The development of methods to study these communities on the basis of 16S rRNA gene sequences analysis was a first step into the molecular analysis of environmental communities and the study of biodiversity in natural habitats. A new dimension in this field was reached with the introduction of functional genes of ecological importance and the establishment of genetic tools to study the diversity of functional microbial groups and their responses to environmental factors. Functional gene approaches are excellent tools to study the diversity of a particular function and to demonstrate changes in the composition of prokaryote communities contributing to this function. The phylogeny of many functional genes largely correlates with that of the 16S rRNA gene, and microbial species may be identified on the basis of functional gene sequences. Functional genes are perfectly suited to link culture-based microbiological work with environmental molecular genetic studies. In this review, the development of functional gene studies in environmental microbiology is highlighted with examples of genes relevant for important ecophysiological functions. Examples are presented for bacterial photosynthesis and two types of anoxygenic phototrophic bacteria, with genes of the Fenna-Matthews-Olson-protein ( The structure and functions of microbial communities are highly complex, and environmental factors as well as biological interactions are significant factors in dynamically shaping these structures. Prior to the application of molecular methods, it was almost impossible to even roughly estimate the diversity and structure of these communities. It was the application of methods based on 16S rRNA gene sequences that for the first time enabled studies on microbial diversity in environmental samples and opened our eyes to the tremendous diversity and complexity of environmental communities. http://rdp.cme.msu.edu/index.jsp) [Over the past decades, dramatic progress has been made in the knowledge and understanding on microbial diversity and function in the environment. It is fortunate to be a contemporary witness of the developments over this time, which started with first considerations on prokaryote phylogeny based on oligonucleotide catalogues of the 16S rRNA . Soon thdex.jsp) . The seqToday, the analysis of functional bacterial groups using genes of metabolic key proteins as targets has become a central topic of microbial ecology and brings a new dimension to environmental studies of microbial communities. An increasing number of investigations are concerned with diversity analysis using functional genes and receives support from the exponentially growing number of genome and metagenome sequences emerging along with the tremendously advancing sequencing technologies. These sequence data form a strong backbone for the application of functional genes in environmental studies. This review will focus on the application of functional genes for the analysis of environmental microbial communities and gives examples of selected functions of major ecological significance. It will be the author’s personal view on the developments during the past two decades. The diversity of selected important functional groups based on genes involved in these functions is discussed with special emphasis on marine and hypersaline habitats. Also, a few key studies related to deep sea hot vent communities are highlighted.Dramatic advances in methodology and research strategies for the analysis of prokaryote  phylogeny and their environmental communities have been made over the past two decades. Culture-dependent studies, the only way to approach environmental microbial communities until the 1980s, were inadequate to assess the diversity of bacterial communities. The availability of PCR techniques and extended phylogenetic trees based on 16S rRNA gene sequences from pure cultures not only enabled detailed considerations on microbial phylogeny, but also formed an initial backbone for biodiversity studies in the environment. Together with improved methods of DNA separation and purification using gel electrophoresis and cloning techniques, for the first time studies of mixed bacterial communities were possible by analysis of environmental DNA ,5. The fHowever, these methods allowed identification of the bacterial species based on the 16S rRNA gene sequences only to a limited extent. In addition to biases of DNA extraction and amplification protocols, to a great part this was due to the insufficient separation of complex mixtures in the gradient gels and to the limited sequence information obtained from the short DNA stretches that yielded the best separation within the gels (approx. <500 nt). The lack of phylogenetic resolution due to the consideration of only small sequence stretches was alleviated by selection of primers that cover the complete sequence of the 16S rRNA gene, separation of the PCR products by cloning techniques and sequencing from the clone libraries . These mA major drawback of the molecular approaches in microbial ecology for many years was the failure to identify microorganisms in environmental samples which was related to the discrepancies of results from culture-independent molecular analyses and culture-dependent studies. It was a general tendency that both approaches gave non-congruent views on the environmental communities . The stper se, but only through phylogenetic observations and assumptions. Therefore, more specific approaches have to be applied in order to convincingly unravel the environmental diversity of functional groups having a unifying physiological property. This is achieved by the analysis of suitable functional genes.Another major drawback of studies using 16S rRNA gene sequences is the failure to convincingly depict the diversity of specific functional groups in environmental communities. This is a basic problem of the 16S rRNA gene approach, as the 16S rRNA molecule is not related to metabolic properties 10 bacterial cells with >10.000 different microbial species in a cm3 and surface waters of the oceans have an average of 1 million bacteria, a number of 100 or 1000 clone sequences of a sample (as handled in most biodiversity studies using 16S rRNA gene sequences) could cover only a minor part of the community. Only the most abundant and the most easily amplified sequences could be retrieved from the tremendous diversity of the environmental communities. Although this fraction of the more abundant organisms could be regarded to include the most important players (at least under the conditions and the time of sampling) by no means it represents the potential of a selected sample and habitat under variation of the environmental conditions. In fact, changes of the environmental conditions are expected to provoke significant and complex changes of the community composition with the effect that minor and not detected members of the community become major or even dominant parts of it [In view of the great enthusiasm due to the tremendous advances in environmental microbiology coming along with the use of DNA sequences for microbial biodiversity studies in the 1990s, which caused complete shifts in research approaches and initiated the field of molecular microbial ecology, the limitations of these molecular approaches appeared of minor importance. With the rigorous advances in molecular techniques over the past decade, molecular microbial ecology has grown up and analyses of the complex metagenome of environmental samples became possible. Although a steady increase in sensitivity and resolution in sequencing has been obtained, it should not be overlooked that a basic limitation in studies on the environmental metagenome has remained. This limitation is connected to the generally low number of analyzed sequences of the metagenome from a given environmental sample, with the effect that not the complete community is depicted. (The same holds for metagenetic studies that have been performed with the 16S rRNA gene as target.) If one considers that an individual sample of a marine sediment may contain more than 10Overall, the speed of new methodological developments in sequencing technologies and the progress of knowledge in molecular microbial ecology over the past decades was unbelievable. Since the invention of PCR and sequencing techniques of the Sanger method ,12, the Haemophilus influencae, 1.8 Mb) [Haemophilus influencae), while the throughput of the Illumina technology and similar other technologies is at 5000 Mb/day  [i.e., an information content of 5 × 1012 (5 trillion) base pairs in 1 mL, 1015 base pairs per Liter and 1018 base pairs per 1000 L, with the implication that metagenomic studies before 2010 have used sample volumes from 1–1000 L sea water [15 base pairs of the metagenome), the coverage would be 0.1%. These numbers reflect both, the incredible advances in sequencing technologies on the one hand and the immense complexity of microbial metagenomes on the other hand. At this point it is necessary to recall the breathtaking improvements in the handling of data by computing facilities and software programs over the past 2 decades that all of us have witnessed in the daily life. Without special efforts and specific software programs, the handling of sequence information of a complete metagenome containing 1 billion single prokaryote genomes would not be possible. The evaluation of metagenomic data by bioinformatics tools is another great challenge to metagenome analysis [Sequencing technologies made possible the sequence analysis of the first bacterial genome in 1995 ( 1.8 Mb) . At that 1.8 Mb) . For comme size) . These ame size) ,17. ThisBecause much of the motivation to study bacterial and microbial biodiversity is coming from the desire to understand the functions of the environmental communities and the interactions within these communities, it was a logic step to include functional genes into biodiversity studies and to study functionally defined fractions of the whole microbial community. Functional gene approaches to a great part overcome limitations of resolution and in particular of lacking specificity in microbial community analyses by the 16S rRNA gene approach. They have a number of advantages for investigations of environmental communities. First of all, these approaches focus their view on a selected functional aspect of the environment and on organisms involved in this specific function. Second and most important, these approaches achieve a better resolution regarding the community composition, because the number of genes of a particular function in a given sample is much smaller compared to the number of 16S rRNA genes (and whole genomes) and  less abundant strains may be much easier detected and ideally the whole functional community can be resolved. In addition, many functional genes show faster rates of evolution relative to the 16S rRNA gene, which specifically has been selected to trace prokaryote phylogeny because of its phylogenetic conservative properties . In consThe main challenges at the initiation of functional gene approaches in the late 1990s included the selection of suitable functional genes, the design of appropriate primer sets to amplify sequence stretches of sufficient length and information as well as the establishment of databases with sequences of these genes in order to allow phylogenetic assignment and eventually identification of the players in the environment. Today, it is hardly imaginable, that the lack of appropriate gene sequences in the databases was a major limitation at the initial stages of such investigations and even made it difficult to design proper primer sequences to specifically approach the target gene . The selected genes and the established primers on one hand need to be specific for the selected metabolic pathway on the other hand the primers/selected sequence stretches should enable a complete consideration of the biodiversity of the selected pathway and function. In addition, the selected sequences should contain sufficient phylogenetic information and ideally contain more than 1000 nucleotides to enable good phylogenetic resolution. For a number of functional genes, appropriate primer systems have been described see  and dataAlthough lateral gene transfer may be relevant for most if not all functional genes and potentially may blur the phylogenetic signal as compared to that of the 16S rRNA gene, experience with quite a number of functional genes and the comparison of their phylogeny with that of the 16S rRNA makes clear that events of lateral gene transfer were not frequent events ,22,23,24apr) and dissimilatory sulfite reductase (dsr) playing roles in sulfate reduction as well as in sulfur oxidation [pmo) in methane-oxidizing bacteria and ammonia monooxygenases (amo) in ammonia-oxidizing bacteria [In general, most eco-physiological key functions are found in various phylogenetic lineages and in addition multiple pathways may exist for the same biogeochemical function, as for instance for the oxidation of sulfur compounds, ammonia oxidation, methane oxidation and autotrophic carbon dioxide fixation. The different pathways of the same biochemical function also present different target genes for the molecular analysis. This necessitates the design of different primer systems for each of the target genes of a particular function considered for community analysis, but at the same time it provides the advantage to distinguish the different groups of the studied function. In addition, the same genes may be involved in different functional roles, as exemplified by adenosine-5′phosphosulfate (APS) reductase  the analysis of whole metagenomes and (ii) the selective analysis of functional genes. Even if the coverage of the whole metagenome is small, a tremendous amount of information is becoming available from metagenomic studies, which includes information on functional genes. Thus metagenome studies will continue to provide valuable information for functional aspects in the environment. However, for the specific functional analysis of a particular microbial community, the efforts of metagenomic analysis are not required and the coverage of the functional gene sequences is expected to be much better using up-to-date sequencing techniques for amplicons of specific functional genes. The major advantages of functional metagenetic approaches are the high specificity and the use of a comparable small data volume of sequence information to be analyzed and evaluated.fmoA) as target for the green sulfur bacteria and two reaction center proteins (pufLM) for the phototrophic purple bacteria, adenosine-5′phosphosulfate (APS) reductase (aprA), sulfate thioesterase (soxB) and dissimilatory sulfite reductase (dsrAB) for sulfur oxidizing and sulfate reducing bacteria, ammonia monooxygenase (amoA) for nitrifying/ammonia-oxidizing bacteria, nitrate and nitrite reductases  for denitrifying bacteria and methane monooxygenase (pmoA) for methane oxidizing bacteria , were due to the close phylogenetic relationship between phototrophic and nonphototrophic bacteria within the Proteobacteria. In consequence, specific sequence stretches of 16S rRNA genes that would clearly allow the design of specific primers and the identification of phototrophic representatives in complex mixtures and environmental samples could not be identified. This necessitated the use of functional genes, e.g., those related to photosynthesis to study the environmental diversity of these bacteria. fmoA gene encoding for a bacteriochlorophyll-a protein specific for the green sulfur bacteria and Chloroacidobacteria [pufLM genes encoding for reaction center proteins of the bacterial photosystem II which is specific for all phototrophic purple Proteobacteria and Chloroflexaceae [In order to specifically analyze communities of anoxygenic phototrophic bacteria and their adaptation to different environmental conditions and their geographic distribution, primer systems have been developed that specifically target these bacteria: the bacteria  and the lexaceae ,48. fmoA and pufLM genes [fmoA genes of green sulfur bacteria [pufLM genes of purple sulfur bacteria [Important steps for the possible identification of species in environmental DNA sequences were the establishment of a phylogenetic-based taxonomy supported by 16S rRNA gene sequences and the demonstration of a general congruence of the phylogenies of 16S rRNA genes with those of LM genes ,52,53,54bacteria  and of pbacteria  from mosChloroacidobacterium thermophilum, which occupies special ecological niches in hydrothermal springs and belongs to the Acidobacteria phylum [fmoA is an appropriate target to specifically analyze environmental communities of the green sulfur bacteria [fmoA gene sequences was established including available type strains of the established species [fmoA and 16S rRNA genes was used in the taxonomy of these bacteria to rearrange the strains and species, to redefine some species and in a few cases establish new species and a new genus [fmoA sequence analyses [The FMO protein is a bacteriochlorophyll-a protein that mediates energy transfer between the chlorosomes and the reaction center in the cytoplasmic membrane of green sulfur bacteria  and the a phylum ,37. FMO a phylum . Therefobacteria  and phot species . Remarkaew genus . With thanalyses .pufLM genes were selected, which code for the light (L) and medium (M) subunit of the photosynthetic reaction center type II structural proteins of all phototrophic Proteobacteria  and in addition of the phototrophic members of the Chloroflexi [pufL and pufM genes and in addition, a comprehensive database of pufLM gene sequences of most of the recognized type strains of the purple sulfur bacteria was established [pufLM gene sequences of the purple sulfur bacteria (Gammaproteobacteria) was in good correlation to that of 16S rRNA gene sequences [pufLM genes qualified as a valuable tool for studies of environmental communities of the phototrophic purple bacteria allowing species recognition of these bacteria even in complex mixtures of environmental communities.In order to selectively approach the phylogeny of the phototrophic purple bacteria and to develop tools for the analysis of natural communities of these bacteria, the oroflexi . An imprablished . The phyequences . A phyloequences ,53,59. TpufLM genes have been proposed for the distinction of genera and species, of purple sulfur bacteria, respectively [Sequence information is predestined to link bacterial systematics and functional ecological studies because (i) sequence information is now well established as a property in bacterial systematics and forms a backbone for bacterial phylogeny and systematic treatment; and (ii) sequence information becomes easily available from environmental communities, from individual clones as well as from complete metagenomes and can contain important information about the physiological potential at the level of individual strains and species. Environmental gene sequences (or rather the bacteria associated with the sequences) can be arranged in phylotypes, which comprise sequences above a defined similarity. If a distinction of phylotypes is made at a sequence level that compares to the level of distinction between species with pure cultures, phylotypes can be used to approach the species diversity of environmental communities. If environmental clone sequences are sufficiently similar to known species, represented by their type strains, it is quite likely that they are representatives of this species or close relatives thereof. If considerations concerning sequence similarities as a rough guide for species differentiation of pure cultures are transferred to sequences from the environment, species recognition and an estimate of the species diversity in environmental samples can be achieved with phylotypes as an equivalent to the taxonomic defined species. Similar considerations as have been proposed as guidelines for the use of 16S rRNA gene sequences in taxonomy can be made for functional genes . With thectively ,48.Phototrophic green sulfur bacteria are common in coastal lagoons and marine sediments, where they often form massive colored mass developments, while in freshwater lakes their development is mostly hidden in the depth at the chemoclines. They are dominant primary producers in the Black Sea  and devefmoA and 16S rRNA gene phylogenies of pure cultures of green sulfur bacteria as a solid basis, a first detailed molecular genetic study on the species composition of green sulfur bacteria communities was made with samples from marine and saline habitats of different geographical locations, of the Baltic Sea, the Mediterranean Sea, Sippewissett salt marsh  and Bad Water  using the established primers [fmoA sequences) were associated with salt-dependent phylogenetic lines of green sulfur bacteria which had been previously established with pure cultures [Prosthecochloris [Prosthecochloris aestuarii in enrichments and as isolated cultures from many marine habitats  ,62. In tpufM gene and revealed a remarkable high diversity in different habitats [Rhodoferax and Roseateles (a bacteriochlorophyll-containing aerobic bacterium) and to the aerobic bacteriochlorophyll-containing Alphaproteobacteria Acidiphilium and Bradyrhizobium [Roseobacter and to the facultative aerobic anaerobic phototrophic Rhodobacter/Rhodovulum clades in the open oceans was subject of a number of metagenomic studies and supported the high abundance and great diversity of this group of phototrophic bacteria in ocean waters [pufM gene. However, due to the limitations of separation of bands on the gels and the short sequence fragments (<228 nt) considered, these studies lacked sufficient phylogenetic resolution to identify species or even genera. Nonetheless, they have demonstrated the presence of an immense biodiversity of aerobic phototrophic bacteria in marine habitats, which is to a great part not represented by cultured bacteria and in part not even significantly related to known bacterial species [First molecular genetic studies to characterize the communities of phototrophic purple bacteria were based on sequences of the habitats ,67,68. Mn waters ,69. Thein waters ,71, in sn waters  and in Dn waters . Most of species ,71. Rhodobaca bogoriensis, several purple sulfur bacteria  were isolated [pufM amplicons from environmental DNA. This is in accord with the expected presence of bacteria specifically adapted to the highly sulfidic and also alkaline environment. Sequences from a total of 12 bands (136 nt long) of pufM amplicons could be obtained after reamplification of bands from DGGE [Thiocapsa imhoffii, which apparently is the dominant purple sulfur bacterium at the chemocline, while others showed only distant relations to Rhodoferax, Rhodocyclus and Ectothiorhodospira [pufLM genes. Remarkable are the studies on Soap Lake (Washington), which is a small soda lake with a distinct chemocline (at 20 m depth), a maximum depth of 24 m and high concentrations of sulfide reaching up to 175 mM near the sediment . Both cuisolated . Though isolated  were userom DGGE . Only twodospira . In concpufL and pufM genes (approx. 1400 nt) of most purple bacteria. With these primers, a comprehensive database using the almost complete sequences of most cultured purple sulfur bacteria was established and their phylogenetic relationship was verified [pufLM genes allowed confident identification of species and phylotypes in environmental samples. Two case studies of salt lakes in the Chilean highland and of a coastal lagoon of the Baltic Sea (Germany) may highlight the possibilities of this approach for community studies [Systematic studies with higher phylogenetic resolution and the species-specific analysis of complex environmental communities of phototrophic purple bacteria were enabled by the application of a primer pair that amplified sequences of both verified . The est studies ,75.pufLM gene sequences and the impact of changes in temperature and salinity were determined under controlled conditions in the laboratory by using RFLP, cloning and sequencing [pufLM phylotypes of the community of purple sulfur bacterial of this brackish water lagoon affiliated to genera and species of phototrophic purple sulfur bacteria typically isolated from such habitats including Marichromatium, Thiocystis, Thiorhodococcus, Allochromatium, Thiocapsa, Thiorhodovibrio; but some sequences were related to moderately halophilic Halochromatium and Thiohalocapsa species [pufLM sequence similarity to the closest known type strain and therefore might be regarded as representing new genera [The phototrophic bacterial community of a brackish water Baltic Sea coastal lagoon was characterized on the basis of almost complete quencing ,48. Thisquencing . Accordi species ,76,77,78w genera . Thus, tpufLM clone libraries and compared to that from the natural habitat. The highest diversity of identified phylotypes was observed in the natural sample . Most phylotypes in the habitat were tolerating the complete range of salt concentrations and developed up to 7.5% NaCl. While the community at 0% NaCl was clearly dominated by only two Allochromatium phylotypes, the community was more divers at all other conditions [Thiorhodococcus mannitoliphagus (99.8%), Thiorhodococcus kakinadensis (98.2%), and Marichromatium gracile (100%). This is quite remarkable and indicates an even higher diversity in the environmental sample than resolved by its metagenetic analysis and also demonstrates that media and culture conditions were quite appropriate for almost all purple sulfur bacteria recognized in the environmental sample by the genetic approach.Both temperature and salinity had a significant influence on the community structure . In expenditions . With thMarichromatium gracile, which has been repeatedly isolated from marine coastal habitats [Marichromatium gracile became most prominent, which was not detected at lower temperatures and not in the habitat sample. The clear preference of Marichromatium gracile for elevated temperatures points to its obvious competitive advantage in the shallow-water habitats if heated during daytime by the sun and supports findings of Serrano et al. who characterized a slightly thermophilic strain of this species [An interesting property of habitats ,62, was  species .Roseobacter/Rhodobacter clade are common to these lakes [pufLM primer system of [The salt lakes of Chilean Salares represent an extraordinary and extreme habitat with special conditions regarding salt concentration and composition, irradiation and drastic diurnal changes in temperature ,81. Anoxse lakes . Like otystem of  with focystem of . The greystem of . Chromatiaceae such as Halochromatium salexigens, Halochromatium glycolicum, Thiohalocapsa halophila, Ectothiorhodospira mobilis, Ectothiorhodospira variabilis and Halorhodospira halophila as closest relatives [b-containing anoxygenic phototrophic bacteria distant to (<80% sequence similarity) the genera Thiococcus, Thioflavicoccus and Thioalkalicoccus, that form a separate phylogenetic branch among the purple sulfur bacteria [bchl-b [The communities of purple sulfur bacteria from both salt lakes were characterized by the presence of representatives related to the type strains of moderately and extremely halophilic elatives ,77,78,83bacteria ,52,84. T [bchl-b ,86,87. TEctothiorhodospira mobilis and Thiohalocapsa halophila), whereas the great majority of phylotypes had a pufLM sequence of such a low similarity (<80%) to known purple sulfur bacteria that quite likely they might represent new genera. Most remarkable was the dominance and diversity (11 phylotypes) of a novel, so far unknown lineage of pufLM containing Gammaproteobacteria, which was highly diverse and prevalent in different lakes of the Salar de Atacama [Only two out of 24 phylotypes identified as phototrophic Gammaproteobacteria could be clearly identified on the species level  or as electron donors for energy generation in chemo- and photolithotrophic ways of life. The cycling of sulfur compounds by oxidation and reduction processes is governed by a number of prokaryotes from different phylogenetic lineages that use different biochemical pathways. In marine sediments sulfate reduction is a major process in organic matter degradation ,91 and pAllochromatium vinosum, the sulfate-reducing deltaproteobacterium Desulfovibrio vulgaris and the sulfate-reducing archaeon Archaeoglobus fulgidus strongly supported the hypothesis that these systems are true homologues and common ancestral genes for dissimilatory sirohaem sulfite reductases as well as APS reductases exist [Archaeoglobus has gained a true bacterial dsr gene by lateral gene transfer presumably from a bacterial ancestor [aprA) and of the dissimilatory sulfite reductase (dsrAB) [Different biochemical pathways are involved in the sulfur cycling. One of the pathways for oxidation of sulfur compounds is the well-known reaction sequence including the formation and oxidation of adenosine-5′-phosphosulfate (APS) as intermediate with the involvement of disulfite reductase and APS reductase as key enzymes. First important insight into the evolution of these pathways was obtained from the comparable analysis of enzymes involved in the dissimilatory reduction of sulfate and in the oxidation of reduced sulfur compounds. Key enzymes of sulfate-reducing bacteria are APS reductase and disulfite reductase and already Peck  concludees exist . Today wancestor . Suitabl (dsrAB) ,92,93.soxB gene encoding the sulfate thioesterase was established as a suitable marker gene for this pathway [A second important and widely distributed pathway of sulfur oxidation is the so-called Sox-pathway, which includes complex reaction sequences ,95. The  pathway ,96,97,98aprA gene was applied to a large number of reference strains from sulfate-reducing and sulfur-oxidizing prokaryotes [aprA genes have in general been transmitted vertically during evolution, which supports their usage as functional gene markers in microbial diversity surveys”. By using this aprA gene-based approach, a high diversity of sulfur-oxidizers was detected and clear differences noted in communities of the studied habitats with putative chemolithoautotrophic sulfur oxidizers of Beta- and Gammaproteobacteria being dominant in the hydrothermal sphere while sulfur-oxidizing Alphaproteobacteria were the major component in non-hydrothermal samples [Polymastia corticata by comparing dsr, apr and 16S rRNA gene sequences [The adenosine-5′-phosphosulfate reductase (APS reductase) is a key enzyme of microbial sulfate reduction and also of sulfur oxidation processes and is common to sulfate-reducing bacteria and archaea and also to an important group of sulfur-oxidizing bacteria. A modified primer pair targeting the karyotes . This stkaryotes . These a samples . The samequences .aprA sequences [aprA gene clones. Though composition and diversity varied largely between the sites and with the sediment depths, Desulfobacteraceae in general represented the major group of sulfate-reducers in most samples, followed by Desulfobulbaceae in several and Desulfotomaculum-related phylotypes present only in a few samples [aprA reference gene sequences [Also deep-sea subseafloor communities of sulfur-oxidizing and sulfur-reducing microorganisms from sediments of three sites of the Pacific margin off Japan revealed a markedly diverse community of equences . 135 dis samples . At leasequences . aprA and dsrAB gene libraries [dsrAB sequences from this study revealed a large hidden diversity of so far unrecognized sulfur-oxidizers both of Alpha- and of Gammaproteobacteria, part of which was supposed to perform a chemoautotrophic way of life [Novel groups of sulfur-oxidizing Gamma- and Alphaproteobacteria were recognized in studies of marine intertidal sediments from the German Wadden Sea by analysis of the diversity of ibraries . The phy of life .aprA and dsrA present in the samples affiliated to corresponding phylogenetic clusters [aprA and dsrA clone libraries from the Black Sea sediment where almost all aprA and all dsrA sequences affiliated either to Desulfobulbus elongatus or to Desulfotalea psychrophila [The analysis of sediments of the Black Sea and the deep biosphere of the Peru margin revealed that the sequences of clusters . The authrophila .apr gene sequences are valuable tools to analyze the environmental communities of sulfate-reducing as well as sulfur-oxidizing bacteria. In both groups in addition to aprA the sequences of dsrAB give additional significance to the analyses.Altogether, these few investigations have demonstrated that Thiobacillus denitrificans [dsrAB gene of analyzed reference strains were consistent with those from the 16S rRNA gene, including the archaeal sulfate reducer Archaeoglobus fulgidus [dsrAB and 16S rRNA gene sequences [The dissimilatory siroheme sulfite reductase is a key enzyme in sulfate reduction and catalyzes the six-electron reduction of sulfite to sulfide. It is operating in the reversed reaction in sulfur oxidation as well and was first characterized from the sulfur-oxidizing rificans . The enzfulgidus . This viequences . Though equences ,22. dsr sequence analysis to evaluate the diversity of SRB, a highly diverse population of sulfate-reducing bacteria was found [Desulfonema and Desulfococcus group. Though several SRB were known to be oxygen tolerant and some may have limited capacity to respire with oxygen [Desulfonema-like SRB in the oxic part gave evidence for a possibly specific adaptation of these bacteria to life at the oxic/anoxic interface or even under microoxic conditions [In a study of a microbial mat of the hypersaline Solar Lake using as found . Specialh oxygen , the hignditions .dsrAB genes was also applied to study the diversity of sulfate-reducing bacteria in the Guaymas Basin [Desulfobacter species. In addition, a new group of sulfate-reducers forming a cluster separate from all known sulfate-reducers was found that could not be identified in clone libraries of the 16S rRNA gene [The analysis of as Basin , in whicRNA gene . aprA and dsrAB as functional genes.The sulfate reducing bacteria represent a good example to demonstrate the advances in our understanding of the biology and ecology of a microbial group made over the past decades. Until the 1970s these bacteria were regarded as a highly specialized group, strictly dependent on anoxic conditions and highly restricted to the metabolism of a few organic molecules only. The pioneering work of Fritz Widdel had initiated a new vision on the biology of these bacteria . He cultdsrAB by Müller et al. [dsrAB sequences, these authors constructed a consensus tree including more than 1200 sequences with approx. 1.9 kb length. Four major branches of dsrAB families were recognized: the reductive bacterial type, the oxidative bacterial type, the reductive archaeal type and a branch represented so far only by a second copy of this gene in Moorella thermoacetica [dsrAB genes in sulfate-reducing and sulfur-oxidizing bacteria are largely congruent [The advances achieved by functional gene studies over the past decade are well represented in the extensive work on r et al. . In ordeoacetica . With a ongruent ,22. dsrAB, these studies established so-called superclusters, the Deltaproteobacteria, the Nitrospirae supercluster (previously named Thermodesulfovibrio supercluster), the Firmicutes group, and an environmental supercluster 1 [dsrAB sequences of the euryarchaeal Archaeoglobus formed a separate branch in this reductive bacterial-type dsrAB family tree [dsrAB sequences are represented by the genera Pyrobaculum, Vulcanisaeta and Caldivirga, though sulfate reduction so far has been demonstrated only for Caldivirga maquilingensis.Among the reductive bacterial type of luster 1 . The dsrily tree . The trudsrAB tree forms a monophyletic enzyme family that is phylogenetically distinct from all “reductive dsrAB sequences” and contains branches of Alpha-, Beta- and Gammaproteobacteria and Chlorobi. It is considered from the branching pattern of the tree that this branch evolved by functional adaptation from an ancient reductive dsrAB. With one significant exception , the sequence-based relationships of dsrAB are in good accordance with the 16S rRNA based phylogeny [The oxidative hylogeny . dsrAB sequence identity  was recommended to distinguish species. With this assumption the core dataset represented 647 species-level phylotypes of the reductive and 118 of the oxidative bacterial dsrAB type. A total of 299 environmental sequences of the core dataset did not affiliate with members of any described taxonomic family, but formed 13 stable, monophyletic lineages equivalent to the family level [dsrAB sequences are useful phylogenetic markers to identify recognized taxa (genera/species) on the basis of environmental dsrAB sequences or in environmental metagenomes [With this background, the species identification in environmental samples was attempted. A threshold of 90% ly level . It has agenomes . The laragenomes . Thiosulfate is an important and stable intermediate in the oxidation of reduced sulfur compounds and according to its central position within the biological sulfur cycle, it can undergo a number of transformations both in oxidative as well as reductive pathways. Among the oxidative pathways a central role has been given to the Sox enzyme system, which apparently is widespread among sulfur-oxidizing bacteria (SOB) and has primarily been discussed in regard to thiosulfate oxidation although it may be involved more generally in reactions of sulfur oxidation ,95. Paracoccus pantotrophus. The SoxB enzyme complex appears to be widespread among various phylogenetic groups of sulfur-oxidizing photo- and chemotrophic bacteria that oxidize thiosulfate to sulfate and is involved in two pathways of sulfur oxidation [Paracoccus sulfur oxidation pathway (PSO) catalyzes the oxidation of thiosulfate to sulfate without the formation of sulfur intermediates involving the SoxCD enzyme complex; (ii) The so-called branched pathway lacks the SoxCD complex and catalyzes the oxidation of reduced sulfur compounds to intermediate sulfur compounds with the characteristic deposition of intra- or extracellular sulfur globules [A key enzyme of the thiosulfate-oxidizing multi-enzyme complex is SoxB containing a prosthetic manganese cluster in the reaction center and being essential for thiosulfate oxidation by xidation ,94: (i) globules ,109,110.soxB gene, the phylogeny of the sox-pathways has been evaluated for use in biodiversity studies [soxB fragment of approx. 1000 bp from sulfur-oxidizing bacteria. The study of the primary and secondary structure of the amino acid sequences revealed, that the selected priming sites were highly conserved and had only a few amino acid changes among the available sequences [soxB genes demonstrating an orthologous relationship of the genes from the various bacteria included in this study [soxB from a small number of reference strains, new isolates and environmental DNA from a hydrothermal vent habitat in the North Fiji Basin were compared and used to infer relationships of soxB between sulfur-oxidizing bacteria belonging to various 16S rRNA-based phylogenetic groups [soxB phylogeny, indications of lateral gene transfer were also visible [Halothiobacillus, Thiobacillus, Starkeya.Based on the  studies ,96. A prsoxB gene in different phylogenetic groups of phototrophic and chemotrophic SOB was confirmed [soxB phylogeny [In a study with more than 120 sulfur-oxidizing bacteria, the wide distribution of the onfirmed . Though hylogeny .soxB-based phylotypes, which correlated well with environmental gradients [soxB gene sequences varied with the geochemical conditions. Sequence differences of the soxB gene were found to correlate to the utilization of the branched or the PSO Sox pathway and it was concluded that the spatial distribution of the different soxB gene variations along the outflow channel at Rattlesnake Spring could indicate niche partitioning among the bacteria that oxidize reduced sulfur compounds [Thiobacillus, Thiomonas, Chlorobium, Allochromatium, Thiothrix, Halothiobacillus) and others to genera using the PSO pathway  [Thiothrix almost as sole component, followed by varying abundances of the different members of the community downside the channel starting with major sequence numbers of Halothiobacillus and Thiobacillus and ending with a community dominated by Rhodovulum, Bradyrhizobium and Thiobacillus phylotypes [An interesting case study alongside geochemical gradients of the outflow of Rattlesnake Spring (Oklahoma) demonstrated the spatial distribution of radients . It coulompounds . Some phizobium) . Specifiylotypes .soxB gene analyses revealed that salinity played a key role in controlling the diversity and distribution of SOB populations [Studies on the abundance and diversity of SOB populations in four Qinghai-Tibetan lakes based on ulations . In fresulations . soxB gene in pure cultures of various halophilic and haloalkaliphilic sulfur-oxidizing bacteria (SOB) and environmental samples of salt and soda lakes in southwestern Siberia and in the Wadi Natrun (Egypt) was studied by Tourova et al. [soxB phylotypes were well related to sequences from known sulfur-oxidizing Gamma- and Alphaproteobacteria and also to isolates from the investigated lakes, others represented new uncultured lineages and were significantly distant from those present in pure cultures. These may represent new taxa at least at the species or genus level [Thiohalorhabdus and phylotypes of Alphaproteobacteria related to Roseinatronobacter and Roseobacter were found in one and phylotypes of Marinobacter and Halothiobacillus species in another one. In contrast, phylotypes belonging to the Thioalkalivibrio cluster were common to the soda lakes and represented major or single phylotypes in two of them (including a mix of samples from different soda lakes of the Wadi Natrun). The third soda lake was dominated by a phylotype related to Thiohalomonas denitrificans and in addition showed representatives related to Thioalkalibacter, Halochromatium,Thioalkalivibrio, and to the alphaproteobacterium Roseinatronobacter [Halorhodospira species are well known as dominant members of sulfur-oxidizing phototrophic bacteria in these lakes [et al. [soxB was successfully amplified from close relatives of the genus Ectothiorhodospira [The diversity of the a et al. . Though us level . Most sinobacter . Though se lakes ,116,117, [et al.  to be caodospira  and thernar) and a periplasmic nitrate reductase (nap) and two types of nitrite reductases (nirK and nirS). The search for suitable primer binding sites in genes involved in the denitrification process led to the construction of specific primers for narH, narG, napA, nirS and nirK and a more detailed study using the narH gene [Denitrifying bacteria are a highly diverse group of facultative anaerobic bacteria and are able to switch from aerobic respiration with oxygen as terminal electron acceptor to anaerobic respiration with nitrate, nitrite and nitrogen oxides as terminal electron acceptors. Several key enzymes involved in the respiratory pathway of dissimilatory nitrate reduction are characteristic for the denitrification process and may qualify for analysis of denitrifying bacterial diversity. Most attention has been paid to a membrane-bound  to the preferred gene for biodiversity studies [narH sequences. The Firmicutes with Bacillus, Staphylococcus and Paenibacillus formed a distinct cluster, as did the Enterobacteria , the Betaproteobacteria  and the Gammaproteobacteria (Pseudomonas). These results were a strong motivation for further application of the narH gene in environmental studies of denitrifying bacteria and to consider the narH gene as primary target for monitoring diversity and activity of denitrifying bacteria.The membrane-bound nitrate reductase catalyzes the first step in nitrate reduction. A detailed analysis of the secondary structure of NarH revealed several conserved parts of the membrane-bound nitrate reductase ß-subunit (containing four iron-sulfur clusters as prosthetic groups which are fixed in the protein by four cysteine binding motifes) as possible priming sites for amplification and made  studies . The spe studies . Major pnarH primers, valuable insight into denitrifying communities in different parts of the Baltic Sea was obtained [narH sequence analysis in the environmental metagenome using an advanced double gradient denaturing gradient electrophoresis (DG-DGGE) combined with sequencing of the separated bands from the DGGE gels. Well resolved gradient gels nicely demonstrated, that over an annual time period  at a reference station in the Baltic Sea (“Boknis Eck”) the community of nitrate reducing bacteria was quite stable at the sediment surface but showed great variation in the bottom-near water [In a first case study using these obtained ,118. SevnarH sequences correlated well with a minimum in the nitrate concentrations and a maximum in the activity of nitrate reduction alongside the depth profile (from 2–4 cm depth) [Bacillus and Pseudomonas represented approx. 60% of the isolated denitrifiers from Kiel Bight, none of these were found in the environmental sequences [A clear stratification was seen in the denitrifying communities with different populations in the upper (1–3 cm depth) and lower (3–5 cm depth) compartments of the top sediment layers of the Kiel bight (Boknis Eck and Kiel Bight) and the Gulf of Finland. In these sediments a clear maximum of bands of m depth) . Signifiequences . Part ofParacoccus denitrificans [In contrast to the two sediments of shallow areas of the Baltic Sea, other sites located in the deep Gotland Basin (at 240 and 190 m depth) were characterized by anoxic and sulfidic water bodies above the sediments. Also in these sediments, the nitrate profiles had clear minima between 2–4 cm depth and most of the bands of nitrate reductase were found in these layers. However, all sequences retrieved from the DGGE gel bands were associated with Alphaproteobacteria and formed clusters in some distance to the denitrifying rificans .Paracoccus denitrificans and the assumption that these bacteria would be able to link sulfide and sulfur oxidation to nitrate reduction as is known from P. denitrificans.It was concluded that different environmental conditions select different communities of nitrate reducing bacteria in upper and lower sediment layers below the oxic water bodies (Kiel Bight and Gulf of Finland) and below the almost permanent anoxic water bodies in the Gotland Basin. It was suggested that a possible coupling between nitrification and denitrification in the transition between oxic and anoxic parts of the stratified sediments selects specific groups of denitrifiers different from those that predominate in the deeper anoxic parts of these sediments, while the supposed competition between sulfate reducers and nitrate reducers governs the development in the mostly anoxic and sulfidic sediments in the deep Gotland Basin . This conarG were applied for studies on nitrate reducing bacterial communities in sediments of a fresh water lake [narH the majority of the sequences from environmental DNA were related to Gammaproteobacteria while a number of Gram-positive bacteria were isolated from the sediments.Gene sequences of the α-subunit of nitrate reductase ter lake . Similarnap genes is an alternate widely distributed enzyme of dissimilatory nitrate reduction and is expressed in the presence of nitrate. The core part of this enzyme is encoded by napA and is highly conserved in chemoautotrophic nitrate-reducing Epsilonproteobacteria [napA genes from the vent sites were from Epsilonproteobacteria [A periplasmic nitrate reductase encoded by bacteria . Epsilonbacteria .nirK or a cytochrome-containing (cyt c and cyt d) enzyme encoded by nirS [nirK or nirS. Much emphasis has been given to the application of genes coding for nitrite reductases in denitrifying bacteria. In general, denitrifying bacteria carry one of the two types of nitrite reductases, either a copper-containing enzyme encoded by  by nirS . This ofCommunities of denitrifying bacteria in a number of freshwater and marine habitats have been studied by applying the genes of nitrite reductases ,124,125.nirK and nirS also are involved in aerobic microbial nitrification and anaerobic ammonia oxidation, respectively. Quite interestingly nirK genes  have been found in aerobic ammonia-oxidizing bacteria [nirK phylogenetic tree of nitrifying Betaproteobacteria corresponded to that of amoA and the 16S rRNA gene [nirK sequences retrieved from the environment may include sequences from ammonia-oxidizing bacteria and that nirK has coevolved with amoA in these bacteria. Because nirK and norB sequences from Nitrosospira spp. were phylogenetically not clearly separated from those of denitrifiers, it was suggested that they have been subjected to lateral gene transfer [It should be mentioned, that bacteria  and poinRNA gene . These rtransfer . nirS in anammox bacteria and nirK in AOA see the following paragraphs.For the involvement of nirK appear to be adapted to different environmental conditions alongside the stratified water column, whereas such a specific adaptation could not be seen with bacteria carrying nirS [nirK being associated to specific salinities, but those carrying nirS were not [nirK and nirS was reported from the suboxic zone of the Black Sea, where nirK sequences showed much greater variation with depth while nirS-bacteria were more homogenously distributed [nirK show different responses to a variety of environmental conditions and occupy different ecological niches compared to those having nirS. It would certainly be interesting to learn more about the strategic differences of the two groups of denitrifiers.According to T-RFLP analyses and gene sequences of denitrifying bacterial communities from Lake Kinneret, a monomictic freshwater lake in Israel, it could be demonstrated that different clades of bacteria using ing nirS . A similwere not . Anothertributed . ApparenThe oxidation of ammonia is a key step in the global nitrogen cycle and ammonia-oxidizing microorganisms (AOM) are widely distributed in environments and have received a lot of attention over the past decades. Three important groups of microorganisms are contributing to the oxidation of ammonia: the aerobic ammonia-oxidizing bacteria (AOB), the anaerobic ammonia-oxidizing bacteria (anammox) and the archaeal ammonia oxidizers (AOA). The isolation and cultivation of all three groups in the laboratory are quite problematic, in particular due to their slow growth rates and poor growth yields. For decades, these circumstances severely limited progress of our knowledge in the biology of the AOB. After the recognition of archaeal ammonia oxidation, the major interest moved to ammonia-oxidizing archaea (AOA), to their role in the environment and to the relative importance of both groups in the nitrification process ,137,138.amoA sequences, AOB are found in two monophyletic groups, one is belonging to the Betaproteobacteria with the genera Nitrosomonas and Nitrosospira (including Nitrosolobus and Nitrosovibrio) and the other belonging to the Gammaproteobacteria, including Nitrosococcus oceani and Nitrosococcus halophilus [amoCAB operon. The amo operon was found in multiple almost identical copies in Betaproteobacteria but in single copies in the Gammaproteobacteria of AOB [Traditionally, ammonia oxidation was known as a property of the aerobic ammonia-oxidizing bacteria (AOB), which are typically chemoautotrophic bacteria. Based on 16S rRNA gene and lophilus . These ba of AOB . amoA turned out to be a suitable marker for the whole group of aerobic ammonia-oxidizing bacteria and amoA was applied to characterize representative environmental communities. A number of PCR primers to amplify amoA have been used, though considerable differences were found in their performance and specificity [amoA gene as a molecular marker [amoA used is highly conserved and in addition is relatively short (around 450 bp), it provides less resolution than the 16S rRNA gene [amoCAB operon was used for molecular studies and a nested PCR approach was applied to amplify environmental amoCAB sequences, including the complete amoA gene and providing an increase of sensitivity in detecting amo genes in samples even with low abundances of AOB [The ammonia monooxygenase gene cificity ,141. Manr marker ,141. As RNA gene . Indeed,s of AOB . amoA retrieved from an acidic hot spring in a Japanese gold mine revealed AOB highly similar to Nitrosomonas spp. [amoA and 16S rRNA gene sequence analysis of enrichment cultures from Salar de Huasco [Nitrosomonas were identified and sequences of amoA related to N. halophila and N. nitrosa were frequently found at all salinities. Ammonia-oxidizing Gammaproteobacteria were not found in Salar de Huasco. Apparently, AOB are also adapted to a variety of extreme environmental conditions. Sequences of nas spp. . AOB were Huasco . SeveralamoA sequences affiliated with ammonia-oxidizing Betaproteobacteria, but Gammaproteobacteria and AOA could not be found [Nitrosomonas halophila from other soda lakes, a Mongolian soda lake, and Qinghai Lake (China) and of a Nitrosomonas isolate from a Greenland alkaline tufa column. Others were related to Nitrosococcus europaea and Nitrosomonas eutropha [Studies on Mono Lake demonstrated that all be found . Most ofeutropha .amo-like genes in archaeal DNA from metagenomic libraries from seawater [Nitrosopumilus maritimus was isolated as the first ammonia-oxidizing archaeon [Candidatus Nitrosocaldus yellowstonii [Candidatus Nitrososphaera gargensis [Soon after the first discovery of seawater  and soilseawater , Nitrosoarchaeon . This waowstonii  and Candargensis . In factargensis . Our knoargensis ,148,149.amoA  have been identified [anirKa being found throughout the water column while two different variants of nirKb predominated in the upper water layers and in the lower ones [anirKb, but not of anirKa, was highly similar to that of the archaeal amoA [nirKb and amoA. Two distinct ecotypes of archaeal ammonia-oxidizers in the ocean were also recognized by using two amoA primer sets. One ecotype is adapted to high concentrations (1.2 µM in average) and the other to low concentrations (<0.01 µM) of ammonia [In nitrifying Archaea (Thaumarchaeota) two variants of archaeal entified . These vwer ones . In addieal amoA , which i ammonia . This di ammonia , which inirS), a hydrazine synthase (hzs) and a hydrazine oxidase (hzo) but not an ammonia monooxygenase [nirS gene was used as molecular marker to study the presence and potential activity of anammox in the environment by RT-PCR [hzo gene was applied to identify anammox bacteria from marine sediments, coastal wetland, and the deep ocean [hzsA) has been identified as a unique marker for studies of anammox bacteria and was applied to anammox enrichment cultures and various environmental samples including marine samples from the Barent Sea and Northeast Greenland [Another group of ammonia oxidizing bacteria is known that performs an anaerobic ammonia oxidation (anammox). These bacteria were identified as new unique Planctomycetes  and appaxygenase . The niry RT-PCR . Also thep ocean . In addireenland . These spmoA gene and 16S rRNA gene have been shown to be congruent and pmoA is well established as a phylogenetic marker gene for studies of methanotrophic bacteria in the environment [amoA and pmoA may amplify both genes. Aerobic methanotrophic bacteria perform an important ecological function in the global carbon cycle by oxidation and assimilation of methane. Much of our “early knowledge” from the 1970s to 1990s on these bacteria comes from studies in paddy soils and rice fields ,166,167.ironment . It has ironment , which ipmoA gene have been frequently applied in environmental studies since many years, e.g., to study methanotrophic communities in Lake Washington [Nitrosospira were amplified in an untreated soil, after the addition of methane a shift in the community favored the development of both type I and type II methanotrophic bacteria [Primers of the shington , in riceshington , in a bishington  and in mshington ,173. Indshington ,26. Whilbacteria , which ipmoA sequences was applied to study the diversity of methanotrophic bacteria from environmental samples and from cultures of methane-oxidizing bacteria and to define phylogenetic groups and phylotypes [pmoA sequences has been used to define major phylogenetic groups of methanotrophic bacteria, to refine the established grouping and to define habitat specificity at different systematic levels of these bacteria [Amplicon pyrosequencing techniques using ylotypes . Though bacteria . Since their discovery in 1977 and first pioneering microbiological studies ,175, hydThe Logatchev hydrothermal field is an ultramafic-hosted system on the Mid-Atlantic Ridge, with the Irina II complex as main structure consisting of a large mound with several black smoker chimneys at the top and mussel fields surrounding the base of the chimneys . VentingsoxB, aprA) and autotrophic CO2-fixation by the Calvin-Benson-Bassham cycle  and the reductive tricarboxylic acid cycle (sequences coding for ATP citrate lyase aclB) as functional markers and the 16S rRNA gene as an additional phylogenetic marker [2-fixation [aprA, soxB,cbbM, cbbL and aclB, both groups used different pathways for these processes  and most of the sequences were similar to aprA sequences known from epibionts and endosymbionts from vent habitats [The microbial community structure within hydrothermal fluids from the Irina II structure of the Logatchev vent field was analyzed using genes involved in different pathways of sulfur metabolism  and the reductive tricarboxylic acid cycle (acl). Tubeworms of the genus Lamellibrachia are common to hydrothermal vent and cold-seep communities in the Pacific and other hydrothermal habitats. Comparison with tubeworm species  from cold seeps in the Gulf of Mexico revealed the presence of acl and cbbL/M genes in these species as well [Riftia pachyptila [A functional gene study of endosymbionts of vestimentiferan tube worms from a hydrothermal vent site in the Mediterranean Sea identified as symbiont . Geneticsymbiont . However as well . These rchyptila  suggest Rimicaris exoculata which dominates the faunal biomass at some locations of deep-sea hydrothermal vent sites at the Mid-Atlantic Ridge [aclB, ccbL, cbbM), sulfur  and hydrogen (hynL) metabolism [Desulfocapsa. Based on functional gene analyses, it was concluded that the Gamma- and Epsilonproteobacteria associated to the shrimp are capable of autotrophic growth by oxidizing reduced sulfur compounds, and that the Deltaproteobacteria as reducers of sulfate may enable the cycling of sulfur compounds in a microscale dimension within the gill [Sulfurovum lithotrophicum as the closest cultured relative [Leucothrix mucor as the closest cultured bacterium [hynL), which is involved in the oxidation of molecular hydrogen for energy generation, were identified. The relation of these genes to sulfate reducers and Epsilonproteobacteria highlights the potential of both groups to grow with hydrogen as an electron source. In relation to the genes of sulfur oxidation and carbon dioxide fixation it appears quite likely that in addition to Epsilon- and Gammaproteobacteria also the sulfate-reducing Gammaproteobacteria may contribute to autotrophic carbon fixation and productivity of the epibiotic community [in situ hybridization and this visualization supports the assumption of physiological interactions between the groups of sulfate-reducing and sulfur-oxidizing bacteria [A multiple functional gene study was made with the epibacterial community associated with the shrimp ic Ridge . This sptabolism . The comthe gill . The phyrelative . The Gamacterium . Similaracterium ,180,183.ommunity . The locbacteria . Molecular studies using functional genes as targets have not only presented important phylogenetic information of major biochemical pathways, but also gave crucial information on the competitive nature of the considered pathways in the environment, or rather their relevance for the competition of the organisms employing these pathways. Furthermore, detailed information on the functional diversity in many habitats and on the selection of and preference for specific habitats by microorganisms has accumulated in recent years. In this context, aspects of geographical distribution were also of major concern. Quite importantly, for most functional genes the general congruence with the phylogeny of the 16S rRNA gene has been established, and growing databases of functional gene sequences make it possible to trace their phylogeny and to identify species or phylotypes in the environment on the basis of functional gene sequences. Undoubtedly, a new era of microbial ecology was started with the introduction of 16S rRNA gene analyses in environmental studies and the established molecular microbial ecology opened up completely new possibilities to this research field. For the first time, microbial biodiversity studies were possible based on 16S rRNA gene sequences. A new dimension is given to microbial ecology with the introduction of functional genes as targets for biodiversity studies which enables the identification of strains and species on the functional gene level with the perspective of bioactivity studies under environmental conditions using functional gene transcripts.nirS and nirK identify two groups of denitrifying bacteria with different strategies of environmental adaptation [nirS is involved, and on aerobic nitrification, in which nirK is involved [anirKa and anirKb genes as well as to different variants of the archaeal amoA gene [dsr gene analysis in microbial mats of Solar Lake with a clear dominance of relatives of Desulfonema in this niche [One of the most significant findings resulting from the application of functional genes in environmental studies clearly points out the importance of biochemical pathways for performance and competition of microorganisms in the environment and for the selection of and preference for ecological niches by the microorganisms. In this context, the success of sulfur-oxidizing Epsilonproteobacteria in the gradient systems of deep sea hydrothermal vents is explained by the employment of energy efficient pathways such as the reversed TCA for carbon fixation and the Sox-pathway for sulfur oxidation as compared to the Calvin cycle and the APS pathway by sulfur-oxidizing Gammaproteobacteria ,98. Simiaptation ,127,136.involved ,134,135.moA gene . Althougis niche .Functional gene studies have convincingly and repeatedly demonstrated that small changes in the environmental conditions significantly can change the community composition and that individual species or phylotypes may have a narrow range of suitable conditions for development and successful competition. pufLM and fmoA genes to the study of environmental communities of phototrophic purple and green sulfur bacteria has pointed out the relevance of salt relations and temperature responses to the distribution of species in the environment. A decent part of the environmental diversity of these bacteria is available in pure cultures and, depending on the habitat, a varying part of the diversity can be identified at the level of species and genera using fmoA or pufLM gene sequences. It was realized that coastal lagoons to a significant part harbor marine, salt tolerant and moderately halophilic species, most of which are known at the genus level and that in part drastic changes in the community composition occur as response to temperature and salinity changes. This can be taken as an indication that salt tolerance is an important property to adapt and survive in these habitats which can undergo large variations in salt content depending on evaporation and supply of new freshwater or seawater. In two Chilean salt lakes situated in close distance to each other , species and phylotypes were identified with the next relatives known to be specifically adapted to the high salt concentrations in the habitat . Despite this unifying aspect of the two salares, it was quite remarkable to see that even different samples from the same lake demonstrated a largely different composition of the communities of purple sulfur bacteria [soxB sequences, and in addition to realize that a characteristic and typical community structure can be found in individual soda lakes and salt lakes [The application of bacteria . Thus enlt lakes . narH gene sequences, occupy clearly different niches in strongly sulfidic marine sediments and in stratified sediments with an oxic surface layer. On top of this a clear preference for different denitrifiers is seen in different layers of the stratified sediment horizons [soxB-based phylotypes alongside the environmental gradients was demonstrated [Likewise, it is interesting to see that denitrifying Alpha- and Gammaproteobacteria, as demonstrated by horizons . Generalnstrated .pufLM genes [In view of the tremendous advances in molecular microbial ecology which was established only two decades ago, there is much enthusiasm in this research field. Nevertheless it must be considered that also the most advanced methods have at least some limitations. Although we are convinced that the use of specific functional primers for gene amplification gives the most comprehensive picture on the specific function’s diversity, we still have to consider biases, e.g., due to methods of DNA extraction from the environment and primer specificity. We also have to consider that a complete record of the species diversity may not be achieved, which is, however, more relevant to the analysis of complete metagenomes than to specific functional metagenetic analyses, no matter how accurate and extensive the sequence analyses may be (see the previous discussion to this aspect). In this sense, we have to accept that the true diversity of a given sample is higher than shown by the metagenetic and metagenomic analyses and that some species not seen under one condition may increase in importance or even become dominant under changed conditions. Such an incomplete coverage of environmental communities as well as the selective property of salt concentrations and temperature was nicely demonstrated in culture experiments with phototrophic purple sulfur bacteria from a coastal Baltic Sea lagoon using the photosynthetic LM genes . In cons3, while the total number of prokaryotes in this sediment exceeded 1010 cells cm3 . It is quite obvious that the analysis of environmental DNA of this sediment would not have given a sign of these green sulfur bacteria, as their abundance is far below the detection limit of these approaches (even using currently available high throughput sequencing techniques) and they are considered as an unimportant part of the community. However, whenever such sediments undergo environmental changes that allow the sulfide produced by sulfate-reducing bacteria to stay in the top layer of the sediment illuminated by the sun or even to penetrate into the water body of small lagoons, this minor portion of the community may rapidly form massive developments that are indeed often seen in suitable coastal habitats. The principal advantage of enrichment experiments is the high selectivity in regard to a particular functional property and this may be specifically used to determine the diversity of a given function, i.e., anoxygenic phototrophic sulfur oxidation [in situ experiments under environmental conditions or in experimental enrichment studies in the laboratory.One of the oldest and strongest selective conditions employed by microbiologists is the use of specific culture media for enrichment and cultivation of various bacteria with different physiological demands . The spexidation  or aerobxidation  either bin situ also maybe found after enrichment with culture conditions and media properly established, as exemplified for the phototrophic sulfur bacteria [Though we are far from being able to systematically describe the species diversity of natural communities, even of the well characterized anoxygenic phototrophic bacteria, results of the work discussed here demonstrate that progress is made with high speed and that this might well be achieved in the future. An important point to support this optimistic view is the fact that the combination of cultivation-independent and cultivation-dependent approaches demonstrated that most of the phylotypes seen bacteria ,186,187.bacteria ,116,117 bacteria . As outlbacteria . Increasin-situ meta-transcriptomic analyses and laboratory cultivation studies will provide deeper insights into the understanding of microbial communities and will add another new dimension to microbial ecology, the species-specific in situ measurement of activities in complex environmental communities. Activities of selected genes and dynamic changes thereof will enable analysis of the dynamic adaptation of communities to changes in the environmental conditions, including diurnal and long term changes, and relate these changes to the functional transcript and the phylotype. Functional gene studies as highlighted in this review are certainly forming the basis for such meta-transcriptomic studies.For the future, the combination of


Skilled individuals are characterized by fine-tuned perceptual and motor functions. Here, we tested the idea that the sensory and motor functions of highly-trained individuals are coupled. We assessed the relationships among multifaceted somatosensory and motor functions of expert pianists. The results demonstrated a positive covariation between the acuity of weight discrimination and the precision of force control during piano keystrokes among the pianists but not among the non-musicians. However, neither the age of starting musical training nor the total amount of life-long piano practice was correlated with these sensory-motor functions in the pianists. Furthermore, a difference between the pianists and non-musicians was absent for the weight discrimination acuity but present for precise force control during keystrokes. The results suggest that individuals with innately superior sensory function had finer motor control only in a case of having undergone musical training. Intriguingly, the tactile spatial acuity of the fingertip was superior in the pianists compared with the non-musicians but was not correlated with any functions representing fine motor control among the pianists. The findings implicate the presence of two distinct mechanisms of sensorimotor learning elicited by musical training, which occur either independently in individual sensorimotor modalities or through interacting between modalities. Plasticity of the nervous system changes sensory and motor functions through training. Musicians provide unique opportunities to shed light on the complex biological mechanism underlying the interaction of neuroplasticity of the human sensorimotor system with long-term multimodal training from childhood12468Recent research has revealed a shared mechanism between motor and perceptual learning11121416171922232425The purpose of the study was to assess the relationships among the somatosensory and motor functions of musicians. A spectrum of these functions were evaluated and compared within and between expert musicians and non-musicians. We postulated superior somatosensory and motor functions in musicians compared with non-musicians and a correlation between somatosensory acuity and fine motor control in musicians. The study also sought to assess the effects of early and deliberate musical practice on somatosensory and motor functions.Forty participants including twenty-one pianists and nineteen non-musicians underwent five independent sensorimotor function tests . Each of the tests was performed with the participant’s non-dominant ring finger, and assessed a variety of sensorimotor functions.To assess the relationships among the somatosensory and motor abilities in the music-relevant and music-irrelevant tests, a multiple linear regression analysis was performed for each of the variables evaluated among the individuals of the musician and non-musician groups. The analysis was performed separately for each of the two groups owing both to the aforementioned group difference in some of the variables and to no hypothesis of differences in the regression coefficient between the groups. The questionnaire that evaluated the participants’ history of musical training was only administered to the group of pianists. The results indicated that the average age at which musical training commenced was 4.1 ± 0.9 (range: 3–6) years old and that the total amount of piano practice during a lifetime was 21915 ± 11561 (range: 6570–53290) hours. A multiple linear regression analysis predicting each of the somatosensory and motor functions assessed by the individual tests yielded no significant covariation with each of the two variables representing the history of musical training (p > 0.05). Therefore, these results indicate that the individual differences in musical training history among the pianists failed to account for the inter-individual variability of the sensory and motor functions evaluated in the study.The present study demonstrated superior abilities in several particular somatosensory and motor functions in trained pianists compared with musically untrained individuals. In agreement with several previous findings6In the motor domain, the precision of sustained force production was superior for the pianists compared with the non-musicians , which s3128331In contrast with the group difference between the pianists and the non-musicians, the inter-individual difference in the fine control of force production during the piano keystrokes among the pianists was accounted for by the individuals’ differences in weight discrimination ability . A possiOne may expect that there can be some particular genetic predisposition that commonly determines both the somatosensory and motor functions of pianists, which can explain a correlation of sensory and motor functions across pianists. Several recent studies reported the presence of genetic factors that determine musical expertise24An intriguing observation was the correlation between each of the weight and spatial discrimination abilities and precision of sustained force production across the non-musicians . By contThe variability in the timing of the constrained piano keystrokes covaried negatively and positively with the loudness variability of the keystrokes in the pianists and the non-musicians, respectively. Because constrained keystrokes require independent control of movements between the fingers, a lack of independent movement control therefore compromises the control of both timing and forceIn conclusion, the present study demonstrated that the inter-individual difference in the active force perception ability, which was independent of early and extensive piano training, was associated with that in fine motor control in the pianists. The result suggests that individuals with innately superior sensory function benefited to a greater extent from extensive multimodal musical training and thereby acquired finer motor control. Foster and Zatorre (2010) showed a relationship between structural cortical differences in musicians and the performance on musical transformation tasks, which, however, remained when accounting for the individual amount of musical training3738Forty participants took part in the experiments. The participants included twenty-one right-handed pianists  aged between nineteen and thirty-four years (24.3 ± 4.7 years) and nineteen age-matched right-handed non-musicians  aged between 19 and 39 years (21.6 ± 5.2 years). All pianists majored in piano playing and underwent formal musical education at music conservatories, whereas the non-musicians had very little or no experience studying piano playing (less than three years). In accord with the Declaration of Helsinki, the experimental procedures were explained to all participants. Informed consent was obtained from all participants prior to participation in the experiment. The experimental protocol was approved by the ethics committee at Sophia University, and all methods were performed in accordance with the relevant guidelines and regulations. A sample size was determined prior to initiating the whole data collection by means of a power analysis using G-Power.The experiments consisted of five sensorimotor function tests . Each of the tests was performed with the participant’s non-dominant ring finger to minimize confounding linear additive effects of hand use during daily and recreational activities.To evaluate spatial discrimination ability, the discrimination threshold of the left ring fingertip was measured via a two-alternative forced-choice simultaneous spatial two-point discrimination taskμs) were used as stimuli. An experimenter pressed a monofilament carefully and gently against the surface of each participant’s left ring fingertip for three seconds. Two series of ascending and descending pressures were tested. Each participant closed his/her eyes and was asked to report whether he/she could perceive contact with the filament.To evaluate passive force discrimination ability, the cutaneous pressure threshold of the left ring fingertip was measured via a two-alternative forced-choice cutaneous pressure discrimination task using monofilaments . A total of four Semmes – Weinstein monofilaments . A total of eight different weights  were used. The experimenter placed two weights on a table in front of the participant. Each participant was asked to lift up each weight with the fingertip of the left ring finger as slowly as possible, hold for three seconds, and return the weight to the initial position. Each participant then answered whether each of them was heavier or both were the same weight in a two-alternative forced-choice fashion.We evaluated music-irrelevant motor functions of the left ring finger using a custom-made force sensor system . The resolution and maximum measurable force of the sensor were 0.05 and 49N, respectively. A target trajectory of isometric force production was visually displayed on the screen and had a shape of trapezium consisting of three successive phases: increase, hold, and decrease of force. Each of these three phases lasted for eight seconds  and had a peak of 1.6 N. Each participant exerted force on the force sensor with the left ring fingertip in an isometric manner to trace the targeted force trajectory as accurately as possible over five trials. The force data were sampled at 1 kHz. During the isometric force production, the participants were instructed to keep the remaining four digits immobilized.staccato touch to synchronize with the metronome and elicit the target loudness of tones. Each participant performed the task either by keeping the other four digits immobilized and depressing the adjacent keys (“constrained condition”) or without any explicit requirements regarding the motions of the non-striking fingers (“unconstrained condition”). The participants performed each of the two conditions over five trials.Using a digital piano with wooden keys (Yamaha DGP-5), we evaluated music-relevant motor functions of the left ring finger. An experimenter sounded a metronome , and each participant struck a piano key with the left ring fingertip with a A short questionnaire that included questions about his/her history of musical training  was completed by each participant.To identify a sensory discrimination threshold according to the results of the individual sensory tests, a logistic regression analysis was performed on the derived datasets. The threshold was defined as the effectiveness level computed from a logistic regression.For the music-irrelevant motor test , the absolute value of the difference between the target force trajectory and the force trajectory produced by a participant was computed every millisecond and was then averaged over time for each of the increase, hold, and decrease phases to yield an index for the erroneous force production amount. The computed value was then averaged across five trials.For the music-relevant motor test , the absolute value of the difference in each of loudness and timing between the targeted tone provided by the metronome and the tone produced by the participant was computed every second and was then averaged over time for each of the constrained and unconstrained conditions. We used these variables as the indices of timing and force variability of the piano keystrokes.For each of the individual variables, a two-sample t-test was performed to assess the group difference between the pianists and the non-musicians (p < 0.05). Neither multivariate analysis of variance (MANOVA) nor p-value correction for multiple comparisons was carried out, firstly because each of the individual sensory and motor tests were performed separately and independently, and secondly because we did not hypothesize a difference between any pair of the sensory and motor functions. In addition, a multiple regression analysis was performed to evaluate the covariation between the individual sensory and motor abilities for each of the pianists and non-musicians. The regression model predicted each of the sensory and motor variables based on the remaining variables (p < 0.05). We performed the regression analysis for each of the two groups separately, because we did not have any hypothesis regarding a difference in the regression coefficient between the groups.How to cite this article: Hosoda, M. and Furuya, S. Shared somatosensory and motor functions in musicians. Sci. Rep.6, 37632; doi: 10.1038/srep37632 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


A coprime array is capable of achieving more degrees-of-freedom for direction-of-arrival (DOA) estimation than a uniform linear array when utilizing the same number of sensors. However, existing algorithms exploiting coprime array usually adopt predefined spatial sampling grids for optimization problem design or include spectrum peak search process for DOA estimation, resulting in the contradiction between estimation performance and computational complexity. To address this problem, we introduce the Estimation of Signal Parameters via Rotational Invariance Techniques (ESPRIT) to the coprime coarray domain, and propose a novel coarray ESPRIT-based DOA estimation algorithm to efficiently retrieve the off-grid DOAs. Specifically, the coprime coarray statistics are derived according to the received signals from a coprime array to ensure the degrees-of-freedom (DOF) superiority, where a pair of shift invariant uniform linear subarrays is extracted. The rotational invariance of the signal subspaces corresponding to the underlying subarrays is then investigated based on the coprime coarray covariance matrix, and the incorporation of ESPRIT in the coarray domain makes it feasible to formulate the closed-form solution for DOA estimation. Theoretical analyses and simulation results verify the efficiency and the effectiveness of the proposed DOA estimation algorithm. Direction-of-arrival (DOA) estimation aims at retrieving the directional information of sources from the array received signals, and plays a fundamental role in a variety of practical application fields including radar, sonar, acoustics, radio astronomy, and wireless communications ,9,10,11.Recently, the coprime array has been proposed as the realization of coprime sampling in the spatial domain, and attracted tremendous interests in the field of array signal processing . On one The MUltiple SIgnal Classification (MUSIC) technique  is one oIn this paper, we propose a novel coprime array DOA estimation algorithm by processing the equivalent coprime coarray received signal with Estimation of Signal Parameters via Rotational Invariance Techniques (ESPRIT), where off-grid DOAs can be efficiently resolved without spectrum peak search. Unlike the ESPRIT-like algorithms carried out in ,39, whicWe derive the coarray statistics of the coprime array, and introduce the idea of ESPRIT to the coarray domain for retrieving the DOAs with an increased number of DOFs.We extract a pair of shift invariant uniform linear subarrays from the coprime coarray, and investigate the rotational invariance of the corresponding coarray domain signal subspaces.We provide the closed-form solution for efficient DOA estimation, which enables performing off-grid DOA estimation without predefined spatial sampling grids or the spectrum peak search process.The main contributions of this paper can be summarized as follows:The rest of this paper is organized as follows. Notations: Throughout this paper, vectors and matrices are respectively represented by lower-case boldface and upper-case boldface characters. M and N are coprime integers with N sensors spaced d equals a half-wavelength. Collocating these two ULAs with the sensor at the zeroth position aligned, as shown in The coprime array consists of two ULAs as illustrated in K uncorrelated narrowband plane-wave signals from the directions l-th snapshot can be modeled ask-th columnAssuming there are K sources L available snapshots asThe theoretical covariance matrix of the coprime array received signals Obviously, the sample covariance matrix In this section, we elaborate on the proposed coarray ESPRIT-based DOA estimation algorithm for coprime array, where off-grid DOA can be resolved without the predefined spatial sampling grids or the spectrum peak search process. The coprime array received signals are firstly transformed to the equivalent second-order received signal of an augmented coprime coarray, such that the number of DOFs can be effectively increased. Since the ESPRIT requires a shift invariant array geometry to perform, a pair of uniform linear subarrays is extracted from the derived coprime coarray, where the shift invariance is investigated based on the signal statistics in the coarray domain. Based on the rotational invariance of the subspaces corresponding to the subarray pair, the closed-form solution is formulated for efficiently estimating the DOAs of each source.The coarray domain equivalent received signal can be derived by vertically stacking each column of the sample covariance matrix Since the elements Although the coprime coarray is non-uniform, it has been revealed in  that theℓ-th subarray consists of On the other hand, although ancement ,35. In p R˜yy is Here, the theoretical coprime coarray covariance matrix can be represented asroved in  by rearrTo employ the ESPRIT in the coarray domain, a pair of translationally separated coprime coarray subarrays with the same geometry is required, such that the corresponding coarray statistic characteristics imposed by the shift invariance can be investigated. While the mathematically derived coprime coarray is practically nonexistent, the available statistics in the coarray domain are the second-order received signal d is existed for the corresponding virtual sensors in each subarray.Based on the observations mentioned above, a pair of uniform linear subarrays consisting of Similar to Equation (1), the received signals of the pair of coprime coarray subarrays can be theoretically modeled asd between the doublet virtual sensors, the received signals While the subarray pair K largest eigenvalues contained in the diagonal of The shift invariance of the coprime coarray subarray pair results in the rotational invariance of the underlying signal subspaces in Since the column space of Combining the relationship revealed in Equation (17), which is imposed by the shift invariance, we haveK. Hence, there exists a Since the column spaces spanned by Substituting the relationship between the signal space and the manifold matrix established in Equation (23), Equation (25) can be rewritten asDefining While the matrix k-th source can be readily formulated ask-th eigenvalue of Hence, K to ensure the estimation resolution and accuracy. Therefore, the proposed coarray ESPRIT-based algorithm has a superior performance in terms of computational complexity, and enables efficiently resolving off-grid DOAs.The computational complexity of the proposed DOA estimation algorithm is lgorithm  has a colgorithm  do not rThe steps of the proposed coarray ESPRIT-based DOA estimation algorithm are listed in In our simulations, the pair of coprime integers is selected to be lgorithm , the spalgorithm , and thelgorithm . The prey-axis of In the first example, we compare the DOA estimation performance of each algorithm using coprime array in It is clear that all of the algorithms we considered are capable of identifying the 15 sources with only 10 sensors, where the DOF superiority offered by the coprime array is demonstrated. The additional peaks appeared in the spatial spectrum of the SSR algorithm are caused by the undetermined regularization parameter, which is utilized to balance the sparsity and the reconstruction accuracy in the optimization problem for signal reconstruction. These irregular spurious peaks, especially those that closely approach the peaks corresponding to the true DOAs, lead to the difficulties in determining DOA estimations. The coprime virtual array interpolation-based algorithm has a better spatial spectrum characteristic than the SS-MUSIC algorithm since the array interpolation process makes full use of the information contained in the non-uniform coprime coarray In the second example, we compare the Root Mean Square Error (RMSE) of the estimated DOAs for each algorithm, where the RMSE criterion is defined ask-th source in the q-th Monte Carlo trial, and the RMSE is averaged from Here, We can observe from Meanwhile, we also consider a more generalized case, where the direction of the incident source is randomly selected from the uniform distribution on the interval In the third example, we compare the resolution performance of each algorithm in It can be observed from In the fourth example, we compare the RMSE performance of each algorithm in It is clear from In the last example, we depict the RMSE performance of the proposed algorithm in It is clear from We have proposed a novel coarray ESPRIT-based DOA estimation algorithm for coprime array, where off-grid DOAs can be efficiently resolved with increased number of DOFs. The coprime coarray is firstly derived as well as its corresponding statistics, where more virtual sensors can be utilized by processing the equivalent second-order received signal. By extracting a pair of shift invariant uniform linear subarrays from the coprime coarray, the rotational invariance of their signal subspaces is investigated in the coarray domain, and the closed-form solution for DOA estimation is formulated based on the idea of ESPRIT. Neither the predefined spatial sampling grids nor the spectrum peak search process is required for the proposed algorithm, ensuring a good balance between the resolution performance and the computational complexity. Theoretical analyses and simulation results demonstrate the superiority of the proposed algorithm in terms of achievable DOFs, estimation accuracy, spatial resolution and computational efficiency.