Engineering a highly durable adeno-associated virus receptor for analytical applications.

Adeno-associated virus (AAV) is a major viral vector used in gene therapy. There are multiple AAV serotypes, and many engineered AAV serotypes are developed to alter their tissue tropisms with capsid modification. The universal AAV receptor (AAVR) is an essential receptor for multiple AAV serotypes. Since most AAV serotypes used in gene therapy infect cells via interaction with AAVR, the quantification of the vector-binding ability of AAV to AAVR could be an important quality check for therapeutic AAV vectors. To enable a steady evaluation of the AAV-AAVR interaction, we created an engineered AAVR through mutagenesis. Engineered AAVR showed high durability against acid while retaining its AAV-binding activity. An affinity chromatography column with the engineered AAVR was also developed. This column enabled repeated binding and acid dissociation measurements of AAVR with various AAV serotypes. Our data showed that the binding affinities of AAV2 to AAVR were diverse among serotypes, providing insight into the relationship with the infection efficiency of AAV vectors. Thus, this affinity column can be used in process development for quality checks, quantitating capsid titers, and affinity purification of AAV vectors. Furthermore, this column may serve as a useful tool in novel AAV vector capsid engineering.

Improved electrochemical properties of graphite electrodes incubated with iron powders in rice-paddy fields boost power outputs from microbial fuel cells.

Studies have shown that the supplementation of anode-surrounding soil with zero-valent iron (ZVI) boosts power outputs from rice paddy-field microbial fuel cells (RP-MFCs). In order to understand mechanisms by which ZVI boosts outputs from RP-MFCs, the present study operated RP-MFCs with and without ZVI, and compositions of anode-associated bacteria and electrochemical properties of graphite anodes were analyzed after 3-month operation. Metabarcoding using 16S rRNA gene fragments showed that bacterial compositions did not largely differ among these RP-MFCs. Cyclic voltammetry showed improved electrochemical properties of anodes recovered from ZVI-supplemented RP-MFCs, and this was attributed to the adhesion of iron-oxide films onto graphite surfaces. Bioelectrochemical devices equipped with graphite anodes recovered from ZVI-supplemented RP-MFCs generated higher currents than those with fresh graphite anodes. These results suggest that ZVI is oxidized to iron oxides in paddy-field soil and adheres onto graphite anodes, resulting in the boost of power outputs from RP-MFCs.

Periodontal therapy for localized severe periodontitis in a patient receiving fixed orthodontic treatment: a case report.

BACKGROUND: Orthodontic treatment involves movement of teeth by compression and resorption of the alveolar bone using orthodontic forces. These movements are closely linked to the interactions between the teeth and the periodontal tissues that support them. Owing to an increase in adults seeking orthodontic treatment, orthodontists increasingly encounter patients with periodontal diseases, in whom orthodontic treatment is contraindicated. In rare cases, periodontitis may develop after treatment initiation. However, no approach for treating periodontitis after the initiation of orthodontic treatment has been established. Here, we present an approach for managing localized severe periodontitis manifesting after initiating orthodontic treatment. CASE PRESENTATION: A 32-year-old Japanese woman was referred to the Department of Dentistry and Oral Surgery by an orthodontist who observed symptoms of acute periodontitis in the maxillary molars that required periodontal examination and treatment. A detailed periodontal examination, including oral bacteriological examination, revealed localized severe periodontitis (stage III, grade B) in the maxillary left first and second molars and in the mandibular right second molar. After consultation with the orthodontist, the orthodontic treatment was suspended based on the results of the bacteriological examination to allow for periodontal treatment. Full-mouth disinfection was performed with adjunctive oral sitafloxacin. Periodontal and bacteriological examinations after treatment revealed regression of the localized periodontitis with bone regeneration. Thereafter, orthodontic treatment was resumed, and good progress was achieved. CONCLUSIONS: Orthodontists should recognize the risk of acute severe periodontitis in young adults. Asymptomatic patients with localized severe periodontitis may clear a screening test before orthodontic treatment but develop acute symptoms with bone resorption during orthodontic treatment. Therefore, patients requiring orthodontic treatment should be examined by their family dentist or a periodontist to rule out periodontal issues that may impede orthodontic treatment. The patients should also be informed of age-related risks. Further, periodontists, family dentists, and orthodontists who treat adults should be informed about periodontitis and the need for interdisciplinary collaboration. In patients who develop periodontitis after orthodontic treatment initiation, temporary interruption of orthodontic treatment and aggressive periodontal intervention may facilitate recovery.

Proposed protocol for treatment of severe periodontitis without platelet transfusion in patients with aplastic anemia: a case report.

BACKGROUND: Aplastic anemia is an intractable disease characterized by pancytopenia, susceptibility to infection, and difficulty in achieving hemostasis. In patients with severe periodontal disease and aplastic anemia, spontaneous bleeding from the gingival tissue due to thrombocytopenia and during brushing is common, which may further exacerbate dental issues. Comprehensive periodontal treatment for patients with aplastic anemia is highly challenging and requires collaboration with a hematologist. Here, we discuss the case of a patient with aplastic anemia and severe periodontitis who was successfully treated in collaboration with our hematology department. CASE PRESENTATION: A 36-year-old Japanese woman with chief complaints of spontaneous gingival bleeding, pain, and increasing tooth mobility consulted our department. She had developed pancytopenia at age 11 years and was later diagnosed with aplastic anemia, making her susceptible to infection due to leukopenia. The results of the initial periodontal examination led to a diagnosis of severe generalized periodontitis (generalized stage IV grade C periodontitis) caused by leukopenia and poor oral hygiene. We adopted a comprehensive treatment plan, including invasive dental procedures. The patient exhibited no postoperative bleeding due to aplastic anemia-induced thrombocytopenia and experienced a good outcome. CONCLUSIONS: Both physicians and dentists should be aware that immunocompromised patients with aplastic anemia are at risk of developing severe periodontitis with severe alveolar bone resorption if the condition is combined with poor oral hygiene. Even in the presence of aplastic anemia, patients with severe periodontitis can undergo comprehensive dental treatment, including dental extraction and periodontal surgery, if bleeding and susceptibility to infection are controlled. This requires the cooperation of the patient and hematologists and can ultimately contribute to improving the patient's quality of life.

Enhanced electricity generation in rice paddy-field microbial fuel cells supplemented with iron powders.

Microbial fuel cells installed in rice paddy fields (RP-MFCs) are able to serve as on-site batteries for operating low-power environmental sensors. In order to increase the utility and reliability of RP-MFCs, however, further research is necessary for boosting the power output. Here we examined several powdered iron species, including zero valent iron (ZVI), goethite, and magnetite, for their application to increasing power outputs from RP-MFCs. Soil around anodes was supplemented with either of these iron species, and RP-MFCs were operated for several months during the transplanting and harvesting. It was found that power outputs from RP-MFCs supplemented with ZVI were more than double the outputs from control (not supplemented with iron species) and other RP-MFCs, even after iron corrosion was ceased, and the maximum power density reached 130 mW/m2 (per projected area of the anode). Metabarcoding of 16S rRNA gene amplicons suggested that several taxa represented by fermentative and exoelectrogenic bacteria were substantially increased in MFCs supplemented with ZVI. Results suggest that ZVI lowers oxidation/reduction potential around anodes, activates anaerobic microbes involved in the conversion of organic matter into electricity and increases power output from RP-MFCs.

Overexpression of the adenylate cyclase gene cyaC facilitates current generation by Shewanella oneidensis in bioelectrochemical systems.

Electrochemically active bacteria (EAB) are capable of electrochemical interactions with electrodes via extracellular electron transfer (EET) pathways and serve as essential components in bioelectrochemical systems. Previous studies have suggested that EAB, such as Shewanella oneidensis MR-1, use cyclic AMP (cAMP) receptor proteins to coordinately regulate the expression of catabolic and EET-related genes, prompting us to hypothesize that the intracellular cAMP concentration is an important factor determining the electrochemical activities of EAB. The present study constructed an MR-1 mutant, cyaC-OE, that overexpressed cyaC, a gene encoding a membrane-bound class III adenylate cyclase, and examined its electrochemical and transcriptomic characteristics. We show that the intracellular cAMP concentration in cyaC-OE is more than five times that in wild-type MR-1, and that cya-OE generates approximately two-fold higher current in BES than the wild-type strain. In addition, the expression of genes involved in EET and anaerobic carbon catabolism is up-regulated in cya-OE compared to that in the wild-type strain. These results suggest that increasing the intracellular cAMP level is a promising approach for constructing EAB with high catabolic and electrochemical activities.

Does Quick Sepsis-Related Organ Failure Assessment Suggest the Use of Initial Empirical Carbapenem Therapy in Bacteremia Caused by Extended-Spectrum ß-Lactamase-Producing Bacteria? :A Multicenter Case-Control Study.

We hypothesized that quick Sequential Organ Failure Assessment (qSOFA) would be associated with 30-day mortality in bacteremia caused by extended-spectrum ß-lactamase (ESBL)-producing bacteria and might be a selection criterion for the use of carbapenem as initial empirical therapy. A multicenter retrospective study was conducted in six hospitals. All patients who had bacteremia due to ESBL-producing bacteria were included in the study. Multivariable logistic regression analysis was performed to analyze 30-day mortality as the main outcome. A total of 203 adult patients were identified with bacteremia caused by ESBL-producing Escherichia coli, Klebsiella pneumoniae, or Proteus mirabilis. In multivariate logistic regression analysis, bacteremia caused by ESBL-producing K. pneumoniae or P. mirabilis (odds ratio [OR] 5.07, 95% confidence interval [CI] 1.64-15.56), underlying liver disease (OR 3.38, 95% CI 1.09-10.00), and underlying solid cancer (OR 3.45, 95% CI 1.27-9.69) were associated with 30-day mortality. In a subgroup analysis, empirical non-carbapenem therapy was associated with 30-day mortality in bacteremia caused by ESBL-producing K. pneumoniae or P. mirabilis. Our results suggest that the qSOFA score is not a selection criterion for the use of carbapenem in initial empirical therapy.

CpdA is involved in amino acid metabolism in Shewanella oneidensis MR-1.

Cyclic 3',5'-adenosine monophosphate (cAMP) phosphodiesterase (CPD) is an enzyme that catalyzes the hydrolysis of cAMP, a signaling molecule affecting diverse cellular and metabolic processes in bacteria. Some CPDs are also known to function in cAMP-independent manners, while their physiological roles remain largely unknown. Here, we investigated physiological roles of CPD in Shewanella oneidensis MR-1, a model environmental bacterium, and report that CPD is involved in amino-acid metabolism. We found that a CPD-deficient mutant of MR-1 (ΔcpdA) showed decreased expression of genes for the synthesis of methionine, S-adenosylmethionine, and histidine and required these three compounds to grow in minimal media. Interestingly, deletion of adenylate cyclases in ΔcpdA did not restore the ability to grow in minimal media, indicating that the amino acid requirements were not due to the accumulation of cAMP. These results suggest that CPD is involved in the regulation of amino acid metabolism in MR-1 in a cAMP-independent manner.

Poly iron sulfate flocculant as an effective additive for improving the performance of microbial fuel cells.

Laboratory microbial fuel cells were supplied with artificial wastewater and used to examine how supplementation with poly iron sulfate, an inorganic polymer flocculant widely used in wastewater-treatment plants, affects electricity generation and anode microbiomes. It is shown that poly iron sulfate substantially increases electric outputs from microbial fuel cells. Microbiological analyses show that iron and sulfate separately affect anode microbiomes, and the increase in power output is associated with the increases in bacteria affiliated with the families Geobacteraceae and/or Desulfuromonadaceae. We suggest that poly iron sulfate is an effective additive for increasing the electric output from microbial fuel cells. Other utilities of poly iron sulfate in microbial fuel cells are also discussed.

Missing Iron-Oxidizing Acidophiles Highly Sensitive to Organic Compounds.

The genus Acidithiobacillus includes iron-oxidizing lithoautotrophs that thrive in acidic mine environments. Acidithiobacillus ferrooxidans is a representative species and has been extensively studied for its application to the bioleaching of precious metals. In our attempts to cultivate the type strain of A. ferrooxidans (ATCC 23270(T)), repeated transfers to fresh inorganic media resulted in the emergence of cultures with improved growth traits. Strains were isolated from the resultant culture by forming colonies on inorganic silica-gel plates. A representative isolate (strain NU-1) was unable to form colonies on agarose plates and was more sensitive to organics, such as glucose, than the type strain of A. ferrooxidans. Strain NU-1 exhibited superior growth traits in inorganic iron media to those of other iron-oxidizing acidithiobacilli, suggesting its potential for industrial applications. A draft genome of NU-1 uncovered unique features in catabolic enzymes, indicating that this strain is not a mutant of the A. ferrooxidans type strain. Our results indicate that the use of inorganic silica-gel plates facilitates the isolation of as-yet-unexamined iron-oxidizing acidithiobacilli from environmental samples and enrichment cultures.

Evaluation of microbial fuel cells for electricity generation from oil-contaminated wastewater.

Large quantities of oils and fats are discharged into wastewater from food industries. We evaluated the possibility of using microbial fuel cells (MFCs) for the generation of electricity from food-industry wastewater containing vegetable oils. Single-chamber MFCs were supplied with artificial wastewater containing soybean oil, and oil removal and electric output were examined under several different conditions. We found that MFC performance could be improved by supplementing wastewater with an emulsifier, inoculating MFCs with oil-contaminated soil, and coating the graphite-felt anodes with carbon nanotubes, resulting in a power output of more than 2 W m-2 (based on the projected area of the anode). Sequencing of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments detected abundant amount of Burkholderiales bacteria (known to include oil degraders) in the oil-contaminated soil and anode biofilm, whereas those affiliated with the genus Geobacter were only detected in the anode biofilm. These results suggest that MFCs can be used for energy recovery from food industry wastewater containing vegetable oils.

Iron-oxide minerals affect extracellular electron-transfer paths of Geobacter spp.

Some bacteria utilize (semi)conductive iron-oxide minerals as conduits for extracellular electron transfer (EET) to distant, insoluble electron acceptors. A previous study demonstrated that microbe/mineral conductive networks are constructed in soil ecosystems, in which Geobacter spp. share dominant populations. In order to examine how (semi)conductive iron-oxide minerals affect EET paths of Geobacter spp., the present study grew five representative Geobacter strains on electrodes as the sole electron acceptors in the absence or presence of (semi)conductive iron oxides. It was found that iron-oxide minerals enhanced current generation by three Geobacter strains, while no effect was observed in another strain. Geobacter sulfurreducens was the only strain that generated substantial amounts of currents both in the presence and absence of the iron oxides. Microscopic, electrochemical and transcriptomic analyses of G. sulfurreducens disclosed that this strain constructed two distinct types of EET path; in the absence of iron-oxide minerals, bacterial biofilms rich in extracellular polymeric substances were constructed, while composite networks made of mineral particles and microbial cells (without polymeric substances) were developed in the presence of iron oxides. It was also found that uncharacterized c-type cytochromes were up-regulated in the presence of iron oxides that were different from those found in conductive biofilms. These results suggest the possibility that natural (semi)conductive minerals confer energetic and ecological advantages on Geobacter, facilitating their growth and survival in the natural environment.

Anaerocella delicata gen. nov., sp. nov., a strictly anaerobic bacterium in the phylum Bacteroidetes isolated from a methanogenic reactor of cattle farms.

A strictly anaerobic bacterial strain (WN081(T)) was isolated from rice-straw residue in a methanogenic reactor treating waste from cattle farms in Japan. Cells were Gram-staining negative, non-motile, non-spore-forming straight rods. The strain grew rather well on PY agar slants supplemented with a B-vitamin mixture as well as sugars (PYV4S medium) and made translucent and glossy colonies. Growth in liquid medium with the same composition, however, was scanty, and growth was not improved in spite of various additives to the medium. Strain WN081(T) produced small amounts of acetate, propionate, isobutyrate, butyrate, isovalerate and H(2) from PYV liquid medium. The strain did not use carbohydrates or organic acids. The pH range for growth was narrow (pH 6.8-8.2), having a pH optimum at 6.8-7.5. The temperature range for growth was 10-37°C, the optimum being 25-30°C. The strain was sensitive to bile, and did not have catalase or oxidase activities. Hydrogen sulfide was produced from L-cysteine and L-methionine as well as peptone. Indole was produced from L-tryptophan and peptone. The strain had iso-C(15:0) as the exclusively predominant cellular fatty acid (70%) together with some branched chain components (such as iso-C(15:0) DMA, iso-C(17:0) 3-OH and iso-C(15:0) aldehyde) as minor components. The genomic DNA G+C content was 32.3 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence placed strain WN081(T) in the phylum Bacteroidetes with rather low sequence similarities with the related species such as Rikenella microfusus (85.7% sequence similarity), Alistipes putredinis (85.5%) and Alistipes finegoldii (85.5%) in the family Rikenellaceae. Based on the phylogenetic, physiological and chemotaxonomic analyses, the novel genus and species Anaerocella delicata gen. nov., sp. nov. is proposed to accommodate the strain. The type strain is WN081(T) (= JCM 17049(T) = DSM 23595(T)).

Methanogenesis facilitated by electric syntrophy via (semi)conductive iron-oxide minerals.

Methanogenesis is an essential part of the global carbon cycle and a key bioprocess for sustainable energy. Methanogenesis from organic matter is accomplished by syntrophic interactions among different species of microbes, in which interspecies electron transfer (IET) via diffusive carriers (e.g. hydrogen and formate) is known to be the bottleneck step. We report herein that the supplementation of soil microbes with (semi)conductive iron-oxide minerals creates unique interspecies interactions and facilitates methanogenesis. Methanogenic microbes were enriched from rice paddy field soil with either acetate or ethanol as a substrate in the absence or presence of (semi)conductive iron oxides (haematite or magnetite). We found that the supplementation with either of these iron oxides resulted in the acceleration of methanogenesis in terms of lag time and production rate, while the supplementation with an insulative iron oxide (ferrihydrite) did not. Clone-library analyses of 16S rRNA gene fragments PCR-amplified from the enrichment cultures revealed that the iron-oxide supplementation stimulated the growth of Geobacter spp. Furthermore, the addition of a specific inhibitor for methanogenesis suppressed the growth of Geobacter spp. These results suggest that Geobacter grew under syntrophic association with methanogens, and IET could occur via electric currents through (semi)conductive iron-oxide minerals (termed 'electric syntrophy'). Given the ubiquity of conductive minerals in nature, such energetic interactions may occur widely in soil and sediments and can be used to develop efficient bioenergy processes.

Respiratory interactions of soil bacteria with (semi)conductive iron-oxide minerals.

Pure-culture studies have shown that dissimilatory metal-reducing bacteria are able to utilize iron-oxide nanoparticles as electron conduits for reducing distant terminal acceptors; however, the ecological relevance of such energy metabolism is poorly understood. Here, soil microbial communities were grown in electrochemical cells with acetate as the electron donor and electrodes (poised at 0.2 V versus Ag/AgCl) as the electron acceptors in the presence and absence of iron-oxide nanoparticles, and respiratory current generation and community structures were analysed. Irrespective of the iron-oxide species (hematite, magnetite or ferrihydrite), the supplementation with iron-oxide minerals resulted in large increases (over 30-fold) in current, while only a moderate increase (∼10-fold) was observed in the presence of soluble ferric/ferrous irons. During the current generation, insulative ferrihydrite was transformed into semiconductive goethite. Clone-library analyses of 16S rRNA gene fragments PCR-amplified from the soil microbial communities revealed that iron-oxide supplementation facilitated the occurrence of Geobacter species affiliated with subsurface clades 1 and 2. We suggest that subsurface-clade Geobacter species preferentially thrive in soil by utilizing (semi)conductive iron oxides for their respiration.

Analysis of gene transcripts in a crude oil-degrading marine microbial community.

We established a procedure for analyzing gene transcripts in a microbial community of unknown genomic background. Analysis of a crude oil-degrading marine microbial community detected the expression of genes related to the biodegradation of fatty acids and the biosynthesis of glycolipids probably involved in the emulsification of crude oil.

Identification of the electron transfer flavoprotein as an upregulated enzyme in the benzoate utilization of Desulfotignum balticum.

Desulfotignum balticum utilizes benzoate coupled to sulfate reduction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis was conducted to detect proteins that increased more after growth on benzoate than on butyrate. A comparison of proteins on 2D gels showed that at least six proteins were expressed. The N-terminal sequences of three proteins exhibited significant identities with the alpha and beta subunits of electron transfer flavoprotein (ETF) from anaerobic aromatic-degraders. By sequence analysis of the fosmid clone insert (37,590 bp) containing the genes encoding the ETF subunits, we identified three genes, whose deduced amino acid sequences showed 58%, 74%, and 62% identity with those of Gmet_2267 (Fe-S oxidoreductase), Gmet_2266 (ETF beta subunit), and Gmet_2265 (ETF alpha subunit) respectively, which exist within the 300-kb genomic island of aromatic-degradation genes from Geobacter metallireducens GS-15. The genes encoding ETF subunits found in this study were upregulated in benzoate utilization.

Degradative capacities and bioaugmentation potential of an anaerobic benzene-degrading bacterium strain DN11.

Azoarcus sp. strain DN11 is a denitrifying bacterium capable of benzene degradation under anaerobic conditions. The present study evaluated strain DN11 for its application to bioaugmentation of benzene-contaminated underground aquifers. Strain DN11 could grow on benzene, toluene, m-xylene, and benzoate as the sole carbon and energy sources under nitrate-reducing conditions, although o- and p-xylenes were transformed in the presence of toluene. Phenol was not utilized under anaerobic conditions. Kinetic analysis of anaerobic benzene degradation estimated its apparent affinity and inhibition constants to be 0.82 and 11 microM, respectively. Benzene-contaminated groundwater taken from a former coal-distillation plant site was anaerobically incubated in laboratory bottles and supplemented with either inorganic nutrients (nitrogen, phosphorus, and nitrate) alone, or the nutrients plus strain DN11, showing that benzene was significantly degraded only when DN11 was introduced. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments, and quantitative PCR revealed that DN11 decreased after benzene was degraded. Following the decrease in DN11 16S rRNA gene fragments corresponding to bacteria related to Owenweeksia hongkongensis and Pelotomaculum isophthalicum, appeared as strong bands, suggesting possible metabolic interactions in anaerobic benzene degradation. Results suggest that DN11 is potentially useful for degrading benzene that contaminates underground aquifers at relatively low concentrations.

Sulfurospirillum cavolei sp. nov., a facultatively anaerobic sulfur-reducing bacterium isolated from an underground crude oil storage cavity.

A novel facultatively anaerobic sulfur-reducing bacterium, designated strain Phe91(T), was isolated from petroleum-contaminated groundwater in an underground crude oil storage cavity at Kuji in Iwate, Japan. Cells of strain Phe91(T) were slightly curved rods with single polar flagella. Optimum growth was observed at pH 7.0 and 30 degrees C. The novel strain utilized elemental sulfur, thiosulfate, sulfite, dithionite, arsenate, nitrate and DMSO as electron acceptors with lactate as an energy and carbon source, but nitrite was not utilized. Microaerophilic growth was also observed. Fumarate, pyruvate, lactate, malate, succinate, hydrogen (with acetate as a carbon source) and formate (with acetate) could serve as electron donors. Fumarate, pyruvate and malate were fermented. The DNA G+C content was 42.7 mol%. On the basis of 16S rRNA gene sequence phylogeny, strain Phe91(T) was affiliated with the genus Sulfurospirillum in the class Epsilonproteobacteria and was most closely related to Sulfurospirillum deleyianum (sequence similarity 97 %). However, the DNA-DNA hybridization value between strain Phe91(T) and S. deleyianum was only 14 %. Based on the physiological and phylogenetic data, Phe91(T) should be classified as a representative of a novel species in the genus Sulfurospirillum; the name Sulfurospirillum cavolei sp. nov. is proposed, with Phe91(T) (=JCM 13918(T)=DSM 18149(T)) as the type strain.

Rapid intrinsic biodegradation of benzene, toluene, and xylenes at the boundary of a gasoline-contaminated plume under natural attenuation.

A groundwater plume contaminated with gasoline constituents [mainly benzene, toluene, and xylenes (BTX)] had been treated by pumping and aeration for approximately 10 years, and the treatment strategy was recently changed to monitored natural attenuation (MNA). To gain information on the feasibility of using MNA to control the spread of BTX, chemical and microbiological parameters in groundwater samples obtained inside and outside the contaminated plume were measured over the course of 73 weeks. The depletion of electron acceptors (i.e., dissolved oxygen, nitrate, and sulfate) and increase of soluble iron were observed in the contaminated zone. Laboratory incubation tests revealed that groundwater obtained immediately outside the contaminated zone (the boundary zone) exhibited much higher potential for BTX degradation than those in the contaminated zone and in uncontaminated background zones. The boundary zone was a former contaminated area where BTX were no longer detected. Denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified bacterial 16S rRNA gene fragments revealed that DGGE profiles for groundwater samples obtained from the contaminated zone were clustered together and distinct from those from uncontaminated zones. In addition, unique bacterial rRNA types were observed in the boundary zone. These results indicate that the boundary zone in the contaminant plumes served as a natural barrier for preventing the BTX contamination from spreading out.