Novel small multidrug resistance protein Tmt endows the Escherichia coli with triphenylmethane dyes bioremediation capability.

Dye contamination in printing and dyeing wastewater has long been a major concern due to its serious impact on both the environment and human health. In the quest for bioremediation of these hazardous dyes, biological resources such as biodegradation bacteria and enzymes have been investigated in severely polluted environments. In this context, the triphenylmethane transporter gene (tmt) was identified in six distinct clones from a metagenomic library of the printing and dyeing wastewater treatment system. Escherichia coli expressing tmt revealed 98.1% decolorization efficiency of triphenylmethane dye malachite green within 24 h under shaking culture condition. The tolerance to malachite green was improved over eightfold in the Tmt strain compared of the none-Tmt expressed strain. Similarly, the tolerance of Tmt strain to other triphenylmethane dyes like crystal violet and brilliant green, was improved by at least fourfold. Site-directed mutations, including A75G, A75S and V100G, were found to reinforce the tolerance of malachite green, and double mutations of these even further improve the tolerance. Therefore, the tmt has been demonstrated to be a specific efflux pump for triphenylmethane dyes, particularly the malachite green. By actively pumping out toxic triphenylmethane dyes, it significantly extends the cells tolerance in a triphenylmethane dye-rich environment, which may provide a promising strategy for bioremediation of triphenylmethane dye pollutants in the environments.

Selenium-oxidizing Agrobacterium sp. T3F4 decreases arsenic uptake by Brassica rapa L. under a native polluted soil.

Toxic arsenic (As) and trace element selenium (Se) are transformed by microorganisms but their complex interactions in soil-plant systems have not been fully understood. An As- and Se- oxidizing bacterium, Agrobacterium sp. T3F4, was applied to a native seleniferous As-polluted soil to investigate As/Se uptake by the vegetable Brassica rapa L. and As-Se interaction as mediated by strain T3F4. The Se content in the aboveground plants was significantly enhanced by 34.1%, but the As content was significantly decreased by 20.5% in the T3F4-inoculated pot culture compared to the control (P < 0.05). Similar result was shown in treatment with additional 5 mg/kg of Se(IV) in soil. In addition, the As contents in roots were significantly decreased by more than 35% under T3F4 or Se(IV) treatments (P<0.05). Analysis of As-Se-bacterium interaction in a soil simulation experiment showed that the bioavailability of Se significantly increased and As was immobilized with the addition of the T3F4 strain (P < 0.05). Furthermore, an As/Se co-exposure hydroponic experiment demonstrated that As uptake and accumulation in plants was reduced by increasing Se(IV) concentrations. The 50% growth inhibition concentration (IC50) values for As in plants were increased about one-fold and two-fold under co-exposure with 5 and 10 µmol/L Se(IV), respectively. In conclusion, strain T3F4 improves Se uptake but decreases As uptake by plants via oxidation of As and Se, resulting in decrease of soil As bioavailability and As/Se competitive absorption by plants. This provides a potential bioremediation strategy for Se biofortification and As immobilization in As-polluted soil.

Toxic response of antimony in the Comamonas testosteroni and its application in soil antimony bioremediation.

Antimony (Sb) is toxic to ecosystems and potentially to public health via its accumulation in the food chain. Bioavailability and toxicity of Sb have been reduced using various methods for the remediation of Sb-contaminated soil in most studies. However, Sb-contaminated soil remediation by microbial agents has been rarely evaluated. In this study, we evaluated the potential for the use of Comamonas testosteroni JL40 in the bioremediation of Sb-contamination. Strain JL40 immobilized more than 30 % of the Sb(III) in solution and oxidized over 18 % to Sb(V) for detoxification. Meanwhile, strain JL40 responds to Sb toxicity through such as Sb efflux, intracellular accumulation, biofilm production, and scavenging of reactive oxygen species (ROS), etc. The results of the pot experiment showed the average Sb content of the brown rice was decreased by 59.1%, 38.8%, and 48.4%, for 1.8, 50, and 100 mg/kg Sb spiked soils, respectively. In addition, the results of plant, soil enzyme activity, and rice agronomic trait observations showed that the application of strain JL40 could maintain the health of plants and soil and improve rice production. The single-step and sequential extraction of Sb from rhizosphere soil showed that strain JL40 also plays a role in Sb immobilization and oxidation in the soil environment. During rice potted cultivation, bacterial community analysis and plate counting showed that the strain JL40 could still maintain 103 CFU/g after 30 days of inoculation. With phenotypic and differential proteomics analysis, strain JL40 conferred Sb(III) tolerance by a combination of immobilization, oxidation, efflux and scavenging of ROS, etc. Our study demonstrates the application of Sb-immobilizing and oxidizing bacteria to lower soil Sb and reduce accumulation of Sb in rice. Our results provide guidance for bacterial remediation of Sb-contaminated soil.

Croceibacterium selenioxidans sp. nov., isolated from seleniferous soil.

A Gram-negative, aerobic bacterial strain, designated LX-88T, was isolated from seleniferous soil in Enshi, Hubei Province, PR China. Strain LX-88Toxidized elemental selenium to selenite, and produced carotenoids but not bacteriochlorophyll. The isolate grew optimally at 28 °C, pH 8.0 and with 0.5 % (w/v) NaCl. Phylogenetic analysies of the organism's 16S rRNA and bacterial core gene set sequences indicated that LX-88T belongs to the genus Croceibacterium, and has the highest degree of 16S rRNA gene sequence similarity to Croceibacterium soli MN-1T (97.4 %). The LX-88T genome was 3.4 Mbp and had a G+C content of 63.6 mol%. The average nucleotide identity and digital DNA-DNA hybridization values showed low relatedness (below 95 and 70 %, respectively) between strain LX-88T and other strains in the genus Croceibacterium. Ubiquinone-10 was the predominant quinone. The polar lipid profile was dominated by diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, an unidentified aminolipid, an unidentified phospholipid and an unidentified lipid. The major fatty acid was summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). These physiological and biochemical tests facilitated the differentiation of strain LX-88T from other members of the genus Croceibacterium. The results of this multifaceted taxonomic study indicate that strain LX-88T represents a novel species in the genus Croceibacterium, for which the name Croceibacterium selenioxidans sp. nov. is proposed. The type strain is LX-88T (=MCCC 1K08007T=LMG 32570T).

Microbial oxidation of organic and elemental selenium to selenite.

Selenium (Se) is an essential trace element for life. Se reduction has attracted much attention in the microbial Se cycle, but there is less evidence for Se oxidation. In particular, it is unknown whether microorganisms oxidise organic Se(-II). In this study, four strains of bacteria, namely Dyella spp. LX-1 and LX-66, and Rhodanobacter spp. LX-99 and LX-100, isolated from seleniferous soil, were involved in the oxidation of selenomethionine (SeMet), selenocystine (SeCys2), selenourea and Se(0) to selenite (Se(IV)) in pure cultures. The oxidation rates of organic Se were more rapidly than those of Se(0) in liquid media. Then Se(0) and SeMet were used as examples, microbial oxidation was the predominant process for both additional Se(0) and SeMet in sterilised alkaline or acidic soils. The Se(IV) concentrations were significantly higher at pH 8.56 than at pH 5.25. In addition, water-soluble Se (SOLSe) and exchangeable and carbonate-bound Se (EXC-Se) fractions increased dramatically with these four Se-oxidising bacteria in unsterilised seleniferous soil. To our knowledge, this is the first study to find that various bacteria are involved in the oxidation of organic Se to Se oxyanions, bridging the gap of Se redox in the Se biogeochemical cycle.

Functional and structural characterization of AntR, an Sb(III) responsive transcriptional repressor.

The ant operon of the antimony-mining bacterium Comamonas testosterone JL40 confers resistance to Sb(III). The operon is transcriptionally regulated by the product of the first gene in the operon, antR. AntR is a member of ArsR/SmtB family of metal/metalloid-responsive repressors resistance. We purified and characterized C. testosterone AntR and demonstrated that it responds to metalloids in the order Sb(III) = methylarsenite (MAs(III) >> As(III)). The protein was crystallized, and the structure was solved at 2.1 Å resolution. The homodimeric structure of AntR adopts a classical ArsR/SmtB topology architecture. The protein has five cysteine residues, of which Cys103a from one monomer and Cys113b from the other monomer, are proposed to form one Sb(III) binding site, and Cys113a and Cys103b forming a second binding site. This is the first report of the structure and binding properties of a transcriptional repressor with high selectivity for environmental antimony.

Comamonas testosteroni antA encodes an antimonite-translocating P-type ATPase.

Antimony, like arsenic, is a toxic metalloid widely distributed in the environment. Microbial detoxification of antimony has recently been identified. Here we describe a novel bacterial P1B-type antimonite (Sb(III))-translocating ATPase from the antimony-mining bacterium Comamonas testosterone JL40 that confers resistance to Sb(III). In a comparative proteomics analysis of strain JL40, an operon (ant operon) was up-regulated by Sb(III). The ant operon includes three genes, antR, antC and antA. AntR belongs to the ArsR/SmtB family of metalloregulatory proteins that regulates expression of the ant operon. AntA belongs to the P1B family of the P-type cation-translocating ATPases. It has both similarities to and differences from other members of the P1B-1 subfamily and appears to be the first identified member of a distinct subfamily that we designate P1B-8. Expression AntA in E. coli AW3110 (Δars) conferred resistance to Sb(III) and reduced the intracellular concentration of Sb(III) but not As(III) or other metals. Everted membrane vesicles from cells expressing antA accumulated Sb(III) but not As(III), where uptake in everted vesicles reflects efflux from cells. AntC is a small protein with a potential Sb(III) binding site, and co-expression of AntC with AntA increased resistance to Sb(III). We propose that AntC functions as an Sb(III) chaperone to AntA, augmenting Sb(III) efflux. The identification of a novel Sb(III)-translocating ATPase enhances our understanding of the biogeochemical cycling of environmental antimony by bacteria.

Luteimonas gilva sp. nov., isolated from farmland soil.

A Gram-stain-negative, rod-shaped bacterium, strain H23T, was isolated from farmland soil sampled in Enshi City, Hubei Province, PR China. The isolate grew optimally at 28-32 °C, pH 8.0 and with 0.5 % (w/v) NaCl. Based on the results of 16S rRNA gene sequence and phylogenetic analyses, strain H23T belonged to the genus Luteimonas with the highest degree of 16S rRNA gene sequence similarity to Luteimonas cucumeris Y4T (97.41 %). The DNA G+C content was 65.88 mol%. The average nucleotide identity and the Genome-to-Genome Distance Calculator results also showed low relatedness (below 95 and 70 %, respectively) between strain H23T and type strains in the genus Luteimonas. Ubiquinone-8 was the predominant quinone. The major fatty acids were iso-C15 : 0, iso-C16 : 0, iso-C17 : 0 and iso-C17 : 1 ω9c. Polar lipids were dominated by diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unidentified phospholipids. Low digital DNA-DNA hybridization values, as well as physiological and biochemical differences, such as no casein hydrolysis, being catalase-negative, and tesing positive for cystine arylamidase, α-chymotrypsin and N-acetyl-ß-glucosaminidase, could distinguish strain H23T from its closely related species. Strain H23T is considered to represent a novel species in the genus Luteimonas, for which the name Luteimonas gilva sp. nov. is proposed, with strain H23T (=CCTCC AB 2019255T=KCTC 72593T) as the type strain.