Relationships between Colony Morphotypes and Oil Tolerance in Rhodococcus rhodochrous

A mucoidal strain of Rhodococcus rhodochrous was resistant to 10% (vol/vol) n-hexadecane, while its rough derivatives were sensitive. When the extracellular polysaccharide (EPS) produced by the mucoidal strain was added to cultures of the rough strains, the rough strains gained resistance to n-hexadecane. Thus, EPS confer tolerance to n-hexadecane in members of the genus Rhodococcus.     

Metabolism of acetylene and acetaldehyde by Rhodococcus rhodochrous

We studied the ability of a soil bacterium, identified as Rhodococcus rhodochrous, to grow on acetylene and to accumulate acetaldehyde. Its maximum growth rate on acetylene was obtained at about 30 degrees C (mu = 0.11 h-1) and was independent of the concentration of this gas in air from 0.14 to 16% (v/v). During growth, acetylene was quantitatively transformed to acetaldehyde, ethanol, acetate, CO2, and biomass in proportions which varied with culture age and temperature. Growth was completely inhibited by acetaldehyde at a concentration of 10 mM. The inhibitory effect was relieved by addition of acetate. Growth on ethanol up to 140 mM did not result in acetaldehyde accumulation. Acetylene consumption was constitutive with apparent Km and Vmax equal to 250 microM and 800 nmol.min-1.(mg protein)-1, respectively. In resting cell suspensions, acetylene consumption rates decreased more rapidly under air than under nitrogen. The inhibitory effect of acetaldehyde was enhanced in the presence of oxygen. Acetaldehyde accumulation in aerobic resting cell conditions did not exceed 10 mM (440 mg/L), but under anaerobic conditions it attained more than 70 mM (3.08 g/L).     

Fluorene cometabolism by Rhodococcus rhodochrous and Pseudomonas fluorescens

The transformation of fluorene by Rhodococcus rhodochrous strain 172 grown on sucrose and Pseudomonas fluorescens strain 26K grown on glycerol was studied as a function of the substrate concentration and the growth phase. Under certain cultivation conditions, fluorene was completely consumed from the medium. The specific transformation rate of fluorene was considerably higher when it was transformed in the presence of the cosubstrates than when it served as the sole carbon source. An approach to the evaluation of the specific transformation rate of fluorene during batch cultivations is proposed.     

Dimethylformamide is a novel nitrilase inducer in Rhodococcus rhodochrous

Nitrilases are of commercial interest in the selective synthesis of carboxylic acids from nitriles. Nitrilase induction was achieved here in three bacterial strains through the incorporation of a previously unrecognised and inexpensive nitrilase inducer, dimethylformamide (DMF), during cultivation of two Rhodococcus rhodochrous strains (ATCC BAA-870 and PPPPB BD-1780), as well as a closely related organism (Pimelobacter simplex PPPPB BD-1781). Benzonitrile, a known nitrilase inducer, was ineffective in these strains. Biocatalytic product profiling, enzyme inhibition studies and protein sequencing were performed to distinguish the nitrilase activity from that of sequential nitrile hydratase-amidase activity. The expressed enzyme, a 40-kDa protein with high sequence similarity to nitrilase protein Uniprot Q-03217, hydrolyzed 3-cyanopyridine to produce nicotinic acid exclusively in strains BD-1780 and BD-1781. These strains were capable of synthesising both the vitamin nicotinic acid as well as β-amino acids, a compound class of pharmaceutical interest. The induced nitrilase demonstrated high enantioselectivity (> 99%) in the hydrolysis of 3-amino-3-phenylpropanenitrile to the corresponding carboxylic acid.

     

Fluorene Cometabolism by Rhodococcus rhodochrous and Pseudomonas fluorescens Cultures

The transformation of fluorene by Rhodococcus rhodochrous strain 172 grown on sucrose and Pseudomonas fluorescens strain 26K grown on glycerol was studied as a function of the substrate concentration and the growth phase. Under certain cultivation conditions, fluorene was completely consumed from the medium. The specific transformation rate of fluorene was considerably higher when it was transformed in the presence of the cosubstrates than when it served as the sole carbon source. An approach to the evaluation of the specific transformation rate of fluorene during batch cultivations is proposed.     

Nitrilase of Rhodococcus rhodochrous J1. Purification and characterization

Nitrilase was purified from an extract of isovaleronitrile-induced cells of Rhodococcus rhodochrous J1 in seven steps. In the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous by polyacrylamide electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 78 kDa and consists of two subunits identical in molecular mass. The purified enzyme exhibits a pH optimum of 7.6 and a temperature optimum of 45 degrees C. The enzyme catalyzed stoichiometrically the hydrolysis of benzonitrile to benzoic acid and ammonia, and no formation of amide was detected. The enzyme required thiol compounds such as dithiothreitol, L-cysteine or reduced glutathione to exhibit maximum activity. The enzyme was specific for nitrile groups attached to an aromatic or heteroaromatic ring, e.g. benzonitrile, 3-chlorobenzonitrile, 4-tolunitrile, 2-furonitrile and 2-thiophenecarbonitrile. The comparison of the properties of the enzyme with other nitrilases and nitrile hydratases has been also discussed.     

产腈水解酶菌株的诱变及培养优化

对实验室保存的1株产腈水解酶的Rhodococcus rhodochrous菌株采用氯化锂进行诱变处理,筛选得到了1株产酶活力较高的菌株tg1-A6。经过优化得到培养基的配方为(g.L-1):葡萄糖10,谷氨酸钠10,酵母膏3,己内酰胺7,MgSO40.5,K2HPO40.75,KH2PO40.75。当培养温度28℃,摇床转速200 r.min-1,初始pH值7.0,通过补加葡萄糖,该菌的腈水解酶酶活可达到26.77 U.mL-1。     

Isolation and primary characterization of an amidase from Rhodococcus rhodochrous

Amidase (EC 3.5.1.4) was purified to homogeneity from Rhodococcus rhodochrous M8 using isopropanol fractionation and exchange chromatography on Mono Q. The isolated amidase consists of four identical subunits with molecular weight 42+/-2 kD. The activity of the enzyme is maximal at 55-60 degrees C and within the pH range 5-8. The amidase from R. rhodochrous M8 is highly sensitive to such sulfhydryl reagents as Hg2+ and Cu2+. Chelators (EDTA and o-phenanthroline) and serine proteinase inhibitors (PMSF and DIFP) did not inhibit the activity of the enzyme. The enzyme exhibits hydrolytic and acyl transferase activity and does not possess urease activity. Aliphatic amides (acetamide and propionamide) were the best substrates for the amidase from R. rhodochrous M8, whereas bulky aromatic amides were poor substrates of this enzyme. The properties of the isolated enzyme are similar to those found in the corresponding amidase from Arthrobacter sp. J-1 and an amidase with wide substrate specificity from Brevibacterium sp. R312.     

Occurrence of Amidases in the Industrial Microbe Rhodococcus rhodochrous J1

Rhodococcus rhodochrous J1, of which the high-M_r nitrile hydratase has been used for the industrial manufacture of acrylamide from acrylonitrile, produced at least two amidases differing in substrate specificity, judging from the effects of various amides on amidase activity in this strain. These amidases seemed to be inducible enzymes depending on amide compounds.     

Sterols and diacylglycerophosphocholines in the lipids of the hydrocarbon-utilizing prokaryote Rhodococcus rhodochrous

S orkhoh , N.A., G hannoum , M.A., I brahim , A.S., S tretton , R.J. & R adwan , S.S. 1990. Sterols and diacylglycerophosphocholines in the lipids of the hydrocarbon-utilizing prokaryote Rhodococcus rhodochrous. Journal of Applied Bacteriology 69 , 856–863.
Two strains of Rhodococcus rhodochrous were grown in an inorganic medium containing either glucose or dodecane as sole sources of carbon and their total lipids were extracted and analysed. Dodecane-grown cells contained more total lipids than glucose-grown cells. Lipid classes that increased in dodecane-grown cells were sterols, monoacylglycerols, diacylglycerophosphocholines and an unknown glyco-lipid. Sterols and diacylglycerophosphocholines were unequivocally identified in this species. The major acyl moieties in total lipids from glucose-grown cells were palmitic and octadecanoic acids, in addition to small amounts of linoleic and linolenic acids and traces of tuberculostearic acid. The latter was confined to diacyl-glycerophosphoglycerols and the unknown glycolipid. In addition to these acids, dodecane-grown cells contained relatively large proportions of lauric and myristic acids in their total lipids, which were esterified mainly in diacylglycerophosphocholines and the unknown glycolipid.     

A 2-Hydroxypyridine Catabolism Pathway in Rhodococcus rhodochrous Strain PY11

Rhodococcus rhodochrous PY11 (DSM 101666) is able to use 2-hydroxypyridine as a sole source of carbon and energy. By investigating a gene cluster (hpo) from this bacterium, we were able to reconstruct the catabolic pathway of 2-hydroxypyridine degradation. Here, we report that in Rhodococcus rhodochrous PY11, the initial hydroxylation of 2-hydroxypyridine is catalyzed by a four-component dioxygenase (HpoBCDF). A product of the dioxygenase reaction (3,6-dihydroxy-1,2,3,6-tetrahydropyridin-2-one) is further oxidized by HpoE to 2,3,6-trihydroxypyridine, which spontaneously forms a blue pigment. In addition, we show that the subsequent 2,3,6-trihydroxypyridine ring opening is catalyzed by the hypothetical cyclase HpoH. The final products of 2-hydroxypyridine degradation in Rhodococcus rhodochrous PY11 are ammonium ion and α-ketoglutarate.     

Biodesulfurization of dibenzothiophene and gas oil using a bioreactor containing a catalytic bed with Rhodococcus rhodochrous immobilized on silica

Biodesulfurization (BDS) in a bioreactor packed with a catalytic bed of silica containing immobilized Rhodococcus rhodochrous was studied. Various bed lengths and support particle sizes were evaluated for BDS of dibenzothiophene (DBT) and gas oil. The sulfur-containing substrates were introduced separately into the bioreactor at different feed flows. Higher removal of sulfur from DBT and gas oil was achieved with a long bed, lower substrate flow, and larger sizes of immobilization particles. The packed bed bioreactor containing metabolic active cells was recycled and maintained BDS activity.     

Occurrence of a cobalt-induced and cobalt-containing nitrile hydratase in Rhodococcus rhodochrous J1

The formation of nitrile hydratase required cobalt ions in Rhodococcus rhodochrous J1. No other transition-metals could replace the cobalt ion. The Rhodococcus nitrile hydratase was purified to homogeneity and found to contain a cobalt atom. The occurrence of a cobalt-induced and cobalt-containing nitrile hydratase, different from the nitrile hydratases in Pseudomonas chlororaphis B23 and Brevibacterium R312 containing a ferric ion in their active center, has been demonstrated here for the first time.     

Cobalt-dependent transcription of the nitrile hydratase gene in Rhodococcus rhodochrous M8

Abstract The effects of cobalt ions on the activities of Rhodococcus rhodochrous M8 enzymes for nitrile utilization, nitrile hydratase and amidase, were investigated. In contrast to amidase, synthesis of nitrile hydratase and its activity required cobalt ions in the growth medium. Northern blot analysis showed that in the presence of cobalt ions, the level of mRNA for nitrile hydratase genes was several times higher than that under cobalt-limited conditions. It was assumed that the low nitrile hydratase activity in cells grown in the absence of cobalt ions is connected either with the weak expression of nitrile hydratase genes or with the rapid degradation of nitrile hydratase mRNA.     

Extracellular polysaccharides of Rhodococcus rhodochrous S-2 stimulate the degradation of aromatic components in crude oil by indigenous marine bacteria

Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing D-glucose, D-galactose, D-mannose, D-glucuronic acid, and lipids, which is important to the tolerance of this strain to an aromatic fraction of (AF) Arabian light crude oil (N. Iwabuchi, N. Sunairi, H. Anzai, M. Nakajima, and S. Harayama, Appl. Environ. Microbiol. 66:5073-5077, 2000). In the present study, we examined the effects of S-2 EPS on the growth of indigenous marine bacteria on AF. Indigenous bacteria did not grow significantly in seawater containing AF even when nitrogen, phosphorus, and iron nutrients were supplemented. The addition of S-2 EPS to seawater containing nutrients and AF resulted in the emulsification of AF, promotion of the growth of indigenous bacteria, and enhancement of the degradation of AF by the bacteria. PCR-denaturing gradient gel electrophoresis analyses show that addition of S-2 EPS to the seawater containing nutrients and AF changed the composition of the bacterial populations in the seawater and that bacteria closely related to the genus Cycloclasticus became the major population. These results suggest that Cycloclasticus was responsible for the degradation of hydrocarbons in AF. The effects of 15 synthetic surfactants on the degradation of AF by indigenous marine bacteria were also examined, but enhancement of the degradation of AF was not significant. S-2 EPS was hence the most effective of the surfactants tested in promoting the biodegradation of AF and may thus be an attractive agent to use in the bioremediation of oil-contaminated marine environments.     

Evidence for an inducible nucleotide-dependent acetone carboxylase in Rhodococcus rhodochrous B276

The metabolism of acetone was investigated in the actinomycete Rhodococcus rhodochrous (formerly Nocardia corallina) B276. Suspensions of acetone- and isopropanol-grown R. rhodochrous readily metabolized acetone. In contrast, R. rhodochrous cells cultured with glucose as the carbon source lacked the ability to metabolize acetone at the onset of the assay but gained the ability to do so in a time-dependent fashion. Chloramphenicol and rifampin prevented the time-dependent increase in this activity. Acetone metabolism by R. rhodochrous was CO2 dependent, and 14CO2 fixation occurred concomitant with this process. A nucleotide-dependent acetone carboxylase was partially purified from cell extracts of acetone-grown R. rhodochrous by DEAE-Sepharose chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the acetone carboxylase was composed of three subunits with apparent molecular masses of 85, 74, and 16 kDa. Acetone metabolism by the partially purified enzyme was dependent on the presence of a divalent metal and a nucleoside triphosphate. GTP and ITP supported the highest rates of acetone carboxylation, while CTP, UTP, and XTP supported carboxylation at 10 to 50% of these rates. ATP did not support acetone carboxylation. Acetoacetate was determined to be the stoichiometric product of acetone carboxylation. The longer-chain ketones butanone, 2-pentanone, 3-pentanone, and 2-hexanone were substrates. This work has identified an acetone carboxylase with a novel nucleotide usage and broader substrate specificity compared to other such enzymes studied to date. These results strengthen the proposal that carboxylation is a common strategy used for acetone catabolism in aerobic acetone-oxidizing bacteria.     

Stereo- and regiospecific cis,cis-muconate cycloisomerization by Rhodococcus rhodochrous N75

cis,cis-Muconate cycloisomerase was purified to homogeneity from cells of Rhodococcus rhodochrous N75 grown at the expense of benzoate and p-toluate as the sole sources of carbon. A single cycloisomerase was found to be induced in this organism with no isoforms being detected when R. rhodochrous N75 was grown on either benzoate or p-toluate as the sole source of carbon. The enzyme is hexameric with a single subunit Mr of 40,000. cis,cis-Muconate cycloisomerase from R. rhodochrous N75 displayed strict regio- and stereospecificity whereby cis,cis-muconate is cycloisomerized to (4S)-muconolactone and 2-methyl- and 3-methyl-substituted muconates are cycloisomerized to 2-methyl- and 4-methyl-substituted muconolactones by 1,4- and 3,6-cycloisomerization, respectively.     

Nitrile hydratase and amidase from Rhodococcus rhodochrous hydrolyze acrylic fibers and granular polyacrylonitriles

Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein(-1)) and amidase activity (38.4 nkat mg of protein(-1)) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C. I. Basic Blue 9.     

Hydroaromatic metabolism in Rhodococcus rhodochrous: purification and characterisation of its NAD-dependent quinate dehydrogenase

Quinate grown cells of Rhodococcus rhodochrous N75 metabolized both quinate and shikimate via protocatechuate to succinate and acetyl CoA. The initial enzyme of the hydroaromatic pathway, quinate dehydrogenase was purified 188-fold to electrophoretic homogeneity. The enzyme is a monomer with a native relative molecular mass of 44,000 and is NAD-dependent. The enzyme is highly stereospecific with regard to hydroaromatic substrates, oxidising only the axial hydroxyl group at C-3 of (-)-isomers of quinate, shikimate, dihydroshikimate and t-3,t-4-dihydroxycyclohexane-c-1-carboxylate, but shows activity with several NAD analogues.     

Nitrile Hydratase and Amidase from Rhodococcus rhodochrous Hydrolyze Acrylic Fibers and Granular Polyacrylonitriles

Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein⁻¹) and amidase activity (38.4 nkat mg of protein⁻¹) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrous enzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/S value) for C.I. Basic Blue 9.