Ginro Endo

Potential of Aerobic Denitrification by Pseudomonas stutzeri TR2 To Reduce Nitrous Oxide Emissions from Wastewater Treatment Plants

ABSTRACT In contrast to most denitrifiers studied so far, Pseudomonas stutzeri TR2 produces low levels of nitrous oxide (N2O) 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 N2O 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 N2O were present, strain TR2 reduced N2O 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 cd1 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 N2O 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 N2O emissions when applied to sewage disposal fields.

Structure analysis of a class II transposon encoding the mercury resistance of the Gram-positive Bacterium bacillus megaterium MB1, a strain isolated from minamata bay, Japan.

A unique transposon was found in the chromosome of Bacillus megaterium MB1, a Gram-positive bacterium isolated from mercury-polluted sediments of Minamata Bay, Japan. The transposon region of a 14.5kb DNA fragment was amplified by PCR using a single PCR primer designed from the nucleotide sequence of an inverted repeat of class II transposons. The molecular analysis revealed that the PCR-amplified DNA fragment encodes a transposition module similar to that of Tn21. The transposon also encodes a broad-spectrum mercury resistance region having a restriction endonuclease map identical to that of Bacillus cereus RC607, a strain isolated from Boston Harbor, USA. The result of a phylogenetic analysis of the amino acid sequence of putative resolvase of the transposon showed that the transposon is phylogenetically closer to the transposons of Gram-positive bacteria than those of Gram-negative bacteria. Besides the transposition module and mer operon, the transposon encodes a mobile genetic element of bacterial group II introns between the resolvase gene and mer operon. The intron, however, does not intervene in any exon gene. The discovery of this newly found combination of the complex mobile elements may offer a clue to understanding the horizontal dissemination of broad-spectrum mercury resistance among microbes.

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.

Diversity of mercury resistance determinants among Bacillus strains isolated from sediment of Minamata Bay

Thirty mercury-resistant (Hg R) Bacillus strains were isolated from mercury-polluted sediment of Minamata Bay, Japan. Mercury resistance phenotypes were classified into broad-spectrum (resistant to inorganic Hg(2+) and organomercurials) and narrow-spectrum (resistant to inorganic Hg(2+) and sensitive to organomercurials) groups. Polymerase chain reaction (PCR) product sizes and the restriction nuclease site maps of mer operon regions from all broad-spectrum Hg R Bacillus were identical to that of Bacillus megaterium MB1. On the other hand, the PCR products of the targeted merP (extracellular mercury-binding protein gene) and merA (intracellular mercury reductase protein gene) regions from the narrow-spectrum Hg R Bacillus were generally smaller than those of the B. megaterium MB1 mer determinant. Diversity of gene structure configurations was also observed by restriction fragment length polymorphism (RFLP) profiles of the merA PCR products from the narrow-spectrum Hg R Bacillus. The genetic diversity of narrow-spectrum mer operons was greater than that of broad-spectrum ones.

Identification of three merB genes and characterization of a broad-spectrum mercury resistance module encoded by a class II transposon of Bacillus megaterium strain MB1

The complete structure of a broad-spectrum mercury resistance module was shown by sequencing the Gram-positive bacterial transposon TnMERI1 of Bacillus megaterium MB1. The regions encoding organomercury resistance were identified. Upstream of a previously identified organomercurial lyase merB (merB1) region of TnMERI1, a second merR (merR2) and a second merB gene (merB2) were found. These genes constitute a second operon (mer operon 2) following a promoter/operator (P(merR2)) region. A third organomercurial lyase gene (merB3) was found immediately upstream of the mer operon (mer operon 1) followed by a promoter/operator (P(merB3)) region homologous to that of the mer operon 1 (P(merR1)-merR1-merE-like-merT-merP-merA). The complete genetic structure of the mercury resistance module is organized as P(merB3)-merB3-P(merR1)-merR1-merE-like-merT+ ++ -merP-merA-P(merR2)-merR2 -merB2-merB1. The subcloning analysis of these three merB genes showed distinct substrate specificity as different organomercury lyase genes.

Dissemination of TnMERI1-like mercury resistance transposons among Bacillus isolated from worldwide environmental samples

Fifty-six mercury-resistant (Hg(R)) Bacillus strains were isolated from natural environments at various sites of the world. Southern hybridisation and polymerase chain reaction (PCR) analysis showed that 21 of the 56 isolates have closely related or identical mer operons to that of Bacillus megaterium MB1. These 21 isolates displayed a broad-spectrum mercury resistance and volatilised Hg(0). PCR amplification with a single primer and restriction fragment length polymorphism analysis showed that these 21 isolates had TnMERI1-like class II transposons. These transposons can be classified into Tn5084, Tn5085, or TnMERI1. From these results, at least three types of class II mercury resistance transposons exist in Hg(R)Bacillus and these transposons may contribute the worldwide distribution and horizontal dissemination of the mer operons among Bacillus strains in natural environments.

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.

Mercury removal and recovery by immobilized Bacillus megaterium MB1

From several mercury removing microorganisms, we selected Bacillus megaterium MB1, which is nonpathogenic, broad-spectrum mercury resistant, mercuric ion reducing, heat tolerant, and spore-forming, as a useful bacterium for bioremediation of mercury pollution. In this study, mercury removal performance of the immobilized B. megaterium MB1 was investigated to develop safe, efficient and stable catalytic bio-agent for mercury bioremediation. The results showed that the alginate gel immobilized B. megaterium MB1 cells efficiently removed 80% of mercury from the solution containing 10 mg/L mercuric chloride within 24 h. These cells still had high activity of mercury removal even after mercuric ion loading was repeated for nine times. The analysis of mercury contents of the alginate beads with and without immobilized B. megaterium MB1 suggested that a large portion of reduced metallic mercury was trapped in the gel beads. It was concluded that the alginate gel immobilized B. megaterium MB1 cells have potential to remove and recover mercury from mercury-containing water.

Characterization of two regulatory genes of the mercury resistance determinants from TnMERI1 by luciferase-based examination.

The broad-spectrum mercury resistance transposon, TnMERI1, of Bacillus megaterium strain MB1, contains three proposed operator/promoter (O/P) transcriptional start sites and two regulatory genes (merR1 and merR2). A series of luciferase (lux)-based transcriptional fusion plasmids were studied in Escherichia coli to show that both merR1 and merR2 gene products repressed transcription from O/PmerB3, O/PmerR1, and O/PmerR2 under uninduced conditions. Derepression occurred when the merR1 gene was present and Hg(2+) functioned as an inducer. In the presence of organomercurial compounds, basal transcription of merB3 was needed to produce inorganic Hg(2+) as the inducer of expression regulated by MerR1 at O/PmerB3. The presence of merR2 repressed transcription from all three O/Pmer sites under both non-induced conditions and when inorganic Hg(2+) or organomercurials were added. These results show that MerR1 functions as a repressor in the absence of Hg(2+) and as an activator in the presence of Hg(2+), while MerR2 functions as a repressor.

Simultaneous detection and removal of organomercurial compounds by using the genetic expression system of an organomercury lyase from the transposon TnMERI1

Abstract. Using a newly identified organomercury lyase gene (merB3) expression system from TnMERI1, the mercury resistance transposon first found in Gram-positive bacteria, a dual-purpose system to detect and remove organomercurial contamination was developed. A plasmid was constructed by fusing the promoterless luxAB genes as bioluminescence reporter genes downstream of the merB3 gene and its operator/promoter region. Another plasmid, encoding mer operon genes from merR1 to merA, was also constructed to generate an expression regulatory protein, MerR1, and a mercury reductase enzyme, MerA. These two plasmids were transformed into Escherichia coli cells to produce a biological system that can detect and remove environmental organomercury contamination. Organomercurial compounds, such as neurotoxic methylmercury at nanomolar levels, were detected using the biomonitoring system within a few minutes and were removed during the next few hours.