Nasreen Bano

Depth Distribution of Microbial Diversity in Mono Lake, a Meromictic Soda Lake in California

ABSTRACT We analyzed the variation with depth in the composition of members of the domain Bacteria in samples from alkaline, hypersaline, and currently meromictic Mono Lake in California. DNA samples were collected from the mixolimnion (2 m), the base of the oxycline (17.5 m), the upper chemocline (23 m), and the monimolimnion (35 m). Composition was assessed by sequencing randomly selected cloned fragments of 16S rRNA genes retrieved from the DNA samples. Most of the 212 sequences retrieved from the samples fell into five major lineages of the domain Bacteria: α- and γ-Proteobacteria (6 and 10%, respectively), Cytophaga-Flexibacter-Bacteroides (19%), high-G+C-content gram-positive organisms (Actinobacteria; 25%), and low-G+C-content gram-positive organisms (Bacillus and Clostridium; 19%). Twelve percent were identified as chloroplasts. The remaining 9% represented β- and δ-Proteobacteria, Verrucomicrobiales, and candidate divisions. Mixolimnion and oxycline samples had low microbial diversity, with only 9 and 12 distinct phylotypes, respectively, whereas chemocline and monimolimnion samples were more diverse, containing 27 and 25 phylotypes, respectively. The compositions of microbial assemblages from the mixolimnion and oxycline were not significantly different from each other (P = 0.314 and 0.877), but they were significantly different from those of chemocline and monimolimnion assemblages (P < 0.001), and the compositions of chemocline and monimolimnion assemblages were not significantly different from each other (P = 0.006 and 0.124). The populations of sequences retrieved from the mixolimnion and oxycline samples were dominated by sequences related to high-G+C-content gram-positive bacteria (49 and 63%, respectively) distributed in only three distinct phylotypes, while the population of sequences retrieved from the monimolimnion sample was dominated (52%) by sequences related to low-G+C-content gram-positive bacteria distributed in 12 distinct phylotypes. Twelve and 28% of the sequences retrieved from the chemocline sample were also found in the mixolimnion and monimolimnion samples, respectively. None of the sequences retrieved from the monimolimnion sample were found in the mixolimnion or oxycline samples. Elevated diversity in anoxic bottom water samples relative to oxic surface water samples suggests a greater opportunity for niche differentiation in bottom versus surface waters of this lake.

Phylogenetic Composition of Bacterioplankton Assemblages from the Arctic Ocean

ABSTRACT We analyzed the phylogenetic composition of bacterioplankton assemblages in 11 Arctic Ocean samples collected over three seasons (winter-spring 1995, summer 1996, and summer-fall 1997) by sequencing cloned fragments of 16S rRNA genes. The sequencing effort was directed by denaturing gradient gel electrophoresis (DGGE) screening of samples and the clone libraries. Sequences of 88 clones fell into seven major lineages of the domain Bacteria: α (36%)-, γ (32%)-, δ (14%)-, and ε (1%)-Proteobacteria; Cytophaga-Flexibacter-Bacteroides spp. (9%); Verrucomicrobium spp. (6%); and green nonsulfur bacteria (2%). A total of 34% of the cloned sequences (excluding clones in the SAR11 and Roseobacter groups) had sequence similarities that were <94% compared to previously reported sequences, indicating the presence of novel sequences. DGGE fingerprints of the selected samples showed that most of the bands were common to all samples in all three seasons. However, additional bands representing sequences related to Cytophaga and Polaribacter species were found in samples collected during the summer and fall. Of the clones in a library generated from one sample collected in spring of 1995, 50% were the same and were most closely affiliated (99% similarity) with Alteromonas macleodii, while 50% of the clones in another sample were most closely affiliated (90 to 96% similarity) with Oceanospirillum sp. The majority of the cloned sequences were most closely related to uncultured, environmental sequences. Prominent among these were members of the SAR11 group. Differences between mixed-layer and halocline samples were apparent in DGGE fingerprints and clone libraries. Sequences related to α-Proteobacteria (dominated by SAR11) were abundant (52%) in samples from the mixed layer, while sequences related to γ-proteobacteria were more abundant (44%) in halocline samples. Two bands corresponding to sequences related to SAR307 (common in deep water) and the high-G+C gram-positive bacteria were characteristic of the halocline samples.

Anaerobic Oxidation of Arsenite in Mono Lake Water and by a Facultative, Arsenite-Oxidizing Chemoautotroph, Strain MLHE-1

ABSTRACT Arsenite [As(III)]-enriched anoxic bottom water from Mono Lake, California, produced arsenate [As(V)] during incubation with either nitrate or nitrite. No such oxidation occurred in killed controls or in live samples incubated without added nitrate or nitrite. A small amount of biological As(III) oxidation was observed in samples amended with Fe(III) chelated with nitrolotriacetic acid, although some chemical oxidation was also evident in killed controls. A pure culture, strain MLHE-1, that was capable of growth with As(III) as its electron donor and nitrate as its electron acceptor was isolated in a defined mineral salts medium. Cells were also able to grow in nitrate-mineral salts medium by using H2 or sulfide as their electron donor in lieu of As(III). Arsenite-grown cells demonstrated dark 14CO2 fixation, and PCR was used to indicate the presence of a gene encoding ribulose-1,5-biphosphate carboxylase/oxygenase. Strain MLHE-1 is a facultative chemoautotroph, able to grow with these inorganic electron donors and nitrate as its electron acceptor, but heterotrophic growth on acetate was also observed under both aerobic and anaerobic (nitrate) conditions. Phylogenetic analysis of its 16S ribosomal DNA sequence placed strain MLHE-1 within the haloalkaliphilic Ectothiorhodospira of the γ-Proteobacteria. Arsenite oxidation has never been reported for any members of this subgroup of the Proteobacteria.

Ammonia oxidation and ammonia-oxidizing bacteria and archaea from estuaries with differing histories of hypoxia

Nitrification, the oxidation of NH4+ to NO2− and subsequently to NO3−, plays a central role in the nitrogen cycle and is often a critical first step in nitrogen removal from estuarine and coastal environments. The first and rate-limiting step in nitrification is catalyzed by the enzyme ammonia monooxygenase (AmoA). We evaluate the relationships between the abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) amoA genes; potential nitrification rates and environmental variables to identify factors influencing AOA abundance and nitrifier activity in estuarine sediments. Our results showed that potential nitrification rates increased as abundance of AOA amoA increased. In contrast, there was no relationship between potential nitrification rates and AOB amoA abundance. This suggests that AOA are significant in estuarine nitrogen cycling. Surprisingly, more of the variability in potential nitrification rates was predicted by salinity and pore water sulfide than by dissolved oxygen history.

Phylogenetic Composition of Arctic Ocean Archaeal Assemblages and Comparison with Antarctic Assemblages

ABSTRACT Archaea assemblages from the Arctic Ocean and Antarctic waters were compared by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified using the Archaea-specific primers 344f and 517r. Inspection of the DGGE fingerprints of 33 samples from the Arctic Ocean (from SCICEX submarine cruises in 1995, 1996, and 1997) and 7 Antarctic samples from Gerlache Strait and Dallman Bay revealed that the richness of Archaea assemblages was greater in samples from deep water than in those from the upper water column in both polar oceans. DGGE banding patterns suggested that most of the Archaea ribotypes were common to both the Arctic Ocean and the Antarctic Ocean. However, some of the Euryarchaeota ribotypes were unique to each system. Cluster analysis of DGGE fingerprints revealed no seasonal variation but supported depth-related differences in the composition of the Arctic Ocean Archaea assemblage. The phylogenetic composition of the Archaea assemblage was determined by cloning and then sequencing amplicons obtained from the Archaea-specific primers 21f and 958r. Sequences of 198 clones from nine samples covering three seasons and all depths grouped with marine group I Crenarchaeota (111 clones), marine group II Euryarchaeota (86 clones), and group IV Euryarchaeota (1 clone). A sequence obtained only from a DGGE band was similar to those of the marine group III Euryarchaeota.

Analysis of microbial gene transcripts in environmental samples.

ABSTRACT We analyzed gene expression in marine and freshwater bacterioplankton communities by the direct retrieval and analysis of microbial transcripts. Environmental mRNA, obtained from total RNA by subtractive hybridization of rRNA, was reverse transcribed, amplified with random primers, and cloned. Approximately 400 clones were analyzed, of which ∼80% were unambiguously mRNA derived. mRNAs appeared to be from diverse taxonomic groups, including both Bacteria (mainly α- and γ-Proteobacteria) and Archaea (mainly Euryarchaeota). Many transcripts could be linked to environmentally important processes such as sulfur oxidation (soxA), assimilation of C1 compounds (fdh1B), and acquisition of nitrogen via polyamine degradation (aphA). Environmental transcriptomics is a means of exploring functional gene expression within natural microbial communities without bias toward known sequences, and provides a new approach for obtaining community-specific variants of key functional genes.

Diversity and Distribution of DNA Sequences with Affinity to Ammonia-Oxidizing Bacteria of the β Subdivision of the Class Proteobacteria in the Arctic Ocean

ABSTRACT The spatial distribution and diversity of ammonia-oxidizing bacteria of the β subdivision of the class Proteobacteria(hereinafter referred to as ammonia oxidizers) in the Arctic Ocean were determined. The presence of ammonia oxidizers was detected by PCR amplification of 16S rRNA genes using a primer set specific for this group of organisms (nitA and nitB, which amplifies a 1.1-kb fragment between positions 137 and 1234, corresponding to Escherichia coli 16S rDNA numbering). We analyzed 246 samples collected from the upper water column (5 to 235 m) during March and April 1995, September and October 1996, and September 1997. Ammonia oxidizers were detected in 25% of the samples from 5 m, 80% of the samples from 55 m, 88% of the samples from 133 m, and 50% of the samples from 235 m. Analysis of nitA-nitB PCR product by nested PCR-denaturing gradient gel electrophoresis (DGGE) showed that all positive samples contained the same major band (band A), indicating the presence of a dominant, ubiquitous ammonia oxidizer in the Arctic Ocean basin. Twenty-two percent of the samples contained additional major bands. These samples were restricted to the Chukchi Sea shelf break, the Chukchi cap, and the Canada basin; areas likely influenced by Pacific inflow. The nucleotide sequence of the 1.1-kb nitA-nitB PCR product from a sample that contained only band A grouped with sequences designated group 1 marine Nitrosospira-like sequences. PCR-DGGE analysis of 122 clones from four libraries revealed that 67 to 71% of the inserts contained sequences with the same mobility as band A. Nucleotide sequences (1.1 kb) of another distinct group of clones, found only in 1995 samples (25%), fell into the group 5 marineNitrosomonas-like sequences. Our results suggest that the Arctic Ocean β-proteobacterial ammonia oxidizers have low diversity and are dominated by marine Nitrosospira-like organisms. Diversity appears to be higher in Western Arctic Ocean regions influenced by inflow from the Pacific Ocean through the Bering and Chukchi seas.

Ammonia‐oxidizing Archaea in the Arctic Ocean and Antarctic coastal waters

We compared abundance, distributions and phylogenetic composition of Crenarchaeota and ammonia-oxidizing Archaea (AOA) in samples collected from coastal waters west of the Antarctic Peninsula during the summers of 2005 and 2006, with samples from the central Arctic Ocean collected during the summer of 1997. Ammonia-oxidizing Archaea and Crenarchaeota abundances were estimated from quantitative PCR measurements of amoA and 16S rRNA gene abundances. Crenarchaeota and AOA were approximately fivefold more abundant at comparable depths in the Antarctic versus the Arctic Ocean. Crenarchaeota and AOA were essentially absent from the Antarctic Summer Surface Water (SSW) water mass (0-45 m depth). The ratio of Crenarchaeota 16S rRNA to archaeal amoA gene abundance in the Winter Water (WW) water mass (45-105 m depth) of the Southern Ocean was much lower (0.15) than expected and in sharp contrast to the ratio (2.0) in the Circumpolar Deep Water (CDW) water mass (105-3500 m depth) immediately below it. We did not observe comparable segregation of this ratio by depth or water mass in Arctic Ocean samples. A ubiquitous, abundant and polar-specific crenarchaeote was the dominant ribotype in the WW and important in the upper halocline of the Arctic Ocean. Our data suggest that this organism does not contain an ammonia monooxygenase gene. In contrast to other studies where Crenarchaeota populations apparently lacking amoA genes are found in bathypelagic waters, this organism appears to dominate in well-defined, ammonium-rich, near-surface water masses in polar oceans.

Metatranscriptomic analysis of ammonia-oxidizing organisms in an estuarine bacterioplankton assemblage

Quantitative PCR (qPCR) analysis revealed elevated relative abundance (1.8% of prokaryotes) of marine group 1 Crenarchaeota (MG1C) in two samples of southeastern US coastal bacterioplankton, collected in August 2008, compared with samples collected from the same site at different times (mean 0.026%). We analyzed the MG1C sequences in metatranscriptomes from these samples to gain an insight into the metabolism of MG1C population growing in the environment, and for comparison with ammonia-oxidizing bacteria (AOB) in the same samples. Assemblies revealed low diversity within sequences assigned to most individual MG1C open reading frames (ORFs) and high homology with ‘Candidatus Nitrosopumilus maritimus’ strain SCM1 genome sequences. Reads assigned to ORFs for ammonia uptake and oxidation accounted for 37% of all MG1C transcripts. We did not recover any reads for Nmar_1354–Nmar_1357, proposed to encode components of an alternative, nitroxyl-based ammonia oxidation pathway; however, reads from Nmar_1259 and Nmar_1667, annotated as encoding a multicopper oxidase with homology to nirK, were abundant. Reads assigned to two homologous ORFs (Nmar_1201 and Nmar_1547), annotated as hypothetical proteins were also abundant, suggesting that their unknown function is important to MG1C. Superoxide dismutase and peroxiredoxin-like transcripts were more abundant in the MG1C transcript pool than in the complete metatranscriptome, suggesting an enhanced response to oxidative stress by the MG1C population. qPCR indicated low AOB abundance (0.0010% of prokaryotes), and we found no transcripts related to ammonia oxidation and only one RuBisCO transcript among the transcripts assigned to AOB, suggesting they were not responding to the same environmental cues as the MG1C population.

Widespread Distribution in Polar Oceans of a 16S rRNA Gene Sequence with Affinity to Nitrosospira-Like Ammonia-Oxidizing Bacteria

ABSTRACT We analyzed the phylogenetic compositions of ammonia-oxidizing bacteria of the β subclass of Proteobacteria from 42 Southern Ocean samples. We found a Nitrosospira-like 16S rRNA gene sequence in all 20 samples that yielded PCR products (8 of 30 samples from the Ross Sea and 12 of 12 samples from the Palmer Peninsula). We also found this sequence in Arctic Ocean samples, indicating a transpolar, if not global, distribution; however, slight differences between Arctic and Antarctic sequences may be evidence of polar endemism.