Nocardioides Sp Research Articles

Gentisate 1,2-Dioxygenase fromXanthobacter polyaromaticivorans127W

A putative gentisate 1,2-dioxygenase was encoded in the dibenzothiophene degradation gene cluster (dbd) from Xanthobacter polyaromaticivorans 127W. The deduced amino acid sequence showed high sequence similarity with gentisate dioxygenases from Pseudomonas alcaligenes (AAD49427, 65% identical), Bradyrhizobium japonicum (NP_766750, 64%), and P. aeruginosa (ZP_00135722, 54%), and moderate similarity with 1-hydroxy-2-naphthoate dioxygenase from Nocardioides sp. KP7 (BAA31235, 33%) and salicylate dioxygenase from Pseudaminobacter salicylatoxidans (AAQ91293, 33%). The enzyme, GDOxp, was heterologously produced in Escherichia coli and purified to homogeneity. GDOxp formed a tetramer and exhibited high dioxygenase activity against 1,4-dihydroxy 2-naphthoate as well as gentisate, suggesting unusually broad substrate specificity. GDOxp easily released ferrous ion under unfavorable temperature and pH conditions to become an inactive monomer protein. An inactive monomer protein can reconstitute a tetramer structure and restore enzyme activity in a cooperative manner upon the addition of ferrous ion. Chymotryptic digestion and protein truncation experiments suggested that the N-terminal region is important for the tetramerization of GDOxp.

Isolation and characterization of Arthrobacter sp. strain MCM B-436, an atrazine-degrading bacterium, from rhizospheric soil

Atrazine is one of the most environmentally prevalent s-triazine-ring herbicides. The widespread use of atrazine and its toxicity necessitates search for remediation technology. As atrazine is still used in India as a major herbicide, exploration of atrazine-degrading bacterial community is of immense importance. Considering lack of reports on well characterized atrazine-degrading bacterial cultures from India and wide diversity and density of microorganisms in rhizosphere, soil sample from rhizosphere of atrazine-resistant plant was studied. Arthrobacter sp. strain isolated in this investigation utilizes atrazine as the sole nitrogen source. In addition, the bacterium degrades other triazines such as ametryn, cyanizine, propazine and simazine. PCR analysis confirms the presence of atzBCD and triazine hydrolase ( trzN) genes on chromosomal DNA. Sequencing of the trzN gene reveals high sequence similarity with trzN from Nocardioides sp. C190. An inducible and intracellular atrazine chlorohydrolase enzyme was isolated and partially purified from this isolate. This study confirms the presence of atrazine-degrading microbial population in Indian soils and could be used efficiently for remediation of contaminated soils. Presence of trzN gene indicates possible presence of bacterial community with more efficient and novel enzymatic capabilities. Comparison of enzyme and gene structure of this isolate with other geographically distinct atrazine-degrading strains will help us in the better understanding of gene transfer and evolution.

Trichloroethylene degradation by butane-oxidizing bacteria causes a spectrum of toxic effects

The physiological consequences of trichloroethylene (TCE) transformation by three butane oxidizers were examined. Pseudomonas butanovora, Mycobacterium vaccae, and Nocardioides sp. CF8 utilize distinctly different butane monooxygenases (BMOs) to initiate degradation of the recalcitrant TCE molecule. Although the primary toxic event resulting from TCE cometabolism by these three strains was loss of BMO activity, species differences were observed. P. butanovora and Nocardioides sp. CF8 maintained only 4% residual BMO activity following exposure to 165 microM TCE for 90 min and 180 min, respectively. In contrast, M. vaccae maintained 34% residual activity even after exposure to 165 microM TCE for 300 min. Culture viability was reduced 83% in P. butanovora, but was unaffected in the other two species. Transformation of 530 nmol of TCE by P. butanovora (1.0 mg total protein) did not affect the viability of BMO-deficient P. butanovora cells, whereas transformation of 482 nmol of TCE by toluene-grown Burkholderia cepacia G4 caused 87% of BMO-deficient P. butanovora cells to lose viability. Together, these results contrast with those previously reported for other bacteria carrying out TCE cometabolism and demonstrate the range of cellular toxicities associated with TCE cometabolism.

Isolation and characterization of microorganisms capable of hydrolysing the herbicide mefenacet

Three strains— Nocardioides sp. MFC-A, Rhodococcus rhodochrous MFC-B, and Stenotrophomonas sp. MFC-C—were newly isolated from rice soil or bark compost as microorganisms capable of cometabolically degrading mefenacet (2-benzothiazol-2-yloxy- N-methyl-acetanilide). Several other species belonging to the genera Nocardioides, Rhodococcus, and Stenotrophomonas were also capable of degrading mefenacet, suggesting that the ability is not specific to these isolates, but common at least in these genera. N-methylaniline and 2-benzothiazoloxy acetic acid were identified as the metabolites by liquid chromatography-mass spectrometry. Besides mefenacet, the isolates could degrade one or more other amide pesticides, such as flutolanil, mepronil, metolachlor, and pretilachlor. These results indicate that the strains Nocardioides sp. MFC-A, Rhodococcus rhodochrous MFC-B, and Stenotrophomonas sp. MFC-C degrade mefenacet via hydrolysis of the amide bond, but their substrate specificities differ.

Glycolipids produced by Antarctic Nocardioides sp. during growth on n-paraffin

An actinomycete isolated from Antarctic soil (Dewart Island) and identified as Nocardioides sp. A-8 grown on n-paraffin as a carbon source produced biosurfactants. Hemolysis of erythrocytes, growth inhibition of Bacillus subtilis and thin-layer chromatography studies revealed that the secreted biosurfactants are rhamnolipids. They lowered the surface tension of the medium below 35 mN/m and efficiently emulsified aromatic hydrocarbons and n-paraffin. The results showed that the Antarctic Nocardioides sp. A-8 and its produced glycolipids with effective surface and emulsifying properties represent a promising potential for application in bioremediation of soil environments polluted with hydrocarbons.

Biochemical and Molecular Characterization of a Ring Fission Dioxygenase with the Ability to Oxidize (Substituted) Salicylate(s) from Pseudaminobacter salicylatoxidans

The gene coding for a dioxygenase with the ability to cleave salicylate by a direct ring fission mechanism to 2-oxohepta-3,5-dienedioic acid was cloned from Pseudaminobacter salicylatoxidans strain BN12. The deduced amino acid sequence encoded a protein with a molecular mass of 41,176 Da, which showed 28 and 31% sequence identity, respectively, to a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 and a 1-hydroxy-2-naphthoate 1,2-dioxygenase from Nocardioides sp. KP7. The highest degree of sequence identity (58%) was found to a presumed gentisate 1,2-dioxygenase from Corynebacterium glutamicum. The enzyme from P. salicylatoxidans BN12 was heterologously expressed in Escherichia coli and purified as a His-tagged enzyme variant. The purified enzyme oxidized in addition to salicylate, gentisate, 5-aminosalicylate, and 1-hydroxy-2-naphthoate also 3-amino- and 3- and 4-hydroxysalicylate, 5-fluorosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-bromosalicylate, 3-, 4-, and 5-methylsalicylate, and 3,5-dichlorosalicylate. The reactions were analyzed by high pressure liquid chromatography/mass spectrometry, and the reaction products were tentatively identified. For comparison, the putative gentisate 1,2-dioxygenase from C. glutamicum was functionally expressed in E. coli and shown to convert gentisate but not salicylate or 1-hydroxy-2-naphthoate.

Piericidins C5 and C6: New 4‐Pyridinol Compounds Produced by Streptomyces sp. and Nocardioides sp.

Piericidins C5 (1) and C6 (2), two new members of the piericidin family, were isolated from a Streptomyces sp. and a Nocardioides sp., together with known piericidins C1 (3), C2 (4), C3 (5), C4 (6), D1 (7), and A3 (8). The structures were determined on the basis of their spectroscopic data. Both new compounds inhibited cell division of fertilized starfish (Asterina pectinifera) eggs at the minimum inhibitory concentration of 0.09 microg/mL.

Piericidins C 5 and C 6: new 4-pyridinol compounds produced by Streptomyces sp. and Nocardioides sp.

Piericidins C 5 ( 1) and C 6 ( 2), two new members of the piericidin family, were isolated from a Streptomyces sp. and a Nocardioides sp., together with known piericidins C 1 ( 3), C 2 ( 4), C 3 ( 5), C 4 ( 6), D 1 ( 7), and A 3 ( 8). The structures were determined on the basis of their spectroscopic data. Both new compounds inhibited cell division of fertilized starfish ( Asterina pectinifera) eggs at the minimum inhibitory concentration of 0.09 μg/mL.

Isolation and characterisation of Nocardioides sp. SP12, an atrazine-degrading bacterial strain possessing the gene trzN from bulk- and maize rhizosphere soil

We report the characterisation of Nocardioides sp. SP12, an atrazine-degrading bacteria isolated from atrazine-treated bulk- and maize rhizosphere soil. Based on 16S rDNA alignment, strain SP12 showed close phylogenic relationships with Nocardioides sp. C157 and Nocardioides simplex. Internal transcribed spacer (ITS) sequences of strain SP12 were longer than those of other Nocardioides sp. and present Ala- and Ile-tRNA unlike Actinomycetales. Nocardioides sp. SP12 presents a novel atrazine catabolic pathway combining trzN with atzB and atzC. Atrazine biodegradation ends in a metabolite that co-eluted in HPLC with cyanuric acid. This metabolite shows an absorption spectrum identical to that of cyanuric acid with a maximal absorption at 214.6 nm. The mass of the atrazine metabolite is in concordance with that of cyanuric acid according to mass spectrometry analysis. Quantitative PCR revealed that the ITS sequence of Nocardioides sp. SP12 was at a lower number than the one of trzN in atrazine-treated soil samples. It suggests that trzN could also be present in other atrazine degrading bacteria. The numbers of trzN and ITS sequences of Nocardioides sp. SP12 were higher in the maize rhizosphere than in bulk soil.

Isolation from Nocardioides sp. strain CT16, purification, and characterization of a deoxycytidine deaminase extremely thermostable in the presence of D,L-dithiothreitol.

A deoxycytidine deaminase that was extremely thermostable in the presence of dithiothreitol was found in a mesophilic bacterium isolated from soil. The bacterium was classified as a Nocardioides sp. The enzyme was purified to a homogeneous protein by treatment at 100 degrees C, ammonium sulfate precipitation, and chromatography on DEAE-Toyopearl, hydroxyapatite, and then Sephacryl S-100. Twenty micrograms of the pure enzyme was obtained from 811 mg of the starting crude protein. After treatment at 50 degrees C for 15 min in the absence of dithiothreitol, enzyme activity was 44% of the starting activity; after treatment at 100 degrees C for 2 h in the presence of 50 mm dithiothreitol, activity was 56% of the starting activity. Dithiothreitol greatly stabilized the enzyme. Activity was maximum at 99 degrees C. The Km values for deoxycytidine, cytidine, and methyl-deoxycytidine were 55.2, 140, and 130 microM, respectively. The molecular mass was estimated to be 52 kDa by gel permeation chromatography. The enzyme molecule was dissociated into two subunits each of 18 kDa subunit when reduced with mercaptoethanol.

Obtaining of a High-Density Culture of Nocardioides sp.

Selection of carbon sources demonstrated ethanol to be the best substrate for a high-density Nocardioides sp. culture. A strategy for control over high-density fed-batch culture production was developed, which permitted maximizing the yield of biomass (21 g/l). The control, based on the ExpoDense algorithm, should be predetermined at the first phase and adaptive in the second phase of the two-phase process of high-density culture production.

Degradation of 2,3,4,6-tetrachlorophenol at low temperature and low dioxygen concentrations by phylogenetically different groundwater and bioreactor bacteria.

Effects of low temperature and low oxygen partial pressure on the occurrence and activity of 2,3,4,6-tetrachlorophenol degrading bacteria in a boreal chlorophenol contaminated groundwater and a full-scale fluidized-bed bioreactor were studied using four polychlorophenol degrading bacterial isolates of different phylogenetic backgrounds. These included an alpha-proteobacterial Sphingomonas sp. strain MT1 isolated from the full-scale bioreactor and three isolates from the contaminated groundwater which were identified as beta-proteobacterial Herbaspirillum sp. K1, a Gram-positive bacterium with high G + C content Nocardioides sp. K44 and an alpha-proteobacterial Sphingomonas sp. K74. The Sphingomonas strains K74 and MT1 and Nocardioides sp. K44 degraded 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol as the sole carbon and energy sources. Close to stoichiometric inorganic chloride release with the 2,3,4,6-tetrachlorophenol removal and the absence of methylation products indicated mineralization. Tetrachlorophenol degradation by the Herbaspirillum sp. K1 was enhanced by yeast extract, malate, glutamate, pyruvate, peptone and casitone. At 8 degrees C, Sphingomonas sp. K74 had the highest specific degradation rate (mu(max) = 4.9 x 10(-2) mg h(-1) cell(-1)) for 2,3,4,6-tetrachlorophenol. The Nocardioides strain K44 had the highest affinity (K(s) = 0.46 mg l(-1)) fortetrachlorophenol. K1 and MT1 grew microaerophilically in semisolid glucose medium. Furthermore, the growth of MT1 was inhibited in liquid glucose medium at high oxygen partial pressure indicating sensitivity to accumulating toxic oxygen species. On the other hand, trichlorophenol degradation was not affected by oxygen concentration (2-21%). The isolates K44, K74 and MT1, with optimum growth temperatures between 23 and 25 degrees C, degraded tetrachlorophenol faster at 8 degrees C than at room temperature indicating distinctly different temperature optima for chlorophenol degradation and growth on complex media. These results show efficient polychlorophenol degradation by the isolates at the boreal groundwater conditions, i.e., at low temperature and low oxygen concentrations. Differences in chlorophenol degradation and sensitivities to chlorophenols and oxygen among the isolates indicate that the phylogenetically different chlorophenol degraders have found different niches in the contaminated groundwater and thus potential for contaminant degradation under a variety of saturated subsurface conditions.

Influence of phenol on biodegradation of p-nitrophenol by freely suspended and immobilized Nocardioides sp. NSP41.

The effect of the presence of an alternate toxic compound (phenol) on the p-nitrophenol (PNP)-degrading activity of freely suspended and calcium alginate immobilized Nocardioides sp. NSP41 was investigated. In the single substrate experiments, when the concentration of phenol and PNP was increased to 1400 mg l(-1) and 400 mg l(-1), respectively, the initial cell concentrations in the freely suspended cell culture should be higher than 1.5 g dry cell weight l(-1) for complete degradation. In the simultaneous degradation experiment, when the initial concentration of phenol was increased from 100 to 400 mg l(-1), the specific PNP degradation rate at the concentration of 200 mg l(-1) was decreased from 0.028 to 0.021 h(-1). A freely suspended cell culture with a high initial cell concentration resulted in a high volumetric degradation rate, suggesting the potential use of immobilized cells for simultaneous degradation. In the immobilized cell cultures, although simultaneous degradation of PNP and phenol was maintained, the specific PNP and phenol degradation rate decreased. However, a high volumetric PNP and phenol degradation rate could be achieved by immobilization because of the high cell concentration. Furthermore, when the immobilized cells were reused in the simultaneous degradation of PNP and phenol, they did not lose their PNP- and phenol-degrading activity for 12 times in semi-continuous cultures. Taken together, the use of immobilized Nocardioides sp. NSP41 for the simultaneous degradation of PNP and phenol at high concentrations is quite feasible because of the high volumetric PNP and phenol degradation rate and the reusability of immobilized cells.

Classification of 'Nocardioides fulvus' IFO 14399 and Nocardioides sp. ATCC 39419 in Kribbella gen. nov., as Kribbella flavida sp. nov. and Kribbella sandramycini sp. nov.

By 16S rDNA sequence analysis, two strains identified previously as members of the genus Nocardioides, namely 'Nocardioides fulvus' IFO 14399 and Nocardioides sp. ATCC 39419, were found to form lines of descent distinct from the genus Nocardioides. Therefore, the two strains were studied taxonomically in detail by phenotypic, particularly chemotaxonomic, characterization, and by phylogenetic analysis including other related taxa. The genomic DNA G + C contents of 'N. fulvus' IFO 14399 and Nocardioides sp. ATCC 39419 are 70 and 68 mol%, respectively. The two strains contain LL-diaminopimelic acid as the diamino acid in their peptidoglycan, together with L-alanine at position 1 of the peptide subunit. The predominant menaquinone found in the two strains is MK-9(H4). The major cellular fatty acid found in the two strains is anteiso-C15:0' which is a minor component of members of the genus Nocardioides. The diagnostic polar lipid of the two strains is phosphatidylcholine. A phylogenetic tree was constructed which showed that the two strains from evolutionary lineages distinct from other LL-diaminopimelic-acid-containing taxa such as, for example, the genus Nocardioides. On the basis of our data, a new genus, Kribbella gen. nov., and two new species, Kribbella flavida gen. nov., sp. nov. and Kribbellla sandramycini gen. nov., sp. nov., are proposed. It is also proposed that the new genus be included in the family Nocardioidaceae.

Characterization of genes for enzymes involved in the phenanthrene degradation in Nocardioides sp. KP7

The nucleotide sequence of the gene cluster, phdEFABGHCD, encoding enzymes responsible for the transformation of phenanthrene to 1-hydroxy-2-naphthoate in Nocardioides sp. strain KP7 was determined. This gene cluster, which may constitute a single operon, resided at 6.1-kb downstream of the phdIJK gene cluster encoding the enzymes for the transformation of 1-hydroxy-2-naphthoate to o-phthalate. In general, the phd products exhibited moderate degrees of homology with isofunctional enzymes found in pathways for the degradation of other aromatic compounds. Remarkably, the phdC gene product had features of the [3Fe4S] type ferredoxin, which has not been found so far as a component of the ring-hydroxylating dioxygenase. Escherichia coli carrying the genes for phenanthrene dioxygenase, phdABCD, was capable to oxidize phenanthrene.

Biochemical and Molecular Characterization of 1-Hydroxy-2-naphthoate Dioxygenase from Nocardioides sp. KP7

1-Hydroxy-2-naphthoate dioxygenase, which cleaves the singly hydroxylated aromatic ring, was purified from phenanthrene-degrading Nocardioides sp. strain KP7. The purified enzyme had a molecular mass of 45 kDa by SDS-polyacrylamide gel electrophoresis and 270 kDa by gel filtration chromatography. The apparent Km and kcat values of this enzyme for 1-hydroxy-2-naphthoate were 10 microM and 114 s-1, respectively. One mole of molecular oxygen was consumed when 1 mol of 1-hydroxy-2-naphthoate was oxidized. This enzyme contained 1 mol of Fe(II)/mol of the subunit and was inactivated by o-phenanthroline. The enzyme that had been inactivated by o-phenanthroline was reactivated by incubating with FeSO4 and ascorbic acid. Thus, Fe(II) was required for the enzyme to exhibit activity. The structural gene for this enzyme was screened from a cosmid library and then sequenced, the length of the 1-hydroxy-2-naphthoate gene being 1161 base pairs. The deduced amino acid sequence of this enzyme was different from those of other ring-cleaving dioxygenases that cleave the doubly hydroxylated aromatic ring.

Inter- and intraspecific phylogenetic analysis of the genus Nocardioides and related taxa based on 16S rDNA sequences.

The 16S rDNAs from 38 strains of the genus Nocardioides, two Aeromicrobium species and Terrabacter tumescens were directly sequenced and then analysed. The mean nucleotide similarity value between the type strains of validly described Nocardioides species was 96.1 +/- 3.0%. The mean nucleotide similarity value between the type strains of validly described Nocardioides species and the two Aeromicrobium species was 93.7 +/- 1.4% T. tumescens was distantly related to the genera Nocardioides and Aeromicrobium. The mean intraspecific nucleotide similarity value of 16S rDNA sequences from Nocardioides albus was 99.5 +/- 0.5%. The mean intraspecific nucleotide similarity of 16S rDNA sequences from Nocardioides simplex was 100%, except for N. simplex strains ATCC 13260, ATCC 19565 and ATCC 19566, which were shown not to be members of the genus Nocardioides. 'Nocardioides flavus' strains IFO 14396T and IFO 14397, and 'Nocardioides fulvus' JCM 3335T showed a 16S rDNA similarity value of 100% with Nocardioides luteus KCTC 9575T and Nocardioides albus JCM 5854. 'N. fulvus' IFO 14399 shared its highest 16S rDNA similarity with Nocardioides sp. ATCC 39419 at 99%. 'N. fulvus' IFO 14399 and Nocardioides sp. ATCC 39419 formed a phylogenetic lineage distinct from the genera Nocardioides and Aeromicrobium. 'Nocardioides thermolilacinus' strains IFO 14335T and IFO 14336 displayed a close relationship to the genus Streptomyces. From 16S rDNA sequence analyses, it is considered that some strains that have been attributed to the genus Nocardioides should be taxonomically re-evaluated.

Simultaneous degradation of p-nitrophenol and phenol by a newly isolated Nocardioides sp.

A p-nitrophenol (PNP)- and phenol-mineralizing bacterium (strain NSP41) was isolated from an industrial wastewater and identified as a member of the genus Nocardioides. PNP was degraded via a hydroquinone pathway, and phenol was degraded through a catechol pathway in strain NSP41. Both enzyme systems for the degradation of PNP and phenol were induced simultaneously in the presence of both compounds. Although both enzyme systems were induced at the same time, PNP and phenol were degraded by the hydroquinone and catechol pathway, respectively. However, during the simultaneous degradation in the low phenol concentration, after the exhaustion of phenol, some PNP was transformed by the catechol pathway and 4-nitrocatechol was transiently accumulated. Kinetically, the addition of phenol greatly enhanced the apparent PNP degradation rate, which may be due to the increased cell mass by the assimilation of phenol.

Monitoring of an Alkaline 2,4,6-Trichlorophenol-Degrading Enrichment Culture by DNA Fingerprinting Methods and Isolation of the Responsible Organism, Haloalkaliphilic Nocardioides sp. Strain M6

A site situated near Alkali Lake (Oregon) and highly contaminated by chloroaromatic compounds was chosen for isolation of alkaliphilic chlorophenol-degrading bacteria. Prolonged cultivation of an enrichment culture followed by successive transfers resulted in a strong increase in the 2,4,6-trichlorophenol (2,4,6-TCP) degradation rate. Repetitive extragenic palindromic PCR and amplified ribosomal DNA restriction analysis were applied to distinguish members of the enrichment culture and monitor them during the enrichment procedure. Comparison of the fingerprints of the isolates obtained from the enrichment culture and its total DNA fingerprint indicated the presence of an unidentified bacterium in the enrichment culture, assisting in its isolation. The 2,4,6-TCP-degrading isolate, M6, was tentatively identified as a Nocardioides sp. strain based on its partial 16S RNA sequence and fatty acid profile. Strain M6 was capable of utilizing up to 1.6 g of 2,4,6-TCP per liter as a sole carbon and energy source and could also grow on 2,4-dichlorophenol and 2,4,5-trichlorophenol. A high-cell-density suspension of this strain degraded a wide range of chlorinated phenols from di- to pentachlorophenol while showing a clear preference for phenols containing chlorine substituents in positions 2 plus 4. Based on its optimal pH (9.0 to 9.4) and sodium ion concentration (0.2 to 0.4 M) for growth, Nocardioides sp. strain M6 is a slightly halophilic alkaliphile.

Influence of incubation period on the plasmid content of proliferating Nocardioides sp.

S. SANDHYA. 1996. Nocardioides sp., isolated for organic sulphur degradation, harbours catabolic plasmid pSB1 of molecular weight 34.2 kb. A correlation was noticed between the plasmid content and degradation of the organic sulphur compound dibenzothiophene (DBT). The maximum oxidation of DBT was only after 64 h, during which the content of plasmid DNA was also optimum.