A new D-stereospecific amino acid amidase from Ochrobactrum anthropi

A new D-stereospecific amino acid amidase has been partially purified from Ochrobactrum anthropi SCRC SV3, which had been isolated and selected from soil. The Mr of the enzyme was estimated to be about 38,000, and its isoelectric point was 5.3. The enzyme catalyzes the stereospecific hydrolysis of D-amino acid amide to yield D-amino acid and ammonia. The major substrates included D-phenylalanine amide, D-tyrosine amide, D-tryptophan amide, D-leucine amide, and D-alanine amide.     

Simple method for transformation of Ochrobactrum anthropi

A rapid and simple method for preparation of highly efficient Ochrobactrum anthropi electrocompetent cells has been developed. The efficiency of transformation increased 200-fold when the cells were prepared from liquid culture compared to agar plates. Effects of different conditions, including cell density, electric field strength, resistance and plasmid size were evaluated to develop an electroporation protocol. The electrocompetent O. anthropi prepared by this method were 9-fold more efficient than commercial sources of competent Escherichia coli. The method described here will enhance the genetic manipulation of Ochrobactrum as a bioremediation tool and a biopesticide agent.     

Genotyping of Ochrobactrum anthropi by recA-based comparative sequence, PCR-RFLP, and 16S rRNA gene analysis

A recA-PCR restriction fragment length polymorphism assay was developed to study intraspecies variation among Ochrobactrum anthropi. Primers deduced from the known recA gene sequence of the genetically closely related genus Brucella allowed the specific amplification of a 1065 bp recA fragment from each of the 38 O. anthropi and the eight Brucella strains investigated. RecA was also amplified from the type strains of O. intermedium, O. tritici, and O. lupini but could not be generated from O. grignonense and O. gallinifaecis. Subsequent comparative recA sequence- and HaeIII-recA restriction fragment length polymorphism analysis identified nine different genospecies among the tested 38 O. anthropi isolates, whereas the recA sequences of the Brucella spp. were indistinguishable. Furthermore, Brucella spp., O. anthropi, O. intermedium, and O. tritici were clearly separated from each other by means of their recA sequences and HaeIII restriction patterns. Five strains of uncertain species status listed in the Culture Collection University of G?teborg bacterial culture collection as O. anthropi were characterized by recA analysis, and their phylogenetic position within the Brucella-Ochrobactrum group was determined. In summary, recA-sequence analysis provides a new reliable molecular subtyping tool to study the phylogeny of the Ochrobactrum taxon at both the inter- and intraspecies level.     

Establishment of a Gene Expression System in Ochrobactrum anthropi

Genetic studies of Ochrobactrum anthropi are hindered by the lack of a suitable gene expression system. We constructed a set of vectors containing several promoters and a His tag fusion in the N terminus to facilitate protein detection and purification. The new vectors should significantly enhance the genetic manipulation and characterization of O. anthropi.     

D,L-Hydantoinase activity of an Ochrobactrum anthropi strain

AIMS: A microorganism with the ability to release methionine from D,L-(2-methylthioethyl) hydantoin (strain 245) was isolated from soil. The aim of this study was the identification of the strain and the adjustment of the conditions of growth and of the enzymatic reaction, in order to achieve high specific activities of bioconversion of the hydantoin. METHODS AND RESULTS: Strain 245 was identified as Ochrobactrum anthropi. The strain grew at alkaline pH (up to 10.0) and its hydantoinase activity was found to be inducible by the substrate D,L-(2-methylthioethyl) hydantoin. The enzyme is also alkalostable, with a pH optimum of 9.0. Under these conditions, hydantoinase activity was significantly enhanced and its half life prolonged when 200 mmol l-1 ammonium and phosphate were added. The addition of Ca2+, Na+, Cu2+, Co2+, Mg2+, Zn2+ or Fe3+ (0.5 mmol l-1) to the reaction mixture increased the hydantoinase activity of strain 245 up to tenfold after 24 h of incubation, compared with unamended controls. CONCLUSION: The adequate adjustment of some environmental parameters (pH, addition of inducer, presence of ammonium, phosphate, heavy metals and other ions) can considerably increase the D, L-hydantoinase activity of strain 245. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here set up the initial conditions for a further application of strain 245 in the production of methionine from hydantoine.     

Establishment of a gene expression system in Ochrobactrum anthropi

Genetic studies of Ochrobactrum anthropi are hindered by the lack of a suitable gene expression system. We constructed a set of vectors containing several promoters and a His tag fusion in the N terminus to facilitate protein detection and purification. The new vectors should significantly enhance the genetic manipulation and characterization of O. anthropi.     

Heavy metal biosorption by biomass of Ochrobactrum anthropi producing exopolysaccharide in activated sludge

The removal of chromium, cadmium and copper, toxic metals of high environmental priority due to their toxicity, from dilute aqueous solutions has been studied in the present work, applying a dead exopolysaccharide producing bacterium, Ochrobactrum anthropi, isolated from activated sludge. Particularly, the effect of pH, metal concentration and the effects of contact time were considered. Optimum adsorption pH values of chromium(VI), cadmium(II) and copper(II) were 2.0, 8.0 and 3.0 respectively. Experimental results also showed the influence of initial metal concentration on the metal uptake for dried biomass. Both the Freundlich and Langmuir adsorption models were suitable for describing the short-term biosorption of chromium(VI), cadmium(II) and copper(II) by O. anthropi.     

Reporter genes for real-time in vive monitoring of Ochrobactrum anthropi infection

Despite the increasing interest in Ochrobactrum anthropi as an emerging nosocomial pathogen resistant to most commonly used antimicrobials, relatively little is known about the pathogenesis and factors contributing to its virulence. Also, many aspects of interaction between Ochrobactrum spp. and their hosts remain unclear. The ability to monitor O. anthropi infection in the host will facilitate our understanding of the pathogenic mechanisms and will lead to better choices of antimicrobial or additional therapeutic strategies. We have demonstrated the ability to stably express three reporter genes (green fluorescence protein GFP, red fluorescence protein RFP and luciferase Lux) and track the infection in a J774A.1 murine macrophage cell line as well as in the BALB/c mouse. Our results suggest that these reporter genes should improve genetic studies in O. anthropi , particularly those aimed at understanding pathogenesis, virulence factors and host interaction.     

Effects of atrazine on Ochrobactrum anthropi membrane fatty acids.

Ochrobactrum anthropi is a gram-negative bacillus recognized as a human opportunist pathogen isolated in clinical specimens and not of clinical significance. We report a new aspect of this bacterium, that it has been isolated from activated sludge. In fact, it is able to grow on atrazine (2-chloro-4-ethylamino-6-isopropyl-amine-s-triazine) by utilizing it as the only source of carbon. Our results show that atrazine (0.03 g/liter) causes a dramatical increase in the degree of saturation of membrane fatty acids. Analysis and identification of bacterial fatty acids were performed by gas chromatography and gas chromatography-mass spectrometry techniques.     

Paraquat resistance of transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene

Transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene, which encodes a membrane transporter mediating resistance to paraquat, were generated. Transgenic plants displayed higher resistance against paraquat than wild-type plants, as estimated by plant viability, ion leakage and chlorophyll loss, but no resistance against other active oxygen generators, such as H2O2 and menadione. Moreover, lower levels of paraquat accumulated in transgenic plants, compared to wild-type plants, indicating that the PqrA protein detoxifies paraquat either via increased efflux or decreased uptake of the herbicide, but not by removing active oxygen species. The results collectively demonstrate that the bacterial paraquat resistance gene, pqrA, can be functionally expressed in plant cells, and utilized for the development of paraquat-resistant crop plants.     

Ammonium and methylammonium uptake in a fertilizer-degrading strain of Ochrobactrum anthropi

The transport of ammonium and methylammonium was studied in a strain of Ochrobactrum anthropi, a microorganism isolated from garden soil and able to degrade methyleneureas which are used as slow-release nitrogen fertilizer. The activity of both transport systems was determined using [¹⁴C]methylammonium. Differences between the two transport systems were observed with regard to their pH- and temperature dependence as well as their kinetic parameters and regulation during growth with various nitrogen sources. Ammonium transport was subject to repression by ammonium and to derepression in its absence, while the methylammonium carrier was induced in the presence of methylamine. The ammonium but not the methylammonium transport system was severely inhibited by ammonium, and metabolic poisons inhibited both uptake systems. The analysis of intracellular metabolites using thin-layer chromatography and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry indicated that methylammonium was rapidly metabolized to N-methylglutamate via -N-methylglutamine.     

Purification, Characterization, and Gene Cloning of Purine Nucleosidase from Ochrobactrum anthropi

A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K_m values being 11.8 to 56.3 μM. The optimal activity temperature and pH were 50°C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K_i and K_i′ values of 0.455 to 11.2 μM. Metal ion chelators inhibited activity, and the addition of Zn²⁺ or Co²⁺ restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH₂-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.     

Microbial conversion of xylose into useful bioproducts

Microorganisms can produce a number of different bioproducts from the sugars in plant biomass. One challenge is devising processes that utilize all of the sugars in lignocellulosic hydrolysates. D-xylose is the second most abundant sugar in these hydrolysates. The microbial conversion of D-xylose to ethanol has been studied extensively; only recently, however, has conversion to bioproducts other than ethanol been explored. Moreover, in the case of yeast, D-xylose may provide a better feedstock for the production of bioproducts other than ethanol, because the relevant pathways are not subject to glucose-dependent repression. In this review, we discuss how different microorganisms are being used to produce novel bioproducts from D-xylose. We also discuss how D-xylose could be potentially used instead of glucose for the production of value-added bioproducts.

     

Multidimensional proteomic analysis of the soluble subproteome of the emerging nosocomial pathogen Ochrobactrum anthropi

We report the first large-scale gel-free proteomic analysis of the soluble subproteome of the emerging pathogen Ochrobactrum anthropi. Utilizing our robust offline multidimensional protein identification protocol, a total of 57 280 peptides were initially identified utilizing automated MS/MS analysis software. We describe our investigation of the heuristic protein validation tool PROVALT and demonstrate its ability to increase the speed and accuracy of the curation process of large-scale proteomic datasets. PROVALT reduced our peptide list to 8517 identified peptides and further manual curation of these peptides led to a final list of 984 uniquely identified peptides that resulted in the positive identification of 249 proteins. These identified proteins were functionally classified and physiochemically characterized. A variety of typical "housekeeping" functions identified within the proteome included nucleic acid, amino and fatty acid anabolism and catabolism, glycolysis, TCA cycle, and pyruvate and selenoamino acid metabolism. In addition, a number of potential virulence factors of relevance to both plant and human disease were identified.     

Microbial oxidases catalyzing conversion of glycolaldehyde into glyoxal

The present paper reviews oxidases catalyzing conversion of glycolaldehyde into glyoxal. The enzymatic oxidation of glycolaldehyde into glyoxal was first reported in alcohol oxidases (AODs) from methylotrophic yeasts such as Candida and Pichia, and glycerol oxidase (GLOD) from Aspergillus japonicus, although it had been reported that these enzymes are specific to short-chain linear aliphatic alcohols and glycerol, respectively. These enzymes continuously oxidized ethylene glycol into glyoxal via glycolaldehyde. The AODs produced by Aspergillus ochraceus and Penicillium purpurescens also oxidized glycolaldehyde. A new enzyme exhibiting oxidase activity for glycolaldehyde was reported from a newly isolated bacterium, Paenibacillus sp. AIU 311. The Paenibacillus enzyme exhibited high activity for aldehyde alcohols such as glycolaldehyde and glyceraldehyde, but not for methanol, ethanol, ethylene glycol or glycerol. The deduced amino acid sequence of the Paenibacillus AOD was similar to that of superoxide dismutases (SODs), but not to that of methylotrophic yeast AODs. Then, it was demonstrated that SODs had oxidase activity for aldehyde alcohols including glycolaldehyde. The present paper describes characteristics of glycolaldehyde oxidation by those enzymes produced by different microorganisms.     

Isolation from Ochrobactrum anthropi of a Novel Class II 5-Enopyruvylshikimate-3-Phosphate Synthase with High Tolerance to Glyphosate

Applying the genomic library construction process and colony screening, a novel aroA gene encoding 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified, cloned, and overexpressed, and the enzyme was purified to homogeneity. Furthermore, site-directed mutagenesis was employed to assess the role of single amino acid residues in glyphosate resistance.The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19) is the sixth enzyme in the shikimate pathway, which is essential for the synthesis of aromatic amino acids and many aromatic metabolites in plants, fungi, and microorganisms (2, 11, 16), including apicomplexan parasites (22). It converts shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) to 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate. Interest in the characterization of EPSPS has increased significantly since the enzyme was identified as the primary target of the broad-spectrum, nonselective herbicide glyphosate [N-(phosphonomethyl)glycine] (25). Glyphosate is a competitive inhibitor with respect to PEP and binds adjacent to S3P in the active site of EPSPS, thereby mimicking an intermediate state of the ternary enzyme-substrate complex (23).Two classes of EPSPS, class I and II enzymes, sharing less than 30% amino acid similarity have been reported (9). Class I includes those found in plants and bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium, whose catalytic activity is inhibited at low micromolar concentrations of glyphosate (8). Class II EPSPS, found in Pseudomonas sp. strain PG2982, Agrobacterium tumefaciens strain CP4, Streptococcus pneumoniae, and Staphylococcus aureus, was distinguished by its ability to sustain efficient catalysis in the presence of high glyphosate concentrations (6, 9).Although a large number of AroA enzymes (EPSPS) have been cloned, identified, and tested as glyphosate resistant, only AroA variants derived from the A. tumefaciens strain CP4 have been successfully used commercially (9). To find a new enzyme similar to that of the AroA_{A. tumefaciens CP4}, in this study a highly glyphosate-tolerant strain from the rhizosphere of rice in a field where glyphosate is frequently used has been selected and identified on M9 minimal medium containing 200 mM glyphosate, and its 16S rRNA gene sequence confirmed that this strain was strongly related to Ochrobactrum anthropi (99.9%). Additionally, the aroA_{O. anthropi} gene was isolated and kinetic characteristics of the Ochrobactrum anthropi strain EPSP synthase were determined in this study.     

Role of Ser11 in the stabilization of the structure of Ochrobactrum anthropi glutathione transferase

GSTs (glutathione transferases) are a multifunctional group of enzymes, widely distributed and involved in cellular detoxification processes. In the xenobiotic-degrading bacterium Ochrobactrum anthropi, GST is overexpressed in the presence of toxic concentrations of aromatic compounds such as 4-chlorophenol and atrazine. We have determined the crystal structure of the GST from O. anthropi (OaGST) in complex with GSH. Like other bacterial GSTs, OaGST belongs to the Beta class and shows a similar binding pocket for GSH. However, in contrast with the structure of Proteus mirabilis GST, GSH is not covalently bound to Cys10, but is present in the thiolate form. In our investigation of the structural basis for GSH stabilization, we have identified a conserved network of hydrogen-bond interactions, mediated by the presence of a structural water molecule that links Ser11 to Glu198. Partial disruption of this network, by mutagenesis of Ser11 to alanine, increases the K(m) for GSH 15-fold and decreases the catalytic efficiency 4-fold, even though Ser11 is not involved in GSH binding. Thermal- and chemical-induced unfolding studies point to a global effect of the mutation on the stability of the protein and to a central role of these residues in zippering the terminal helix of the C-terminal domain to the starting helix of the N-terminal domain.