Purification and characterization of the enzymes from Brevundimonas naejangsanensis that degrade ochratoxin A and B.

Ochratoxin A (OTA) and Ochratoxin B (OTB) co-contaminate many types of agricultural products. Screening enzymes that degrade both OTA and OTB has significance in food safety. In this study, four novel OTA and OTB degrading enzymes, namely BnOTase1, BnOTase2, BnOTase3, and BnOTase4, were purified from the metabolites of the Brevundimonas naejangsanensis ML17 strain. These four enzymes hydrolyzed OTA into OTα and hydrolyzed OTB into OTß. BnOTase1, BnOTase2, BnOTase3, and BnOTase4 have the apparent Km values for hydrolyzing OTA of 19.38, 0.92, 12.11, 1.09 µmol/L and for hydrolyzing OTB of 0.76, 2.43, 0.60, 0.64 µmol/L respectively. OTα and OTß showed no significant cytotoxicity to HEK293 cells, suggesting that these enzymes mitigate the toxicity of OTA and OTB. The discovery of the novel OTA and OTB degrading enzymes enriches the research on ochratoxin control and provides objects for protein rational design.

High arsenic tolerance in Brevundimonas aurantiaca PFAB1 from an arsenic-rich Indian hot spring

BACKGROUND Arsenic contamination in the ground water of rural India is a recurrent problem and decon tamination is mostly based on the chemical or physical treatments until now. Microbial bioremediation is eco-friendly, cheap, time-efficient and does not produce any toxic by-products. RESULT In the present study, a high arsenic tolerant bacteria Brevundimonas aurantiaca PFAB1 was iso lated from Panifala hot spring located in West Bengal, India. Previously Panifala was also reported to be an arsenic-rich hot spring. B. aurantiaca PFAB1 exhibited both positive arsenic reductase and arsenite oxidase activity. It was tolerant to arsenite up to 90 mM and arsenate up to 310 mM. Electron microscopy has proved significant changes in cellular micromorphology and stalk appearance under the presence of arsenic in growth medium. Bioaccumulation of arsenic in As (III) treated cells were 0.01% of the total cell weight, while 0.43% in case of As (V) treatment. CONCLUSIONS All experimental lines of evidence prove the uptake/accumulation of arsenic within the bac terial cell. All these features will help in the exploitation of B. aurantiaca PFAB1 as a potent biological weapon to fight arsenic toxicity in the near future

Inhibition of an organophosphate-detoxifying bacterial phosphotriesterase by albumin and plasma thiol components.

The phosphotriesterase of the bacterium Brevundimonas diminuta (BdPTE) is a naturally occurring enzyme that catalyzes the hydrolysis of organophosphate (OP) nerve agents as well as pesticides and offers a potential treatment of corresponding intoxications. While BdPTE mutants with improved catalytic efficiencies against several OPs have been described, unexpectedly, less efficient breakdown of an OP was observed upon application in an animal model compared with in vitro measurements. Here, we describe detailed inhibition studies with the high-activity BdPTE mutant 10-2C3(C59M/C227A) by human plasma components, indicating that this enzyme is inhibited by serum albumin. The inhibitory activity is mediated by depletion of crucial zinc ions from the BdPTE active site, either via the known high-affinity zinc binding site of albumin or via chemical complex formation with its free thiol side chain at position Cys34. Albumin pre-charged with zinc ions or carrying a chemically blocked Cys34 side chain showed significantly reduced inhibitory activity; in fact, the combination of both measures completely abolished BdPTE inhibition. Consequently, the available zinc ion concentration in blood plays an important role for BdPTE activity in vivo and should be taken into account for therapeutic development and application of a catalytic OP scavenger.

Lasso Peptide Benenodin-1 Is a Thermally Actuated [1]Rotaxane Switch.

Mechanically interlocked molecules that change their conformation in response to stimuli have been developed by synthetic chemists as building blocks for molecular machines. Here we describe a natural product, the lasso peptide benenodin-1, which exhibits conformational switching between two distinct threaded conformers upon actuation by heat. We have determined the structures of both conformers and have characterized the kinetics and energetics of the conformational switch. Single amino acid substitutions to benenodin-1 generate peptides that are biased to a single conformer, showing that the switching behavior is potentially an evolvable trait in these peptides. Lasso peptides such as benenodin-1 can be recognized and cleaved by enzymes called lasso peptide isopeptidases. We show that only the native conformer of benenodin-1 is cleaved by its cognate isopeptidase. Thus, thermally induced conformational switching of benenodin-1 may also be relevant to the biological function of these molecules.

Aquidulcibacter paucihalophilus gen. nov., sp. nov., a novel member of family Caulobacteraceae isolated from cyanobacterial aggregates in a eutrophic lake.

A bacterial strain, TH1-2T, was isolated from cyanobacterial aggregates in the eutrophic Lake Taihu, Jiangsu Province, China. Cells were observed to be Gram-negative, slightly curved and rod-shaped. Optimal growth was obtained at pH 7.0 (range 5.5-8.5) and 30 °C (range 20-37 °C) in trypticase soy broth (TSB) without NaCl. Growth was not observed in TSB with 1.0% (w/v) NaCl added. The cells were found to be positive for oxidase and catalase activities. The major fatty acids were identified as 3-hydroxy hexadecanoic acid (C16:0 3-OH), C16:1 ω5c and summed feature 8 (consisting of C18:1 ω6c and cis-11-Octadecenoic acid (C18:1 ω7c). The major polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The 16S rRNA gene sequence of strain TH1-2T was found to be phylogenetically related to those of Glycocaulis abyssi MCS 33T and Brevundimonas naejangsanensis BIO-TAS2-2T (90.5 and 90.0% similarity, respectively). The genomic G+C content of strain TH1-2T was 55.6 mol% based on whole genome calculations. Average nucleotide identities (ANI) and the digital DNA-DNA hybridizations (DDH) for complete genomes ranged from 66.84 to 67.32 and 21.3 to 31.8% between strain TH1-2T and type strains within the family Caulobacteraceae, higher than those between strain TH1-2T and the strains within the family Hyphomonadaceae. The phenotypic, chemotaxonomic and phylogenetic properties, and genome analysis, indicate that strain TH1-2T (=CGMCC 1.12979T = LMG 28362T) represents a novel species in a new genus within the family Caulobacteraceae; thus, the name Aquidulcibacter paucihalophilus gen. nov., sp. nov., is proposed.

Phenylobacterium composti sp. nov., isolated from cotton waste compost in Korea.

A light-yellow-coloured bacterium, designated strain 4T-6(T), was isolated from cotton waste compost in Korea. Cells of the strain were strictly aerobic, Gram-negative, motile (by means of single polar flagella) and rod-shaped. Optimal growth occurred at 30 degrees C and at pH 7.0-8.0. The major fatty acids were 11-methyl C(18 : 1)omega7c (23.8 %), C(18 : 1)omega7c (23.2 %), C(16 : 0) (19.8 %) and C(17 : 0) (14.8 %). The DNA G+C content was 67.5 mol%. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 4T-6(T) was a member of the genus Phenylobacterium, showing the highest sequence similarities with those of 'Phenylobacterium zucineum' HLK1 (98.8 %) and Phenylobacterium lituiforme DSM 14363(T) (98.4 %). However, the values for DNA-DNA relatedness between strain 4T-6(T) and 'P. zucineum' HLK1 and P. lituiforme DSM 14363(T) were 45 and 43 %, respectively. Phylogenetic characteristics, physiological properties and DNA-DNA hybridization data indicate that strain 4T-6(T) represents a novel species of the genus Phenylobacterium, for which the name Phenylobacterium composti sp. nov. is proposed. The type strain is 4T-6(T) (=KACC 12597(T)=DSM 19425(T)).

Brevundimonas aveniformis sp. nov., a stalked species isolated from activated sludge.

A Gram-negative, rod-like, stalk-producing bacterium, designated strain EMB102(T), was isolated from activated sludge that performed enhanced biological phosphorus removal in a sequencing batch reactor. Cells without stalks were motile with single polar flagella, but cells that did produce stalks were non-motile and lacked polar flagella. Growth of strain EMB102(T) was observed at temperatures between 15 and 35 degrees C (optimum, 30 degrees C) and between pH 6.0 and 9.0 (optimum, pH 7.5-8.5). The predominant fatty acids of strain EMB102(T) were C(18 : 1) omega 7c, C(16 : 0) and C(15 : 0). The predominant polar lipid was phosphatidylglycerol. The G+C content of the genomic DNA was 64.1 mol% and the major quinone was Q-10. Comparative 16S rRNA gene sequence analyses showed that strain EMB102(T) formed a distinct phyletic lineage within the genus Brevundimonas. The levels of 16S rRNA gene sequence similarity between the type strains of Brevundimonas species ranged from 95.8 to 97.5 %. DNA-DNA relatedness levels between the EMB102(T) and closely related Brevundimonas species were below 15.0 %. On the basis of chemotaxonomic data and molecular properties, strain EMB102(T) represents a novel species within the genus Brevundimonas, for which the name Brevundimonas aveniformis sp. nov. is proposed. The type strain is EMB102(T) (=KCTC 12609(T)=DSM 17977(T)).

Brevundimonas kwangchunensis sp. nov., isolated from an alkaline soil in Korea.

Two Gram-negative, rod-shaped bacterial strains, KSL-102T and KSL-110, were isolated from an alkaline soil in Korea, and their taxonomic positions were investigated by use of a polyphasic study. The two strains grew optimally at pH 7.0-8.0 and 30 degrees C without NaCl. They contained Q-10 as the predominant ubiquinone. The major fatty acids were C(18:1)omega7c and C(16:0) on trypticase soy agar, but 11-methyl C(18:1)omega7c was also a major component when the two strains were cultivated on LMG medium no. 221. Their DNA G + C contents were 68.4-68.7 mol%. Strains KSL-102T and KSL-110 exhibited three nucleotide differences in their 16S rRNA gene sequences and a mean DNA-DNA relatedness value of 85 %. Phylogenetic trees based on 16S rRNA gene sequences showed that the two strains fell within the evolutionary radiation encompassed by the genus Brevundimonas. Levels of 16S rRNA gene sequence similarity between the two strains and the type strains of recognized Brevundimonas species ranged from 96.3 to 98.4%. DNA-DNA relatedness levels between the two strains and recognized Brevundimonas species were 8-21%. On the basis of phenotypic, phylogenetic and genetic data, strains KSL-102T and KSL-110 were classified in the genus Brevundimonas as members of a novel species, for which the name Brevundimonas kwangchunensis sp. nov. is proposed. The type strain is KSL-102T (= KCTC 12380T = DSM 17033T).

Brevundimonas mediterranea sp. nov., a non-stalked species from the Mediterranean Sea.

Six strains of Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from the Mediterranean Sea. 16S rRNA gene sequence analysis indicated that the strains were affiliated within the alphaproteobacterial genus Brevundimonas, with Brevundimonas intermedia (99.4 %) and Brevundimonas vesicularis (99.2 %) as their closest relatives. This affiliation was supported by chemotaxonomic data (major polar lipids: phosphatidyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidyl glucopyranosyl diacylglycerol; major fatty acids: C(18 : 1), C(16 : 0), C(16 : 1), C(15 : 0), C(17 : 1)omega8c, 11-Me-C(18 : 1)omega5t). The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the strains from all recognized Brevundimonas species. The strains therefore represent a novel species, for which the name Brevundimonas mediterranea sp. nov. is proposed, with the type strain V4.BO.10T)(=LMG 21911T=CIP 107934T).