The Microbiology of Biological Soil Crusts.

Biological soil crusts are thin, inconspicuous communities along the soil atmosphere ecotone that, until recently, were unrecognized by ecologists and even more so by microbiologists. In its broadest meaning, the term biological soil crust (or biocrust) encompasses a variety of communities that develop on soil surfaces and are powered by photosynthetic primary producers other than higher plants: cyanobacteria, microalgae, and cryptogams like lichens and mosses. Arid land biocrusts are the most studied, but biocrusts also exist in other settings where plant development is constrained. The minimal requirement is that light impinge directly on the soil; this is impeded by the accumulation of plant litter where plants abound. Since scientists started paying attention, much has been learned about their microbial communities, their composition, ecological extent, and biogeochemical roles, about how they alter the physical behavior of soils, and even how they inform an understanding of early life on land. This has opened new avenues for ecological restoration and agriculture.

Beneficial Cyanosphere Heterotrophs Accelerate Establishment of Cyanobacterial Biocrust.

Biological soil crusts (biocrusts) are communities of microbes that inhabit the surface of arid soils and provide essential services to dryland ecosystems. While resistant to extreme environmental conditions, biocrusts are susceptible to anthropogenic disturbances that can deprive ecosystems of these valuable services for decades. Until recently, culture-based efforts to produce inoculum for cyanobacterial biocrust restoration in the southwestern United States focused on producing and inoculating the most abundant primary producers and biocrust pioneers, Microcoleus vaginatus and members of the family Coleofasciculaceae (also called Microcoleus steenstrupii complex). The discovery that a unique microbial community characterized by diazotrophs, known as the cyanosphere, is intimately associated with M. vaginatus suggests a symbiotic division of labor in which nutrients are traded between phototrophs and heterotrophs. To probe the potential use of such cyanosphere members in the restoration of biocrusts, we performed coinoculations of soil substrates with cyanosphere constituents. This resulted in cyanobacterial growth that was more rapid than that seen for inoculations with the cyanobacterium alone. Additionally, we found that the mere addition of beneficial heterotrophs enhanced the formation of a cohesive biocrust without the need for additional phototrophic biomass within native soils that contain trace amounts of biocrust cyanobacteria. Our findings support the hitherto-unknown role of beneficial heterotrophic bacteria in the establishment and growth of biocrusts and allow us to make recommendations concerning biocrust restoration efforts based on the presence of remnant biocrust communities in disturbed areas. Future biocrust restoration efforts should consider cyanobacteria and their beneficial heterotrophic community as inoculants. IMPORTANCE The advancement of biocrust restoration methods for cyanobacterial biocrusts has been largely achieved through trial and error. Successes and failures could not always be traced back to particular factors. The investigation and application of foundational microbial interactions existing within biocrust communities constitute a crucial step toward informed and repeatable biocrust restoration methods.

What Could Explain δ13C Signatures in Biocrust Cyanobacteria of Drylands?

Dryland ecosystems are increasing in geographic extent and contribute greatly to interannual variability in global carbon dynamics. Disentangling interactions among dominant primary producers, including plants and autotrophic microbes, can help partition their contributions to dryland C dynamics. We measured the δ13C signatures of biological soil crust cyanobacteria and dominant plant species (C3 and C4) across a regional scale in the southwestern USA to determine if biocrust cyanobacteria were coupled to plant productivity (using plant-derived C mixotrophically), or independent of plant activity (and therefore purely autotrophic). Cyanobacterial assemblages located next to all C3 plants and one C4 species had consistently more negative δ13C (by 2‰) than the cyanobacteria collected from plant interspaces or adjacent to two C4 Bouteloua grass species. The differences among cyanobacterial assemblages in δ13C could not be explained by cyanobacterial community composition, photosynthetic capacity, or any measured leaf or root characteristics (all slopes not different from zero). Thus, microsite differences in abiotic conditions near plants, rather than biotic interactions, remain a likely mechanism underlying the observed δ13C patterns to be tested experimentally.

Coleofasciculaceae, a Monophyletic Home for the Microcoleus steenstrupii Complex and Other Desiccation-tolerant Filamentous Cyanobacteria.

Cyanobacteria classified as Microcoleus steenstrupii play a significant role as pioneers of biological soil crusts (biocrusts), but this taxon is recognized to constitute a diverse complex of strains and field populations. With the aim of clarifying its systematics, we conducted a polyphasic characterization of this and allied taxa. A 16S ribosomal gene meta-analysis of published environmental sequences showed that the complex encompasses a variety of well supported genus-level clades with clade-specific environmental preferences, indicating significant niche differentiation. Fifteen strains in the M. steenstrupii complex were selected as representative of naturally occurring clades and studied using 16S rRNA gene phylogeny, morphology, and niche delineation with respect to temperature and rainfall. Bayesian phylogenetic reconstructions within a comprehensive, curated database of long 16S rRNA cyanobacterial sequences (1,000 base pairs or more) showed that they all belonged in a monophyletic, family-level clade (91.4% similarity) that included some other known genera of desiccation-resistant, largely terrestrial, filamentous, nonheterocystous cyanobacteria, including Coleofasciculus, the type genus for the family Coleofasciculaceae. To accommodate this biodiversity, we redescribe the Coleofasciculaceae, now composed of 11 genera, among which six are newly described herein (Funiculus, Parifilum, Arizonema, Crassifilum, Crustifilum, and Allocoleopsis), and five were previously recognized (Porphyrosiphon, Coleofasciculus, Pycnacronema, Potamolinea, and Wilmottia). We provide an evaluation of their respective niches and global distributions within biocrusts based on published molecular data. This new systematics treatment should help simplify and improve our understanding of the biology of terrestrial cyanobacteria.

A Fog-Irrigated Soil Substrate System Unifies and Optimizes Cyanobacterial Biocrust Inoculum Production.

Biological soil crusts (biocrusts) are globally important microbial communities inhabiting the top layer of soils. They provide multiple services to dryland ecosystems but are particularly vulnerable to anthropogenic disturbance from which they naturally recover only slowly. Assisted inoculation with cyanobacteria is held as a promising approach to promote biocrust regeneration. Two different methodologies have been developed for this purpose: mass cultivation of biocrust pioneer species (such as the cyanobacteria Microcoleus spp.) on cellulose supports, and polymicrobial cultivation of biocrusts in soils within greenhouse settings. Here, we aimed to test a novel method to grow cyanobacterial biocrust inoculum based on fog irrigation of soil substrates (FISS) that can be used with either culture-based or mixed-community approaches. We found that the FISS system presents clear advantages over previous inoculum production methodologies; overall, FISS eliminates the need for specialized facilities and decreases user effort. Specifically, there were increased microbial yields and simplification of design compared to those of the culture-based and mixed-community approaches, respectively. Its testing also allows us to make recommendations on underexplored aspects of biocrust restoration: (i) field inoculation levels should be equal to or greater than the biomass found in the substrate and (ii) practices regarding evaluation of cyanobacterial biomass should, under certain circumstances, include proxies additional to chlorophyll aIMPORTANCE Biocrust inoculum production for use in dryland rehabilitation is a powerful tool in combating the degradation of dryland ecosystems. However, the facilities and effort required to produce high-quality inoculum are often a barrier to effective large-scale implementation by land managers. By unifying and optimizing the two foremost methods for cyanobacterial biocrust inoculum production, our work improves on the ease and cost with which biocrust restoration technology can be translated to practical widespread implementation.

What's in a name? The case of cyanobacteria.

A redefinition of the cyanobacterial lineage has been proposed based on phylogenomic analysis of distantly related nonphototrophic lineages. We define Cyanobacteria here as "Organisms in the domain bacteria able to carry out oxygenic photosynthesis with water as an electron donor and to reduce carbon dioxide as a source of carbon, or those secondarily evolved from such organisms."

Optimizing the Production of Nursery-Based Biological Soil Crusts for Restoration of Arid Land Soils.

Biological soil crusts (biocrusts) are topsoil communities formed by cyanobacteria or other microbial primary producers and are typical of arid and semiarid environments. Biocrusts promote a range of ecosystem services, such as erosion resistance and soil fertility, but their degradation by often anthropogenic disturbance brings about the loss of these services. This has prompted interest in developing restoration techniques. One approach is to source biocrust remnants from the area of interest for scale-up cultivation in a microbial "nursery" that produces large quantities of high-quality inoculum for field deployment. However, growth dynamics and the ability to reuse the produced inoculum for continued production have not been assessed. To optimize production, we followed nursery growth dynamics of biocrusts from cold (Great Basin) and hot (Chihuahuan) deserts. Peak phototrophic biomass was attained between 3 and 7 weeks in cold desert biocrusts and at 12 weeks in those from hot deserts. We also reused the resultant biocrust inoculum to seed successive incubations, tracking both phototroph biomass and cyanobacterial community structure using 16S rRNA gene amplicon sequencing. Hot desert biocrusts showed little to no viability upon reinoculation, while cold desert biocrusts continued to grow, but at the expense of progressive shifts in species composition. This leads us to discourage the reuse of nursery-grown inoculum. Surprisingly, growth was highly variable among replicates, and overall yields were low, a fact that we attribute to the demonstrable presence of virulent and stochastically distributed but hitherto unknown cyanobacterial pathogens. We provide recommendations to avoid pathogen incidence in the process.IMPORTANCE Biocrust communities provide important ecosystem services for arid land soils, such as soil surface stabilization promoting erosion resistance and contributing to overall soil fertility. Anthropogenic degradation to biocrust communities (through livestock grazing, agriculture, urban sprawl, and trampling) is common and significant, resulting in a loss of those ecosystem services. Losses impact both the health of the native ecosystem and the public health of local populations due to enhanced dust emissions. Because of this, approaches for biocrust restoration are being developed worldwide. Here, we present optimization of a nursery-based approach to scaling up the production of biocrust inoculum for field restoration with respect to temporal dynamics and reuse of biological materials. Unexpectedly, we also report on complex population dynamics, significant spatial variability, and lower than expected yields that we ascribe to the demonstrable presence of cyanobacterial pathogens, the spread of which may be enhanced by some of the nursery production standard practices.

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.

Extreme cellular adaptations and cell differentiation required by a cyanobacterium for carbonate excavation.

Some cyanobacteria, known as euendoliths, excavate and grow into calcium carbonates, with their activity leading to significant marine and terrestrial carbonate erosion and to deleterious effects on coral reef and bivalve ecology. Despite their environmental relevance, the mechanisms by which they can bore have remained elusive and paradoxical, in that, as oxygenic phototrophs, cyanobacteria tend to alkalinize their surroundings, which will encourage carbonate precipitation, not dissolution. Therefore, cyanobacteria must rely on unique adaptations to bore. Studies with the filamentous euendolith, Mastigocoleus testarum, indicated that excavation requires both cellular energy and transcellular calcium transport, mediated by P-type ATPases, but the cellular basis for this phenomenon remains obscure. We present evidence that excavation in M. testarum involves two unique cellular adaptations. Long-range calcium transport is based on active pumping at multiple cells along boring filaments, orchestrated by the preferential localization of calcium ATPases at one cell pole, in a ring pattern, facing the cross-walls, and by repeating this placement and polarity, a pattern that breaks at branching and apical cells. In addition, M. testarum differentiates specialized cells we call calcicytes, that which accumulate calcium at concentrations more than 500-fold those found in other cyanobacteria, concomitantly and drastically lowering photosynthetic pigments and enduring severe cytoplasmatic alkalinization. Calcicytes occur commonly, but not exclusively, in apical parts of the filaments distal to the excavation front. We suggest that calcicytes allow for fast calcium flow at low, nontoxic concentrations through undifferentiated cells by providing buffering storage for excess calcium before final excretion to the outside medium.

Exposure to predicted precipitation patterns decreases population size and alters community structure of cyanobacteria in biological soil crusts from the Chihuahuan Desert.

Cyanobacteria typically colonize the surface of arid soils, building biological soil crust (biocrusts) that provide a variety of ecosystem benefits, ranging from fertilization to stabilization against erosion. We investigated how future scenarios in precipitation anticipated for the Northern Chihuahuan Desert affected abundance and composition of biocrust cyanobacteria in two grassland ecosystems. Scenarios included a decrease in precipitation and a delay of monsoon rainfall. After three years, both treatments negatively affected cyanobacteria, although the effects of monsoon delay were milder than those of decreased precipitation. Mature biocrusts in black grama grassland suffered severe losses in cyanobacterial biomass and diversity, but compositionally simpler biocrusts in blue grama-dominated grassland maintained biomass, only suffering diversity losses. This could be partially explained by the differential sensitivity of cyanobacterial taxa: nitrogen-fixing Scytonema spp. were the most sensitive, followed by phylotypes in the Microcoleus steenstrupii complex. Microcoleus vaginatus was the least affected in all cases, but is known to be very sensitive to warming. We predict that altered precipitation will tend to prevent biocrusts from reaching successional maturity, selecting for M. vaginatus over competing M. steenstrupii, among pioneer biocrust-formers. A shift towards heat-sensitive M. vaginatus could ultimately destabilize biocrusts when precipitation changes are combined with global warming.

A New Niche for Anoxygenic Phototrophs as Endoliths.

Anoxygenic phototrophic bacteria (APBs) occur in a wide range of aquatic habitats, from hot springs to freshwater lakes and intertidal microbial mats. Here, we report the discovery of a novel niche for APBs: endoliths within marine littoral carbonates. In a study of 40 locations around Isla de Mona, Puerto Rico, and Menorca, Spain, 16S rRNA high-throughput sequencing of endolithic community DNA revealed the presence of abundant phylotypes potentially belonging to well-known APB clades. An ad hoc phylogenetic classification of these sequences enabled us to refine the assignments more stringently. Even then, all locations contained such putative APBs, often reaching a significant proportion of all phototrophic sequences. In fact, in some 20% of samples, their contribution exceeded that of oxygenic phototrophs, previously regarded as the major type of endolithic microbe in carbonates. The communities contained representatives of APBs in the Chloroflexales, various proteobacterial groups, and Chlorobi The most abundant phylotypes varied with geography: on Isla de Mona, Roseiflexus and Chlorothrix-related phylotypes dominated, whereas those related to Erythrobacter were the most common in Menorca. The presence of active populations of APBs was corroborated through an analysis of photopigments: bacteriochlorophylls were detected in all samples, bacteriochlorophyll c and a being most abundant. We discuss the potential metabolism and geomicrobial roles of endolithic APBs. Phylogenetic inference suggests that APBs may be playing a role as photoheterotrophs, adding biogeochemical complexity to our understanding of such communities. Given the global extent of coastal carbonate platforms, they likely represent a very large and unexplored habitat for APBs.IMPORTANCE Endolithic microbial communities from carbonates, which have been explored for over 2 centuries in predominantly naturalistic studies, were thought to be primarily composed of eukaryotic algae and cyanobacteria. Our report represents a paradigm shift in this regard, at least for the marine environment, demonstrating the presence of ubiquitous and abundant populations of APBs in this habitat. It raises questions about the role of these organisms in the geological dynamics of coastal carbonates, including coral reefs.

Microbial Nursery Production of High-Quality Biological Soil Crust Biomass for Restoration of Degraded Dryland Soils.

Biological soil crusts (biocrusts) are slow-growing, phototroph-based microbial assemblages that develop on the topsoils of drylands. Biocrusts help maintain soil fertility and reduce erosion. Because their loss through human activities has negative ecological and environmental health consequences, biocrust restoration is of interest. Active soil inoculation with biocrust microorganisms can be an important tool in this endeavor. We present a culture-independent, two-step process to grow multispecies biocrusts in open greenhouse nursery facilities, based on the inoculation of local soils with local biocrust remnants and incubation under seminatural conditions that maintain the essence of the habitat but lessen its harshness. In each of four U.S. Southwest sites, we tested and deployed combinations of factors that maximized growth (gauged as chlorophyll a content) while minimizing microbial community shifts (assessed by 16S rRNA sequencing and bioinformatics), particularly for crust-forming cyanobacteria. Generally, doubling the frequency of natural wetting events, a 60% reduction in sunlight, and inoculation by slurry were optimal. Nutrient addition effects were site specific. In 4 months, our approach yielded crusts of high inoculum quality reared on local soil exposed to locally matched climates, acclimated to desiccation, and containing communities minimally shifted in composition from local ones. Our inoculum contained abundant crust-forming cyanobacteria and no significant numbers of allochthonous phototrophs, and it was sufficient to treat ca. 6,000 m2 of degraded dryland soils at 1 to 5% of the typical crust biomass concentration, having started from a natural crust remnant as small as 6 to 30 cm2 IMPORTANCE: Soil surface crusts can protect dryland soils from erosion, but they are often negatively impacted by human activities. Their degradation causes a loss of fertility, increased production of fugitive dust and intensity of dust storms with associated traffic problems, and provokes general public health hazards. Our results constitute an advance in the quest to actively restore biological soil covers by providing a means to obtain high-quality inoculum within a reasonable time (a few months), thereby allowing land managers to recover essential, but damaged, ecosystem services in a sustainable, self-perpetuating way as provided by biocrust communities.

Description of Deinococcus oregonensis sp. nov., from biological soil crusts in the Southwestern arid lands of the United States of America.

Biological soil crusts are distinct habitats, harbor unique prokaryotic diversity and gave an impetus to isolate novel species. In the present study, a pink-pigmented bacterium, (OR316-6T), was isolated from biological soil crusts using oligotrophic BG11-PGY medium. Strain OR316-6T was Gram-positive, short rods, non-motile and non-spore forming. Cells were positive for catalase, oxidase and ß-galactosidase and negative for most of the enzymatic activities. The major fatty acids present were C16:0, C17:0, and C16:1ω7c and contained MK-8 and MK-10 as the predominant menaquinones. The cell wall peptidoglycan was of A3ß variant with L-ornithine as the diamino acid. Based on the above characteristics, strain OR316-6T was assigned to the genus Deinococcus. The phylogenetic analysis indicated that strain OR316-6T was closely related to D. aquatilis DSM 23025T with a 16S rRNA gene similarity of 99.3 % and clustered with a bootstrap value of 100 %. DNA-DNA similarity between strain OR316-6T and D. aquatilis DSM 23025T was 37.0 % indicating that strain OR316-6T was a novel species. Further, DNA fingerprinting of stains OR316-6T and D. aquatilis DSM 23035T demonstrated that both strains were related to each other with a similarity coefficient of only 0.32 and supported the species status to strain OR316-6T. In addition, phenotypic characteristics distinguished strain OR316-6T from D. aquatilis DSM 23025T. Based on the cumulative differences, strain OR316-16T exhibited with its closely related species, it was identified as a novel species and proposed the name Deinococcus oregonensis sp. nov. The type strain is D. oregonensis sp. nov. (OR316-6T = JCM 13503T = DSM 17762T).

Hydrogen export from intertidal cyanobacterial mats: sources, fluxes and the influence of community composition.

Microbial mats from marine intertidal settings have been reported to release significant quantities of H2 , in a unique trait among other mats and microbial communities. However, the H2 source and ecophysiological mechanisms that enable its export are not well understood. We examined H2 accumulation and export in three types of greenhouse-reared mats, from the intertidal region of Guerrero Negro, Mexico, and kept under natural light-dark conditions and wetting and drying cycles simulating low-, mid- and high-tidal height periodicity. All mats released H2 reproducibly and sustainably for 1.5 years. Net H2 export took place in a pulsed daily manner, starting after dusk, and waning in the morning, as photosynthesis resumed. Mid- and low-tidal mats developed high concentrations, capable of sustaining export fluxes that represented 2-4% of the water split through primary productivity. Neither N2 fixation nor direct photolytic hydrogenogenesis was significant to this H2 export, which was fermentative in origin, variable among mats, originating from cyanobacterial photosynthate. Analyses of community composition by pyrosequencing of 16S rRNA and hoxH genes indicate that filamentous non-heterocystous cyanobacteria (e.g. Lyngbya, Microcoleus) were important in the process of H2 export, as was the relatively low abundance and activity of methanogens and sulfate reducers.

Nimble vs. torpid responders to hydration pulse duration among soil microbes.

Environmental parameters vary in time, and variability is inherent in soils, where microbial activity follows precipitation pulses. The expanded pulse-reserve paradigm (EPRP) contends that arid soil microorganisms have adaptively diversified in response to pulse regimes differing in frequency and duration. To test this, we incubate Chihuahuan Desert soil microbiomes under separate treatments in which 60 h of hydration was reached with pulses of different pulse duration (PD), punctuated by intervening periods of desiccation. Using 16S rRNA gene amplicon data, we measure treatment effects on microbiome net growth, growth efficiency, diversity, and species composition, tracking the fate of 370 phylotypes (23% of those detected). Consistent with predictions, microbial diversity is a direct, saturating function of PD. Increasingly larger shifts in community composition are detected with decreasing PD, as specialist phylotypes become more prominent. One in five phylotypes whose fate was tracked responds consistently to PD, some preferring short pulses (nimble responders; NIRs) and some longer pulses (torpid responders; TORs). For pulses shorter than a day, microbiome growth efficiency is an inverse function of PD, as predicted. We conclude that PD in pulsed soil environments constitutes a major driver of microbial community assembly and function, largely consistent with the EPRP predictions.

Spatial organization of a soil cyanobacterium and its cyanosphere through GABA/Glu signaling to optimize mutualistic nitrogen fixation.

Soil biocrusts are characterized by the spatial self-organization of resident microbial populations at small scales. The cyanobacterium Microcoleus vaginatus, a prominent primary producer and pioneer biocrust former, relies on a mutualistic carbon (C) for nitrogen (N) exchange with its heterotrophic cyanosphere microbiome, a mutualism that may be optimized through the ability of the cyanobacterium to aggregate into bundles of trichomes. Testing both environmental populations and representative isolates, we show that the proximity of mutualistic diazotroph populations results in M. vaginatus bundle formation orchestrated through chemophobic and chemokinetic responses to gamma-aminobutyric acid (GABA) /glutamate (Glu) signals. The signaling system is characterized by: a high GABA sensitivity (nM range) and low Glu sensitivity (µM to mM), the fact that GABA and Glu are produced by the cyanobacterium as an autoinduction response to N deficiency, and by the presence of interspecific signaling by heterotrophs in response to C limitation. Further, it crucially switches from a positive to a negative feedback loop with increasing GABA concentration, thus setting maximal bundle sizes. The unprecedented use of GABA/Glu as an intra- and interspecific signal in the spatial organization of microbiomes highlights the pair as truly universal infochemicals.

An evolutionary optimum amid moderate heritability in prokaryotic cell size.

We investigate the distribution and evolution of prokaryotic cell size based on a compilation of 5,380 species. Size spans four orders of magnitude, from 100 nm (Mycoplasma) to more than 1 cm (Thiomargarita); however, most species congregate heavily around the mean. The distribution approximates but is distinct from log normality. Comparative phylogenetics suggests that size is heritable, yet the phylogenetic signal is moderate, and the degree of heritability is independent of taxonomic scale (i.e., fractal). Evolutionary modeling indicates the presence of an optimal cell size to which most species gravitate. The size is equivalent to a coccus of 0.70 µm in diameter. Analyses of 1,361 species with sequenced genomes show that genomic traits contribute to size evolution moderately and synergistically. Given our results, scaling theory, and empirical evidence, we discuss potential drivers that may expand or shrink cells around the optimum and propose a stability landscape model for prokaryotic cell size.

Description of Hymenobacter arizonensis sp. nov. from the southwestern arid lands of the United States of America.

Strain OR362-8(T) was isolated from a biological soil crust sample collected from the southwestern arid lands of the United States of America, using BG11-PGY medium. Cells of OR362-8(T) were found to be rod shaped; occur singly, as pairs and in groups; non-motile; positive for catalase, oxidase, phosphatase and gelatinase; hydrolyze starch; contain iso-C(15:0), anteiso-C(15:0), iso-C(15:1)G, C(16:1ω5c) and summed feature 3 (C(16:1(ω7c))/iso-C(15:0) 2OH as defined by the MIDI system) as the major fatty acids; and MK-7 as the sole respiratory quinone. A BLAST sequence similarity search using 16S rRNA gene sequence of OR362-8(T) identified Hymenobacter as the nearest genus with a similarity of 90.4-96.9 %. The phylogenetic analyses based on the phenetic methods UPGMA, NJ, ME and DNA parsimony resulted in the clustering of OR362-8(T) with Clade 1 Hymenobacter species represented by Hymenobacter glaciei, Hymenobacter antarcticus, Hymenobacter flocculans, Hymenobacter metalli and Hymenobacter soli with the closest being the Hymenobacter glaciei (96.9 % 16S rRNA gene sequence similarity). Besides the strong phylogentic affiliation, OR362-8(T) also exhibited significant phenotypic and chemotaxonomic differences with the members of Clade 1 Hymenobacter spp. More importantly, the DNA G+C content (mol%) of OR362-8(T) is very high (70 %) compared to the nearest species identified by phylogenetic analysis. Based on the phylogenetic, phenotypic and chemotaxonomic characteristics, OR362-8(T) was assigned to a novel species for which we propose here the name Hymenobacter arizonensis sp. nov., with OR362-8(T) (=ATCC BAA 1266(T) = DSM 17860(T) = JCM 13504(T)) as the type strain.

Functional genomics of novel secondary metabolites from diverse cyanobacteria using untargeted metabolomics.

Mass spectrometry-based metabolomics has become a powerful tool for the detection of metabolites in complex biological systems and for the identification of novel metabolites. We previously identified a number of unexpected metabolites in the cyanobacterium Synechococcus sp. PCC 7002, such as histidine betaine, its derivatives and several unusual oligosaccharides. To test for the presence of these compounds and to assess the diversity of small polar metabolites in other cyanobacteria, we profiled cell extracts of nine strains representing much of the morphological and evolutionary diversification of this phylum. Spectral features in raw metabolite profiles obtained by normal phase liquid chromatography coupled to mass spectrometry (MS) were manually curated so that chemical formulae of metabolites could be assigned. For putative identification, retention times and MS/MS spectra were cross-referenced with those of standards or available sprectral library records. Overall, we detected 264 distinct metabolites. These included indeed different betaines, oligosaccharides as well as additional unidentified metabolites with chemical formulae not present in databases of metabolism. Some of these metabolites were detected only in a single strain, but some were present in more than one. Genomic interrogation of the strains revealed that generally, presence of a given metabolite corresponded well with the presence of its biosynthetic genes, if known. Our results show the potential of combining metabolite profiling and genomics for the identification of novel biosynthetic genes.

Microbial excavation of solid carbonates powered by P-type ATPase-mediated transcellular Ca2+ transport.

Some microbes, among them a few species of cyanobacteria, are able to excavate carbonate minerals, from limestone to biogenic carbonates, including coral reefs, in a bioerosive activity that directly links biological and geological parts of the global carbon cycle. The physiological mechanisms that enable such endolithic cyanobacteria to bore, however, remain unknown. In fact, their boring constitutes a geochemical paradox, in that photoautotrophic metabolism will tend to precipitate carbonates, not dissolve them. We developed a stable microbe/mineral boring system based on a cyanobacterial isolate, strain BC008, with which to study the process of microbial excavation directly in the laboratory. Measurements of boring into calcite under different light regimes, and an analysis of photopigment content and photosynthetic rates along boring filaments, helped us reject mechanisms based on the spatial or temporal separation of alkali versus Acid-generating metabolism (i.e., photosynthesis and respiration). Instead, extracellular Ca(2+) imaging of boring cultures in vivo showed that BC008 was able to take up Ca(2+) at the excavation front, decreasing the local extracellular ion activity product of calcium carbonate enough to promote spontaneous dissolution there. Intracellular Ca(2+) was then transported away along the multicellular cyanobacterial trichomes and excreted at the distal borehole opening into the external medium. Inhibition assays and gene expression analyses indicate that the uptake and transport was driven by P-type Ca(2+)-ATPases. We believe such a chemically simple and biologically sophisticated mechanism for boring to be unparalleled among bacteria.