Characterization of free and immobilized (<Emphasis Type="Italic">S</Emphasis>)-aminotransferase for acetophenone production

Gordonia amicalis F.5.25.8 has the unique ability to desulfurize dibenzothiophene and to metabolize carbazole [Santos et al., Appl Microbiol Biotechnol 71:355–362, 2006]. Efforts to amplify the dsz genes from G. amicalis F.5.25.8 based on polymerase chain reaction (PCR) primers designed using the dsz gene sequences of Rhodococcus erythropolis IGTS8 were mostly unsuccessful. A comparison of the protein sequences of dissimilar desulfurization enzymes (DszABC, BdsABC, and TdsABC) revealed multiple conserved regions. PCR primers targeting some of the most highly conserved regions of the desulfurization genes allowed us to amplify dsz genes from G. amicalis F.5.25.8. DNA sequence data that include nearly the entirety of the desulfurization operon as well as the promoter region were obtained. The most closely related dsz genes are those of G. alkinovorans strain 1B at 85% identity. The PCR primers reported here should be useful in microbial ecology studies and the amplification of desulfurization genes from previously uncharacterized microbial cultures.     

Desulfurization of dibenzothiophene by Bacillus subtilis recombinants carrying dszABC and dszD genes

The desulfurization (dsz) genes from Rhodococcus erythropolis DS-3 were successfully integrated into the chromosomes of Bacillus subtilis ATCC 21332 and UV1 using an integration vector pDGSDN, yielding two recombinant strains, B. subtilis M29 and M28 in which the integrated dsz genes were expressed efficiently under the promoter, Pspac. The dibenzothiophene (DBT) desulfurization efficiency of M29 was 16.2 mg DBT l⁻¹ h⁻¹ at 36 h, significantly higher than that of R. erythropolis DS−3 and B. subtilis M28 and also showed no product inhibition. The interfacial tension of the supernatant fermented by M29 varied from 48 mN m⁻¹ to 4.2 mN m⁻¹, lower than that of the recombinant strain, M28, reveals that the biosurfactant secreted from M29 may have an important function in the DBT desulfurization process.     

Cloning of a rhodococcal promoter using a transposon for dibenzothiophene biodesulfurization

The expression of biodesulfurization genes (dsz) in Rhodococcus erythropolis strain KA2-5-1 is repressed by sulfate which is the product of biodesulfurization. The application of a sulfate non-repressible promoter could be effective in enhancing biodesulfurization. A promoter-probe transposon was constructed using the promoterless, red-shifted green fluorescence protein gene (rsgfp). A 340 bp putative promoter element, designated kap1, was isolated from a strain KA2-5-1 recombinant that had shown high fluorescence intensity. The activity of kap1 was not affected by 1 mM sulfate. It gave about a 2-fold greater activity than the 16S ribosomal RNA promoter in R. erythropolis strain KA2-5-1 and is therefore useful for expressing desulfurization genes in rhodococcal strains.     

Identification and Kinetic Characteristics of an Indigenous Diesel-degrading Gordonia alkanivorans Strain

Summary An indigenous strain Gordonia alkanivorans CC-JG39 was isolated from oil-contaminated sludge of a local gas station located in central Taiwan. The bacterial isolate was able to grow on diesel-containing Bushnell–Haas medium and also tolerate various chemical additives frequently used in petroleum products (e.g. BETX, methyl-tert-butyl ether, and naphthalene). Kinetics of diesel-limited cell growth and biodegradation of diesel followed a Monod-type model. The kinetic constants for cell growth (μ_max and K_S,G) were 0.158 h⁻¹ and 3196 mg/l, respectively, while those for biodegradation of diesel (v_{max, diesel} and K_S,D) were 3.59 mg/h/mg cell and 2874 mg/l, respectively. G. alkanivorans CC-JG39 produced extracellular surface-active material, leading to a low surface tension of nearly 33 mN/m. The CC-JG39 strain also possessed the ability to float towards the oil/water interface. These features might play some roles in enhancing the mass transfer efficiency between oil substrate and the bacterial cells. Therefore, G. alkanivorans CC-JG39 may have potential applications in bioremediation of oil pollution sites.     

Biodesulfurization of dibenzothiophene by recombinant Gordonia alkanivorans RIPI90A

The dszABC genes from newly reported dibenzothiophene biodesulfurizing bacterium, Gordonia alkanivorans RIPI90A were cloned and sequenced. The overall nucleotide sequence similarity between the dszABC genes of G. alkanivorans RIPI90A and those of Rhodococcus erythropolis IGTS8 and Gordonia nitida were 83.1% and 83.2%, respectively. A gene transfer system for G. alkanivorans RIPI90A was established employing the Escherichia coli-Rhodococcus shuttle vector pRSG43 as suitable cloning vector, resulting in transformation efficiencies up to 1.6 x 10(5)CFUs microg(-1) plasmid DNA. This stable vector was applied to cloning and efficient expression of the dsz genes under the control of lac promoter. The recombinant strain was able to desulfurize dibenzothiophene in the presence of inorganic sulfate and sulfur-containing amino acids. The maximum desulfurization activity by recombinant resting cells (131.8 microM2-hydroxybiphenylg(dry cell weight)(-1)h(-1)) was increased 2.67-fold in comparison to the highest desulfurization activity of native resting cells.     

Evidence for the role of zinc on the performance of dibenzothiophene desulfurization by <Emphasis Type="Italic">Gordonia alkanivorans</Emphasis> strain 1B

Gordonia alkanivorans strain 1B is able to desulfurize dibenzothiophene (DBT) to 2-hydroxybiphenyl (2-HBP), the final product of the 4S pathway. However, both the cell growth and the rate of desulfurization can be largely affected by the nutrient composition of the growth medium due to cofactor requirements of many enzymes involved in the biochemical pathways. In this work, the effect of several metal ions on the growth and DBT desulfurization by G. alkanivorans was studied. From all the metal ions tested, only the absence of zinc significantly affected the cell growth and the desulfurization rate. By increasing the concentration of Zn from 1 to 10 mg L⁻¹, 2-HBP productivity was improved by 26%. The absence of Zn²⁺, when sulfate was also used as the only sulfur source, did not cause any difference in the bacterial growth. Resting cells grown in the presence of Zn²⁺ exhibited a 2-HBP specific productivity of 2.29 μmol g⁻¹ (DCW) h⁻¹, 7.6-fold higher than the specific productivity obtained by resting cells grown in the absence of Zn²⁺ (0.30 μmol g⁻¹ (DCW) h⁻¹). These data suggests that zinc might have a key physiological role in the metabolism of DBT desulfurization.     

Comparative studies of phenotypic and genetic characteristics between two desulfurizing isolates of Rhodococcus erythropolis and the well-characterized R. erythropolis strain IGTS8

Two Rhodococcus erythropolis isolates, named A66 and A69, together with the well-characterized R. erythropolis strain IGTS8 were compared biochemically and genetically. Both isolates, like strain IGTS8, desulfurized DBT to 2-hydroxybiphenyl (2-HBP), following the 4S pathway of desulfurization. Strain IGTS8 showed the highest (81.5%) desulfurization activity in a medium containing DBT at 30 °C. Strain A66 showed approximately the same desulfurization activity either when incubated at 30 °C or at 37 °C, while strain A69 showed an increase of desulfurization efficiency (up to 79%) when incubated at 37 °C. Strains A66 and A69 were also able to grow using various organosulfur or organonitrogen-compounds as the sole sulfur or nitrogen sources. The biological responses of A66, A69 and IGTS8 strains to a series of mutagens and environmental agents were evaluated, trying to mimic actual circumstances involved in exposure/handling of microorganisms during petroleum biorefining. The results showed that strains A69 and IGTS8 were much more resistant to UVC treatment than A66. The three desulfurization genes (dszA, dszB and dszC) present in strains A66 and A69 were partially characterized. They seem to be located on a plasmid, not only in the strain IGTS8, but also in A66 and A69. PCR amplification was observed using specific primers for dsz genes in all the strains tested; however, no amplification product was observed using primers for carbazole (car) or quinoline (qor) metabolisms. All this information contributes to broaden our knowledge concerning both the desulfurization of DBT and the degradation of organonitrogen compounds within the R. erythropolis species.     

Cloning the amidase gene from Rhodococcus rhodochrous M8 and its expression in Escherichia coli

The amidase gene from Rhodococcus rhodochrous M8 was cloned by PCR amplification with primers developed by use of peptide amino acid sequences obtained after treating amidase with trypsin. Nucleotide sequence analysis of this gene revealed high homology with aliphatic amidases from R. erythropolis R312 and Pseudomonas aeruginosa. Considering the substrate specificity and the results of DNA analysis, amidase from R. rhodochrous M8 was assigned to the group of aliphatic amidases preferentially hydrolyzing short-chain aliphatic amides. The amidase gene was expressed in cells of Escherichia coli from the self promoter and from the lac promoter. To clone a fragment of R. rhodochrous M8 chromosome (approximately 9 kb), containing the entire structural gene and its flanking regions, plasmid pRY1 that can be integrated into the chromosome via homology regions was used. No sequences of the nitrile hydratase gene, the second key gene of nitrile degradation in strain R. rhodochrous M8, were detected. Thus, genes encoding amidase and nitrile hydratase in strain R. rhodochrous M8 are not organized into a single operon despite their common regulation.     

Isolation and preliminary characterization of a respiratory nitrate reductase from hydrocarbon-degrading bacterium <Emphasis Type="Italic">Gordonia alkanivorans</Emphasis> S7

Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors. The respiratory nitrate reductase (Nar) from this organism has been isolated using ion exchange chromatography and gel filtration, and then preliminarily characterized. PAGE, SDS-PAGE and gel filtration chromatography revealed that Nar consisted of three subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40°C. K _m values for NO₃ ⁻ (110 μM) and for ClO₃ ⁻ (138 μM) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from Gordonia species.     

De-repression and comparison of oil–water separation activity of the dibenzothiophene desulfurizing bacterium, <Emphasis Type="Italic">Mycobacterium</Emphasis> sp. G3

Expression of the desulfurization genes (dsz) in Mycobacterium sp. G3 is repressed by sulfate, which is the product of biodesulfurization. An expression clone, pSMTABC, was constructed by placing the dsz genes downstream of the hsp60 promoter and the constructed plasmid was electroporated into G3. The recombinant strain G3-1 desulfurized dibenzothiophene in the presence of 0.5 mM sulfate while the Dsz phenotype was completely repressed in the wild-type strain. However, there was no significant increase in the amount of desulfurization enzymes in G3-1. In addition, G3 had superior separation of diesel oil–water separation activity compared to E. coli, which is superior to desulfurizing rhodococci.     

Temperature effect on bacterial azo bond reduction kinetics: an Arrhenius plot analysis

Studied was the effect of temperature in the range 12–46 °C on the rate of bacterial decolorization of the mono-azo dye Acid Orange 7 by Alcaligenes faecalis 6132 and Rhodococcus erythropolis 24. With both strains the raise of temperature led to a corresponding raise of decolorization rate better manifested by R. erythropolis. The analysis of the Arrhenius plot revealed a break near the middle of the temperature range. The regression analysis showed practically complete identity of the observed break point temperatures (T _BP): 20.7 °C for Alc. faecalis and 20.8 °C for R. erythropolis. The values of the activation energy of the decolorization reaction (E _a) were found to depend on both the organism and the temperature range. In the range below T _BP the estimated values of E _a were 138 ± 7 kJ mol⁻¹ for Alc. faecalis and 160 ± 8 kJ mol⁻¹ for R. erythropolis. In the range above T _BP they were 54.2 ± 1.8 kJ mol⁻¹ for Alc. faecalis and 37.6 ± 4.1 kJ mol⁻¹ for R. erythropolis. Discussed are the possible reasons for the observed abrupt change of the activation energy.     

Toxicity evaluation of 2-hydroxybiphenyl and other compounds involved in studies of fossil fuels biodesulphurisation

The acute toxicity of some compounds used in fossil fuels biodesulphurisation studies, on the respiration activity, was evaluated by Gordonia alkanivorans and Rhodococcus erythropolis. Moreover, the effect of 2-hydroxybiphenyl on cell growth of both strains was also determined, using batch (chronic bioassays) and continuous cultures. The IC₅₀ values obtained showed the toxicity of all the compounds tested to both strains, specially the high toxicity of 2-HBP. These results were confirmed by the chronic toxicity data. The toxicity data sets highlight for a higher sensitivity to the toxicant by the strain presenting a lower growth rate, due to a lower cells number in contact with the toxicant. Thus, microorganisms exhibiting faster generation times could be more resistant to 2-HBP accumulation during a BDS process. The physiological response of both strains to 2-HBP pulse in a steady-state continuous culture shows their potential to be used in a future fossil fuel BDS process.     

Cell adaptation to solvent, substrate and product: a successful strategy to overcome product inhibition in a bioconversion system

Carvone has previously been found to highly inhibit its own production at concentrations above 50 mM during conversion of a diastereomeric mixture of (−)-carveol by whole cells of Rhodococcus erythropolis. Adaptation of the cells to the presence of increasing concentrations of carveol and carvone in n-dodecane prior to biotransformation proved successful in overcoming carvone inhibition. By adapting R. erythropolis cells for 197 h, an 8.3-fold increase in carvone production rate compared to non-adapted cells was achieved in an air-driven column reactor. After an incubation period of 268 h, a final carvone concentration of 1.03 M could be attained, together with high productivity [0.19 mg carvone h⁻¹ (ml organic phase)⁻¹] and high yield (0.96 g carvone g carveol⁻¹).     

Mapping of the resistance genes of the R plasmid NR1.

Summary The drug resistance genes on the r-determinants component of the composite R plasmid NR1 were mapped on the EcoRI restriction endonuclease fragments of the R plasmid by cloning the fragments using the plasmid RSF2124 as a vector. The sulfonamide (Su) and streptomycin/spectinomycin (Sm/Sp) resistance genes are located on EcoRI fragment G of NR1. The expression of resistance to mercuric ions (Mer) requires both EcoRI fragment H and I of NR1. The expression of chloramphenicol (Cm) and fusidic acid (Fus) resistance requires EcoRI fragments A and J of NR1. The kan fragment of the related R plasmid R6-5 can substitute for EcoRI fragment J of NR1 in the expression of Cm and Fus resistance. The structural genes for Cm and Fus resistance appear to be a part of an operon whose expression is controlled by the same promoter.     

Recombinant Rhodococcus sp. strain T09 can desulfurize DBT in the presence of inorganic sulfate

The putative Rhodococcus rrn promoter region was cloned from the benzothiophene desulfurizing Rhodococcus sp. strain T09, and the dibenzothiophene desulfurizing gene, dsz, was expressed under the control of the putative rrn promoter in the strain T09 using a Rhodococcus–E.coli shuttle vector. Strain T09 harboring the expression vector, pNT, could desulfurize dibenzothiophene in the presence of inorganic sulfate, methionine, or cysteine, while the Dsz phenotype was completely repressed in recombinant cells carrying the gene under the control of the native dsz promoter under the same conditions. Among the sulfur sources examined, no intermediates were detected and only the final desulfurized product, 2-hydroxy-biphenyl, was produced using ammonium sulfate as the sulfur source. Received: 4 December 2001 / Accepted: 7 January 2002     

Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes

Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.