mmol photon m-2s-1 during Antarctic summer days and ARA was likely to be light saturated for much of the time. We estimate that N2 fixation represented on average a N input into the ponds of over 1 g m-2y-1. This value appears to be the highest N input to this Antarctic ecosystem.

Environmental Factors Controlling N2 Fixation in Mediterranean Rice Fields

A bstractThe objectives of this study were to analyze the environmental controls on N2 fixation in Spanish rice fields. Nitrogenase activity, measured as the acetylene-reducing activity (ARA), was estimated in situ during different intervals of the cropping period. At the same time, physical and chemical variables and cyanobacterial occurrence were determined in water and soil. Nitrogen fixation was measurable at all sampling sites, being higher in July and lower in June after a short dry period. The ARA values ranged from 0.23 to 75.5 kg N Na−1 year−1. Because blooms or other conspicuous cyanobacterial forms were not included in the measurements, maximum rates of nitrogen fixation may have been higher. Environmental variables that correlated with ARA varied on a seasonal basis. Water properties such as calcium, hardness, or conductivity, and soil properties such as conductivity and sodium correlated positively with N2 fixation; however, nutrient parameters such as dissolved inorganic nitrogen or soluble reactive phosphorus were negatively correlated. Cyanobacterial abundance, in general, did not correlate with ARA. The overall conclusion is that nitrogen fixation may be an important N input in the N cycle of rice fields, and could lessen pollution problems by lowering the demand for chemical fertilizers.

Acclimation of photosynthetic pigments and photosynthesis of the cyanobacterium Nostoc sp. strain UAM206 to combined fluctuations of irradiance, pH, and inorganic carbon availability

Summary We analysed the combined effect of pH, irradiance, and inorganic carbon availability on growth and pigment composition of the cyanobacterium Nostoc sp. strain UAM206, isolated from rice fields. This cyanobacterium contains phycoerythrin in its phycobilisomes and can show chromatic acclimation. Under inorganic carbon limitation, the growth rate of Nostoc sp. strain UAM206 was affected by pH, but not by irradiance. Chlorophyll a phycoerythrin (PE), and phycocyanin (PC) contents were inversely correlated to irradiance. Chlorophyll a (Chla) content was not affected by pH; however, with increasing pH, phycocyanin, phycoerythrin, and allophycocyanin (APC) content increased. Inorganic carbon availability masked or decreased some of the effects of pH under inorganic carbon limitation; however, the significant effect of pH on the allophycocyanin contents was clearly independent of the inorganic carbon availability. Analysis of our results indicates that elevation of external pH and available inorganic carbon results in an increase in the number of phycobilisomes along with a decrease in their size (PC+PE/APC ratio), while irradiance mostly affects the size of the phycobilisome. Likewise, increasing irradiance, pH, and available inorganic carbon results in an increase in the PSII/PSI ratio (APC/Chla ratio). Finally, Nostoc sp. strain UAM206 seems able to acclimate its photosynthetic apparatus to variations of the three studied environmental factors that are known to occur in rice fields.

pbpB, a Gene Coding for a Putative Penicillin-Binding Protein, Is Required for Aerobic Nitrogen Fixation in the Cyanobacterium Anabaena sp. Strain PCC7120

Transposon mutagenesis of Anabaena sp. strain PCC7120 led to the isolation of a mutant strain, SNa1, which is unable to fix nitrogen aerobically but is perfectly able to grow with combined nitrogen (i. e., nitrate). Reconstruction of the transposon mutation of SNa1 in the wild-type strain reproduced the phenotype of the original mutant. The transposon had inserted within an open reading frame whose translation product shows significant homology with a family of proteins known as high-molecular-weight penicillin-binding proteins (PBPs), which are involved in the synthesis of the peptidoglycan layer of the cell wall. A sequence similarity search allowed us to identify at least 12 putative PBPs in the recently sequenced Anabaena sp. strain PCC7120 genome, which we have named and organized according to predicted molecular size and the Escherichia coli nomenclature for PBPs; based on this nomenclature, we have denoted the gene interrupted in SNal as pbpB and its product as PBP2. The wild-type form of pbpB on a shuttle vector successfully complemented the mutation in SNa1. In vivo expression studies indicated that PBP2 is probably present when both sources of nitrogen, nitrate and N(2), are used. When nitrate is used, the function of PBP2 either is dispensable or may be substituted by other PBPs; however, under nitrogen deprivation, where the differentiation of the heterocyst takes place, the role of PBP2 in the formation and/or maintenance of the peptidoglycan layer is essential.

Relationship Between Abundance of N2-fixing Cyanobacteria and Environmental Features of Spanish Rice Fields

In order to estimate the potential utilization of N2-fixing (heterocystous) cyanobacteria as natural biofertilizers in the Valencian rice fields (Spain), the distribution and seasonal variation of these microorganisms in water and sediment samples were evaluated, and the relationships among cyanobacterial abundance and physical and chemical characteristics of soil and water were investigated. N2-fixing cyanobacteria were present in all the samples analyzed (25 sampling points sampled three times per year during two years). The relative cyanobacterial abundance in soil and water followed contrasting patterns, maximum presence in soil coincided with minimum abundance in water. Correlation analysis showed that cyanobacterial abundance in the two phases (water and sediment) was influenced more by water than by soil properties. Salinity, mineralization variables, and soluble reactive phosphate (SRP) correlated positively with heterocystous cyanobacteria presence. Furthermore, dissolved inorganic nitrogen (DIN) and the ratio DIN: SRP correlated negatively with cyanobacterial abundance. However DIN: SRP ratio better described the cyanobacterial distribution, with a threshold effect: below the Redfield ratio value (7.2 in mass units) cyanobacterial abundance was clearly higher.

Wide variation in the cyanobacterial complement of presumptive penicillin-binding proteins

A genomic analysis of putative penicillin-binding proteins (PBPs) that are involved in the synthesis of the peptidoglycan layer of the cell wall and are encoded in 12 cyanobacterial genomes was performed in order to help elucidate the role(s) of these proteins in peptidoglycan synthesis, especially during cyanobacterial cellular differentiation. The analysis suggested that the minimum set of PBPs needed to assemble the peptidoglycan layer in cyanobacteria probably does not exceed one bifunctional transpeptidase–transglycosylase Class A high-molecular-weight PBP; two Class B high-molecular-weight PBPs, one of them probably involved in cellular elongation and the other in septum formation; and one low-molecular-weight PBP. The low-molecular-weight PBPs of all of the cyanobacteria analyzed are putative endopeptidases and are encoded by fewer genes than in Escherichia coli. We show that in Anabaena sp. strain PCC 7120, predicted proteins All2981 and Alr4579, like Alr5101, are Class A high-molecular-weight PBPs that are required for the functional differentiation of aerobically diazotrophic heterocysts, indicating that some members of this class of PBPs are required specifically for cellular developmental processes.

Screening of Soil Cyanobacteria for Antifungal and Antibacterial Activity

Abstract Soil cyanobacteria isolated from the paddy fields of seven provinces in Iran was evaluated for antimicrobial activity. Aqueous, petroleum ether, and methanol extracts from 76 microalgae were examined for antimicrobial properties against four bacteria and two fungi. Of total microalgae, 22.4% (17 cyanobacteria) exhibited antimicrobial effects. Selected cyanobacteria with positive antimicrobial activities were members of the families Stigonemataceae, Nostocaceae, Oscillatoriaceae, and Chroococcaceae. Growth of Bacillus subtilis Persian Type Culture Collection (PTCC) 1204 and Staphylococcus epidermidis PTCC 1114 were inhibited by 12 and 14 species of cyanobacteria, respectively. Also, eight cyanobacteria inhibited the growth of Escherichia coli PTCC 1047, and two species inhibited the growth of Salmonella typhi PTCC 1108. Considering fungi, six species inhibited the growth of Candida kefyr ATCC 1140, and one cyanobacterium species inhibited the growth of Candida albicans ATCC 14053. Furthermore, one of the paddy fields (Khozestan) showed no antimicrobial activity.

Acclimation of Cyanobacterial Communities in Rice Fields and Response of Nitrogenase Activity to Light Regime

A bstractThe short-term and long-term effects of light regime on nitrogenase activity (NA) and cyanobacterial communities in rice fields (Valencia, Spain) were examined. Daily variation in nitrogen fixation was measured during three periods of the crop cycle: tillering (formation of secondary stems in the rice plants), heading (formation of reproductive structures), and maturity. Two locations were examined over two consecutive years (1994 and 1995). Despite differences in the crop-cycle periods, location, and year, a consistent pattern of nitrogen fixation was observed, with a main activity peak in the morning and another in the late evening. Short-term experiments, performed on two cyanobacterial blooms (Nostoc sp. and Anabaena sp.) exposed to natural light under plant canopy (7% incidence irradiance), and to different light intensities under neutral density screens without plant cover (full sunlight, 43%, 26%, and 13% of incident irradiance), indicated that nitrogenase activity (NA) was dependent on both light intensity and quality. In long-term experiments, where natural communities of cyanobacteria were exposed to one month of different light intensities, changes in the species composition of the three main genera of heterocystous cyanobacteria (Nostoc, Anabaena, and Calothrix) were observed. The light intensity at which communities were exposed for one month became the optimum irradiance for NA for each cyanobacterial community. Assays performed at higher or lower irradiances showed lower NA. Nitrogen fixation followed a pattern of seasonal variation along the crop cycle. Values were low at the beginning of the crop (May), reached a maximum value at the end of the tillering stage (June), and declined thereafter until the end of the cultivation cycle (September).

Environmental factors affectingin vitro nitrogenase activity of cyanobacteria isolated from rice-fields

The nitrogen-fixing capacity of four cyanobacterial strains was tested in relation to heterotrophic ability, tolerance to combined nitrogen and adaptive capacity to changes in light intensity and pH. Strains (Anabaena variabilis UAM 202;Calothrix marchica UAM 214;Nodularia spumigena UAM 204,Nostoc punctiforme UAM 205) were isolated from the rice-fields of Valencia (Spain).C. marchica, was the only strain able to grow and to fix dinitrogen under heterotrophic conditions, with fructose and glucose. Fructose was the best substrate supporting growth and dinitrogen fixation in mixotrophy (growth in the light under conditions where CO2 and organic carbon are assimilated simultaneously), photoheterotrophy (growth in the light on an organic compound in the absence of net CO2 fixation) and heterotrophy (growth on an organic compound in the dark). Ammonium repressed nitrogenase more than nitrate. Full repression was observed only at concentrations of combined nitrogen higher than those usually found in rice-fields. Carbohydrates had a protective effect on nitrogenase against ammonium inhibition inC. marchica. All four strains showed increased nitrogenase activity when the light intensity was increased during assays. Variations of pH normally occurring in rice fields led to no important changes in nitrogenase activity inC. marchica. This fact, together with its potential for heterotrophic growth and tolerance to combined nitrogen, make this the most suitable of the four strains for inoculation experiments in rice fields.