Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.     

Three color cDNA microarrays: quantitative assessment through the use of fluorescein-labeled probes

Gene expression studies using microarrays have great potential to generate new insights into human disease pathogenesis, but data quality remains a major obstacle. In particular, there does not exist a method to determine prior to hybridization whether an array will yield high quality data, given good study design and target preparation. We have solved this problem through development of a three-color cDNA microarray platform where printed probes are fluorescein labeled, but are spectrally compatible with Cy3 and Cy5 dye-labeled targets when using confocal laser scanners possessing narrow bandwidths. This approach enables prehybridization evaluation of array/spot morphology, DNA deposition and retention and background levels. By using these measurements and the intra-slide coefficient of variation for fluorescence intensity we show that slides in the same batch are not equivalent and measurable prehybridization parameters can be predictive of hybridization performance as determined by replicate consistency. When hybridizing target derived from two cell lines to high and low quality replicate pairs (n = 50 pairs), a direct and significant relationship between prehybridization signal-to-background noise and post-hybridization reproducibility (R² = 0.80, P < 0.001) was observed. We therefore conclude that slide selection based upon prehybridization quality scores will greatly benefit the ability to generate reliable gene expression data.     

Elevated Abundance of Bacteriophage Infecting Bacteria in Soil

Here we report the first direct counts of soil bacteriophage and show that substantial populations of these viruses exist in soil (grand mean = 1.5 × 10⁷ g⁻¹), at least 350-fold more than the highest numbers estimated from traditional viable plaque counts. Adding pure cultures of a Serratia phage to soil showed that the direct counting methods with electron microscopy developed here underestimated the added phage populations by at least eightfold. So, assuming natural phages were similarly underestimated, virus numbers in soil averaged 1.5 × 10⁸ g⁻¹, which is equivalent to 4% of the total population of bacteria. This high abundance was to some extent confirmed by hybridizing colonies grown on Serratia and Pseudomonas selective media with cocktails of phage infecting these bacteria. This showed that 8.9 and 3.9%, respectively, hybridized with colonies from the two media and confirmed the presence of phage DNA sequences in the cultivable fraction of the natural population. Thus, soil phage, like their aquatic counterparts, are likely to be important in controlling bacterial populations and mediating gene transfer in soil.     

The ancestral eutherian karyotype is present in Xenarthra

Molecular studies have led recently to the proposal of a new super-ordinal arrangement of the 18 extant Eutherian orders. From the four proposed super-orders, Afrotheria and Xenarthra were considered the most basal. Chromosome-painting studies with human probes in these two mammalian groups are thus key in the quest to establish the ancestral Eutherian karyotype. Although a reasonable amount of chromosome-painting data with human probes have already been obtained for Afrotheria, no Xenarthra species has been thoroughly analyzed with this approach. We hybridized human chromosome probes to metaphases of species (Dasypus novemcinctus, Tamandua tetradactyla, and Choloepus hoffmanii) representing three of the four Xenarthra families. Our data allowed us to review the current hypotheses for the ancestral Eutherian karyotype, which range from 2n = 44 to 2n = 48. One of the species studied, the two-toed sloth C. hoffmanii (2n = 50), showed a chromosome complement strikingly similar to the proposed 2n = 48 ancestral Eutherian karyotype, strongly reinforcing it.     

Detection of Genes Involved in Biodegradation and Biotransformation in Microbial Communities by Using 50-Mer Oligonucleotide Microarrays

To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance. This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences. Based on artificial probes, our results showed that under hybridization conditions of 50°C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was ~5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 × 10⁷ cells in the presence of background RNA. Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r² = 0.95 to 0.99). Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms. In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation. The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r² = 0.74). In addition, application of the arrays to both polyaromatic-hydrocarbon- and benzene-toluene-ethylbenzene-xylene-contaminated and uncontaminated soils indicated that the developed microarrays appeared to be useful for profiling differences in microbial community structures. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of biodegradation genes and the microbial community in contaminated environments, although more work is needed to improve detection sensitivity.     

Dissimilatory Arsenate and Sulfate Reduction in Sediments of Two Hypersaline, Arsenic-Rich Soda Lakes: Mono and Searles Lakes, California

A radioisotope method was devised to study bacterial respiratory reduction of arsenate in sediments. The following two arsenic-rich soda lakes in California were chosen for comparison on the basis of their different salinities: Mono Lake (~90 g/liter) and Searles Lake (~340 g/liter). Profiles of arsenate reduction and sulfate reduction were constructed for both lakes. Reduction of [⁷³As]arsenate occurred at all depth intervals in the cores from Mono Lake (rate constant [k] = 0.103 to 0.04 h⁻¹) and Searles Lake (k = 0.012 to 0.002 h⁻¹), and the highest activities occurred in the top sections of each core. In contrast, [³⁵S]sulfate reduction was measurable in Mono Lake (k = 7.6 ×10⁴ to 3.2 × 10⁻⁶ h⁻¹) but not in Searles Lake. Sediment DNA was extracted, PCR amplified, and separated by denaturing gradient gel electrophoresis (DGGE) to obtain phylogenetic markers (i.e., 16S rRNA genes) and a partial functional gene for dissimilatory arsenate reduction (arrA). The amplified arrA gene product showed a similar trend in both lakes; the signal was strongest in surface sediments and decreased to undetectable levels deeper in the sediments. More arrA gene signal was observed in Mono Lake and was detectable at a greater depth, despite the higher arsenate reduction activity observed in Searles Lake. A partial sequence (about 900 bp) was obtained for a clone (SLAS-3) that matched the dominant DGGE band found in deeper parts of the Searles Lake sample (below 3 cm), and this clone was found to be closely related to SLAS-1, a novel extremophilic arsenate respirer previously cultivated from Searles Lake.     

Light-directed 5′→3′ synthesis of complex oligonucleotide microarrays

Light-directed synthesis of high-density microarrays is currently performed in the 3′→5′ direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5′→3′ direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5′→3′ direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150 000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R² = 0.998). We have also shown that the 3′ ends of array probes are available for sequence-specific primer extension and ligation reactions.     

An Improved Protocol for Quantification of Freshwater Actinobacteria by Fluorescence In Situ Hybridization

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml⁻¹) followed by achromopeptidase (60 U ml⁻¹) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.     

Abundance and Distribution of Ostreococcus sp. in the San Pedro Channel, California, as Revealed by Quantitative PCR

Ostreococcus is a genus of widely distributed marine phytoplankton which are picoplanktonic in size (<2 μm) and capable of rapid growth. Although Ostreococcus has been detected around the world, little quantitative information exists on its contribution to planktonic communities. We designed and implemented a genus-specific TaqMan-based quantitative PCR (qPCR) assay to investigate the dynamics and ecology of Ostreococcus at the USC Microbial Observatory (eastern North Pacific). Samples were collected from 5 m and the deep chlorophyll maximum (DCM) between September 2000 and August 2002. Ostreococcus abundance at 5 m was generally <5.0 × 10³ cells ml⁻¹, with a maximum of 8.2 × 10⁴ cells ml⁻¹. Ostreococcus abundance was typically higher at the DCM, with a maximum of 3.2 × 10⁵ cells ml⁻¹. The vertical distribution of Ostreococcus was examined in March 2005 and compared to the distribution of phototrophic picoeukaryotes (PPE) measured by flow cytometry. The largest contribution to PPE abundance by Ostreococcus was ~70% and occurred at 30 m, near the DCM. Despite its relatively low abundance, the depth-integrated standing stock of Ostreococcus in March 2005 was ~30 mg C m⁻². Our work provides a new technique for quantifying the abundance of Ostreococcus and demonstrates the seasonal dynamics of this genus and its contribution to picoeukaryote biomass at our coastal sampling station.     

Abundances and Distributions of the Dominant nifH Phylotypes in the Northern Atlantic Ocean

Understanding the factors that influence the distribution and abundance of marine diazotrophs is important in order to assess their role in the oceanic nitrogen cycle. Environmental DNA samples from four cruises to the North Atlantic Ocean, covering a sampling area of 0°N to 42°N and 67°W to 13°W, were analyzed for the presence and amount of seven nifH phylotypes using real-time quantitative PCR and TaqMan probes. The cyanobacterial phylotypes dominated in abundance (94% of all nifH copies detected) and were the most widely distributed. The filamentous cyanobacterial type, which included both Trichodesmium and Katagnymene, was the most abundant (51%), followed by group A, an uncultured unicellular cyanobacterium (33%), and gamma A, an uncultured gammaproteobacterium (6%). Group B, unicellular cyanobacterium Crocosphaera, and group C Cyanothece-like phylotypes were not often detected (6.9% and 2.3%, respectively), but where present, could reach high concentrations. Gamma P, another uncultured gammaproteobacterium, was seldom detected (0.5%). Water temperature appeared to influence the distribution of many nifH phylotypes. Very high (up to 1 × 10⁶ copies liter⁻¹) nifH concentrations of group A were detected in the eastern basin (25 to 17°N, 27 to 30°W), where the temperature ranged from 20 to 23°C. The highest concentrations of filamentous phylotypes were measured between 25 and 30°C. The uncultured cluster III phylotype was uncommon (0.4%) and was associated with mean water temperatures of 18°C. Diazotroph abundance was highest in regions where modeled average dust deposition was between 1 and 2 g/m²/year.     

Use of a three-color cDNA microarray platform to measure and control support-bound probe for improved data quality and reproducibility

Construction methodologies for cDNA microarrays lack the ability to determine array integrity prior to hybridization, leaving the array itself a source of uncontrolled experimental variation. We solved this problem through development of a three-color cDNA array platform whereby printed probes are tagged with fluorescein and are compatible with Cy3 and Cy5 target labeling dyes when using confocal laser scanners possessing narrow bandwidths. Here we use this approach to: (i) develop a tracking system to monitor the printing of probe plates at predicted coordinates; (ii) define the quantity of immobilized probe necessary for quality hybridized array data to establish pre-hybridization array selection criteria; (iii) investigate factors that influence probe availability for hybridization; and (iv) explore the feasibility of hybridized data filtering using element fluorescein intensity. A direct and significant relationship (R² = 0.73, P < 0.001) between pre-hybridization average fluorescein intensity and subsequent hybridized replicate consistency was observed, illustrating that data quality can be improved by selecting arrays that meet defined pre-hybridization criteria. Furthermore, we demonstrate that our three-color approach provides a means to filter spots possessing insufficient bound probe from hybridized data sets to further improve data quality. Collectively, this strategy will improve microarray data and increase its utility as a sensitive screening tool.     

Environmental Investigations of Vibrio parahaemolyticus in Oysters after Outbreaks in Washington, Texas, and New York (1997 and 1998)

Total Vibrio parahaemolyticus densities and the occurrence of pathogenic strains in shellfish were determined following outbreaks in Washington, Texas, and New York. Recently developed nonradioactive DNA probes were utilized for the first time for direct enumeration of V. parahaemolyticus in environmental shellfish samples. V. parahaemolyticus was prevalent in oysters from Puget Sound, Wash.; Galveston Bay, Tex.; and Long Island Sound, N.Y., in the weeks following shellfish-associated outbreaks linked to these areas. However, only two samples (one each from Washington and Texas) were found to harbor total V. parahaemolyticus densities exceeding the level of concern of 10,000 g⁻¹. Pathogenic strains, defined as those hybridizing with tdh and/or trh probes, were detected in a few samples, mostly Puget Sound oysters, and at low densities (usually <10 g⁻¹). Intensive sampling in Galveston Bay demonstrated relatively constant water temperature (27.8 to 31.7°C) and V. parahaemolyticus levels (100 to 1,000 g⁻¹) during the summer. Salinity varied from 14.9 to 29.3 ppt. A slight but significant (P < 0.05) negative correlation (−0.25) was observed between V. parahaemolyticus density and salinity. Based on our data, findings of more than 10,000 g⁻¹ total V. parahaemolyticus or >10 g⁻¹ tdh- and/or trh-positive V. parahaemolyticus in environmental oysters should be considered extraordinary.     

Dynamics and Distribution of Cyanophages and Their Effect on Marine Synechococcus spp.

Cyanophages infecting marine Synechococcus cells were frequently very abundant and were found in every seawater sample along a transect in the western Gulf of Mexico and during a 28-month period in Aransas Pass, Tex. In Aransas Pass their abundance varied seasonally, with the lowest concentrations coincident with cooler water and lower salinity. Along the transect, viruses infecting Synechococcus strains DC2 and SYN48 ranged in concentration from a few hundred per milliliter at 97 m deep and 83 km offshore to ca. 4 × 10⁵ ml^-1 near the surface at stations within 18 km of the coast. The highest concentrations occurred at the surface, where salinity decreased from ca. 35.5 to 34 ppt and Synechococcus concentrations were greatest. Viruses infecting strains SNC1, SNC2, and 838BG were distributed in a similar manner but were much less abundant (<10 to >5 × 10³ ml^-1). When Synechococcus concentrations exceeded ca. 10³ ml^-1, cyanophage concentrations increased markedly (ca. 10² to > 10⁵ ml^-1), suggesting that a minimum host density was required for efficient viral propagation. Data on the decay rate of viral infectivity d (per day), as a function of solar irradiance I (millimoles of quanta per square meter per second), were used to develop a relationship (d = 0.2610I - 0.00718; r² = 0.69) for conservatively estimating the destruction of infectious viruses in the mixed layer of two offshore stations. Assuming that virus production balances losses and that the burst size is 250, ca. 5 to 7% of Synechococcus cells would be infected daily by viruses. Calculations based on contact rates between Synechococcus cells and infectious viruses produce similar results (5 to 14%). Moreover, balancing estimates of viral production with contact rates for the farthest offshore station required that most Synechococcus cells be susceptible to infection, that most contacts result in infection, and that the burst size be about 324 viruses per lytic event. In contrast, in nearshore waters, where ca. 80% of Synechococcus cells would be contacted daily by infectious cyanophages, only ca. 1% of the contacts would have to result in infection to balance the estimated virus removal rates. These results indicate that cyanophages are an abundant and dynamic component of marine planktonic communities and are probably responsible for lysing a small but significant portion of the Synechococcus population on a daily basis.     

Diazotrophic Diversity and Distribution in the Tropical and Subtropical Atlantic Ocean

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3°N and 56.6 to 18.5°W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as γ-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and γ-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30°C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30°C, more often in waters with temperature of <26°C, and were sometimes recovered from waters with detectable nitrate concentrations.     

A Novel Strain D5 Isolated from Acacia confusa

We isolated a novel strain D5 from nodules of Acacia confusa. Under strict sterile conditions the strain could successfully nodulate Acacia confusa, A. crassicarpa and A. mangium, with nitrogenase activity ranging from 18.90 to 19.86 nmol·g⁻¹·min⁻¹. In the phylogenetic tree based on a complete 16S rRNA gene sequence, the sequence of strain D5 shared 99% homology with that of four species of genus Pseudomonas. The 685 bp nodA fragment amplified from strain D5 shared 95% homology with the nodA sequence of 9 species of genus Bradyrhizobium, with a genetic distance of 0.01682. The 740 bp nifH gene fragment was amplified from strain D5. This strain D5 nifH gene and Bradyrhizobium spp. formed a branch, showing 98% homology and a genetic distance of 0. The homology between this branch and the Bradyrhizobium spp. DG in another branch was 99%, with a genetic distance of 0.007906. These results indicate that this strain D5 is a new type of nitrogen-fixing bacterium.     

Coliform Bacteria and Nitrogen Fixation in Pulp and Paper Mill Effluent Treatment Systems

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N₂ fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations. In situ N₂ fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N₂-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N₂ fixation (0.87 to 4.90 mg of N liter⁻¹ day⁻¹) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.