New and highly active microbial phosphotriesterase sources.

Many toxic insecticides used worldwide as well as some chemical warfare agents are phosphotriester derivatives. Therefore, detoxification of organophosphorus compounds has become the subject of many studies and in particular bioremediation, based on the phosphotriesterase catalysed hydrolysis of these compounds, has shown to be an effective and ecological methodology. In order to identify new bacterial phosphotriesterases, a simple and sensitive fluorimetric screening method on solid media was employed that allowed the selection of six strains with phosphotriesterase activity. Since pH and temperature are important parameters for bioremediation of contaminated soils and waters, the influence of these variables on the rate of the enzymatic hydrolysis was assessed. This study afforded notable results, being the most remarkable one the increased activity exhibited by Nocardia asteroides and Streptomyces setonii strains at 50°C, 7 and 30 times higher than at 30°C, respectively. Compared with the results obtained with Brevundimonas diminuta, whose activity is usually considered as reference, an increase of 26 and 75 times is observed, respectively.

Exploration of respiratory chain of Nocardia asteroides: purification of succinate quinone oxidoreductase.

Nocardia asteroides is a pathogenic bacterium that causes severe pulmonary infections and plays a vital role in HIV development. Its electron transport chain containing cytochromes as electron carriers is still undiscovered. Information regarding cytochromes is important during drug synthesis based on cytochrome inhibitions. In this study we explored the electron transport of N. asteroides. Spectroscopic analysis of cytoplasm and membranes isolated from N. asteroides indicates the presence of soluble cytochrome-c, complex-II and the modified a(1)c(1) complex as the terminal oxidase. The molecular weight of the respiratory complex-II isolated and purified from the given bacterium was 103 kDa and was composed of three subunits, of 14, 26 and 63 kDa. Complex-II showed symmetrical α-absorption peaks at 561 nm in the reduced state. Spectral analysis revealed the presence of only one heme b molecule (14-kDa subunit) in complex-II, which was confirmed by heme staining. Heme b content was found to be 9.5 nmol/mg in complex-II. The electron transport chain of N. asteroides showed the presence of soluble cytochrome-c, cytochrome-a(1)c(1) and cytochrome-b.

Characterization of dopamine-depleting activity of Nocardia asteroides strain GUH-2 culture filtrate on PC12 cells.

Experimental infection of BALB/c mice with the Gram-positive bacterium Nocardia asteroides (strain GUH-2) results in life-long movement abnormalities including head shaking and spinning when held by the tail. The head shaking is temporarily inhibited by treatment with dopamine's precursor levodopa, suggesting that abnormalities in dopaminergic neurotransmission may be involved in these movement abnormalities. Cell-free filtrates from N. asteroides cultures induce > 70% dopamine depletion in rat pheochromocytoma PC12 cells, suggesting that Nocardia's effects on dopamine neurons may result in part from secreted factors. The nature of this dopamine-depleting activity was examined in the present study. Dopamine-depleting activity in N. asteroides culture filtrate was resistant to heat (100 degrees C x 30 min), proteases, and chloroform extraction, and was present in a low molecular mass (< 3 kDa) fraction. It was partially inhibited by decreasing (to 4.0) or increasing (to 10.0) the filtrate pH. GUH-2 filtrate increased cellular lactate dehydrogenase release by only 2%, and induced apoptotic morphology in only 11% of PC12 cells, suggesting that dopamine-depleting activity was not due to either cell injury or induction of apoptosis. These results suggest that a protease-resistant, low molecular mass substance secreted by N. asteroides may be responsible for its dopamine-depleting effects.

Molecular and biochemical analysis of AST-1, a class A beta-lactamase from Nocardia asteroides sensu stricto.

A beta-lactamase gene was cloned from a Nocardia asteroides sensu stricto clinical isolate. A recombinant plasmid, pAST-1, expressed the beta-lactamase AST-1 in Escherichia coli JM109. Its pI was 4.8, and its relative molecular mass was 31 kDa. E. coli JM109(pAST-1) was resistant to penicillins and narrow-spectrum cephalosporins. The beta-lactamase AST-1 had a restricted hydrolytic activity spectrum. Its activity was partially inhibited by clavulanic acid but not by sulbactam and tazobactam. AST-1 is an Ambler class A beta-lactamase sharing 65% amino acid identity with beta-lactamase FAR-1, the most closely related enzyme.

Antimicrobial susceptibility profile of soil isolates of Nocardia asteroides from Kuwait.

OBJECTIVES: To find the antimicrobial susceptibility profile of 42 soil isolates of Nocardia asteroides against 14 antimicrobial agents representing beta-lactams, aminoglycosides, ciprofloxacin, minocycline, erythromycin and third generation cephalosporins. METHODS: The antimicrobial susceptibility was determined by the disk diffusion method using Mueller-Hinton agar medium. A homogeneous suspension giving an inoculum of 106-108 CFU/mL was used to streak the plates. The zone of inhibition was read after 36-48 h of incubation at 37 degrees C. RESULTS: All the soil isolates of N. asteroides were susceptible to amikacin, imipenem and tobramycin. Susceptibility to cephalosporins was quite variable; 86% of the isolates were susceptible to cefotaxime, 57% to ceftriaxone and 40% to cefamandole. Fifty-seven per cent of the isolates showed intermediate susceptibility to cefamandole, 33% to ceftriaxone and 5% to cefotaxime. Ninety-three per cent of the isolates were resistant to sulfamethoxazole alone or in combination with trimethoprim. CONCLUSIONS: The study reports a wide variation in the antimicrobial susceptibility profile of soil isolates of N. asteroides originating from a single geographical area. Of interest is the finding that over 90% of N. asteroides isolates were resistant to sulfamethoxazole without any previous exposure to this drug. This may have serious therapeutic implications as sulphonamides or the combination of trimethoprim-sulfamethoxazole is the therapy of choice for nocardiosis. Demonstration of resistance to beta-lactam antibiotics may be attributed to the presence of beta-lactamases which was detectable in > 90% of the soil strains of N. asteroides. The study underscores the importance of antimicrobial susceptibility testing for clinical isolates of Nocardia since individual strains show considerable differences in their susceptibility patterns necessitating therapeutic adjustments.

A study of the enzymatic profile of soil isolates of Nocardia asteroides.

In this study, using the API-ZYM system, we have reported the enzyme profile of 42 soil strains and 2 clinical strains of Nocardia asteroides isolated locally. Of the 19 enzymes tested, only 7 were demonstrable in over 90% of the soil isolates. These included alkaline phosphatase, esterase lipase, leucine arylamidase, acid phosphatase, phosphohydrolase, alpha-glucosidase and beta-glucosidase. In addition, beta-galactosidase activity was demonstrated in all the strains by the O-nitrophenyl-beta-D-galactopyranoside (ONPG) test. The enzymes which were not demonstrable in > 95% of the strains included valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, alpha-galactosidase, beta-glucoronidase, N-acetyl-beta-glucosaminidase, alpha-mannosidase and alpha-fucosidase. With the exception of valine arylamidase, which was lacking in all but one isolate, the enzyme profiles of the soil isolates were comparable with the clinical isolates of N. asteroides reported in previous studies. The reasons for this difference in the two sets of isolates is not clear. The study reinforces the view that specific differences in the enzymatic profiles of Nocardia species could be used for their rapid identification. However, more extensive studies are needed to establish the reproducibility of this method. To the best of our knowledge, this is the first study of the enzymatic profile of soil isolates of N. asteroides originating from a single geographic region.

Non-inducible, mainly cell-associated beta-lactamase from Nocardia asteroides strain 108.

The beta-lactamase of the soil-borne strain 108 (parental strain) of Nocardia asteroides is a non-inducible enzyme mainly associated with the cells; it can be efficiently extracted by ultrasonication and SDS treatment. Crude enzyme preparations showed penicillinase and cephalosporinase activity. The kinetics of beta-lactamase production and in-vitro susceptibility to combinations of beta-lactam antibiotics plus beta-lactamase inhibitors have been studied in two stable overproducer mutants (A14 and B1) obtained by mutagenization of the parental strain with nitrosoguanidine. The cell-associated enzyme increased with bacterial growth in parental and mutant strains and was particularly abundant in stationary phase cells. The beta-lactamase inhibitors sulbactam and clavulanic acid decreased MIC values of penicillins more efficiently in the parental strain than in mutants, thus indicating some involvement of the enzyme in the resistance of N. asteroides strain 108 to beta-lactam antibiotics.

Novel method for rapid identification of Nocardia species by detection of preformed enzymes.

The purpose of the present study was to devise a method for the identification of Nocardia species that is more technically simple, accurate, and rapid than current standard methods of identification. We focused on a commercial bacteria identification system that contained chromogenic test substrates. Two MicroScan products were selected for use in the study on the basis of their content of chromogenic and conventional substrates. They were the Rapid Anaerobe Identification and the HNID panels. A total of 85 strains of Nocardia representing five species were used in the study. All isolates were identified as Nocardia species by the use of standard methods. The beta-naphthylamide-labeled substrate L-pyrrolidonyl-beta-naphthylamide (PYR), the nitrophenyl-labeled substrate p-nitrophenyl-alpha-D-mannopyranoside (MNP), and indoxyl phosphate were found to be useful for identification purposes. N. farcinica and N. nova were the only species positive for PYR, whereas N. brasiliensis was the only species that hydrolyzed MNP. All strains of N. brasiliensis, N. otitidiscavarium, and N. farcinica were positive for indoxyl phosphate, whereas strains of N. nova and N. asteroides sensu stricto were always negative. Agreement between the standard and enzymatic identification methods was 100%. In summary, detection of preformed enzymes appears to be a simple and reproducible method for the identification of Nocardia spp.

Molecular characterization of a surface-exposed superoxide dismutase of Mycobacterium avium.

Mycobacterium avium is an intracellular pathogen capable of growing inside the phagosomal compartment of macrophages. In this work, we characterized the superoxide dismutase of M. avium, as a putative candidate to resist the oxidative stress. The gene sodA encoding superoxide dismutase (SOD:EC1.15.1.1) from Mycobacterium avium TMC724 was cloned and sequenced. It encodes a 23 kDa protein (207 aminoacids) showing identity with the Mycobacterium leprae SOD (91%) and the M. tuberculosis SOD (83%). This enzyme was functionally expressed in both Escherichia coli and Mycobacterium smegmatis, and identified as a manganese (Mn) SOD on the basis of sequence comparison with other MnSODs from different organisms, and by activity inhibition studies. By indirect immunogold labeling of M. avium with a mAb directed against M. leprae SOD, the enzyme was found to be exposed at the cell surface of M. avium. It was also shown that SOD was released in supernates of M. avium TMV724 during exponential growth, suggesting a role of this enzyme during interactions with the environment. When SOD was expressed in the non-pathogenic M. smegmatis, it was also exposed at the surface of bacteria and released in supernates, but this was not sufficient to protect this recombinant mycobacterium from the killing mechanisms of macrophages.

Isolation, sequencing and expression of the superoxide dismutase-encoding gene (sod) of Nocardia asteroides strain GUH-2.

Nocardia asteroides (Na) superoxide dismutase (SOD) has been implicated as a virulence factor that allows the organism to survive intracellular killing by phagocytic cells. A full-length Na sod gene from a pathogenic strain of Na (strain GUH-2) was cloned from a recombinant phage library using the Mycobacterium tuberculosis (Mt) sod gene (Mt sod) as a probe. The promoter region and structural gene (624 bp) of Na sod was sequenced and nucleotide sequence comparisons reveal 77% homology with Mt sod. The Na sod gene also shares considerable sequence homology with sod of other mycobacterial species. In addition, conserved amino acid (aa) sequences important for metal binding indicate that Mn2+ is the preferred metal ion ligand for Na SOD. An Na sod expression plasmid, pYEX1, under transcriptional control of the Mt hsp70 promoter (pY6013), produced a 25-kDa protein product which showed SOD activity when stained in a native polyacrylamide gel and reacted with rabbit polyclonal antibody specific for Na SOD by Western blot. pYEX1, via transformation, was able to complement an Escherichia coli double sodAB mutant deficient in SOD production in the presence of paraquat (methyl viologen) which stimulates the production of superoxide radicals.

Purification and properties of glutamine synthetase from Nocardia asteroides.

Glutamine synthetase (GS, EC 6.3.1.2) from Nocardia asteroides was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-150, and DEAE-Sepharose chromatography. The native molecular weight of the purified enzyme was determined to be 720 kDa. SDS-PAGE analysis of the purified preparation revealed a single band corresponding to 59 kDa, indicating the possible presence of 12 identical subunits. The divalent cations Mn2+ and Mg2+ were found to be essential for optimal transferase and biosynthetic activity, respectively. The optimal pH and temperature for both activities of the enzyme were found to be 7.2 and 50 degrees C. Amino acids such as L-alanine, glycine, and aspartate inhibited the GS activity. The Km values for the substrates of the biosynthetic reaction ATP, glutamate, and ammonium chloride were found to be 400 microM, 7.7 mM, and 200 microM, respectively. Addition of ammonium chloride to the nitrogen-limited culture resulted in a decrease of GS transferase and biosynthetic activities. Phosphodiesterase treatment of the extract from ammonia-shocked cultures showed an increase in GS transferase activity. The results indicate the possible regulation of GS by covalent modification.

Application of the API YeastIdent system in determining the enzymatic profiles of Nocardia asteroides isolates.

Glucose represses production of ammonium in many clinical isolates of Nocardia asteroides growing on bromcresolpurple casein glucose agar. Strains exhibiting this property are designated as group A, while group B represents isolates showing a high degree of proteolytic activity and a resulting rapid increase in pH. Twenty isolates of N. asteroides were characterized as group A or B. Enzymatic profiles obtained using the API YeastIdent system showed significant enzymatic variation between 12 group B and 8 group A isolates. Proteolytic enzymes which most varied in activity between groups were glycine aminopeptidase, histidine aminopeptidase and leucyl glycine aminopeptidase. As some of the N. asteroides isolates were isolated from asymptomatic patients, it is of interest to consider the possibility of one group being of low virulence while the other is more strongly associated with infection.

IS204: an insertion sequence from Nocardia asteroides (mexicana) YP21.

An insertion sequence was found in Nocardia asteroides YP21. The element, designated IS204, is 1452 bp long and has 19/23 bp imperfect inverted repeats at its ends. The sequences of IS204 terminal inverted repeats have high homology with those of IS1096 from Mycobacterium smegmatis. An 8-bp duplication of target sequence was found at the insertion site. Sequence analysis revealed that IS204 contains an open reading frame of 1134 bp, which encodes a putative transposase similar to those found in IS1096 and in Tn4652 from Pseudomonas sp. EST1001. At least nine copies of IS204 are present in the genome of N asteroides YP21. The plasmid pCY 104::IS204 could be inserted with another copy of IS204 at a different site. The possible mechanisms are discussed.

Ammonium assimilation in Nocardia asteroides.

Specific enzymes of ammonium assimilation were measured in cell-free extracts of Nocardia asteroides grown in a synthetic medium with glutamate as the nitrogen source. Cell-free extracts had active glutamine synthetase (GS) and glutamate synthase (GOGAT) and alanine dehydrogenase (ADH) but glutamate dehydrogenase (GDH) could not be detected in the enzyme preparation. This shows that GS/GOGAT is the major pathway of ammonium assimilation in N. asteroides.

Purification and characterization of a novel FMN-dependent enzyme. Membrane-bound L-(+)-pantoyl lactone dehydrogenase from Nocardia asteroides.

Membrane-bound L-(+)-pantoyl lactone dehydrogenase, an enzyme that catalyzes the formation of ketopantoyl lactone from L-(+)-pantoyl lactone, was solubilized with Brij 35 and purified 78-fold to apparent homogeneity, with a 3.7% overall recovery, from Nocardia asteroides through purification procedures including successive ammonium sulfate fractionation, and DEAE-Sephacel, Sepharose CL-6B and Cellulofine GC-700-m column chromatography in the presence of Brij 35. The relative molecular mass of the native enzyme, as estimated on high-performance gel-permeation chromatography, is at least more than 600 kDa and its subunit molecular mass is 42 kDa. The enzyme shows high specificity for L-(+)-pantoyl lactone as a substrate (Km = 26.8 mM; Vmax = 4.22 mumol.min-1.mg protein-1). Brij 35 acts as a stabilizer and also as an efficient activator of the enzyme. The prosthetic group of L-(+)-pantoyl lactone dehydrogenase was identified as noncovalently bound FMN.

Influence of nutritional factors on growth and hydrolytic enzyme production in Nocardia asteroides.

The growth and production of hydrolytic enzymes such as alpha-amylase, esterase and peroxidase as influenced by the type of media, carbon and nitrogen sources and C:N ratio were monitored in Nocardia asteroides at 37 degrees C. Sabouraud dextrose and the synthetic media yielded maximum growth compared with tryptic soy broth. Among the carbon sources (dextrose, fructose, sucrose, maltose, starch and citrate), monosaccharides supported maximum growth and induced higher alpha-amylase activity but repressed the peroxidase activity. On the other hand, the disaccharides and starch produced less growth but induced maximum esterase and peroxidase activities. Glutamate among the nitrogen sources (nitrate, nitrite, ammonium, hydroxylamine, glutamate and casein) supported maximum growth. Glutamate, nitrate and casein induced alpha-amylase and esterase activities but suppressed peroxidase activity. Nitrite, ammonium and hydroxylamine stimulated peroxidase activity to the maximum but repressed alpha-amylase and esterase activities. Low, medium and high C:N ratios induced maximum peroxidase, esterase and alpha-amylase activities, respectively.

Purification and properties of lactate dehydrogenase from Nocardia asteroides.

The lactate dehydrogenase (LDH) from Nocardia asteroides was purified to homogeneity by ammonium sulphate precipitation, gel filtration on Sephadex G-150 and DEAE-Sepharose column chromatography. The purified enzyme showed a single band in native condition which indicated its homogeneity. SDS-PAGE of the purified enzyme showed the presence of three bands which correspond to molecular weights of 60, 66 and 74 kDa. The pH and temperature optima of the purified enzyme were 9.5 and 50 degrees C, respectively. The metal ions Mn++, Fe++, Co++, Mg++ and Ca++, increased the purified LDH activity. On the other hand, enzyme activity was completely inhibited by CuCl2. Potassium chloride, ammonium sulphate and sodium chloride did not alter the enzyme activity. The purified enzyme exhibited a Km value of 1.6 x 10(-5) M for pyruvate.

Urease-negative Nocardia asteroides causing cutaneous nocardiosis.

We report a case of cutaneous nocardiosis, caused by a urease-negative strain of Nocardia asteroides, in a patient who had undergone long-term corticosteroid therapy. The microbiological characteristics of the isolate were typical of N. asteroides except for the failure of the isolate to hydrolyze urea. During the course of routine identification, laboratory specialists should be aware of the occasional occurrence of atypical strains of N. asteroides.

Acid phosphatase activity in Nocardia brasiliensis and Nocardia asteroides.

Nocardia asteroides and N. brasiliensis strains were found to possess acid phosphatase activity. This enzyme was found to be cell-associated and its activity paralleled the cell mass increase seen during the Nocardia growth cycle. Of the strains tested, N. brasiliensis exhibited the highest enzymatic activity. Implications of these findings are related to current evidence which indicates that other microbial acid phosphatases may constitute potential pathogenic factors for humans.