Structural analysis of mycolic acids from phenol-degrading strain of Rhodococcus erythropolis by liquid chromatography–tandem mass spectrometry

We used reversed phase liquid chromatography?Celectrospray ionization tandem mass spectrometry for direct analysis of mycolic acids (MAs) from four different cultivations of Rhodococcus erythropolis. This technique enabled us to identify and quantify the specific molecular species of MAs directly from lipid extracts of the bacterium, including the determination of their basic characteristics such as retention time and mass spectra. We identified a total of 60 molecular species of MAs by means of LC/MS. In collision-induced dissociation tandem mass spectrometry, the [M-H]^? ions eliminated two residues, i.e., meroaldehyde and carboxylate anions containing ??-alkyl chains. The structural information from these fragment ions affords structural assignment of the mycolic acids, including the lengths and number of double bond(s). Two strains, i.e., R. erythropolis CCM 2595 and genetically modified strain CCM 2595 pSRK 21 phe were cultivated on two different substrates (phenol and phenol with addition of humic acids as a sole carbon source). The addition of humic acids showed that there is a marked increase of unsaturated mycolic acids, mostly in the range of 20?C100?%. This effect is more pronounced in the R. erythropolis CCM 2595 strain.     

Degradation of polychlorinated biphenyls (PCBs) by four bacterial isolates obtained from the PCB-contaminated soil and PCB-contaminated sediment

We investigated the PCB-degrading abilities of four bacterial strains isolated from long-term PCB-contaminated soil (Alcaligenes xylosoxidans and Pseudomonas stutzeri) and sediments (Ochrobactrum anthropi and Pseudomonas veronii) that were co-metabolically grown on glucose plus biphenyl which is an inducer of the PCB catabolic pathway. The aim of study was to determine the respective contribution of biomass increase and expression of degrading enzymes on the PCB degrading abilities of each isolate. Growth on 5 g l⁻¹ glucose alone resulted in the highest stimulation of the growth of bacterial strains, whereas grown on 10 mg l⁻¹, 100 mg l⁻¹, 1 g l⁻¹, or 5 g l⁻¹ biphenyl did not effected the bacterial growth. None of the strains used in this study was able to grow on PCBs as the sole carbon source. Cells grown on glucose exhibited enhanced degradation ability due to an increased biomass. Addition of biphenyl at concentrations of 1 or 5 g l⁻¹ did not increase total PCB degradation, but stimulated the degradation of highly chlorinated congeners for some of the strains. The degradation of di- and tri-chlorobiphenyls was significantly lower for cells grown on 5 g l⁻¹ biphenyl independently on glucose addition. The highest degradation of the PCBs was obtained for A. xylosoxidans grown in the presence of glucose. Thus A. xylosoxidans appears to be the most promising among the four bacterial isolates for the purpose of bioremediation.     

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.     

Potential of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> as a bioremediation organism

Summary The capability of Rhodococcus erythropolis CCM 2595(ATCC 11048) to utilize phenol, pyrocatechol, resorcinol, p-nitrophenol, p-chlorophenol, hydroquinone and hydroxybenzoate, respectively, or as respective binary mixtures with phenol, was described. This capability was found to depend on the substrate and its initial concentration. Some monoaromatic compounds had a suppressive effect on the strain’s ability to utilize phenol in a binary mixture and easily utilizable monoaromatics were strong inducers of the phenol 2-monooxygenase (EC 1.14.13.7). The capacity of R. erythropolis to colonize a synthetic zeolite was demonstrated and the enhancement of phenol tolerance of biofilms utilizing phenol was observed. The effect of humic acids on phenol killing was described and discussed as well. To allow use of recombinant DNA technology for strain improvement, methods of genetic transfer (transformation and conjugation) in R. erythropolis were established.     

Versatile aromatic compound-degrading capacity and microdiversity of <Emphasis Type="Italic">Thauera</Emphasis> strains isolated from a coking wastewater treatment bioreactor

Bacteria of the Thauera genus have been described as important aromatic compound degraders and have attracted increased attention. In this study, three Thauera strains (Q4, Q20-C, and 3–35) were isolated from a coking wastewater treatment plant (WWTP) with a high abundance of Thauera. The 16S rRNA, nitrite reductase, and phenol hydroxylase (LmPH) genes and pollutant-degrading capacity of these strains were characterized and compared. Their 16S rRNA gene sequences were identical, but the genomic structures differed, as demonstrated by distinct enterobacterial repetitive intergenic consensus sequence PCR profiles with a similarity of less than 0.65. The analysis of degradation of coking wastewater by these strains showed that most of the main organic pollutants—phenol, methylphenol, and indole, but not quinoline—were degraded under aerobic conditions. These strains contained different LmPHs genes and showed different phenol degradation rates (Q4 > 3–35 > Q20-C). The presence of a microdiversity of Thauera spp. implies the existence of various finely differentiated niches in the industrial WWTP. The capacity of the Thauera strains to degrade a wide spectrum of aromatic compounds suggests their potential in bioremediation applications targeting aromatic pollutant-containing wastewater.     

Horizontal transfer of catabolic plasmids in the process of naphthalene biodegradation in model soil systems

The process of naphthalene degradation by indigenous, introduced, and transconjugant strains was studied in laboratory soil microcosms. Conjugation transfer of catabolic plasmids was demonstrated in naphthalene-contaminated soil. Both indigenous microorganisms and an introduced laboratory strain BS394 (pNF142::TnMod-OTc) served as donors of these plasmids. The indigenous bacterial degraders of naphthalene isolated from soil were identified as Pseudomonas putida and Pseudomonas fluorescens. The frequency of plasmid transfer in soil was 10^?5–10^?4 per donor cell. The activity of the key enzymes of naphthalene biodegradation in indigenous and transconjugant strains was studied. Transconjugant strains harboring indigenous catabolic plasmids possessed high salicylate hydroxylase and low catechol-2,3-dioxygenase activities, in contrast to indigenous degraders, which had a high level of catechol-2,3-dioxygenase activity and a low level of salicylate hydroxylase. Naphthalene degradation in batch culture in liquid mineral medium was shown to accelerate due to cooperation of the indigenous naphthalene degrader P. fluorescens AP1 and the transconjugant strain P. putida KT2442 harboring the indigenous catabolic plasmid pAP35. The role of conjugative transfer of naphthalene biodegradation plasmids in acceleration of naphthalene degradation was demonstrated in laboratory soil microcosms.     

Bacterium Hafnia alvei secretes l-methioninase enzyme: Optimization of the enzyme secretion conditions

I isolated bacteria from blue cheese in order to find bacterial strains secreting l-methioninase enzyme, and optimized the conditions for the most efficient enzyme secretion. The efficient isolate, identified according to the 16S rRNA gene sequence analysis, was Hafnia alvei belonging to Enterobacteriaceae. I confirmed that the H. alvei strain harbored the methionase gene, mdeA (1194 bp). The environmental (pH, temperature) and nutritional (carbon and nitrogen sources and Mg concentration) factors influencing the l-methioninase production of H. alvei were optimized. The highest yield of l-methioninase enzyme was reached after 48 h of incubation when the acidity of the growing medium was adjusted to pH 7.5 and the temperature was 35 °C. The following concentrations of the supplements increased the l-methioninase yield in the medium: galactose (2.0 g L⁻¹), MgSO₄ (0.25 g L⁻¹), l-methionine as an inducer (2.0 g L⁻¹), and l-asparagine as an additional N source (1.5 g L⁻¹). I introduce a bacterial strain of H. alvei that is previously unreported to secrete l-methioninase enzyme and show that a carbon source is a mandatory supplement whereas l-methionine is not a mandatory supplement for l-methioninase enzyme production of H. alvei.     

Biodegradation of phenol using hairy roots of Helianthus annuus L.

Phenol is a common pollutant which is found in wastewater of many industries and removal of phenol from the industrial effluents is a major challenge. Recently, the use of hairy roots has been probed for the removal of phenol. In the present study, phenol at various concentrations (100–500 mg L⁻¹) was treated with hairy roots of Helianthus annuus (sunflower hairy roots, SHRs). SHRs removed 100 mg L⁻¹ of phenol after 144 h of incubation. The effect of polyethylene glycol (PEG), l-proline and d-glucose on the rate of phenol removal was also studied. l-proline enhanced the removal efficiency of SHRs resulting in the removal of 100 mg L⁻¹ of phenol after 24 h while PEG did not show any effect on removal. Peroxidase activity was induced after 24 h of phenol addition. Phenol metabolism to generate catechol as a major metabolite was confirmed using HPLC and GC–MS analyses. The detection of small amounts of cis-cis muconic acid and fumaric acid in the reaction medium suggests that these metabolites are produced from the ring cleavage of catechol. The phytotoxicity and cytotoxicity results suggest the non-toxic nature of the resulting phenol metabolites.     

Systems metabolic engineering of Corynebacterium glutamicum eliminates all by-products for selective and high-yield production of the platform chemical 5-aminovalerate

5-aminovalerate (AVA) is a platform chemical of substantial commercial value to derive nylon-5 and five-carbon derivatives like δ-valerolactam, 1,5-pentanediol, glutarate, and 5-hydroxyvalerate. De novo bio-production synthesis of AVA using metabolically engineered cell factories is regarded as exemplary route to provide this chemical in a sustainable way. So far, this route is limited by low titers, rates and yields and suffers from high levels of by-products. To overcome these limitations, we developed a novel family of AVA producing C. glutamicum cell factories. Stepwise optimization included (i) improved AVA biosynthesis by expression balancing of the heterologous davBA genes from P. putida, (ii) reduced formation of the by-product glutarate by disruption of the catabolic y-aminobutyrate pathway (iii), increased AVA export, and (iv) reduced AVA re-import via native and heterologous transporters to account for the accumulation of intracellular AVA up to 300 mM. Strain C. glutamicum AVA-5A, obtained after several optimization rounds, produced 48.3 g L⁻¹ AVA in a fed-batch process and achieved a high yield of 0.21 g g⁻¹. Surprisingly in later stages, the mutant suddenly accumulated glutarate to an extent equivalent to 30% of the amount of AVA formed, tenfold more than in the early process, displaying a severe drawback toward industrial production. Further exploration led to the discovery that ArgD, naturally aminating N-acetyl-l-ornithine during l-arginine biosynthesis, exhibits deaminating side activity on AVA towards glutarate formation. This promiscuity became relevant because of the high intracellular AVA level and the fact that ArgD became unoccupied with the gradually stronger switch-off of anabolism during production. Glutarate formation was favorably abolished in the advanced strains AVA-6A, AVA-6B, and AVA-7, all lacking argD. In a fed-batch process, C. glutamicum AVA-7 produced 46.5 g L⁻¹ AVA at a yield of 0.34 g g⁻¹ and a maximum productivity of 1.52 g L⁻¹ h⁻¹, outperforming all previously reported efforts and stetting a milestone toward industrial manufacturing of AVA. Notably, the novel cell factories are fully genome-based, offering high genetic stability and requiring no selection markers     

Purification and characterization of four key enzymes from a feather-degrading Bacillus subtilis from the gut of tarantula Chilobrachys guangxiensis

A feather-degrading bacterium was isolated from the gut of the tarantula Chilobrachys guangxiensis, and was classified as Bacillus subtilis (named Bacillus subtilis CH-1) according to both the phenotypic characteristics and 16S rRNA profile. The improved culture conditions for feather-degrading were 10.0 g l⁻¹ mannitol, 10.0 g l⁻¹ tryptone, 0.1 g l⁻¹ MgCl₂, 0.4 g l⁻¹ KH₂PO₄, 0.3 g l⁻¹ K₂HPO₄, 0.5 g l⁻¹ NaCl, and 2.0 g l⁻¹ intact feather, with pH 8.5 and 37 °C. In the optimized medium, the intact black feather was completely degraded by Bacillus subtilis CH-1 in 24 h. Furthermore, four kinds of enzymes which include extracellular protease Vpr, peptidase T, γ-glutamyl transpeptidase and glyoxalmethylglyoxal reductase were identified as having principal roles. Simultaneously, the relationship between the disulfide bond reducing activity (DRT) and the keratinase activity (KT) in B. subtilis CH-1 fermentation system was discussed. This is the first report for a feather-degrading enteric bacterium from tarantula. The identification of the enzymes shines a light on further understanding the molecular mechanism of feather-degrading by microbes.     

Identification of Rhodococcuserythropolis isolates capable of degrading the fungicide carbendazim

Continuous enrichment cultures were used to identify bacterial isolates capable of degrading the fungicide carbendazim. The bacteria originated from sites that had been repeatedly treated with the structurally related parent fungicide, benomyl, over a period of several years. Six isolates were identified as carbendazim degraders on the basis of their ability to produce diffusion-clearing zones on a minimal salts solid medium spray-coated with a 0.1% solution of carbendazim, their ability to grow in a minimal salts broth supplemented with carbendazim as the sole carbon source, and their ability to reduce carbendazim levels in liquid cultures. All six isolates were identified as Rhodococcus erythropolis or a closely related species by analyses of nutritional utilization and whole-cell fatty acid methyl ester profiles. A chemically induced mutant of R. erythropolis isolate B2E was identified that was no longer capable of degrading carbendazim, as determined by negative results in all three assays. Further characterization of these strains provides an opportunity for their development in bioremediation of the compound. Received: 22 July 1996 / Received revision: 16 December 1996 / Accepted: 10 January 1997     

Mutation of Candida tropicalis by Irradiation with a He-Ne Laser To Increase Its Ability To Degrade Phenol

Candida tropicalis isolated from acclimated activated sludge was used in this study. Cell suspensions with 5 × 10⁷ cells ml⁻¹ were irradiated by using a He-Ne laser. After mutagenesis, the irradiated cell suspension was diluted and plated on yeast extract-peptone-dextrose (YEPD) medium. Plates with approximately 20 individual colonies were selected, and all individual colonies were harvested for phenol biodegradation. The phenol biodegradation stabilities for 70 phenol biodegradation-positive mutants, mutant strains CTM 1 to 70, ranked according to their original phenol biodegradation potentials, were tested continuously during transfers. Finally, mutant strain CTM 2, which degraded 2,600 mg liter⁻¹ phenol within 70.5 h, was obtained on the basis of its capacity and hereditary stability for phenol biodegradation. The phenol hydroxylase gene sequences were cloned in wild and mutant strains. The results showed that four amino acids were mutated by irradiation with a laser. In order to compare the activity of phenol hydroxylase in wild and mutant strains, their genes were expressed in Escherichia coli BL21(DE3) and enzyme activities were spectrophotometrically determined. It was clear that the activity of phenol hydroxylase was promoted after irradiation with a He-Ne laser. In addition, the cell growth and intrinsic phenol biodegradation kinetics of mutant strain CTM 2 in batch cultures were also described by Haldane's kinetic equation with a wide range of initial phenol concentrations from 0 to 2,600 mg liter⁻¹. The specific growth and degradation rates further demonstrated that the CTM 2 mutant strain possessed a higher capacity to resist phenol toxicity than wild C. tropicalis did.     

Development of a microtitre plate method for determination of phenol utilization,biofilm formation and respiratory activity by environmental bacterial isolates

Twenty-five aerobic phenol-degrading bacteria, isolated from different environmental samples on phenol agar after several subcultures in phenol broth, utilized phenol (0.2 g l⁻¹) within 24 h, but removal of phenol was more rapid when other carbon sources were also present. A microtitre plate method was developed to determine growth rate, biofilm formation and respiratory activity of the strains isolated. Pseudomonas putida strains C5 and D6 showed maximum growth (as O.D. at 600 nm), P. putida D6 and unidentified bacterial strain M1 were more stable at high concentrations of phenol (0.8 g l⁻¹), and P. putida C5 formed the greatest amount of biofilm in 0.5 g phenol l⁻¹ medium. Measurement of dehydrogenase activity as reduction of triphenyl tetrazolium chloride supported data on growth rate and biofilm formation. The microtitre plate method provided a selective method for detection of the best phenol degrading and biofilm-forming microorganisms, and was also a rapid, convenient means of studying the effect of phenol concentration on growth rate and biofilm formation.     

Biodegradation of phenol and its derivatives by engineered bacteria: current knowledge and perspectives

Biodegradation of phenolic compounds is a promising alternative to physical and chemical methods used to remove these toxic pollutants from the environment. The ability of various microorganisms to metabolize phenol and its derivatives (alkylphenols, nitrophenols and halogenated derivatives) has therefore been intensively studied. Knowledge of the enzymes catalyzing the individual reactions, the genes encoding these enzymes and the regulatory mechanisms involved in the expression of the respective genes in bacteria serves as a basis for the development of more efficient degraders of phenols via genetic engineering methods. Engineered bacteria which efficiently degrade phenolic compounds were constructed in laboratories using various approaches such as cloning the catabolic genes in multicopy plasmids, the introduction of heterologous genes or broadening the substrate range of key enzymes by mutagenesis. Efforts to apply the engineered strains in in situ bioremediation are problematic, since engineered strains often do not compete successfully with indigenous microorganisms. New efficient degraders of phenolic compounds may be obtained by complex approaches at the organism level, such as genome shuffling or adaptive evolution. The application of these engineered bacteria for bioremediation will require even more complex analysis of both the biological characteristics of the degraders and the physico-chemical conditions at the polluted sites.     

Resorcinol degradation by a <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> strain under osmotic stress: mono and binary substrate matrices with phenol

A phenol-degrading Penicillium chrysogenum strain previously isolated from a salt mine was able to grow at 1,000 mg l⁻¹ of resorcinol on solid medium. The aerobic degradation of resorcinol by P. chrysogenum CLONA2 was studied in batch cultures in minimal mineral medium with 58.5 g l⁻¹ of sodium chloride using resorcinol as the sole carbon source. The fungal strain showed the ability to degrade up to 250 mg l⁻¹ of resorcinol. Resorcinol and phenol efficiency degradation by P. chrysogenum CLONA2 was compared. This strain removes phenol faster than resorcinol. When phenol and resorcinol were in binary substrate matrices, phenol enhanced resorcinol degradation, and organic load decreased with respect to the mono substrate matrices. The acute toxicity of phenol and resorcinol, individually and in combination, to Artemia franciscana larvae has been verified before and after the bioremediation process with P. chrysogenum CLONA2. The remediation process was effective in mono and binary substrate systems.     

Description of Massilia rubra sp. nov., Massilia aquatica sp. nov., Massilia mucilaginosa sp. nov., Massilia frigida sp. nov., and one Massilia genomospecies isolated from Antarctic streams,lakes and regoliths

Bacteria of the genus Massilia often colonize extreme ecosystems, however, a detailed study of the massilias from the Antarctic environment has not yet been performed. Here, sixty-four Gram-stain-negative, aerobic, motile rods isolated from different environmental samples on James Ross Island (Antarctica) were subjected to a polyphasic taxonomic study. The psychrophilic isolates exhibited slowly growing, moderately slimy colonies revealing bold pink-red pigmentation on R2A agar. The set of strains exhibited the highest 16S rRNA gene sequence similarities (99.5–99.9%) to Massilia violaceinigra B2^T and Massilia atriviolacea SOD^T and formed several phylogenetic groups based on the analysis of gyrB and lepA genes. Phenotypic characteristics allowed four of them to be distinguished from each other and from their closest relatives. Compared to the nearest phylogenetic neighbours the set of six genome-sequenced representatives exhibited considerable phylogenetic distance at the whole-genome level. Bioinformatic analysis of the genomic sequences revealed a high number of putative genes involved in oxidative stress response, heavy-metal resistance, bacteriocin production, the presence of putative genes involved in nitrogen metabolism and auxin biosynthesis. The identification of putative genes encoding aromatic dioxygenases suggests the biotechnology potential of the strains. Based on these results four novel species and one genomospecies of the genus Massilia are described and named Massilia rubra sp. nov. (P3094^T = CCM 8692^T = LMG 31213^T), Massilia aquatica sp. nov. (P3165^T = CCM 8693^T = LMG 31211^T), Massilia mucilaginosa sp. nov. (P5902^T = CCM 8733^T = LMG 31210^T), and Massilia frigida sp. nov. (P5534^T = CCM 8695^T = LMG 31212^T).     

Phenol phycoremediation by Haematococcus pluvialis coupled with enhanced astaxanthin and lipid production under rac-GR24 supplementation for enhanced biodiesel production

The present study evaluated the impact of rac-GR24 on biomass and astaxanthin production under phenol stress coupled with biodiesel recovery from Haematococcus pluvialis. Phenol supplementation showed negative impact on growth, where the lowest biomass productivity of 0.027 g L^-1 day⁻¹ was recorded at 10 µM phenol, while 0.4 µM rac-GR24 supplementation showed the highest recorded biomass productivity of 0.063 g L^-1 day⁻¹. Coupling 0.4 µM rac-GR24 at different phenol concentrations confirmed the potential of rac-GR24 to mitigate the toxic effect of phenol by enhancing yield of PSII yield, RuBISCo activity, and antioxidant efficiency, which resulted in improved phenol phycoremediation efficiency. In addition, results suggested a synergistic action by rac-GR24 supplementation under phenol treatment where rac-GR24 enhanced lipid accumulation, while phenol enhanced astaxanthin production. Dual supplementation of rac-GR24 and phenol showed the highest recorded FAMEs content, which was 32.6% higher than the control, with improved biodiesel quality. The suggested approach could enhance the economic feasibility of triple-purpose application of microalgae in wastewater treatment, astaxanthin recovery, and biodiesel production.     

Diversity of Nitrile Hydratase and Amidase Enzyme Genes in Rhodococcus erythropolis Recovered from Geographically Distinct Habitats

A molecular screening approach was developed in order to amplify the genomic region that codes for the α- and β-subunits of the nitrile hydratase (NHase) enzyme in rhodococci. Specific PCR primers were designed for the NHase genes from a collection of nitrile-degrading actinomycetes, but amplification was successful only with strains identified as Rhodococcus erythropolis. A hydratase PCR product was also obtained from R. erythropolis DSM 43066^T, which did not grow on nitriles. Southern hybridization of other members of the nitrile-degrading bacterial collection resulted in no positive signals other than those for the R. erythropolis strains used as positive controls. PCR-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS) analysis of the hydratases in the R. erythropolis strains revealed unique patterns that mostly correlated with distinct geographical sites of origin. Representative NHases were sequenced, and they exhibited more than 92.4% similarity to previously described NHases. The phylogenetic analysis and deduced amino acid sequences suggested that the novel R. erythropolis enzymes belonged to the iron-type NHase family. Some different residues in the translated sequences were located near the residues involved in the stabilization of the NHase active site, suggesting that the substitutions could be responsible for the different enzyme activities and substrate specificities observed previously in this group of actinomycetes. A similar molecular screening analysis of the amidase gene was performed, and a correlation between the PRS patterns and the geographical origins identical to the correlation found for the NHase gene was obtained, suggesting that there was coevolution of the two enzymes in R. erythropolis. Our findings indicate that the NHase and amidase genes present in geographically distinct R. erythropolis strains are not globally mixed.