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Characteristics of a Microcystin-Degrading Bacterium under Alkaline Environmental Conditions.

ABSTRACT: The pH of the water associated with toxic blooms of cyanobacteria is typically in the alkaline range; however, previously only microcystin-degrading bacteria growing in neutral pH conditions have been isolated. Therefore, we sought to isolate and characterize an alkali-tolerant microcystin-degrading bacterium from a water bloom using microcystin-LR. Analysis of the 16S rRNA gene sequence revealed that the isolated bacterium belonged to the genus Sphingopyxis, and the strain was named C-1. Sphingopyxis sp. C-1 can grow; at pH 11.0; however, the optimum pH for growth was pH 7.0. The microcystin degradation activity of the bacterium was the greatest between pH 6.52 and pH 8.45 but was also detected at pH 10.0. The mlrA homolog encoding the microcystin-degrading enzyme in the C-1 strain was conserved. We concluded that alkali-tolerant microcystin-degrading bacterium played a key role in triggering the rapid degradation of microcystin, leading to the disappearance of toxic water blooms in aquatic environments.

SUBMITTER: Okano K

PROVIDER: S-EPMC2826094 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Characteristics of a Microcystin-Degrading Bacterium under Alkaline Environmental Conditions.

Okano Kunihiro K Shimizu Kazuya K Kawauchi Yukio Y Maseda Hideaki H Utsumi Motoo M Zhang Zhenya Z Neilan Brett A BA Sugiura Norio N

Journal of toxicology 20090101

The pH of the water associated with toxic blooms of cyanobacteria is typically in the alkaline range; however, previously only microcystin-degrading bacteria growing in neutral pH conditions have been isolated. Therefore, we sought to isolate and characterize an alkali-tolerant microcystin-degrading bacterium from a water bloom using microcystin-LR. Analysis of the 16S rRNA gene sequence revealed that the isolated bacterium belonged to the genus Sphingopyxis, and the strain was named C-1. Sphing ...[more]

PMID: 20224628

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Project description:Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS-) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (the Halomonas group for the heterotrophs and the genus Thioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus Thioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase activity which converted cyanate (CNO-) to ammonia and CO2. On the other hand, cyanase activity either was absent or was present at very low levels in the autotrophic strains grown on thiocyanate as the sole energy and N source. As a result, large amounts of cyanate were found to accumulate in the media during utilization of thiocyanate at pH 10 in batch and thiocyanate-limited continuous cultures. This is a first direct proof of a "cyanate pathway" in pure cultures of thiocyanate-degrading bacteria. Since it is relatively stable under alkaline conditions, cyanate is likely to play a role as an N buffer that keeps the alkaliphilic bacteria safe from inhibition by free ammonia, which otherwise would reach toxic levels during dissimilatory degradation of thiocyanate.

Project description:Biocontainment is a safeguard strategy for preventing uncontrolled proliferation of genetically engineered microorganisms (GEMs) in the environment. Biocontained GEMs are designed to survive only in the presence of a specific molecule. The design of a pollutant-degrading and pollutant-dependent GEM prevents its proliferation after cleaning the environment. In this study, we present a biocontained toluene-degrading bacterium based on Acinetobacter sp. Tol 5. The bamA gene, which encodes an essential outer membrane protein, was deleted from the chromosome of Tol 5 but complemented with a plasmid carrying a bamA gene regulated by the Pu promoter and the regulatory protein XylR. The resultant strain (PuBamA) degraded toluene, similarly to the wild-type Tol 5. Although the cell growth of the PuBamA strain was remarkably inhibited after toluene depletion, escape mutants emerged at a frequency of 1 per 5.3 × 10-7 cells. Analyses of escape mutants revealed that insertion sequences (ISs) carrying promoters were inserted between the Pu promoter and the bamA gene on the complemented plasmid. MinION deep sequencing of the plasmids extracted from the escape mutants enabled the identification of three types of ISs involved in the emergence of escape mutants, suggesting a strategy for reducing it. IMPORTANCE GEMs are beneficial for various applications, including environmental protection. However, the risks of GEM release into the environment have been debated for a long time. If a pollutant is employed as a specific molecule for a biocontainment system, GEMs capable of degrading pollutants are available for environmental protection. Nevertheless, to our knowledge, biocontained degraders for real pollutants have not been reported in academic journals so far. This is possibly due to the difficulty in the expression of enzymes for degrading pollutants in a tractable bacterium such as Escherichia coli. On the other hand, bacteria with enzymes for degrading pollutants are often intractable as a host of GEMs due to the shortage of tools for genetic manipulation. This study reports the feasibility of a biocontainment strategy for a toluene degrader. Our results provide useful insights into the construction of a GEM biocontainment system for environmental protection.

Dataset Information

Characteristics of a Microcystin-Degrading Bacterium under Alkaline Environmental Conditions.

Publications

Characteristics of a Microcystin-Degrading Bacterium under Alkaline Environmental Conditions.

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