Diversity of ndo Genes in Mangrove Sediments Exposed to Different Sources of Polycyclic Aromatic Hydrocarbon Pollution

Polycyclic aromatic hydrocarbon (PAH) pollutants originating from oil spills and wood and fuel combustion are pollutants which are among the major threats to mangrove ecosystems. In this study, the composition and relative abundance in the sediment bacterial communities of naphthalene dioxygenase (ndo) genes which are important for bacterial adaptation to environmental PAH contamination were investigated. Three urban mangrove sites which had characteristic compositions and levels of PAH compounds in the sediments were selected. The diversity and relative abundance of ndo genes in total community DNA were assessed by a newly developed ndo denaturing gradient gel electrophoresis (DGGE) approach and by PCR amplification with primers targeting ndo genes with subsequent Southern blot hybridization analyses. Bacterial populations inhabiting sediments of urban mangroves under the impact of different sources of PAH contamination harbor distinct ndo genotypes. Sequencing of cloned ndo amplicons comigrating with dominant DGGE bands revealed new ndo genotypes. PCR-Southern blot analysis and ndo DGGE showed that the frequently studied nah and phn genotypes were not detected as dominant ndo types in the mangrove sediments. However, ndo genotypes related to nagAc-like genes were detected, but only in oil-contaminated mangrove sediments. The long-term impact of PAH contamination, together with the specific environmental conditions at each site, may have affected the abundance and diversity of ndo genes in sediments of urban mangroves.     

Pleiotropic and Epistatic Behavior of a Ring-Hydroxylating Oxygenase System in the Polycyclic Aromatic Hydrocarbon Metabolic Network from Mycobacterium vanbaalenii PYR-1

Despite the considerable knowledge of bacterial high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) metabolism, the key enzyme(s) and its pleiotropic and epistatic behavior(s) responsible for low-molecular-weight (LMW) PAHs in HMW PAH-metabolic networks remain poorly understood. In this study, a phenotype-based strategy, coupled with a spray plate method, selected a Mycobacterium vanbaalenii PYR-1 mutant (6G11) that degrades HMW PAHs but not LMW PAHs. Sequence analysis determined that the mutant was defective in pdoA2, encoding an aromatic ring-hydroxylating oxygenase (RHO). A series of metabolic comparisons using high-performance liquid chromatography (HPLC) analysis revealed that the mutant had a lower rate of degradation of fluorene, anthracene, and pyrene. Unlike the wild type, the mutant did not produce a color change in culture media containing fluorene, phenanthrene, and fluoranthene. An Escherichia coli expression experiment confirmed the ability of the Pdo system to oxidize biphenyl, the LMW PAHs naphthalene, phenanthrene, anthracene, and fluorene, and the HMW PAHs pyrene, fluoranthene, and benzo[a]pyrene, with the highest enzymatic activity directed toward three-ring PAHs. Structure analysis and PAH substrate docking simulations of the Pdo substrate-binding pocket rationalized the experimentally observed metabolic versatility on a molecular scale. Using information obtained in this study and from previous work, we constructed an RHO-centric functional map, allowing pleiotropic and epistatic enzymatic explanation of PAH metabolism. Taking the findings together, the Pdo system is an RHO system with the pleiotropic responsibility of LMW PAH-centric hydroxylation, and its epistatic functional contribution is also crucial for the metabolic quality and quantity of the PAH-MN.     

Genotypic and Phenotypic Responses of a Riverine Microbial Community to Polycyclic Aromatic Hydrocarbon Contamination

The phenotypic and genotypic adaptation of a freshwater sedimentary microbial community to elevated (22 to 217 μg g [dry weight] of sediment⁻¹) levels of polycyclic aromatic hydrocarbons (PAHs) was determined by using an integrated biomolecular approach. Central to the approach was the use of phospholipid fatty acid (PLFA) profiles to characterize the microbial community structure and nucleic acid analysis to quantify the frequency of degradative genes. The study site was the Little Scioto River, a highly impacted, channelized riverine system located in central Ohio. This study site is a unique lotic system, with all sampling stations having similar flow and sediment characteristics both upstream and downstream from the source of contamination. These characteristics allowed for the specific analysis of PAH impact on the microbial community. PAH concentrations in impacted sediments ranged from 22 to 217 μg g (dry weight) of sediment⁻¹, while PAH concentrations in ambient sediments ranged from below detection levels to 1.5 μg g (dry weight) of sediment⁻¹. Total microbial biomass measured by phospholipid phosphate (PLP) analysis ranged from 95 to 345 nmol of PLP g (dry weight) of sediment⁻¹. Nucleic acid analysis showed the presence of PAH-degradative genes at all sites, although observed frequencies were typically higher at contaminated sites. Principal component analysis of PLFA profiles indicated that moderate to high PAH concentrations altered microbial community structure and that seasonal changes were comparable in magnitude to the effects of PAH pollution. These data indicate that this community responded to PAH contamination at both the phenotypic and the genotypic level.     

Characterization of Novel Polycyclic Aromatic Hydrocarbon Dioxygenases from the Bacterial Metagenomic DNA of a Contaminated Soil

Ring-hydroxylating dioxygenases (RHDs) play a crucial role in the biodegradation of a range of aromatic hydrocarbons found on polluted sites, including polycyclic aromatic hydrocarbons (PAHs). Current knowledge on RHDs comes essentially from studies on culturable bacterial strains, while compelling evidence indicates that pollutant removal is mostly achieved by uncultured species. In this study, a combination of DNA-SIP labeling and metagenomic sequence analysis was implemented to investigate the metabolic potential of main PAH degraders on a polluted site. Following in situ labeling using [¹³C]phenanthrene, the labeled metagenomic DNA was isolated from soil and subjected to shotgun sequencing. Most annotated sequences were predicted to belong to Betaproteobacteria, especially Rhodocyclaceae and Burkholderiales, which is consistent with previous findings showing that main PAH degraders on this site were affiliated to these taxa. Based on metagenomic data, four RHD gene sets were amplified and cloned from soil DNA. For each set, PCR yielded multiple amplicons with sequences differing by up to 321 nucleotides (17%), reflecting the great genetic diversity prevailing in soil. RHDs were successfully overexpressed in Escherichia coli, but full activity required the coexpression of two electron carrier genes, also cloned from soil DNA. Remarkably, two RHDs exhibited much higher activity when associated with electron carriers from a sphingomonad. The four RHDs showed markedly different preferences for two- and three-ring PAHs but were poorly active on four-ring PAHs. Three RHDs preferentially hydroxylated phenanthrene on the C-1 and C-2 positions rather than on the C-3 and C-4 positions, suggesting that degradation occurred through an alternate pathway.     

Predicting the Efficacy of Polycyclic Aromatic Hydrocarbon Bioremediation in Creosote-Contaminated Soil Using Bioavailability Assays

Nonexhaustive extraction (propanol, butanol, hydroxypropyl-β-cyclodextrin [HPCD]), persulfate oxidation and biodegradability assays were employed to determine the bioavailability of polycyclic aromatic hydrocarbons (PAHs) in creosote-contaminated soil. After 16 weeks incubation, greater than 89% of three-ring compounds (acenaphthene, anthracene, fluorene, and phenanthrene) and 21% to 79% of four-ring compounds (benz[a]anthracene, chrysene, fluoranthene, and pyrene) were degraded by the indigenous microorganisms under biopile conditions. No significant decrease in five- (benzo[a]pyrene, benzo[b+k]fluoranthene) and six-ring compounds (benz[g,h,i]perylene, indeno[1,2,3-c,d]pyrene) was observed. Desorption of PAHs using propanol or butanol could not predict PAH biodegradability: low-molecular-weight PAH biodegradability was underestimated whereas high-molecular-weight PAH biodegradability was overestimated. Persulfate oxidation and HPCD extraction of creosote-contaminated soil was able to predict three- and four-ring PAH biodegradability; however, the biodegradability of five-ring PAHs was overestimated. These results demonstrate that persulfate oxidation and HPCD extraction are good predictors of PAH biodegradability for compounds with octanol-water partitioning coefficients of < 6.     

Molecular Characterization and Substrate Preference of a Polycyclic Aromatic Hydrocarbon Dioxygenase from Cycloclasticus sp. Strain A5

Cycloclasticus sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated phn genes, although the sequence and order of these phn genes were significantly different from the sequence and order of the known PAH-degrading genes. The phnA1, phnA2, phnA3, and phnA4 genes, which encode the α and β subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The phnA4A3 gene cluster was located 3.7 kb downstream of the phnA2 gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the α and β subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the [2Fe-2S] cluster ligands were conserved. Escherichia coli cells possessing the phnA1A2A3A4 genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the E. coli cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases.     

Detection and Enumeration of Aromatic Oxygenase Genes by Multiplex and Real-Time PCR

Our abilities to detect and enumerate pollutant-biodegrading microorganisms in the environment are rapidly advancing with the development of molecular genetic techniques. Techniques based on multiplex and real-time PCR amplification of aromatic oxygenase genes were developed to detect and quantify aromatic catabolic pathways, respectively. PCR primer sets were identified for the large subunits of aromatic oxygenases from alignments of known gene sequences and tested with genetically well-characterized strains. In all, primer sets which allowed amplification of naphthalene dioxygenase, biphenyl dioxygenase, toluene dioxygenase, xylene monooxygenase, phenol monooxygenase, and ring-hydroxylating toluene monooxygenase genes were identified. For each primer set, the length of the observed amplification product matched the length predicted from published sequences, and specificity was confirmed by hybridization. Primer sets were grouped according to the annealing temperature for multiplex PCR permitting simultaneous detection of various genotypes responsible for aromatic hydrocarbon biodegradation. Real-time PCR using SYBR green I was employed with the individual primer sets to determine the gene copy number. Optimum polymerization temperatures for real-time PCR were determined on the basis of the observed melting temperatures of the desired products. When a polymerization temperature of 4 to 5°C below the melting temperature was used, background fluorescence signals were greatly reduced, allowing detection limits of 2 × 10² copies per reaction mixture. Improved in situ microbial characterization will provide more accurate assessment of pollutant biodegradation, enhance studies of the ecology of contaminated sites, and facilitate assessment of the impact of remediation technologies on indigenous microbial populations.     

Soil Microbial Community Responses to a Decade of Warming as Revealed by Comparative Metagenomics

Soil microbial communities are extremely complex, being composed of thousands of low-abundance species (<0.1% of total). How such complex communities respond to natural or human-induced fluctuations, including major perturbations such as global climate change, remains poorly understood, severely limiting our predictive ability for soil ecosystem functioning and resilience. In this study, we compared 12 whole-community shotgun metagenomic data sets from a grassland soil in the Midwestern United States, half representing soil that had undergone infrared warming by 2°C for 10 years, which simulated the effects of climate change, and the other half representing the adjacent soil that received no warming and thus, served as controls. Our analyses revealed that the heated communities showed significant shifts in composition and predicted metabolism, and these shifts were community wide as opposed to being attributable to a few taxa. Key metabolic pathways related to carbon turnover, such as cellulose degradation (∼13%) and CO₂ production (∼10%), and to nitrogen cycling, including denitrification (∼12%), were enriched under warming, which was consistent with independent physicochemical measurements. These community shifts were interlinked, in part, with higher primary productivity of the aboveground plant communities stimulated by warming, revealing that most of the additional, plant-derived soil carbon was likely respired by microbial activity. Warming also enriched for a higher abundance of sporulation genes and genomes with higher G+C content. Collectively, our results indicate that microbial communities of temperate grassland soils play important roles in mediating feedback responses to climate change and advance the understanding of the molecular mechanisms of community adaptation to environmental perturbations.     

Parental Smoking and Risk of Childhood Brain Tumors by Functional Polymorphisms in Polycyclic Aromatic Hydrocarbon Metabolism Genes

Background

A recent meta-analysis suggested an association between exposure to paternal smoking during pregnancy and childhood brain tumor risk, but no studies have evaluated whether this association differs by polymorphisms in genes that metabolize tobacco-smoke chemicals.

Methods

We assessed 9 functional polymorphisms in 6 genes that affect the metabolism of polycyclic aromatic hydrocarbons (PAH) to evaluate potential interactions with parental smoking during pregnancy in a population-based case-control study of childhood brain tumors. Cases (N = 202) were ≤10 years old, diagnosed from 1984–1991 and identified in three Surveillance, Epidemiology, and End Results (SEER) registries in the western U.S. Controls in the same regions (N = 286) were frequency matched by age, sex, and study center. DNA for genotyping was obtained from archived newborn dried blood spots.

Results

We found positive interaction odds ratios (ORs) for both maternal and paternal smoking during pregnancy, EPHX1 H139R, and childhood brain tumors (P _interaction = 0.02; 0.10), such that children with the high-risk (greater PAH activation) genotype were at a higher risk of brain tumors relative to children with the low-risk genotype when exposed to tobacco smoke during pregnancy. A dose-response pattern for paternal smoking was observed among children with the EPHX1 H139R high-risk genotype only (OR_{no exposure} = 1.0; OR_{≤3 hours/day} = 1.32, 95% CI: 0.52–3.34; OR_>3hours/day = 3.18, 95% CI: 0.92–11.0; P _trend = 0.07).

Conclusion

Parental smoking during pregnancy may be a risk factor for childhood brain tumors among genetically susceptible children who more rapidly activate PAH in tobacco smoke.     

Detection of New cry Genes of Bacillus thuringiensis by Use of a Novel PCR Primer System

On the basis of the known cry gene sequences of Bacillus thuringiensis, three sets of primers were designed from four conserved blocks found in the delta-endotoxin-coding region. The primer pairs designed amplify the regions between blocks 1 and 5, 2 and 5, and 1 and 4. In silico analyses indicated that 100% of the known three-domain cry gene sequences can be amplified by these sets of primers. To test their ability to amplify known and unknown cry gene sequences, 27 strains from the CINVESTAV (LBIT series) collection showing atypical crystal morphology were selected. Their DNA was used as the template with the new primer system, and after a systematic amplification and sequencing of the amplicons, each strain showed one or more cry-related sequences, totaling 54 different sequences harbored by the 27 strains. Seven sequences were selected on the basis of their low level of identity to the known cry sequences, and once cloning and sequencing of the complete open reading frames were done, three new cry-type genes (primary ranks) were identified and the toxins that they encode were designated Cry57Aa1, Cry58Aa1, and Cry59Aa1 by the B. thuringiensis Toxin Nomenclature Committee. The rest of the seven sequences were classified Cry8Ka2, Cry8-like, Cry20Ba1, and Cry1Ma1 by the committee. The crystal morphology of the selected strains and analysis of the new Cry protein sequences showed interesting peculiarities.Bacillus thuringiensis has been safely used for the control of insect pests within the orders Lepidoptera, Coleoptera, and Diptera for the last 50 years (19). It is an aerobic, Gram-positive bacterium whose insecticidal activity is based on the presence of parasporal crystalline inclusions formed during the sporulation process. These parasporal bodies or crystals are assembled by the so-called Cry proteins, expressed by the cry genes (21).B. thuringiensis shows great variability, as has been demonstrated by the huge number of strains isolated around the world (19), by the number of serotypes known to date (a total of 84) (20), and by the great number of different cry gene sequences accumulated so far (a total of 492) (8), as well as by the number of molecular characterization tools that have been developed, such as sequencing of the flagellin gene and of the gyrB and aroE genes, the band patterns from repetitive extragenic palindromic-PCR analyses, and the plasmid patterns, among others (17, 18, 25, 27), all indicating the great variability within this species.In spite of this variability, some similarity has been found in the three-domain Cry proteins, evidenced by the presence of three conserved domains in the tertiary structure of the active toxin (delta-endotoxin), even from Cry toxins showing low levels of identity (i.e., the Cry1-type, Cry3-type, and Cry4-type toxins): one domain with a bundle of α helices and two domains of β sheets (3). This uniformity is, at least in part, a reflection of the five conserved blocks in the gene structure, which are present in almost all the cry genes (10).The great number of sequences known to date is mostly a result of the strong interest in finding novel Cry proteins, with the foci being on three main purposes: (i) the search for a new range of activities, (ii) the search for higher levels of toxicity, and (iii) the search for alternative toxins in case of resistance development. The search for novel Cry toxins has followed different strategies, such as (i) the development of DNA libraries and their expression in acrystalliferous mutants of B. thuringiensis (24); (ii) hybridization with degenerate oligonucleotides, partial sequences, or complete cry genes used as probes (5, 12); and (iii) PCR amplification of putative novel cry genes by using multiple primers (14) or a combination of general primers, followed either by restriction analysis (26) or by a second amplification using a mixture of general and specific primers (13).These strategies have shown certain levels of efficiency in detecting new cry genes, but they also have some limitations. For example, the screening of a large library is time-consuming and requires a high level serendipity, and the use of Southern analysis with either degenerate or specific probes is limited to finding only sequences related to known cry genes. The amplification of putatively new cry genes by PCR has been widely used in a series of different strategies. However, most of them are limited to finding genes with sequences similar to those used in the primer design. Previous attempts to design universal primers used only nine cry types, which significantly limits the search for actual new genes. Besides, the new sequences described were limited to the amplicons and no new cry genes were reported (6).This report describes the design of a system of three sets of primers, based on conserved regions of the cry family, potentially able to amplify all the known three-domain cry genes. The use of this strategy with B. thuringiensis strains showing atypical crystal morphology allowed the identification of three new cry types and four more new cry holotypes.     

Molecular Cloning, Nucleotide Sequence, and Expression of Genes Encoding a Polycyclic Aromatic Ring Dioxygenase from Mycobacterium sp. Strain PYR-1

Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5′ to the 3′ direction, were a dehydrogenase, the dioxygenase small (β)-subunit, and the dioxygenase large (α)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large α subunit did not cluster with most of the known α-subunit sequences but rather with three newly described α subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.     

Abundance and Diversity of Bacterial Nitrifiers and Denitrifiers and Their Functional Genes in Tannery Wastewater Treatment Plants Revealed by High-Throughput Sequencing

Biological nitrification/denitrification is frequently used to remove nitrogen from tannery wastewater containing high concentrations of ammonia. However, information is limited about the bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants (WWTPs) due to the low-throughput of the previously used methods. In this study, 454 pyrosequencing and Illumina high-throughput sequencing, combined with molecular methods, were used to comprehensively characterize structures and functions of nitrification and denitrification bacterial communities in aerobic and anaerobic sludge of two full-scale tannery WWTPs. Pyrosequencing of 16S rRNA genes showed that Proteobacteria and Synergistetes dominated in the aerobic and anaerobic sludge, respectively. Ammonia-oxidizing bacteria (AOB) amoA gene cloning revealed that Nitrosomonas europaea dominated the ammonia-oxidizing community in the WWTPs. Metagenomic analysis showed that the denitrifiers mainly included the genera of Thauera, Paracoccus, Hyphomicrobium, Comamonas and Azoarcus, which may greatly contribute to the nitrogen removal in the two WWTPs. It is interesting that AOB and ammonia-oxidizing archaea had low abundance although both WWTPs demonstrated high ammonium removal efficiency. Good correlation between the qPCR and metagenomic analysis is observed for the quantification of functional genes amoA, nirK, nirS and nosZ, indicating that the metagenomic approach may be a promising method used to comprehensively investigate the abundance of functional genes of nitrifiers and denitrifiers in the environment.     

Effect of Sulfadiazine on Abundance and Diversity of Denitrifying Bacteria by Determining nirK and nirS Genes in Two Arable Soils

Sulfadiazine (SDZ) is an antibiotic frequently used in agricultural husbandry. Via manuring of excrements of medicated animals, the drug reaches the soil and might impair important biochemical transformation processes performed by microbes, e.g., the nitrogen turnover. We studied the effect of pig manure and SDZ-spiked pig manure on denitrifying bacteria by quantifying nirK and nirS nitrite reductase genes in two arable soils. Addition of manure entailed mainly an increase of nirK-harboring denitrifiers in both soils, whereas in the SDZ-amended treatments, primarily the nirS denitrifiers increased in abundance after the bioavailable SDZ had declined. However, the community composition of nirS nitrite reducers investigated by denaturing gradient gel electrophoresis did not change despite the observed alterations in abundance.     

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.     

Improved Enrichment and Isolation of Polycyclic Aromatic Hydrocarbons (PAH)-Degrading Microorganisms in Soil Using Anthracene as a Model PAH

Lack of attention to soil and microbial characteristics that influence PAHs degradation has been a leading cause of failures in isolation of efficient PAH degraders and bioaugumentation processes with microbial consortia. This study compared the classic method of isolation of PAHs-degraders with a modified method employing a pre-enrichment respirometric analysis. The modified enrichment of PAH degrading microorganisms using in vitro microcosm resulted to reduced enrichment period and more efficient PAH-degrading microbial consortia. Results indicate that natural soils with strong heterotrophic microbial activity determined through pre-enrichment analysis, are better suited for the isolation of efficient PAH degrading microorganisms with significant reduction of the enrichment period.     

Non-Protein Coding RNA Genes as the Novel Diagnostic Markers for the Discrimination of Salmonella Species Using PCR

Salmonellosis, a communicable disease caused by members of the Salmonella species, transmitted to humans through contaminated food or water. It is of paramount importance, to generate accurate detection methods for discriminating the various Salmonella species that cause severe infection in humans, including S. Typhi and S. Paratyphi A. Here, we formulated a strategy of detection and differentiation of salmonellosis by a multiplex polymerase chain reaction assay using S. Typhi non-protein coding RNA (sRNA) genes. With the designed sequences that specifically detect sRNA genes from S. Typhi and S. Paratyphi A, a detection limit of up to 10 pg was achieved. Moreover, in a stool-seeding experiment with S. Typhi and S. Paratyphi A, we have attained a respective detection limit of 15 and 1.5 CFU/mL. The designed strategy using sRNA genes shown here is comparatively sensitive and specific, suitable for clinical diagnosis and disease surveillance, and sRNAs represent an excellent molecular target for infectious disease.     

Genomic Diversity within the Genus Pediococcus as Revealed by Randomly Amplified Polymorphic DNA PCR and Pulsed-Field Gel Electrophoresis

The genomic diversity of 33 previously assigned strains from six species within the genus Pediococcus was assessed by randomly amplified polymorphic DNA (RAPD) PCR and pulsed-field-gel electrophoresis (PFGE). The RAPD PCR patterns produced by two separate random primers, termed P1 (ACGCGCCCT) and P2 (ATGTAACGCC), were compared by the Pearson correlation coefficient and the unweighted pair group method with arithmetic averages clustering algorithm. Pattern variations between repeat samples set a strain discrimination threshold of less than 70% similarity. P1 and P2 primers alone and in combination produced 14, 21, and 28 distinct patterns, respectively. When each strain was assigned with a type strain with which it shared the highest level of similarity, both primers grouped 17 of the 27 strains to their proposed species. PFGE following genomic digestion with the restriction enzymes ApaI, NotI, and AscI produced 30, 32, and 28 distinct macrorestriction patterns, respectively. Specific DNA fragments within the NotI and AscI macrorestriction patterns for each strain were observed that allowed 27 of the 33 strains to be assigned to their proposed species. For example, following digestion with AscI, all Pediococcus parvulus strains were characterized by two DNA fragments, one of approximately 220 kb and another between 700 and 800 kb. The exceptions correlated with those observed with both RAPD PCR primers and included three P. damnosus and two P. pentosaceus strains that grew at temperatures regarded as nonpermissive for their proposed species but not for those with which they grouped.     

Detection of Microbial Growth on Polycyclic Aromatic Hydrocarbons in Microtiter Plates by Using the Respiration Indicator WST-1

We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate}, in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.