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With advances in DNA sequencing and miniaturized molecular biology workflows, rapid and affordable sequencing of single-cell genomes has become a reality. Compared to 16S rRNA gene surveys and shotgun metagenomics, large-scale application of single-cell genomics to whole microbial communities provides an integrated snapshot of community composition and function, directly links mobile elements to their hosts, and enables analysis of population heterogeneity of the dominant community members. To that end, we sequenced nearly 500 single-cell genomes from a low diversity hot spring sediment sample from Dewar Creek, British Columbia, and compared this approach to 16S rRNA gene amplicon and shotgun metagenomics applied to the same sample. We found that the broad taxonomic profiles were similar across the three sequencing approaches, though several lineages were missing from the 16S rRNA gene amplicon dataset, likely the result of primer mismatches. At the functional level, we detected a large array of mobile genetic elements present in the single-cell genomes but absent from the corresponding same species metagenome-assembled genomes. Moreover, we performed a single-cell population genomic analysis of the three most abundant community members, revealing differences in population structure based on mutation and recombination profiles. While the average pairwise nucleotide identities were similar across the dominant species-level lineages, we observed differences in the extent of recombination between these dominant populations. Most intriguingly, the creek’s Hydrogenobacter sp. population appeared to be so recombinogenic that it more closely resembled a sexual species than a clonally evolving microbe. Together, this work demonstrates that a randomized single-cell approach can be useful for the exploration of previously uncultivated microbes from community composition to population structure.

Background Metagenomics and single cell genomics provide a window into the genetic repertoire of yet uncultivated microorganisms, but both methods are usually taxonomically untargeted. The combination of fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) has the potential to enrich taxonomically well-defined clades for genomic analyses. Methods Cells hybridized with a taxon-specific FISH probe are enriched based on their fluorescence signal via flow cytometric cell sorting. A recently developed FISH procedure, the hybridization chain reaction (HCR)-FISH, provides the high signal intensities required for flow cytometric sorting while maintaining the integrity of the cellular DNA for subsequent genome sequencing. Sorted cells are subjected to shotgun sequencing, resulting in targeted metagenomes of low diversity. Results Pure cultures of different taxonomic groups were used to (1) adapt and optimize the HCR-FISH protocol and (2) assess the effects of various cell fixation methods on both the signal intensity for cell sorting and the quality of subsequent genome amplification and sequencing. Best results were obtained for ethanol-fixed cells in terms of both HCR-FISH signal intensity and genome assembly quality. Our newly developed pipeline was successfully applied to a marine plankton sample from the North Sea yielding good quality metagenome assembled genomes from a yet uncultivated flavobacterial clade. Conclusions With the developed pipeline, targeted metagenomes at various taxonomic levels can be efficiently retrieved from environmental samples. The resulting metagenome assembled genomes allow for the description of yet uncharacterized microbial clades.

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria. We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2–79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 105 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45–77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria, representing a candidate order (Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O2, suggesting a microaerophilic lifestyle.

With advances in DNA sequencing and miniaturized molecular biology workflows, rapid and affordable sequencing of single-cell genomes has become a reality. Compared to 16S rRNA gene surveys and shotgun metagenomics, large-scale application of single-cell genomics to whole microbial communities provides an integrated snapshot of community composition and function, directly links mobile elements to their hosts, and enables analysis of population heterogeneity of the dominant community members. To that end, we sequenced nearly 500 single-cell genomes from a low diversity hot spring sediment sample from Dewar Creek, British Columbia, and compared this approach to 16S rRNA gene amplicon and shotgun metagenomics applied to the same sample. We found that the broad taxonomic profiles were similar across the three sequencing approaches, though several lineages were missing from the 16S rRNA gene amplicon dataset, likely the result of primer mismatches. At the functional level, we detected a large array of mobile genetic elements present in the single-cell genomes but absent from the corresponding same species metagenome-assembled genomes. Moreover, we performed a single-cell population genomic analysis of the three most abundant community members, revealing differences in population structure based on mutation and recombination profiles. While the average pairwise nucleotide identities were similar across the dominant species-level lineages, we observed differences in the extent of recombination between these dominant populations. Most intriguingly, the creek's Hydrogenobacter sp. population appeared to be so recombinogenic that it more closely resembled a sexual species than a clonally evolving microbe. Together, this work demonstrates that a randomized single-cell approach can be useful for the exploration of previously uncultivated microbes from community composition to population structure.

Assigning a functional role to a microorganism has historically relied on cultivation of isolates or detection of environmental genome-based biomarkers using a posteriori knowledge of function. However, the emerging field of function-driven single-cell genomics aims to expand this paradigm by identifying and capturing individual microbes based on their in situ functions or traits. To identify and characterize yet uncultivated microbial taxa involved in cellulose degradation, we developed and benchmarked a function-driven single-cell screen, which we applied to a microbial community inhabiting the Great Boiling Spring (GBS) Geothermal Field, northwest Nevada. Our approach involved recruiting microbes to fluorescently labeled cellulose particles, and then isolating single microbe-bound particles via fluorescence-activated cell sorting. The microbial community profiles prior to sorting were determined via bulk sample 16S rRNA gene amplicon sequencing. The flow-sorted cellulose-bound microbes were subjected to whole genome amplification and shotgun sequencing, followed by phylogenetic placement. Next, putative cellulase genes were identified, expressed and tested for activity against derivatives of cellulose and xylose. Alongside typical cellulose degraders, including members of the Actinobacteria, Bacteroidetes, and Chloroflexi, we found divergent cellulases encoded in the genome of a recently described candidate phylum from the rare biosphere, Goldbacteria, and validated their cellulase activity. As this genome represents a species-level organism with novel and phylogenetically distinct cellulolytic activity, we propose the name Candidatus ‘Cellulosimonas argentiregionis’. We expect that this function-driven single-cell approach can be extended to a broad range of substrates, linking microbial taxonomy directly to in situ function.

Background Zymomonas mobilis is an aerotolerant α-proteobacterium, which has been genetically engineered for industrial purposes for decades. However, a comprehensive comparison of existing strains on the genomic level in conjunction with phenotype analysis has yet to be carried out. We here performed whole-genome comparison of 17 strains including nine that were sequenced in this study. We then compared 15 available Zymomonas strains for their natural abilities to perform under conditions relevant to biofuel synthesis. We tested their growth in anaerobic rich media, as well as growth, ethanol production and xylose utilization in lignocellulosic hydrolysate. We additionally compared their tolerance to isobutanol, flocculation characteristics, and ability to uptake foreign DNA by electroporation and conjugation. Results Using clustering based on 99% average nucleotide identity (ANI), we classified 12 strains into four clusters based on sequence similarity, while five strains did not cluster with any other strain. Strains belonging to the same 99% ANI cluster showed similar performance while significant variation was observed between the clusters. Overall, conjugation and electroporation efficiencies were poor across all strains, which was consistent with our finding of coding potential for several DNA defense mechanisms, such as CRISPR and restriction–modification systems, across all genomes. We found that strain ATCC31821 (ZM4) had a more diverse plasmid profile than other strains, possibly leading to the unique phenotypes observed for this strain. ZM4 also showed the highest growth of any strain in both laboratory media and lignocellulosic hydrolysate and was among the top 3 strains for isobutanol tolerance and electroporation and conjugation efficiency. Conclusions Our findings suggest that strain ZM4 has a unique combination of genetic and phenotypic traits that are beneficial for biofuel production and propose investing future efforts in further engineering of ZM4 for industrial purposes rather than exploring new Zymomonas isolates.

Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism., Soil ecosystems harbor diverse microorganisms and yet remain only partially characterized as neither single-cell sequencing nor whole-community sequencing offers a complete picture of these complex communities. Thus, the genetic and metabolic potential of this “uncultivated majority” remains underexplored. To address these challenges, we applied a pooled-cell-sorting-based mini-metagenomics approach and compared the results to bulk metagenomics. Informatic binning of these data produced 200 mini-metagenome assembled genomes (sorted-MAGs) and 29 bulk metagenome assembled genomes (MAGs). The sorted and bulk MAGs increased the known phylogenetic diversity of soil taxa by 7.2% with respect to the Joint Genome Institute IMG/M database and showed clade-specific sequence recruitment patterns across diverse terrestrial soil metagenomes. Additionally, sorted-MAGs expanded the rare biosphere not captured through MAGs from bulk sequences, exemplified through phylogenetic and functional analyses of members of the phylum Bacteroidetes. Analysis of 67 Bacteroidetes sorted-MAGs showed conserved patterns of carbon metabolism across four clades. These results indicate that mini-metagenomics enables genome-resolved investigation of predicted metabolism and demonstrates the utility of combining metagenomics methods to tap into the diversity of heterogeneous microbial assemblages. IMPORTANCE Microbial ecologists have historically used cultivation-based approaches as well as amplicon sequencing and shotgun metagenomics to characterize microbial diversity in soil. However, challenges persist in the study of microbial diversity, including the recalcitrance of the majority of microorganisms to laboratory cultivation and limited sequence assembly from highly complex samples. The uncultivated majority thus remains a reservoir of untapped genetic diversity. To address some of the challenges associated with bulk metagenomics as well as low throughput of single-cell genomics, we applied flow cytometry-enabled mini-metagenomics to capture expanded microbial diversity from forest soil and compare it to soil bulk metagenomics. Our resulting data from this pooled-cell sorting approach combined with bulk metagenomics revealed increased phylogenetic diversity through novel soil taxa and rare biosphere members. In-depth analysis of genomes within the highly represented Bacteroidetes phylum provided insights into conserved and clade-specific patterns of carbon metabolism.

Single-cell genome sequencing of individual archaeal and bacterial cells is a vital approach to decipher the genetic makeup of uncultured microorganisms. With this review, we describe single-cell genome analysis with a focus on the unique properties of single-cell sequence data and with emphasis on quality assessment and assurance.

Members of the bacterial candidate phylum WPS-2 (or Eremiobacterota) are abundant in several dry, bare soil environments. In a bare soil deposited by an extinct iron-sulfur spring, we found that WPS-2 comprised up to 24% of the bacterial community and up to 108 cells per g of soil based on 16S rRNA gene sequencing and quantification. A single genus-level cluster (Ca. Rubrimentiphilum) predominated in bare soils but was less abundant in adjacent forest. Nearly complete genomes of Ca. Rubrimentiphilum were recovered as single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs). Surprisingly, given the abundance of WPS-2 in bare soils, the genomes did not indicate any capacity for autotrophy, phototrophy, or trace gas metabolism. Instead, they suggest a predominantly aerobic organoheterotrophic lifestyle, perhaps based on scavenging amino acids, nucleotides, and complex oligopeptides, along with lithotrophic capacity on thiosulfate. Network analyses of the entire community showed that some species of Chloroflexi, Actinobacteria, and candidate phylum AD3 (or Dormibacterota) co-occurred with Ca. Rubrimentiphilum and may represent ecological or metabolic partners. We propose that Ca. Rubrimentiphilum act as efficient heterotrophic scavengers. Combined with previous studies, these data suggest that the phylum WPS-2 includes bacteria with diverse metabolic capabilities.

Microbial communities control numerous biogeochemical processes critical for ecosystem function and health. Most analyses of coastal microbial communities focus on the characterization of bacteria present in either sediment or seawater, with fewer studies characterizing both sediment and seawater together at a given site, and even fewer studies including information about non-bacterial microbial communities. As a result, knowledge about the ecological patterns of microbial biodiversity across domains and habitats in coastal communities is limited-despite the fact that archaea, bacteria, and microbial eukaryotes are present and known to interact in coastal habitats. To better understand microbial biodiversity patterns in coastal ecosystems, we characterized sediment and seawater microbial communities for three sites along the coastline of Puerto Nuevo, Baja California, Mexico using both 16S and 18S rRNA gene amplicon sequencing. We found that sediment hosted approximately 500-fold more operational taxonomic units (OTUs) for bacteria, archaea, and microbial eukaryotes than seawater (p < 0.001). Distinct phyla were found in sediment versus seawater samples. Of the top ten most abundant classes, Cytophagia (bacterial) and Chromadorea (eukaryal) were specific to the sediment environment, whereas Cyanobacteria and Bacteroidia (bacterial) and Chlorophyceae (eukaryal) were specific to the seawater environment. A total of 47 unique genera were observed to comprise the core taxa community across environment types and sites. No archaeal taxa were observed as part of either the abundant or core taxa. No significant differences were observed for sediment community composition across domains or between sites. For seawater, the bacterial and archaeal community composition was statistically different for the Major Outlet site (p < 0.05), the site closest to a residential area, and the eukaryal community composition was statistically different between all sites (p < 0.05). Our findings highlight the distinct patterns and spatial heterogeneity in microbial communities of a coastal region in Baja California, Mexico.

© 2016 International Society for Microbial Ecology All rights reserved. Although little is known regarding microbial life within our planet's rock-hosted deep subseafloor biosphere, boreholes drilled through deep ocean sediment and into the underlying basaltic crust provide invaluable windows of access that have been used previously to document the presence of microorganisms within fluids percolating through the deep ocean crust. In this study, the analysis of 1.7 million small subunit ribosomal RNA genes amplified and sequenced from marine sediment, bottom seawater and basalt-hosted deep subseafloor fluids that span multiple years and locations on the Juan de Fuca Ridge flank was used to quantitatively delineate a subseafloor microbiome comprised of distinct bacteria and archaea. Hot, anoxic crustal fluids tapped by newly installed seafloor sampling observatories at boreholes U1362A and U1362B contained abundant bacterial lineages of phylogenetically unique Nitrospirae, Aminicenantes, Calescamantes and Chloroflexi. Although less abundant, the domain Archaea was dominated by unique, uncultivated lineages of marine benthic group E, the Terrestrial Hot Spring Crenarchaeotic Group, the Bathyarchaeota and relatives of cultivated, sulfate-reducing Archaeoglobi. Consistent with recent geochemical measurements and bioenergetic predictions, the potential importance of methane cycling and sulfate reduction were imprinted within the basalt-hosted deep subseafloor crustal fluid microbial community. This unique window of access to the deep ocean subsurface basement reveals a microbial landscape that exhibits previously undetected spatial heterogeneity.

© 2016 International Society for Microbial Ecology All rights reserved. Over the past decade, high-throughput short-read 16S rRNA gene amplicon sequencing has eclipsed clone-dependent long-read Sanger sequencing for microbial community profiling. The transition to new technologies has provided more quantitative information at the expense of taxonomic resolution with implications for inferring metabolic traits in various ecosystems. We applied single-molecule real-time sequencing for microbial community profiling, generating full-length 16S rRNA gene sequences at high throughput, which we propose to name PhyloTags. We benchmarked and validated this approach using a defined microbial community. When further applied to samples from the water column of meromictic Sakinaw Lake, we show that while community structures at the phylum level are comparable between PhyloTags and Illumina V4 16S rRNA gene sequences (iTags), variance increases with community complexity at greater water depths. PhyloTags moreover allowed less ambiguous classification. Last, a platform-independent comparison of PhyloTags and in silico generated partial 16S rRNA gene sequences demonstrated significant differences in community structure and phylogenetic resolution across multiple taxonomic levels, including a severe underestimation in the abundance of specific microbial genera involved in nitrogen and methane cycling across the Lake's water column. Thus, PhyloTags provide a reliable adjunct or alternative to cost-effective iTags, enabling more accurate phylogenetic resolution of microbial communities and predictions on their metabolic potential.

Archaea are ubiquitous in terrestrial and marine environments and play an important role in biogeochemical cycles. Although air acts as the primary medium for their dispersal among different habitats, their diversity and abundance is not well characterized. The main reason for this lack of insight is that archaea are difficult to culture, seem to be low in number in the atmosphere, and have so far been difficult to detect even with molecular genetic approaches. However, to better understand the transport, residence time, and living conditions of microorganisms in the atmosphere as well as their effects on the atmosphere and vice versa, it is essential to study all groups of bioaerosols. Here we present an in-depth analysis of airborne archaea based on Illumina sequencing of 16S rRNA genes from atmospheric coarse and fine particulate matter samples and show seasonal dynamics and discuss anthropogenic influences on the diversity, composition, and abundance of airborne archaea.The relative proportions of archaea to bacteria, the differences of the community composition in fine and coarse particulate matter, and the high abundance in coarse matter of one typical soil related family, the Nitrososphaeraceae, point to local phyllosphere and soil habitats as primary emission sources of airborne archaea.We found comparable seasonal dynamics for the dominating Euryarchaeota classes and Crenarchaeota orders peaking in summer and fall. In contrast, the omnipresent Cenarchaeales and the Thermoplasmata occur only throughout summer and fall. We also gained novel insights into archaeal composition in fine particulate matter (Nitrosopumilus as the dominating taxa.The seasonal dynamics of methanogenic Euryarchaeota point to anthropogenic activities, such as fertilization of agricultural fields with biogas substrates or manure, as sources of airborne archaea. This study gains a deeper insight into the abundance and composition of archaea in the atmosphere, especially within the fine particle mode, which adds to a better understanding of the overall atmospheric microbiome.

Sequencing of single bacterial and archaeal cells is an important methodology that provides access to the genetic makeup of uncultivated microorganisms. We here describe the high-throughput fluorescence-activated cell sorting-based isolation of single cells from the environment, their lysis and strand displacement-mediated whole genome amplification. We further outline 16S rRNA gene sequence-based screening of single-cell amplification products, their preparation for Illumina sequencing libraries, and finally propose computational methods for read and contig level quality control of the resulting sequence data.

Sequencing of single bacterial and archaeal cells is an important methodology that provides access to the genetic makeup of uncultivated microorganisms. We here describe the high-throughput fluorescence-activated cell sorting-based isolation of single cells from the environment, their lysis and strand displacement-mediated whole genome amplification. We further outline 16S rRNA gene sequence-based screening of single-cell amplification products, their preparation for Illumina sequencing libraries, and finally propose computational methods for read and contig level quality control of the resulting sequence data.

Following publication of the original article [1], the authors pointed out that the figure shown as figure S1 is actually figure S2 and vice versa. Figure S1 should show the barcharts, and figure S2 should shows the heatmaps. Figures captions are in the correct order. 1) "...SILVA-only OTUs (37,066 97% OTUs and 1266 85% clusters) (Additional file 3: Figure S2 )..." - ...SILVA-only OTUs (37,066 97% OTUs and 1266 85% clusters) (Additional file 3: Figure S1 )..." 2) "...in groundwater and soil (Fig. 2, Additional file 2: Figure S1 )." - "in groundwater and soil (Fig. 2, Additional file 2: Figure S2 )." 3) "...from genomes as training data (Additional file 3: Figure S2 , ..." - "...from genomes as training data (Additional file 3: Figure S1 , ..." 4) "...were predicted to be chimeric (Additional file 3: Figure S2 )" - "...were predicted to be chimeric (Additional file 3: Figure S1 )".

Bacteria and fungi are ubiquitous throughout the Earth's lower atmosphere where they often represent an important component of atmospheric aerosols with the potential to impact human health and atmospheric dynamics. However, the diversity, composition, and spatiotemporal dynamics of these airborne microbes remain poorly understood. We performed a comprehensive analysis of airborne microbes across two aerosol size fractions at urban and rural sites in the Colorado Front Range over a 14-month period. Coarse (PM10-2.5) and fine (PM2.5) particulate matter samples were collected at weekly intervals with both bacterial and fungal diversity assessed via high-throughput sequencing. The diversity and composition of the airborne communities varied across the sites, between the two size fractions, and over time. Bacteria were the dominant type of bioaerosol in the collected air samples, while fungi and plants (pollen) made up the remainder, with the relative abundances of fungi peaking during the spring and summer months. As bacteria made up the majority of bioaerosol particles, we analyzed the bacterial communities in greater detail using a bacterial-specific 16S rRNA gene sequencing approach. Overall, bacterial taxonomic richness and the relative abundances of specific bacterial taxa exhibited significant patterns of seasonality. Likewise, airborne bacterial communities varied significantly between sites and across aerosol size fractions. Source-tracking analyses indicate that soils and leaves represented important sources of bacteria to the near-surface atmosphere across all locations with cow fecal bacteria also representing an important source of bioaerosols at the more rural sites during early fall and early spring. Together, these data suggest that a complex set of environmental factors, including changes in atmospheric conditions and shifts in the relative importance of available microbial sources, act to control the composition of microbial bioaerosols in rural and urban environments.

We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.

Generating sequence data of a defined community composed of organisms with complete reference genomes is indispensable for the benchmarking of new genome sequence analysis methods, including assembly and binning tools. Moreover the validation of new sequencing library protocols and platforms to assess critical components such as sequencing errors and biases relies on such datasets. We here report the next generation metagenomic sequence data of a defined mock community (Mock Bacteria ARchaea Community; MBARC-26), composed of 23 bacterial and 3 archaeal strains with finished genomes. These strains span 10 phyla and 14 classes, a range of GC contents, genome sizes, repeat content and encompass a diverse abundance profile. Short read Illumina and long-read PacBio SMRT sequences of this mock community are described. These data represent a valuable resource for the scientific community, enabling extensive benchmarking and comparative evaluation of bioinformatics tools without the need to simulate data. As such, these data can aid in improving our current sequence data analysis toolkit and spur interest in the development of new tools.

© 2016 Salman et al. "Candidatus Achromatium palustre" was recently described as the first marine representative of the Achromatium spp. in the Thiotrichaceae - a sister lineage to the Chromatiaceae in the Gammaproteobacteria. Achromatium spp. belong to the group of large sulfur bacteria as they can grow to nearly 100 μm in size and store elemental sulfur (S0) intracellularly. As a unique feature, Achromatium spp. can accumulate colloidal calcite (CaCO3) inclusions in great amounts. Currently, both process and function of calcite accumulation in bacteria is unknown, and all Achromatium spp. are uncultured. Recently, three single-cell draft genomes of Achromatium spp. from a brackish mineral spring were published, and here we present the first draft genome of a single "Candidatus Achromatium palustre" cell collected in the sediments of the Sippewissett Salt Marsh, Cape Cod, MA. Our draft dataset consists of 3.6 Mbp, has a G + C content of 38.1 % and is nearly complete (83 %). The next closest relative to the Achromatium spp. genomes is Thiorhodovibrio sp. 907 of the family Chromatiaceae, containing phototrophic sulfide-oxidizing bacteria.

Although bacteria are ubiquitous in the near-surface atmosphere, the temporal dynamics of airborne bacterial communities have not been well-studied. We examined seasonal shifts in bacterial abundances, the relative contribution of bacteria to total aerosol loads, and bacterial community structure at a high-elevation research station in northern Colorado, USA. Aerosol samples were collected from the near-surface atmosphere over 5–10 days during each of the four calendar seasons. Bacterial abundances varied by season with the highest concentrations observed during the fall and spring seasons, consistent with the changes in total particle concentrations, with bacterial cells often representing a large fraction (22% on average) of the total near-surface aerosol particles >0.5 μm. Bacterial community composition, determined via barcoded pyrosequencing, also varied significantly by season. The dominant taxa in the spring, winter, and second half of the fall sampling periods (when the ground was snow covered) were bacterial taxa commonly found in other cold environments while the summer and first half of the fall samples contained taxa that were likely derived from soil and leaf-surface environments. Bacteria are clearly an important component of atmospheric aerosols with the abundance and composition of these airborne bacterial communities shaped by seasonal shifts in atmospheric conditions and the conditions of the local terrestrial environment.

Although all plant and animal species harbor microbial symbionts, we know surprisingly little about the specificity of microbial communities to their hosts. Few studies have compared the microbiomes of different species of animals, and fewer still have examined animals in the wild. We sampled four pond habitats in Colorado, USA, where multiple amphibian species were present. In total, 32 amphibian individuals were sampled from three different species including northern leopard frogs (Lithobates pipiens), western chorus frogs (Pseudacris triseriata) and tiger salamanders (Ambystoma tigrinum). We compared the diversity and composition of the bacterial communities on the skin of the collected individuals via barcoded pyrosequencing of the 16S rRNA gene. Dominant bacterial phyla included Acidobacteria, Actinobacteria, Bacteriodetes, Cyanobacteria, Firmicutes and Proteobacteria. In total, we found members of 18 bacterial phyla, comparable to the taxonomic diversity typically found on human skin. Levels of bacterial diversity varied strongly across species: L. pipiens had the highest diversity; A. tigrinum the lowest. Host species was a highly significant predictor of bacterial community similarity, and co-habitation within the same pond was not significant, highlighting that the skin-associated bacterial communities do not simply reflect those bacterial communities found in their surrounding environments. Innate species differences thus appear to regulate the structure of skin bacterial communities on amphibians. In light of recent discoveries that some bacteria on amphibian skin have antifungal activity, our finding suggests that host-specific bacteria may have a role in the species-specific resistance to fungal pathogens.

Summary Large populations of bacteria live on leaf surfaces and these phyllosphere bacteria can have important effects on plant health. However, we currently have a limited understanding of bacterial diversity on tree leaves and the inter- and intra-specific variability in phyllosphere community structure. We used a bar- coded pyrosequencing technique to characterize the bacterial communities from leaves of 56 tree species in Boulder, Colorado, USA, quantifying the intra- and inter-individual variability in the bacterial communi- ties from 10 of these species. We also examined the geographic variability in phyllosphere communities on Pinus ponderosa from several locations across the globe. Individual tree species harboured high levels of bacterial diversity and there was consider- able variability in community composition between trees. The bacterial communities were organized in patterns predictable from the relatedness of the trees as there was significant correspondence between tree phylogeny and bacterial community phylogeny. Inter-specific variability in bacterial community com- position exceeded intra-specific variability, a pattern that held even across continents where we observed minimal geographic differentiation in the bacterial communities on P. ponderosa needles.

In the version of this article initially published, the author A. Murat Eren was listed as A.M. Eren. The corresponding affiliation was given as the Knapp Center for Biomedical Discovery, rather than Department of Medicine, University of Chicago, Chicago, Illinois, USA, and Marine Biological Laboratory, Woods Hole, Massachusetts, USA. The errors have been corrected in the HTML and PDF versions of the article as of 29 November 2017. In the version of this article initially published, the following acknowledgment was omitted: A.L. was supported by the Russian Science Foundation (grant number 14-50-00069). The error has been corrected in the HTML and PDF versions of the article as of 7 December 2017.

Fungal breakdown of plant material rich in lignin and cellulose (i.e. lignocellulose) is of central importance to terrestrial carbon (C) cycling due to the abundance of lignocellulose above and below-ground. Fungal growth on lignocellulose is particularly influential in tropical forests, as woody debris and plant litter contain between 50% and 75% lignocellulose by weight, and can account for 20% of the C stored in these ecosystems. In this study, we evaluated factors affecting fungal growth on a common wood substrate along a wet tropical elevation gradient in the Peruvian Andes. We had three objectives: 1) to determine the temperature sensitivity of fungal growth – i.e. Q10, the factor by which fungal biomass increases given a 10 °C temperature increase; 2) to assess the potential for above-ground fungal colonization and growth on lignocellulose in a wet tropical forest; and 3) to characterize the community composition of fungal wood decomposers across the elevation gradient. We found that fungal growth had a Q10 of 3.93 (95% CI of 2.76–5.61), indicating that fungal biomass accumulation on the wood substrate nearly quadrupled with a 10 °C increase in temperature. The Q10 for fungal growth on wood at our site is higher than Q10 values reported for litter decomposition in other tropical forests. Moreover, we found that above-ground fungal growth on the wood substrate ranged between 37% and 50% of that measured in the soil, suggesting above-ground breakdown of lignocellulose represents an unexplored component of the C cycle in wet tropical forests. Fungal community composition also changed significantly along the elevation gradient, and Ascomycota were the dominant wood decomposers at all elevations. Fungal richness did not change significantly with elevation, directly contrasting with diversity patterns observed for plant and animal taxa across this gradient. Significant variation in fungal community composition across the gradient suggests that the characteristics of fungal decomposer communities are, directly or indirectly, influenced by temperature.

Infectious pancreatic necrosis virus (IPNV) is a major viral pathogen of salmonid fish and causes serious economic losses to salmonid aquaculture. Previously, we demonstrated that the IPNV capsid protein, VP2, expressed in yeast self-assembles into subviral particles (SVPs) and injection of these IPNV rVP2 SVPs into rainbow trout elicits an immune response. Immunized fish had reduced viral loads compared to unimmunized fish when challenged with IPNV. To evaluate the suitability of IPNV rVP2 SVPs for future development of multivalent vaccines, a linear epitope of a human oncogene, c-myc, was cloned into the IPNV rVP2 SVP backbone as a model epitope and expressed in yeast. Western blot analyses with anti-c-myc and anti-IPNV antibodies provided positive identification of both the c-myc and VP2 epitopes on the c-myc VP2 SVPs. Transmission electron microscopy of purified chimeric c-myc VP2 SVPs revealed the formation of approximately 20nm particles. Rainbow trout immunized with c-myc VP2 SVPs elicited both anti-c-myc and anti-IPNV immune responses. When immunized fish were challenged with IPNV, the viral load in the c-myc VP2 SVP immunized fish was significantly lower than the sham-vaccinated controls. The results indicate that IPNV rVP2 SVPs can tolerate the insertion of foreign epitopes without affecting either the antigenic potential of the epitopes of the backbone protein or the inserted foreign epitope. This opens the possibility of using the IPNV rVP2 SVP platform to express epitopes of other viruses, which could pave the way for development of multivalent subunit vaccines or novel marker vaccines.

Although a significant fraction of atmospheric particulate mass is organic carbon, the sources of particulate organic carbon (POC) are not always apparent. One potential source of atmospheric POC is biological particles, such as bacteria, pollen, and fungal spores. Measurements of POC and biological particles, including bacteria, fungal spores, and pollen, were made as part of the Storm Peak Aerosol and Cloud Characterization Study in Steamboat Springs, CO in March–April 2008. Biological particles were identified and characterized using several methods. The results suggest that biological particles could account for an average of 40% of the organic carbon mass in particles with aerodynamic diameters less than 10 μm. These estimates of POC mass from biological particles are highly uncertain; however, the results suggest that biological particles could be a significant source of organic aerosol in the background continental atmosphere and further observations are needed to better constrain these estimates.

Background The rapid development of sequencing technologies has provided access to environments that were either once thought inhospitable to life altogether or that contain too few cells to be analyzed using genomics approaches. While 16S rRNA gene microbial community sequencing has revolutionized our understanding of community composition and diversity over time and space, it only provides a crude estimate of microbial functional and metabolic potential. Alternatively, shotgun metagenomics allows comprehensive sampling of all genetic material in an environment, without any underlying primer biases. Until recently, one of the major bottlenecks of shotgun metagenomics has been the requirement for large initial DNA template quantities during library preparation. Results Here, we investigate the effects of varying template concentrations across three low biomass library preparation protocols on their ability to accurately reconstruct a mock microbial community of known composition. We analyze the effects of input DNA quantity and library preparation method on library insert size, GC content, community composition, assembly quality and metagenomic binning. We found that library preparation method and the amount of starting material had significant impacts on the mock community metagenomes. In particular, GC content shifted towards more GC rich sequences at the lower input quantities regardless of library prep method, the number of low quality reads that could not be mapped to the reference genomes increased with decreasing input quantities, and the different library preparation methods had an impact on overall metagenomic community composition. Conclusions This benchmark study provides recommendations for library creation of representative and minimally biased metagenome shotgun sequencing, enabling insights into functional attributes of low biomass ecosystem microbial communities. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2063-6) contains supplementary material, which is available to authorized users.

The basaltic ocean crust is the largest aquifer system on Earth, yet the rates of biological activity in this environment are unknown. Low-temperature (100°C) fluid samples were investigated from two borehole observatories in the Juan de Fuca Ridge (JFR) flank, representing a range of upper oceanic basement thermal and geochemical properties. Microbial sulfate reduction rates (SRR) were measured in laboratory incubations with (35)S-sulfate over a range of temperatures and the identity of the corresponding sulfate-reducing microorganisms (SRM) was studied by analyzing the sequence diversity of the functional marker dissimilatory (bi)sulfite reductase (dsrAB) gene. We found that microbial sulfate reduction was limited by the decreasing availability of organic electron donors in higher temperature, more altered fluids. Thermodynamic calculations indicate energetic constraints for metabolism, which together with relatively higher cell-specific SRR reveal increased maintenance requirements, consistent with novel species-level dsrAB phylotypes of thermophilic SRM. Our estimates suggest that microbially-mediated sulfate reduction may account for the removal of organic matter in fluids within the upper oceanic crust and underscore the potential quantitative impact of microbial processes in deep subsurface marine crustal fluids on marine and global biogeochemical carbon cycling.

Although bacteria are ubiquitous in the near-surface atmosphere and they can have important effects on human health, airborne bacteria have received relatively little attention and their spatial dynamics remain poorly understood. Owing to differences in meteorological conditions and the potential sources of airborne bacteria, we would expect the atmosphere over different land-use types to harbor distinct bacterial communities. To test this hypothesis, we sampled the near-surface atmosphere above three distinct land-use types (agricultural fields, suburban areas and forests) across northern Colorado, USA, sampling five sites per land-use type. Microbial abundances were stable across land-use types, with ∼10(5)-10(6) bacterial cells per m(3) of air, but the concentrations of biological ice nuclei, determined using a droplet freezing assay, were on average two and eight times higher in samples from agricultural areas than in the other two land-use types. Likewise, the composition of the airborne bacterial communities, assessed via bar-coded pyrosequencing, was significantly related to land-use type and these differences were likely driven by shifts in the sources of bacteria to the atmosphere across the land-uses, not local meteorological conditions. A meta-analysis of previously published data shows that atmospheric bacterial communities differ from those in potential source environments (leaf surfaces and soils), and we demonstrate that we may be able to use this information to determine the relative inputs of bacteria from these source environments to the atmosphere. This work furthers our understanding of bacterial diversity in the atmosphere, the terrestrial controls on this diversity and potential approaches for source tracking of airborne bacteria.

The effect of different templates on generating standard curves that are needed for the absolute quantification of an RNA virus by real-time reverse transcriptase-PCR (RT-PCR) was evaluated. We used infectious pancreatic necrosis virus (IPNV), a major viral pathogen of wild and cultured salmon, as a RNA virus example for the study. A dilution series of four different templates representing the IPNV protease gene (two in vitro transcribed RNAs of 100 bases and 500 bases in length, a plasmid DNA and a DNA oligo) were used as template to produce standard curves to quantify IPNV load in rainbow trout. The slope, the goodness of fit (r(2)), and the efficiency (e) of PCR were statistically equivalent irrespective of the nature of template used in the PCR. Using a factorial ANOVA, no significant difference in IPNV copy number was observed using the four different standard curves for absolute quantification of IPNV in experimentally-challenged rainbow trout. However, when IPNV transcript abundance was less than 100 copies per reaction and when the template size was bigger than the amplicon size amplification was more variable. The data suggests that the size of the template used to generate standard curve should be very similar to the size of the amplicon. A synthetic DNA oligo template would be optimal for this purpose as it can be custom made and only requires the sequence information for its synthesis. However, if the standard curve is generated with template copy number in excess of 100 copies per reaction, the nature of the template has no effect on the standard curve, and therefore, the cheaper template would be the preferred choice of template over the other more expensive options.

Large populations of bacteria live on leaf surfaces and these phyllosphere bacteria can have important effects on plant health. However, we currently have a limited understanding of bacterial diversity on tree leaves and the inter- and intra-specific variability in phyllosphere community structure. We used a bar-coded pyrosequencing technique to characterize the bacterial communities from leaves of 56 tree species in Boulder, Colorado, USA, quantifying the intra- and inter-individual variability in the bacterial communities from 10 of these species. We also examined the geographic variability in phyllosphere communities on Pinus ponderosa from several locations across the globe. Individual tree species harboured high levels of bacterial diversity and there was considerable variability in community composition between trees. The bacterial communities were organized in patterns predictable from the relatedness of the trees as there was significant correspondence between tree phylogeny and bacterial community phylogeny. Inter-specific variability in bacterial community composition exceeded intra-specific variability, a pattern that held even across continents where we observed minimal geographic differentiation in the bacterial communities on P. ponderosa needles.

Bacteria and fungi are ubiquitous in the atmosphere. The diversity and abundance of airborne microbes may be strongly influenced by atmospheric conditions or even influence atmospheric conditions themselves by acting as ice nucleators. However, few comprehensive studies have described the diversity and dynamics of airborne bacteria and fungi based on culture-independent techniques. We document atmospheric microbial abundance, community composition, and ice nucleation at a high-elevation site in northwestern Colorado. We used a standard small-subunit rRNA gene Sanger sequencing approach for total microbial community analysis and a bacteria-specific 16S rRNA bar-coded pyrosequencing approach (4,864 sequences total). During the 2-week collection period, total microbial abundances were relatively constant, ranging from 9.6 × 10 5 to 6.6 × 10 6 cells m −3 of air, and the diversity and composition of the airborne microbial communities were also relatively static. Bacteria and fungi were nearly equivalent, and members of the proteobacterial groups Burkholderiales and Moraxellaceae (particularly the genus Psychrobacter ) were dominant. These taxa were not always the most abundant in freshly fallen snow samples collected at this site. Although there was minimal variability in microbial abundances and composition within the atmosphere, the number of biological ice nuclei increased significantly during periods of high relative humidity. However, these changes in ice nuclei numbers were not associated with changes in the relative abundances of the most commonly studied ice-nucleating bacteria.

To accurately measure the relative expression of a target gene, mRNA expression data is routinely normalized with reference to an internal control gene. We examined the transcriptional stability of four internal control genes, beta-actin, glyceraldehyde-3 phosphate dehydrogenase (GAPDH), elongation factor1-alpha (EF1-alpha), and 18S ribosomal RNA (18S rRNA) while measuring the mRNA expression of a gene encoding a pattern recognition protein, lipopolysaccharide and glucan binding protein (LGBP) gene, in healthy and white spot syndrome virus (WSSV) infected shrimp (Penaeus stylirostris) before and after (4, 8, 16 and 32 h) challenge using real-time RT-PCR. Here, we describe a method to rank the internal control genes based on a linear regression analysis. This analysis enables us to analyze the multivariate data set, e.g. time course study samples with control and treatment groups. Using the linear regression analysis and the WSSV-challenged time course samples, GAPDH was found to be the most stable internal control gene followed by the genes EF1-alpha, 18S rRNA and beta-actin. Using the program geNorm, GAPDH was also found to be the most stable gene followed by the genes EF1-alpha, beta-actin and 18S rRNA. Using the program NormFinder, the ranking of the internal control genes were in the order of EF1-alpha>GAPDH>18S rRNA>beta-actin. The ability to compare the healthy and WSSV infected samples in parallel by the regression analysis makes this method a very useful approach while identifying the optimal reference gene for gene expression analysis.

Infectious pancreatic necrosis virus (IPNV) is a bisegmented double-stranded RNA virus belonging to the family Birnaviridae, genus Aquabirnavirus, which is a major viral pathogen of salmonid fish. The virus infects wild and cultured salmonids, causing high mortality in juvenile trout and salmon. A highly sensitive and specific real-time RT-PCR assay using the fluorogenic dye SYBR((R)) Green I was developed for the detection and quantitation of IPNV in rainbow trout (Oncorhynchus mykiss). Rainbow trout were infected experimentally with IPNV in the laboratory by injection or immersion and then pectoral fin, spleen, and head kidney samples were collected for analysis. The corresponding cDNA was synthesized using DNase I-treated total RNA and then real-time RT-PCR was performed using primers based on the IPNV non-structural protein gene, designated as either NS or VP4. Rainbow trout beta-actin and elongation factor 1alpha (EF-1alpha) genes were used as internal controls. Using real-time RT-PCR, the virus was successfully detected in pectoral fin, spleen, and head kidney tissue samples. The dissociation curves for each amplicon showed a single melting peak at 83, 81.5, and 84 degrees C for IPNV NS, trout beta-actin, and EF-1alpha genes, respectively. The amplicon size and nucleotide sequence was used to confirm the specificity of the products. Using a dilution series of in vitro transcribed RNA, IPNV was reliably detected down to 10 RNA copies and had a dynamic range up to 10(7) RNA copies. A time course assay, using immersion challenged samples, revealed that the virus could be detected in pectoral fin, spleen, and head kidney as early as 24h post-challenge. The average viral load in all three tissues increased over time, reaching its highest level at 21 days post-challenge, which was followed by a slight decrease at 28 days post-challenge. IPNV load in pectoral fin tissue was comparable to the viral load in spleen and head kidney tissues, indicating that pectoral fin could be used for the detection and quantification of IPNV. The development of a non-lethal detection method will be useful for the detection of IPNV and potentially other viruses of finfish in farmed and wild fish.

Infectious pancreatic necrosis virus (IPNV), the etiologic agent of infectious pancreatic necrosis in salmonid fish, causes significant losses to the aquaculture industry. The gene for the viral capsid protein (VP2) was cloned into a yeast expression vector and expressed in Saccharomyces cerevisae. Expression of the capsid gene in yeast resulted in formation of approximately 20 nanometer sub-viral particles composed solely of VP2 protein. Anti-PNV antibodies were detected in rainbow trout vaccinated either by injection of purified VP2- subviral particles (rVP2-SVP) or by feeding recombinant yeast expressing rVP2-SVP. Challenge of rVP2-SVP immunized trout with a heterologous IPNV strain and subsequent viral load determination showed that both injection and orally vaccinated fish had lower DPNV loads than naive or sham-vaccinated fish. This study demonstrates the ability of rVP2-SVPs to induce a specific immune response and the ability of immunized fish to reduce the viral load after an experimentally induced IPNV infection. The invention is not limited to IPNV, and is applicable to other similar viruses for which SVPs can be made and administered to fish.

A real-time reverse transcriptase-polymerase chain reaction assay using the fluorogenic dye SYBR Green I was developed for the detection and quantification of infectious hematopoietic necrosis virus (IHNV) infecting rainbow trout (Oncorhynchus mykiss). Using primers designed for the IHNV nucleocapsid (N) and surface glycoprotein (G) genes, virus was detected in liver, kidney, spleen, adipose tissue, and pectoral fin samples from fish challenged in the laboratory via either injection or immersion and in fish collected from the field. The N- and G-gene amplicons provided melting curves with a single peak at 85.5 and 86.5 degrees C, respectively. Among different tissues tested, overall the N-gene was expressed in greater abundance than the G-gene in both laboratory-challenged and field samples. Kidney, liver, and spleen tissues had higher copies of the N- and G-genes compared to adipose tissue and pectoral fin. In samples from IHNV immersion challenge fish, the virus could be detected in the pectoral fin as early as 1 day post-challenge, and the viral load appears to decline by 6 days post-challenge. To evaluate the usefulness of non-invasive tissue sampling for IHNV detection, pectoral fin samples were collected from fish that were either apparently healthy or showing clinical signs of IHNV infection from commercial operations. Among the apparently healthy fish, using SYBR Green real-time RT-PCR the N-gene was detected in 2 out of 24 (8.3%), while the G-gene was detected in 8 of 24 (33%) fish. Among the fish showing clinical signs of IHNV infection, the N-gene was detected in 15 out of 36 (42%), while the G-gene was detected in 24 of 36 (67%) fish tested. Using a viral plaque assay, virus was detected in 4 of 24 (17%) apparently healthy fish and 33 of 36 (92%) fish showing clinical sign of IHNV infection. The higher level of IHNV detection by plaque assay compared to real-time RT-PCR might be due to the presence of more than one isolate in the field samples, and the inability to detect all the IHNV isolates using the current set of primers used in real-time RT-PCR. In conclusion, we developed a real-time RT-PCR assay for the detection and quantification of IHNV by SYBR Green real-time RT-PCR. This study demonstrates the potential of using fin clip as a non-invasive tissue source for detecting IHNV and possibly other viruses infecting salmonids in commercial aquaculture and in the field.

Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi . Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up- or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase (A. R. Soto, H. Zheng, D. Shoemaker, J. Rodriguez, B. A. Read, and T. M. Wahlund, Appl. Environ. Microbiol. 72:5500-5511, 2006). Differentially regulated genes include those related to cellular metabolism, ion channels, transport proteins, vesicular trafficking, and cell signaling. The putative function of the vast majority of candidate transcripts could not be defined. Nonetheless, the data described herein represent profiles of the transcription changes associated with biomineralization-related pathways in E. huxleyi and have identified novel and potentially useful targets for more detailed analysis.

The marine coccolithophorid Emiliania huxleyi is a cosmopolitan alga intensely studied in relation to global carbon cycling, biogeochemistry, marine ecology, and biomineralization processes. The biomineralization capabilities of coccolithophorids have attracted the attention of scientists interested in exploiting this ability for the development of materials science and biomedical and biotechnological applications. Although it has been well documented that biomineralization in E. huxleyi is promoted by growth under phosphate-limited conditions, the genes and proteins that govern the processes of calcification and coccolithogenesis remain unknown. Suppressive subtractive hybridization (SSH) libraries were constructed from cultures grown in phosphate-limited and phosphate-replete media as tester and driver populations for reciprocal SSH procedures. Positive clones from each of the two libraries were randomly selected, and dot blotting was performed for the analysis of expression patterns. A total of 513 clones from the phosphate-replete library and 423 clones from the phosphate-limited library were sequenced, assembled, and compared to sequences in GenBank using BLASTX. Of the 103 differentially expressed gene fragments from the phosphate-replete library, 34% showed significant homology to other known proteins, while only 23% of the 65 differentially expressed gene fragments from the phosphate-limited library showed homology to other proteins. To further assess mRNA expression, real-time RT-PCR analysis was employed and expression profiles were generated over a 14-day time course for three clones from the phosphate-replete library and five clones from the phosphate-limited library. The fragments isolated provide the basis for future cloning of full-length genes and functional analysis.