A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DhdR.

D-2-Hydroxyglutarate (D-2-HG) is a metabolite involved in many physiological metabolic processes. When D-2-HG is aberrantly accumulated due to mutations in isocitrate dehydrogenase or D-2-HG dehydrogenase, it functions in a pro-oncogenic manner and is thus considered a therapeutic target and biomarker in many cancers. In this study, DhdR from Achromobacter denitrificans NBRC 15125 is identified as an allosteric transcriptional factor that negatively regulates D-2-HG dehydrogenase expression and responds to the presence of D-2-HG. Based on the allosteric effect of DhdR, a D-2-HG biosensor is developed by combining DhdR with amplified luminescent proximity homogeneous assay (AlphaScreen) technology. The biosensor is able to detect D-2-HG in serum, urine, and cell culture medium with high specificity and sensitivity. Additionally, this biosensor is used to identify the role of D-2-HG metabolism in lipopolysaccharide biosynthesis of Pseudomonas aeruginosa, demonstrating its broad usages.

Bioconversion of chitin and concomitant production of chitinase and N-acetylglucosamine by novel Achromobacter xylosoxidans isolated from shrimp waste disposal area.

Marine pollution is a significant issue in recent decades, with the increase in industries and their waste harming the environment and ecosystems. Notably, the rise in shellfish industries contributes to tons of shellfish waste composed of up to 58% chitin. Chitin, the second most ample polymer next to cellulose, is insoluble and resistant to degradation. It requires chemical-based treatment or enzymatic hydrolysis to cleave the chitin polymers. The chemical-based treatment can lead to environmental pollution, so to solve this problem, enzymatic hydrolysis is the best option. Moreover, the resulting biopolymer by-products can be used to boost the fish immune system and also as drug delivery agents. Many marine microbial strains have chitinase producing ability. Nevertheless, we still lack an economical and highly stable chitinase enzyme for use in the industrial sector. So we isolate a novel marine bacterial strain Achromobacter xylosoxidans from the shrimp waste disposal site using chitin minimal medium. Placket-Burman and central composite design statistical models for culture condition optimisation predicted a 464.2 U/ml of chitinase production. The culture conditions were optimised for maximum chitinase production recording up to 467 U/ml. This chitinase from the A. xylosoxidans was 100% active at an optimum temperature of 45 °C (withstand up to 55 °C) and pH 8 with 80% stability. The HPLC analysis of chitinase degraded shellfish waste reveals a major amino acid profile composition-arginine, lysine, aspartic acid, alanine, threonine and low levels of isoleucine and methionine. These chitinase degraded products and by-products can be used as supplements in the aquaculture industry.

Efficient synthesis of ß-lactam antibiotics with in situ product removal by a newly isolated penicillin G acylase.

A penicillin G acylase (PGA) from Achromobacter xylosoxidans PX02 was newly isolated, and site-directed mutagenesis at three important positions αR141, αF142, ßF24 was carried out for improving the enzymatic synthesis of ß-lactam antibiotics. The efficient mutant ßF24A was selected, and the (Ps/Ph)ini (ratio between the initial rate of synthesis and hydrolysis of the activated acyl donor) dramatically increased from 1.42-1.50 to 23.8-24.1 by means of the optimization of reaction conditions. Interestingly, the efficient enzymatic synthesis of ampicillin (99.1% conversion) and amoxicillin (98.7% conversion) from a high concentration (600 mM) of substrate 6-APA in the low acyl donor/nucleus ratio (1.1:1) resulted in a large amount of products precipitation from aqueous reaction solution. Meanwhile, the by-product D-phenylglycine was hardly precipitated, and 93.5% yield of precipitated ampicillin (561 mM) and 94.6% yield of precipitated amoxicillin (568 mM) were achieved with high purity (99%), which significantly simplified the downstream purification. This was the first study to achieve efficient ß-lactam antibiotics synthesis process with in situ product removal, with barely any by-product formation. The effect enzymatic synthesis of antibiotics in aqueous reaction solution with in situ product removal provides a promising model for the industrial semi-synthesis of ß-lactam antibiotics.

A putative enoyl-CoA hydratase contributes to biofilm formation and the antibiotic tolerance of Achromobacter xylosoxidans.

Achromobacter xylosoxidans has attracted increasing attention as an emerging pathogen in patients with cystic fibrosis. Intrinsic resistance to several classes of antimicrobials and the ability to form robust biofilms in vivo contribute to the clinical manifestations of persistent A. xylosoxidans infection. Still, much of A. xylosoxidans biofilm formation remains uncharacterized due to the scarcity of existing genetic tools. Here we demonstrate a promising genetic system for use in A. xylosoxidans; generating a transposon mutant library which was then used to identify genes involved in biofilm development in vitro. We further described the effects of one of the genes found in the mutagenesis screen, encoding a putative enoyl-CoA hydratase, on biofilm structure and tolerance to antimicrobials. Through additional analysis, we find that a fatty acid signaling compound is essential to A. xylosoxidans biofilm ultrastructure and maintenance. This work describes methods for the genetic manipulation of A. xylosoxidans and demonstrated their use to improve our understanding of A. xylosoxidans pathophysiology.

Evaluation of the Immunochromatographic NG-Test Carba 5 for Rapid Identification of Carbapenemase in Nonfermenters.

The immunochromatographic assay NG-Test Carba 5 (NG-Biotech) was evaluated with a collection of 107 carbapenemase-producing nonfermenters (CP-NF) (55 Pseudomonas spp., 51 Acinetobacter spp., and 1 Achromobacter xylosoxidans isolate) and 61 carbapenemase-negative isolates. All KPC, VIM, and NDM carbapenemase producers tested were accurately detected. Of the 16 IMP variants tested, 6 (37.5%) variants were not detected. Considering the epidemiology of CP-NFs in France, the NG-Test Carba 5 would detect 89.4% of CP Pseudomonas spp. but only 12.9% of CP Acinetobacter spp.

Fitting replacement of signal peptide for highly efficient expression of three penicillin G acylases in E. coli.

High level expression of penicillin G acylase (PGA) in Escherichia coli is generally constricted by a complex maturation process and multiple limiting steps. In this study, three PGAs isolated from Providencia rettgeri (PrPGA), Alcaligenes faecalis (AfPGA), and Achromobacter xylosoxidans (AxPGA) were efficiently expressed in E. coli by replacing with applicable signal peptide. Different bottlenecks of the expression process were analyzed for PrPGA, AfPGA, and AxPGA. Subsequently, five efficient signal peptides, including OmpA, pelB, Lpp, PhoA, and MalE, were used to replace the original signal peptides of the PGAs. With respect to AfPGA and AxPGA, translocation was the primary limitation, and the use of pelB signal peptide effectively overcame this barrier. For PrPGA, which was almost not expressed in wild type, the translation initiation efficiency was optimized by replacing with MalE signal peptide. In addition, low temperature (20 °C) slowed down the transcription and translation, thereby facilitating the posttranslational process and preventing the formation of inclusion bodies. Furthermore, combined induction with IPTG and arabinose not only enhanced the cell density but also remarkably improved the expression of PGAs. Final specific activities of the three PGAs reached 2100 (PrPGA), 9200 (AfPGA), and 1400 (AxPGA) U/L/OD600, respectively. This simple and robust strategy by fitting replacement of signal peptide might dramatically improve the expression of PGAs from various bacteria, which was significant in the production of many valuable ß-lactam antibiotics.

Antioxidant enzymes and reactive oxygen species level of the Achromobacter xylosoxidans bacteria during hydrocarbons biotransformation.

The level of catalase and superoxide dismutase induction, as well as generation of superoxide anion radical in cells and accumulation of hydrogen peroxide in the culture medium were researched in three strains of oil-degrading bacteria Achromobacter xylosoxidans at cultivation in rich nutrient medium and in the media with hydrocarbons as the only source of carbon. The effects of pentane, decane, hexadecane, cyclohexane, benzene, naphthalene and diesel fuel were evaluated. It was determined that in the microbial cell on media with hydrocarbons, the generation of superoxide anion radical increases, accumulation of hydrogen peroxide and induction of superoxide dismutase synthesis occur, and catalase activity is reduced. Oxidative stress in the cells of A. xylosoxidans was caused by biotransformation of all the studied hydrocarbons. The most pronounced effect was observed at incubation of bacteria with cyclohexane, pentane, diesel fuel, benzene and naphthalene.

Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding.

Metallo-ß-lactamases (MBLs) threaten the effectiveness of ß-lactam antibiotics, including carbapenems, and are a concern for global public health. ß-Lactam/ß-lactamase inhibitor combinations active against class A and class D carbapenemases are used, but no clinically useful MBL inhibitor is currently available. Tripoli metallo-ß-lactamase-1 (TMB-1) and TMB-2 are members of MBL subclass B1a, where TMB-2 is an S228P variant of TMB-1. The role of S228P was studied by comparisons of TMB-1 and TMB-2, and E119 was investigated through the construction of site-directed mutants of TMB-1, E119Q, E119S, and E119A (E119Q/S/A). All TMB variants were characterized through enzyme kinetic studies. Thermostability and crystallization analyses of TMB-1 were performed. Thiol-based inhibitors were investigated by determining the 50% inhibitory concentrations (IC50) and binding using surface plasmon resonance (SPR) for analysis of TMB-1. Thermostability measurements found TMB-1 to be stabilized by high NaCl concentrations. Steady-state enzyme kinetics analyses found substitutions of E119, in particular, substitutions associated with the penicillins, to affect hydrolysis to some extent. TMB-2 with S228P showed slightly reduced catalytic efficiency compared to TMB-1. The IC50 levels of the new thiol-based inhibitors were 0.66 µM (inhibitor 2a) and 0.62 µM (inhibitor 2b), and the equilibrium dissociation constant (KD ) of inhibitor 2a was 1.6 µM; thus, both were more potent inhibitors than l-captopril (IC50 = 47 µM; KD = 25 µM). The crystal structure of TMB-1 was resolved to 1.75 Å. Modeling of inhibitor 2b in the TMB-1 active site suggested that the presence of the W64 residue results in T-shaped π-π stacking and R224 cation-π interactions with the phenyl ring of the inhibitor. In sum, the results suggest that residues 119 and 228 affect the catalytic efficiency of TMB-1 and that inhibitors 2a and 2b are more potent inhibitors for TMB-1 than l-captopril.

[Cloning and expression of an esterase gene from a new strain capable of enantioselective hydrolyzing methyl (R,S)-N-(2,6-dimethylphenyl) alaninate].

Objective: We screened and identified a strain capable of enantioelectively hydrolyzing methyl (R,S)-N-(2,6-dimethylphenyl) alaninate (MAP), a key intermediate for the synthesis of metalaxyl, followed by cloning and expressing the esterase in E. coli. Methods: We used MAP as the sole carbon source in the medium inoculated with an active sludge specimen to enrich the target microorganism. The strain with the highest hydrolysis activity and enatioelectivity was identified by 16S rRNA sequence analysis, morphological observation and physiological and biochemical properties. From the gene library of the strain, the DNA sequence fragment containing the target gene was found. By DNA sequence analysis and PCR amplification, the esterase gene was obtained. It was ligated with plasmids pET28a (+), then transformed into E. coli BL21Gold (DE3) plysS. Results: We isolated a gram-negative bacterial strain capable of enantioelective hydrolyzing MAP. It was identified as Achromobacter denitrificans. From its gene library, the esterase gene named EHest was found. The recombinant EHest-pET28a(+)-BL21Gold (DE3) plysS was constructed. The recombinant expressed esterase (EHesterase) capable of catalyzing enatioelective hydrolysis of methyl (R,S)-N-(2,6-dimethylphenyl) alaninate. Its size was 27 kDa. The expression activity was 27.1 times as high as that in the original strain. Hydrolysis of MAP (5% M/V) by EHesterase for 1 h at 37℃, the substrate conversion was 29.5% and ee p of the product acid (major in R configuration) was 85.1%. The optimum pH was 9.0 and temperature 50℃. Conclusions: A new isolate Achromobacter denitrificans 1104 capable of enantioelective hydrolyzing MAP was found and identified.

Sequence and structure-based comparative analysis to assess, identify and improve the thermostability of penicillin G acylases.

Penicillin acylases are enzymes employed by the pharmaceutical industry for the manufacture of semi-synthetic penicillins. There is a continuous demand for thermostable and alkalophilic enzymes in such applications. We have carried out a computational analysis of known penicillin G acylases (PGAs) in terms of their thermostable nature using various protein-stabilizing factors. While the presence of disulfide bridges was considered initially to screen putative thermostable PGAs from the database, various other factors such as high arginine to lysine ratio, less content of thermolabile amino acids, presence of proline in ß-turns, more number of ion-pair and other non-bonded interactions were also considered for comparison. A modified consensus approach designed could further identify stabilizing residue positions by site-specific comparison between mesostable and thermostable PGAs. A most likely thermostable enzyme identified from the analysis was PGA from Paracoccus denitrificans (PdPGA). This was cloned, expressed and tested for its thermostable nature using biochemical and biophysical experiments. The consensus site-specific sequence-based approach predicted PdPGA to be more thermostable than Escherichia coli PGA, but not as thermostable as the PGA from Achromobacter xylosoxidans. Experimental data showed that PdPGA was comparatively less thermostable than Achromobacter xylosoxidans PGA, although thermostability factors favored a much higher stability. Despite being mesostable, PdPGA being active and stable at alkaline pH is an advantage. Finally, several residue positions could be identified in PdPGA, which upon mutation selectively could improve the thermostability of the enzyme.

Development of a growth-dependent selection system for identification of L-threonine aldolases.

Threonine aldolases (TAs) are useful enzymes for the synthesis of ß-hydroxy-α-amino acids due to their capability to catalyze asymmetric aldol reactions. Starting from two prochiral compounds, an aldehyde and glycine, two chiral stereocenters were formed in a single step via C-C bond formation. Owing to poor diastereoselectivity and low activity, the enzymatic synthesis of ß-hydroxy-α-amino acids by TAs is still a challenge. For identification of new TAs, a growth-dependent selection system in Pseudomonas putida KT2440 has been developed. This bacterium is able to use aromatic compounds such as benzaldehyde, which is the cleavage product of the TA-mediated retro-aldol reaction of phenylserine, as sole carbon source via the ß-ketoadipate pathway. With DL-threo-ß-phenylserine as sole carbon source, this strain showed only slight growth in minimal medium. This growth deficiency can be restored by introducing and expressing genes encoding TAs. In order to develop a highly efficient selection system, the gene taPp of P. putida KT2440 encoding a TA was successfully deleted by replacement with an antibiotic resistance cassette. Different growth studies were carried out to prove the operability of the selection system. Genes encoding for L- and D-specific TAs (L-TA genes of Escherichia coli (ltaE) and Saccharomyces cerevisiae (gly1) and D-TA gene of Achromobacter xylosoxidans (dtaAX)) were introduced into the selection strain P. putida KT2440ΔtaPp, followed by cultivation on minimal medium supplemented with DL-threo-ß-phenylserine. The results demonstrate that only the selection strains with plasmid-encoded L-TAs were able to grow on this racemic amino acid, whereas the corresponding strain harboring the gene coding for a D-specific TA showed no growth. In summary, it can be stated that a powerful screening tool was developed to identify easily by growth new L-specific threonine aldolases or other enzymes from genomic or metagenomic libraries liberating benzaldehyde.

Elucidation of fluoranthene degradative characteristics in a newly isolated Achromobacter xylosoxidans DN002.

Strain DN002 isolated from petroleum-contaminated soil was identified as Achromobacter xylosoxidans based on morphological and biochemical properties and 16S rRNA phylogeny, and investigated for its potential to utilize numerous polycyclic aromatic hydrocarbons (PAHs) such as fluoranthene and pyrene as sole carbon and energy resource. Biodegradation studies showed that 500 mg(·)l(-1)fluranthene was degraded to 35.6 ± 0.3 mg(·)l(-1) by DN002 after 14 days incubation. During fluoranthene biodegradation, catechol 2,3 dioxygenase (C23O) activity was augmented 1.5 times more than catechol 1,2 dioxygenase (C12O), which indicated that C23O played a major role in fluoranthene degradation by DN002. Protein profiles were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional electrophoresis then analyzed by mass spectrometry induced by fluoranthene; a molecular mass range of 18 ∼ 66 kDa proteins were found upregulated compared with the uninduced control sample, including multiple isoenzymes of ß-oxidation and dehydrogenases as well as dioxygenases. Besides, some new proteins, i.e., dihydrolipoamide succinyltransferase and aldehyde dehydrogenase family proteins and isocitrate lyase were also synthesized.

A novel A3 group aconitase tolerates oxidation and nitric oxide.

Achromobacter denitrificans YD35 is an NO2 (-)-tolerant bacterium that expresses the aconitase genes acnA3, acnA4, and acnB, of which acnA3 is essential for growth tolerance against 100 mm NO2 (-). Atmospheric oxygen inactivated AcnA3 at a rate of 1.6 × 10(-3) min(-1), which was 2.7- and 37-fold lower compared with AcnA4 and AcnB, respectively. Stoichiometric titration showed that the [4Fe-4S](2+) cluster of AcnA3 was more stable against oxidative inactivation by ferricyanide than that of AcnA4. Aconitase activity of AcnA3 persisted against high NO2 (-) levels that generate reactive nitrogen species with an inactivation rate constant of k = 7.8 × 10(-3) min(-1), which was 1.6- and 7.8-fold lower than those for AcnA4 and AcnB, respectively. When exposed to NO2 (-), the acnA3 mutant (AcnA3Tn) accumulated higher levels of cellular citrate compared with the other aconitase mutants, indicating that AcnA3 is a major producer of cellular aconitase activity. The extreme resistance of AcnA3 against oxidation and reactive nitrogen species apparently contributes to bacterial NO2 (-) tolerance. AcnA3Tn accumulated less cellular NADH and ATP compared with YD35 under our culture conditions. The accumulation of more NO by AcnA3Tn suggested that NADH-dependent enzymes detoxify NO for survival in a high NO2 (-) milieu. This novel aconitase is distributed in Alcaligenaceae bacteria, including pathogens and denitrifiers, and it appears to contribute to a novel NO2 (-) tolerance mechanism in this strain.

Presence of OXA-type enzymes in Achromobacter insuavis and A. dolens.

The accurate species identification of Achromobacter isolates is difficult and the clinical isolates of this genus are mostly referred as A. xylosoxidans. Here, we report new OXA variants in 2 isolates identified as A. insuavis (A114, A79) and 1 isolate identified as A. dolens (A336). These results suggest that different bla OXA genes are ubiquitous in the different species of Achromobacter spp. The role of the other species of Achromobacter in clinical samples needs to be reevaluated, and the proper identification is absolutely necessary to understand the epidemiology of this genus.

Efficient synthesis of (R)-3-hydroxypentanenitrile in high enantiomeric excess by enzymatic reduction of 3-oxopentanenitrile.

(R)-3-Hydroxypentanenitrile (HPN) is an important intermediate in the synthesis of an immunosuppressive inosine 5'-monophosphate dehydrogenase inhibitor. An efficient enzymatic procedure for the synthesis of (R)-HPN with over 99 % enantiomeric excess using a novel acetoacetyl-CoA reductase (AdKR) from Achromobacter denitrificans was successfully established. Many microorganisms are known to reduce 3-oxopentannitrile (KPN) to (R)-HPN. An enzyme from A. denitrificans partially purified using ion exchange chromatography reduced KPN to (R)-HPN with high enantioselectivity. The AdKR gene was cloned and sequenced and found to comprise 738 bp and encode a polypeptide of 26,399 Da. The deduced amino acid sequence showed a high degree of similarity to those of other putative acetoacetyl-CoA reductases and putative 3-ketoacyl-ACP reductases. The AdKR gene was singly expressed and coexpressed together with a glucose dehydrogenase (GDH) as a coenzyme regenerator in Escherichia coli under the control of the lac promoter. (R)-HPN was synthesized with over 99 % e.e. using a cell-free extract of recombinant E. coli cells coexpressing AdKR and GDH.

Putative P1B-type ATPase from the bacterium Achromobacter xylosoxidans A8 alters Pb2+/Zn2+/Cd2+-resistance and accumulation in Saccharomyces cerevisiae.

PbtA, a putative P(1B)-type ATPase from the Gram-negative soil bacterium Achromobacter xylosoxidans A8 responsible for Pb(2+)/Zn(2+)/Cd(2+)-resistance in Escherichia coli, was heterologously expressed in Saccharomyces cerevisiae. When present in Zn(2+)- and Pb(2+)/Cd(2+)-hypersensitive S. cerevisiae strains CM137 and DTY168, respectively, PbtA was able to restore Zn(2+)- and Pb(2+)-resistant phenotype. At the same time, the increase of Pb, Zn, and Cd accumulation in yeast was observed. However, Cd(2+)-tolerance of the pbtA-bearing yeasts dramatically decreased. The PbtA-eGFP fusion protein was localized primarily in the tonoplast and also in the plasma membrane and the perinuclear region corresponding to the endoplasmic reticulum at later growth stages. This indicates that PbtA protein is successfully incorporated into membranes in yeasts. Since PbtA caused a substantial increase of Pb(2+)/Zn(2+)-resistance and accumulation in baker's yeast, we propose its further use for the genetic modification of suitable plant species in order to obtain an effective tool for the phytoremediation of sites polluted by toxic transition metals.

Achromobacter denitrificans strain YD35 pyruvate dehydrogenase controls NADH production to allow tolerance to extremely high nitrite levels.

We identified the extremely nitrite-tolerant bacterium Achromobacter denitrificans YD35 that can grow in complex medium containing 100 mM nitrite (NO2(-)) under aerobic conditions. Nitrite induced global proteomic changes and upregulated tricarboxylate (TCA) cycle enzymes as well as antioxidant proteins in YD35. Transposon mutagenesis generated NO2(-)-hypersensitive mutants of YD35 that had mutations at genes for aconitate hydratase and α-ketoglutarate dehydrogenase in the TCA cycle and a pyruvate dehydrogenase (Pdh) E1 component, indicating the importance of TCA cycle metabolism to NO2(-) tolerance. A mutant in which the pdh gene cluster was disrupted (Δpdh mutant) could not grow in the presence of 100 mM NO2(-). Nitrite decreased the cellular NADH/NAD(+) ratio and the cellular ATP level. These defects were more severe in the Δpdh mutant, indicating that Pdh contributes to upregulating cellular NADH and ATP and NO2(-)-tolerant growth. Exogenous acetate, which generates acetyl coenzyme A and then is metabolized by the TCA cycle, compensated for these defects caused by disruption of the pdh gene cluster and those caused by NO2(-). These findings demonstrate a link between NO2(-) tolerance and pyruvate/acetate metabolism through the TCA cycle. The TCA cycle mechanism in YD35 enhances NADH production, and we consider that this contributes to a novel NO2(-)-tolerating mechanism in this strain.

Modification of surface and enzymatic properties of Achromobacter denitrificans and Stenotrophomonas maltophilia in association with diesel oil biodegradation enhanced with alkyl polyglucosides.

The article concerns the influence of selected alkyl polyglucosides on biodegradation, cell surface and enzymatic properties of Stenotrophomonas maltophilia and Achromobacter denitrificans. The biodegradation of diesel oil depends on several factors including type and the amount of surfactant as well as bacterial genera used in the process. Nevertheless, a careful selection of these variables must be made as some bacterial strains prefer to use surfactants as their carbon source. This leads to the lowered biodegradation of diesel oil as can be observed for the tested S. maltophilia strain. Alkyl polyglucosides influenced the cell surface properties of both of the tested strains in slightly different ways. Especially for A. denitrificans, for which the hydrophobicity increased with concentration of both--Lutensol GD 70 and Glucopon 215 in diesel oil-surfactant systems. Moreover, judging by the efficiency of biodegradation, the most effective process was observed in the presence of Lutensol GD 70 (240 and 360 mg L(-1)) with biodegradation rising from 32% (without surfactant) to 68%. No such relation was observed for S. maltophilia.

Cloning, expression and characterization of D-aminoacylase from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173.

D-Aminoacylase catalyzes the conversion of N-acyl-D-amino acids to d-amino acids and fatty acids. The aim of this study was to identify the D-aminoacylase gene from Achromobacter xylosoxidans subsp. denitrificans ATCC 15173 and investigate the biochemical characterization of the enzyme. A previously uncharacterized D-aminoacylase gene (ADdan) from this organism was cloned and sequenced. The open reading frame (ORF) of ADdan was 1467 bp in size encoding a 488-amino acid polypeptide. ADdan, with a high amino acid similarity to N-acyl-D-aspartate amidohydrolase from Alcaligenes A6, showed relatively low sequence similarities to other characterized D-aminoacylases. The recombinant ADdan protein was expressed in Escherichia coli BL21 (DE3) using pET-28a with a T7 promoter. The enzyme was purified in a single chromatographic step using nickel affinity gel column. The molecular mass of the expressed protein, calculated by SDS-PAGE, was about 52 kDa. The purified ADdan showed optimal activity at pH 8.0 and 50°C, and was stable at pH 6.0-8.0 and up to 45°C. Its activity was inhibited by Cu(2+), Fe(2+), Ca(2+), Mn(2+), Ni(2+), Zn(2+) and Hg(2+), whereas Mg(2+) had no significant influence on this recombinant D-aminoacylase. This is the first report on the characterization of D-aminoacylase with activity towards both N-acyl derivatives of neutral D-amino acids and N-acyl-D-aspartate. The characteristics of ADdan could prove to be of interest in industrial production of D-amino acids.

Molecular characterization of IMP-type metallo-ß-lactamases among multidrug-resistant Achromobacter xylosoxidans.

OBJECTIVES: To analyse the metallo-ß-lactamase (MBL) genes and their genetic environments in clinical isolates of Achromobacter xylosoxidans. METHODS: From January 2004 to December 2010, four MBL-producing, multidrug-resistant (MDR) A. xylosoxidans strains were isolated and analysed at a tertiary care university hospital in Japan. Species-level identification was confirmed by 16S ribosomal RNA gene sequencing. Molecular typing was performed using PFGE, the presence of MBL genes was detected using PCR, and integron gene cassettes were examined by cloning and sequence analysis of integron PCR products. The plasmids obtained from individual isolates were analysed based on EcoRI restriction patterns, Southern hybridizations using digoxigenin-labelled probes for bla(IMP-1) and bla(IMP-19) as well as conjugation and transformation experiments. RESULTS: No clonal relationship was found among the four A. xylosoxidans isolates. Three isolates harboured bla(IMP-1) and one isolate harboured bla(IMP-19). These MBL genes were carried on class 1 integrons. Four different class 1 integron gene cassette arrays were found, including orf1-bla(IMP-1)-aacA4, bla(IMP-1)-bla(IMP-1)-nit1/nit2-catB3-bla(IMP-1)-bla(IMP-1), aacA4-bla(IMP-1) and bla(IMP-19). The restriction pattern of the plasmid DNA obtained from each isolate was unique and the hybridization analyses revealed the presence of each MBL gene within a plasmid. Moreover, all of the plasmids carrying an MBL gene could be transformed into Escherichia coli HST08. CONCLUSIONS: This study provides genetic evidence for the existence of IMP-type MBL genes in MDR A. xylosoxidans isolates. Moreover, the present findings provide evidence that A. xylosoxidans can receive IMP-type MBL genes via plasmid-mediated transfer, which contributes to their carbapenem resistance.