High wax ester and triacylglycerol biosynthesis potential in coastal sediments of Antarctic and Subantarctic environments.

The wax ester (WE) and triacylglycerol (TAG) biosynthetic potential of marine microorganisms is poorly understood at the microbial community level. The goal of this work was to uncover the prevalence and diversity of bacteria with the potential to synthesize these neutral lipids in coastal sediments of two high latitude environments, and to characterize the gene clusters related to this process. Homolog sequences of the key enzyme, the wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) were retrieved from 13 metagenomes, including subtidal and intertidal sediments of a Subantarctic environment (Ushuaia Bay, Argentina), and subtidal sediments of an Antarctic environment (Potter Cove, Antarctica). The abundance of WS/DGAT homolog sequences in the sediment metagenomes was 1.23 ± 0.42 times the abundance of 12 single-copy genes encoding ribosomal proteins, higher than in seawater (0.13 ± 0.31 times in 338 metagenomes). Homolog sequences were highly diverse, and were assigned to the Pseudomonadota, Actinomycetota, Bacteroidota and Acidobacteriota phyla. The genomic context of WS/DGAT homologs included sequences related to WE and TAG biosynthesis pathways, as well as to other related pathways such as fatty-acid metabolism, suggesting carbon recycling might drive the flux to neutral lipid synthesis. These results indicate the presence of abundant and taxonomically diverse bacterial populations with the potential to synthesize lipid storage compounds in marine sediments, relating this metabolic process to bacterial survival.

Microbial communities associated with kelp detritus in temperate and subantarctic intertidal sediments.

Kelp forests, among the most productive ecosystems on Earth, cover large areas of the South Atlantic coast. Sediment heterotrophic bacteria have a pivotal role in the degradation of kelp biomass, however, the response of sediment microbial communities to periodic kelp biomass inputs is mostly unknown. Here, we show that kelp biomass induced rapid changes in overlying water chemistry and shifts in sediment microbial communities, which differed in the experimental systems containing Macrocystis pyrifera (M) and Undaria pinnatifida (U) with sediments of the respective regions. We observed results compatible with the degradation of labile, high molecular weight compounds into smaller and more refractory compounds towards the end of the incubations. The capability of microbial communities to degrade alginate, the major component of kelp cell walls, significantly increased with respect to controls after kelp biomass addition (Absorbance at 235 nm 1.2 ± 0.3 and 1.0 ± 0.2 for M and U, respectively, controls <0.2, t = 4 days). Shifts in microbial community structure (based on 16S rRNA gene amplicon sequencing) were tightly related to the kelp treatment and, to a lesser extent, to the sediment provenance (Principal Coordinates Analysis, 80 % of variation explained in the first two axes). Dissolved oxygen, pH, salinity, alginolytic potential, Absorbance at 235 and 600 nm, total N, total C, and SUVA index correlated significantly with community structure. Differentially abundant populations between kelp-amended treatments and controls included members of the Flavobacteriia class (Algibacter and Polaribacter), and Gammaproteobacteria (Psychromonas and Marinomonas), among others. Metagenomes of M and U-amended sediments contained sequences from 18 of the 19 enzyme families related to alginate or fucoidan degradation. Specific taxonomic groups were associated with enzyme classes targeting different substrates, suggesting niche differentiation. This work expands our knowledge on the patterns of microbial assemblages from intertidal sediments in response to kelp biomass inputs.

Microbial assemblages associated with the invasive kelp Undaria pinnatifida in Patagonian coastal waters: Structure and alginolytic potential.

Undaria pinnatifida is a brown algae native to Asia that has settled in various regions worldwide, periodically contributing with large quantities of C and nutrients during its annual cycle. In this work, we analyzed a coastal site in Patagonia (Argentina) that has been colonized for three decades by U. pinnatifida, focusing on associated microbial communities in three different compartments. An important influence of algae was observed in seawater, especially in the bottom of the algal forest during the austral summer (January) at the moment of greater biomass release. This was evidenced by changes in DOC concentration and its quality indicators (higher Freshness and lower Humification index) and higher DIC. Although maximum values of NH4 and PO4 were observed in January, bottom water samples had lower concentrations than surface water, suggesting nutrient consumption by bacteria during algal DOM release. Concomitantly, bacterial abundance peaked, reaching 4.68 ± 1.33 × 105 cells mL -1 (January), showing also higher capability of degrading alginate, a major component of brown algae cell walls. Microbial community structure was influenced by sampling date, season, sampling zone (surface or bottom), and environmental factors (temperature, salinity, pH, dissolved oxygen, nutrients). Samples of epiphytic biofilms showed a distinct community structure compared to seawater, lower diversity, and remarkably high alginolytic capability, suggesting adaptation to degrade algal biomass. A high microdiversity of populations of the genus Leucothrix (Gammaproteobacteria, Thiotrichales) that accounted for a large fraction of epiphytic communities was observed, and changed over time. Epiphytic assemblages shared more taxa with bottom than with surface seawater assemblages, indicating a certain level of exchange between communities in the forest surroundings. This work provides insight into the impact of U. pinnatifida decay on seawater quality, and the role of microbial communities on adapting to massive biomass inputs through rapid DOM turnover.

Predominance and high diversity of genes associated to denitrification in metagenomes of subantarctic coastal sediments exposed to urban pollution.

The aim of this work was to characterize the microbial nitrogen cycling potential in sediments from Ushuaia Bay, a subantarctic environment that has suffered a recent explosive demographic growth. Subtidal sediment samples were retrieved in triplicate from two urban points in the Bay, and analyzed through metagenomic shotgun sequencing. Sequences assigned to genes related to nitrification, nitrate reduction and denitrification were predominant in this environment with respect to metagenomes from other environments, including other marine sediments. The nosZ gene, responsible for nitrous oxide transformation into di-nitrogen, presented a high diversity. The majority of NosZ sequences were classified as Clade II (atypical) variants affiliated to different bacterial lineages such as Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria, Verrucomicrobia, as well as to Archaea. The analysis of a fosmid metagenomic library from the same site showed that the genomic context of atypical variants was variable, and was accompanied by distinct regulatory elements, suggesting the evolution of differential ecophysiological roles. This work increases our understanding of the microbial ecology of nitrogen transformations in cold coastal environments and provides evidence of an enhanced denitrification potential in impacted sediment microbial communities. In addition, it highlights the role of yet overlooked populations in the mitigation of environmentally harmful forms of nitrogen.

Metagenomic Analysis of Subtidal Sediments from Polar and Subpolar Coastal Environments Highlights the Relevance of Anaerobic Hydrocarbon Degradation Processes.

In this work, we analyzed the community structure and metabolic potential of sediment microbial communities in high-latitude coastal environments subjected to low to moderate levels of chronic pollution. Subtidal sediments from four low-energy inlets located in polar and subpolar regions from both Hemispheres were analyzed using large-scale 16S rRNA gene and metagenomic sequencing. Communities showed high diversity (Shannon's index 6.8 to 10.2), with distinct phylogenetic structures (<40% shared taxa at the Phylum level among regions) but similar metabolic potential in terms of sequences assigned to KOs. Environmental factors (mainly salinity, temperature, and in less extent organic pollution) were drivers of both phylogenetic and functional traits. Bacterial taxa correlating with hydrocarbon pollution included families of anaerobic or facultative anaerobic lifestyle, such as Desulfuromonadaceae, Geobacteraceae, and Rhodocyclaceae. In accordance, biomarker genes for anaerobic hydrocarbon degradation (bamA, ebdA, bcrA, and bssA) were prevalent, only outnumbered by alkB, and their sequences were taxonomically binned to the same bacterial groups. BssA-assigned metagenomic sequences showed an extremely wide diversity distributed all along the phylogeny known for this gene, including bssA sensu stricto, nmsA, assA, and other clusters from poorly or not yet described variants. This work increases our understanding of microbial community patterns in cold coastal sediments, and highlights the relevance of anaerobic hydrocarbon degradation processes in subtidal environments.

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach.

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.

Metagenomics unveils the attributes of the alginolytic guilds of sediments from four distant cold coastal environments.

Alginates are abundant polysaccharides in brown algae that constitute an important energy source for marine heterotrophic bacteria. Despite the key role of alginate degradation processes in the marine carbon cycle, little information is available on the bacterial populations involved in these processes. The aim of this work was to gain a better understanding of alginate utilization capabilities in cold coastal environments. Sediment metagenomes from four high-latitude regions of both Hemispheres were interrogated for alginate lyase gene homologue sequences and their genomic context. Sediments contained highly abundant and diverse bacterial assemblages with alginolytic potential, including members of Bacteroidetes and Proteobacteria, as well as several poorly characterized taxa. The microbial communities in Arctic and Antarctic sediments exhibited the most similar alginolytic profiles, whereas brackish sediments showed distinct structures with a higher proportion of novel genes. Examination of the gene neighbourhood of the alginate lyase homologues revealed distinct patterns depending on the potential lineage of the scaffolds, with evidence of evolutionary relationships among alginolytic gene clusters from Bacteroidetes and Proteobacteria. This information is relevant for understanding carbon fluxes in cold coastal environments and provides valuable information for the development of biotechnological applications from brown algae biomass.

Diverse Bacterial Groups Contribute to the Alkane Degradation Potential of Chronically Polluted Subantarctic Coastal Sediments.

We aimed to gain insight into the alkane degradation potential of microbial communities from chronically polluted sediments of a subantarctic coastal environment using a combination of metagenomic approaches. A total of 6178 sequences annotated as alkane-1-monooxygenases (EC 1.14.15.3) were retrieved from a shotgun metagenomic dataset that included two sites analyzed in triplicate. The majority of the sequences binned with AlkB described in Bacteroidetes (32 ± 13 %) or Proteobacteria (29 ± 7 %), although a large proportion remained unclassified at the phylum level. Operational taxonomic unit (OTU)-based analyses showed small differences in AlkB distribution among samples that could be correlated with alkane concentrations, as well as with site-specific variations in pH and salinity. A number of low-abundance OTUs, mostly affiliated with Actinobacterial sequences, were found to be only present in the most contaminated samples. On the other hand, the molecular screening of a large-insert metagenomic library of intertidal sediments from one of the sampling sites identified two genomic fragments containing novel alkB gene sequences, as well as various contiguous genes related to lipid metabolism. Both genomic fragments were affiliated with the phylum Planctomycetes, and one could be further assigned to the genus Rhodopirellula due to the presence of a partial sequence of the 23S ribosomal RNA (rRNA) gene. This work highlights the diversity of bacterial groups contributing to the alkane degradation potential and reveals patterns of functional diversity in relation with environmental stressors in a chronically polluted, high-latitude coastal environment. In addition, alkane biodegradation genes are described for the first time in members of Planctomycetes.

The bacterial community structure of hydrocarbon-polluted marine environments as the basis for the definition of an ecological index of hydrocarbon exposure.

The aim of this study was to design a molecular biological tool, using information provided by amplicon pyrosequencing of 16S rRNA genes, that could be suitable for environmental assessment and bioremediation in marine ecosystems. We selected 63 bacterial genera that were previously linked to hydrocarbon biodegradation, representing a minimum sample of the bacterial guild associated with this process. We defined an ecological indicator (ecological index of hydrocarbon exposure, EIHE) using the relative abundance values of these genera obtained by pyrotag analysis. This index reflects the proportion of the bacterial community that is potentially capable of biodegrading hydrocarbons. When the bacterial community structures of intertidal sediments from two sites with different pollution histories were analyzed, 16 of the selected genera (25%) were significantly overrepresented with respect to the pristine site, in at least one of the samples from the polluted site. Although the relative abundances of individual genera associated with hydrocarbon biodegradation were generally low in samples from the polluted site, EIHE values were 4 times higher than those in the pristine sample, with at least 5% of the bacterial community in the sediments being represented by the selected genera. EIHE values were also calculated in other oil-exposed marine sediments as well as in seawater using public datasets from experimental systems and field studies. In all cases, the EIHE was significantly higher in oiled than in unpolluted samples, suggesting that this tool could be used as an estimator of the hydrocarbon-degrading potential of microbial communities.

Bioprospection of marine microorganisms: potential and challenges for Argentina.

The marine environments of Argentina have a remarkable extension, as well as high biological productivity and biodiversity of both macro- and microorganisms. Despite having a great potential for biotechnological applications, the microorganisms inhabiting these ecosystems remain mostly unexplored and unexploited. In this review, we study the research topics and the interactions among Argentinean laboratories, by analyzing current articles published on biotechnology-related marine microbiology by researchers of this country. In addition, we identify the challenges and opportunities for Argentina to take advantage of the genetic potential of its marine microorganisms. Finally, we suggest possible actions that could improve the development of this research field, as well as the utilization of this knowledge to solve societal needs.

Bioprospection of marine microorganisms: potential and challenges for Argentina / Bioprospección de microorganismos marinos: potencialidades y desafíos para Argentina

The marine environments of Argentina have a remarkable extension, as well as high biological productivity and biodiversity of both macro- and microorganisms. Despite having a great potential for biotechnological applications, the microorganisms inhabiting these ecosystems remain mostly unexplored and unexploited. In this review, we study the research topics and the interactions among Argentinean laboratories, by analyzing current articles published on biotechnology-related marine microbiology by researchers of this country. In addition, we identify the challenges and opportunities for Argentina to take advantage of the genetic potential of its marine microorganisms. Finally, we suggest possible actions that could improve the development of this research field, as well as the utilization of this knowledge to solve societal needs.

El medio ambiente marino de la Argentina tiene una notable extensión, como así también una alta productividad biológica y biodiversidad de macro y microorganismos. A pesar de presentar un gran potencial para aplicaciones biotecnológicas, los microorganismos que habitan estos ecosistemas permanecen mayormente inexplorados y sus propiedades aún no explotadas. En este trabajo de revisión, estudiamos los temas de investigación y las interacciones entre grupos de investigación argentinos, por medio del análisis de los artículos publicados hasta el momento en temáticas relacionadas con la aplicación biotecnológica de microorganismos marinos. Además, identificamos los desafíos y las oportunidades para que la Argentina tome ventaja del potencial genético de sus microorganismos marinos. Por último, sugerimos posibles acciones que podrían mejorar el desarrollo de este campo de estudio, como así también la utilización de este conocimiento para resolver las necesidades de la sociedad.

Alkane biodegradation genes from chronically polluted subantarctic coastal sediments and their shifts in response to oil exposure.

Although sediments are the natural hydrocarbon sink in the marine environment, the ecology of hydrocarbon-degrading bacteria in sediments is poorly understood, especially in cold regions. We studied the diversity of alkane-degrading bacterial populations and their response to oil exposure in sediments of a chronically polluted Subantarctic coastal environment, by analyzing alkane monooxygenase (alkB) gene libraries. Sequences from the sediment clone libraries were affiliated with genes described in Proteobacteria and Actinobacteria, with 67 % amino acid identity in average to sequences from isolated microorganisms. The majority of the sequences were most closely related to uncultured microorganisms from cold marine sediments or soils from high latitude regions, highlighting the role of temperature in the structuring of this bacterial guild. The distribution of alkB sequences among samples of different sites and years, and selection after experimental oil exposure allowed us to identify ecologically relevant alkB genes in Subantarctic sediments, which could be used as biomarkers for alkane biodegradation in this environment. 16 S rRNA amplicon pyrosequencing indicated the abundance of several genera for which no alkB genes have yet been described (Oleispira, Thalassospira) or that have not been previously associated with oil biodegradation (Spongiibacter-formerly Melitea-, Maribius, Robiginitomaculum, Bizionia and Gillisia). These genera constitute candidates for future work involving identification of hydrocarbon biodegradation pathway genes.

Bioprospection of marine microorganisms: biotechnological applications and methods.

Environmental microorganisms constitute an almost inexhaustible reserve of genetic and functional diversity, accumulated during millions of years of adaptive evolution to various selective pressures. In particular, the extent of microbial biodiversity in marine habitats seems to grow larger as new techniques emerge to measure it. This has resulted in novel and more complex approaches for the screening of molecules and activities of biotechnological interest in these environments. In this review, we explore the different partially overlapping biotechnological fields that make use of microorganisms and we describe the different marine habitats that are particularly attractive for bioprospection. In addition, we review the methodological approaches currently used for microbial bioprospection, from the traditional cultivation techniques to state of the art metagenomic approaches, with emphasis in the marine environment.

Bioprospection of marine microorganisms: biotechnological applications and methods

Bioprospección de microorganismos marinos: aplicaciones biotecnológicas y métodos. Los microorganismos ambientales constituyen una reserva prácticamente inagotable de diversidad genética, acumulada durante millones de años de evolución adaptativa a varias presiones selectivas. En particular, la magnitud de la biodiversidad microbiana en hábitats marinos parece crecer al emerger nuevas técnicas para medirla. Como resultado, se han comenzado a utilizar enfoques novedosos y más complejos para la búsqueda de moléculas y actividades de interés biotecnológico en estos ambientes. En este artículo de revisión, nosotros exploramos los diferentes campos de la biotecnología que utilizan microorganismos, los cuales se superponen parcialmente, y describimos los diferentes hábitats marinos que resultan particularmente atractivos para la bioprospección. Además, revisamos los enfoques metodológicos actualmente utilizados para la bioprospección microbiana, desde las técnicas de cultivo tradicionales hasta modernos enfoques metagenómicos, con énfasis en el medio ambiente marino.

Environmental microorganisms constitute an almost inexhaustible reserve of genetic and functional diversity, accumulated during millions of years of adaptive evolution to various selective pressures. In particular, the extent of microbial biodiversity in marine habitats seems to grow larger as new techniques emerge to measure it. This has resulted in novel and more complex approaches for the screening of molecules and activities of biotechnological interest in these environments. In this review, we explore the different partially overlapping biotechnological fields that make use of microorganisms and we describe the different marine habitats that are particularly attractive for bioprospection. In addition, we review the methodological approaches currently used for microbial bioprospection, from the traditional cultivation techniques to state of the art metagenomic approaches, with emphasis in the marine environment.

Abundance, dynamics, and biogeographic distribution of seven polycyclic aromatic hydrocarbon dioxygenase gene variants in coastal sediments of Patagonia.

Novel polycyclic aromatic hydrocarbon dioxygenase gene variants were present in abundances similar to or higher than those of phnA1 from Cycloclasticus spp. at a chronically polluted subantarctic coastal marine environment in Patagonia. These novel gene variants were detected over a 6-year time span and were also present in sediments from temperate Patagonian sites.

Isolation and characterization of biosurfactant-producing Alcanivorax strains: hydrocarbon accession strategies and alkane hydroxylase gene analysis.

Biosurfactant-producing bacteria belonging to the genera Alcanivorax, Cobetia and Halomonas were isolated from marine sediments with a history of hydrocarbon exposure (Aristizábal and Gravina Peninsulas, Argentina). Two Alcanivorax isolates were found to form naturally occurring consortia with strains closely related to Pseudomonas putida and Microbacterium esteraromaticum. Alkane hydroxylase gene analysis in these two Alcanivorax strains resulted in the identification of two novel alkB genes, showing 86% and 60% deduced amino acid sequence identity with those of Alcanivorax sp. A-11-3 and Alcanivorax dieselolei P40, respectively. In addition, a gene homologous to alkB2 from Alcanivorax borkumensis was present in one of the strains. The consortium formed by this strain, Alcanivorax sp. PA2 (98.9% 16S rRNA gene sequence identity with A. borkumensis SK2(T)) and P. putida PA1 was characterized in detail. These strains form cell aggregates when growing as mixed culture, though only PA2 was responsible for biosurfactant activity. During exponential growth phase of PA2, cells showed high hydrophobicity and adherence to hydrocarbon droplets. Biosurfactant production was only detectable at late growth and stationary phases, suggesting that it is not involved in initiating oil degradation and that direct interfacial adhesion is the main hydrocarbon accession mode of PA2. This strain could be useful for biotechnological applications due to its biosurfactant production, catabolic and aggregation properties.

Novel aromatic ring-hydroxylating dioxygenase genes from coastal marine sediments of Patagonia.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs), widespread pollutants in the marine environment, can produce adverse effects in marine organisms and can be transferred to humans through seafood. Our knowledge of PAH-degrading bacterial populations in the marine environment is still very limited, and mainly originates from studies of cultured bacteria. In this work, genes coding catabolic enzymes from PAH-biodegradation pathways were characterized in coastal sediments of Patagonia with different levels of PAH contamination. RESULTS: Genes encoding for the catalytic alpha subunit of aromatic ring-hydroxylating dioxygenases (ARHDs) were amplified from intertidal sediment samples using two different primer sets. Products were cloned and screened by restriction fragment length polymorphism analysis. Clones representing each restriction pattern were selected in each library for sequencing. A total of 500 clones were screened in 9 gene libraries, and 193 clones were sequenced. Libraries contained one to five different ARHD gene types, and this number was correlated with the number of PAHs found in the samples above the quantification limit (r = 0.834, p < 0.05). Overall, eight different ARHD gene types were detected in the sediments. In five of them, their deduced amino acid sequences formed deeply rooted branches with previously described ARHD peptide sequences, exhibiting less than 70% identity to them. They contain consensus sequences of the Rieske type [2Fe-2S] cluster binding site, suggesting that these gene fragments encode for ARHDs. On the other hand, three gene types were closely related to previously described ARHDs: archetypical nahAc-like genes, phnAc-like genes as identified in Alcaligenes faecalis AFK2, and phnA1-like genes from marine PAH-degraders from the genus Cycloclasticus. CONCLUSION: These results show the presence of hitherto unidentified ARHD genes in this sub-Antarctic marine environment exposed to anthropogenic contamination. This information can be used to study the geographical distribution and ecological significance of bacterial populations carrying these genes, and to design molecular assays to monitor the progress and effectiveness of remediation technologies.

Codon optimization of bacterial luciferase (lux) for expression in mammalian cells.

Expression of the bacterial luciferase (lux) system in mammalian cells would culminate in a new generation of bioreporters for in vivo monitoring and diagnostics technology. Past efforts to express bacterial luciferase in mammalian cells have resulted in only modest gains due in part to low overall expression of the bacterial genes. To optimize expression, we have designed and synthesized codon-optimized versions of the luxA and luxB genes from Photorhabdus luminsecens. To evaluate these genes in vivo, stable HEK293 cell lines were created harboring wild type luxA and luxB (WTA/WTB), codon-optimized luxA and wild type luxB (COA/WTB), and codon-optimized versions of both luxA and luxB genes (COA/COB). Although mRNA levels within these clones remained approximately equal, LuxA protein levels increased significantly after codon optimization. On average, bioluminescence levels were increased by more than six-fold [5x10(5) vs 2.9x10(6) relative light units (RLU)/mg total protein] with the codon-optimized luxA and wild type luxB. Bioluminescence was further enhanced upon expression of both optimized genes (2.7x10(7) RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells.

Abundance of dioxygenase genes similar to Ralstonia sp. strain U2 nagAc is correlated with naphthalene concentrations in coal tar-contaminated freshwater sediments.

We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-microl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 +/- 0.7) x 10(3) to (2.9 +/- 0.3) x 10(5) copies of nagAc-like dioxygenase genes per microg of DNA extracted from sediment samples. These values corresponded to (1.2 +/- 0.6) x 10(5) to (5.4 +/- 0.4) x 10(7) copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene.

Evaluation of a plasmid-based 16S-23S rDNA intergenic spacer region array for analysis of microbial diversity in industrial wastewater.

A plasmid-based 16S-23S rDNA intergenic spacer region (ISR) array was developed and optimized for analysis of microbial diversity within complex environmental samples. Plasmid probes with 16S-23S rDNA ISR inserts (800-1500 bp) from industrial wastewater treatment plant (WWTP) microorganisms were arrayed onto glass slides. Hybridization of fluorescently labeled target sequences from two clones from the ISR WWTP library to arrayed probes showed that there was a good linear relationship between hybridization intensity and ISR similarity (r(2)=0.82). Hybridization was highly specific (average background from arrayed probes with less than 80% similarity in ISR sequence was less than 7%). Strong fluorescence intensity corresponded to near-perfect match clones (99% or greater similarity in ISR sequence). A majority of probes (79%) showed no background hybridization. However, weak background (less than 50% for arrayed probes with 90% and 95% similarity in the 16S rRNA genes) was observed from closely related microorganisms. Background fluorescence from the negative control (plasmid vector with no insert) was similar to water and dimethyl sulfoxide (DMSO)-negative controls. Hybridization using fluorescently labeled ISR sequences from a mixed community sample produced strong fluorescent signals with no background from negative controls. A Cy5-labeled reference standard, part of the vector and present in every spotted probe, was used to normalize hybridization values. These results indicate that arrayed plasmid containing ISR probe insert sequences provides specificity and sensitivity for microbial community analysis in a high-throughput array format.