Glyphosate-Induced Phosphonatase Operons in Soil Bacteria of the Genus Achromobacter.

Achromobacter insolitus and Achromobacter aegrifaciens, bacterial degraders of the herbicide glyphosate, were found to induce phosphonatase (phosphonoacetaldehyde hydrolase, EC 3.11.1.1) when grown on minimal media with glyphosate as the sole source of phosphorus. The phosphonatases of the strains were purified to an electrophoretically homogeneous state and characterized. The enzymes differed in their kinetic characteristics and some other parameters from the previously described phosphonatases. The phosphonatase of A. insolitus was first revealed to separate into two stable forms, which had similar kinetic characteristics but interacted differently with affinity and ion-exchange resins. The genomes of the investigated bacteria were sequenced. The phosphonatase genes were identified, and their context was determined: the bacteria were shown to have gene clusters, which, besides the phosphonatase operon, included genes for LysR-type transcription activator (substrate sensor) and putative iron-containing oxygenase PhnHD homologous to monooxygenases PhnY and TmpB of marine organophosphonate degraders. Genes of 2-aminoethylphosphonate aminotransferase (PhnW, EC 2.6.1.37) were absent in the achromobacterial phosphonatase operons; instead, we revealed the presence of genes encoding the putative flavin oxidase HpnW. In silico simulation showed 1-hydroxy-2-aminoethylphosphonate to be the most likely substrate of the new monooxygenase, and a number of glycine derivatives structurally similar to glyphosate to be substrates of flavin oxidase.

Cholesterol Assay Based on Recombinant Cholesterol Oxidase, ABTS, and Horseradish Peroxidase.

Cholesterol determination by cholesterol oxidase reaction is a fast, convenient, and highly specific approach with widespread use in clinical diagnostics. Routinely, endpoint measurements with 4-aminophenazone or 4-aminoantipyrine as chromogens and sodium cholate, surfactants, or alcohols as solubilizing agents are used. Here we describe a novel kinetic method to determine cholesterol in 0.05-0.75 mM range in neutral or acidic buffers by use of recombinant cholesterol oxidase from Nocardioides simplex in a coupled reaction with horseradish peroxidase, ABTS as a chromogen, and methyl-ß-cyclodextrin as a solubilizing agent.

Recombinant Extracellular Cholesterol Oxidase from Nocardioides simplex.

Cholesterol oxidase is a highly demanded enzyme used in medicine, pharmacy, agriculture, chemistry, and biotechnology. It catalyzes oxidation of 3ß-hydroxy-5-ene- to 3-keto-4-ene- steroids with the formation of hydrogen peroxide. Here, we expressed 6xHis-tagged mature form of the extracellular cholesterol oxidase (ChO) from the actinobacterium Nocardioides simplex VKM Ac-2033D (55.6 kDa) in Escherichia coli cells. The recombinant enzyme (ChONs) was purified using affinity chromatography. ChONs proved to be functional towards cholesterol, cholestanol, phytosterol, pregnenolone, and dehydroepiandrosterone. Its activity depended on the structure and length of the aliphatic side chain at C17 atom of the steroid nucleus and was lower with pregnenolone and dehydroepiandrosterone. The enzyme was active in a pH range of 5.25÷6.5 with the pH optimum at 6.0. Kinetic assays and storage stability tests demonstrated that the characteristics of ChONs were generally comparable with or superior to those of commercial ChO from Streptomyces hygroscopicus (ChOSh). The results contribute to the knowledge on microbial ChOs and evidence that ChO from N. simplex VKM Ac-2033D is a promising agent for further applications.

Draft Genome Sequence of Glyphosate-Degrading Achromobacter insolitus Strain Kg 19 (VKM B-3295), Isolated from Agricultural Soil.

The genome of an Achromobacter insolitus strain isolated from an agricultural soil polluted with the herbicide glyphosate is reported. The genome size is 6.4 Mb, with an average G+C content of 65.2%. These genomic data could contribute to a better understanding of the biochemistry and regulatory mechanisms of the microbial degradation of glyphosate and aminomethylphosphonate.

Organophosphonates utilization by soil strains of Ochrobactrum anthropi and Achromobacter sp.

Four bacterial strains from glyphosate- or alkylphosphonates-contaminated soils were tested for ability to utilize different organophosphonates. All studied strains readily utilized methylphosphonic acid and a number of other phosphonates, but differed in their ability to degrade glyphosate. Only strains Ochrobactrum anthropi GPK 3 and Achromobacter sp. Kg 16 utilized this compound after isolation from enrichment cultures with glyphosate. Achromobacter sp. MPK 7 from the same enrichment culture, similar to Achromobacter sp. MPS 12 from methylphosphonate-polluted source, required adaptation to growth on GP. Studied strains varied significantly in their growth parameters, efficiency of phosphonates degradation and characteristic products of this process, as well as in their energy metabolism. These differences give grounds to propose a possible model of interaction between these strains in microbial consortium in phosphonate-contaminated soils.

Glyphosate acetylation as a specific trait of Achromobacter sp. Kg 16 physiology.

The growth parameters of Achromobacter sp. Kg 16 (VKM B-2534 D), such as biomass and maximum specific growth rate, depended only on the source of phosphorus in the medium, but not on the carbon source or the presence of growth factors. With glyphosate as a sole phosphorus source, they were still 40-50 % lower than in media supplemented with orthophosphate or other organophosphonate-methylphosphonic acid. At the first time process of glyphosate acetylation and accumulation of acetylglyphosate in culture medium were revealed in this strain. Acetylglyphosate isolated from cultural liquid was identified by mass spectroscopy; its mass spectrum fully corresponded with that of chemically synthesized acetylglyphosate. Even poorer growth was observed in media with acetylglyphosate: although the strain was able to utilize this compound as a sole source of phosphorus, the maximum biomass was still 58-70 % lower than with glyphosate. The presence of acetylglyphosate in culture medium could also hinder the utilization of glyphosate as a phosphorus source. Therefore, the acetylation of glyphosate may be a specific feature of Achromobacter sp. Kg 16 responsible for its poor growth on this compound.

Distribution of glyphosate and methylphosphonate catabolism systems in soil bacteria Ochrobactrum anthropi and Achromobacter sp.

Bacterial strains capable of utilizing methylphosphonic acid (MP) or glyphosate (GP) as the sole sources of phosphorus were isolated from soils contaminated with these organophosphonates. The strains isolated from MP-contaminated soils grew on MP and failed to grow on GP. One group of the isolates from GP-contaminated soils grew only on MP, while the other one grew on MP and GP. Strains Achromobacter sp. MPS 12 (VKM B-2694), MP degraders group, and Ochrobactrum anthropi GPK 3 (VKM B-2554D), GP degraders group, demonstrated the best degradative capabilities towards MP and GP, respectively, and were studied for the distribution of their organophosphonate catabolism systems. In Achromobacter sp. MPS 12, degradation of MP was catalyzed by C-P lyase incapable of degrading GP (C-P lyase I). Adaptation to growth on GP yielded the strain Achromobacter sp. MPS 12A, which retained its ability to degrade MP via C-P lyase I and was capable of degrading GP with formation of sarcosine, thus suggesting the involvement of a GP-specific C-P lyase II. O. anthropi GPK 3 also degraded MP via C-P lyase I, but degradation of GP in it was initiated by glyphosate oxidoreductase, which was followed by product transformation via the phosphonatase pathway.