Nitric oxide reductase (norB) gene sequence analysis reveals discrepancies with nitrite reductase (nir) gene phylogeny in cultivated denitrifiers

Gene sequence analysis of cnorB and qnorB, both encoding nitric oxide reductases, was performed on pure cultures of denitrifiers, for which previously nir genes were analysed. Only 30% of the 227 denitrifying strains rendered a norB amplicon. The cnorB gene was dominant in Alphaproteobacteria, and dominantly coexisted with the nirK gene, coding for the copper-containing nitrite reductase. Both norB genes were equally present in Betaproteobacteria but no linked distributional pattern of nir and norB genes could be observed. The overall cnorB phylogeny was not congruent with the widely accepted organism phylogeny based on 16S rRNA gene sequence analysis, with strains from different bacterial classes having identical cnorB sequences. Denitrifiers and non-denitrifiers could be distinguished through qnorB gene phylogeny, without further grouping at a higher taxonomic resolution. Comparison of nir and norB phylogeny revealed that genetic linkage of both genes is not widespread among denitrifiers. Thus, independent evolution of the genes for both nitrogen oxide reductases does also occur.     

Phylogenetic analysis of nitric oxide reductase gene homologues from aerobic ammonia-oxidizing bacteria

Nitric oxide (NO) and nitrous oxide (N2O) are climatically important trace gases that are produced by both nitrifying and denitrifying bacteria. In the denitrification pathway, N2O is produced from nitric oxide (NO) by the enzyme nitric oxide reductase (NOR). The ammonia-oxidizing bacterium Nitrosomonas europaea also possesses a functional nitric oxide reductase, which was shown recently to serve a unique function. In this study, sequences homologous to the large subunit of nitric oxide reductase (norB) were obtained from eight additional strains of ammonia-oxidizing bacteria, including Nitrosomonas and Nitrosococcus species (i.e., both beta- and gamma-Proteobacterial ammonia oxidizers), showing widespread occurrence of a norB homologue in ammonia-oxidizing bacteria. However, despite efforts to detect norB homologues from Nitrosospira strains, sequences have not yet been obtained. Phylogenetic analysis placed nitrifier norB homologues in a subcluster, distinct from denitrifier sequences. The similarities and differences of these sequences highlight the need to understand the variety of metabolisms represented within a "functional group" defined by the presence of a single homologous gene. These results expand the database of norB homologue sequences in nitrifying bacteria.     

Diversity of nitrite reductase (nirK and nirS) gene fragments in forested upland and wetland soils

The genetic heterogeneity of nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods. nirK gene fragments could be amplified from both soils, whereas nirS gene fragments could be amplified only from the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of nirK clones was lower than the diversity of nirS clones. Among the 54 distinct nirK RFLP patterns identified in the two soils, only one pattern was found in both soils and in each soil two dominant groups comprised >35% of all clones. No dominance and few redundant patterns were seen among the nirS clones. Phylogenetic analysis of deduced amino acids grouped the nirK sequences into five major clusters, with one cluster encompassing most marsh clones and all upland clones. Only a few of the nirK clone sequences branched with those of known denitrifying bacteria. The nirS clones formed two major clusters with several subclusters, but all nirS clones showed less than 80% identity to nirS sequences from known denitrifying bacteria. Overall, the data indicated that the denitrifying communities in the two soils have many members and that the soils have a high richness of different nir genes, especially of the nirS gene, most of which have not yet been found in cultivated denitrifiers.     

Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in pacific northwest marine sediment communities

Genetic heterogeneity of denitrifying bacteria in sediment samples from Puget Sound and two sites on the Washington continental margin was studied by PCR approaches amplifying nirK and nirS genes. These structurally different but functionally equivalent single-copy genes coding for nitrite reductases, a key enzyme of the denitrification process, were used as a molecular marker for denitrifying bacteria. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially for nirS clones from Puget Sound and a slightly lower level of diversity for nirK and nirS clones from the Washington margin. One group dominated within nirK clones, but no dominance and only a few redundant clones were seen between sediment samples for nirS clones in both habitats. Hybridization and sequencing confirmed that all but one of the 228 putative nirS clones were nirS with levels of nucleotide identities as low as 45.3%. Phylogenetic analysis grouped nirS clones into three distinct subclusters within the nirS gene tree which corresponded to the two habitats from which they were obtained. These sequences had little relationship to any strain with known nirS sequences or to isolates (mostly close relatives of Pseudomonas stutzeri) from the Washington margin sediment samples. nirK clones were more closely related to each other than were the nirS clones, with 78.6% and higher nucleotide identities; clones showing only weak hybridization signals were not related to known nirK sequences. All nirK clones were also grouped into a distinct cluster which could not be placed with any strain with known nirK sequences. These findings show a very high diversity of nir sequences within small samples and that these novel nir clusters, some very divergent from known sequences, are not known in cultivated denitrifiers.     

PCR detection of genes encoding nitrite reductase in denitrifying bacteria

Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships of nir genes were also established. The cd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.     

Diversity of Nitrite Reductase (nirK and nirS) Gene Fragments in Forested Upland and Wetland Soils

The genetic heterogeneity of nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods. nirK gene fragments could be amplified from both soils, whereas nirS gene fragments could be amplified only from the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of nirK clones was lower than the diversity of nirS clones. Among the 54 distinct nirK RFLP patterns identified in the two soils, only one pattern was found in both soils and in each soil two dominant groups comprised >35% of all clones. No dominance and few redundant patterns were seen among the nirS clones. Phylogenetic analysis of deduced amino acids grouped the nirK sequences into five major clusters, with one cluster encompassing most marsh clones and all upland clones. Only a few of the nirK clone sequences branched with those of known denitrifying bacteria. The nirS clones formed two major clusters with several subclusters, but all nirS clones showed less than 80% identity to nirS sequences from known denitrifying bacteria. Overall, the data indicated that the denitrifying communities in the two soils have many members and that the soils have a high richness of different nir genes, especially of the nirS gene, most of which have not yet been found in cultivated denitrifiers.     

Nitric oxide reductase-targeted real-time PCR quantification of denitrifier populations in soil

The quantification of denitrifying bacteria is a component in the further understanding of denitrification processes in the environment. Real-time PCR primers were designed to target two segments of the denitrifier population (cnorB(P) [Pseudomonas mandelii and closely related strains] and cnorB(B) [Bosea, Bradyrhizobium, and Ensifer spp.]) in agricultural soils based on functional cnorB (nitric oxide reductase) gene sequences. Total population numbers were measured using 16S rRNA gene real-time PCR. Two soil microcosm experiments were conducted. Experiment 1 examined the response of the indigenous soil microbial population to the addition of 500 mg/kg glucose-C daily over 7 days in soil microcosms. Changes in the total population were correlated (r = 0.83) between 16S rRNA gene copy numbers and microbial biomass carbon estimates. Members of the cnorB(P) population of denitrifiers showed typical r-strategy by being able to increase their proportion in the total population from starting levels of <0.1% to around 2.4% after a daily addition of 500 mg/kg glucose-C. The cnorB(B) guild was not able to increase its relative percentage of the total population in response to the addition of glucose-C, instead increasing copy numbers only in proportion with the total population measured by 16S rRNA genes. Experiment 2 measured population dynamics in soil after the addition of various amounts of glucose-C (0 to 500 mg/kg) and incubation under denitrifying conditions. cnorB(P) populations increased proportionally with the amount of glucose-C added (from 0 to 500 mg/kg). In soil microcosms, denitrification rates, respiration, and cnorB(P) population densities increased significantly with increasing rates of glucose addition. cnorB(B) guild densities did not increase significantly under denitrifying conditions in response to increasing C additions.     

Diversity of green-like and red-like ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbL) in differently managed agricultural soils

A PCR-based approach was developed to detect ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) form I large-subunit genes (cbbL) as a functional marker of autotrophic bacteria that fix carbon dioxide via the Calvin-Benson-Bassham cycle. We constructed two different primer sets, targeting the green-like and red-like phylogenetic groups of cbbL genes. The diversity of these cbbL genes was analyzed by the use of three differently managed agricultural soils from a long-term field experiment. cbbL gene fragments were amplified from extracted soil DNAs, and PCR products were cloned and screened by restriction fragment length polymorphism analysis. Selected unique cbbL clones were sequenced and analyzed phylogenetically. The green-like cbbL sequences revealed a very low level of diversity, being closely related to the cbbL genes of Nitrobacter winogradskyi and Nitrobacter vulgaris. In contrast, the red-like cbbL gene libraries revealed a high level of diversity in the two fertilized soils and less diversity in unfertilized soil. The majority of environmental red-like cbbL genes were only distantly related to already known cbbL sequences and even formed separate clusters. In order to extend the database of available red-like cbbL sequences, we amplified cbbL sequences from bacterial type culture strains and from bacterial isolates obtained from the investigated soils. Bacterial isolates harboring the cbbL gene were analyzed phylogenetically on the basis of their 16S rRNA gene sequences. These analyses revealed that bacterial genera such as Bacillus, Streptomyces, and Arthrobacter harbor red-like cbbL genes which fall into the cbbL gene clusters retrieved from the investigated soils.     

PCR Detection of Genes Encoding Nitrite Reductase in Denitrifying Bacteria

Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd₁- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships of nir genes were also established. The cd₁ primers were designed to amplify a 778 to 799-bp region of cd₁-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd₁-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd₁-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.     

Nitrite Reductase Genes (nirK and nirS) as Functional Markers To Investigate Diversity of Denitrifying Bacteria in Pacific Northwest Marine Sediment Communities

Genetic heterogeneity of denitrifying bacteria in sediment samples from Puget Sound and two sites on the Washington continental margin was studied by PCR approaches amplifying nirK and nirS genes. These structurally different but functionally equivalent single-copy genes coding for nitrite reductases, a key enzyme of the denitrification process, were used as a molecular marker for denitrifying bacteria. nirS sequences could be amplified from samples of both sampling sites, whereas nirK sequences were detected only in samples from the Washington margin. To assess the underlying nir gene structure, PCR products of both genes were cloned and screened by restriction fragment length polymorphism (RFLP). Rarefraction analysis revealed a high level of diversity especially for nirS clones from Puget Sound and a slightly lower level of diversity for nirK and nirS clones from the Washington margin. One group dominated within nirK clones, but no dominance and only a few redundant clones were seen between sediment samples for nirS clones in both habitats. Hybridization and sequencing confirmed that all but one of the 228 putative nirS clones were nirS with levels of nucleotide identities as low as 45.3%. Phylogenetic analysis grouped nirS clones into three distinct subclusters within the nirS gene tree which corresponded to the two habitats from which they were obtained. These sequences had little relationship to any strain with known nirS sequences or to isolates (mostly close relatives of Pseudomonas stutzeri) from the Washington margin sediment samples. nirK clones were more closely related to each other than were the nirS clones, with 78.6% and higher nucleotide identities; clones showing only weak hybridization signals were not related to known nirK sequences. All nirK clones were also grouped into a distinct cluster which could not be placed with any strain with known nirK sequences. These findings show a very high diversity of nir sequences within small samples and that these novel nir clusters, some very divergent from known sequences, are not known in cultivated denitrifiers.     

Development of PCR Primer Systems for Amplification of Nitrite Reductase Genes (nirK and nirS) To Detect Denitrifying Bacteria in Environmental Samples

A system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128); the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained with nondenitrifying bacteria or with strains of the other nir type. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying one generally amplifying primer combination for each nir gene developed in this study to total DNA preparations from aquatic habitats.     

Two isofunctional nitric oxide reductases in Alcaligenes eutrophus H16.

Two genes, norB and norZ, encoding two independent nitric oxide reductases have been identified in Alcaligenes eutrophus H16. norB and norZ predict polypeptides of 84.5 kDa with amino acid sequence identity of 90%. While norB resides on the megaplasmid pHG1, the norZ gene is located on a chromosomal DNA fragment. Amino acid sequence analysis suggests that norB and norZ encode integral membrane proteins composed of 14 membrane-spanning helices. The region encompassing helices 3 to 14 shows similarity to the NorB subunit of common bacterial nitric oxide reductases, including the positions of six strictly conserved histidine residues. Unlike the Nor enzymes characterized so far from denitrifying bacteria, NorB and NorZ of A. eutrophus contain an amino-terminal extension which may form two additional helices connected by a hydrophilic loop of 203 amino acids. The presence of a NorB/NorZ-like protein was predicted from the genome sequence of the cyanobacterium Synechocystis sp. strain PCC6803. While the common NorB of denitrifying bacteria is associated with a second cytochrome c subunit, encoded by the neighboring gene norC, the nor loci of A. eutrophus and Synechocystis lack adjacent norC homologs. The physiological roles of norB and norZ in A. eutrophus were investigated with mutants disrupted in the two genes. Mutants bearing single-site deletions in norB or norZ were affected neither in aerobic nor in anaerobic growth with nitrate or nitrite as the terminal electron acceptor. Inactivation of both norB and norZ was lethal to the cells under anaerobic growth conditions. Anaerobic growth was restored in the double mutant by introducing either norB or norZ on a broad-host-range plasmid. These results show that the norB and norZ gene products are isofunctional and instrumental in denitrification.     

晋南牛瘤胃中古菌分子多样性的研究

采用3对古菌特异性引物扩增瘤胃古菌16S rRNA基因分别建立克隆库来研究晋南牛瘤胃古菌的多样性.每个克隆库随机挑选100个克隆.引物Arch f364/1386建立的克隆库中,克隆分为四类,分别与四种甲烷短杆菌1Y(61%)、SM9(23%)、NT7(14%)和AK-87(2%)相似.引物1Af/1100Ar建立的克隆库中,克隆分为两类,分别与Methanobacterium aarhusense(72%)和Methanosphaera stadtmanae DSM 3091(28%)相似.引物Met86F/Met1340R建立的克隆库反映的古菌种类较为全面,除以上4种甲烷短杆菌(所占比例分别为47%、26%、11%和3%)外,还有Methanomicrobium mobile(2%)、以及类似Methanobacterium aarhusense(1%)和Methanosphaera stadtmanae(3%)的序列,还有7%的未匹配序列.系统进化分析表明,这些克隆属于Methanobrevibacter、Methanobacterium、Methanosphaera、Methanomicrobium,和未知广域古菌等5个分支.有25类属于广域古菌的未知序列,提示瘤胃中存在大量的未知产甲烷菌.     

Application of group specific amplified rDNA restriction analysis to characterize swine fecal and manure storage pit samples

Group specific amplified ribosomal-DNA restriction analysis was evaluated as a method to rapidly assess microbial community structure in swine fecal and manure storage pit samples. PCR primer sequences were evaluated for their specificity to ribosomal DNA from selected bacterial groups by optimizing annealing temperatures and determining specificity using a set of primer target and non-target organisms. A number of primer sets were identified targeting the following groups: Bacteroides-Prevotella, clostridial clusters I and II, clostridial clusters IX and XI, clostridial clusters XIVa and XIVb, Lactobacillus, Desulfovibrionaceae and Streptococcus-Lactococcus, as well as an universal primer set to represent total populations. Each bacterial group was digested with at least three restriction enzymes. We applied the group specific amplified ribosomal-DNA restriction analysis to swine fecal and manure storage pit samples obtained on two separate occasions. Fecal and manure storage pit samples obtained on the same day were more similar to each other than to any other samples. Results were consistent with 16S ribosomal DNA sequencing data from bacterial isolates and clones obtained from swine feces and manure storage pit. The group specific amplified ribosomal-DNA restriction analysis technique was able to rapid detect gross bacterial community differences among swine fecal and manure storage pit samples and determine groups of interest for more detailed examination.     

Phylogeny of nitrite reductase (nirK) and nitric oxide reductase (norB) genes from Nitrosospira species isolated from soil

Ammonia-oxidizing bacteria are believed to be an important source of the climatically important trace gas nitrous oxide (N(2)O). The genes for nitrite reductase (nirK) and nitric oxide reductase (norB), putatively responsible for nitrous oxide production, have been identified in several ammonia-oxidizing bacteria, but not in Nitrosospira strains that may dominate ammonia-oxidizing communities in soil. In this study, sequences from nirK and norB genes were detected in several cultured Nitrosospira species and the diversity and phylogeny of these genes were compared with those in other ammoniaoxidizing bacteria and in classical denitrifiers. The nirK and norB gene sequences obtained from Nitrosospira spp. were diverse and appeared to be less conserved than 16S rRNA genes and functional ammonia monooxygenase (amoA) genes. The nirK and norB genes from some Nitrosospira spp. were not phylogenetically distinct from those of denitrifiers, and phylogenetic analysis suggests that the nirK and norB genes in ammonia-oxidizing bacteria have been subject to lateral transfer.     

Assessment of fungal diversity in deep-sea sediments by multiple primer approach

Increasing evidence of the fungal diversity in deep-sea sediments has come from amplification of environmental DNA with fungal specific or eukaryote primer sets. In order to assess the fungal diversity in deep-sea sediments of the Central Indian Basin (CIB) at ~5,000 m depth, we amplified sediment DNA with four different primer sets. These were fungal-specific primer pair ITS1F/ITS4 (internal transcribed spacers), universal 18S rDNA primers NS1/NS2, Euk18S-42F/Euk18S-1492R and Euk18S-555F/Euk18S-1269R. One environmental library was constructed with each of the primer pairs, and 48 clones were sequenced per library. These sequences resulted in 8 fungal Operational Taxonomic Units (OTUs) with ITS and 19 OTUs with 18S rDNA primer sets respectively by taking into account the 2% sequence divergence cut-off for species delineation. These OTUs belonged to 20 distinct fungal genera of the phyla Ascomycota and Basidiomycota. Seven sequences were found to be divergent by 79–97% from the known sequences of the existing database and may be novel. A majority of the sequences clustered with known sequences of the existing taxa. The phylogenetic affiliation of a few fungal sequences with known environmental sequences from marine and hypersaline habitat suggests their autochthonous nature or adaptation to marine habitat. The amplification of sequences belonging to Exobasidiomycetes and Cystobasidiomycetes from deep-sea is being reported for the first time in this study. Amplification of fungal sequences with eukaryotic as well as fungal specific primers indicates that among eukaryotes, fungi appear to be a dominant group in the sampling site of the CIB.     

Analysis of denitrification genes and comparison of nosZ, cnorB and 16S rDNA from culturable denitrifying bacteria in potato cropping systems

Bacterial denitrification in agricultural soils is a major source of nitrous oxide, a potent greenhouse gas. This study examined the culturable bacterial population of denitrifiers in arable field soils in potato (Solanum tuberosum L.) production and denitrification genes (nir, nor and nos) and 16S rDNA in those isolates. Enrichments for culturable denitrifiers yielded 31 diverse isolates that were then analysed for denitrification genes. The nitrous oxide reductase (nosZ) gene was found in all isolates. The majority of isolates ( approximately 90%) contained the cnorB nitric oxide reductase gene, with the remainder containing the qnorB gene. Nitrite reductase genes (nirS and nirK) were amplifiable from most of the isolates, and were segregated between species similar to previously isolated denitrifiers. Isolated strains were preliminarily identified using fatty acid methyl ester analysis and further identified using 16S rDNA sequencing. The majority of isolates (21) were classified as Pseudomonas sp., with smaller groups of isolates being most similar to Bosea spp. (4), Achromobacter spp. (4) and two isolates closely related to Sinorhizobium/Ensifer spp. Phylogenetic trees were compared among nosZ, cnorB and 16S rDNA genes for a subset of Pseudomonas strains. The trees were mostly congruent, but some Pseudomonas sp. isolates grouped differently depending on the gene analysed, indicating potential horizontal gene transfer of denitrification genes. Although Bosea spp. are known denitrifiers, to the best of our knowledge this is the first report of isolation and sequencing of denitrification genes from this bacterial genus.     

Morphological and genetic evidence that the cyanobacterium Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck encompasses at least two species

Dense blooms of the cyanobacterium Lyngbya wollei are increasingly responsible for declining water quality and habitat degradation in numerous springs, rivers, and reservoirs. This research represents the first molecular phylogenetic analysis of L. wollei in comparison with the traditional morphological characterization of this species. Specimens were collected from several springs in Florida and a reservoir in North Carolina. Segments of the small-subunit (SSU) rRNA and nifH genes were PCR amplified, cloned, and sequenced. The phylogenetic analysis of the SSU rRNA gene revealed sequences that fell into three distinct subclusters, each with >97% sequence similarity. These were designated operational taxonomic unit 1 (OTU1), OTU2, and OTU3. Similarly, the nifH sequences fell into three distinct subclusters named S1, S2, and S3. When either bulk samples or individual filaments were analyzed, we recovered OTU1 with S1, OTU2 with S2, and OTU3 with S3. The coherence between the three SSU rRNA gene and nifH subclusters was consistent with genetically distinct strains or species. Cells associated with subclusters OTU3 and S3 were significantly wider and longer than those associated with other subclusters. The combined molecular and morphological data indicate that the species commonly identified as L. wollei in the literature represents two or possibly more species. Springs containing OTU3 and S3 demonstrated lower ion concentrations than other collection sites. Geographical locations of Lyngbya subclusters did not correlate with residual dissolved inorganic nitrogen or phosphorus concentrations. This study emphasizes the need to complement traditional identification with molecular characterization to more definitively detect and characterize harmful cyanobacterial species or strains.     

Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification

Denitrification is a facultative respiratory pathway in which nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) are successively reduced to nitrogen gas (N(2)), effectively closing the nitrogen cycle. The ability to denitrify is widely dispersed among prokaryotes, and this polyphyletic distribution has raised the possibility of horizontal gene transfer (HGT) having a substantial role in the evolution of denitrification. Comparisons of 16S rRNA and denitrification gene phylogenies in recent studies support this possibility; however, these results remain speculative as they are based on visual comparisons of phylogenies from partial sequences. We reanalyzed publicly available nirS, nirK, norB, and nosZ partial sequences using Bayesian and maximum likelihood phylogenetic inference. Concomitant analysis of denitrification genes with 16S rRNA sequences from the same organisms showed substantial differences between the trees, which were supported by examining the posterior probability of monophyletic constraints at different taxonomic levels. Although these differences suggest HGT of denitrification genes, the presence of structural variants for nirK, norB, and nosZ makes it difficult to determine HGT from other evolutionary events. Additional analysis using phylogenetic networks and likelihood ratio tests of phylogenies based on full-length sequences retrieved from genomes also revealed significant differences in tree topologies among denitrification and 16S rRNA gene phylogenies, with the exception of the nosZ gene phylogeny within the data set of the nirK-harboring genomes. However, inspection of codon usage and G + C content plots from complete genomes gave no evidence for recent HGT. Instead, the close proximity of denitrification gene copies in the genomes of several denitrifying bacteria suggests duplication. Although HGT cannot be ruled out as a factor in the evolution of denitrification genes, our analysis suggests that other phenomena, such gene duplication/divergence and lineage sorting, may have differently influenced the evolution of each denitrification gene.