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The assembly of single-amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) has led to a surge in genome-based discoveries of members affiliated with Archaea and Bacteria, bringing with it a need to develop guidelines for nomenclature of uncultivated microorganisms. The International Code of Nomenclature of Prokaryotes (ICNP) only recognizes cultures as 'type material', thereby preventing the naming of uncultivated organisms. In this Consensus Statement, we propose two potential paths to solve this nomenclatural conundrum. One option is the adoption of previously proposed modifications to the ICNP to recognize DNA sequences as acceptable type material; the other option creates a nomenclatural code for uncultivated Archaea and Bacteria that could eventually be merged with the ICNP in the future. Regardless of the path taken, we believe that action is needed now within the scientific community to develop consistent rules for nomenclature of uncultivated taxa in order to provide clarity and stability, and to effectively communicate microbial diversity.

Anaerobic microbial hydrocarbon degradation is a major biogeochemical process at marine seeps. Here we studied the response of the microbial community to petroleum seepage simulated for 190 days in a sediment core from the Caspian Sea using a sediment-oil-flow-through (SOFT) system. Untreated (without simulated petroleum seepage) and SOFT sediment microbial communities shared 43% bacterial genus-level 16S rRNA-based operational taxonomic units (OTU0.945) but shared only 23% archaeal OTU0.945. The community differed significantly between sediment layers. The detection of fourfold higher deltaproteobacterial cell numbers in SOFT than in untreated sediment at depths characterized by highest sulfate reduction rates and strongest decrease of gaseous and mid-chain alkane concentrations indicated a specific response of hydrocarbon-degrading Deltaproteobacteria. Based on an increase in specific CARD-FISH cell numbers, we suggest the following groups of sulfate-reducing bacteria to be likely responsible for the observed decrease in aliphatic and aromatic hydrocarbon concentration in SOFT sediments: clade SCA1 for propane and butane degradation, clade LCA2 for mid- to long-chain alkane degradation, clade Cyhx for cycloalkanes, pentane and hexane degradation, and relatives of Desulfobacula for toluene degradation. Highest numbers of archaea of the genus Methanosarcina were found in the methanogenic zone of the SOFT core where we detected preferential degradation of long-chain hydrocarbons. Sequencing of masD, a marker gene for alkane degradation encoding (1-methylalkyl)succinate synthase, revealed a low diversity in SOFT sediment with two abundant species-level MasD OTU0.96.

Microbes hold the key to life. They hold the secrets to our past (as the descendants of the earliest forms of life) and the prospects for our future (as we mine their genes for solutions to some of the planet's most pressing problems, from global warming to antibiotic resistance). However, the piecemeal approach that has defined efforts to study microbial genetic diversity for over 20 years and in over 30,000 genome projects risks squandering that promise. These efforts have covered less than 20% of the diversity of the cultured archaeal and bacterial species, which represent just 15% of the overall known prokaryotic diversity. Here we call for the funding of a systematic effort to produce a comprehensive genomic catalog of all cultured Bacteria and Archaea by sequencing, where available, the type strain of each species with a validly published name (currently∼11,000). This effort will provide an unprecedented level of coverage of our planet's genetic diversity, allow for the large-scale discovery of novel genes and functions, and lead to an improved understanding of microbial evolution and function in the environment.

The diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from Bahamas was studied using comparative sequence analysis of 16S rRNA gene and fluorescence in situ hybridization. The findings show that stable coexistence of multiple endosymbionts is not limited to single species but appears to be a common feature in oligochaete symbioses.

The dissimilatory reduction of sulfate is an ancient metabolic process central to today's biogeochemical cycling of sulfur and carbon in marine sediments. Until now its polyphyletic distribution was most parsimoniously explained by multiple horizontal transfers of single genes rather than by a not-yet-identified 'metabolic island.' Here we provide evidence that the horizontal transfer of a gene cluster may indeed be responsible for the patchy distribution of sulfate-reducing prokaryotes (SRP) in the phylogenetic tree. We isolated three DNA fragments (32 to 41 kb) from uncultured, closely related SRP from DNA directly extracted from two distinct marine sediments. Fosmid ws39f7, and partially also fosmids ws7f8 and hr42c9, harbored a core set of essential genes for the dissimilatory reduction of sulfate, including enzymes for the reduction of sulfur intermediates and synthesis of the prosthetic group of the dissimilatory sulfite reductase. Genome comparisons suggest that encoded membrane proteins universally present among SRP are critical for electron transfer to cytoplasmic enzymes. In addition, novel, conserved hypothetical proteins that are likely involved in dissimilatory sulfate reduction were identified. Based on comparative genomics and previously published experimental evidence, a more comprehensive model of dissimilatory sulfate reduction is presented. The observed clustering of genes involved in dissimilatory sulfate reduction has not been previously found. These findings strongly support the hypothesis that genes responsible for dissimilatory sulfate reduction were concomitantly transferred in a single event among prokaryotes. The acquisition of an optimized gene set would enormously facilitate a successful implementation of a novel pathway.

Knowledge about the role and fate of different populations of heterotrophic picoplankton in marine and inland waters is reviewed. Focus is laid on a growing suite of techniques that link the analysis of bacterial identity with growth, morphology, and various physiological activities at the level of single cells.

An investigation on the morphology of symbiotic bacteria in deep-sea mussels of the genus Bathymodiolus is conducted using transmission electron microscopy (TEM). The results indicate that the Bathymodiolus sp. mussels studied from the Gabon margin harbor two morphologically distinct bacteria that resemble the methanotrophic (5,14,16) and thiotrophic (13,17) symbionts of other Bathymodiolus species.

Comparative 16S rRNA sequence analysis and fluorescence in situ hybridization (FISH) is used to examine the diversity and phylogeny of symbionts in two geographically separated Olavius crassitunicatus populations from the Peru margin. A phylogenetically diverse symbiont population consisting of five or six co-occurring bacterial phylotypes is discovered, which appear to be highly specific and stable in both host populations.

Research has been conducted on bacterioplankton samples. The results of microscopic identification of DNA-synthesizing cells in these samples are reported.

Research has been conducted on fluorescence in situ hybridization of marine bacteria. The preparation and permeabilization procedures and the staining protocols for this hybridization have been modified and are reported.

Research has been conducted on the bacterioplankton from a meromictic lake. The temporal changes of the plankton have been analyzed with the culture-independent techniques and the results indicate that the microbial populations are well adapted to the vertical and temporal gradients of physiochemical conditions.

Research has been conducted on the bacteria and archaea detection techniques used in the costal North Sea and at Monterey Bay, California. The results of the fluorescence in situ hybridization with the rRNA-targeted oligonucleotide probes monolabeled with the cyanin dye or fluorescein-labeled polyribonucleotide probes are discussed.

A clade of alpha-proteobacteria related to Roseobacter spp., a Cytophaga-Flavobacterium cluster, and a cluster of gamma-proteobacteria produce most of the total bacterioplankton metabolic activity in the Celtic Sea. The smallest cells of the gamma-proteobacteria had the highest growth-specific rates, accounting for their dominance in nutrient-limited waters.

Researchers have discovered novel gamma-proteobacteria from the NOR5 lineage in water from the coastal North Sea. These bacteria occurred in high concentrations during the summer.

Research has been conducted on the heterotrophic marin picoplankton. The results of the batch growth experiments with two marine isolates performed in pure culture and coculture on organic carbon of low concentrations are described.

Closely related Prochlorococcus genotypes have been shown to have remarkably different depth distsributions in the Red Sea and the North Atlantic. An in situ hybridization method was used for identification of marine cyanobacteria using horseradish-peroxidase-labelled 16S rRNA targeted oligonucleotide probes in combination with tyramid signal amplification (TSA).

During predation by protists, bacterioplankton form grazing-resistant flocs and filaments that eventually make up most of the biomass. Protist grazing also seems to activate viral activity, which also contributes to bacterioplankton mortality.

The composition and size of freshwater bacteria will vary depending on which predators are present. Many protistan predators preferentially consume larger bacteria, which could explain why most freshwater and marine bacterioplankton are relatively small.

Most soil bacteria are adequately represented in clone libraries prepared from either rRNA or rDNA. These libraries are used to identify bacteria using PCR rather than traditional culture methods.

Analysis of bacteria in a phosphate-removing biofilm from a sequencing batch biofilm reactor shows three species of Nitrosomonas as the predominant organisms. N. europaea and N. oligotropha predominate in the first 100 micrometers, with N. oligotropha found almost exclusively in deeper layers of the biofilm.

Researchers have constructed an accessibility map for the 23S rRNA of E. coli that can be used to develop oligonucleotide probes for fluorescence in situ hybridization. Accessibility to probes varied substantially, with the best signal generated from a probe that was complementary to positions 2018 to 2035.

alpha- and beta-Proteobacteria seem to predominate in lake snow aggregates, where they consume dissolved amino acids in the early phase of the aggregate but release amino acids during the late phase. Lake snow consists of macroscopic aggregates of living and dead algae, zooplankton molts and other organic and inorganic debris.

The phylogenetic composition of Svalbard marine arctic sediment is presented, obtained from rRNA slot blot hybridization and fluorescence in situ hybridization techniques. A large microbial faction was found within the top 5 cm of the sample, with Cytophaga-Flavobacterium and delta-proteobacterial sulfate-reducing bacteria being the most abundant groups.

Researchers have confirmed the global distribution of many lake bacterioplankton by analyzing the microbial communities in a lake in Russia, Germany, and Austria. Members of the alpha, beta, and gamma subclasses of Proteobacteria were represented, as were the Actinobacteria, which made up 63% of the bacterioplankton biomass in one lake.

Marine gamma-proteobacteria adjust to conditions of starvation or plentiful food by altering their intake of proteins. During enrichment experiments, researchers noticed an increase in cell populations with higher protein and DNA content.

Inaccessible regions of ribosomal RNA can be made accessible to FISH probes by using oligonucleotides that are complementary to regions close to the probe target site. These so-called helper oligonucleotides increased the fluorescence of several FISH probes of E. coli 16S rRNA.

Desulfosarcina and Desulfococcus make up about 70% of the marine sulfate-reducing community in the Artic, with Desulfosarcina variabilis making up about half of the community. Sulfate reduction was highest in the top few centimeters of soil.

Research has been conducted on the high abandance and growth of hitherto-uncultured Bacteria from solar salterns. The provisional classification of this bacterial group has been proposed.

Research concerning the culturability of the microorganisms in bacterioplankton from the North Sea is presented. The use of fluorescence in situ hybridization is discussed.

Research has been conducted on the microbiology of manganese and iron reduction. The relationship between sulfate and manganese and iron reduction has been investigated.

Research has been conducted on the Escherichia coli precursor 16S rRNA. The oligonucleotide probes that target the 3' region of the Acinetobacter precursor 16S rRNA have been designed.

Precursor 16S rRNAs of Acinetobacter spp. in activated sludge waste-water treatment systems have been monitored and it appears they undergo a change in growth activity in wastewater treatment systems. Acinetobacter calcoaceticus grown in LB medium had a more substantial and faster increase in precursor 16S rRNA levels vs the increase in total 16S rRNA levels in exponential growth. Precursor 16S rRNa levels of Acinetobacter spp. increased transiently when activated sludge was mixed with influent wastewater in full- and lab-scale wastewater treatment systems.

Research was conducted to examine the phylogenetic affiliation and diversity of the prokaryotic community and to quantify the contribution of sulfate-reducing bacteria to total microbial community. A sample of 30 clones was chosen for sequencing and phylogenetic analysis to investigate diversity in the 16S rDNA clone library. Results indicate that the total diversity evaluated by the 16S rDNA method was very large in the permanently cold sediments and was only partially observed through the screening of 3553 clones.

Research was conducted to examine the phylogenetic affiliation and quantification of psychrophilic sulfate-reducing isolates in marine Arctic sediments. It was shown that psychrohilic sulfate-reducing bacteria are related to mesophic strains and are likely to be phylogenetically diverse as the mesophiles. The abundance of the new group of psychrophilic sulfate-reducing isolates suggests that they may have an important role to play in the sulfur cycle of marine sediments.

According to fluorescence in situ hybridization, members of the Cytophaga-Flavobacterium cluster were the most abundant bacteria in seawater and were also common in freshwater. In freshwater, the beta subclass proteobacteria were the dominant organism.

Microsensors and in situ hybridization can be used to document microbial populations in various parts of a fixed-film treatment plant. At these plants, immobilized microorganisms are used to purify wastewater.

An assay was designed to measure protein biomass of marine planktonic bacteria by flow cytometry. The procedure was calibrated by employing five species of Bacteria isolated from seawater samples and grown in culture at various temperatures. The intensity of SYPRO-protein fluorescence of these bacteria strongly correlated with their total protein content, measured by the bicinchoninic acid method to be in the range of 60 to 330 fg of protein/cell.

Cyanobacterial cells have been identified by using a whole-cell hybridization method that uses horseradish peroxidase (HRP)-labeled, rRNA-targeted oligonucleotides. The technique was used because fluorescent monolabeled oligonucleotides fail to overstain the autofluorescence of the target cells. The genotypic method has adequate sensitivity to detect cyanobacterial cells in the deeper layers of microbial mats. Its cell fixation, specificity and sensitivity were evaluated by using four oligonucleotides to detect four groups of bacteria.

The seasonal and vertical abundance distributions of pelagic members of Bacteria and Archaea in Gossenkollesee were investigated. The population structure at various levels of taxonomic resolution was also studied. Three different populations of uncultivated microbes related to alpha- and beta-proteobacteria were observed through fluorescently labeled oligonucleotide probes. Twenty-five percent of the beta-proteobacteri could be designated to only two phylotypes.

Research was conducted to examine the nitrifying community of a chemolithoautotrophic fluidized bed reactor using microsensors and rRNA-based molecular techniques. The reactor was operated with low ammonia concentrations instead of the high ammonia concentrations it was originally intended for. In situ activity measurements suggest that both ammonia and nitrite oxidizers were active within a 125 micrometer zone at the aggregate surface. Results agree with observations that in natural systems low in ammonium, Nitrosospira spp. can be detected rather than Nitrosomonas spp.

The study investigated the composition and vertical distribution of the microbial community found in the sediments of the Wadden Sea of the German North Sea coast. In situ hybridization with fluorescently-labeled ribosomal RNA-targeted oligonucleotides was used to identify and quantify individual cells. Total cell counts indicate that cells were more concentrated along the beach and mud cores, which decreased with increased depth. Domain-specific probing revealed that bacteria made up most of the microbial community and that Cytophaga-Flavobacterium were the most abundant.

A study was conducted to examine the phylogeny of Nevskia ramosa through the use of ribosomal RNA approach. The gene of the neuston bacteria that were described as a rod-shaped, slightly bent bacteria forming typical flat rosettes in the surface of their habitat, was cloned. Results revealed that the ribosomal RNA sequence of the bacteria was closely related and similar to that of the Xanthomonas species and its relatives.

Three new 16S and 23S rRNA-targeted probes for phylogenetic groups within the beta subclass of the class Proteobacteria were developed for the derivation of a higher taxonomic resolution of the molecular techniques. These were designed in the course of the first-ever application of fluorescent in situ hybridization to examine microbial community structure and dynamics in the fermentor and the bioreactor during start-up, using a laboratory-scale fixed-bed bioreactor degrading a mixture of aromatic compounds.

Flow cytometry criteria were used to sort particular cell types from water, sediment and activated sludge samples to determine if these sorted cells will be useful in molecular analysis. This method will provide investigators with a technique to study less abundant but conspicuous bacterial populations. Further, the method will serve as a screening tool to remove contaminants even in abundant populations.

In-situ hybridization has found increasing use in nucleic acid probes in studying the specific sequences of whole-fixed bacterial cells. The use of fluorescein-tyramide as a substrate for oligonucleotides labeled with horseradish peroxidase can improve the in situ hybridization of bacterial cells. Flow cytometry measurements show that the hybridized cells have a 10- to 20-fold signal amplifications.

The phylogenetic analysis and in situ identification of bacteria in activated sludge was examined using of the ribosomal RNA (rRNA) approach. Full-length genes coding for the small-subunit rRNA were amplified by the polymerase chain reaction and cloned to the pGEM-T vector. Results show that activated sludge sample contains cells from another unsequenced genotype that has just one of two signatures for the genus Arobacter.

The population of Legionella pneumophila strain Philadelphia I JR 32 that was suspended in sterilized tap water was quantified to determine the intracellular environment that promotes the replication of the microbe. Incubation of Legionella pneumophila JR 32 in tap water microcosms induced a viable but nonculturable (VBNC) state after 125 days. However, the dormant bacteria was resuscitated from the VBNC state after the inoculation of the tap water microcosm with Acanthamoeba castellanii.

The alpha- and beta-Proteobacteria in mixed assemblages adopt two specific strategies in response to flagellate predation. The alpha-Proteobacteria increase their growth rate to outgrow predator pressure. The beta-Proteobacteria produce inedible and inactive filaments. The strategies occur within 24 hr post flagellate addition. The production of biomass by the bacteria, after the introduction of flagellate predation, indicates the stimulation of cell elongation.

The primary sequence of the 16S and 23S rRNA of the obligate intracellular bacterial parasites of acanthamoebae is 86 to 87% similar to that of the genus Chlamydia. The parasites probably constitute another genus of Chlamydiaceae rather than another species. The rRNA analysis and in situ hybridization with a horseradish peroxidase-labeled oligonucleotide identifies a site that assigns the 16S rRNA sequence to the respective intracellular parasite. The results of the study show that small amoebae act as environmental reservoirs and vectors for pathogenic bacteria.

Nitrosomonas, ammonia oxidizers, form a thick layer of cell clusters at the top of a nitrifying biofilm in a trickling filter of an aquaculture water recirculation system. Nitrobacter, nitrite oxidizers, form a less-dense layer close to the clusters of ammonia oxidizers on the biofilm. Nitrification is observed only in a narrow zone of 50 micrometer in the upper portion of the biofilm. Both the species are predominant in the oxic zone of the biofilm, but are also present in lesser quantity in anoxic zones.