Triplicate PCR Reactions for 16S rRNA Gene Amplicon Sequencing are Unnecessary
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Marotz, Clarisse↗
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In Vivo Entombment of Bacteria and Fungi during Calcium Oxalate, Brushite, and Struvite Urolithiasis
Human kidney stones form via repeated events of mineral precipitation, partial dissolution, and reprecipitation, which are directly analogous to similar processes in other natural and manmade environments, where resident microbiomes strongly influence biomineralization. High-resolution microscopy and high-fidelity metagenomic (microscopy-to-omics) analyses, applicable to all forms of biomineralization, have been applied to assemble definitive evidence of in vivo microbiome entombment during urolithiasis. Stone fragments were collected from a randomly chosen cohort of 20 patients using standard percutaneous nephrolithotomy (PCNL). Fourier transform infrared (FTIR) spectroscopy indicated that 18 of these patients were calcium oxalate (CaOx) stone formers, whereas one patient formed each formed brushite and struvite stones. This apportionment is consistent with global stone mineralogy distributions. Stone fragments from seven of these 20 patients (five CaOx, one brushite, and one struvite) were thin sectioned and analyzed using brightfield (BF), polarization (POL), confocal, super-resolution autofluorescence (SRAF), and Raman techniques. DNA from remaining fragments, grouped according to each of the 20 patients, were analyzed with amplicon sequencing of 16S rRNA gene sequences (V1–V3, V3–V5) and internal transcribed spacer (ITS1, ITS2) regions. Bulk-entombed DNA was sequenced from stone fragments in 11 of the 18 patients who formed CaOx stones, and the patients who formed brushite and struvite stones. These analyses confirmed the presence of an entombed low-diversity community of bacteria and fungi, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Aspergillus niger. Bacterial cells approximately 1 μm in diameter were also optically observed to be entombed and well preserved in amorphous hydroxyapatite spherules and fans of needle-like crystals of brushite and struvite. In conclusion, these results indicate a microbiome is entombed during in vivo CaOx stone formation. Similar processes are implied for brushite and struvite stones. This evidence lays the groundwork for future in vitro and in vivo experimentation to determine how the microbiome may actively and/or passively influence kidney stone biomineralization.
59 BASIC BIOLOGICAL SCIENCES↗
A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules
A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sublibraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis.
59 BASIC BIOLOGICAL SCIENCES↗
Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline
The dinitrogenase reductase gene ( nifH ) is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for nifH amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. Bioinformatic analysis of marker gene sequences also requires considerable expertise. In this study, we advance the state of the art for nifH analysis by evaluating nifH primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. Furthermore, the developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters. Based on our primer evaluation, we recommend the Ando primer set be used with a modified annealing temperature of 58°C, as this approach captured the largest diversity of nifH templates, including paralog cluster IV/V sequences. To improve nifH sequence analysis, we developed a computational pipeline which infers taxonomy and optionally filters out paralog sequences. In addition, we employed an empirical model to derive optimal operational taxonomic unit (OTU) cutoffs for the nifH gene at the species, genus, and family levels. A comprehensive workflow script named TaxADivA (TAXonomy Assignment and DIVersity Assessment) is provided to ease processing and analysis of nifH amplicons. Our approach is then validated through characterization of diazotroph communities across environmental gradients in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico, in a peat moss-dominated wetland, and in various plant compartments of a sugarcane field. IMPORTANCE: Nitrogen availability often limits ecosystem productivity, and nitrogen fixation, exclusive to prokaryotes, comprises a major source of nitrogen input that sustains food webs. The nifH gene, which codes for the iron protein of the nitrogenase enzyme, is the most widely established molecular marker for the study of nitrogen-fixing microorganisms (diazotrophs) in nature. In this study, a flexible sequencing/analysis pipeline, named TaxADivA, was developed for nifH amplicons produced by Illumina paired-end sequencing, and it enables an inference of taxonomy, performs clustering, and produces output in formats that may be used by programs that facilitate data exploration and analysis. Diazotroph diversity and community composition are linked to ecosystem functioning, and our results advance the phylogenetic characterization of diazotroph communities by providing empirically derived nifH similarity cutoffs for species, genus, and family levels. Thus, the utility of our pipeline is validated for diazotroph communities in a variety of ecosystems, including contaminated beach sands, peatland ecosystems, living plant tissues, and rhizosphere soil.
59 BASIC BIOLOGICAL SCIENCES↗
Shifts of tundra bacterial and archaeal communities along a permafrost thaw gradient in A laska
Abstract Understanding the response of permafrost microbial communities to climate warming is crucial for evaluating ecosystem feedbacks to global change. This study investigated soil bacterial and archaeal communities by Illumina MiSeq sequencing of 16S r RNA gene amplicons across a permafrost thaw gradient at different depths in Alaska with thaw progression for over three decades. Over 4.6 million passing 16S r RNA gene sequences were obtained from a total of 97 samples, corresponding to 61 known classes and 470 genera. Soil depth and the associated soil physical–chemical properties had predominant impacts on the diversity and composition of the microbial communities. Both richness and evenness of the microbial communities decreased with soil depth. Acidobacteria, Verrucomicrobia, Alpha‐ and Gamma‐Proteobacteria dominated the microbial communities in the upper horizon, whereas abundances of Bacteroidetes, Delta‐Proteobacteria and Firmicutes increased towards deeper soils. Effects of thaw progression were absent in microbial communities in the near‐surface organic soil, probably due to greater temperature variation. Thaw progression decreased the abundances of the majority of the associated taxa in the lower organic soil, but increased the abundances of those in the mineral soil, including groups potentially involved in recalcitrant C degradation (Actinomycetales, Chitinophaga , etc.). The changes in microbial communities may be related to altered soil C sources by thaw progression. Collectively, this study revealed different impacts of thaw in the organic and mineral horizons and suggests the importance of studying both the upper and deeper soils while evaluating microbial responses to permafrost thaw.
Deng, Jie↗
Functional Role of Infective Viral Particles on Metal Reduction
A proposed strategy for the remediation of uranium (U) contaminated sites was based on the immobilization of U by reducing the oxidized soluble U, U(VI), to form a reduced insoluble end product, U(IV). Previous studies identified Geobacter sp., including G. sulfurreducens and G. metallireducens, as predominant U(VI)-reducing bacteria under acetate-oxidizing and U(VI)-reducing conditions. Examination of the finished genome sequence annotation of the canonical metal reducing species Geobacter sulfurreducens strain PCA and G. metallireduceans strain GS-15 as well as the draft genome sequence of G. uraniumreducens strain Rf4 identified phage related proteins. In addition, the completed genome for Anaeromyxobacter dehalogenans and the draft genome sequence of Desulfovibrio desulfuricans strain G20, two more model metal-reducing bacteria, also revealed phage related sequences. The presence of these gene sequences indicated that Geobacter spp., Anaeromyxobacter spp., and Desulfovibrio spp. are susceptible to viral infection. Furthermore, viral populations in soils and sedimentary environments in the order of 6.4×10{sup 6}–2.7×10{sup 10} VLP’s cm{sup -3} have been observed. In some cases, viral populations exceed bacterial populations in these environments suggesting that a relationship may exist between viruses and bacteria. Our preliminary screens of samples collected from the ESR FRC indicated that viral like particles were observed in significant numbers. The objective of this study was to investigate the potential functional role viruses play in metal reduction specifically Fe(III) and U(VI) reduction, the environmental parameters affecting viral infection of metal reducing bacteria, and the subsequent effects on U transport.
11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗
Whole Genome Analyses Suggests that Burkholderia sensu lato Contains Two Additional Novel Genera ( Mycetohabitans gen. nov., and Trinickia gen. nov.): Implications for the Evolution of Diazotrophy and Nodulation in the Burkholderiaceae
Burkholderia sensu lato is a large and complex group, containing pathogenic, phytopathogenic, symbiotic and non-symbiotic strains from a very wide range of environmental (soil, water, plants, fungi) and clinical (animal, human) habitats. Its taxonomy has been evaluated several times through the analysis of 16S rRNA sequences, concantenated 4–7 housekeeping gene sequences, and lately by genome sequences. Currently, the division of this group into Burkholderia , Caballeronia , Paraburkholderia , and Robbsia is strongly supported by genome analysis. These new genera broadly correspond to the various habitats/lifestyles of Burkholderia s.l., e.g., all the plant beneficial and environmental (PBE) strains are included in Paraburkholderia (which also includes all the N 2 -fixing legume symbionts) and Caballeronia , while most of the human and animal pathogens are retained in Burkholderia sensu stricto. However, none of these genera can accommodate two important groups of species. One of these includes the closely related Paraburkholderia rhizoxinica and Paraburkholderia endofungorum , which are both symbionts of the fungal phytopathogen Rhizopus microsporus .
59 BASIC BIOLOGICAL SCIENCES↗
Genetic Analysis of the Full-Length gag Gene from the Earliest Korean Subclade B of HIV-1: An Outbreak among Korean Hemophiliacs
We determined the earliest full-length HIV-1 gag gene sequences in 110 patients with HIV-1, including 20 hemophiliacs (HPs) and 90 local controls (LCs). The gag gene from stored sera was amplified using RT-PCR, and was subjected to direct sequencing. Phylogenetic analysis indicated that 94 and 16 sequences belonged to the Korean subclade of HIV-1 subtype B (KSB) and subtype B, respectively. A total of 12 signature pattern amino acids were found within the KSB, distinct from the worldwide consensus of subtype B. Within the KSB, the gag gene sequences from donors O and P and those from the 20 HPs comprised two subclusters. In particular, sequences from donor O strongly clustered with those of eight HPs. Moreover, signature pattern analysis indicated that 14 signature nucleotides were shared between the HPs and LCs within KSB (p < 0.01). Among the 14 nucleotides, positions 9 and 5 belonged to clusters O and P, respectively. In conclusion, signature pattern analysis for the gag gene revealed 12 signature pattern residues within the KSB and also confirmed the previous conclusion that the 20 HPs were infected with viruses due to incompletely inactivated clotting factor IX. This study is the first genetic analysis of the HIV-1 gag gene in Korea.
59 BASIC BIOLOGICAL SCIENCES↗
An Independent Genome Duplication Inferred from Hox Paralogs in the American Paddlefish—A Representative Basal Ray-Finned Fish and Important Comparative Reference
Vertebrates have experienced two rounds of whole-genome duplication (WGD) in the stem lineages of deep nodes within the group and a subsequent duplication event in the stem lineage of the teleosts—a highly diverse group of ray-finned fishes. Here, we present the first full Hox gene sequences for any member of the Acipenseriformes, the American paddlefish, and confirm that an independent WGD occurred in the paddlefish lineage, approximately 42 Ma based on sequences spanning the entire HoxA cluster and eight genes on the HoxD gene cluster. These clusters comprise different HOX loci and maintain conserved synteny relative to bichir, zebrafish, stickleback, and pufferfish, as well as human, mouse, and chick. We also provide a gene genealogy for the duplicated fzd8 gene in paddlefish and present evidence for the first Hox14 gene in any ray-finned fish. Taken together, these data demonstrate that the American paddlefish has an independently duplicated genome. Substitution patterns of the “alpha” paralogs on both the HoxA and HoxD gene clusters suggest transcriptional inactivation consistent with functional diploidization. Further, there are similarities in the pattern of sequence divergence among duplicated Hox genes in paddlefish and teleost lineages, even though they occurred independently approximately 200 Myr apart. We highlight implications on comparative analyses in the study of the “fin-limb transition” as well as gene and genome duplication in bony fishes, which includes all ray-finned fishes as well as the lobe-finned fishes and tetrapod vertebrates.
59 BASIC BIOLOGICAL SCIENCES↗
Mining Thermophile Photosynthesis Genes: A Synthetic Operon Expressing Chloroflexota Species Reaction Center Genes in Rhodobacter sphaeroides
Photosynthesis is the foundation of the vast majority of life systems, and is therefore the most important bioenergetic process on earth. The greatest diversity of photosynthetic systems is found in microorganisms. However, our understanding of the biophysical and biochemical processes that transduce light into chemical energy is derived from a relatively small subset of proteins from microbes that are amenable to cultivation, in contrast to the huge number of predicted proteins that catalyze the initial photochemical reactions deposited in databases, such as from metagenomics. We describe the use of a Rhodobacter sphaeroides laboratory strain for the expression of heterologous photosynthesis genes to demonstrate the feasibility of mining this resource, focusing on hot spring Chloroflexota gene sequences. Using a synthetic operon of genes, we produced a photochemically active complex of reaction center proteins in our biological system. We also present bioinformatic analyses of anoxygenic type II reaction center sequences from metagenomic samples collected from hot (42–90 °C) springs available through the JGI IMG database, to generate a resource of diverse sequences that are potentially adapted to photosynthesis at such temperatures. These data provide a view into the natural diversity of anoxygenic photosynthesis, through a lens focused on high-temperature environments. The approach we took to express such genes can be applied for potential biotechnology purposes as well as for studies of fundamental catalytic properties of these heretofore inaccessible protein complexes.
Chloroflexota↗
jialiu232/MetaFunPrimer_paper_info
Genes belonging to the same functional group may include numerous and variable gene sequences, making characterizing and quantifying difficult. Therefore, high-throughput design tools are needed to simultaneously create primers for improved quantification of target genes. We developed MetaFunPrimer, a bioinformatic pipeline, to design primers for numerous genes of interest. This tool also enables gene target prioritization based on ranking the presence of genes in user-defined references, such as environment-specific metagenomes. Given inputs of protein and nucleotide sequences for gene targets of interest and an accompanying set of reference metagenomes or genomes, MetaFunPrimer generates primers for ranked genes of interest. To demonstrate the usage and benefits of MetaFunPrimer, a total of 78 primer pairs were designed to target observed ammonia monooxygenase subunit A (amoA) genes of ammonia-oxidizing bacteria (AOB) in 1,550 publicly available soil metagenomes. We demonstrate computationally that these amoA-AOB primers can cover 94% of the amoA-AOB genes observed in the 1,550 soil metagenomes compared with a 49% estimated coverage by previously published primers. Finally, we verified the utility of these primer sets in incubation experiments that used long-term nitrogen fertilized or unfertilized soils. High-throughput quantitative PCR (qPCR) results and statistical analyses showed significant differences in relative quantification patterns between the two soils, and subsequent absolute quantifications also confirmed that target genes enumerated by six selected primer pairs were significantly more abundant in the nitrogen-fertilized soils. This new tool gives microbial ecologists a new approach to assess functional gene abundance and related microbial community dynamics quickly and affordably.
Liu, Jia↗
CIP1 polypeptides and their uses
Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.
59 BASIC BIOLOGICAL SCIENCES↗
Trichoderma genes
Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.
59 BASIC BIOLOGICAL SCIENCES↗
Hanseniaspora smithiae sp. nov., a Novel Apiculate Yeast Species From Patagonian Forests That Lacks the Typical Genomic Domestication Signatures for Fermentative Environments
During a survey of Nothofagus trees and their parasitic fungi in Andean Patagonia (Argentina), genetically distinct strains of Hanseniaspora were obtained from the sugar-containing stromata of parasitic Cyttaria spp. Phylogenetic analyses based on the single-gene sequences (encoding rRNA and actin) or on conserved, single-copy, orthologous genes from genome sequence assemblies revealed that these strains represent a new species closely related to Hanseniaspora valbyensis. Additionally, delimitation of this novel species was supported by genetic distance calculations using overall genome relatedness indices (OGRI) between the novel taxon and its closest relatives. To better understand the mode of speciation in Hanseniaspora , we examined genes that were retained or lost in the novel species in comparison to its closest relatives. These analyses show that, during diversification, this novel species and its closest relatives, H. valbyensis and Hanseniaspora jakobsenii , lost mitochondrial and other genes involved in the generation of precursor metabolites and energy, which could explain their slower growth and higher ethanol yields under aerobic conditions. Similarly, Hanseniaspora mollemarum lost the ability to sporulate, along with genes that are involved in meiosis and mating. Based on these findings, a formal description of the novel yeast species Hanseniaspora smithiae sp. nov. is proposed, with CRUB 1602 H as the holotype.
59 BASIC BIOLOGICAL SCIENCES↗
Pseudomonas atagosis sp. nov., and Pseudomonas akappagea sp. nov., New Soil Bacteria Isolated from Samples on the Volcanic Island Izu Oshima, Tokyo
During the exploration of microbial natural resources, two strains of Pseudomonas, PS14 T and PS24 T , were isolated from samples taken from Izu Oshima, a volcanic island located 120 km southwest of central Tokyo. Phylogenetic analysis based on 16S rRNA gene sequences showed that PS14 T was most similar to Pseudomonas baetica a390T (99.6%) and Pseudomonas helmanticensis OHA11 T (99.5%), and that PS24T was most similar to Pseudomonas qingdaonensis JJ3 T (98.8%) and Pseudomonas lutea OK2 T (98.7%). The major fatty acids of these two strains were C 16:0 and C 17:0 cyclo, summed feature 3 (C 16:1 ω6c and/or C 16:1 ω7c), and summed feature 8 (C18:1 ω7c and/or 18:1 ω6c). The phylogenetic analyses, DNA-DNA hybridization results and phenotypic traits indicated that PS14 T and PS24 T constitute two novel species, Pseudomonas atagosis sp. nov. (type strain PS14 T = CECT 9940 T , = LMG 31496 T ) and Pseudomonas akappagea sp. nov. (type strain PS24T = CECT 9941T, = LMG 31497T), respectively. The sequence data of the draft genomes of PS14 T and PS24 T were deposited in the GenBank database under accession numbers VXCA00000000 and VXCP00000000, respectively, and the sequence data of their 16S rRNA genes were deposited in the GenBank database under accession numbers MN396717 and MN382268, respectively.
59 BASIC BIOLOGICAL SCIENCES↗
Previously uncharacterized rectangular bacterial structures in the dolphin mouth
Much remains to be explored regarding the diversity of uncultured, host-associated microbes. Here, we describe rectangular bacterial structures (RBSs) in the mouths of bottlenose dolphins. DNA staining revealed multiple paired bands within RBSs, suggesting the presence of cells dividing along the longitudinal axis. Cryogenic transmission electron microscopy and tomography showed parallel membrane-bound segments that are likely cells, encapsulated by an S-layer-like periodic surface covering. RBSs displayed unusual pilus-like appendages with bundles of threads splayed at the tips. We present multiple lines of evidence, including genomic DNA sequencing of micromanipulated RBSs, 16S rRNA gene sequencing, and fluorescence in situ hybridization, suggesting that RBSs are bacterial and distinct from the genera Simonsiella and Conchiformibius (family Neisseriaceae), with which they share similar morphology and division patterning. Our findings highlight the diversity of novel microbial forms and lifestyles that await characterization using tools complementary to genomics such as microscopy.
59 BASIC BIOLOGICAL SCIENCES↗
Seasonal succession leads to habitat-dependent differentiation in ribosomal RNA:DNA ratios among freshwater lake bacteria
Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA gene sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. Furthermore, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors.
16S rRNA↗
Polynucleobacter meluiroseus sp. nov., a bacterium isolated from a lake located in the mountains of the Mediterranean island of Corsica
Strain AP-Melu-1000-B4 was isolated from a lake located in the mountains of the Mediterranean island of Corsica (France). Phenotypic, chemotaxonomic and genomic traits were investigated. Phylogenetic analyses based on 16S rRNA gene sequencing referred the strain to the cryptic species complex PnecC within the genus Polynucleobacter . The strain encoded genes for biosynthesis of proteorhodopsin and retinal. When pelleted by centrifugation the strain showed an intense rose colouring. Major fatty acids were C 16:1 ω7 c , C 16:0 , C 18:1 ω7 c and summed feature 2 (C 16:1 isoI and C 14:0 - 3OH). The sequence of the 16S rRNA gene contained an indel which was not present in any previously described Polynucleobacter species. Genome sequencing revealed a genome size of 1.89 Mbp and a G+C content of 46.6 mol%. In order to resolve the phylogenetic position of the new strain within subcluster PnecC, its phylogeny was reconstructed from sequences of 319 shared genes. To represent all currently described Polynucleobacter species by whole genome sequences, three type strains were additionally sequenced. Our phylogenetic analysis revealed that strain AP-Melu-100-B4 occupied a basal position compared with previously described PnecC strains. Pairwise determined whole genome average nucleotide identity (gANI) values suggested that strain AP-Melu-1000-B4 represents a new species, for which we propose the name Polynucleobacter meluiroseus sp. nov. with the type strain AP-Melu-1000-B4 T (=DSM 103591 T =CIP 111329 T )
59 BASIC BIOLOGICAL SCIENCES↗