It is time for a new type of type to facilitate naming the microbial world.

Since January 1, 2001, the only acceptable nomenclatural type for species under the International Code of Nomenclature of Prokaryotes (ICNP) has been pure cultures. Here, we argue that this requirement is discordant with the more inclusive nature of nomenclatural types accepted under other codes of nomenclature and posit that the unique rigidity of the ICNP has failed to serve the broad research community and has stifled progress. This case is based on the axiom that many archaea and bacteria are interdependent in nature and therefore difficult, if not impossible, to grow, preserve, and distribute as pure cultures. As such, a large proportion of Earth's biodiversity cannot be named under the current system, which limits our ability to communicate about microbial diversity within and beyond the microbiology research community. Genome sequence data are now encouraged for valid publication of new taxa in microbial systematics journals, and metagenome-assembled genomes and single cell-amplified genomes are being generated rapidly from every biome on Earth. Thus, genome sequences are available for both cultivated and uncultivated microorganisms and can readily serve as a new category of nomenclatural type, allowing for a unified nomenclature for all archaea and bacteria, whether or not they are available as pure cultures. Ideally this would be under a single code of nomenclature but, as we review here, the newly established SeqCode will operate in parallel with the ICNP as a first step toward this goal.

Potential role of Thermus thermophilus and T. oshimai in high rates of nitrous oxide (N2O) production in ∼80 °C hot springs in the US Great Basin.

Ambient nitrous oxide (N(2)O) emissions from Great Boiling Spring (GBS) in the US Great Basin depended on temperature, with the highest flux, 67.8 ± 2.6 µmol N(2)O-N m(-2) day(-1) , occurring in the large source pool at 82 °C. This rate of N(2)O production contrasted with negligible production from nearby soils and was similar to rates from soils and sediments impacted with agricultural fertilizers. To investigate the source of N(2)O, a variety of approaches were used to enrich and isolate heterotrophic micro-organisms, and isolates were screened for nitrate reduction ability. Nitrate-respiring isolates were identified by 16S rRNA gene sequencing as Thermus thermophilus (31 isolates) and T. oshimai (three isolates). All isolates reduced nitrate to N(2)O but not to dinitrogen and were unable to grow with N(2)O as a terminal electron acceptor. Representative T. thermophilus and T. oshimai strains contained genes with 96-98% and 93% DNA identity, respectively, to the nitrate reductase catalytic subunit gene (narG) of T. thermophilus HB8. These data implicate T. thermophilus and T. oshimai in high flux of N(2)O in GBS and raise questions about the genetic basis of the incomplete denitrification pathway in these organisms and on the fate of biogenic N(2)O in geothermal environments.

Microbiology and geochemistry of Little Hot Creek, a hot spring environment in the Long Valley Caldera.

A culture-independent community census was combined with chemical and thermodynamic analyses of three springs located within the Long Valley Caldera, Little Hot Creek (LHC) 1, 3, and 4. All three springs were approximately 80 degrees C, circumneutral, apparently anaerobic and had similar water chemistries. 16S rRNA gene libraries constructed from DNA isolated from spring sediment revealed moderately diverse but highly novel microbial communities. Over half of the phylotypes could not be grouped into known taxonomic classes. Bacterial libraries from LHC1 and LHC3 were predominantly species within the phyla Aquificae and Thermodesulfobacteria, while those from LHC4 were dominated by candidate phyla, including OP1 and OP9. Archaeal libraries from LHC3 contained large numbers of Archaeoglobales and Desulfurococcales, while LHC1 and LHC4 were dominated by Crenarchaeota unaffiliated with known orders. The heterogeneity in microbial populations could not easily be attributed to measurable differences in water chemistry, but may be determined by availability of trace amounts of oxygen to the spring sediments. Thermodynamic modeling predicted the most favorable reactions to be sulfur and nitrate respirations, yielding 40-70 kJ mol(-1) e(-) transferred; however, levels of oxygen at or below our detection limit could result in aerobic respirations yielding up to 100 kJ mol(-1) e(-) transferred. Important electron donors are predicted to be H(2), H(2)S, S(0), Fe(2+) and CH(4), all of which yield similar energies when coupled to a given electron acceptor. The results indicate that springs associated with the Long Valley Caldera contain microbial populations that show some similarities both to springs in Yellowstone and springs in the Great Basin.

Remarkable morphological diversity of viruses and virus-like particles in hot terrestrial environments.

Electron microscopic studies of the viruses in two hot springs (85 degrees C, pH 1.5-2.0, and 75-93 degrees C, pH 6.5) in Yellowstone National Park revealed particles with twelve different morphotypes. This diversity encompassed known viruses of hyperthermophilic archaea, filamentous Lipothrixviridae, rod-shaped Rudiviridae, and spindle-shaped Fuselloviridae, and novel morphotypes previously not observed in nature. Two virus types resembled head-and-tail bacteriophages from the families Siphoviridae and Podoviridae, and constituted the first observation of these viruses in a hydrothermal environment. Viral hosts in the acidic spring were members of the hyperthermophilic archaeal genus Acidianus.

Marinobacter strain NCE312 has a Pseudomonas-like naphthalene dioxygenase.

One strain of bacteria, designated NCE312, was isolated from a naphthalene-digesting chemostat culture that was inoculated with creosote-contaminated marine sediment. The strain was isolated based on its ability to grow using naphthalene as a sole carbon source. In addition, the strain degraded 2-methylnaphthalene and 1-methylnaphthalene. Analysis of a 16S rRNA gene sequence from NCE312 placed the isolate in the genus Marinobacter. Degenerate PCR primers were used to amplify a fragment of a naphthalene 1,2-dioxygenase large subunit gene. A phylogenetic analysis indicated the Marinobacter naphthalene dioxygenase is similar to those from Pseudomonas and Burkholderia strains suggesting that the dioxygenase gene may have been transferred horizontally between these lineages of bacteria.

Vibrio cyclotrophicus sp. nov., a polycyclic aromatic hydrocarbon (PAH)-degrading marine bacterium.

Strain P-2P44T was isolated from creosote-contaminated marine sediments by using a most-probable number procedure in which phenanthrene was the sole carbon and energy source. Growth experiments showed that P-2P44T utilized several two- and three-ring polycyclic aromatic hydrocarbons (PAHs) as substrates, including naphthalene, 2-methylnaphthalene and phenanthrene. Additionally, gas-chromatography experiments showed that P-2P44T degraded several other PAHs, though it was unable to use them as sole sources of carbon and energy. Phylogenetic analyses confirmed that strain P-2P44T is a member of the genus Vibrio, most closely related to Vibrio splendidus. However, strain P-2P44T shared only 98.3% 16S rDNA identity and 35% DNA-DNA reassociation with the type strain of V. splendidus. Strain P-2P44T differed phenotypically from V. splendidus. Together, these differences indicated that strain P-2P44T represents a novel species in the genus Vibrio, for which the name Vibrio cyclotrophicus sp. nov. is proposed; the type strain is P-2P44T (= ATCC 700982T = PICC 106644T).

Anaerobic naphthalene degradation by microbial pure cultures under nitrate-reducing conditions.

Pure bacterial cultures were isolated from a highly enriched denitrifying consortium previously shown to anaerobically biodegrade naphthalene. The isolates were screened for the ability to grow anaerobically in liquid culture with naphthalene as the sole source of carbon and energy in the presence of nitrate. Three naphthalene-degrading pure cultures were obtained, designated NAP-3-1, NAP-3-2, and NAP-4. Isolate NAP-3-1 tested positive for denitrification using a standard denitrification assay. Neither isolate NAP-3-2 nor isolate NAP-4 produced gas in the assay, but both consumed nitrate and NAP-4 produced significant amounts of nitrite. Isolates NAP-4 and NAP-3-1 transformed 70 to 90% of added naphthalene, and the transformation was nitrate dependent. No significant removal of naphthalene occurred under nitrate-limited conditions or in cell-free controls. Both cultures exhibited partial mineralization of naphthalene, representing 7 to 20% of the initial added (14)C-labeled naphthalene. After 57 days of incubation, the largest fraction of the radiolabel in both cultures was recovered in the cell mass (30 to 50%), with minor amounts recovered as unknown soluble metabolites. Nitrate consumption, along with the results from the (14)C radiolabel study, are consistent with the oxidation of naphthalene coupled to denitrification for NAP-3-1 and nitrate reduction to nitrite for NAP-4. Phylogenetic analyses based on 16S ribosomal DNA sequences of NAP-3-1 showed that it was closely related to Pseudomonas stutzeri and that NAP-4 was closely related to Vibrio pelagius. This is the first report we know of that demonstrates nitrate-dependent anaerobic degradation and mineralization of naphthalene by pure cultures.

Comparative phylogenetic analyses of members of the order Planctomycetales and the division Verrucomicrobia: 23S rRNA gene sequence analysis supports the 16S rRNA gene sequence-derived phylogeny.

Almost complete 23S rRNA gene sequences were obtained from 13 planctomycete strains, the fimbriated, prosthecate bacterium Verrucomicrobium spinosum and two strains of the genus Prosthecobacter. The 23S rRNA genes were amplified by the PCR, using modified primers. The majority of the planctomycete strains investigated were shown to have 23S rRNA genes that were not linked to the 16S rRNA genes. Amplification of the 5'-termini of these genes was achieved using a novel primer-design strategy. Comparative phylogenetic analyses were performed using the 23S rRNA gene sequences determined in this study and previously determined 16S rRNA gene sequences. The phylogenetic dendrograms constructed from both datasets showed that the planctomycetes form a coherent group and distinct lineage within the domain Bacteria. Analysis of 23S rRNA gene sequences of Verrucomicrobium spinosum, Prosthecobacter fusiformis and Prosthecobacter sp. strain FC-2 showed that these organisms cluster together, as was also shown here and previously by analysis of 16S rRNA gene sequences. The distinct phylogenetic position of the division Verrucomicrobia was also supported by analysis of the 23S rRNA gene sequences, and no statistically significant phylogenetic relationship between the division Verrucomicrobia and the planctomycetes was found. The analyses presented in this study also provide further evidence that the chlamydiae are no more related to members of the order Planctomycetales and the division Verrucomicrobia than they are to members of other bacterial lineages.

Polycyclic aromatic hydrocarbon degradation by a new marine bacterium, Neptunomonas naphthovorans gen. nov., sp. nov.

Two strains of bacteria were isolated from creosote-contaminated Puget Sound sediment based on their ability to utilize naphthalene as a sole carbon and energy source. When incubated with a polycyclic aromatic hydrocarbon (PAH) compound in artificial seawater, each strain also degraded 2-methylnaphthalene and 1-methylnaphthalene; in addition, one strain, NAG-2N-113, degraded 2,6-dimethylnaphthalene and phenanthrene. Acenaphthene was not degraded when it was used as a sole carbon source but was degraded by both strains when it was incubated with a mixture of seven other PAHs. Degenerate primers and the PCR were used to isolate a portion of a naphthalene dioxygenase iron-sulfur protein (ISP) gene from each of the strains. A phylogenetic analysis of PAH dioxygenase ISP deduced amino acid sequences showed that the genes isolated in this study were distantly related to the genes encoding naphthalene dioxygenases of Pseudomonas and Burkholderia strains. Despite the differences in PAH degradation phenotype between the new strains, the dioxygenase ISP deduced amino acid fragments of these organisms were 97.6% identical. 16S ribosomal DNA-based phylogenetic analysis placed these bacteria in the gamma-3 subgroup of the Proteobacteria, most closely related to members of the genus Oceanospirillum. However, morphologic, physiologic, and genotypic differences between the new strains and the oceanospirilla justify the creation of a novel genus and species, Neptunomonas naphthovorans. The type strain of N. naphthovorans is strain NAG-2N-126.

Isolation of marine polycyclic aromatic hydrocarbon (PAH)-degrading Cycloclasticus strains from the Gulf of Mexico and comparison of their PAH degradation ability with that of puget sound Cycloclasticus strains.

Phenanthrene- and naphthalene-degrading bacteria were isolated from four offshore and nearshore locations in the Gulf of Mexico by using a modified most-probable-number technique. The concentrations of these bacteria ranged from 10(2) to 10(6) cells per ml of wet surficial sediment in mildly contaminated and noncontaminated sediments. A total of 23 strains of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were obtained. Based on partial 16S ribosomal DNA sequences and phenotypic characteristics, these 23 strains are members of the genus Cycloclasticus. Three representatives were chosen for a complete phylogenetic analysis, which confirmed the close relationship of these isolates to type strain Cycloclasticus pugetii PS-1, which was isolated from Puget Sound. PAH substrate utilization tests which included high-molecular-weight PAHs revealed that these isolates had similar, broad substrate ranges which included naphthalene, substituted naphthalenes, phenanthrene, biphenyl, anthracene, acenaphthene, and fluorene. Degradation of pyrene and fluoranthene occurred only when the strains were incubated with phenanthrene. Two distinct partial PAH dioxygenase iron sulfur protein (ISP) gene sequences were PCR amplified from Puget Sound and Gulf of Mexico Cycloclasticus strains. Phylogenetic analyses of these sequences revealed that one ISP type is related to the bph type of ISP sequences, while the other ISP type is related to the nah type of ISP sequences. The predicted ISP amino acid sequences for the Gulf of Mexico and Puget Sound strains are identical, which supports the hypothesis that these geographically separated isolates are closely related phylogentically. Cycloclasticus species appear to be numerically important and widespread PAH-degrading bacteria in both Puget Sound and the Gulf of Mexico.

Verrucomicrobia div. nov., a new division of the bacteria containing three new species of Prosthecobacter.

Four strains of nonmotile, prosthecate bacteria were isolated in the 1970s and assigned to the genus Prosthecobacter. These strains were compared genotypically by DNA/DNA reassociation and 16S rDNA based phylogenetic analyses. Genotypic comparisons were complemented with phenotypic characterizations. Together, these studies clearly indicate each Prosthecobacter strain represents a novel species of bacteria. We propose three new species of Prosthecobacter, P. dejongeii strain FC1, P. vanneervenii strain FC2, and P. debontii strain FC3; P. fusiformis is reserved for the type strain of the genus, strain FC4. Additionally, we propose the genera Prosthecobacter and Verrucomicrobium, currently members of the order Verrucomicrobiales, to comprise a novel higher order taxonomic group, the division Verrucomicrobia div. nov. and the class Verrumicrobiae class nov. Many novel members of the Verrucomicrobia, as revealed by molecular ecology studies, await isolation and description.

Phylogeny of Prosthecobacter, the fusiform caulobacters: members of a recently discovered division of the bacteria.

Prosthecobacter fusiformis is morphologically similar to caulobacters; however, it lacks a dimorphic life cycle. To determine the relatedness of the genus Prosthecobacter to dimorphic caulobacters and other prosthecate members of the alpha subgroup of the Proteobacteria (alpha-Proteobacteria), we isolated and sequenced 16S rRNA genes from four Prosthecobacter strains. Surprisingly, the results of phylogenetic analyses placed the fusiform caulobacters in a deeply rooted division of the Bacteria that was most closely affiliated with the Planctomyces-Chlamydia group and only distantly related to the alpha-Proteobacteria. The genus Prosthecobacter shares a common lineage in this division with Verrucomicrobium spinosum, a polyprosthecate, heterotrophic bacterium. Consistent with this phylogenetic placement, menaquinones were isolated from Prosthecobacter strains and menaquinones have been isolated from Verrucomicrobium strains and planctomycetes but not from members of the alpha-Proteobacteria. Thus, the genus Prosthecobacter is a second genus in the recently described order Verrucomicrobiales. Members of the genus Prosthecobacter are susceptible to beta-lactam antibiotics and contain mesodiaminopimelic acid, indicating that they, unlike members of the Planctomycetales or Chlamydiales, have peptidoglycan cell walls. This major phenotypic difference, together with the phylogenetic independence of the verrucomicrobia, indicates that these bacteria and the sources of related 16S ribosomal DNAs obtained from soils, freshwater, and the marine pelagic environment represent an unrecognized division of the Bacteria.