MicroNiche: an R package for assessing microbial niche breadth and overlap from amplicon sequencing data.

Niche is a fundamental concept in ecology. It integrates the sum of biotic and abiotic environmental requirements that determines a taxon's distribution. Microbiologists currently lack quantitative approaches to address niche-related hypotheses. We tested four approaches for the quantification of niche breadth and overlap of taxa in amplicon sequencing datasets, with the goal of determining generalists, specialists and environmental-dependent distributions of community members. We applied these indices to in silico training datasets first, and then to real human gut and desert biological soil crust (biocrust) case studies, assessing the agreement of the indices with previous findings. Implementation of each approach successfully identified a priori conditions within in silico training data, and we found that by including a limit of quantification based on species rank, one could identify taxa falsely classified as specialists because of their low, sparse counts. Analysis of the human gut study offered quantitative support for Bacilli, Gammaproteobacteria and Fusobacteria specialists enriched after bariatric surgery. We could quantitatively characterise differential niche distributions of cyanobacterial taxa with respect to precipitation gradients in biocrusts. We conclude that these approaches, made publicly available as an R package (MicroNiche), represent useful tools to assess microbial environment-taxon and taxon-taxon relationships in a quantitative manner.

Prevalence of Ca²âº-ATPase-mediated carbonate dissolution among cyanobacterial euendoliths.

Recent physiological work has shown that the filamentous euendolithic cyanobacterium Mastigocoleus testarum (strain BC008) is able to bore into solid carbonates using Ca²âº-ATPases to take up Ca²âº from the medium at the excavation front, promoting dissolution of CaCO3 there. It is not known, however, if this is a widespread mechanism or, rather, a unique capability of this model strain. To test this, we undertook a survey of multispecies euendolithic microbial assemblages infesting natural carbonate substrates in marine coastal waters of the Caribbean, Mediterranean, South Pacific, and Sea of Cortez. Microscopic examination revealed the presence of complex assemblages of euendoliths, encompassing 3 out of the 5 major cyanobacterial orders. 16S rRNA gene clone libraries detected even greater diversity, particularly among the thin-filamentous forms, and allowed us to categorize the endoliths in our samples into 8 distinct phylogenetic clades. Using real-time Ca²âº imaging under a confocal laser scanning microscope, we could show that all communities displayed light-dependent formation of Ca²âº-supersaturated zones in and around boreholes, a staple of actively boring phototrophs. In 3 out of 4 samples, boring activity was sensitive to at least one of two inhibitors of Ca²âº-ATPase transporters (thapsigargin or tert-butylhydroquinone), indicating that the Ca²âº-ATPase mechanism is widespread among cyanobacterial euendoliths but perhaps not universal. Function-community structure correlations point to one particular clade of baeocyte-forming euendoliths as the potential exception.

The biogenicity of modern terrestrial roll-up structures and its significance for ancient life on land.

Roll-up structures (Roll-ups) are sedimentary structures formed by the desiccation-mediated curling of a surface, cohesive layer into a subcylindrical, coiled shape. Their origin in terrestrial environments has been attributed to the shrinking effect of argillaceous components, while microbes are thought to be the curling agent in intertidal marine settings. Roll-ups also exist in terrestrial environments and the rock record, but their genesis is unclear. Proving a biogenic origin of terrestrial roll-ups would make them excellent biosignatures to track ancient life on land. In this study, we tested the biogenicity of modern roll-ups from arid terrestrial environments, showing that, regardless of their geographic location and textural properties, they invariably contained large and distinct cyanobacterial populations compared to adjacent, non-rolled surface soil. Cyanobacterial populations inhabiting these roll-ups were genetically diverse, but consistently dominated by filamentous, non-heterocystous forms. We could also recreate roll-ups artificially by desiccating clay and organic polysaccharide slurries on sandy substrates, and show that clay roll-ups were less prone to re-form after wetting-and-drying cycles and less resistant to erosion than organically bound or naturally occurring ones. All this evidence suggests that fossil roll-ups found in ancient terrestrial deposits are biogenic features.

Effect of biological soil crusts on soil elemental concentrations: implications for biogeochemistry and as traceable biosignatures of ancient life on land.

Biological soil crusts (BSCs) are topsoil biosedimentary structures built by photosynthetic microbes commonly found today on arid soils. They play a role in soil stabilization and the fertility of arid lands, and are considered modern analogues of ancient terrestrial microbial communities. We determined the concentrations of four biogenic and 21 other elements, mostly metals, in surface soils that hosted BSCs, in the soils underneath those crusts, and in proximate but non-crusted surface soils. The samples were from six sites in the Colorado Plateau highlands and the Sonoran Desert lowlands. In spite of the variability in climate and geologic setting, we found statistically significant overall trends of enrichment in biogenic elements and depletion in non-biogenic elements when BSCs were compared with non-crusted soils. The differences between crusted and non-crusted soils were statistically significant at approximately 95% confidence for C, N (enrichments) and for Ca, Cr, Mn, Cu, Zn, As, and Zr (depletions). These trends are best explained by the activity of microbes. As expected, no differences in the concentrations of C, N, P, and S were detected between the soils underneath the crusts and the non-crusted soils, but the former showed depletion of non-biogenic elements, indicating that the leaching effect of crust microbes extends downward in the soil. These patterns speak to the need for a sustained input of allochthonous material, possibly dust, to maintain BSC fertility. These elemental patterns can be considered a biosignature that may be preserved in the rock record and might help identify ancient microbial communities on land.

Molecular genetics and genomic analysis of scytonemin biosynthesis in Nostoc punctiforme ATCC 29133.

The indole-alkaloid scytonemin is the most common and widespread sunscreen among cyanobacteria. Previous research has focused on its nature, distribution, ecology, physiology, and biochemistry, but its molecular genetics have not been explored. In this study, a scytonemin-deficient mutant of the cyanobacterium Nostoc punctiforme ATCC 29133 was obtained by random transposon insertion into open reading frame NpR1273. The absence of scytonemin under conditions of induction by UV irradiation was the single phenotypic difference detected in a comparative analysis of the wild type and the mutant. A cause-effect relationship between the phenotype and the mutation in NpR1273 was demonstrated by constructing a second scytoneminless mutant through directed mutagenesis of that gene. The genomic region flanking the mutation revealed an 18-gene cluster (NpR1276 to NpR1259). Four putative genes in the cluster, NpR1274 to NpR1271, with no previously known functions, are likely to be involved in the assembly of scytonemin. Also in this cluster, there is a redundant set of genes coding for shikimic acid and aromatic amino acid biosynthesis enzymes, leading to the production of tryptophan and tyrosine, which are likely to be biosynthetic precursors of the sunscreen.

Hydrotaxis of cyanobacteria in desert crusts.

We studied the migration of cyanobacteria in desert crusts from Las Bárdenas Reales (Spain). The crusts were almost exclusively colonized by the filamentous cyanobacterium Oscillatoria, which formed a dense layer approximately 600 microm thick located between 1.5 and 2.1 mm deep. Laboratory and field experiments showed that saturation of the crust with liquid water induced a migration of the cyanobacteria leading to a significant greening of the surface within a few minutes. Under light and rapid evaporation, the green color rapidly disappeared and the crust surface was completely devoid of filaments within 60 min. In contrast, 260 min was required to recover the original white color of the crust when slow evaporation was experimentally imposed. The up and down migration following wetting and drying occurred also in the dark. This demonstrates that light was not a required stimulus. Addition of ATP synthesis inhibitors prevented the cyanobacterium from migrating down into the crust, with filaments remaining on the surface. Therefore, the disappearance of the green color observed during desiccation can only be attributed to an active cyanobacterial motility response to the decrease in the water content. The simplest explanation that can account for the evidence gathered is the presence of a mechanism that links, directly or indirectly, these motility responses to gradients in water content, namely a form of hydrotaxis.

Small-scale vertical distribution of bacterial biomass and diversity in biological soil crusts from arid lands in the Colorado plateau.

We characterized, at millimeter resolution, bacterial biomass, diversity, and vertical stratification of biological soil crusts in arid lands from the Colorado Plateau. Microscopic counts, extractable DNA, and plate counts of viable aerobic copiotrophs (VAC) revealed that the top centimeter of crusted soils contained atypically large bacterial populations, tenfold larger than those in uncrusted, deeper soils. The plate counts were not always consistent with more direct estimates of microbial biomass. Bacterial populations peaked at the immediate subsurface (1-2 mm) in light-appearing, young crusts, and at the surface (0-1 mm) in well-developed, dark crusts, which corresponds to the location of cyanobacterial populations. Bacterial abundance decreased with depth below these horizons. Spatially resolved DGGE fingerprints of Bacterial 16S rRNA genes demonstrated the presence of highly diverse natural communities, but we could detect neither trends with depth in bacterial richness or diversity, nor a difference in diversity indices between crust types. Fingerprints, however, revealed the presence of marked stratification in the structure of the microbial communities, probably a result of vertical gradients in physicochemical parameters. Sequencing and phylogenetic analyses indicated that most of the naturally occurring bacteria are novel types, with low sequence similarity (83-93%) to those available in public databases. DGGE analyses of the VAC populations indicated communities of lower diversity, with most types having sequences more than 94% similar to those in public databases. Our study indicates that soil crusts represent small-scale mantles of fertility in arid ecosystems, harboring vertically structured, little-known bacterial populations that are not well represented by standard cultivation methods.

Cyanobacterial diversity in extreme environments in Baja California, Mexico: a polyphasic study

Cyanobacterial diversity from two geographical areas of Baja California Sur, Mexico, were studied: Bahia Concepcion, and Ensenada de Aripez. The sites included hypersaline ecosystems, sea bottom, hydrothermal springs, and a shrimp farm. In this report we describe four new morphotypes, two are marine epilithic from Bahia Concepcion, Dermocarpa sp. and Hyella sp. The third, Geitlerinema sp., occurs in thermal springs and in shrimp ponds, and the fourth, Tychonema sp., is from a shrimp pond. The partial sequences of the 16S rRNA genes and the phylogenetic relationship of four cyanobacterial strains (Synechococcus cf. elongatus, Leptolyngbya cf. thermalis, Leptolyngbya sp., and Geitlerinema sp.) are also presented. Polyphasic studies that include the combination of light microscopy, cultures and the comparative analysis of 16S rRNA gene sequences provide the most powerful approach currently available to establish the diversity of these oxygenic photosynthetic microorganisms in culture and in nature (AU)

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Phylogenetic and morphological diversity of cyanobacteria in soil desert crusts from the Colorado plateau.

We compared the community structures of cyanobacteria in four biological desert crusts from Utah's Colorado Plateau developing on different substrata. We analyzed natural samples, cultures, and cyanobacterial filaments or colonies retrieved by micromanipulation from field samples using microscopy, denaturing gradient gel electrophoresis, and sequencing of 16S rRNA genes. While microscopic analyses apparently underestimated the biodiversity of thin filamentous cyanobacteria, molecular analyses failed to retrieve signals for otherwise conspicuous heterocystous cyanobacteria with thick sheaths. The diversity found in desert crusts was underrepresented in currently available nucleotide sequence databases, and several novel phylogenetic clusters could be identified. Morphotypes fitting the description of Microcoleus vaginatus Gomont, dominant in most samples, corresponded to a tight phylogenetic cluster of probable cosmopolitan distribution, which was well differentiated from other cyanobacteria traditionally classified within the same genus. A new, diverse phylogenetic cluster, named "Xeronema," grouped a series of thin filamentous Phormidium-like cyanobacteria. These were also ubiquitous in our samples and probably correspond to various botanical Phormidium and Schizothrix spp., but they are phylogenetically distant from thin filamentous cyanobacteria from other environments. Significant differences in community structure were found among soil types, indicating that soil characteristics may select for specific cyanobacteria. Gypsum crusts were most deviant from the rest, while sandy, silt, and shale crusts were relatively more similar among themselves.

Long-term compositional changes after transplant in a microbial mat cyanobacterial community revealed using a polyphasic approach.

Using a polyphasic approach that included microscopy, cultivation and 16S rRNA-based cultivation-independent molecular fingerprinting, we compared the cyanobacterial composition of Solar Lake microbial mats and samples thereof transplanted and maintained in new settings for extended periods of time. Significant changes in community composition, with clear replacement of the dominant cyanobacterium, Microcoleus chthonoplastes, were detected in all cases. The most dramatic shifts occurred in a sample kept in the laboratory for 3 years, which resulted in dominance by an Oscillatoria-like cyanobacterium whose 16S rRNA closely matched that of a morphologically similar isolate from mats in Mexico. Transfer of Solar Lake mat to an artificial experimental pond with incubation under seminatural conditions resulted in an increase in cyanobacterial diversity. Judging from the molecular signatures, two novel, previously unrecognized and phylogenetically well-delimited cyanobacterial populations became dominant. Through cultivation, one population was shown to correspond to a filamentous, non-heterocystous group of Cyanobacteria with very narrow trichomes (approximately equals 0.75-1.5 microm). The most dominant novel molecular signature, however, could not be identified by cultivation efforts or correlation with microscopy and, upon phylogenetic analyses, its 16S rRNA genes showed no particular close association to known cyanobacterial groups.

Cyanobacterial diversity in extreme environments in Baja California, Mexico: a polyphasic study.

Cyanobacterial diversity from two geographical areas of Baja California Sur, Mexico, were studied: Bahia Concepcion, and Ensenada de Aripez. The sites included hypersaline ecosystems, sea bottom, hydrothermal springs, and a shrimp farm. In this report we describe four new morphotypes, two are marine epilithic from Bahia Concepcion, Dermocarpa sp. and Hyella sp. The third, Geitlerinema sp., occurs in thermal springs and in shrimp ponds, and the fourth, Tychonema sp., is from a shrimp pond. The partial sequences of the 16S rRNA genes and the phylogenetic relationship of four cyanobacterial strains (Synechococcus cf. elongatus, Leptolyngbya cf. thermalis, Leptolyngbya sp., and Geitlerinema sp.) are also presented. Polyphasic studies that include the combination of light microscopy, cultures and the comparative analysis of 16S rRNA gene sequences provide the most powerful approach currently available to establish the diversity of these oxygenic photosynthetic microorganisms in culture and in nature.

Monitoring of oxygenic and anoxygenic photosynthesis in a unicyanobacterial biofilm, grown in benthic gradient chamber.

In order to assess the role of cyanobacteria in the formation and dynamics of microenvironments in microbial mats, we studied an experimental biofilm of a benthic, halotolerant strain, belonging to the Halothece cluster of cyanobacteria. The 12-week-old biofilm developed in a sand core incubated in a benthic gradient chamber under opposing oxygen and sulfide vertical concentration gradients. At the biofilm surface, and as a response to high light irradiances, specific accumulation of myxoxanthophyll was detected in the cells, consistent with the typical vertical distribution of sun versus shade species in nature. The oxygen turn-over in terms of gross photosynthesis and net productivity rates was comparable to oxygen dynamics in natural microbial mats. Sulfide blocked O(2) production at low irradiances in deep biofilm layers but the dynamics of H(2)S and pH demonstrated that sulfide removal by anoxygenic photosynthesis was taking place. At higher irradiances, as soon as H(2)S was depleted, the cells switched to oxygenic photosynthesis as has been postulated for natural communities. The similarities between this experimental biofilm and natural benthic microbial mats demonstrate the central role of cyanobacteria in shaping microenvironmental gradients and processes in other complex microbial communities.

The halotolerance and phylogeny of cyanobacteria with tightly coiled trichomes (Spirulina Turpin) and the description of Halospirulina tapeticola gen. nov., sp. nov.

The morphologies, halotolerances, temperature requirements, pigment compositions and 16S rRNA gene sequences of five culture collection strains and six novel isolates of cyanobacteria with helical, tightly coiled trichomes were investigated. All strains were very similar morphologically and could be assigned to the genus Spirulina (or section Euspirulina sensu Geitler), according to traditional classification. However, the isolates showed significantly different requirements for salinity and temperature, which were in accordance with their respective environmental origins. The genetic divergence among the strains investigated was large. The results indicate the drastic underestimation of the physiological and phylogenetic diversity of these cyanobacteria by the current morphology-based classification and the clear need for new taxa. Three of the isolates originated from hypersaline waters and were similar with respect to their high halotolerance, broad euryhalinity and elevated temperature tolerance. By phylogenetic analyses, they were placed in a tight monophyletic cluster apart from all other cyanobacteria. Thus it is proposed to reclassify highly halotolerant cyanobacteria with tightly coiled trichomes in Halospirulina gen. nov., with the type species Halospirulina tapeticola sp. nov.

A novel prokaryotic UVB photoreceptor in the cyanobacterium Chlorogloeopsis PCC 6912.

We present evidence for the presence and nature of a UVB-specific photoreceptor in the cyanobacterium Chlorogloeopsis PCC 6912. The photoreceptor mediates at least the photosensory induction of mycosporine-like amino acid (MAA) synthesis. Because MAA synthesis in this organism can also be induced under salt stress, we could distinguish between the photosensory and the purely biochemical requirements of MAA synthesis. Neither visible light nor UV radiation was necessary for the biosynthetic process, thus indicating that the UVB (280-320 nm) dependence of biosynthesis is based on a UV photosensory capacity of the organism. An action spectrum of the MAA synthesis showed a distinct peak at 310 nm tailing down into the UVA (320-400 nm) region with no detected activity above 340 nm. We found that radiation below 300 nm caused significant inhibition of synthesis of MAAs indicating that the action spectrum at these wavelengths may not have been satisfactorily resolved. We propose that a pterin is a good candidate for a photoreceptor chromophore as (1) reduced pterins present absorption spectra congruent with the action spectrum obtained; and (2) an inhibitor of the biosynthetic pathway of pterins and an antagonist of excited states of pterins, both depressed the photosensory efficiency of induction but not its chemosensory efficiency.

Vibrational raman spectroscopic study of scytonemin, the UV-protective cyanobacterial pigment.

The Raman spectrum of the photoprotective pigment scytonemin found in cyanobacterial sheaths has been obtained for the first time. Its skeletal structure is extensively conjugated and unique in nature. Detailed molecular vibrational assignments are proposed and a distinctive group of four corroborative vibrational bands have been identified as unique indicators for the compound. These bands, especially a prominent feature at wavenumber 1590 cm(-1), are sufficiently conspicuous to be detectable in the mixed biomolecular pools of undisturbed natural microbial communities. This has been confirmed by demonstrating the Raman spectral bands for scytonemin in a sample of an intact intertidal cyanobacterial mat.

Matching molecular diversity and ecophysiology of benthic cyanobacteria and diatoms in communities along a salinity gradient.

The phylogenetic diversity of oxygenic phototrophic microorganisms in hypersaline microbial mats and their distribution along a salinity gradient were investigated and compared with the halotolerances of closely related cultivated strains. Segments of 16S rRNA genes from cyanobacteria and diatom plastids were retrieved from mat samples by DNA extraction and polymerase chain reaction (PCR), and subsequently analysed by denaturing gradient gel electrophoresis (DGGE). Sequence analyses of DNA from individual DGGE bands suggested that the majority of these organisms was related to cultivated strains at levels that had previously been demonstrated to correlate with characteristic salinity responses. Proportional abundances of amplified 16S rRNA gene segments from phylogenetic groupings of cyanobacteria and diatoms were estimated by image analysis of DGGE gels and were generally found to correspond to abundances of the respective morphotypes determined by microscopic analyses. The results indicated that diatoms accounted for low proportions of cells throughout, that the cyanobacterium Microcoleus chthonoplastes and close relatives dominated the communities up to a salinity of 11% and that, at a salinity of 14%, the most abundant cyanobacteria were related to highly halotolerant cultivated cyanobacteria, such as the recently established phylogenetic clusters of Euhalothece and Halospirulina. Although these organisms in cultures had previously demonstrated their ability to grow with close to optimal rates over a wide range of salinities, their occurrence in the field was restricted to the highest salinities investigated.

Ultraviolet and osmotic stresses induce and regulate the synthesis of mycosporines in the cyanobacterium chlorogloeopsis PCC 6912

The cyanobacterium Chlorogloeopsis PCC 6912 was found to synthesize and accumulate two putative UV sunscreen compounds of the mycosporine (mycosporine-like amino acid; MAA) type: mycosporine-glycine and shinorine. These MAAs were not constitutively present in the cells; their synthesis could be induced specifically either by exposure to UVB radiation (280-320 nm) or by osmotic stress, but not by other stress factors such as heat or cold shock, nutrient limitation, or photooxidative stress. A significant synergistic enhancement of MAA synthesis was observed when both stress factors were applied in combination. Although osmotic stress could induce MAA synthesis, comparison of the intracellular contents of MAAs with those of sugar osmolytes (glucose and trehalose) indicated that MAAs play no significant role in attaining osmotic homeostasis. UVB strongly enhanced the accumulation of shinorine, whereas osmotic stress had a more pronounced effect on mycosporine-glycine. This differential effect on the steady-state contents of each MAA could be explained either by differential regulation of biosynthesis or by differential loss rates of MAAs (leakage) under each condition. A preferential leakage of mycosporine-glycine from the cells after a hypoosmotic shock was detected. The results are interpreted in terms of an adaptive necessity for a combined regulatory control responding to both UV and external osmotic conditions in organisms that accumulate water-soluble sunscreens intracellularly.

Quantifying microbial diversity: morphotypes, 16S rRNA genes, and carotenoids of oxygenic phototrophs in microbial mats.

We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied.

Solar ultraviolet and the evolutionary history of cyanobacteria.

On the basis of photobiological, evolutionary, paleontological, paleoenvironmental and physiological arguments, a time course for the role of solar ultraviolet radiation (UVR, wavelengths below 400 nm) in the ecology and evolution of cyanobacteria is proposed in which three main periods can be distinguished. An initial stage, before the advent of oxygenic photosynthesis, when high environmental fluxes of UVC (wavelengths below 280 nm) and UVB (280-320 nm) may have depressed the ability of protocyanobacteria to develop large populations or restricted them to UVR refuges. A second stage lasting between 500 and 1500 Ma (million years), started with the appearance of true oxygen-evolving cyanobacteria and the concomitant formation of oxygenated (micro)environments under an oxygen free-atmosphere. In this second stage, the age of UV, the overall importance of UVR must have increased substantially, since the incident fluxes of UVC and UVB remained virtually unchanged, but additionally the UVA portion of the spectrum (320-400 nm) suddenly became biologically injurious and extremely reactive oxygen species must have formed wherever oxygen and UVR spatially coincided. The last period began with the gradual oxygenation of the atmosphere and the formation of the stratospheric ozone shield. The physiological stress due to UVC all but disappeared and the effects of UVB were reduced to a large extent. Evidence in support of this dynamics is drawn from the phylogenetic distribution of biochemical UV-defense mechanisms among cyanobacteria and other microorganisms. The specific physical characteristics of UVR and oxygen exposure in planktonic, sedimentary and terrestrial habitats are used to explore the plausible impact of UVR in each of the periods on the ecological distribution of cyanobacteria.