High-Resolution Differentiation of Cyanobacteria by Using rRNA-Internal Transcribed Spacer Denaturing Gradient Gel Electrophoresis

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3′ end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis. Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.     

High-resolution differentiation of Cyanobacteria by using rRNA-internal transcribed spacer denaturing gradient gel electrophoresis

For many ecological studies of cyanobacteria, it is essential that closely related species or strains can be discriminated. Since this is often not possible by using morphological features, cyanobacteria are frequently studied by using DNA-based methods. A powerful method for analysis of the diversity and dynamics of microbial populations and for checking the purity and affiliation of cultivated strains is denaturing gradient gel electrophoresis (DGGE). We realized high-resolution discrimination of a variety of cyanobacteria by means of DGGE analysis of sections of the internal transcribed spacer between the 16S and 23S rRNA genes (rRNA-ITS). A forward primer specific for cyanobacteria, targeted at the 3' end of the 16S rRNA gene, was designed. The combination of this primer and three different reverse primers targeted to the rRNA-ITS or to the 23S rRNA gene yielded PCR products of different sizes from cultures of all 16 cyanobacterial genera that were tested but not from other bacteria. DGGE profiles produced from the shortest section of rRNA-ITS consisted of one band for all but one cyanobacterial genera, and those generated from longer stretches of rRNA-ITS yielded DGGE profiles containing one to four bands. The suitability of DGGE for detecting intrageneric and intraspecific variation was tested by using strains of the genus Microcystis: Many strains could be discriminated by means of rRNA-ITS DGGE, and the resolution of this method was strikingly higher than that obtained with previously described methods. The applicability of the developed DGGE assays for analysis of cyanobacteria in field samples was demonstrated by using samples from freshwater lakes. The advantages and disadvantages associated with the use of each developed primer set are discussed.     

Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community.

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments was used to profile microbial populations inhabiting different temperature regions in the microbial mat community of Octopus Spring, Yellowstone National Park. DGGE allowed a rapid evaluation of the distributions of amplifiable sequence types. Profiles were essentially identical within regions of the mat defined by one temperature range but varied between sites with different temperature ranges. Individual DGGE bands were sequenced, and the sequences were compared with those previously obtained from the mat by cloning and from cultivated Octopus Spring isolates. Two known cyanobacterial populations and one known green nonsulfur bacterium-like population were detected by DGGE, as were many new cyanobacterial and green nonsulfur and green sulfur bacterium-like populations and a novel bacterial population of uncertain phylogenetic affiliation. The distributions of several cyanobacterial populations compared favorably with results obtained previously by oligonucleotide probe analyses and suggest that adaptation to temperature has occurred among cyanobacteria which are phylogenetically very similar.     

Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing.

The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 10(4) recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the gamma subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the alpha subdivision and the other distinct from known proteobacterial subdivisions. The rRNA sequences of the alpha-related group are nearly identical to those of some Sargasso Sea picoplankton, suggesting a global distribution of these organisms.     

Identification of and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis

The microbial assemblages of Lake Cisó and Lake Vilar (Banyoles, northeast Spain) were analyzed in space and time by microscopy and by performing PCR-denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA gene fragments. Samples obtained from different water depths and at two different times of the year (in the winter during holomixis and in the early spring during a phytoplankton bloom) were analyzed. Although the lakes have the same climatic conditions and the same water source, the limnological parameters were different, as were most of the morphologically distinguishable photosynthetic bacteria enumerated by microscopy. The phylogenetic affiliations of the predominant DGGE bands were inferred by performing a comparative 16S rRNA sequence analysis. Sequences obtained from Lake Cisó samples were related to gram-positive bacteria and to members of the division Proteobacteria. Sequences obtained from Lake Vilar samples were related to members of the Cytophaga-Flavobacterium-Bacteroides phylum and to cyanobacteria. Thus, we found that like the previously reported differences between morphologically distinct inhabitants of the two lakes, there were also differences among the community members whose morphologies did not differ conspicuously. The changes in the species composition from winter to spring were also marked. The two lakes both contained sequences belonging to phototrophic green sulfur bacteria, which is consistent with microscopic observations, but these sequences were different from the sequences of cultured strains previously isolated from the lakes. Euryarchaeal sequences (i.e., methanogen- and thermoplasma-related sequences) also were present in both lakes. These euryarchaeal group sequences dominated the archaeal sequences in Lake Cisó but not in Lake Vilar. In Lake Vilar, a new planktonic population related to the crenarchaeota produced the dominant archaeal band. The phylogenetic analysis indicated that new bacterial and archaeal lineages were present and that the microbial diversity of these assemblages was greater than previously known. We evaluated the correspondence between the abundances of several morphotypes and DGGE bands by comparing microscopy and sequencing results. Our data provide evidence that the sequences obtained from the DGGE fingerprints correspond to the microorganisms that are actually present at higher concentrations in the natural system.     

Composition of humic acid-degrading estuarine and marine bacterial communities

We examined the bacterial decomposition of humic acids (HA) in two flow-through culture experiments, one inoculated by marine and one by estuarine bacterial communities. In both experiments, the cultures were fed with HA media of salinities of 28 and 14, close to their ambient and a distinctly different, foreign salinity. HA were decomposed to >?60% of the initial concentration within 70?days, and the foreign salinity yielded the highest decomposition. A detrended correspondence analysis of denaturing gradient gel electrophoresis (DGGE) banding patterns showed that during incubation, the bacterial community composition underwent distinct changes. A phylogenetic analysis of DGGE bands excised and bacteria isolated at the end on HA as the sole carbon source showed that Alphaproteobacteria and Gammaproteobacteria largely dominated the communities in the marine flow-through cultures, whereas Gammaproteobacteria, Actinobacteria and Alphaproteobacteria dominated the estuarine communities. Eleven of 13 isolates obtained from both experiments were able to grow on HA as the sole carbon source, seven on phenol and three, affiliated to the Roseobacter clade, on various aromatic acids. The bacteria retrieved from the flow-through cultures were closely (96-99%) affiliated to organisms capable of degrading humic matter, aromatic and aliphatic compounds and also to other bacteria reported previously from the Wadden Sea and Weser estuary.     

Bacterioplankton community composition along a salinity gradient of sixteen high-mountain lakes located on the Tibetan Plateau, China

The influence of altitude and salinity on bacterioplankton community composition (BCC) in 16 high-mountain lakes located at altitudes of 2,817 to 5,134 m on the Eastern Qinghai-Xizang (Tibetan) Plateau, China, spanning a salinity gradient from 0.02% (freshwater) to 22.3% (hypersaline), was investigated. Three different methods, fluorescent in situ hybridization, denaturing gradient gel electrophoresis (DGGE) with subsequent band sequencing, and reverse line blot hybridization (RLB) with probes targeting 17 freshwater bacterial groups, were used for analysis of BCC. Furthermore, the salt tolerances of 47 strains affiliated with groups detected in or isolated from the Tibetan habitats were investigated. Altitude was not found to influence BCC significantly within the investigated range. Several groups of typical freshwater bacteria, e.g., the ACK-M1 cluster and the Polynucleobacter group, were detected in habitats located above 4,400 m. Salinity was found to be the dominating environmental factor controlling BCC in the investigated lakes, resulting in only small overlaps in the BCCs of freshwater and hypersaline lakes. The relative abundances of different classes of Proteobacteria showed a sharp succession along the salinity gradient. Both DGGE and RLB demonstrated that a few freshwater bacterial groups, e.g., GKS98 and LD2, appeared over wide salinity ranges. Six freshwater isolates affiliated with the GKS98 cluster grew in ecophysiological experiments at maximum salinities of 0.3% to 0.7% (oligosaline), while this group was detected in habitats with salinities up to 6.7% (hypersaline). This observation indicated ecologically significant differences in ecophysiological adaptations among members of this narrow phylogenetic group and suggested ecological significance of microdiversity.     

Identification of and Spatio-Temporal Differences between Microbial Assemblages from Two Neighboring Sulfurous Lakes: Comparison by Microscopy and Denaturing Gradient Gel Electrophoresis

The microbial assemblages of Lake Cisó and Lake Vilar (Banyoles, northeast Spain) were analyzed in space and time by microscopy and by performing PCR-denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA gene fragments. Samples obtained from different water depths and at two different times of the year (in the winter during holomixis and in the early spring during a phytoplankton bloom) were analyzed. Although the lakes have the same climatic conditions and the same water source, the limnological parameters were different, as were most of the morphologically distinguishable photosynthetic bacteria enumerated by microscopy. The phylogenetic affiliations of the predominant DGGE bands were inferred by performing a comparative 16S rRNA sequence analysis. Sequences obtained from Lake Cisó samples were related to gram-positive bacteria and to members of the division Proteobacteria. Sequences obtained from Lake Vilar samples were related to members of the Cytophaga-Flavobacterium-Bacteroides phylum and to cyanobacteria. Thus, we found that like the previously reported differences between morphologically distinct inhabitants of the two lakes, there were also differences among the community members whose morphologies did not differ conspicuously. The changes in the species composition from winter to spring were also marked. The two lakes both contained sequences belonging to phototrophic green sulfur bacteria, which is consistent with microscopic observations, but these sequences were different from the sequences of cultured strains previously isolated from the lakes. Euryarchaeal sequences (i.e., methanogen- and thermoplasma-related sequences) also were present in both lakes. These euryarchaeal group sequences dominated the archaeal sequences in Lake Cisó but not in Lake Vilar. In Lake Vilar, a new planktonic population related to the crenarchaeota produced the dominant archaeal band. The phylogenetic analysis indicated that new bacterial and archaeal lineages were present and that the microbial diversity of these assemblages was greater than previously known. We evaluated the correspondence between the abundances of several morphotypes and DGGE bands by comparing microscopy and sequencing results. Our data provide evidence that the sequences obtained from the DGGE fingerprints correspond to the microorganisms that are actually present at higher concentrations in the natural system.     

Diversity and distribution in hypersaline microbial mats of bacteria related to Chloroflexus spp

Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of "green nonsulfur bacteria." PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.     

Spatial distribution of diatom and cyanobacterial mats in the Dead Sea is determined by response to rapid salinity fluctuations

Cyanobacteria and diatom mats are ubiquitous in hypersaline environments but have never been observed in the Dead Sea, one of the most hypersaline lakes on Earth. Here we report the discovery of phototrophic microbial mats at underwater freshwater seeps in the Dead Sea. These mats are either dominated by diatoms or unicellular cyanobacteria and are spatially separated. Using in situ and ex situ O₂ microsensor measurements we show that these organisms are photosynthetically active in their natural habitat. The diatoms, which are phylogenetically associated to the Navicula genus, grew in culture at salinities up to 40 % Dead Sea water (DSW) (14 % total dissolved salts, TDS). The unicellular cyanobacteria belong to the extremely halotolerant Euhalothece genus and grew at salinities up to 70 % DSW (24.5 % TDS). As suggested by a variable O₂ penetration depth measured in situ, the organisms are exposed to drastic salinity fluctuations ranging from brackish to DSW salinity within minutes to hours. We could demonstrate that both phototrophs are able to withstand such extreme short-term fluctuations. Nevertheless, while the diatoms recover better from rapid fluctuations, the cyanobacteria cope better with long-term exposure to DSW. We conclude that the main reason for the development of these microbial mats is a local dilution of the hypersaline Dead Sea to levels allowing growth. Their spatial distribution in the seeping areas is a result of different recovery rates from short or long-term fluctuation in salinity.     

Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.     

Diversity and Distribution in Hypersaline Microbial Mats of Bacteria Related to Chloroflexus spp.

Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of “green nonsulfur bacteria.” PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.     

Microbial biogeography along an estuarine salinity gradient: combined influences of bacterial growth and residence time

Shifts in bacterioplankton community composition along the salinity gradient of the Parker River estuary and Plum Island Sound, in northeastern Massachusetts, were related to residence time and bacterial community doubling time in spring, summer, and fall seasons. Bacterial community composition was characterized with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA. Average community doubling time was calculated from bacterial production ([(14)C]leucine incorporation) and bacterial abundance (direct counts). Freshwater and marine populations advected into the estuary represented a large fraction of the bacterioplankton community in all seasons. However, a unique estuarine community formed at intermediate salinities in summer and fall, when average doubling time was much shorter than water residence time, but not in spring, when doubling time was similar to residence time. Sequencing of DNA in DGGE bands demonstrated that most bands represented single phylotypes and that matching bands from different samples represented identical phylotypes. Most river and coastal ocean bacterioplankton were members of common freshwater and marine phylogenetic clusters within the phyla Proteobacteria, Bacteroidetes, and ACTINOBACTERIA: Estuarine bacterioplankton also belonged to these phyla but were related to clones and isolates from several different environments, including marine water columns, freshwater sediments, and soil.     

Culture-independent analysis of bacterial species from an anaerobic mat from Lake Fryxell, Antarctica: prokaryotic diversity revisited.

Using a lake sediment mat sample from Lake Fryxell, Antarctica, different DNA extraction and purification methods were compared by denaturing gradient gel electrophoresis (DGGE). Based on the analyses of cloned 16S rRNA gene sequences a high degree of as yet uncultured prokaryotes have been reported in this sample. Although the vast majority of these as yet uncultured organisms seem to be classified as representatives of Firmicutes, Proteobacteria and Bacteriodetes, many of these taxa should be regarded novel species as judged from the distance of their gene sequences to those of their nearest cultured phylogenetic neighbours. The physiological properties of cultured strains from Lake Fryxell and of those of described species that are phylogenetically affiliated to the as yet uncultured species from this environment, suggest the presence of a well developed food web of primary producers, anaerobic degraders and fermenters, and aerobes. The few novel species described from this sample add to the increasing number of species characterized from various Antarctic habitats. Determination of the phylogenetic relatedness of the mat clone sequences of Clostridia with recent entries into public databases revealed that many of the putative species are closely related to other putative species detected in a broad range of environments, ranging from rumen and gut, anaerobic and polluted soil to sediment and groundwater samples.     

Detection and characterization of fungal infections of Ammophila arenaria (marram grass) roots by denaturing gradient gel electrophoresis of specifically amplified 18s rDNA.

Marram grass (Ammophila arenaria L.), a sand-stabilizing plant species in coastal dune areas, is affected by a specific pathosystem thought to include both plant-pathogenic fungi and nematodes. To study the fungal component of this pathosystem, we developed a method for the cultivation-independent detection and characterization of fungi infecting plant roots based on denaturing gradient gel electrophoresis (DGGE) of specifically amplified DNA fragments coding for 18S rRNA (rDNA). A nested PCR strategy was employed to amplify a 569-bp region of the 18S rRNA gene, with the addition of a 36-bp GC clamp, from fungal isolates, from roots of test plants infected in the laboratory, and from field samples of marram grass roots from both healthy and degenerating stands from coastal dunes in The Netherlands. PCR products from fungal isolates were subjected to DGGE to examine the variation seen both between different fungal taxa and within a single species. DGGE of the 18S rDNA fragments could resolve species differences from fungi used in this study yet was unable to discriminate between strains of a single species. The 18S rRNA genes from 20 isolates of fungal species previously recovered from A. arenaria roots were cloned and partially sequenced to aid in the interpretation of DGGE data. DGGE patterns recovered from laboratory plants showed that this technique could reliably identify known plant-infecting fungi. Amplification products from field A. arenaria roots also were analyzed by DGGE, and the major bands were excised, reamplified, sequenced, and subjected to phylogenetic analysis. Some recovered 18S rDNA sequences allowed for phylogenetic placement to the genus level, whereas other sequences were not closely related to known fungal 18S rDNA sequences. The molecular data presented here reveal fungal diversity not detected in previous culture-based surveys.     

SALINITY TOLERANCE OF DIATOMS FROM THALASSIC HYPERSALINE ENVIRONMENTS

Thirty-four benthic diatom strains were isolated from thalassic hypersaline marine environments and their salinity tolerance characterized in growth experiments conducted at salinities ranging from 0.5% to 17.5% (weight of total salts per volume, g·100 mL ^{− 1}). The results were compared with the patterns of diatom species distribution and abundance in hypersaline evaporation ponds and tidal channels of Guerrero Negro, Baja California Sur, Mexico. The isolated strains were representative of the diatom assemblages present in the saltern ponds but were less so of natural assemblages in tidal channels. In general, we found a clear decreasing trend of diatom diversity in the field and in the isolated strains with increasing salinity. With some exceptions, the upper limit of salinity tolerance in cultivated strains corresponded to their distribution in field samples. However, the relative abundance of species in the field was not correlated with growth rates achieved in culture for the same salinities. Most cultured strains exhibited extreme euryhalinity growing well from brackish to hypersaline conditions, but the particulars of salt tolerance were quite diverse among strains. The most halotolerant taxa, two Amphora species, Amphora cf. subacutiuscula Schoeman, Nitzschia fusiformis Grunow, and Entomoneis sp., grew well in salinities ranging from 0.5% to 15%. Three strains of Pleurosigma strigosum W. Smith that were unable to grow in salinities less than 5% total salts represent the only true halophilic diatoms ever reported. The fact that many strains displayed a remarkable halotolerance, with optimal or near-optimal growth rates at salinities as high as three times that of seawater, implies that diatoms from hypersaline environments are evolutionarily highly adapted to such environments.     

Microbialite response to an anthropogenic salinity gradient in Great Salt Lake,Utah

A railroad causeway across Great Salt Lake, Utah (GSL), has restricted water flow since its construction in 1959, resulting in a more saline North Arm (NA; 24%–31% salinity) and a less saline South Arm (SA; 11%–14% salinity). Here, we characterized microbial carbonates collected from the SA and the NA to evaluate the effect of increased salinity on community composition and abundance and to determine whether the communities present in the NA are still actively precipitating carbonate or if they are remnant features from prior to causeway construction. SSU rRNA gene abundances associated with the NA microbialite were three orders of magnitude lower than those associated with the SA microbialite, indicating that the latter community is more productive. SSU rRNA gene sequencing and functional gene microarray analyses indicated that SA and NA microbialite communities are distinct. In particular, abundant sequences affiliated with photoautotrophic taxa including cyanobacteria and diatoms that may drive carbonate precipitation and thus still actively form microbialites were identified in the SA microbialite; sequences affiliated with photoautotrophic taxa were in low abundance in the NA microbialite. SA and NA microbialites comprise smooth prismatic aragonite crystals. However, the SA microbialite also contained micritic aragonite, which can be formed as a result of biological activity. Collectively, these observations suggest that NA microbialites are likely to be remnant features from prior to causeway construction and indicate a strong decrease in the ability of NA microbialite communities to actively precipitate carbonate minerals. Moreover, the results suggest a role for cyanobacteria and diatoms in carbonate precipitation and microbialite formation in the SA of GSL.     

Changes in the community structure and activity of betaproteobacterial ammonia-oxidizing sediment bacteria along a freshwater-marine gradient

To determine whether the distribution of estuarine ammonia-oxidizing bacteria (AOB) was influenced by salinity, the community structure of betaproteobacterial ammonia oxidizers (AOB) was characterized along a salinity gradient in sediments of the Ythan estuary, on the east coast of Scotland, UK, by denaturant gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rRNA gene fragments. Ammonia-oxidizing bacteria communities at sampling sites with strongest marine influence were dominated by Nitrosospira cluster 1-like sequences and those with strongest freshwater influence were dominated by Nitrosomonas oligotropha-like sequences. Nitrosomonas sp. Nm143 was the prevailing sequence type in communities at intermediate brackish sites. Diversity indices of AOB communities were similar at marine- and freshwater-influenced sites and did not indicate lower species diversity at intermediate brackish sites. The presence of sequences highly similar to the halophilic Nitrosomonas marina and the freshwater strain Nitrosomonas oligotropha at identical sampling sites indicates that AOB communities in the estuary are adapted to a range of salinities, while individual strains may be active at different salinities. Ammonia-oxidizing bacteria communities that were dominated by Nitrosospira cluster 1 sequence types, for which no cultured representative exists, were subjected to stable isotope probing (SIP) with 13C-HCO3-, to label the nucleic acids of active autotrophic nitrifiers. Analysis of 13C-associated 16S rRNA gene fragments, following CsCl density centrifugation, by cloning and DGGE indicated sequences highly similar to the AOB Nitrosomonas sp. Nm143 and Nitrosomonas cryotolerans and to the nitrite oxidizer Nitrospira marina. No sequence with similarity to the Nitrosospira cluster 1 clade was recovered during SIP analysis. The potential role of Nitrosospira cluster 1 in autotrophic ammonia oxidation therefore remains uncertain.