Lithic cyanobacterial communities in the polyextreme Sahara Desert: implications for the search for the limits of life.

The hyperarid Sahara Desert presents extreme and persistent dry conditions with a limited number of hours during which the moisture availability, temperature and light allow phototrophic growth. Some cyanobacteria can live in these hostile conditions by seeking refuge under (hypolithic) or inside (endolithic) rocks, by colonizing porous spaces (cryptoendoliths) or fissures in stones (chasmoendoliths). Chroococcidiopsis spp. have been reported as the dominant or even the only phototrophs in these hot desert lithic communities. However, the results of this study reveal the high diversity of and variability in cyanobacteria among the sampled habitats in the Sahara Desert. The chasmoendolithic samples presented high coccoid cyanobacteria abundances, although the dominant cyanobacteria were distinct among different locations. A high predominance of a newly described cyanobacterium, Pseudoacaryochloris sahariense, was found in hard, compact, and more opaque stones with cryptoendolithic colonization. On the other hand, the hypolithic samples were dominated by filamentous, non-heterocystous cyanobacteria. Thermophysiological bioassays confirmed desiccation and extreme temperature tolerance as drivers in the cyanobacterial community composition of these lithic niches. The results of the present study provide key factors for understanding life strategies under polyextreme environmental conditions. The isolated strains, especially the newly described cyanobacterium P. sahariense, might represent suitable microorganisms in astrobiology studies aimed at investigating the limits of life.

Microenvironmental Conditions Drive the Differential Cyanobacterial Community Composition of Biocrusts from the Sahara Desert.

The Sahara Desert is characterized by extreme environmental conditions, which are a unique challenge for life. Cyanobacteria are key players in the colonization of bare soils and form assemblages with other microorganisms in the top millimetres, establishing biological soil crusts (biocrusts) that cover most soil surfaces in deserts, which have important roles in the functioning of drylands. However, knowledge of biocrusts from these extreme environments is limited. Therefore, to study cyanobacterial community composition in biocrusts from the Sahara Desert, we utilized a combination of methodologies in which taxonomic assignation, for next-generation sequencing of soil samples, was based on phylogenetic analysis (16S rRNA gene) in parallel with morphological identification of cyanobacteria in natural samples and isolates from certain locations. Two close locations that differed in microenvironmental conditions were analysed. One was a dry salt lake (a "chott"), and the other was an extension of sandy, slightly saline soil. Differences in cyanobacterial composition between the sites were found, with a clear dominance of Microcoleus spp. in the less saline site, while the chott presented a high abundance of heterocystous cyanobacteria as well as the filamentous non-heterocystous Pseudophormidium sp. and the unicellular cf. Acaryochloris. The cyanobacteria found in our study area, such as Microcoleus steenstrupii, Microcoleus vaginatus, Scytonema hyalinum, Tolypothrix distorta, and Calothrix sp., are also widely distributed in other geographic locations around the world, where the conditions are less severe. Our results, therefore, indicated that some cyanobacteria can cope with polyextreme conditions, as confirmed by bioassays, and can be considered extremotolerant, being able to live in a wide range of conditions.

Analysis of molecular diversity within single cyanobacterial colonies from environmental samples.

Attached or floating macroscopic cyanobacteria can be found in shallow waters and can be easily hand-collected, but their identification is often challenging due to their high morphological variability. In addition, many members of environmental samples lose their morphological adaptations under controlled conditions, making the integration of analyses of field populations and derived isolated cultures necessary in order to evaluate phenotypic plasticity for identification purposes. Therefore, in this study, twenty-nine macroscopic field samples were analyzed by Illumina sequencing and parallel optical microscopy. Some colonies showed the typical morphological characteristics of Rivularia biasolettiana, and others showed those of Rivularia haematites. However, other Rivularia-like colonies showed ambiguous morphologies, and some of them showed the phenotypic features of the new genus Cyanomargarita, which is virtually indistinguishable from Rivularia in the field. In all of the colonies, phylotype composition was highly heterogeneous, with abundances varying depending on the analyzed sample. Some colonies were dominated (97-99%) by a single phylotype, while in others, the percentage of the dominant phylotype decreased to approximately 50-60%. Surprisingly, the same dominant phylotype was found in R. biasolettiana and R. haematites colonies. The relationships between environmental and/or biological factors and morphological variability in these colonies are discussed.

Differences in the Cyanobacterial Community Composition of Biocrusts From the Drylands of Central Mexico. Are There Endemic Species?

In drylands worldwide, biocrusts, topsoil microbial communities, are prevalent, contributing to the biostabilization of soils and allowing the subsequent establishment and growth of vascular plants. In early successional biocrusts, cyanobacteria are the first dominant colonizers of bare ground, largely determining their functioning. However, there are large gaps in our knowledge of the cyanobacterial diversity in biocrusts, particularly in understudied geographic regions, such as the tropical latitudes. We analyzed the diversity of the cyanobacteria inhabiting the biocrusts of semideserts from Central Mexico in two localities belonging to the same desert system (Chihuahuan Desert) that are separated by a cordillera that crosses the center of Mexico. Morphological identification of the cyanobacteria was carried out after cultivation in parallel with the direct observation of the environmental samples and was supported by genetic characterization through analysis of the 16S rRNA gene of the isolated strains and by next-generation sequencing of the soil samples. Taxonomic assignment revealed a clear dominance of heterocystous cyanobacteria at one of the studied locations (Actopan, Hidalgo state). Although heterocystous forms were abundant at the other location (Atexcac, Puebla state), almost a third of the cyanobacterial phylotypes were represented by unicellular/colonial cyanobacteria, mostly Chroococcidiopsis spp. Only 28.4% of the phylotypes were found to be common to both soils. Most of the other taxa, however, were biocrust-type specific, and approximately 35% of the phylotypes were found to be unique to the soil they were collected in. In addition, differences in the abundances of the shared cyanobacteria between the locations were also found. These differences in the cyanobacterial distribution were supported by the distinct responses of the isolated strains representative of the sites to extreme heat and desiccation in bioassays. Some cyanobacteria with high abundance or only present at the hottest Actopan site, such as Scytonema hyalinum, Scytonema crispum, Nostoc commune, Nostoc sp., and Calothrix parietina, survived extreme heat and desiccation. However, Tolypothrix distorta and Chroococcidiopsis spp. were clearly sensitive to these extreme conditions in relation to their lower abundances at Actopan as opposed to Atexcac. Since novel biocrust-associated phylotypes were also found, the emergence of endemic cyanobacterial taxa is discussed.

Polyphasic evaluation of key cyanobacteria in biocrusts from the most arid region in Europe.

Cyanobacteria are key microbes in topsoil communities that have important roles in preventing soil erosion, carbon and nitrogen fixation, and influencing soil hydrology. However, little is known regarding the identity and distribution of the microbial components in the photosynthetic assemblages that form a cohesive biological soil crust (biocrust) in drylands of Europe. In this study, we investigated the cyanobacterial species colonizing biocrusts in three representative dryland ecosystems from the most arid region in Europe (SE Spain) that are characterized by different soil conditions. Isolated cyanobacterial cultures were identified by a polyphasic approach, including 16S rRNA gene sequencing, phylogenetic relationship determination, and morphological and ecological habitat assessments. Three well-differentiated groups were identified: heterocystous-cyanobacteria (Nostoc commune, Nostoc calcicola, Tolypothrix distorta and Scytonema hyalinum), which play an important role in N and C cycling in soil; nonheterocystous bundle-forming cyanobacteria (Microcoleus steenstrupii, Trichocoleus desertorum, and Schizothrix cf. calcicola); and narrow filamentous cyanobacteria (Leptolyngbya frigida and Oculatella kazantipica), all of which are essential genera for initial biocrust formation. The results of this study contribute to our understanding of cyanobacterial species composition in biocrusts from important and understudied European habitats, such as the Mediterranean Basin, a hotspot of biodiversity, where these species are keystone pioneer organisms.

Cyanobacterial biocrust diversity in Mediterranean ecosystems along a latitudinal and climatic gradient.

Cyanobacteria are a key biotic component as primary producers in biocrusts, topsoil communities that have important roles in the functioning of drylands. Yet, major knowledge gaps exist regarding the composition of biocrust cyanobacterial diversity and distribution in Mediterranean ecosystems. We describe cyanobacterial diversity in Mediterranean semiarid soil crusts along an aridity gradient by using next-generation sequencing and bioinformatics analyses, and detect clear shifts along it in cyanobacterial dominance. Statistical analyses show that temperature and precipitation were major parameters determining cyanobacterial composition, suggesting the presence of differentiated climatic niches for distinct cyanobacteria. The responses to temperature of a set of cultivated, pedigreed strains representative of the field populations lend direct support to that contention, with psychrotolerant vs thermotolerant physiology being strain dependent, and consistent with their dominance along the natural gradient. Our results suggest a possible replacement, as global warming proceeds, of cool-adapted by warm-adapted nitrogen-fixing cyanobacteria (such as Scytonema) and a switch in the dominance of Microcoleus vaginatus by thermotolerant, novel phylotypes of bundle-forming cyanobacteria. These differential sensitivities of cyanobacteria to rising temperatures and decreasing precipitation, their ubiquity, and their low generation time point to their potential as bioindicators of global change.

Diversity of biocrust-forming cyanobacteria in a semiarid gypsiferous site from Central Spain.

Cyanobacteria are a key constituent of biocrusts, communities dominated by lichens, mosses and associated microorganisms, which are prevalent in drylands worldwide and that largely determine their functioning. Despite their importance, there are large gaps in our knowledge of the composition and diversity of cyanobacteria associated with biocrusts, particularly in areas such as the Mediterranean Basin. We studied the diversity of these cyanobacteria in a gypsiferous grassland from Central Spain using both morphological identification after cultivation and genetic analyses with the 16S rRNA gene. Nine different morphotypes were observed, eight corresponding to filamentous, and one to unicellular cyanobacteria. We found cyanobacterial genera typical of biocrust communities, such as Microcoleus and Trichocoleus, and N-fixing cyanobacteria such as Scytonema and Nostoc. Genetic information allowed us to identify cultures belonging to recently described genera such as Roholtiella, Nodosilinea and Mojavia. We also describe two new phylotypes of Microcoleus and Scytonema, which are key genera contributing to ecosystem functioning in biocrust-dominated ecosystems worldwide.

Nitrogen fixation in a non-heterocystous cyanobacterial mat from a mountain river.

In situ nitrogen fixation was investigated in a cyanobacterial mat growing on the bed of rocks of the Muga River, Spain. The filamentous non-heterocystous cyanobacterium Schizothrix dominated the mat, showing nitrogenase activity in the light at similar rates to those found in nearby heterocystous Rivularia colonies. N2 fixation in the light was significantly increased by an inhibitor of PSII and oxygen evolution, DCMU (3-[3,4-dichlorophenyl]-1,1-dimethylurea), and anaerobic conditions. However, no nitrogenase activity was found in the dark. Addition of fructose as a respiratory substrate induced nitrogenase activity in samples incubated under aerobic conditions in the dark but not in anaerobic conditions. Microelectrode oxygen profiles showed internal microaerobic microzones where nitrogen fixation might concentrate. Analyses of the 16S rRNA gene revealed only the presence of sequences belonging to filamentous non-heterocystous cyanobacteria. nifH gene diversity showed that the major phylotypes also belonged to this group. One of the three strains isolated from the Schizothrix mat was capable of fixing N2 and growing in the absence of combined N. This was consistent with the nifH gene analysis. These results suggest a relevant contribution of non-heterocystous cyanobacteria to nitrogen fixation in these mats.

Characterization and responses to environmental cues of a photosynthetic antenna-deficient mutant of the filamentous cyanobacterium Anabaena sp. PCC 7120.

The cyanobacterial phycobilisome (PBS) is a giant pigment-protein complex which harvests light energy for photosynthesis and comprises two structures: a core and peripheral rods. Most studies on PBS structure and function are based on mutants of unicellular strains. In this report, we describe the phenotypic and genetic characterization of a transposon mutant of the filamentous Anabaena sp. strain PCC 7120, denoted LC1, which cannot synthesize the phycobiliprotein phycocyanin (PC), the main component of the rods; in this mutant, the transposon had inserted into the cpcB gene (orf alr0528) which putatively encodes PC-ß chain. Mutant LC1 was able to synthesize phycoerythrocyanin (PEC), a phycobiliprotein (PBP) located at the terminal region of the rods; but in the absence of PC, PEC did not attach to the PBSs that only retained the allophycocyanin (APC) core; ferredoxin: NADP+-oxidoreductase (FNR) that is associated with the PBS in the wild type, was not found in isolated PBSs from LC1. The performance of the mutant exposed to different environmental conditions was evaluated. The mutant phenotype was successfully complemented by cloning and transfer of the wild type complete cpc operon to mutant LC1. Interestingly, LC1 compensated its mutation by significantly increasing the number of its core-PBS and the effective quantum yield of photosystem II (PSII) photochemistry; this feature suggests a more efficient energy conversion in the mutant which may be useful for biotechnological applications.

Monitoring bioavailable phosphorus in lotic systems: a polyphasic approach based on cyanobacteria.

Conventional assays to measure phosphorus in freshwater systems are sometimes not sufficient to quantify the actual bioavailable P for aquatic biota since some inorganic or organic P species may not be detected by chemical methods, and their bioavailability can be affected by a range of environmental factors. This situation could lead regulatory agencies to be unable to detect imminent ecosystem-degrading phenomena such as cyanobacterial blooms. It could also be an obstacle in studying the ecophysiological requirements of freshwater communities. P bioavailability in five rivers located in central Spain was analysed by a polyphasic approach (combinations of different marker types) based on cyanobacteria. This approach included a parallel study with the use of a self-luminescent P-cyanobacterial bioreporter based on a phosphatase alkaline promoter, determination of in situ alkaline phosphatase activities from cyanobacteria found at sampling sites, and the characterisation of cyanobacterial morphological features related to P bioavailability (hairs, polyphosphate granules and calyptras). An inverse relationship was found between values of bioavailable P, measured by the bioreporter and phosphatase activities. Cyanobacteria from sampling sites with low bioavailable P showed high phosphatase activity and vice versa, although some differences in values of this activity were observed in different cyanobacteria found at the same place, in relation to different growth strategies. Morphological characteristics associated with P limitation or P enrichment also varied between sampling locations. Cyanobacteria collected from sampling sites with reduced P bioavailability, measured by bioreporter and phosphatase activity, had a lower abundance of polyphosphate granules; those cyanobacteria capable of developing hairs or calyptras showed a greater abundance of these structures. Conversely, polyphosphate granules in cyanobacteria increased as P bioavailability increased as measured by the bioreporter and phosphatase activity. The study shows that the results of genetic, physiological and microscopic analyses based on these methods complement each other, implying that combining their findings would provide a more complete analysis of the nutrient status of running waters.

A battery of bioreporters of nitrogen bioavailability in aquatic ecosystems based on cyanobacteria.

Cyanobacteria are the main primary producers and are responsible for the blooms and eutrophication processes caused by excess nutrients in surface waters. The aim of this paper is to use cyanobacteria to monitor the presence and bioavailability of different chemical species of nitrogen in freshwater. Cyanobacteria have mechanisms which can detect the presence of nutrients in their environment and can activate or repress specific genes, or operons, depending on nutrient bioavailability. Therefore, monitoring the expression of these genes can facilitate measurement of the availability of nutrients. To achieve this we have constructed self-bioluminescent reporter strains of the filamentous nitrogen-fixing cyanobacterium Nostoc sp. PCC 7120 expressing promoters of genes responsive to nitrogen fused to the luxCDABE operon. Three promoters were selected to direct the expression of luxCDABE: The first, the glnA promoter, is activated in the absence of combined nitrogen. We found that it responded linearly to the addition of known amounts of combined N in the range 50-500 µM NH4(+) or NO3(-). The second, the nirA operon promoter, turns on in the presence of nitrate being inhibited by ammonium. The bioreporter responded linearly in the range of 10-100 µM NO3. The third, the gifA promoter, is activated in the presence of ammonium, responding linearly in the range 100-600 µM NH4(+). We also used a previously described strain of Synechococcus elongatus PCC 7942 expressing glnN (glutamine synthetase type III) fused to luxAB. We found that the glnN promoter responded linearly to the addition of known amounts of N in the range 50-500 µM NH4(+) or NO3(-). These cyanobacterial bioreporters were tested in real environmental samples (i.e. river waters) which confirmed their validity and showed a broad spectrum response. They are therefore useful in the detection of both total N-bioavailability and specific nitrogen species.

Novel cyanobacterial bioreporters of phosphorus bioavailability based on alkaline phosphatase and phosphate transporter genes of Anabaena sp. PCC 7120.

There is heterogeneity in the way cyanobacteria respond to P starvation and subsequently how they adapt to environments with low or fluctuating P concentrations. In this study, we have fused the promoterless lux operon luxCDABE to the promoter regions of Anabaena sp. PCC 7120 phoA genes putatively encoding alkaline phosphatases, phoA (all2843) and phoA-like (alr5291) and to the promoter region of one operon putatively encoding a high affinity phosphate transporter pst1 (all4575-4572). The self-bioluminescent strains constructed in this way, Anabaena AP (phoA promoter), Anabaena AP-L (phoA-like promoter), and Anabaena PST (pst1 promoter) have been used to study the expression of these genes in response to P starvation and P re-feeding with inorganic and organic phosphate sources. Our data showed that the pst1 promoter was activated at much higher level than the phoA-like promoter following P starvation; however, we did not observe activation of the phoA promoter. The P re-feeding experiments revealed that both strains, Anabaena (A.) PST and A. AP-L could be used as novel bioreporters of P availability in environmental samples. Both strains were used to estimate bioavailable P in environmental samples (fresh- and wastewaters) with a wide range of soluble P concentrations. The results indicated that most of the P in the water samples was in chemical forms available to the cyanobacterium; however there were some differences in the estimates given by both strains as A. PST appeared to be more adequate for the samples with the lowest P load while A. AP-L gave similar or even higher values of P concentrations than those chemically measured in samples with higher P load.

Activation of geminivirus V-sense promoters in roots is restricted to nematode feeding sites.

Obligate sedentary endoparasitic nematodes, such as the root-knot and cyst nematodes, elicit the differentiation of specialized nematode nurse or feeding cells [nematode feeding sites (NFS), giant cells and syncytia, respectively]. During NFS differentiation, marked changes in cell cycle progression occur, partly similar to those induced by some geminiviruses. In this work, we describe the activation of V-sense promoters from the Maize streak virus (MSV) and Wheat dwarf virus (WDV) in NFS formed by root-knot and cyst nematodes. Both promoters were transiently active in microinjection experiments. In tobacco and Arabidopsis transgenic lines carrying promoter-beta-glucuronidase fusions, the MSV V-sense promoter was activated in the vascular tissues of aerial plant parts, primarily leaf and cotyledon phloem tissue and some floral structures. Interestingly, in roots, promoter activation was restricted to syncytia and giant cells tested with four different nematode populations, but undetectable in the rest of the root system. As the activity of the promoter in transgenic rootstocks should be restricted to NFS only, the MSV promoter may have utility in engineering grafted crops for nematode control. Therefore, this study represents a step in the provision of some of the much needed additional data on promoters with restricted activation in NFS useful in biotechnological nematode control strategies.