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Clark, Daniel D., and Scott A. Ensign. "Evidence for an Inducible Nucleotide-Dependent Acetone Carboxylase in Rhodococcus rhodochrousB276." Journal of Bacteriology 181, no. 9 (May 1, 1999): 2752–58. http://dx.doi.org/10.1128/jb.181.9.2752-2758.1999.
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ABSTRACT The metabolism of acetone was investigated in the actinomyceteRhodococcus rhodochrous (formerly Nocardia corallina) B276. Suspensions of acetone- and isopropanol-grownR. rhodochrous readily metabolized acetone. In contrast,R. rhodochrous cells cultured with glucose as the carbon source lacked the ability to metabolize acetone at the onset of the assay but gained the ability to do so in a time-dependent fashion. Chloramphenicol and rifampin prevented the time-dependent increase in this activity. Acetone metabolism by R. rhodochrous was CO2 dependent, and 14CO2 fixation occurred concomitant with this process. A nucleotide-dependent acetone carboxylase was partially purified from cell extracts of acetone-grownR. rhodochrous by DEAE-Sepharose chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the acetone carboxylase was composed of three subunits with apparent molecular masses of 85, 74, and 16 kDa. Acetone metabolism by the partially purified enzyme was dependent on the presence of a divalent metal and a nucleoside triphosphate. GTP and ITP supported the highest rates of acetone carboxylation, while CTP, UTP, and XTP supported carboxylation at 10 to 50% of these rates. ATP did not support acetone carboxylation. Acetoacetate was determined to be the stoichiometric product of acetone carboxylation. The longer-chain ketones butanone, 2-pentanone, 3-pentanone, and 2-hexanone were substrates. This work has identified an acetone carboxylase with a novel nucleotide usage and broader substrate specificity compared to other such enzymes studied to date. These results strengthen the proposal that carboxylation is a common strategy used for acetone catabolism in aerobic acetone-oxidizing bacteria.
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Krum, Jonathan G., and Scott A. Ensign. "Heterologous Expression of Bacterial Epoxyalkane:Coenzyme M Transferase and Inducible Coenzyme M Biosynthesis in Xanthobacter Strain Py2 andRhodococcus rhodochrous B276." Journal of Bacteriology 182, no. 9 (May 1, 2000): 2629–34. http://dx.doi.org/10.1128/jb.182.9.2629-2634.2000.
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ABSTRACT Coenzyme M (CoM) (2-mercaptoethanesulfonic acid) biosynthesis is shown to be coordinately regulated with the expression of the enzymes of alkene and epoxide metabolism in the propylene-oxidizing bacteriaXanthobacter strain Py2 and Rhodococcus rhodochrous strain B276. These results provide the first evidence for the involvement of CoM in propylene metabolism by R. rhodochrous and demonstrate for the first time the inducible nature of eubacterial CoM biosynthesis.
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STRACHAN, Philip D., Andrew A. FREER, and Charles A. FEWSON. "Purification and characterization of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 and cloning and sequencing of its catA gene." Biochemical Journal 333, no. 3 (August 1, 1998): 741–47. http://dx.doi.org/10.1042/bj3330741.
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A method was developed for the purification of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 that had been grown in the presence of benzyl alcohol. The enzyme has very similar apparent Km (1–2 µM) and Vmax (13–19 units/mg of protein) values for the intradiol cleavage of catechol, 3-methylcatechol and 4-methylcatechol and it is optimally active at pH 9. Cross-linking studies indicate that the enzyme is a homodimer. It contains 0.6 atoms of Fe per subunit. The enzyme was crystallized with 15% (w/v) poly(ethylene glycol) 4000/0.33 M CaCl2/25 mM Tris (pH 7.5) by using a microseeding technique. Preliminary X-ray characterization showed that the crystals are in space group C2 with unit-cell dimensions a = 111.9 Å, b = 78.1 Å, c = 134.6 Å, β = 100 °. An oligonucleotide probe, made by hemi-nested PCR, was used to clone the gene encoding catechol 1,2-dioxygenase (catA). The deduced 282-residue sequence corresponds to a protein of molecular mass 31539 Da, close to the molecular mass of 31558 Da obtained by electrospray MS of the purified enzyme. catA was subcloned into the expression vector pTB361, allowing the production of catechol 1,2-dioxygenase to approx. 40% of the total cellular protein. The deduced amino acid sequence of the enzyme has 56% and 75% identity with the catechol 1,2-dioxygenases of Arthrobacter mA3 and Rhodococcus erythropolis AN-13 respectively, but less than 35% identity with intradiol catechol and chlorocatechol dioxygenases of Gram-negative bacteria. The nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number AF043741.
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Poelarends, Gerrit J., Leonid A. Kulakov, Michael J. Larkin, Johan E. T. van Hylckama Vlieg, and Dick B. Janssen. "Roles of Horizontal Gene Transfer and Gene Integration in Evolution of 1,3-Dichloropropene- and 1,2-Dibromoethane-Degradative Pathways." Journal of Bacteriology 182, no. 8 (April 15, 2000): 2191–99. http://dx.doi.org/10.1128/jb.182.8.2191-2199.2000.
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ABSTRACT The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseudomonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct bacteria and an analysis of their flanking sequences. The dhaA gene of the 1-chlorobutane-degrading strain NCIMB13064 was found to reside within a 1-chlorobutane catabolic gene cluster, which also encodes a putative invertase (invA), a regulatory protein (dhaR), an alcohol dehydrogenase (adhA), and an aldehyde dehydrogenase (aldA). The latter two enzymes may catalyze the oxidative conversion of n-butanol, the hydrolytic product of 1-chlorobutane, to n-butyric acid, a growth substrate for many bacteria. The activity of the dhaR gene product was analyzed in Pseudomonas sp. strain GJ1, in which it appeared to function as a repressor of dhaA expression. The 1,2-dibromoethane-degrading strain GP1 contained a conserved DNA segment of 2.7 kb, which included dhaR, dhaA, and part of invA. A 12-nucleotide deletion indhaR led to constitutive expression of dhaA in strain GP1, in contrast to the inducible expression of dhaAin strain NCIMB13064. The 1,3-dichloropropene-degrading strain 170 possessed a conserved DNA segment of 1.3 kb harboring little more than the coding region of the dhaA gene. In strains 170 and GP1, a putative integrase gene was found next to the conserveddhaA segment, which suggests that integration events were responsible for the acquisition of these DNA segments. The data indicate that horizontal gene transfer and integrase-dependent gene acquisition were the key mechanisms for the evolution of catabolic pathways for the man-made chemicals 1,3-dichloropropene and 1,2-dibromoethane.
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Boyd, Jeffrey M., Ashley Ellsworth, and Scott A. Ensign. "Characterization of 2-Bromoethanesulfonate as a Selective Inhibitor of the Coenzyme M-Dependent Pathway and Enzymes of Bacterial Aliphatic Epoxide Metabolism." Journal of Bacteriology 188, no. 23 (September 22, 2006): 8062–69. http://dx.doi.org/10.1128/jb.00947-06.
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ABSTRACT Bacterial growth with short-chain aliphatic alkenes requires coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which serves as the nucleophile for activation and conversion of epoxide products formed from alkene oxidation to central metabolites. In the present work the CoM analog 2-bromoethanesulfonate (BES) was shown to be a specific inhibitor of propylene-dependent growth of and epoxypropane metabolism by Xanthobacter autotrophicus strain Py2. BES (at low [millimolar] concentrations) completely prevented growth with propylene but had no effect on growth with acetone or n-propanol. Propylene consumption by cells was largely unaffected by the presence of BES, but epoxypropane accumulated in the medium in a time-dependent fashion with BES present. The addition of BES to cells resulted in time-dependent loss of epoxypropane degradation activity that was restored upon removal of BES and addition of CoM. Exposure of cells to BES resulted in a loss of epoxypropane-dependent CO2 fixation activity that was restored only upon synthesis of new protein. Addition of BES to cell extracts resulted in an irreversible loss of epoxide carboxylase activity that was restored by addition of purified 2-ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), the terminal enzyme of epoxide carboxylation, but not by addition of epoxyalkane:CoM transferase or 2-hydroxypropyl-CoM dehydrogenase, the enzymes which catalyze the first two reactions of epoxide carboxylation. Comparative studies of the propylene-oxidizing actinomycete Rhodococcus rhodochrous strain B276 showed that BES is an inhibitor of propylene-dependent growth in this organism as well but is not an inhibitor of CoM-independent growth with propane. These results suggest that BES inhibits propylene-dependent growth and epoxide metabolism via irreversible inactivation of the key CO2-fixing enzyme 2-KPCC.
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Toraya, Tetsuo, Takayuki Oka, Manabu Ando, Mamoru Yamanishi, and Hiroshi Nishihara. "Novel Pathway for Utilization of Cyclopropanecarboxylate by Rhodococcus rhodochrous." Applied and Environmental Microbiology 70, no. 1 (January 2004): 224–28. http://dx.doi.org/10.1128/aem.70.1.224-228.2004.
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ABSTRACT A new strain isolated from soil utilizes cyclopropanecarboxylate as the sole source of carbon and energy and was identified as Rhodococcus rhodochrous (H. Nishihara, Y. Ochi, H. Nakano, M. Ando, and T. Toraya, J. Ferment. Bioeng. 80:400-402, 1995). A novel pathway for the utilization of cyclopropanecarboxylate, a highly strained compound, by this bacterium was investigated. Cyclopropanecarboxylate-dependent reduction of NAD+ in cell extracts of cyclopropanecarboxylate-grown cells was observed. When intermediates accumulated in vitro in the absence of NAD+ were trapped as hydroxamic acids by reaction with hydroxylamine, cyclopropanecarboxohydroxamic acid and 3-hydroxybutyrohydroxamic acid were formed. Cyclopropanecarboxyl-coenzyme A (CoA), 3-hydroxybutyryl-CoA, and crotonyl-CoA were oxidized with NAD+ in cell extracts, whereas methacrylyl-CoA and 3-hydroxyisobutyryl-CoA were not. When both CoA and ATP were added, organic acids corresponding to the former three CoA thioesters were also oxidized in vitro by NAD+, while methacrylate, 3-hydroxyisobutyrate, and 2-hydroxybutyrate were not. Therefore, it was concluded that cyclopropanecarboxylate undergoes oxidative degradation through cyclopropanecarboxyl-CoA and 3-hydroxybutyryl-CoA. The enzymes catalyzing formation and ring opening of cyclopropanecarboxyl-CoA were shown to be inducible, while other enzymes involved in the degradation were constitutive.
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Cincilei, Angela G., Svetlana A. Tolocichina, Inna O. Rastimesina, Ion P. Dragalin, Veronica Dumbraveanu, Nina V. Streapan, and Vera C. Mamaliga. "Preparation of Microbiological Agents for Organic Pollutants Removal in Wastewater." Chemistry Journal of Moldova 4, no. 2 (December 2009): 40–43. http://dx.doi.org/10.19261/cjm.2009.04(2).13.
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The authors have investigated the biochemical aspects of degradation processes of persistent organic compound benzothiazole by immobilized Rhodococcus rhodochrous cells, such as entrapped in Ca-alginate beads, or as being immobilized on some solid carries. The mineralization of toxicant was complete and biodestructive capacity of entrapped in alginate bacteria increased with each new experimental cycle.
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Arriaga, José Miguel, Noah D. Cohen, James N. Derr, M. Keith Chaffin, and Ronald J. Martens. "Detection of Rhodococcus Equi by Polymerase Chain Reaction Using Species-Specific Nonproprietary Primers." Journal of Veterinary Diagnostic Investigation 14, no. 4 (July 2002): 347–53. http://dx.doi.org/10.1177/104063870201400416.
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Species-specific primers for the polymerase chain reaction (PCR) for the detection of Rhodococcus equi were developed. These primers were based on unique DNA fragments produced from R. equi reference strains and field isolates. Following random amplification of polymorphic DNA from R. equi and R. rhodochrous with a set of 40 arbitrary 10–base pair (bp) primers, a pair of species-specific primers was designed to detect a unique 700-bp fragment of R. equi chromosomal DNA. This PCR product was limited to R. equi and was not detectable in other Rhodococcus species or in a panel of additional gram-positive and gram-negative bacteria.
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Obidi, O. F. "Comparative Fatty Acid Profiling of Klebsiella pneumoniae and Rhodococcus rhodochrous Isolated from Spoilt Paints by Gas Chromatography." Nigerian Journal of Biotechnology 37, no. 2 (March 12, 2021): 47–55. http://dx.doi.org/10.4314/njb.v37i2.5.
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The use of fatty acids to study the differences in un-related microbes is limited. This study analyzes the fatty acids produced by two unrelated microorganisms: Klebsiella pneumoniae (Gram-negative, aerobic, non-endospore forming, usually encapsulated rod-shaped bacteria of the family Enterobacteriaceae) and Rhodococcus rhodochrous (metabolically versatile, non-spore-forming, non-motile actinomycete) isolated from spoilt paints. Fatty acids produced by the organisms were analyzed using an efficient MIDI-Sherlock gas chromatography method . K. pneumoniae was characterized by a high content of straight chain, branched chain, hydroxyl and cyclo-fatty acids made up of C12: 0, C13:0, C14:0 iso, C14:0, C15:0 iso, C15:0 anteiso, C15:1 ω 8c, C15:0, C16:0 iso, C16:1w5c, C16:0, C15:03OH, C17:1 ω 8c, C17:0 cyclo, C17:0, C18:1 ω5c and C18:0. R. rhodochrous was dominated by straight chain, monounsaturated and 10-methyl fatty acids. The inability to synthesize branched, cyclo- and hydroxyl- fatty acids, was observed in R. rhodochrous which composed mainly of C14: 0, C15: 1 ω 5c, C15:0, C16:1 ω 9c, C16:0, C17:1 ω 8c, C17:0, C17:0 10-methyl, C18: 1 ω 9c, C18.0, 10 methyl-C18:0 TBSA, C20:1 ω 9c, and C20:0. Descriptive statistics reveal a mean of 2.53, 15.10 and 15.15 for retention time (RT), equivalent chain length (ECL) and Peak name, respectively. Possible implications of the variations in fatty acid distribution may include differences in their abilities to produce various secondary metabolites and potentials to degrade a variety of xenobiotics.
Keywords: Fatty acids, paints, Rhodococcus rhodochrous, Klebsiella pneumoniae
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Tauber, M. M., A. Cavaco-Paulo, K. H. Robra, and G. M. Gübitz. "Nitrile Hydratase and Amidase fromRhodococcus rhodochrous Hydrolyze Acrylic Fibers and Granular Polyacrylonitriles." Applied and Environmental Microbiology 66, no. 4 (April 1, 2000): 1634–38. http://dx.doi.org/10.1128/aem.66.4.1634-1638.2000.
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ABSTRACT Rhodococcus rhodochrous NCIMB 11216 produced nitrile hydratase (320 nkat mg of protein−1) and amidase activity (38.4 nkat mg of protein−1) when grown on a medium containing propionitrile. These enzymes were able to hydrolyze nitrile groups of both granular polyacrylonitriles (PAN) and acrylic fibers. Nitrile groups of PAN40 (molecular mass, 40 kDa) and PAN190 (molecular mass, 190 kDa) were converted into the corresponding carbonic acids to 1.8 and 1.0%, respectively. In contrast, surfacial nitrile groups of acrylic fibers were only converted to the corresponding amides. X-ray photoelectron spectroscopy analysis showed that 16% of the surfacial nitrile groups were hydrolyzed by the R. rhodochrousenzymes. Due to the enzymatic modification, the acrylic fibers became more hydrophilic and thus, adsorption of dyes was enhanced. This was indicated by a 15% increase in the staining level (K/Svalue) for C.I. Basic Blue 9.
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Li, Chunyan, Yue Li, Xiaosong Cheng, Liping Feng, Chuanwu Xi, and Ying Zhang. "Immobilization of Rhodococcus rhodochrous BX2 (an acetonitrile-degrading bacterium) with biofilm-forming bacteria for wastewater treatment." Bioresource Technology 131 (March 2013): 390–96. http://dx.doi.org/10.1016/j.biortech.2012.12.140.
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Iwabuchi, Noriyuki, Michio Sunairi, Makoto Urai, Chiaki Itoh, Hiroshi Anzai, Mutsuyasu Nakajima, and Shigeaki Harayama. "Extracellular Polysaccharides of Rhodococcus rhodochrous S-2 Stimulate the Degradation of Aromatic Components in Crude Oil by Indigenous Marine Bacteria." Applied and Environmental Microbiology 68, no. 5 (May 2002): 2337–43. http://dx.doi.org/10.1128/aem.68.5.2337-2343.2002.
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ABSTRACT Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing d-glucose, d-galactose, d-mannose, d-glucuronic acid, and lipids, which is important to the tolerance of this strain to an aromatic fraction of (AF) Arabian light crude oil (N. Iwabuchi, N. Sunairi, H. Anzai, M. Nakajima, and S. Harayama, Appl. Environ. Microbiol. 66:5073-5077, 2000). In the present study, we examined the effects of S-2 EPS on the growth of indigenous marine bacteria on AF. Indigenous bacteria did not grow significantly in seawater containing AF even when nitrogen, phosphorus, and iron nutrients were supplemented. The addition of S-2 EPS to seawater containing nutrients and AF resulted in the emulsification of AF, promotion of the growth of indigenous bacteria, and enhancement of the degradation of AF by the bacteria. PCR-denaturing gradient gel electrophoresis analyses show that addition of S-2 EPS to the seawater containing nutrients and AF changed the composition of the bacterial populations in the seawater and that bacteria closely related to the genus Cycloclasticus became the major population. These results suggest that Cycloclasticus was responsible for the degradation of hydrocarbons in AF. The effects of 15 synthetic surfactants on the degradation of AF by indigenous marine bacteria were also examined, but enhancement of the degradation of AF was not significant. S-2 EPS was hence the most effective of the surfactants tested in promoting the biodegradation of AF and may thus be an attractive agent to use in the bioremediation of oil-contaminated marine environments.
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Zhao, Hongyan, Kejian Tian, Qing Qiu, Yu Wang, Hongyan Zhang, Shuang Ma, Shenbao Jin, and Hongliang Huo. "Genome Analysis of Rhodococcus Sp. DSSKP-R-001: A Highly Effective β-Estradiol-Degrading Bacterium." International Journal of Genomics 2018 (October 28, 2018): 1–11. http://dx.doi.org/10.1155/2018/3505428.
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We screened bacteria that use E2 as its sole source of carbon and energy for growth and identified them as Rhodococcus, and we named them DSSKP-R-001. For a better understanding of the metabolic potential of the strain, whole genome sequencing of Rhodococcus DSSKP-R-001 and annotation of the functional genes were performed. The genomic sketches included a predicted protein-coding gene of approximately 5.4 Mbp with G + C content of 68.72% and 5180. The genome of Rhodococcus strain DSSKP-R-001 consists of three replicons: one chromosome and two plasmids of 5.2, 0.09, and 0.09, respectively. The results showed that there were ten steroid-degrading enzymes distributed in the whole genome of the strain. The existence and expression of estradiol-degrading enzymes were verified by PCR and RTPCR. Finally, comparative genomics was used to compare multiple strains of Rhodococcus. It was found that Rhodococcus DSSKP-R-001 had the highest similarity to Rhodococcus sp. P14 and there were 2070 core genes shared with Rhodococcus sp. P14, Rhodococcus jostii RHA1, Rhodococcus opacus B4, and Rhodococcus equi 103S, showing evolutionary homology. In summary, this study provides a comprehensive understanding of the role of Rhodococcus DSSKP-R-001 in estradiol-efficient degradation of these assays for Rhodococcus. DSSKP-R-001 in bioremediation and evolution within Rhodococcus has important meaning.
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Petrusma, M., G. Hessels, L. Dijkhuizen, and R. van der Geize. "Multiplicity of 3-Ketosteroid-9 -Hydroxylase Enzymes in Rhodococcus rhodochrous DSM43269 for Specific Degradation of Different Classes of Steroids." Journal of Bacteriology 193, no. 15 (June 3, 2011): 3931–40. http://dx.doi.org/10.1128/jb.00274-11.
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Lee, Ju-Hoon, and Daniel J. O’Sullivan. "Sequence Analysis of Two Cryptic Plasmids from Bifidobacterium longum DJO10A and Construction of a Shuttle Cloning Vector." Applied and Environmental Microbiology 72, no. 1 (January 2006): 527–35. http://dx.doi.org/10.1128/aem.72.1.527-535.2006.
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ABSTRACT 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.
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Eliaz, Noam, Eliora Ron, Michael Gozin, Sara Younger, Dvora Biran, and Noam Tal. "Microbial Degradation of Epoxy." Materials 11, no. 11 (October 29, 2018): 2123. http://dx.doi.org/10.3390/ma11112123.
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Epoxy resins have a wide range of applications, including in corrosion protection of metals, electronics, structural adhesives, and composites. The consumption of epoxy resins is predicted to keep growing in the coming years. Unfortunately, thermoset resins cannot be recycled, and are typically not biodegradable. Hence, they pose environmental pollution risk. Here, we report degradation of epoxy resin by two bacteria that are capable of using epoxy resin as a sole carbon source. These bacteria were isolated from soil samples collected from areas around an epoxy and polyurethanes manufacturing plant. Using an array of molecular, biochemical, analytical, and microscopic techniques, they were identified as Rhodococcus rhodochrous and Ochrobactrum anthropi. As epoxy was the only carbon source available for these bacteria, their measured growth rate reflected their ability to degrade epoxy resin. Bacterial growth took place only when the two bacteria were grown together, indicating a synergistic effect. The surface morphology of the epoxy droplets changed significantly due to the biodegradation process. The metabolic pathway of epoxy by these two microbes was investigated by liquid chromatography mass spectrometry. Bisphenol A, 3,3′-((propane-2,2-diylbis(4,1-phenylene))bis(oxy))bis(propane-1,2-diol) and some other constituents were identified as being consumed by the bacteria.
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Nahar, Akhikun, Anthony L. Baker, David S. Nichols, John P. Bowman, and Margaret L. Britz. "Benchmarking DNA Extraction Methods for Phylogenomic Analysis of Sub-Antarctic Rhodococcus and Williamsia Species." Microorganisms 9, no. 6 (June 9, 2021): 1253. http://dx.doi.org/10.3390/microorganisms9061253.
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Bacteria containing mycolic acids in their cell envelope are often recalcitrant to cell lysis, so extracting DNA of sufficient quality for third-generation sequencing and high-fidelity genome assembly requires optimization, even when using commercial kits with protocols for hard-to-lyse bacteria. We benchmarked three spin-column-based kits against a classical DNA extraction method employing lysozyme, proteinase K and SDS for six lysozyme-resistant, sub-Antarctic strains of Corynebaceriales. Prior cultivation in broths containing glycine at highly growth-inhibitory concentrations (4.0–4.5%) improved cell lysis using both classical and kit methods. The classical method produced DNA with average fragment sizes of 27–59 Kbp and tight fragment size ranges, meeting quality standards for genome sequencing, assembly and phylogenomic analyses. By 16S rRNA gene sequencing, we classified two strains as Williamsia and four strains as Rhodococcus species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed Rhodococcus sp. 1163 and 1168 and Williamsia sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as R. qingshengii and R. erythropolis, respectively, using ANI similarity of >98% and AF >60% for species delineation. On this basis, some members of the R. erythropolis genome cluster groups, including strains currently named as R. enclensis, R. baikonurensis, R. opacus and R. rhodochrous, would be reclassified either as R. erythropolis or R. qingshengii.
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Blackall, Linda L. "Molecular Identification of Activated Sludge Foaming Bacteria." Water Science and Technology 29, no. 7 (April 1, 1994): 35–42. http://dx.doi.org/10.2166/wst.1994.0299.
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The formation of a stable, dark, viscous foam or scum on activated sludge aeration basin surfaces is a recent and increasing problem. Microscopically, the foam is comprised of a predominance of one of a few different types of filamentous bacteria. The diversity of filaments found in the foam is increasing with time. However, two prominent foaming organisms in Australia are Nocardia amarae and Nocardia pinensis. Although these nocardioforms can be microscopically differentiated, N. amarae is indistinguishable from other foaming filaments such as Rhodococcus rhodochrous and Tsukamurella paurometabolum. Morphology is the cardinal character used in differentiating bulking filamentous microorganisms. However, microorganism morphology is a notoriously poor descriptive attribute that can vary widely depending upon nutritional conditions. The past decade has witnessed the use of molecular biological techniques as the dominant approach for the detection of microorganisms that are difficult to identify by conventional culture techniques or microscopy. The most common nucleic acid target sequences for molecular probes are found in conserved genes such as 16S rDNA. Genomic DNA was isolated from strains of N. amarae and N. pinensis. The 16S rDNA was amplified by the polymerase chain reaction and sequenced using an automated DNA sequencing machine. The sequences were compared and regions that could be exploited for oligonucleotide probes were highlighted. These regions can differentiate the two species and by comparison with sequences available in Genbank, they can differentiate N. amarae from other foaming nocardioforms for which 16S rDNA sequence information is available.
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Shaposhnikova, Anastasiia, Michal Kuty, Radka Chaloupkova, Jiri Damborsky, Ivana Kuta Smatanova, Babak Minofar, and Tatyana Prudnikova. "Stabilization of Haloalkane Dehalogenase Structure by Interfacial Interaction with Ionic Liquids." Crystals 11, no. 9 (September 1, 2021): 1052. http://dx.doi.org/10.3390/cryst11091052.
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Ionic liquids attracted interest as green alternatives to replace conventional organic solvents in protein stability studies. They can play an important role in the stabilization of enzymes such as haloalkane dehalogenases that are used for biodegradation of warfare agents and halogenated environmental pollutants. Three-dimensional crystals of haloalkane dehalogenase variant DhaA80 (T148L+G171Q+A172V+C176F) from Rhodococcus rhodochrous NCIMB 13064 were grown and soaked with the solutions of 2-hydroxyethylammonium acetate and 1-butyl-3-methylimidazolium methyl sulfate. The objective was to study the structural basis of the interactions between the ionic liquids and the protein. The diffraction data were collected for the 1.25 Å resolution for 2-hydroxyethylammonium acetate and 1.75 Å resolution for 1-butyl-3-methylimidazolium methyl sulfate. The structures were used for molecular dynamics simulations to study the interactions of DhaA80 with the ionic liquids. The findings provide coherent evidence that ionic liquids strengthen both the secondary and tertiary protein structure due to extensive hydrogen bond interactions.
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Uroz, Stéphane, Phil M. Oger, Emilie Chapelle, Marie-Thérèse Adeline, Denis Faure, and Yves Dessaux. "A Rhodococcus qsdA-Encoded Enzyme Defines a Novel Class of Large-Spectrum Quorum-Quenching Lactonases." Applied and Environmental Microbiology 74, no. 5 (January 11, 2008): 1357–66. http://dx.doi.org/10.1128/aem.02014-07.
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ABSTRACT A gene involved in N-acyl homoserine lactone (N-AHSL) degradation was identified by screening a genomic library of Rhodococcus erythropolis strain W2. This gene, named qsdA (for quorum-sensing signal degradation), encodes an N-AHSL lactonase unrelated to the two previously characterized N-AHSL-degrading enzymes, i.e., the lactonase AiiA and the amidohydrolase AiiD. QsdA is related to phosphotriesterases and constitutes the reference of a novel class of N-AHSL degradation enzymes. It confers the ability to inactivate N-AHSLs with an acyl chain ranging from C6 to C14, with or without substitution at carbon 3. Screening of a collection of 15 Rhodococcus strains and strains closely related to this genus clearly highlighted the relationship between the ability to degrade N-AHSLs and the presence of the qsdA gene in Rhodococcus. Bacteria harboring the qsdA gene interfere very efficiently with quorum-sensing-regulated functions, demonstrating that qsdA is a valuable tool for developing quorum-quenching procedures.
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Nagababu, Peddinti, and Vanga Umamaheswara Rao. "Biosynthesis, Characterization and Antibacterial activity of Silver nanoparticles of Excoecaria agallocha L. fruit extract." International Journal of Drug Delivery 9, no. 1 (June 21, 2017): 03. http://dx.doi.org/10.5138/09750215.2094.
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<p>In this present study, <em>Excoecaria agallocha </em>fruit aqueous extract was used to synthesize Silver Nano Particles (Ag NPs/SNPs) which has proven as eco-friendly, nontoxic, less time consuming and energy saving. The synthesized SNPs were characterized by UV-Visible spectroscopy, FTIR and SEM studies. The SNPs were checked for the antibacterial activity against both Gram positive and Gram negative bacteria. The characterization studies clearly revealed the formation and synthesis of SNPs which also showed the inhibitory activity on the tested bacteria. SNPs of <em>Excoecaria agallocha </em>fruit showed higher zone of inhibition against <em>Micrococcus luteus</em>, <em>Arthrobacter protophormiae</em>, <em>Rhodococcus rhodochrous</em>, <em>Bacillus subtilis</em>, <em>Alcaligens faecalis</em>, <em>Enterobacter aerogenes</em>, <em>Proteus mirabilis</em> and <em>Salmonella enterica </em>when compared to that of standard antibiotic, Streptomycin.</p>
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Alvarez, H. M., and A. Hernández. "NADP+-dependent malic enzymes as a tool for improving oil production in Rhodococcus bacteria." New Biotechnology 44 (October 2018): S89—S90. http://dx.doi.org/10.1016/j.nbt.2018.05.941.
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Wilbrink, M. H., M. Petrusma, L. Dijkhuizen, and R. van der Geize. "FadD19 of Rhodococcus rhodochrous DSM43269, a Steroid-Coenzyme A Ligase Essential for Degradation of C-24 Branched Sterol Side Chains." Applied and Environmental Microbiology 77, no. 13 (May 20, 2011): 4455–64. http://dx.doi.org/10.1128/aem.00380-11.
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ABSTRACTThe actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode β-oxidation enzymes with a putative role in sterol side chain degradation. We investigated the physiological roles of several genes, i.e.,fadD17,fadD19,fadE26,fadE27, andro04690DSM43269, by gene inactivation studies in mutant strain RG32 ofRhodococcus rhodochrousDSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9α-hydroxylase (KSH) activity and was constructed following the identification, cloning, and sequential inactivation of fivekshAgene homologs in strain DSM43269. We show that mutant strain RG32 is fully blocked in steroid ring degradation but capable of selective sterol side chain degradation. Except for RG32ΔfadD19, none of the mutants constructed in RG32 revealed an aberrant phenotype on sterol side chain degradation compared to parent strain RG32. Deletion offadD19in strain RG32 completely blocked side chain degradation of C-24 branched sterols but interestingly not that of cholesterol. The additional inactivation offadD17in mutant RG32ΔfadD19also did not affect cholesterol side chain degradation. Heterologously expressed FadD19DSM43269nevertheless was active toward steroid-C26-oic acid substrates. Our data identified FadD19 as a steroid-coenzyme A (CoA) ligase with an essentialin vivorole in the degradation of the side chains of C-24 branched-chain sterols. This paper reports the identification and characterization of a CoA ligase with anin vivorole in sterol side chain degradation. The high similarity (67%) between the FadD19DSM43269and FadD19H37Rvenzymes further suggests that FadD19H37Rvhas anin vivorole in sterol metabolism ofMycobacterium tuberculosisH37Rv.
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Aislabie, J., and G. Lloyd-Jones. "A review of bacterial-degradation of pesticides." Soil Research 33, no. 6 (1995): 925. http://dx.doi.org/10.1071/sr9950925.
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Pesticide fate in the environment is affected by microbial activity. Some pesticides are readily degraded by microorganisms, others have proven to be recalcitrant. A diverse group of bacteria, including members of the genera Alcaligenes, Flavobacterium, Pseudomonas and Rhodococcus, metabolize pesticides. Microbial degradation depends not only on the presence of microbes with the appropriate degradative enzymes, but also on a wide range of environmental parameters. This review describes recent advances in biodegradation of pesticides by addressing the biology and molecular characterization of some pesticide degrading bacteria.
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Denger, Karin, Jürgen Ruff, David Schleheck, and Alasdair M. Cook. "Rhodococcus opacus expresses the xsc gene to utilize taurine as a carbon source or as a nitrogen source but not as a sulfur source." Microbiology 150, no. 6 (June 1, 2004): 1859–67. http://dx.doi.org/10.1099/mic.0.27077-0.
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The Gram-positive bacteria Rhodococcus opacus ISO-5 and Rhodococcus sp. RHA1 utilized taurine (2-aminoethanesulfonate) as the sole source of carbon or of nitrogen or of sulfur for growth. Different gene clusters and enzymes were active under these different metabolic situations. Under carbon- or nitrogen-limited conditions three enzymes were induced, though to different levels: taurine-pyruvate aminotransferase (Tpa), alanine dehydrogenase (Ald) and sulfoacetaldehyde acetyltransferase (Xsc). The specific activities of these enzymes in R. opacus ISO-5 were sufficient to explain the growth rates under the different conditions. These three enzymes were purified and characterized, and the nature of each reaction was confirmed. Analyses of the genome of Rhodococcus sp. RHA1 revealed a gene cluster, tauR-ald-tpa, putatively encoding regulation and oxidation of taurine, located 20 kbp from the xsc gene and separate from two candidate phosphotransacetylase (pta) genes, as well as many candidate ABC transporters (tauBC). PCR primers allowed the amplification and sequencing of the tauR-ald-tpa gene cluster and the xsc gene in R. opacus ISO-5. The N-terminal sequences of the three tested proteins matched the derived amino acid sequences of the corresponding genes. The sequences of the four genes found in each Rhodococcus strain shared high degrees of identity (>95 % identical positions). RT-PCR studies proved transcription of the xsc gene when taurine was the source of carbon or of nitrogen. Under sulfur-limited conditions no xsc mRNA was generated and no Xsc was detected. Taurine dioxygenase (TauD), the enzyme catalysing the anticipated desulfonative reaction when taurine sulfur is assimilated, was presumed to be present because oxygen-dependent taurine disappearance was demonstrated with taurine-grown cells only. A putative tauD gene (with three other candidates) was detected in strain ISO-5. Regulation of the different forms of metabolism of taurine remains to be elucidated.
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Costa, Leonardo Emanuel de Oliveira, Thamy Lívia Ribeiro Corrêa, Janaina Aparecida Teixeira, Elza Fernandes de Araújo, and Marisa Vieira de Queiroz. "Endophytic bacteria isolated from Phaseolus vulgaris produce phytases with potential for biotechnology application." Brazilian Journal of Biological Sciences 5, no. 11 (2018): 657–71. http://dx.doi.org/10.21472/bjbs.051105.
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Currently, endophytic microorganisms have become a good source of different enzymes and others metabolites of industrial interest. Among a huge spectral of molecules, enzymes as phytases have been emphasized by the ability to hydrolyze the phytic acid that represents the largest storage form of inorganic phosphorus in cereals, which are the staple diet of monogastric animals such as swine and poultry. Moreover, phytic acid acts as an antinutrient by chelating divalent metal ions, and it is interesting provide phytase as an animal feed supplement for those monogastric animals. In the current study, 158 endophytic bacteria isolated from the leaves of three cultivars of Phaseolus vulgaris were assessed for the ability to produce phytase. Among them, four isolates belonging to the Pseudomonas, Stenotrophomonas, Microbacterium and Rhodococcus genera were highlighted, due their phytase production. The phytase produced by Microbacterium foliorum BAC1157 exhibited activity at 70 oC and stability in the presence of divalent cations, indicating that this phytase has a promising use in the animal feed industry. To the authors' knowledge, this is the first report on phytase production by bacteria of the Microbacterium genera.
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Standing, Taryn-Ann, Erika du Plessis, Stacey Duvenage, and Lise Korsten. "Internalisation potential of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica subsp. enterica serovar Typhimurium and Staphylococcus aureus in lettuce seedlings and mature plants." Journal of Water and Health 11, no. 2 (April 11, 2013): 210–23. http://dx.doi.org/10.2166/wh.2013.164.
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The internalisation potential of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7 and Salmonella enterica subsp. enterica serovar Typhimurium in lettuce was evaluated using seedlings grown in vermiculite in seedling trays as well as hydroponically grown lettuce. Sterile distilled water was spiked with one of the four human pathogenic bacteria (105 CFU/mL) and used to irrigate the plants. The potential for pathogen internalisation was investigated over time using light microscopy, transmission electron microscopy and viable plate counts. Additionally, the identities of the pathogens isolated from internal lettuce plant tissues were confirmed using polymerase chain reaction with pathogen-specific oligonucleotides. Internalisation of each of the human pathogens was evident in both lettuce seedlings and hydroponically grown mature lettuce plants. To our knowledge, this is the first report of S. aureus internalisation in lettuce plants. In addition, the levels of background microflora in the lettuce plants were determined by plate counting and the isolates identified using matrix-assisted laser ionisation–time of flight (MALDI–TOF). Background microflora assessments confirmed the absence of the four pathogens evaluated in this study. A low titre of previously described endophytes and soil inhabitants, i.e., Enterobacter cloacae, Enterococcus faecalis, Lysinibacillus fusiformis, Rhodococcus rhodochrous, Staphylococcus epidermidis and Staphylococcus hominis were identified.
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Navarro-Llorens, Juana María, Marianna A. Patrauchan, Gordon R. Stewart, Julian E. Davies, Lindsay D. Eltis, and William W. Mohn. "Phenylacetate Catabolism in Rhodococcus sp. Strain RHA1: a Central Pathway for Degradation of Aromatic Compounds." Journal of Bacteriology 187, no. 13 (July 1, 2005): 4497–504. http://dx.doi.org/10.1128/jb.187.13.4497-4504.2005.
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ABSTRACT In gram-negative bacteria, a pathway for aerobic degradation of phenylacetic acid (PAA) that proceeds via phenylacetyl-coenzyme A (CoA) and hydrolytic ring fission plays a central role in the degradation of a range of aromatic compounds. In contrast, the PAA pathway and its role are not well characterized in gram-positive bacteria. A cluster including 13 paa genes encoding enzymes orthologous to those of gram-negative bacteria was identified on the chromosome of Rhodococcus sp. strain RHA1. These genes were transcribed during growth on PAA, with 11 of the genes apparently in an operon yielding a single transcript. Quantitative proteomic analyses revealed that at least 146 proteins were more than twice as abundant in PAA-grown cells of RHA1 than in pyruvate-grown cells. Of these proteins, 29 were identified, including 8 encoded by the paa genes. Knockout mutagenesis indicated that paaN, encoding a putative ring-opening enzyme, was essential for growth on PAA. However, paaF, encoding phenylacetyl-CoA ligase, and paaR, encoding a putative regulator, were not essential. paaN was also essential for growth of RHA1 on phenylacetaldehyde, phenylpyruvate, 4-phenylbutyrate, 2-phenylethanol, 2-phenylethylamine, and l-phenylalanine. In contrast, growth on 3-hydroxyphenylacetate, ethylbenzene, and styrene was unaffected. These results suggest that the range of substrates degraded via the PAA pathway in RHA1 is somewhat limited relative to the range in previously characterized gram-negative bacteria.
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Yam, Katherine C., Sachi Okamoto, Joseph N. Roberts, and Lindsay D. Eltis. "Adventures inRhodococcus — from steroids to explosivesThis article is based on a presentation by Dr. Lindsay Eltis at the 60th Annual Meeting of the Canadian Society of Microbiologists in Hamilton, Ontario, 14 June 2010. Dr. Eltis was the recipient of the 2010 Norgen Biotek Corporation / CSM Award, an annual award sponsored by Norgen Biotek and the Canadian Society of Microbiologists intended to recognize outstanding scientific work in microbiology by a Canadian researcher." Canadian Journal of Microbiology 57, no. 3 (March 2011): 155–68. http://dx.doi.org/10.1139/w10-115.
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Rhodococcus is a genus of mycolic-acid-containing actinomycetes that utilize a remarkable variety of organic compounds as growth substrates. This degradation helps maintain the global carbon cycle and has increasing applications ranging from the biodegradation of pollutants to the biocatalytic production of drugs and hormones. We have been using Rhodococcus jostii RHA1 as a model organism to understand the catabolic versatility of Rhodococcus and related bacteria. Our approach is exemplified by the discovery of a cluster of genes specifying the catabolism of cholesterol. This degradation proceeds via β-oxidative degradation of the side chain and O2-dependent cleavage of steroid ring A in a process similar to bacterial degradation of aromatic compounds. The pathway is widespread in Actinobacteria and is critical to the pathogenesis of Mycobacterium tuberculosis , arguably the world’s most successful pathogen. The close similarity of some of these enzymes with biphenyl- and polychlorinated-biphenyl-degrading enzymes that we have characterized is facilitating inhibitor design. Our studies in RHA1 have also provided important insights into a number of novel metalloenzymes and their biosynthesis, such as acetonitrile hydratase (ANHase), a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. Molecular genetic and biochemical studies have identified AnhE as a dimeric metallochaperone that delivers cobalt to ANHase, enabling its maturation in vivo. Other metalloenzymes we are characterizing include N-acetylmuramic acid hydroxylase, which catalyzes an unusual hydroxylation of the rhodococcal and mycobacterial peptidoglycan, and 2 RHA1 dye-decolorizing peroxidases. Using molecular genetic and biochemical approaches, we have demonstrated that one of these enzymes is involved in the degradation of lignin. Overall, our studies are providing fundamental insights into a range of catabolic processes that have a wide variety of applications.
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Barney, Brett M., Bradley D. Wahlen, EmmaLee Garner, Jiashi Wei, and Lance C. Seefeldt. "Differences in Substrate Specificities of Five Bacterial Wax Ester Synthases." Applied and Environmental Microbiology 78, no. 16 (June 8, 2012): 5734–45. http://dx.doi.org/10.1128/aem.00534-12.
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ABSTRACTWax esters are produced in certain bacteria as a potential carbon and energy storage compound. The final enzyme in the biosynthetic pathway responsible for wax ester production is the bifunctional wax ester synthase/acyl-coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT), which utilizes a range of fatty alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. We report here the isolation and substrate range characterization for five WS/DGAT enzymes from four different bacteria:Marinobacter aquaeoleiVT8,Acinetobacter baylyi,Rhodococcus jostiiRHA1, andPsychrobacter cryohalolentisK5. The results from kinetic studies of isolated enzymes reveal a differential activity based on the order of substrate addition and reveal subtle differences between the substrate selectivity of the different enzymes. Thesein vitroresults are compared to the wax ester and triacylglyceride product profiles obtained from each organism grown under neutral lipid accumulating conditions, providing potential insights into the role that the WS/DGAT enzyme plays in determining the final wax ester products that are produced under conditions of nutrient stress in each of these bacteria. Further, the analysis revealed that one enzyme in particular fromM. aquaeoleiVT8 showed the greatest potential for future study based on rapid purification and significantly higher activity than was found for the other isolated WS/DGAT enzymes. The results provide a framework to test prospective differences between these enzymes for potential biotechnological applications such as high-value petrochemicals and biofuel production.
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Poelarends, Gerrit J., Marga Wilkens, Michael J. Larkin, Jan Dirk van Elsas, and Dick B. Janssen. "Degradation of 1,3-Dichloropropene by Pseudomonas cichorii 170." Applied and Environmental Microbiology 64, no. 8 (August 1, 1998): 2931–36. http://dx.doi.org/10.1128/aem.64.8.2931-2936.1998.
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ABSTRACT The gram-negative bacterium Pseudomonas cichorii 170, isolated from soil that was repeatedly treated with the nematocide 1,3-dichloropropene, could utilize low concentrations of 1,3-dichloropropene as a sole carbon and energy source. Strain 170 was also able to grow on 3-chloroallyl alcohol, 3-chloroacrylic acid, and several 1-halo-n-alkanes. This organism produced at least three different dehalogenases: a hydrolytic haloalkane dehalogenase specific for haloalkanes and two 3-chloroacrylic acid dehalogenases, one specific for cis-3-chloroacrylic acid and the other specific for trans-3-chloroacrylic acid. The haloalkane dehalogenase and thetrans-3-chloroacrylic acid dehalogenase were expressed constitutively, whereas the cis-3-chloroacrylic acid dehalogenase was inducible. The presence of these enzymes indicates that 1,3-dichloropropene is hydrolyzed to 3-chloroallyl alcohol, which is oxidized in two steps to 3-chloroacrylic acid. The latter compound is then dehalogenated, probably forming malonic acid semialdehyde. The haloalkane dehalogenase gene, which is involved in the conversion of 1,3-dichloropropene to 3-chloroallyl alcohol, was cloned and sequenced, and this gene turned out to be identical to the previously studieddhaA gene of the gram-positive bacterium Rhodococcus rhodochrous NCIMB13064. Mutants resistant to the suicide substrate 1,2-dibromoethane lacked haloalkane dehalogenase activity and therefore could not utilize haloalkanes for growth. PCR analysis showed that these mutants had lost at least part of the dhaA gene.
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Gon�alves, Edmilson R., Hirofumi Hara, Daisuke Miyazawa, Julian E. Davies, Lindsay D. Eltis, and William W. Mohn. "Transcriptomic Assessment of Isozymes in the Biphenyl Pathway of Rhodococcus sp. Strain RHA1." Applied and Environmental Microbiology 72, no. 9 (September 2006): 6183–93. http://dx.doi.org/10.1128/aem.00947-06.
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ABSTRACT Rhodococcus sp. RHA1 grows on a broad range of aromatic compounds and vigorously degrades polychlorinated biphenyls (PCBs). Previous work identified RHA1 genes encoding multiple isozymes for most of the seven steps of the biphenyl (BPH) pathway, provided evidence for coexpression of some of these isozymes, and indicated the involvement of some of these enzymes in the degradation of BPH, ethylbenzene (ETB), and PCBs. To investigate the expression of these isozymes and better understand how they contribute to the robust degradative capacity of RHA1, we comprehensively analyzed the 9.7-Mb genome of RHA1 for BPH pathway genes and characterized the transcriptome of RHA1 growing on benzoate (BEN), BPH, and ETB. Sequence analyses revealed 54 potential BPH pathway genes, including 28 not previously reported. Transcriptomic analysis with a DNA microarray containing 70-mer probes for 8,213 RHA1 genes revealed a suite of 320 genes of diverse functions that were upregulated during growth both on BPH and on ETB, relative to growth on the control substrate, pyruvate. By contrast, only 65 genes were upregulated during growth on BEN. Quantitative PCR assays confirmed microarray results for selected genes and indicated that some of the catabolic genes were upregulated over 10,000-fold. Our analysis suggests that up to 22 enzymes, including 8 newly identified ones, may function in the BPH pathway of RHA1. The relative expression levels of catabolic genes did not differ for BPH and ETB, suggesting a common regulatory mechanism. This study delineated a suite of catabolic enzymes for biphenyl and alkyl-benzenes in RHA1, which is larger than previously recognized and which may serve as a model for catabolism in other environmentally important bacteria having large genomes.
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Powthong, Pannapa, Apichai Sripean, and Pattra Suntornthiticharoen. "Screening of active antimicrobial and biological enzymes of microbial isolated from soil in Thailand." Asian Journal of Pharmaceutical and Clinical Research 10, no. 4 (April 1, 2017): 73. http://dx.doi.org/10.22159/ajpcr.2017.v10i4.15454.
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Objective: The objectives of this study were to isolate microorganisms and screen for potential antimicrobial activities from the soil. Methods: In this study, a total of 425 isolates were isolated from 100 soil samples. The preliminary screening for antimicrobial activities of these isolates was performed by modified cross-streak, agar diffusion, and modified icrodilution technique against 16 pathogenic bacteria and fungi.Results: In the anti-microbial activity, there were three isolates, namely, 277, 303, and 307 exhibited inhibitory activity against methicillin-resistantStaphylococcus aureus and Salmonella typhimurium respectively. This study also examined the various enzymes producing from soil microorganisms including chitinase, chitosanase, amylase, cellulose, caseinase, gelatinase, esterase, and lipase production of different selective media for 24 and 48hrs using the direct spot method. The results revealed that 28 isolates could produce various enzymes with strong activity. Most of them produced gelatinase (5.65%) and caseinase (5.18%). There were four isolates that produce broad-spectrum enzyme. In addition, the investigation of selectedmicroorganism identification showed that they can be divided into three groups: Burkholderia spp., Pseudomonas spp., and Rhodococcus spp.Conclusion: This study demonstrated that the microorganisms from soil are capable of producing potential, antibacterial, and bioactive enzymes.Keywords: Antimicrobial activity, Extracellular enzyme, Soil microbial, Drug-resistant bacteria.
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Baratto, Maria Camilla, David A. Lipscomb, Michael J. Larkin, Riccardo Basosi, Christopher C. R. Allen, and Rebecca Pogni. "Spectroscopic Characterisation of the Naphthalene Dioxygenase from Rhodococcus sp. Strain NCIMB12038." International Journal of Molecular Sciences 20, no. 14 (July 11, 2019): 3402. http://dx.doi.org/10.3390/ijms20143402.
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Polycyclic aromatic hydrocarbons (PAHs), such as naphthalene, are potential health risks due to their carcinogenic and mutagenic effects. Bacteria from the genus Rhodococcus are able to metabolise a wide variety of pollutants such as alkanes, aromatic compounds and halogenated hydrocarbons. A naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038 has been characterised for the first time, using electron paramagnetic resonance (EPR) spectroscopy and UV-Vis spectrophotometry. In the native state, the EPR spectrum of naphthalene 1,2-dioxygenase (NDO) is formed of the mononuclear high spin Fe(III) state contribution and the oxidised Rieske cluster is not visible as EPR-silent. In the presence of the reducing agent dithionite a signal derived from the reduction of the [2Fe-2S] unit is visible. The oxidation of the reduced NDO in the presence of O2-saturated naphthalene increased the intensity of the mononuclear contribution. A study of the “peroxide shunt”, an alternative mechanism for the oxidation of substrate in the presence of H2O2, showed catalysis via the oxidation of mononuclear centre while the Rieske-type cluster is not involved in the process. Therefore, the ability of these enzymes to degrade recalcitrant aromatic compounds makes them suitable for bioremediative applications and synthetic purposes.
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Tian, Kejian, Fanxing Meng, Qi Meng, Yan Gao, Lili Zhang, Le Wang, Yuqing Wang, Xue Li, and Hongliang Huo. "The Analysis of Estrogen-Degrading and Functional Metabolism Genes in Rhodococcus equi DSSKP-R-001." International Journal of Genomics 2020 (August 26, 2020): 1–13. http://dx.doi.org/10.1155/2020/9369182.
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Estrogen contamination is recognized as one of the most serious environmental problems, causing widespread concern worldwide. Environmental estrogens are mainly derived from human and vertebrate excretion, drugs, and agricultural activities. The use of microorganisms is currently the most economical and effective method for biodegradation of environmental estrogens. Rhodococcus equi DSSKP-R-001 (R-001) has strong estrogen-degrading capabilities. Our study indicated that R-001 can use different types of estrogen as its sole carbon source for growth and metabolism, with final degradation rates above 90%. Transcriptome analysis showed that 720 (E1), 983 (E2), and 845 (EE2) genes were significantly upregulated in the estrogen-treated group compared with the control group, and 270 differentially expressed genes (DEGs) were upregulated across all treatment groups. These DEGs included ABC transporters; estrogen-degrading genes, including those that perform initial oxidation and dehydrogenation reactions and those that further degrade the resulting substrates into small molecules; and metabolism genes that complete the intracellular transformation and utilization of estrogen metabolites through biological processes such as amino acid metabolism, lipid metabolism, carbohydrate metabolism, and the tricarboxylic acid cycle. In summary, the biodegradation of estrogens is coordinated by a metabolic network of estrogen-degrading enzymes, transporters, metabolic enzymes, and other coenzymes. In this study, the metabolic mechanisms by which Rhodococcus equi R-001 degrades various estrogens were analyzed for the first time. A new pollutant metabolism system is outlined, providing a starting point for the construction of engineered estrogen-degrading bacteria.
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Charnock, Colin. "Norwegian Soils and Waters Contain Mesophilic, Plastic-Degrading Bacteria." Microorganisms 9, no. 1 (January 3, 2021): 94. http://dx.doi.org/10.3390/microorganisms9010094.
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Plastic pollution has become one of the most critical environmental issues, as rapidly increasing production, compounded by persistence of plastic wastes in the environment, are outpacing efforts to keep ecosystems plastic-free. A switch to plastics more amenable to microbial attack is one of several possible responses. Against this background, the current study describes the isolation, enumeration and polyphasic characterization of plastic-degrading bacteria present in Norwegian terrestrial and aquatic habits. It shows that these bacteria are present in relatively high numbers, and that plastic-degrading capabilities are found in several taxa, most especially Streptomyces. Some isolates wereable to degrade several plastics. Notably, a Rhodococcus sp. and a Streptomyces sp. degraded, respectively, four and six of the eight plastics investigated and a number of other polymers relevant for plastic blends. The paper also has a methodological aspect, presenting various approaches for assaying plastic-degrading properties and a PCR/sequencing-based approach for the identification of potential polyethylene terephthalate-degrading genes. A candidate gene was detected in several Streptomyces isolates. The study shows that Norwegian environments are a rich source of bacteria with the ability to degrade bioplastics possibly representing a natural remediation capacity, as well as a potential source of useful enzymes.
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Ohhata, Naoko, Nobuyuki Yoshida, Hiroshi Egami, Tohoru Katsuragi, Yoshiki Tani, and Hiroshi Takagi. "An Extremely Oligotrophic Bacterium, Rhodococcus erythropolis N9T-4, Isolated from Crude Oil." Journal of Bacteriology 189, no. 19 (August 3, 2007): 6824–31. http://dx.doi.org/10.1128/jb.00872-07.
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ABSTRACT Rhodococcus erythropolis N9T-4, which was isolated from crude oil, showed extremely oligotrophic growth and formed its colonies on a minimal salt medium solidified using agar or silica gel without any additional carbon source. N9T-4 did not grow under CO2-limiting conditions but could grow on a medium containing NaHCO3 under the same conditions, suggesting that the oligotrophic growth of N9T-4 depends on CO2. Proteomic analysis of N9T-4 revealed that two proteins, with molecular masses of 45 and 55 kDa, were highly induced under the oligotrophic conditions. The primary structures of these proteins exhibited striking similarities to those of methanol: N,N′-dimethyl-4-nitrosoaniline oxidoreductase and an aldehyde dehydrogenase from Rhodococcus sp. These enzyme activities were three times higher under oligotrophic conditions than under n-tetradecane-containing heterotrophic conditions, and gene disruption for the aldehyde dehydrogenase caused a lack of growth on the minimal salt medium. Furthermore, 3-hexulose 6-phosphate synthase and phospho-3-hexuloisomerase activities, which are key enzymes in the ribulose monophosphate pathway in methylotrophic bacteria, were detected specifically in the cell extract of oligotrophically grown N9T-4. These results suggest that CO2 fixation involves methanol (formaldehyde) metabolism in the oligotrophic growth of R. erythropolis N9T-4.
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Villalba, María S., and Héctor M. Alvarez. "Identification of a novel ATP-binding cassette transporter involved in long-chain fatty acid import and its role in triacylglycerol accumulation in Rhodococcus jostii RHA1." Microbiology 160, no. 7 (July 1, 2014): 1523–32. http://dx.doi.org/10.1099/mic.0.078477-0.
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Members of the genus Rhodococcus are specialists in the biosynthesis and accumulation of triacylglycerols (TAGs). As no transport protein related to TAG metabolism has yet been characterized in these bacteria, we used the available genomic information of Rhodococcus jostii RHA1 to perform a broad survey of genes coding for putative lipid transporter proteins in this oleaginous micro-organism. Among the seven genes encoding putative lipid transporters, ro05645 (now called ltp1: lipid transporter protein) coding for an ATP-binding cassette protein was found clustered with others genes encoding enzymes catalysing the three putative acylation reactions of the Kennedy pathway for TAG synthesis. Overexpression of ltp1 in the RHA1 strain led to an increase of approximately sixfold and threefold in biomass and TAG production, respectively, when cells were cultivated on palmitic acid and oleic acid. Moreover, overexpression of ltp1 also promoted a significant increase in the uptake of a fluorescently labelled long-chain fatty acid (LCFA), as compared with the WT strain RHA1, and its further incorporation into the TAG fraction. Gluconate-grown cells showed increasing amounts of intracellular free fatty acids, but not of TAG, after overexpressing ltp1. Thus, for the first time to our knowledge, a transporter functionally related to TAG metabolism was identified in oleaginous rhodococci. Our results suggested that Ltp1 is an importer of LCFAs that plays a functional role in lipid homeostasis of R. jostii RHA1.
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Li, Xingchun, Wei He, Meijin Du, Jin Zheng, Xianyuan Du, and Yu Li. "Design of a Microbial Remediation Inoculation Program for Petroleum Hydrocarbon Contaminated Sites Based on Degradation Pathways." International Journal of Environmental Research and Public Health 18, no. 16 (August 20, 2021): 8794. http://dx.doi.org/10.3390/ijerph18168794.
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This paper analyzed the degradation pathways of petroleum hydrocarbon degradation bacteria, screened the main degradation pathways, and found the petroleum hydrocarbon degradation enzymes corresponding to each step of the degradation pathway. Through the Copeland method, the best inoculation program of petroleum hydrocarbon degradation bacteria in a polluted site was selected as follows: single oxygenation path was dominated by Streptomyces avermitilis, hydroxylation path was dominated by Methylosinus trichosporium OB3b, secondary oxygenation path was dominated by Pseudomonas aeruginosa, secondary hydroxylation path was dominated by Methylococcus capsulatus, double oxygenation path was dominated by Acinetobacter baylyi ADP1, hydrolysis path was dominated by Rhodococcus erythropolis, and CoA path was dominated by Geobacter metallireducens GS-15 to repair petroleum hydrocarbon contaminated sites. The Copeland method score for this solution is 22, which is the highest among the 375 solutions designed in this paper, indicating that it has the best degradation effect. Meanwhile, we verified its effect by the Cdocker method, and the Cdocker energy of this solution is −285.811 kcal/mol, which has the highest absolute value. Among the inoculation programs of the top 13 petroleum hydrocarbon degradation bacteria, the effect of the best inoculation program of petroleum hydrocarbon degradation bacteria was 18% higher than that of the 13th group, verifying that this solution has the best overall degradation effect. The inoculation program of petroleum hydrocarbon degradation bacteria designed in this paper considered the main pathways of petroleum hydrocarbon pollutant degradation, especially highlighting the degradability of petroleum hydrocarbon intermediate degradation products, and enriching the theoretical program of microbial remediation of petroleum hydrocarbon contaminated sites.
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Eulberg, Dirk, Elena M. Kourbatova, Ludmila A. Golovleva, and Michael Schlömann. "Evolutionary Relationship between Chlorocatechol Catabolic Enzymes from Rhodococcus opacus 1CP and Their Counterparts in Proteobacteria: Sequence Divergence and Functional Convergence." Journal of Bacteriology 180, no. 5 (1998): 1082–94. http://dx.doi.org/10.1128/jb.180.5.1082-1094.1998.
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Biochemical investigations of the muconate and chloromuconate cycloisomerases from the chlorophenol-utilizing strainRhodococcus opacus (erythropolis) 1CP had previously indicated that the chlorocatechol catabolic pathway of this strain may have developed independently from the corresponding pathways of proteobacteria. To test this hypothesis, we cloned the chlorocatechol catabolic gene cluster of strain 1CP by using PCR with primers derived from sequences of N termini and peptides of purified chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase. Sequencing of the clones revealed that they comprise different parts of the same gene cluster in which five open reading frames have been identified. The clcB gene for chloromuconate cycloisomerase is transcribed divergently from a gene which codes for a LysR-type regulatory protein, the presumed ClcR. Downstream of clcRbut separated from it by 222 bp, we detected the clcA andclcD genes, which could unambiguously be assigned to chlorocatechol 1,2-dioxygenase and dienelactone hydrolase. A gene coding for a maleylacetate reductase could not be detected. Instead, the product encoded by the fifth open reading frame turned out to be homologous to transposition-related proteins of IS1031 and Tn4811. Sequence comparisons of ClcA and ClcB to other 1,2-dioxygenases and cycloisomerases, respectively, clearly showed that the chlorocatechol catabolic enzymes of R. opacus 1CP represent different branches in the dendrograms than their proteobacterial counterparts. Thus, while the sequences diverged, the functional adaptation to efficient chlorocatechol metabolization occurred independently in proteobacteria and gram-positive bacteria, that is, by functionally convergent evolution.
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Calvo-Bado, Leo A., Tim R. Pettitt, Nick Parsons, Geoff M. Petch, J. Alun W. Morgan, and John M. Whipps. "Spatial and Temporal Analysis of the Microbial Community in Slow Sand Filters Used for Treating Horticultural Irrigation Water." Applied and Environmental Microbiology 69, no. 4 (April 2003): 2116–25. http://dx.doi.org/10.1128/aem.69.4.2116-2125.2003.
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ABSTRACT An experimental slow sand filter (SSF) was constructed to study the spatial and temporal structure of a bacterial community suppressive to an oomycete plant pathogen, Phytophthora cryptogea. Passage of water through the mature sand column resulted in complete removal of zoospores of the plant pathogen. To monitor global changes in the microbial community, bacterial and fungal numbers were estimated on selective media, direct viable counts of fungal spores were made, and the ATP content was measured. PCR amplification of 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) were used to study the dynamics of the bacterial community in detail. The top layer (1 cm) of the SSF column was dominated by a variable and active microbial population, whereas the middle (50 cm) and bottom (80 cm) layers were dominated by less active and diverse bacterial populations. The major changes in the microbial populations occurred during the first week of filter operation, and these populations then remained to the end of the study. Spatial and temporal nonlinear mapping of the DGGE bands provided a useful visual representation of the similarities between SSF samples. According to the DGGE profile, less than 2% of the dominating bands present in the SSF column were represented in the culturable population. Sequence analysis of DGGE bands from all depths of the SSF column indicated that a range of bacteria were present, with 16S rRNA gene sequences similar to groups such as Bacillus megaterium, Cytophaga, Desulfovibrio, Legionella, Rhodococcus rhodochrous, Sphingomonas, and an uncharacterized environmental clone. This study describes the characterization of the performance, and microbial composition, of SSFs used for the treatment of water for use in the horticultural industry. Utilization of naturally suppressive population of microorganisms either directly or by manipulation of the environment in an SSF may provide a more reproducible control method for the future.
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Schwarz, Jenny, Georg Hubmann, Katrin Rosenthal, and Stephan Lütz. "Triaging of Culture Conditions for Enhanced Secondary Metabolite Diversity from Different Bacteria." Biomolecules 11, no. 2 (January 30, 2021): 193. http://dx.doi.org/10.3390/biom11020193.
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Over the past decade, the one strain many compounds (OSMAC) approach has been established for the activation of biosynthetic gene clusters (BGCs), which mainly encode the enzymes of secondary metabolite (SM) biosynthesis pathways. These BGCs were successfully activated by altering various culture conditions, such as aeration rate, temperature, and nutrient composition. Here, we determined the biosynthetic potential of 43 bacteria using the genome mining tool antiSMASH. Based on the number of BGCs, biological safety, availability of deposited cultures, and literature coverage, we selected five promising candidates: Bacillus amyloliquefaciens DSM7, Corallococcus coralloides DSM2259, Pyxidicoccus fallax HKI727, Rhodococcus jostii DSM44719, and Streptomyces griseochromogenes DSM40499. The bacteria were cultivated under a broad range of OSMAC conditions (nutrient-rich media, minimal media, nutrient-limited media, addition of organic solvents, addition of biotic additives, and type of culture vessel) to fully assess the biosynthetic potential. In particular, we investigated so far scarcely applied OSMAC conditions to enhance the diversity of SMs. We detected the four predicted compounds bacillibactin, desferrioxamine B, myxochelin A, and surfactin. In total, 590 novel mass features were detected in a broad range of investigated OSMAC conditions, which outnumber the predicted gene clusters for all investigated bacteria by far. Interestingly, we detected mass features of the bioactive compounds cyclo-(Tyr-Pro) and nocardamin in extracts of DSM7 and DSM2259. Both compounds were so far not reported for these strains, indicating that our broad OSMAC screening approach was successful. Remarkably, the infrequently applied OSMAC conditions in defined medium with and without nutrient limitation were demonstrated to be very effective for BGC activation and for SM discovery.
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Turnaev, Igor I., Konstantin V. Gunbin, Valentin V. Suslov, Ilya R. Akberdin, Nikolay A. Kolchanov, and Dmitry A. Afonnikov. "The Phylogeny of Class B Flavoprotein Monooxygenases and the Origin of the YUCCA Protein Family." Plants 9, no. 9 (August 25, 2020): 1092. http://dx.doi.org/10.3390/plants9091092.
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YUCCA (YUCCA flavin-dependent monooxygenase) is one of the two enzymes of the main auxin biosynthesis pathway (tryptophan aminotransferase enzyme (TAA)/YUCCA) in land plants. The evolutionary origin of the YUCCA family is currently controversial: YUCCAs are assumed to have emerged via a horizontal gene transfer (HGT) from bacteria to the most recent common ancestor (MRCA) of land plants or to have inherited it from their ancestor, the charophyte algae. To refine YUCCA origin, we performed a phylogenetic analysis of the class B flavoprotein monooxygenases and comparative analysis of the sequences belonging to different families of this protein class. We distinguished a new protein family, named type IIb flavin-containing monooxygenases (FMOs), which comprises homologs of YUCCA from Rhodophyta, Chlorophyta, and Charophyta, land plant proteins, and FMO-E, -F, and -G of the bacterium Rhodococcus jostii RHA1. The type IIb FMOs differ considerably in the sites and domain composition from the other families of class B flavoprotein monooxygenases, YUCCAs included. The phylogenetic analysis also demonstrated that the type IIb FMO clade is not a sibling clade of YUCCAs. We have also identified the bacterial protein group named YUC-like FMOs as the closest to YUCCA homologs. Our results support the hypothesis of the emergence of YUCCA via HGT from bacteria to MRCA of land plants.
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Pirog, T. P., T. A. Shevchuk, L. V. Nikituk, D. A. Lutsai, and O. I. Paliichuk. "INFLUENCE OF CULTIVATION CONDITIONS ON ANTIMICROBIAL AND ANTI-ADHESIVE ACTIVITY OF SURFACTANTS OF BACTERIA OF ACINETOBACTER, RHODOCOCCUS AND NOCARDIA GENERA." Proceedings of the National Academy of Sciences of Belarus, Biological Series 63, no. 3 (August 25, 2018): 307–15. http://dx.doi.org/10.29235/1029-8940-2018-63-3-307-315.
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Microbial surfactants are multifunctional products because they cannot only reduce the surface tension at the interface and emulsify various substrates, but also display antimicrobial and anti-adhesion activity (including the ability to destroy biofilms). However, under various conditions of producer’s cultivation the surfactant composition and their properties can vary. One of the approaches to increasing antimicrobial and anti-adhesion activity of the surfactant can be an increase in medium of producer cultivation content of activators of key enzymes biosynthesis of aminolipids − the most effective antimicrobial agents. Activators of NADP+-dependent glutamate dehydrogenase in Acinetobacter calcoaceticus IMV B-7241 are cations of calcium, magnesium and zinc, Rhodococcus erythropolis IMV Ac-5017 and Nocardia vaccinii IMV B-7405 – calcium.Surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2:1). Antimicrobial activity of surfactants was determined by index of minimum inhibitory concentration (MIC), antiadhesive − by spectrophotometry. The degree of biofilm destruction was determined as difference between the number of adhered cells in untreated and treated with surfactant holes of polystyrene immunological plate containing pre-formed biofilm of test cultures and was expressed as a percentage. It was found that addition of CaCl2 (0.1 g/l) into medium cultivation of R. erythropolis IMV Ac-5017, increasing concentration of this salt to 0.4 g/l in medium for N. vaccinii IMV B-7405 growth, introduction of CaCl2(0.1 g l) and increasing MgSO4·7H2O content to 0.2 g/l, or adding Zn2+(38 μM) into medium cultivation of A. calcoaceticus IMV B-7241 was accompanied by synthesis of surfactants MICs of which against test cultures were 1.2–13 times lower, their adhesion on abiotic surfaces treated with such surfactants was on average 10−40 % lower, and the degree of biofilms destruction was 7−20 % higher than indicators established for surfactants obtained on the base medium. The obtained data indicate the possibility of regulating antimicrobial and anti-adhesion activity of microbial surfactants under producer cultivation.
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Ringelberg, David B., Jeffrey W. Talley, Edward J. Perkins, Samuel G. Tucker, Richard G. Luthy, Edward J. Bouwer, and Herbert L. Fredrickson. "Succession of Phenotypic, Genotypic, and Metabolic Community Characteristics during In Vitro Bioslurry Treatment of Polycyclic Aromatic Hydrocarbon-Contaminated Sediments." Applied and Environmental Microbiology 67, no. 4 (April 1, 2001): 1542–50. http://dx.doi.org/10.1128/aem.67.4.1542-1550.2001.
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ABSTRACT Dredged harbor sediment contaminated with polycyclic aromatic hydrocarbons (PAHs) was removed from the Milwaukee Confined Disposal Facility and examined for in situ biodegradative capacity. Molecular techniques were used to determine the successional characteristics of the indigenous microbiota during a 4-month bioslurry evaluation. Ester-linked phospholipid fatty acids (PLFA), multiplex PCR of targeted genes, and radiorespirometry techniques were used to define in situ microbial phenotypic, genotypic, and metabolic responses, respectively. Soxhlet extractions revealed a loss in total PAH concentrations of 52%. Individual PAHs showed reductions as great as 75% (i.e., acenapthene and fluorene). Rates of 14C-PAH mineralization (percent/day) were greatest for phenanthrene, followed by pyrene and then chrysene. There was no mineralization capacity for benzo[a]pyrene. Ester-linked phospholipid fatty acid analysis revealed a threefold increase in total microbial biomass and a dynamic microbial community composition that showed a strong correlation with observed changes in the PAH chemistry (canonicalr 2 of 0.999). Nucleic acid analyses showed copies of genes encoding PAH-degrading enzymes (extradiol dioxygenases, hydroxylases, and meta-cleavage enzymes) to increase by as much as 4 orders of magnitude. Shifts in gene copy numbers showed strong correlations with shifts in specific subsets of the extant microbial community. Specifically, declines in the concentrations of three-ring PAH moieties (i.e., phenanthrene) correlated with PLFA indicative of certain gram-negative bacteria (i.e., Rhodococcus spp. and/or actinomycetes) and genes encoding for naphthalene-, biphenyl-, and catechol-2,3-dioxygenase degradative enzymes. The results of this study suggest that the intrinsic biodegradative potential of an environmental site can be derived from the polyphasic characterization of the in situ microbial community.
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Jafra, S., J. Przysowa, R. Czajkowski, A. Michta, P. Garbeva, and J. M. Van der Wolf. "Detection and characterization of bacteria from the potato rhizosphere degradingN-acyl-homoserine lactone." Canadian Journal of Microbiology 52, no. 10 (October 1, 2006): 1006–15. http://dx.doi.org/10.1139/w06-062.
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Quorum sensing plays a role in the regulation of soft rot diseases caused by the plant pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. The signal molecules involved in quorum sensing in P. carotovorum subsp. carotovorum belong to the group of N-acyl homoserine lactones (AHLs). In our study, we screened bacteria isolated from the potato rhizosphere for the ability to degrade AHLs produced by P. carotovorum subsp. carotovorum. Six isolates able to degrade AHLs were selected for further studies. According to 16S rDNA sequence analysis and fatty acid methyl ester profiling, the isolates belonged to the genera Ochrobactrum, Rhodococcus, Pseudomonas, Bacillus, and Delftia. For the genera Ochrobactrum and Delftia, for the first time AHL-degrading isolates were found. Data presented in this study revealed for the first time that Ochrobactrum sp. strain A44 showed the capacity to inactivate various synthetic AHL molecules; the substituted AHLs were inactivated with a lower efficiency than the unsubstituted AHLs. Compared with the other isolates, A44 was very effective in the degradation of AHLs produced by P. carotovorum subsp. carotovorum. It was verified by polymerase chain reaction, DNA–DNA hybridization, and a lactone ring reconstruction assay that Ochrobactrum sp. strain A44 did not possess AHL lactonase activity. AHL degradation in Ochrobactrum sp. strain A44 occurred intracellularly; it was not found in the culture supernatant. AHL-degrading activity of A44 was thermo sensitive. Experiments in planta revealed that Ochrobactrum sp. strain A44 significantly inhibited the maceration of potato tuber tissue. Since A44 did not produce antibiotics, the attenuation of the decay might be due to the quenching of quorum- sensing-regulated production of pectinolytic enzymes. The strain can potentially serve to control P. carotovorum subsp. carotovorum in potato.Key words: AHL degradation, Ochrobactrum sp., Pectobacterium carotovorum.
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Luo, Dan, Jixiang Chen, Gang Xie, Liang Yue, and Yonggang Wang. "Enzyme characterization and biological activities of a resuscitation promoting factor from an oil degrading bacterium Rhodococcus erythropolis KB1." PeerJ 7 (May 21, 2019): e6951. http://dx.doi.org/10.7717/peerj.6951.
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Resuscitation-promoting factors (Rpf) are a class of muralytic enzymes, which participate in recovery of dormant cells and promoting bacteria growth in poor media. In the present study the expression vector of the rpf-1 gene from an oil-degrading bacterium Rhodococcus erythropolis KB1 was constructed and expressed in Escherichia coli. The expressed protein was purified by Ni2+-affinity chromatography, and showed muralytic activity when measured with 4-methylumbelliferyl-β-D-N,N′,N″-triacetyl chitotrioside as substrate. Addition of purified Rpf-1 to R. erythropolis culture efficiently improved bacterial cell growth. The purified protein also increased resuscitation of viable but nonculturable cells of R. erythropolis to culturable state. The conserved amino acid residues including Asp45, Glu51, Cys50, Thr60, Gln69, Thr74, Trp75 and Cys114 of the Rpf-1 were replaced with different amino acids. The mutant proteins were also expressed and purified with Ni2+-affinity chromatography. The muralytic activities of the mutant proteins decreased to different extents when compared with that of the wild type Rpf-1. Gln69 was found to play the most important role in the enzyme activity, substitution of Gln69 with lysine (Q69K) resulted in the greatest decrease of muralytic activity. The other amino acid residues such as Asp45, Glu51, Cys50 and Cys114 were also found to be very important in maintaining muralytic activity and biological function of the Rpf-1. Our results indicated that Rpf-1 from R. erythropolis showed muralytic activities and weak protease activity, but the muralytic activity was responsible for its growth promotion and resuscitation activity.
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Tratsiak, Katsiaryna, Tatyana Prudnikova, Ivana Drienovska, Lukas Chrast, Jiri Damborsky, Pavlina Rezacova, Michal Kuty, Radka Chaloupkova, and Ivana Kuta Smatanova. "Crystal structure of the novel haloalkane dehalogenases." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1678. http://dx.doi.org/10.1107/s2053273314083211.
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Haloalkane dehalogenases (EC 3.8.1.5; HLDs) are microbial enzymes with catalytic activity for the hydrolytic conversion of xenobiotic and highly toxic halogenated aliphatic compounds to the corresponding alcohols. Biodegradation, biosensing, biocatalysis and cellular imaging are potentially practical applications for the HLDs. Two newly isolated and purified psychrophilic haloalkane dehalogenases, exhibiting interesting catalytic properties, DpcA from Psychrobacter cryohalolentis K5 and DmxA from Marinobacter sp. ELB17, were used for the crystallization experiments and structure determination. Diffracted crystals of DpcA(left) and DmxA(right) (see figure, the scale bar -100μm) were refined up to the 1.05 Å and 1.45 Å resolutions, respectively. Diffraction data for DpcA were collected on beamline 14.2 at the BESSY II electron-storage ring (Helmholtz-Zentrum Berlin (HZB), Germany) and equipped with a Rayonics MX-225 CCD detector at the wavelengths of 0.978 Å, and for DmxA were collected using Pilatus 6M-F detector at the wavelengths of 0.972 Å on the beamline ID29, at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France). Crystals of DpcA belonged to P21 space group with unit-cell parameters: a = 41.3, b = 79.4, c = 43.5 A °, α = β = 90.0, γ = 95.0 and contained 1 molecule in the asymmetric unit. Crystals of DmxA belonged to P212121 space group, with unit-cell parameters: a = 43.371, b = 78.343, c = 150.51; α = γ = β = 90.0 and contained 2 molecules in the asymmetric unit. The structures were solved by molecular replacement with MOLREP from the CCP4 software suite. The coordinates of Xanthobacter autotrophicus (PDB code: 1B6G; 40% sequence identities for 121 residues and 53% sequence similarity was used as search model for DpcA structure and for DmxA from Rhodococcus rhodochrous (PDB entry 4E46; 48% sequence identity for 142 residues and 63% sequence similarity). Belonging to the superfamily of α/β - hydrolases, according to the catalytic pentad, HLDs are subdivided onto the three subfamilies. DpcA belongs to the HLD - I: Asp- His - Asp + Trp - Trp and DmxA to the HLD – II: Asp - His - Glu + Asn - Trp. We thank M. Weiss and S. Pühringer (BESSY). This work is supported by the Grant Agency of the Czech Republic (P207/12/0775).Also by the Ministry of Education of the Czech Republic (CZ.1.05/2.1.00/01.0024 and CZ.1.05/2.1.00/01.0001). The support of the Academy of Sciences of the Czech Republic is acknowledged as well.
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Duarte, Gabriela Frois, Alexandre Soares Rosado, Lucy Seldin, Welington de Araujo, and Jan Dirk van Elsas. "Analysis of Bacterial Community Structure in Sulfurous-Oil-Containing Soils and Detection of Species Carrying Dibenzothiophene Desulfurization (dsz) Genes." Applied and Environmental Microbiology 67, no. 3 (March 1, 2001): 1052–62. http://dx.doi.org/10.1128/aem.67.3.1052-1062.2001.
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ABSTRACT The selective effects of sulfur-containing hydrocarbons, with respect to changes in bacterial community structure and selection of desulfurizing organisms and genes, were studied in soil. Samples taken from a polluted field soil (A) along a concentration gradient of sulfurous oil and from soil microcosms treated with dibenzothiophene (DBT)-containing petroleum (FSL soil) were analyzed. Analyses included plate counts of total bacteria and of DBT utilizers, molecular community profiling via soil DNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and detection of genes that encode enzymes involved in the desulfurization of hydrocarbons, i.e., dszA, dszB, and dszC.Data obtained from the A soil showed no discriminating effects of oil levels on the culturable bacterial numbers on either medium used. Generally, counts of DBT degraders were 10- to 100-fold lower than the total culturable counts. However, PCR-DGGE showed that the numbers of bands detected in the molecular community profiles decreased with increasing oil content of the soil. Analysis of the sequences of three prominent bands of the profiles generated with the highly polluted soil samples suggested that the underlying organisms were related to Actinomyces sp.,Arthrobacter sp., and a bacterium of uncertain affiliation.dszA, dszB, and dszC genes were present in all A soil samples, whereas a range of unpolluted soils gave negative results in this analysis. Results from the study of FSL soil revealed minor effects of the petroleum-DBT treatment on culturable bacterial numbers and clear effects on the DBT-utilizing communities. The molecular community profiles were largely stable over time in the untreated soil, whereas they showed a progressive change over time following treatment with DBT-containing petroleum. Direct PCR assessment revealed the presence of dszB-related signals in the untreated FSL soil and the apparent selection of dszA- and dszC-related sequences by the petroleum-DBT treatment. PCR-DGGE applied to sequential enrichment cultures in DBT-containing sulfur-free basal salts medium prepared from the A and treated FSL soils revealed the selection of up to 10 distinct bands. Sequencing a subset of these bands provided evidence for the presence of organisms related to Pseudomonas putida, a Pseudomonassp., Stenotrophomonas maltophilia, and Rhodococcus erythropolis. Several of 52 colonies obtained from the A and FSL soils on agar plates with DBT as the sole sulfur source produced bands that matched the migration of bands selected in the enrichment cultures. Evidence for the presence of dszB in 12 strains was obtained, whereas dszA and dszC genes were found in only 7 and 6 strains, respectively. Most of the strains carrying dszA or dszC were classified asR. erythropolis related, and all revealed the capacity to desulfurize DBT. A comparison of 37 dszA sequences, obtained via PCR from the A and FSL soils, from enrichments of these soils, and from isolates, revealed the great similarity of all sequences to the canonical (R. erythropolis strain IGTS8)dszA sequence and a large degree of internal conservation. The 37 sequences recovered were grouped in three clusters. One group, consisting of 30 sequences, was minimally 98% related to the IGTS8 sequence, a second group of 2 sequences was slightly different, and a third group of 5 sequences was 95% similar. The first two groups contained sequences obtained from both soil types and enrichment cultures (including isolates), but the last consisted of sequences obtained directly from the polluted A soil.
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Frederick, Joni, Fritha Hennessy, Uli Horn, Pilar de la Torre Cortés, Marcel van den Broek, Ulrich Strych, Richard Willson, et al. "The complete genome sequence of the nitrile biocatalyst Rhodococcus rhodochrous ATCC BAA-870." BMC Genomics 21, no. 1 (January 2, 2020). http://dx.doi.org/10.1186/s12864-019-6405-7.
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Abstract Background Rhodococci are industrially important soil-dwelling Gram-positive bacteria that are well known for both nitrile hydrolysis and oxidative metabolism of aromatics. Rhodococcus rhodochrous ATCC BAA-870 is capable of metabolising a wide range of aliphatic and aromatic nitriles and amides. The genome of the organism was sequenced and analysed in order to better understand this whole cell biocatalyst. Results The genome of R. rhodochrous ATCC BAA-870 is the first Rhodococcus genome fully sequenced using Nanopore sequencing. The circular genome contains 5.9 megabase pairs (Mbp) and includes a 0.53 Mbp linear plasmid, that together encode 7548 predicted protein sequences according to BASys annotation, and 5535 predicted protein sequences according to RAST annotation. The genome contains numerous oxidoreductases, 15 identified antibiotic and secondary metabolite gene clusters, several terpene and nonribosomal peptide synthetase clusters, as well as 6 putative clusters of unknown type. The 0.53 Mbp plasmid encodes 677 predicted genes and contains the nitrile converting gene cluster, including a nitrilase, a low molecular weight nitrile hydratase, and an enantioselective amidase. Although there are fewer biotechnologically relevant enzymes compared to those found in rhodococci with larger genomes, such as the well-known Rhodococcus jostii RHA1, the abundance of transporters in combination with the myriad of enzymes found in strain BAA-870 might make it more suitable for use in industrially relevant processes than other rhodococci. Conclusions The sequence and comprehensive description of the R. rhodochrous ATCC BAA-870 genome will facilitate the additional exploitation of rhodococci for biotechnological applications, as well as enable further characterisation of this model organism. The genome encodes a wide range of enzymes, many with unknown substrate specificities supporting potential applications in biotechnology, including nitrilases, nitrile hydratase, monooxygenases, cytochrome P450s, reductases, proteases, lipases, and transaminases.