Bacterium Rhodococcus Erythropolis Research Articles

A Flavor Lactone Mimicking AHL Quorum-Sensing Signals Exploits the Broad Affinity of the QsdR Regulator to Stimulate Transcription of the Rhodococcal qsd Operon Involved in Quorum-Quenching and Biocontrol Activities.

In many Gram-negative bacteria, virulence, and social behavior are controlled by quorum-sensing (QS) systems based on the synthesis and perception of N-acyl homoserine lactones (AHLs). Quorum-quenching (QQ) is currently used to disrupt bacterial communication, as a biocontrol strategy for plant crop protection. In this context, the Gram-positive bacterium Rhodococcus erythropolis uses a catabolic pathway to control the virulence of soft-rot pathogens by degrading their AHL signals. This QS signal degradation pathway requires the expression of the qsd operon, encoding the key enzyme QsdA, an intracellular lactonase that can hydrolyze a wide range of substrates. QsdR, a TetR-like family regulator, represses the expression of the qsd operon. During AHL degradation, this repression is released by the binding of the γ-butyrolactone ring of the pathogen signaling molecules to QsdR. We show here that a lactone designed to mimic quorum signals, γ-caprolactone, can act as an effector ligand of QsdR, triggering the synthesis of qsd operon-encoded enzymes. Interaction between γ-caprolactone and QsdR was demonstrated indirectly, by quantitative RT-PCR, molecular docking and transcriptional fusion approaches, and directly, in an electrophoretic mobility shift assay. This broad-affinity regulatory system demonstrates that preventive or curative quenching therapies could be triggered artificially and/or managed in a sustainable way by the addition of γ-caprolactone, a compound better known as cheap food additive. The biostimulation of QQ activity could therefore be used to counteract the lack of consistency observed in some large-scale biocontrol assays.

Response surface-based optimization of the biodegradation of a simulated vegetable oily ballast wastewater under temperate conditions using the Antarctic bacterium Rhodococcus erythropolis ADL36

Response surface-based optimization of the biodegradation of a simulated vegetable oily ballast wastewater under temperate conditions using the Antarctic bacterium Rhodococcus erythropolis ADL36

Biochemical Characterization of DszD, the Flavin Reductase Involved in Bacterial Biodesulfurization

Dibenzothiophene (DBT) is a compound found in crude oil that upon combustion releases sulfur dioxide. The current chemical process used to remove sulfur from crude oil, hydrodesulfurization (HDS), is inefficient because it fails to work effectively on heterocyclic organosulfur compounds, such as DBT. Biodesulfurization (BDS) is a promising alternative to HDS for industrial sulfur removal. Microorganisms, like the bacterium Rhodococcus erythropolis, utilize BDS enzymes to remove sulfur from these organosulfur compounds while retaining the hydrocarbon structure of the fossil fuel components. In our work, we focus on the flavin reductase from the 4S BDS pathway, dibenzothiophene flavin reductase (DszD) from R. erythropolis, which catalyzes the reduction of flavin for subsequent use of dibenzothiophene monooxygenases DszC and DszA. This project entails the use of biochemical and structural experimentation to fully comprehend catalysis of DszD. DszD has been successfully overexpressed and purified to homogeneity from E. coli. Kinetic parameters have been determined using steady‐state and transient‐state kinetics. Weare currently looking at binding interactions between DszD and its substrate, DBT. Our preliminary experiments suggest that DBT may be acting as an effector; which means that DBT is selectively binding to DszD and regulating its biological activity. We plan to follow similar experiments with other sulfur‐containing compounds found in crude oil to see there is a direct interaction between substrate and DszD. Our current work will ultimately propel our research towards the long‐term goal of improving the BDS pathway for widespread industrial use.Support or Funding InformationThis work was funded by the Research Corporation through CCSA 22672 and the NIH through Grant 5SC2AI109500This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of biosurfactants produced by the oil-degrading bacterium Rhodococcus erythropolis S67 at low temperature.

Production of trehalolipid biosurfactants by Rhodococcus erythropolis S67 depending on the growth temperature was studied. R. erythropolis S67 produced glycolipid biosurfactants such as 2,3,4-succinoyl-octanoyl-decanoyl-2'-decanoyl trehalose and 2,3,4-succinoyl-dioctanoyl-2'-decanoyl trehalose during the growth in n-hexadecane medium at 26 and 10°C, despite the different aggregate state of the hydrophobic substrate at low temperature. The surface tension of culture medium was found being reduced from 72 to 27 and 45mNm-1, respectively. Production of trehalolipid biosurfactants by R. erythropolis S67 at low temperature could be useful for the biodegradation of petroleum hydrocarbons at low temperatures by enhancing the bioremediation performance in cold regions.

Rhodococcus erythropolis Oleate Hydratase: a New Member in the Oleate Hydratase Family Tree—Biochemical and Structural Studies

AbstractRecently, the enzyme family of oleate hydratases (OHs: EC 4.2.1.53) has gained increasing scientific and economic interest, as these FAD‐binding bacterial enzymes do not require cofactor recycling and possess high thermal and pH stability. Their products, hydroxy fatty acids, are used in specialty chemical applications including surfactant and lubricant formulations. The “oleate hydratase engineering database”, established by Schmid et al. (2017), divides all OHs into 11 families (HFam1 to 11). To date, only two crystal structures of homodimeric OHs from the families HFam2 and HFam11 have been reported. In this study, we biophysically characterized an OH belonging to the HFam3 family, originating from the marine bacterium Rhodococcus erythropolis, for the first time. The crystal structure revealed that this new OH (OhyRe) surprisingly is a monomer in its active form. This particular feature provides new avenues for enzyme engineering and recycling through immobilization.

Biofiltration of benzo[α]pyrene, toluene and formaldehyde in air by a consortium of Rhodococcus erythropolis and Fusarium solani: Effect of inlet loads, gas flow and temperature

Volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs) are air contaminants with serious effects on human health. They include compounds with very different physicochemical properties, ranging from low to high volatility and low to high hydrophobicity. The objective of this research is to assess the feasibility of the simultaneous abatement of formaldehyde, a soluble and slightly polar VOC, toluene a hydrophobic and volatile VOC and benzo[α]pyrene (BaP), a representative PAH in a biofiltration reactor inoculated with the fungi Fusarium solani and the bacteria Rhodococcus erythropolis. Results obtained at an extended range of inlet loads: 3.7 to 447.7, 9.0 to 273.1 and 6.9 to 247.4gm−3h−1 of toluene, formaldehyde and BaP, respectively, show that the elimination capacity and removal efficiencies of the contaminants were largely independent of each other. Moreover, the system can accommodate a fivefold increase in inlet gas flow maintaining removal efficiencies close to 60% for all the contaminants tested when the inlet loads of contaminants were kept constant. The most dramatic decrease in elimination capacity and removal efficiency in the system was obtained by changing the temperature of the system, where a decrease from 25°C to 17°C reduced the formaldehyde removal efficiency from 67% to 43%. BaP and toluene removal efficiencies were less affected by the decrease in system’s temperature. This study shows the high flexibility of a biofiltration system inoculated with F. solani and R. erythropolis for the abatement of toluene, formaldehyde and BaP.

Biodegradation of 7-Ketocholesterol by Rhodococcus erythropolis MTCC 3951: Process optimization and enzymatic insights

The oxidation of cholesterol results in the formation of oxysterols such as 7-ketocholesterol (7KC), which are implicated in a number of age-related disorders such as atherosclerosis, Alzheimers’ disease and macular degeneration. Current modalities use antioxidants and other natural or synthetic molecules to reduce 7KC-induced cytotoxity. The alternative application of enzymes from microbial sources to degrade oxysterols in vitro and in vivo is an innovative approach. The present study aims to assess the potential of the bacteria Rhodococcus erythropolis MTCC 3951 in degrading 7KC and mining relevant enzymes involved. This strain has been previously reported to be a degrader of xenobiotics such as polyphenols, toluene and catechol. Under optimized conditions, Rhodococcus erythropolis MTCC 3951 is found to degrade 93% of 1g/l concentration of 7KC within 15days of incubation. The extra- and intra-cellular extracts were also able to hydrolyse the compound indicating the involvement of enzymatic systems in the process. The strain produced cholesterol oxidase, lipase, dehydrogenase and reductase in the presence of 7KC. We have also identified a few intermediate products to predict the degradation pathway.

Rhodococcus lactonase with organophosphate hydrolase (OPH) activity and His6-tagged OPH with lactonase activity: evolutionary proximity of the enzymes and new possibilities in their application

Decontamination of soils with complex pollution using natural strains of microorganisms is a matter of great importance. Here we report that oil-oxidizing bacteria Rhodococcus erythropolis AC-1514D and Rhodococcus ruber AC-1513D can degrade various organophosphorous pesticides (OP). Cell-mediated degradation of five different OP is apparently associated with the presence of N-acylhomoserine lactonase, which is pronouncedly similar (46-50 %) to the well-known enzyme organophosphate hydrolase (OPH), a hydrolysis catalyst for a wide variety of organophosphorous compounds. Additionally, we demonstrated the high lactonase activity of hexahistidine-tagged organophosphate hydrolase (His6-OPH) with respect to various N-acylhomoserine lactones, and we determined the catalytic constants of His6-OPH towards these compounds. These experimental data and theoretical analysis confirmed the hypothesis about the evolutionary proximity of OPH and lactonases. Using Rhodococcus cells, we carried out effective simultaneous biodegradation of pesticide paraoxon (88 mg/kg) and oil hydrocarbon hexadecane (6.3 g/kg) in the soil. Furthermore, the discovered high lactonase activity of His6-OPH offers new possibilities for developing an efficient strategy of combating resistant populations of Gram-negative bacterial cells.

A new multicopper oxidase from Gram-positive bacterium Rhodococcus erythropolis with activity modulating methionine rich tail

Multicopper oxidases are involved in a wide variety of physiological tasks in nature. They are part of the lignin formation/decomposition system in plants and fungi. In bacteria they are part of developmental processes and the heavy metal resistance apparatus. A well characterised example is the copper tolerance protein CueO of Escherichia coli (CueO(EC)). Here, we report the heterologous expression of the apo- and holo-form of CueO(RE), a homologue to CueO(EC) from Rhodococcus erythropolis. Upon incubation with copper(II) ions, low active apo-CueO(RE) was converted into the active holo-CueO(RE) in vivo. The holo-form was physico-chemically characterised using a copper(I) BCA complex and the model substrate 2,6-dimethoxyphenol. The spectroscopic and catalytic properties are different from CueO(EC), revealing a high catalytic efficiency (k(cat)/K(m)) of 115 min(-1)mM(-1) with physiological K(m) of 80 μM for the cuprous oxidase activity. At the C-terminus of CueO(RE) a methionine rich tail region was identified which can be found in a variety of actinobacteria. Chimeras of the E. coli and R. erythropolis enzymes were constructed to investigate the influence of this tail regarding kinetic parameters. It was shown that the tail did not have the same function as the corresponding methionine rich loop in CueO(EC). However, it modulated the kinetic properties of the enzyme.

Nitrile hydratase of Rhodococcus erythropolis: characterization of the enzyme and the use of whole cells for biotransformation of nitriles

The intracellular cobalt-type nitrile hydratase was purified from the bacterium Rhodococcuserythropolis. The pure enzyme consisted of two subunits of 29 and 30 kDa. The molecular weight of the native enzyme was estimated to be 65 kDa. At 25 °C the enzyme had a half-life of 25 h. The Michaelis–Menten constants Km and vmax for the enzyme were 0.624 mM and 5.12 μmol/min/mg, respectively, using 3-cyanopyridine as the substrate. The enzyme-containing freely-suspended bacterial cells and the cells immobilized within alginate beads were evaluated for converting the various nitriles to amides. In a packed bed reactor, alginate beads (2 % alginate; 3 mm bead diameter) containing 200 mg/mL of cells, achieved a conversion of >90 % for benzonitrile and 4-cyanopyridine in 38 h (25 °C, pH 7.0) at a feed substrate concentration of 100 mM. The beads could be reused for up to six reaction cycles.

Mathematic Modeling for Optimum Conditions on Aflatoxin B1 Degradation by the Aerobic Bacterium Rhodococcus erythropolis

Response surface methodology was employed to optimize the degradation conditions of AFB1 by Rhodococcus erythropolis in liquid culture. The most important factors that influence the degradation, as identified by a two-level Plackett-Burman design with six variables, were temperature, pH, liquid volume, inoculum size, agitation speed and incubation time. Central composite design (CCD) and response surface analysis were used to further investigate the interactions between these variables and to optimize the degradation efficiency of R. erythropolis based on a second-order model. The results demonstrated that the optimal parameters were: temperature, 23.2 °C; pH, 7.17; liquid volume, 24.6 mL in 100-mL flask; inoculum size, 10%; agitation speed, 180 rpm; and incubation time, 81.9 h. Under these conditions, the degradation efficiency of R. erythropolis could reach 95.8% in liquid culture, which was increased by about three times as compared to non-optimized conditions. The result by mathematic modeling has great potential for aflatoxin removal in industrial fermentation such as in food processing and ethanol production.

Optimization of lipid production in the oleaginous bacterium Rhodococcus erythropolis growing on glycerol as the sole carbon source

The growth of an oleaginous bacteria strain Rhodococcus erythropolis on glycerol was studied. Lipid accumulation was influenced by the glycerol concentration, nitrogen source, nitrogen concentration, pH medium, incubation time and aeration rate. Under the best optimized flask culture medium; 30 g/L glycerol plus 0.75 g/L urea, incubation temperature at 30°C, agitation rate of 150 rpm and 96 h of cultivation time, 3.93 g/L of dry biomass, 1.84 g/L cellular lipid accumulation and 45.8% accumulated lipid of total dry biomass were produced. The scaling up of R. erythropolis in bioreactors yielded the biomass and lipid content of 11.74 g/L and 14.1% of total dry biomass, respectively. The lipid composition of oleaginous microorganisms contained a high proportion of C16 and C18 fatty acids. The extracted lipids were mainly 16.48% C14:1, 16.69% C16:1, 20.16% C16:0, 18.90% C18:1. R. erythropolis could be directed to using raw glycerol obtained from biodiesel by-product as substrate, in order to accumulate lipids for biodiesel production. Key words : Rhodococcus eryhropolis , glycerol, lipid accumulation, biodiesel, fatty acid composition.

Effect of surfactants on the biofilm of Rhodococcus erythropolis, a potent degrader of aromatic pollutants

Bioremediation processes based on biofilms are usually very effective. The presence of (bio)surfactants in such processes can increase bioavailability of hydrophobic pollutants in aqueous phase. However, surfactants can affect the biofilm as well as individual microbial cells in different ways. Biosurfactants produced by a microbial population can be involved in the final structure of biofilm. An external application of synthetic surfactants or 'foreign' biosurfactants often results in partial or complete destruction of the biofilm and their high concentrations also have a toxic effect on microbial cells. Finding a suitable surfactant and its concentration, which would minimize the negative effects mentioned above, would allow to construct effective bioremediation processes using the benefits of both the biofilm and the surfactant. In this context, G(+) bacterium Rhodococcus erythropolis, which has a wide potential for biodegradation of aromatic compounds, was studied. High surface hydrophobicity of its cells, given mainly by the presence of mycolic acids in the cell envelopes, allows formation of stable biofilms. Three synthetic surfactants (Spolapon AOS 146, Novanik 0633A, Tween 80) and rhamnolipid isolated from Pseudomonas aeruginosa were used. Changes in initial adhesion and biofilm formation caused by the surfactants were monitored in a flow cell equipped with hydrophilic/hydrophobic carriers and analyzed by image analysis.

Shoot the messages not the messengers

Microbes use quorum sensing (QS) as a mechanism to regulate host colonization and virulence in the rhizosphere. Various Gram-positive and Gram-negative bacteria engage in quorum sensing as a mode of communication to inflict pathogenicity on hosts. Of late, the use of various microbial biocontrol agents to restrict pathogenic fungi and bacteria has gained some pace. Although, not much is known about direct antagonistic mechanisms adapted by various biocontrol agents on pathogens, it still represents a sustainable technique to control pathogenesis. Crepin et al. (2012) in this issue of Plant Soil address, for the first time, the question of regulating quorum sensing (QS) by quorum-quenching (QQ) techniques. Crepin et al. show that a rhizosphere bacteria Rhodococcus erythropolis catabolizes the N-acylhomoserine lactones (N-AHLs) produced by Pectobacterium atrosepticum, thus attenuating its virulence. Their experimental results strongly support the involvement of inter-bacterial communication in the rhizosphere. This knowledge is of crucial importance for putting into practice sustainable disease-protection strategies for biocontrol technologies.

Assessment of Substrate Inhibition of Bacterial Oligopeptidase B

Oligopeptidase B (OPB; EC 3.4.21.83) from 2 Gram-negative bacteria, Stenotrophomonas maltophilia (Stm) and Serratia marcescens (Sem), and the Gram-positive bacterium Rhodococcus erythropolis (Re) were cloned and characterized to clarify their activities and substrate specificities using peptidyl-MCA substrates containing Arg or Lys. The cloned enzymes, Stm, Sem and ReOPBs, in addition to Escherichia coli OPB (EcOPB) were expressed using a pET expression system. Although the Stm and SemOPBs share 45% sequence identity to each other and up to 60% identity with respect to their catalytic domains, their activities towards MCA substrates were quite different. StmOPB is approximately 100-500 times more active than SemOPB and 3-30 times more active than EcOPB. The activity of ReOPB is comparable to that of StmOPB and it shares 40% and 36% identity to StmOPB and SemOPB, respectively. Some features of Stm, Re and EcOPBs are similar to those of previously cloned OPBs, which were also strongly inhibited by substrates, but SemOPB differs from all other OPBs in that it is not inhibited by substrates; even substrates containing double arginine at 35 µM did not inhibit SemOPB. On the other hand, the same substrates at only 5 µM inhibited the activity of the Stm, Re, and EcOPB. This phenomenon was not observed with substrates containing single or double lysine.

Nonlinear changes in the activity of the oxygen-dependent demethylase system in Rhodococcus erythropolis cells in the presence of low and very low doses of formaldehyde

The effect of exogenous, highly diluted formaldehyde on the rate of demethylation/re-methylation of veratric acid by the bacteria Rhodococcus erythropolis was studied using electrophoretic and microscopic techniques. The activity of 4-O-demethylase, responsible for accumulation of vanillic acid, and the levels of veratric and vanillic acids were determined using capillary electrophoresis. Formaldehyde was serially diluted at 1:100 ratios, and the total number of iterations was 20. After incubation of the successive dilutions of formaldehyde with the bacteria, demethylase activity oscillated in a sinusoidal manner. It was established using capillary electrophoresis that methylation of vanillic acid to veratric acid occurred at a double rate, as shown by the doubled fluctuation in the concentration of veratrate. There were also changes in the NADH oxidase activity, which is associated with methylation processes. Microscopic observations revealed the presence of numerous enlarged vacuoles in bacterial cells during the accumulation of large amounts of vanillic acid, and their disappearance together with a decrease in 4-O-demethylase activity. The presented results give evidence for the ability of living cells to detect the presence of submolecular concentrations of biological effectors in their environment and provide a basis for a scientific explanation of the law of hormesis and the therapeutic effect of homeopathic dilutions.

The genus Dracunculus – A source of triacylglycerols containing odd-numbered ω-phenyl fatty acids

Reversed phase liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/MS-APCI) was used to identify and quantify triacylglycerols (TAGs) having odd-numbered ω-phenylalkanoic acids from seeds of the flower plant Dracunculus vulgaris, and TAGs from the bacterium Rhodococcus erythropolis prepared by precursor directed biosynthesis from phenylalanine and having the corresponding even-numbered ω-phenylalkanoic acids. Model compounds, which are not commercially available, were prepared by organic synthesis and this allowed us to extend the number of identified natural TAGs to nearly 140 molecular species. Both synthetic and natural compounds containing ω-phenylalkanoic acids were found to have antioxidant and free radical scavenging properties.

Biodegradation of sulfamethoxazole by individual and mixed bacteria

Antibiotic compounds, like sulfamethoxazole (SMX), have become a concern in the aquatic environment due to the potential development of antibacterial resistances. Due to excretion and disposal, SMX has been frequently detected in wastewaters and surface waters. SMX removal in conventional wastewater treatment plants (WWTPs) ranges from 0% to 90%, and there are opposing results regarding its biodegradability at lab scale. The objective of this research was to determine the ability of pure cultures of individual and mixed consortia of bacteria (Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas putida, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus rhodocrous, and Rhodococcus zopfii) known to exist in WWTP activated sludge to remove SMX. Results showed that R. equi alone had the greatest ability to remove SMX leading to 29% removal (with glucose) and the formation of a metabolite. Degradation pathways and metabolite structures have been proposed based on the potential enzymes produced by R. equi. When R. equi was mixed with other microorganisms, a positive synergistic effect was not observed and the maximum SMX removal achieved was 5%. This indicates that pure culture results cannot be extrapolated to mixed culture conditions, and the methodology developed here to study the biodegradability of compounds under controlled mixed culture conditions offers an alternative to conventional studies using pure bacterial cultures or inocula from activated sludge sources consisting of unknown and variable microbial populations.

Enzymes Hydrolyzing Structural Components and Ferrous Ion Cause Rusty-root Symptom on Ginseng (Panax ginseng)

Microbial induction of rusty-root was proved in this study. The enzymes hydrolyzing plant structural materials, including pectinase, pectolyase, ligninase, and cellulase, caused the rusty-root in ginseng. Pectinase and pectolyase produced the highest rusty-color formation. Ferrous ion (Fe+++) caused the synergistic effect on rusty-root formation in ginseng when it was used with pectinase. The effect of ferric ion (Fe++) on rusty-root formation was slow, compared with Fe+++, probably due to gradual oxidation to Fe+++. Other metal ions including the ferric ion (Fe++) did not affect rusty-root formation. The endophytic bacteria Agrobacterium tumefaciens, Lysobacter gummosus, Pseudomonas veronii, Pseudomonas marginalis, Rhodococcus erythropolis, and Rhodococcus globerulus, and the rotten-root forming phytophathogenic fungus Cylindrocarpon destructans, caused rusty-root. The polyphenol formation (rusty color) was not significantly different between microorganisms. The rotten-root-forming C. destructans produced large quantities of external cellulase activity (about 2.3 U[micronM/min/mg protein]), which indicated the pathogenecity of the fungus, whereas the bacteria produced 0.1-0.7 U. The fungal external pectinase activities (0.05 U) and rusty-root formation activity were similar to those of the bacteria. In this report, we proved that microbial hydrolyzing enzymes caused rusty-root (Hue value 15 degrees) of ginseng, and ferrous ion worsened the symptom.

Biphenyl and ethylbenzene dioxygenases of Rhodococcus jostii RHA1 transform PBDEs

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants that have been widely used in consumer products, but that are problematic because of their environmental persistence and endocrine-disrupting properties. To date, very little is known about PBDE degradation by aerobic microorganisms and the enzymes involved in PBDE transformation. Resting cells of the polychlorinated biphenyl-degrading actinomycete, Rhodococcus jostii RHA1, depleted nine mono- through penta-BDEs in separate assays. Extensive depletion of PBDEs occurred with cells grown on biphenyl, ethylbenzene, propane, or styrene, whereas very limited depletion occurred with cells grown on pyruvate or benzoate. In RHA1, expression of bphAa encoding biphenyl dioxygenase (BPDO) and etbAa1 and etbAc encoding ethylbenzene dioxygenase (EBDO) was induced 30- to 3,000-fold during growth on the substrates that supported PBDE depletion. The BPDO and EBDO enzymes had gene expression profiles that matched the PBDE-depletion profiles exhibited by RHA1 grown on different substrates. Using the non-PBDE-degrading bacterium Rhodococcus erythropolis as a host, two recombinant strains were developed by inserting the eth and bph genes of RHA1, respectively. The resultant EBDO extensively depleted mono- through penta-BDEs, while the BPDO depleted only mono-, di-, and one tetra-BDE. A dihydroxylated-BDE was detected as the primary metabolite of 4-bromodiphenyl ether in both recombinant strains. These results indicate that although both dioxygenases are capable of transforming PBDEs, EBDO more potently transforms the highly brominated congeners. The availability of substrates or inducing compounds can markedly affect total PBDE removal as well as patterns of removal of individual congeners.