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PubMed2005

Desulphurization of dibenzothiophene and diesel oils by bacteria.

AIMS: To study the desulphurization of dibenzothiophene (DBT), a recalcitrant thiophenic component of fossil fuels, by two bacteria namely Rhodococcus sp. and Arthrobacter sulfureus isolated from oil-contaminated soil/sludge in order to use them for reducing the sulphur content of diesel oil in compliance with environmental regulations. METHODS AND RESULTS: The desulphurization pathway of DBT by the two bacteria was determined by gas chromatography (GC) and GC-mass spectrometry. Both organisms were found to produce 2-hydroxy biphenyl (2-HBP), the desulphurized product of DBT. Sulphur contents of culture supernatants of Rhodococcus sp. and A. sulfureus grown with DBT as sole sulphur source were analysed by X-ray fluorescence indicating sulphur levels of 8 and 10 ppm, respectively, as compared with 27 ppm in control. In order to study desulphurization of diesel oils obtained from an oil refinery, resting cell studies were carried out which showed a decrease of about 50% in sulphur content of the oil obtained from the hydrodesulphurization (HDS) unit of the refinery. CONCLUSIONS: Rhodococcus sp. and A. sulfureus selectively remove sulphur from DBT to form 2-HBP. Application of these bacteria for desulphurization of diesel showed promising potential for decreasing the sulphur content of diesel oil. SIGNIFICANCE AND IMPACT OF THE STUDY: The process of microbial desulphurization described herein can be used for significantly reducing the sulphur content of oil, particularly, after the process of HDS which would help in meeting the regulatory standards for sulphur level in diesel oil.

Arthrobacter↗

PubMed2006

Replication and inheritance of Nocardia plasmid pC1.

Nocardia sp. C-14-1, isolated from acrylic fiber wastewater, can degrade long-chain alkanes and succinonitrile efficiently. Here we report the characterization of an indigenous plasmid pC1. The overall nucleotide sequence of pC1 consisted of 5841 bp. The five ORFs, encoding a DNA recombinase, replication protein (Rep(pC1)) and three proteins of unknown function, were predicted on pC1. The Rep(pC1) displayed its homology with the Rep of Rhodococcus large plasmid p33701, suggesting a theta type of replication. An Escherichia coli plasmid (containing the single rep(pC1) gene) propagated autonomously in low copy number in Nocardia or Rhodococcus, suggesting that rep(pC1) was an essential gene for plasmid replication. The plasmid (containing the single rep(pC1) gene) presented as inheritance unstable hints that other pC1 loci were required for the stable inheritance of plasmids. By comparison of the plasmid-borne Rep proteins, we classify Rhodococcus or Nocardia plasmids into four groups.

DNA Replication↗

PubMed2002

[Study on the reclassification of two deposited strains AS4.1186 and AS4.1187 of Nocardia].

Polyphasic evidence for the reclassification of Nocardia nostocoides AS4.1186 and Nocardia salmonicolor var. aurantiaca AS4.1187 indicated that strain AS4.1186 was closely related to Saccharothrix texasensis and strain AS4.1187 was very closely related to Rhodococcus ruber. Binary 16S rDNA sequence for the pair AS4.1186 and Saccharothrix texasensis NRRL B-16134T, and the pair AS4.1187 and Rhodococcus ruber DSM 43338T exhibited 99.3% and 99.5% similarity, respectively. Corresponding DNA-DNA reassociation values were significantly higher than 70%, 77.6% and 82.9%, respectively. Results of chemotaxonomic analyses of cell wall, mycolic acid, principal menaquinones, phospholipid type and the G + C content of the DNA supported the conclusions of the genotypic analyses. The very similar morphological and physiological characteristics agreed with the high degree of relatedness. On the basis of phylogenetic analyses, DNA-DNA reassociation values, chemotaxonomic properties, morphological and physiological characteristics, it is concluded that strain AS4.1186 and AS4.1187 should be removed from the genus Nocardia. We propose to transfer Nocardia nostocoides AS4.1186 to the genus Saccharothrix, combine with the species S. texasensis, and combine Nocardia salmonicolor var. aurantiaca AS4.1187 with the species Rhodococcus ruber.

Actinomycetales↗

PubMed2005

[Selectively desulfurizing organic sulfur of diesel oil by resting cells].

Rhodococcus sp. FS-1 ,which can specially break the C[single bond]S bond of dibenzothiophene (DBT) and convert DBT into 2-hydrobenzophene by "4S" pathway, is used to decrease the sulfur content in diesel oil. This research focuses on the diesel oil desulfurization using resting cells of Rhodococcus sp. FS-1. The research result indicate that the desulfurization activity of the resting cells of Rhodococcus sp. FS-1 was strongly high. When the concentration of DBT was 0.5 - 1.0mmol/L and the ratio of oil/water was 1:5, the effect of desulfurization was highest. The desulfurization rate was over 85 % when desulfurizing the diesel oil twice. GC analysis proves that this process did not break the carbon-carbon backbone. The calorific value of diesel oil is reserved.

Biodegradation, Environmental↗

PubMed2004

Bacterial communities and enzyme activities of PAHs polluted soils.

Three soils (i.e. a Belgian soil, B-BT, a German soil, G, and an Italian agricultural soil, I-BT) with different properties and hydrocarbon-pollution history with regard to their potential to degrade phenanthrene were investigated. A chemical and microbiological evaluation of soils was done using measurements of routine chemical properties, bacterial counts and several enzyme activities. The three soils showed different levels of polycyclic aromatic hydrocarbons (PAHs), being their contamination strictly associated to their pollution history. High values of enzyme activities and culturable heterotrophic bacteria were detected in the soil with no or negligible presence of organic pollutants. Genetic diversity of soil samples and enrichment cultures was measured as bands on denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences from the soil and enrichment community DNAs. When analysed by Shannon index (H'), the highest genetic biodiversity (H'=2.87) was found in the Belgian soil B-BT with a medium-term exposition to PAHs and the poorest biodiversity (H'=0.85) in the German soil with a long-term exposition to alkanes and PAHs and where absence, or lower levels of enzyme activities were measured. For the Italian agricultural soil I-BT, containing negligible amounts of organic pollutants but the highest Cu content, a Shannon index=2.13 was found. The enrichment of four mixed cultures capable of degrading solid phenanthrene in batch liquid systems was also studied. Phenanthrene degradation rates in batch systems were culture-dependent, and simple (one-slope) and complex (two-slope) kinetic behaviours were observed. The presence of common bands of microbial species in the cultures and in the native soil DNA indicated that those strains could be potential in situ phenanthrene degraders. Consistent with this assumption are the decrease of PAH and phenanthrene contents of Belgian soil B-BT and the isolation of phenanthrene-degrading bacteria. From the fastest phenanthrene-degrading culture C(B-BT), representative strains were identified as Achromobacter xylosoxidans (100%), Methylobacterium sp. (99%), Rhizobium galegae (99%), Rhodococcus aetherovorans (100%), Stenotrophomonas acidaminiphila (100%), Alcaligenes sp. (99%) and Aquamicrobium defluvium (100%). DGGE-profiles of culture C(B-BT) showed bands attributable to Rhodococcus, Achromobacter, Methylobacterium rhizobium, Alcaligenes and Aquamicrobium. The isolation of Rhodococcus aetherovorans and Methylobacterium sp. can be consistent with the hypothesis that different phenanthrene-degrading strategies, cell surface properties, or the presence of xenobiotic-specific membrane carriers could play a role in the uptake/degradation of solid phenanthrene.

Bacteria↗

PubMed2001

A novel gene encoding a 54 kDa polypeptide is essential for butane utilization by Pseudomonas sp. IMT37.

Twenty-three propane- and butane-utilizing bacteria were isolated from soil samples collected from oilfields. Three of them have been identified as Rhodococcus sp. IMT35, Pseudomonas sp. IMT37 and Pseudomonas sp. MT40. SDS-PAGE analysis of the membrane of Rhodococcus sp. IMT35 revealed the presence of at least four polypeptides induced by propane. Polyclonal antibody raised against a 58 kDa polypeptide from Rhodococcus sp. IMT35 specifically detected bacteria which were actively utilizing propane or butane. Immunoscreening of a genomic library in lambdagt11 with this antibody resulted in isolation of a clone containing a 4.9 kb EcoRI genomic DNA fragment. This 4.9 kb DNA fragment was found to hybridize specifically with organisms which could grow on propane or butane. This fragment could therefore be used as a probe for detection of such bacteria. A 2.3 kb fragment having an ORF encoding a polypeptide of 54 kDa was identified by screening a genomic library of Pseudomonas sp. IMT37 with this 4.9 kb EcoRI fragment. The sequence of the ORF (designated orf54) was found to be novel. Primer extension and S1 nuclease mapping showed that transcription of the ORF starts at base 283 and it had sequences upstream similar to that of a Pseudomonas promoter (-12, -24 type). Disruption of the ORF by a kanamycin ('kan') cassette prevented the organism from growing on any alkane but did not affect its ability to utilize the respective alkanols and acids, indicating that alcohol dehydrogenase and subsequent steps in the pathway remained unaltered. The mutants had no detectable level of butane monooxygenase activity. Therefore, the product of this gene plays a crucial role in the first step of the pathway and is an essential component of monooxygenase. The findings imply that this bacterium either employs a common genetic and metabolic route or at least shares the product of this gene for utilization of many alkanes.

Bacterial Proteins↗

PubMed2002

Plasmid-borne genes code for an angular dioxygenase involved in dibenzofuran degradation by Terrabacter sp. strain YK3.

The genes responsible for angular dioxygenation of dibenzofuran in actinomycetes were cloned by using a degenerate set of PCR primers designed by using conserved sequences of the dioxygenase alpha subunit genes. One sequence of alpha subunit genes was commonly amplified from four dibenzofuran-utilizing actinomycetes: Terrabacter sp. strains YK1 and YK3, Rhodococcus sp. strain YK2, and Microbacterium sp. strain YK18. A 5.2-kb PstI fragment encoding the alpha and beta subunits of the terminal dioxygenase, ferredoxin, and ferredoxin reductase (designated dfdA1 to dfdA4, respectively) was cloned from the large circular plasmid pYK3 isolated from Terrabacter sp. strain YK3. We confirmed that transcription of the dfdA1 gene was induced by dibenzofuran in Terrabacter sp. strain YK3. Southern blot hybridization analysis revealed that this type of dioxygenase gene is distributed among diverse dibenzofuran-utilizing actinomycetes. However, genes homologous to dfdA1 were not detected in dibenzofuran utilization-deficient mutants of Terrabacter, Rhodococcus, and Microbacterium species. When the dfdA1 to dfdA4 genes were introduced into a non-dibenzofuran-degrading mutant of Rhodococcus sp. strain YK2, strain YK2-RD2, which had spontaneously lost the gene homologous to dfdA1, the ability to degrade dibenzofuran was restored. Analysis of the breakdown products indicated that DfdA has angular dioxygenase activity. This dfdA transformant degraded several aromatic compounds, indicating that the novel angular dioxygenase possesses broad substrate specificity.

Actinomycetales↗

PubMed2003

Characterization of hydrocarbon-degrading microbial populations in contaminated and pristine Alpine soils.

Biodegradation of petroleum hydrocarbons in cold environments, including Alpine soils, is a result of indigenous cold-adapted microorganisms able to degrade these contaminants. In the present study, the prevalence of seven genotypes involved in the degradation of n-alkanes (Pseudomonas putida GPo1 alkB; Acinetobacter spp. alkM; Rhodococcus spp. alkB1, and Rhodococcus spp. alkB2), aromatic hydrocarbons (P. putida xylE), and polycyclic aromatic hydrocarbons (P. putida ndoB and Mycobacterium sp. strain PYR-1 nidA) was determined in 12 oil-contaminated (428 to 30,644 mg of total petroleum hydrocarbons [TPH]/kg of soil) and 8 pristine Alpine soils from Tyrol (Austria) by PCR hybridization analyses of total soil community DNA, using oligonucleotide primers and DNA probes specific for each genotype. The soils investigated were also analyzed for various physical, chemical, and microbiological parameters, and statistical correlations between all parameters were determined. Genotypes containing genes from gram-negative bacteria (P. putida alkB, xylE, and ndoB and Acinetobacter alkM) were detected to a significantly higher percentage in the contaminated (50 to 75%) than in the pristine (0 to 12.5%) soils, indicating that these organisms had been enriched in soils following contamination. There was a highly significant positive correlation (P < 0.001) between the level of contamination and the number of genotypes containing genes from P. putida and Acinetobacter sp. but no significant correlation between the TPH content and the number of genotypes containing genes from gram-positive bacteria (Rhodococcus alkB1 and alkB2 and Mycobacterium nidA). These genotypes were detected at a high frequency in both contaminated (41.7 to 75%) and pristine (37.5 to 50%) soils, indicating that they are already present in substantial numbers before a contamination event. No correlation was found between the prevalence of hydrocarbon-degradative genotypes and biological activities (respiration, fluorescein diacetate hydrolysis, lipase activity) or numbers of culturable hydrocarbon-degrading soil microorganisms; there also was no correlation between the numbers of hydrocarbon degraders and the contamination level. The measured biological activities showed significant positive correlation with each other, with the organic matter content, and partially with the TPH content and a significant negative correlation with the soil dry-mass content (P < 0.05 to 0.001).

Altitude↗

PubMed2006

Sequence analysis of two cryptic plasmids from Bifidobacterium longum DJO10A and construction of a shuttle cloning vector.

Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other B. longum plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused rep gene and an RCR-type ori consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJH10S had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a Rhodococcus rhodochrous plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in B. longum DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of Rhodococcus rhodochrous (67%) and significantly higher than that of B. longum (60.1%), it may have been acquired through horizontal gene transfer from a Rhodococcus species, as both genera are members of the Actinomycetes and are intestinal inhabitants. An Escherichia coli-B. longum shuttle cloning vector was constructed from pDOJH10S and the E. coli ori region of p15A, a lacZ gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into E. coli and B. longum. It could not be introduced into lactic acid bacteria (Lactococcus and Lactobacillus), showing it was not very promiscuous. It was stably maintained in B. longum in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.

Amino Acid Sequence↗

PubMedDate not supplied

[Production of oil-processing compounds by microorganisms from the Daqing oil field, China].

Twenty pure cultures isolated from formation waters of the Daqing oil field were studied with respect to their capacity to produce surface-active compounds in media with individual hydrocarbons, lower alcohols, and fatty acids. Aerobic saprotrophic bacteria belonging to the genera Bacillus, Brevibacillus, Rhodococcus, Dietzia, Kocuria, Gordonia, Cellulomonas, Clavibacter, Pseudomonas, and Acinetobacter decreased the surface tension of cultivation media from 55-63 to 28-44 mN/m. Strains of Bacillus cereus, Rhodococcus ruber, and Bacillus licheniformis produced biosurfactants most actively. Bacteria of the genera Rhodococcus, Dietzia, Kocuria, and Gordonia produced exopolysaccharides in media with hydrocarbons. Culture liquids of the strains of R. ruber and B. licheniformis exhibited oil-releasing effect. Thus, the Daqing oil field is inhabited by aerobic bacteria capable of producing effective oil-releasing agents.

Alcohols↗

PubMedDate not supplied

[Use of claydite-immobilized oil-oxidizing microbial cells for purification of water from oil].

Oil-oxidizing bacteria were isolated from oil-polluted soil and water samples and identified as Acinetobacter calcoaceticus K-4, Nocardia vaceinii K-8, Rhodococcus erythropolis EK-1, and Mycobacterium sp. K-2. It was found that immobilization of the bacteria on an expanded clay aggregate accelerated their growth and consumption of hydrocarbon substrates. It was also found that water polluted with 100 mg/l oil could be purified with Rhodococcus erythropolis EK-1 and Nocardia vaceinii K-8 cells immobilized in this way. The dependence of the degree of water purification on its flow rate, aeration, and availability of nitrogen and phosphorus sources was determined. The efficiency of water purification from oil by immobilized Rhodococcus erythropolis EK-1 cells at high flow rates (of up to 0.68 l/h), low aeration (of 0.1 l/l per min) and an intermittent supply of 0.01% diammonium phosphate reached 99.5-99.8%.

Aluminum Silicates↗

PubMed2006

[Identification of mycobacteria by sequencing of rpoB gene and 16S rRNA].

PURPOSE: To classify a specific Mycobacterium among various mycobacteria utilizing sequencing of rpoB gene. To classify mycobacteria not identified by DNA-DNA hybridization (DDH) using sequencing of rpoB and 16S rRNA gene. OBJECTS AND METHODS: Classification of 106 Mycobacteria strains, one Nocardia strain, one Rhodococcus strain, four Gordona strains was made by using partial sequencing of rpoB and 16S rRNA (RIDOM). Thereafter, 38 mycobacteria clinical strains not identified by DDH were classified utilizing the DNA sequencing data. RESULTS: Pairs of M. kansasii and M. gastri, M. abscessus and M. chelonae, M.fortuitum (ATCC49404) and M. polcinum, M. peregrinum and M. septicum, M. farucinogense and M. senegalense and M. fortuitum (ATCC49403), Rhodococcus, Nocardia and Gordona strains were classified using sequencing of rpoB gene. Even though sequencing of rpoB and 16S rRNA gene was utilized, it was impossible to classify M. tuberculosis complex, M. avium family, M. marinum and M. ulcerans, and M. fortitum subsp. fortuitum and M. fortuitum subsp. acetamidolyticus. The 38 mycobacteria clinical strains not identified by DDH were successfully classified using sequencing of both rpoB and 16S rRNA. These sequencing analyses showed that M. heckeshornense, M. branderi, M. intermedium, M. shimoidei, M. wolinskyi, M. malmoense and M. lentiflavum could be identified. Thirty six clinical isolates (94.7%) and 32 clinical isolates (84.2%) were identified by rpoB sequencing and 16S rRNA sequencing (RIDOM), respectively. CONCLUSION: The classification ratio of mycobacteria including Nocardia, Rhodococcus and Gordona is 69.6% for sequencing of 16S rRNA and 89.3% for sequencing of rpoB gene. Sequencing of rpoB is useful for classification of mycobacteria due to its genetic diversity, but has some limitation in its application. In order to classify mycobacteria more accurately, it is important to combine sequencing of rpoB and 16S rRNA and biochemical/biological tests.

Bacterial Proteins↗

PubMed2002

Application of multi-parameter flow cytometry using fluorescent probes to study substrate toxicity in the indene bioconversion.

The bioconversion of indene to cis-(1S,2R) indandiol, a potential key intermediate in the synthesis of Merck's HIV protease inhibitor, CRIXIVAN trade mark, can be achieved using a Rhodococcus strain. This study using Rhodococcus I24 reports on the application of multiparameter flow cytometry for the measurement of cell physiological properties based on cytoplasmic membrane (CM) integrity and membrane depolarization as indicators of toxic effects of the substrate, indene. Quantification of intact polarized CM, intact depolarized CM and permeabilized CM of a large population of bacterial cells has been conducted using specific intracellular and membrane-binding fluorescent stains. Measurements of oxygen uptake rate (OUR) and optical density (OD) as indicators of metabolic activity and biomass growth, respectively, were also made. Indene concentrations of up to 0.25 g/L (0.037 g indene/g dry cell weight) did not significantly (<5% compared to control) affect cell light-scattering properties, intact CM, membrane polarization, respiratory activity, or biomass growth. Between this value and 1.5 g/L (0.221 g indene/g dry cell weight), the changes in intact CM, respiratory activity and biomass growth were relatively insignificant (<5% compared to control), although dissipation of the membrane potential of a significant proportion of the cell population occurred at 0.50 g/L (0.074 g indene/g dry cell weight). At 2.5 g/L (0.368 g indene/g dry cell weight) there was a significant increase in the dead cell population, accompanied by changes in the extracellular cationic concentrations and substantial decrease in respiratory activity. The primary effect of indene toxicity was the disruption of the proton motive force across the cytoplasmic membrane which drives the formation of ATP. The disruption of the proton motive force may have been due to the measured changes in proton permeability across the membrane. In addition, indene may have directly inhibited the membrane-bound enzymes related to respiratory activity. The overall consequence of this was reduced respiratory activity and biomass growth. The cell physiological properties measured via flow cytometry are important for understanding the effects of toxicity at the cellular level which neither measurements of biomass growth or indandiol formation rates can provide since both are cell averaged measurements. The technique described here can also be used as a generic tool for measuring cell membrane properties in response to toxicity of other indene-resistant strains that may be possible to use as recombinant hosts to perform the biotransformation of indene. This study has demonstrated that flow cytometry is a powerful tool for the measurement of cell physiological properties to assess solvent toxicity on whole cell biocatalysts.

Adenosine Triphosphate↗

PubMed2006

Proteomic and transcriptional characterization of aromatic degradation pathways in Rhodoccocus sp. strain TFB.

Rhodococcus sp. strain TFB is a versatile gram-positive bacterium able to grow on a wide variety of aromatic compounds as carbon and energy sources. Since the strain is refractory to genetic analysis, a proteomic approach was used to study the metabolic pathways involved in the catabolism of such compounds by analyzing differentially induced proteins. The most marked difference was observed when the proteome profiles of phthalate-grown cells were compared with those cultured in the presence of tetralin- or naphthalene, suggesting that different metabolic pathways are involved in the degradation of mono- and polyaromatic compounds. Comparison with the proteome of glucose-grown cells indicated that each pathway was specifically induced by the corresponding aromatic compound. A combination of proteomics and molecular biology led to the identification of 14 proteins (65-80% identical to known Pht proteins) that describe a complete pathway for the catabolism of phthalate to central metabolites via intradiol cleavage of protochatechuic acid. Chaperonins were also induced in phthalate-grown cells, indicating that growth on this compound induces a stress response. Absence of catabolite repression by glucose was observed by both transcriptional and proteome analysis, suggesting that Rhodococcus sp. strain TFB may have advantages over other tightly regulated strains in bioremediation.

Amino Acid Sequence↗

PubMed1999

The Glu residue in the conserved Asn-Glu-Pro sequence of endoglycoceramidase is essential for enzymatic activity.

Endoglycoceramidase (EGCase) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. We previously cloned the gene encoding EGCase II of Rhodococcus sp. M-777 and reported that the deduced amino acid sequence contained the Asn-Glu-Pro (NEP) sequence, conserved as part of the active site of family A cellulases (endo-1,4-beta-glucanases) (J. Biol. Chem. 272, 19846, 1997). The NEP sequence was also found in the deduced amino acid sequence of the newly cloned EGCase gene of Rhodococcus sp. C9. Replacement of the Glu residue in the NEP sequence with Gln or Asp by site-directed mutagenesis caused marked loss of enzymatic activity in both the M-777 and C9 EGCases but did not affect the expression of EGCase protein. This result clearly indicated that the NEP sequence is part of the active site of EGCase, in which the Glu residue plays an important role in the catalytic reaction, possibly in the same manner as in endo-1,4-beta-glucanase.

Amino Acid Sequence↗

PubMed1998

Late events in the assembly of 20S proteasomes.

Electron microscopy and STEM mass measurements have been used to characterize late intermediates in the assembly pathway of wildtype and mutant Rhodococcus proteasomes. A proteolytically inactive and processing-incompetent mutant, betaK33A, allowed a short-lived late intermediate of the pathway to be captured, the preholoproteasome. In this fully assembled 20S complex the 14 propeptides with an aggregate mass of 100 kDa fill the whole central cavity and most of the two antechambers. It is further shown that in wildtype Rhodococcus proteasomes the propeptides are degraded in a processive manner undergoing multiple cleavages before the products are discharged and the inner cavities are cleared. It appears that the docking of two half-proteasomes, i.e., preholoproteasome formation, is sufficient to trigger autocleavage of the Gly-1/Thr1 bond necessary for active site formation and the subsequent degradation of the propeptides.

Amino Acid Substitution↗

PubMedDate not supplied

Linear alkanesulfonates as carbon and energy sources for gram-positive and gram-negative bacteria.

Several bacteria from soil and rainwater samples were enriched and isolated with propanesulfonate or butanesulfonate as sole carbon and energy source. Most of the strains isolated utilized nonsubstituted alkanesulfonates with a chain length of C3-C6 and the substituted sulfonates taurine and isethionate as carbon and energy source. A gram-positive isolate, P40, and a gram-negative isolate, P53, were characterized in more detail. Phylogenetic analysis grouped strain P40 within group IV of the genus Rhodococcus and showed a close relationship with Rhodococcus opacus. After phylogenetic and physiological analyses, strain P53 was identified as Comamonas acidovorans. Both bacteria also utilized a wide range of sulfonates as sulfur source. Strain P40, but not strain P53, released sulfite into the medium during dissimilation of sulfonated compounds. Cell-free extracts of strain P53 exhibited high sulfite oxidase activity [2.34 U (mg protein)-1] when assayed with ferricyanide, but not with cytochrome c. Experiments with whole-cell suspensions of both strains showed that the ability to dissimilate 1-propanesulfonate was specifically induced during growth on this substrate and was not present in cells grown on propanol, isethionate or taurine. Whole-cell suspensions of both strains accumulated acetone when oxidizing the non-growth substrate 2-propanesulfonate. Strain P40 cells also accumulated sulfite under these conditions. Stoichiometric measurements with 2-propanesulfonate as substrate in oxygen electrode experiments indicate that the nonsubstituted alkanesulfonates were degraded by a monooxygenase. When strain P53 grew with nonsubstituted alkanesulfonates as carbon and energy source, cells expressed high amounts of yellow pigments, supporting the proposition that an oxygenase containing iron sulfur centres or flavins was involved in their degradation.

Acinetobacter↗

PubMed2002

P450(camr), a cytochrome P450 catalysing the stereospecific 6- endo-hydroxylation of (1 R)-(+)-camphor.

Rhodococcus sp. NCIMB 9784 accumulated 6- endo-hydroxycamphor 3 when grown on (1 R)-(+)-camphor 1 as sole carbon source. The structure of 3 has been unambiguously assigned for the first time using X-ray crystallography. A soluble cytochrome P450 hydroxylase, induced by growth on (1 R)-(+)-camphor and designated P450(camr), has been isolated from the bacterium Rhodococcus sp. NCIMB 9784. Using authentic 6- endo hydroxycamphor as standard, a cell-free system consisting of pure P450(camr) and putidaredoxin and putidaredoxin reductase from Pseudomonas putida confirmed that the enzyme hydroxylates (1 R)-(+)-camphor specifically in the 6- endoposition, in contrast to the 5- exo hydroxylation catalysed by the well-studied P450(cam) from P. putida. P450(camr) has a molecular mass of approximately 44 kDa, and a pI of 4.8.

Camphor↗