The recent advent of DNA sequencing technologies facilitates the use of genome sequencing data that provide means for more informative and precise classification and identification of members of the Bacteria and Archaea. Because the current species definition is based on the comparison of genome sequences between type and other strains in a given species, building a genome database with correct taxonomic information is of paramount need to enhance our efforts in exploring prokaryotic diversity and discovering novel species as well as for routine identifications. Here we introduce an integrated database, called EzBioCloud, that holds the taxonomic hierarchy of the Bacteria and Archaea, which is represented by quality-controlled 16S rRNA gene and genome sequences. Whole-genome assemblies in the NCBI Assembly Database were screened for low quality and subjected to a composite identification bioinformatics pipeline that employs gene-based searches followed by the calculation of average nucleotide identity. As a result, the database is made of 61 700 species/phylotypes, including 13 132 with validly published names, and 62 362 whole-genome assemblies that were identified taxonomically at the genus, species and subspecies levels. Genomic properties, such as genome size and DNA G+C content, and the occurrence in human microbiome data were calculated for each genus or higher taxa. This united database of taxonomy, 16S rRNA gene and genome sequences, with accompanying bioinformatics tools, should accelerate genome-based classification and identification of members of the Bacteria and Archaea. The database and related search tools are available at www.ezbiocloud.net/.

Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies.

Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for "consolidated bioprocessing" (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

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Microbial cellulose utilization: fundamentals and biotechnology.

Evolution of Protein Molecules

The human oral cavity contains a number of different habitats, including the teeth, gingival sulcus, tongue, cheeks, hard and soft palates, and tonsils, which are colonized by bacteria. The oral microbiome is comprised of over 600 prevalent taxa at the species level, with distinct subsets predominating at different habitats. The oral microbiome has been extensively characterized by cultivation and culture-independent molecular methods such as 16S rRNA cloning. Unfortunately, the vast majority of unnamed oral taxa are referenced by clone numbers or 16S rRNA GenBank accession numbers, often without taxonomic anchors. The first aim of this research was to collect 16S rRNA gene sequences into a curated phylogeny-based database, the Human Oral Microbiome Database (HOMD), and make it web accessible (www.homd.org). The HOMD includes 619 taxa in 13 phyla, as follows: Actinobacteria, Bacteroidetes, Chlamydiae, Chloroflexi, Euryarchaeota, Firmicutes, Fusobacteria, Proteobacteria, Spirochaetes, SR1, Synergistetes, Tenericutes, and TM7. The second aim was to analyze 36,043 16S rRNA gene clones isolated from studies of the oral microbiota to determine the relative abundance of taxa and identify novel candidate taxa. The analysis identified 1,179 taxa, of which 24% were named, 8% were cultivated but unnamed, and 68% were uncultivated phylotypes. Upon validation, 434 novel, nonsingleton taxa will be added to the HOMD. The number of taxa needed to account for 90%, 95%, or 99% of the clones examined is 259, 413, and 875, respectively. The HOMD is the first curated description of a human-associated microbiome and provides tools for use in understanding the role of the microbiome in health and disease.

The Human Oral Microbiome

The naturally occurring genetic heterogeneity of autotrophic ammonia-oxidizing populations belonging to the beta subclass of the Proteobacteria was studied by using a newly developed PCR-based assay targeting a partial stretch of the gene which encodes the active-site polypeptide of ammonia monooxygenase (amoA). The PCR yielded a specific 491-bp fragment with all of the nitrifiers tested, but not with the homologous stretch of the particulate methane monooxygenase, a key enzyme of methane-oxidizing bacteria. The assay also specifically detected amoA in DNA extracted from various aquatic and terrestrial environments. The resulting PCR products retrieved from rice roots, activated sludge, a freshwater sample, and an enrichment culture were used for the generation of amoA gene libraries. No false positives were detected in a set of 47 randomly selected clone sequences that were analyzed further. The majority of the environmental sequences retrieved from rice roots and activated sludge grouped within the phylogenetic radiation defined by cultured strains of the genera Nitrosomonas and Nitrosospira. The comparative analysis identified members of both of these genera in activated sludge; however, only Nitrosospira-like sequences with very similar amino acid patterns were found on rice roots. Further differentiation of these molecular isolates was clearly possible on the nucleic acid level due to the accumulation of synonymous mutations, suggesting that several closely related but distinct Nitrosospira-like populations are the main colonizers of the rhizosphere of rice. Each of the amoA gene libraries obtained from the freshwater sample and the enrichment culture was dominated by a novel lineage that shared a branch with the Nitrosospira cluster but could not be assigned to any of the known pure cultures. Our data suggest that amoA represents a very powerful molecular tool for analyzing indigenous ammonia-oxidizing communities due to (i) its specificity, (ii) its fine-scale resolution of closely related populations, and (iii) the fact that a functional trait rather than a phylogenetic trait is detected.

The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations.

A new hierarchic classification structure for the taxa between the taxonomic levels of genus and class is Proposed for the actinomycete line of descent as defined by analysis of small subunit (16S) rRNA and genes coding for this molecule (rDNA). While the traditional circumscription of a genus of the actinomycete subphylum is by and large in accord with the 16S rRNA/rDNA-based phylogenetic clustering of these organisms. most of the higher taxa proposed in the past do not take into account the phylogenetic clustering of genera. The rich chemical, morphological and physiological diversity of phylogenetically closely related genera makes the description of families and higher taxa so broad that they become meaningless for the description of the enclosed taxa. Here we present a classification system in which phylogenetically neighboring taxa at the genus level are clustered into families, suborders, orders, subclasses, and a class irrespective of those phenotypec characteristics on which the delineation of taxa has been based in the past. Rather than being based on a listing of a wide array of chemotaxonomic, morphological, and physiological properties, the delineation is based solely on 16S rDNA/rRNA sequence-based phylogenetic clustering and the presence of taxon-specific 16S rDNA RNA signature nucleotides.

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Proposal for a New Hierarchic Classification System, Actinobacteria classis nov.

The genus Nocardiopsis was shown to be phylogenetically coherent and to represent a distinct lineage within the radiation of the order Actinomycetales. The closest relatives of the genus Nocardiopsis are members of the genera Actinomadura, Thermomonospora, Streptosporangium, and Microtetraspora. The intrageneric structure of the genus Nocardiopsis is shown to consist of a highly related species group containing Nocardiopsis dassonvillei, Nocardiopsis alborubida, and Nocardiopsis antarctica and a second group of less highly related species comprising Nocardiopsis alba subsp. alba, Nocardiopsis alba subsp. prasina, and Nocardiopsis listeri. Nocardiopsis lucentensis occupies a position intermediate between the two species groups. The results of a 16S ribosomal DNA sequence analysis are generally consistent with the available chemotaxonomic, phenotypic, and DNA-DNA hybridization data. The phylogenetic position and the morpho- and chemotaxonomic properties of Nocardiopsis species support the creation of a family for the genus Nocardiopsis, Nocardiopsaceae fam. nov.

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The Genus Nocardiopsis Represents a Phylogenetically Coherent Taxon and a Distinct Actinomycete Lineage: Proposal of Nocardiopsaceae fam. nov.

In order to assess the effect of genome size and number of 16S rRNA genes (rDNAs) on the quantities of PCR-generated partial 16S rDNA fragments, equimolar amounts of DNA from pairs of different species for which these parameters are known were subjected to gene amplification. The experimentally determined ratio of PCR products obtained, as determined by image analysis of SYBR-Green I-stained amplification products, corresponded well with the predicted ratio calculated from the number of rrn genes per equimolar amounts of DNA in mixtures of Escherichia coli and "Thermus thermophilus" and of Pseudomonas aeruginosa and "T. thermophilus." The values for the pair of Bacillus subtilis and "T. thermophilus" showed greater deviations from the predicted value. The dependence of the amount of 16S rDNA amplification product on these two parameters makes it impossible to quantify the number of species represented in 16S rDNA clone libraries of environmental samples as long as these two parameters are unknown for the species present.

Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species

Two strains of a new gram-positive coryneform bacterium isolated from soil and from a sandstone surface are described. Strain 2002-39/1T (T = type strain) is a coccoid, nonmotile, non-acid-fast, microaerophilic organism. The menaquinones of this strain are MK-12 and MK-11, and the main components of the whole-cell sugars are glucose and rhamnose. No mycolic acids are present. The G+C content of the DNA is 74 mol%. Comparative 16S ribosomal DNA studies and a cell wall analysis revealed that this strain represents a new genus belonging to the group of actinomycetes that have diaminobutyric acid in their peptidoglycans. The second strain, strain ST54, which was isolated from a sandstone surface, had the same characteristic features as strain 2002-39/1T. The name Agrococcus jenensis gen. nov., sp. nov., is proposed for these organisms. The type strain is strain 2002-39/1, which has been deposited in the German Collection of Microorganisms and Cell Cultures as strain DSM 9580.

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Agrococcus jenensis gen. nov., sp. nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall.

Many crude oil constituents are biodegradable in the presence of oxygen; however, a substantial anaerobic degradation has never been demonstrated. An unusually low content of n-alkanes in oils of certain deposits is commonly attributed to selective utilization of these hydrocarbons by aerobic microorganisms. On the other hand, oil wells and production fluids were shown to harbour anaerobic sulphate-reducing bacteria, but their actual electron donors and carbon sources were unknown. On the basis of nutritional properties of various bacterial isolates it was assumed that fatty acids and H2 are potential electron donors for sulphate reduction in situ. Here we demonstrate that hydrocarbons in crude oil are used directly by sulphate-reducing bacteria growing under strictly anoxic conditions. A moderately thermophilic pure culture selectively utilizes n-alkanes in oil for sulphate reduction to sulphide. In addition, a mesophilic sulphate-reducing enrichment culture is shown to oxidize alkylbenzenes in oil. Thus, sulphate-reducing bacteria utilizing aliphatic and aromatic hydrocarbons as electron donors may present a significant source of sulphide in oil deposits and oil production plants.

Fred A. Rainey

Papers

Proposal for a New Hierarchic Classification System, Actinobacteria classis nov.

The Genus Nocardiopsis Represents a Phylogenetically Coherent Taxon and a Distinct Actinomycete Lineage: Proposal of Nocardiopsaceae fam. nov.

Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species

Agrococcus jenensis gen. nov., sp. nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall.

Anaerobic oxidation of hydrocarbons in crude oil by new types of sulphate-reducing bacteria