Arsenic uptake by Pteris vittata in a subarctic arsenic-contaminated agricultural field in Japan: An 8-year study.

In this study, the phytoremediation potential of tropical and subtropical arsenic (As) hyperaccumulating fern Pteris vittata in an As contaminated farmland field near an abandoned goldmine was investigated. The tested field is located in a subarctic area of northeast Japan. This study was aimed at decreasing the risk of As in the soil (water-soluble As) with nurturing the soil and respecting the plant life cycle for the sustainable phytoremediation for 8 years. The field was tilled and planted with new seedlings of the fern every spring and the grown fern was harvested every autumn. The biomass and As concentration in fronds, rhizomes and roots of the fern were analyzed separately after harvesting each year. The biomass of the fronds of P. vittata was significantly affected by the yearly change of the weather condition, but As concentration in fronds was kept at 100-150 mg/kg dry weight. The accumulated As in P. vittata was higher than that of As-hyperaccumulator fern Pteris cretica, the native fern in the field trial area. Harvested biomass of P. vittata per plant was also higher than that of P. cretica. More than 43.5 g As/154 m2 (convertible to 2.82 kg of As per hectare) was removed from the farmland field by P. vittata phytoremediation at the end of the 8-year experiment. Because of the short-term plant growth period and soil tilling process, total As in soil did not show significant depletion. However, the water-soluble As in the surface and deeper soil, which is phytoavailable and easily taken in cultivated plants, decreased to 10 µg/L (Japan Environmental Quality Standard for water-soluble As in soil) by the 8-year phytoremediation using P. vittata. These research data elucidate that the tropical and subtropical As hyperaccumulating fern, P. vittata, is applicable for As phytoremediation in the subarctic climate area.

New evidence of arsenic translocation and accumulation in Pteris vittata from real-time imaging using positron-emitting 74As tracer.

Pteris vittata is an arsenic (As) hyperaccumulator plant that accumulates a large amount of As into fronds and rhizomes (around 16,000 mg/kg in both after 16 weeks hydroponic cultivation with 30 mg/L arsenate). However, the sequence of long-distance transport of As in this hyperaccumulator plant is unclear. In this study, we used a positron-emitting tracer imaging system (PETIS) for the first time to obtain noninvasive serial images of As behavior in living plants with positron-emitting 74As-labeled tracer. We found that As kept accumulating in rhizomes as in fronds of P. vittata, whereas As was retained in roots of a non-accumulator plant Arabidopsis thaliana. Autoradiograph results of As distribution in P. vittata showed that with low As exposure, As was predominantly accumulated in young fronds and the midrib and rachis of mature fronds. Under high As exposure, As accumulation shifted from young fronds to mature fronds, especially in the margin of pinna, which resulted in necrotic symptoms, turning the marginal color to gray and then brown. Our results indicated that the function of rhizomes in P. vittata was As accumulation and the regulation of As translocation to the mature fronds to protect the young fronds under high As exposure.

Identification of A Novel Arsenic Resistance Transposon Nested in A Mercury Resistance Transposon of Bacillus sp. MB24.

A novel TnMERI1-like transposon designated as TnMARS1 was identified from mercury resistant Bacilli isolated from Minamata Bay sediment. Two adjacent ars operon-like gene clusters, ars1 and ars2, flanked by a pair of 78-bp inverted repeat sequences, which resulted in a 13.8-kbp transposon-like fragment, were found to be sandwiched between two transposable genes of the TnMERI1-like transposon of a mercury resistant bacterium, Bacillus sp. MB24. The presence of a single transcription start site in each cluster determined by 5'-RACE suggested that both are operons. Quantitative real time RT-PCR showed that the transcription of the arsR genes contained in each operon was induced by arsenite, while arsR2 responded to arsenite more sensitively and strikingly than arsR1 did. Further, arsenic resistance complementary experiments showed that the ars2 operon conferred arsenate and arsenite resistance to an arsB-knocked out Bacillus host, while the ars1 operon only raised arsenite resistance slightly. This transposon nested in TnMARS1 was designated as TnARS1. Multi-gene cluster blast against bacteria and Bacilli whole genome sequence databases suggested that TnMARS1 is the first case of a TnMERI1-like transposon combined with an arsenic resistance transposon. The findings of this study suggested that TnMERI1-like transposons could recruit other mobile elements into its genetic structure, and subsequently cause horizontal dissemination of both mercury and arsenic resistances among Bacilli in Minamata Bay.

Comparative geochemical evaluation of toxic metals pollution and bacterial communities of industrial effluent tributary and a receiving estuary in Nigeria.

Toxic metals/metalloid contaminations of estuarine sediments due to compromised tributaries arouse significant interest in studying bacterial community that triggers natural attenuation processes. Geo-accumulation index (Igeo), contamination factor (CF), pollution load index (PLI), and Hakanson potential ecological risk index (RI) as a sum of risk factors (Er) were used to quantify toxic metal/metalloid-pollution status of Lagos Lagoon (2W) and 'Iya-Alaro' tributary (4W) sediments in comparison with pristine 'Lekki Conservation Centre' sediment (L1-B). Bacteriology of the ecosystems was based on culture-independent analyses using pyrosequencing. 2W and 4W were extremely contaminated with mercury (Igeo > 7), whereas, cadmium contamination was only observed in 4W. The two ecosystems were polluted with toxic metal based on PLI, where mercury (Er = 2900 and 1900 for 4W and 2W, respectively) posed very high ecological risks. Molecular fingerprinting revealed that Proteobacteria, Firmicutes, and Acidobacteria predominately contributed the 20 most abundant genera in the two ecosystems. The 240 and 310 species present in 2W and 4W, respectively, but absent in L1-B, thrive under the metal concentrations in the polluted hydrosphere. Whereas, the 58,000 species missing in 2W and 4W but found in L1-B would serve as indicators for systems impacted with metal eco-toxicity. Despite toxic metal pollution of the ecosystems understudied, bacterial communities play vital roles in self-recovery processes occurring in the hydrosphere.

Bio-oxidation of elemental mercury during growth of mercury resistant yeasts in simulated hydrosphere.

Transformation of metallic mercury (Hg°) to mercuric ion (Hg2+) in hydrosphere is the entrance of mercury cycle in water environments and leads to toxicological impact of serious global concern. Two yeast strains of Yarrowia (Idd1 and Idd2) isolated from Hg-contaminated sediments were studied for their mediating role in Hg° dissolution and oxidation. Growth of the Yarrowia cells in Hg-free liquid medium, incubated for 5 d in closed air-tight systems containing Hg°, produced extracellular polymeric substances (EPS). Approximately 230 (±5.7) ng and 120 (±6.8) ng of the dissolved Hg° were oxidized to Hg2+ by the cultures of Idd1 and Idd2, respectively, 5 day post-inoculation. Transmission electron microscopy (TEM) and X-ray energy dispersive spectrophotometry (XEDS) analysis of the EPS and cell mass revealed the presence of extracellular Hg nanoparticles, presumably HgS, as an indication of EPS-Hg complexation that is useful for Hg° dissolution and its eventual oxidation to Hg2+ by the cells. Fourier transmission infra-red (FTIR) analyses of the EPS and cell-mass during Hg-oxidation revealed that amine and carbonyl groups were used by EPS for Hg complexation. Our findings provided information about mediatory role played by Yarrowia (Idd1 and Idd2) in hydrosphere in biogeochemical cycling of Hg.

Mercury bioremediation by mercury resistance transposon-mediated in situ molecular breeding.

Mercury-resistant (HgR) bacteria occur in various bacterial species from a wide variety of environmental sources. Resistance is conferred by a set of operon genes termed the mer operon. Many HgR bacteria have been isolated from diverse environments and clinical samples, and it is recognized that mer operons are often localized on transposons. Previous research reports have suggested that HgR transposons participate in the horizontal gene transfer of mer operons among bacteria. This was confirmed by a study that found that mer operons were distributed worldwide in Bacilli with dissemination of TnMERI1-like transposons. In this mini review, possible strategies for transposon-mediated in situ molecular breeding (ISMoB) of HgR bacteria in their natural habitat are discussed. In ISMoB, the target microorganisms for breeding are indigenous bacteria that are not HgR but that are dominant and robust in their respective environments. Additionally, we propose a new concept of bioremediation technology for environmental mercury pollution by applying transposon-mediated ISMoB for environmental mercury pollution control.

Mercury removal during growth of mercury tolerant and self-aggregating Yarrowia spp.

Ecotoxicological implications of mercury (Hg) pollution of hydrosphere require effective Hg-removal strategies as antidote to the environmental problems. Mercury-tolerant yeasts, Yarrowia spp. Idd1 and Idd2 strains, were studied for intracellular accumulation and extracellular micro-precipitation of Hg during growth stage of the yeast strains. In a liquid medium containing 870 (±23.6) µg of bioavailable Hg2+, 419.0 µg Hg2+ (approx.) was taken up by the wet biomasses of the yeast strains after 48 h post-inoculation. Large portion of the adsorbed Hg was found in cell wall (approx. 49-83 %) and spheroplast (approx. 62-89 %). Negligible quantities of Hg were present in the mitochondria (0.02-0.02 %), and appreciable amount of Hg was observed in nuclei and cell debris (15.2-65.3 %) as evidence of bioaccumulation. Extracellular polymeric substances (EPS) produced by the growing Yarrowia cells was a complex of protein, carbohydrates and other substances, immobilizing 43.8 (±0.7)-58.7 (±1.0) % of initial Hg in medium as micro-precipitates, while 10.13 ± 0.4-39.2 ± 4.3 % Hg content was volatilized. Transmission electron microscopy coupled with X-ray energy dispersive spectrophotometry confirmed the cellular removal of Hg and formation of EPS-Hg complex colloids in the surrounding bulk solution as micro-precipitates in form of extracellular Hg-nanoparticles. Hg mass balance in the bio-sequestration experiment revealed excellent Hg removal (>97 %) from the medium (containing ≤16 µg ml-1 Hg2+) by the yeast strains via bioaccumulation, volatilization and micro-precipitation. The yeast strains are also effectively applicable in biological purification technology for Hg contaminated water because of their high self-aggregation activity and separatability from the aquatic environments. Graphical abstract Yarrowia species are oligotrophic marine yeasts that exhibited great potentials for mercuric ion remediation technologies, which are classified into four categories based on the process acting on the metal. These include immobilization through biosorption, compartmentation via bioaccumulation, separation from bulk solution via micro-precipitation upon EPS-Hg complex formation, and destruction that is a process to reduce the mercuric ion to metallic mercury.

Extracellular mercury sequestration by exopolymeric substances produced by Yarrowia spp.: Thermodynamics, equilibria, and kinetics studies.

Exopolymeric substances (EPS) produced by highly mercury-resistant strains of the yeast Yarrowia spp. (Idd1 and Idd2) were isolated and studied for their mercury binding potential. Excellent yield (approximately 0.3 g EPS per gram biomass) of soluble EPS in medium with 3% glucose was observed in the Yarrowia cultures 7 day post-inoculation. A gram dry weight of the EPS consists mainly of carbohydrates (0.4 g), protein (0.3-0.4 g), uronic acid (0.02 g), and nucleic acids (0.002 g). Mercury interactions with the biopolymer were measured as uptake kinetics from a simulated aquatic system and modelled with thermodynamics and calculated mass action equilibria. The EPS forms a complex with Hg2+ in water with small activation energy (≤2 kJ mol-1), achieving about 30 mg Hg2+ adsorption per gram dry weight of EPS. The adsorption models confirmed complexation of Hg2+ by the EPS via heterogeneous multilayer adsorption that obey second-order kinetics at constant rate of 4.0 and 8.1 mg g-1 min-1. The EPS used chemisorption as rate-limiting step that controls the uptake of Hg2+ from aquatic systems during micro-precipitation as bio-removal strategy. The EPS are promising biotechnological tools to design bioreactors for treatment of mercury-rich industrial wastewater.

Mercury resistance transposons in Bacilli strains from different geographical regions.

A total of 65 spore-forming mercury-resistant bacteria were isolated from natural environments worldwide in order to understand the acquisition of additional genes by and dissemination of mercury resistance transposons across related Bacilli genera by horizontal gene movement. PCR amplification using a single primer complementary to the inverted repeat sequence of TnMERI1-like transposons showed that 12 of 65 isolates had a transposon-like structure. There were four types of amplified fragments: Tn5084, Tn5085, Tn(d)MER3 (a newly identified deleted transposon-like fragment) and Tn6294 (a newly identified transposon). Tn(d)MER3 is a 3.5-kb sequence that carries a merRETPA operon with no merB or transposase genes. It is related to the mer operon of Bacillus licheniformis strain FA6-12 from Russia. DNA homology analysis shows that Tn6294 is an 8.5-kb sequence that is possibly derived from Tn(d)MER3 by integration of a TnMERI1-type transposase and resolvase genes and in addition the merR2 and merB1 genes. Bacteria harboring Tn6294 exhibited broad-spectrum mercury resistance to organomercurial compounds, although Tn6294 had only merB1 and did not have the merB2 and merB3 sequences for organomercurial lyases found in Tn5084 of B. cereus strain RC607. Strains with Tn6294 encode mercuric reductase (MerA) of less than 600 amino acids in length with a single N-terminal mercury-binding domain, whereas MerA encoded by strains MB1 and RC607 has two tandem domains. Thus, Tn(d)MER3 and Tn6294 are shorter prototypes for TnMERI1-like transposons. Identification of Tn6294 in Bacillus sp. from Taiwan and in Paenibacillus sp. from Antarctica indicates the wide horizontal dissemination of TnMERI1-like transposons across bacterial species and geographical barriers.

Development of suitable hydroponics system for phytoremediation of arsenic-contaminated water using an arsenic hyperaccumulator plant Pteris vittata.

In this study, we found that high-performance hydroponics of arsenic hyperaccumulator fern Pteris vittata is possible without any mechanical aeration system, if rhizomes of the ferns are kept over the water surface level. It was also found that very low-nutrition condition is better for root elongation of P. vittata that is an important factor of the arsenic removal from contaminated water. By the non-aeration and low-nutrition hydroponics for four months, roots of P. vittata were elongated more than 500 mm. The results of arsenate phytofiltration experiments showed that arsenic concentrations in water declined from the initial concentrations (50 µg/L, 500 µg/L, and 1000 µg/L) to lower than the detection limit (0.1 µg/L) and about 80% of arsenic removed was accumulated in the fern fronds. The improved hydroponics method for P. vittata developed in this study enables low-cost phytoremediation of arsenic-contaminated water and high-affinity removal of arsenic from water.

Mercury bioremoval by Yarrowia strains isolated from sediments of mercury-polluted estuarine water.

Difference in mercuric ion removal by resting and growing cells of two mercury-resistant yeast strains, identified as Yarrowia spp. (strains Idd1 and Idd2), were studied. Resting cells of strain Idd2 exhibited high maximum Hg(2+) removal capacity (59 mg mercury per g dry cell weight [gdw(-1)]) by adsorption than those of resting cells of strain Idd1 (32 mg gdw(-1)). The resting cells of strain Idd2 exhibited a higher Hg(2+) desorption capacity using CaCl2 (68 %) and EDTA (48 %) than strain Idd1, depicting weaker binding of Hg(2+) onto strain Idd2 unlike strain Idd1. The actively growing yeast cells showed opposite Hg removal characteristics to those of the resting cells. Strain Idd1 adsorbed less Hg(2+) from culture medium supplemented with Hg(2+) than strain Idd2. However, the growing strain Idd1 reduced and vaporized 27 % of supplemented Hg(2+) as metallic mercury (Hg(0)), while the growing strains Idd2 vaporized 15 % of the supplemented Hg(2+). These two yeast strains are potential biotechnological tools for the eventual bioremediation of polluted aquatic systems.

Evaluation of the effectiveness and salt stress of Pteris vittata in the remediation of arsenic contamination caused by tsunami sediments.

On March 11, 2011, one of the negative effects of the tsunami phenomenon that devastated the Pacific coast of the Tohoku district in Japan was the deposition of a wide range of arsenic (As) contamination to the soil. To remediate such a huge area of contamination, phytoremediation by Pteris vittata, an As-hyperaccumulator, was considered. To evaluate the efficacy of applying P. vittata to the area, the salt tolerance of P. vittata and the phytoextraction of As from soil samples were investigated. For the salt tolerance test, spore germination was considerably decreased at an NaCl level of more than 100 mM. At 200 mM, the gametophytes exhibited a morphological defect. Furthermore, the growth inhibition of P. vittata was observed with a salinity that corresponded to 66.2 mS/m of electric conductivity (EC) in the soil. A laboratory phytoremediation experiment was conducted using As-contaminated soils for 166 days. P. vittata grew and accumulated As at 264 mg/kg-DW into the shoots. Consequently, the soluble As in the soil was evidently decreased. These results showed that P. vittata was applicable to the phytoremediation of As-contaminated soil with low salinity as with the contamination caused by the 2011 tsunami.

Effectiveness of heat treatment to protect introduced denitrifying bacteria from eukaryotic predatory microorganisms in a pilot-scale bioreactor.

Bioaugmentation of bioreactor systems with pre-cultured bacteria has proven difficult because inoculated bacteria are easily eliminated by predatory eukaryotic-microorganisms. Here, we demonstrated an intermediate thermal treatment was effective for protecting introduced denitrifying bacteria from eukaryotic predators and consequently allowed the inoculated bacteria to survive longer in a denitrification reactor.

Community structure and population dynamics of ammonia oxidizers in composting processes of ammonia-rich livestock waste.

This study investigated the relationship between the population dynamics of ammonia-oxidizing bacteria (AOB) and archaea (AOA), and changes in the concentrations of nitrogenous compounds during ammonia-rich livestock waste-composting processes. The data showed that ammonia in beef and dairy cow livestock waste-composting piles was slowly oxidized to nitrite and nitrate after approximately 21-35 days under thermophilic or moderately thermophilic and mesophilic conditions. Real-time quantitative PCR (qPCR) assays showed a relative abundance of betaproteobacterial AOB during ammonia oxidation but did not detect AOA in any composting stage. Furthermore, real-time qPCR and terminal-restriction fragment length polymorphism (T-RFLP) analyses for the AOB in two composting processes (beef and dairy cow livestock waste) out of the three studied found that thermophilic or moderately thermophilic uncultured betaproteobacterial AOB from the "compost AOB cluster" contributed to ammonia oxidation during hot composting stages. Non-metric multidimensional scaling analyses of the data from T-RFLP showed that only a few analogous species predominated during composting of beef, dairy cow and pig livestock wastes, and thus, the AOB community structures in the three composting piles operating under different conditions were similar. AOB-targeted clone library analyses revealed that uncultured members of the "compost AOB cluster", which could be clearly distinguished from the authentic species of the genus Nitrosomonas, were the major constituents of the AOB populations. These results suggested that a limited and unique species of AOB played a role in ammonia oxidation during the composting of ammonia-rich livestock waste.

Bioaugmentation of a wastewater bioreactor system with the nitrous oxide-reducing denitrifier Pseudomonas stutzeri strain TR2.

In bioaugmentation technology, survival of inoculant in the treatment system is prerequisite but remains to be a crucial hurdle. In this study, we bioaugmented the denitrification tank of a piggery wastewater treatment system with the denitrifying bacterium Pseudomonas stutzeri strain TR2 in two pilot-scale experiments, with the aim of reducing nitrous oxide (N(2)O), a gas of environmental concern. In the laboratory, strain TR2 grew well and survived with high concentrations of nitrite (5-10 mM) at a wide range of temperatures (28-40°C). In the first augmentation of the pilot-scale experiment, strain TR2 inoculated into the denitrification tank with conditions (30°C, ~0.1 mM nitrite) survived only 2-5 days. In contrast, in the second augmentation with conditions determined to be favorable for the growth of the bacterium in the laboratory (40-45°C, 2-5 mM nitrite), strain TR2 survived longer than 32 days. During the time when the presence of strain TR2 was confirmed by quantitative real-time PCR, N(2)O emission was maintained at a low level even under nitrite-accumulating conditions in the denitrification and nitrification tanks, which provided indirect evidence that strain TR2 can reduce N(2)O in the pilot-scale system. Our results documented the effective application of growth conditions favorable for strain TR2 determined in the laboratory to maintain growth and performance of this strain in the pilot-scale reactor system and the decrease of N(2)O emission as the consequence.

Survival of the aerobic denitrifier Pseudomonas stutzeri strain TR2 during co-culture with activated sludge under denitrifying conditions.

The aerobic denitrifier Pseudomonas stutzeri TR2 (strain TR2) has the potential to reduce nitrous oxide emissions during the wastewater treatment process. In this application, it is important to find the best competitive survival conditions for strain TR2 in complex ecosystems. To that end, we examined co-cultures of strain TR2 with activated sludge via five passage cultures in a medium derived from treated piggery wastewater that contained a high concentration of ammonium. The results are as follows: (i) The medium supported the proliferation of strain TR2 (P. stutzeri strains) under denitrifying conditions. (ii) Nitrite was a better denitrification substrate than nitrate for TR2 survival. (iii) Strain TR2 also demonstrated strong survival even under aerobic conditions. This suggests that strain TR2 is effectively augmented to the wastewater treatment process, aiding in ammonium-nitrogen removal and reducing nitrous oxide production with a partial nitrification technique in which nitrite accumulates.

Organomercurials removal by heterogeneous merB genes harboring bacterial strains.

Organomercury lyase (MerB) is a key enzyme in bacterial detoxification and bioremediation of organomercurials. However, the merB gene is often considered as an ancillary component of the mer operon because there is zero to three merB genes in different mer operons identified so far. In this study, organomercurials' removal abilities of native mercury-resistant bacteria that have one or multiple merB genes were examined. Each heterogeneous merB genes from these bacteria was further cloned into Escherichia coli to investigate the substrate specificity of each MerB enzyme. The merB1 gene from Bacillus megaterium MB1 conferred the highest volatilization ability to methylmercury chloride, ethylmercury chloride, thimerosal and p-chloromercuribenzoate, while the merB3 from B. megaterium MB1 conferred the fastest mercury volatilization activity to p-chloromercuribenzoate. The substrate specificities among these MerB enzymes show the necessity for selecting the appropriate bacteria strains or MerB enzymes to apply them in bioremediation engineering for cleaning up specific organomercurial contaminations.

Potential of aerobic denitrification by Pseudomonas stutzeri TR2 to reduce nitrous oxide emissions from wastewater treatment plants.

In contrast to most denitrifiers studied so far, Pseudomonas stutzeri TR2 produces low levels of nitrous oxide (N(2)O) even under aerobic conditions. We compared the denitrification activity of strain TR2 with those of various denitrifiers in an artificial medium that was derived from piggery wastewater. Strain TR2 exhibited strong denitrification activity and produced little N(2)O under all conditions tested. Its growth rate under denitrifying conditions was near comparable to that under aerobic conditions, showing a sharp contrast to the lower growth rates of other denitrifiers under denitrifying conditions. Strain TR2 was tolerant to toxic nitrite, even utilizing it as a good denitrification substrate. When both nitrite and N(2)O were present, strain TR2 reduced N(2)O in preference to nitrite as the denitrification substrate. This bacterial strain was readily able to adapt to denitrifying conditions by expressing the denitrification genes for cytochrome cd(1) nitrite reductase (NiR) (nirS) and nitrous oxide reductase (NoS) (nosZ). Interestingly, nosZ was constitutively expressed even under nondenitrifying, aerobic conditions, consistent with our finding that strain TR2 preferred N(2)O to nitrite. These properties of strain TR2 concerning denitrification are in sharp contrast to those of well-characterized denitrifiers. These results demonstrate that some bacterial species, such as strain TR2, have adopted a strategy for survival by preferring denitrification to oxygen respiration. The bacterium was also shown to contain the potential to reduce N(2)O emissions when applied to sewage disposal fields.

Expressing a bacterial mercuric ion binding protein in plant for phytoremediation of heavy metals.

A specific mercuric ion binding protein (MerP) originating from transposon TnMERI1 of Bacillus megaterium strain MB1 isolated from Minamata Bay displayed good adsorption capability for a variety of heavy metals. In this study, the Gram-positive MerP protein was expressed in transgenic Arabidopsis to create a model system for phytoremediation of heavy metals. Under control of an actin promoter, the transgenic Arabidpsis showed higher tolerance and accumulation capacity for mercury, cadium and lead when compared with the control plant. Results from confocal microscopy analysis also indicate that MerP was localized at the cell membrane and vesicles of plant cells. The developed transgenic plants possessing excellent metal-accumulative ability could have potential applications in decontamination of heavy metals.

Splicing of a bacterial group II intron from bacillus megaterium is independent of intron-encoded protein.

The splicing of a bacterial group II subclass B intron B.me.I1 from Bacillus megaterium chromosomes was investigated. RT-PCR and nucleic acid hybridization methods were used to understand the role of the intron-encoded protein (IEP) in the splicing of B.me.I1. An in vivo assay showed that the splicing occurred in the absence of IEP. An in vitro assay showed that B.me.I1 was spliced under conditions similar to those of the intracellular environment with no help from other biological molecules. Because all group II introns previously reported needed IEPs for their splicing in vivo, our results suggest that B.me.I1 is an "actual" self-splicing group II intron. This is also the first report to recognize the existence of group II introns that independently splice mRNA in vivo. The self-splicing of a bacterial intron may support that eukaryotic spliceosomal introns originated in bacterial genomes.