Cloning, expression and mutation of a triazophos hydrolase gene from Burkholderia sp. SZL-1.

Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA, cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C-50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of Kcat and Kcat/Km for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research.

Pedobacter nanyangensis sp. nov., isolated from herbicide-contaminated soil.

A Gram-stain-negative, strictly aerobic, non-spore-forming, motile, rod-shaped bacterium, designated Q-4T, was isolated from a herbicide-contaminated soil sample in Nanyang, Henan province, China. Strain Q-4T grew optimally in the LB medium without NaCl supplement at a pH range of 6.0­7.0 and a temperature of 30 °C. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Q-4T was most closely related to 'Pedobacter zeaxanthinifaciens' TDMA-5 (97.4 % 16S rRNA gene sequence similarity), followed by Pedobacter xixiisoli S27T (95.8 %). The genomic DNA G+C content of strain Q-4T was 41.8 mol%. MK-7 was the major respiratory quinone. Phosphatidylethanolamine and phosphoaminolipid were the major polar lipids. The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1ω6c and/or C16 : 1ω7c (summed feature 3) and C16 : 1ω7c/C16 : 1ω6c (summed feature 3). Strain Q-4T showed low DNA­DNA relatedness with 'P. zeaxanthinifaciens' TDMA-5 (21.4 ± 0.6 %). Physiological and biochemical characteristics are able to distinguish strain Q-4T from the most closely related species of the genus Pedobacter. On the basis of genotypic and phenotypic data, strain Q-4T is considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter nanyangensis sp. nov. is proposed. The type strain is Q-4T ( = KCTC 42442T = ACCC 19798T).

Mangrovibacter yixingensis sp. nov., isolated from farmland soil.

A Gram-staining-negative, facultatively anaerobic, rod-shaped, nitrogen-fixing bacterial strain, designated TULL-AT, was isolated from a farmland soil sample in Yixing, China. The optimal conditions for growth were 30 °C, pH 7.0-8.0 and 0% (w/v) NaCl. Q8 was the dominant respiratory quinone and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and aminophospholipid. Phylogenetic analysis of 16S rRNA gene sequences showed that strain TULL-AT was most closely related to Mangrovibacter plantisponsor MSSRF40T (99.6%), followed by Salmonella enterica subsp. diarizonae DSM 14847T (96.8%) and Cronobacter condimenti 1330T (96.8 %). Sequence analysis of the genes rpoB, gyrB and hsp60 revealed that those of strain TULL-AT also exhibit high sequence similarity with those of the species M. plantisponsor MSSRF40T (95.5, 94.1 and 93.4%). The genomic DNA G+C content was 52 mol%. The major fatty acids of strain TULL-AT were C16 : 0, C16 : 1ω7c and/or C16 : 1ω6c, C18 : 1ω7c /C18 : 1ω6c, C14 : 0, C14 : 0 3-OH/iso-C16 : 1 I and iso-C17 : 1 I and/or anteiso-C17 : 1 B. Strain TULL-AT showed low DNA-DNA relatedness with M. plantisponsor MSSRF40T (35.10 ± 1.41%). Based on the multiple genotypic and phenotypic data, strain TULL-AT is considered to represent a novel species of the genus Mangrovibacter, for which the name Mangrovibacter yixingensis sp. nov. is proposed. The type strain is TULL-AT ( = ACCC 19709T = KCTC 42181T).

Flavobacterium suzhouense sp. nov., isolated from farmland river sludge.

A Gram-stain-negative bacterium, designated XIN-1(T), was isolated from a farmland river sludge sample in Suzhou, China. Cells of strain XIN-1(T) were strictly aerobic, non-motile and rod-shaped. Strain XIN-1(T) grew optimally at pH 7.0 and 28 °C. Phylogenetic analysis of the 16S rRNA gene sequences showed that strain XIN-1(T) was most closely related to Flavobacterium hauense BX12(T) (98.2 % sequence similarity), followed by Flavobacterium beibuense F44-8(T) (96.3 %). The major respiratory quinone was menaquinone-6 and the major polar lipid was phosphatidylethanolamine. The major fatty acids (>5 %) were summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), summed feature 4 (comprising iso-C17 : 1 I and/or anteiso-C17 : 1 B), iso-C15 : 0, C16 : 0 and iso-C17 : 0 3-OH. The genomic DNA G+C content of strain XIN-1(T) was 39.8 mol%. Strain XIN-1(T) showed low DNA-DNA relatedness with F. hauense BX12(T) (38.7±0.5 %). On the basis of genotypic and phenotypic data, strain XIN-1(T) is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium suzhouense sp. nov. is proposed. The type strain is XIN-1(T) ( = CCTCC AB 2014200(T) = KCTC 42107(T)).