scholarly journals Phenol Degradation in the Strictly Anaerobic Iron-Reducing Bacterium Geobacter metallireducens GS-15

2009 ◽  
Vol 75 (12) ◽  
pp. 3912-3919 ◽  
Author(s):  
Kathleen M. Schleinitz ◽  
Sirko Schmeling ◽  
Nico Jehmlich ◽  
Martin von Bergen ◽  
Hauke Harms ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Posttranscriptional Regulation ◽  
Phenol Degradation ◽  
Regulation Mechanism ◽  
Strictly Anaerobic ◽  
Carboxylase Activity ◽  
Transcription Analysis ◽  
Geobacter Metallireducens ◽  
Isotope Exchange Reaction ◽  
Cell Extracts

ABSTRACT Information on anaerobic phenol metabolism by physiological groups of bacteria other than nitrate reducers is scarce. We investigated phenol degradation in the strictly anaerobic iron-reducing deltaproteobacterium Geobacter metallireducens GS-15 using metabolite, transcriptome, proteome, and enzyme analyses. The results showed that the initial steps of phenol degradation are accomplished by phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) as known from Thauera aromatica, but they also revealed some distinct differences. The pps-ppc gene cluster identified in the genome is functional, as shown by transcription analysis. In contrast to T. aromatica, transcription of the pps- and ppc-like genes was induced not only during growth on phenol, but also during growth on benzoate. In contrast, proteins were detected only during growth on phenol, suggesting the existence of a posttranscriptional regulation mechanism for these initial steps. Phenylphosphate synthase and phenylphosphate carboxylase activities were detected in cell extracts. The carboxylase does not catalyze an isotope exchange reaction between 14CO2 and 4-hydroxybenzoate, which is characteristic of the T. aromatica enzyme. Whereas the enzyme of T. aromatica is encoded by ppcABCD, the pps-ppc gene cluster of G. metallireducens contains only a ppcB homologue. Nearby, but oriented in the opposite direction, is a ppcD homologue that is transcribed during growth on phenol. Genome analysis did not reveal obvious homologues of ppcA and ppcC, leaving open the question of whether these genes are dispensable for phenylphosphate carboxylase activity in G. metallireducens or are quite different from the Thauera counterparts and located elsewhere in the genome.

scholarly journals Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans

2010 ◽  
Vol 192 (19) ◽  
pp. 5237-5244 ◽  
Author(s):  
Diliana Dancheva Simeonova ◽  
Marlena Marie Wilson ◽  
William W. Metcalf ◽  
Bernhard Schink
Keyword(s):  
Sequence Analysis ◽  
Gene Cluster ◽  
Electron Donor ◽  
Genomic Library ◽  
Amino Acid Sequences ◽  
Strictly Anaerobic ◽  
Oxidation Activity ◽  
Cell Extracts ◽  
Expression Of Genes ◽  
Phosphorus Source

ABSTRACT Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO2 reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.

scholarly journals Draft Genome Sequence of Comamonas testosteroni TA441, a Bacterium That Has a Cryptic Phenol Degradation Gene Cluster

2019 ◽  
Vol 8 (39) ◽  
Author(s):  
Hiroyuki Arai ◽  
Masaharu Ishii
Keyword(s):  
Gene Cluster ◽  
Genome Sequence ◽  
Phenol Degradation ◽  
Draft Genome ◽  
Draft Genome Sequence ◽  
Comamonas Testosteroni ◽  
Spontaneous Mutations ◽  
Content Type ◽  
Repressor Gene ◽  
Mutant Strains

Comamonas testosteroni TA441 has a complete phenol degradation gene cluster but does not degrade phenol because the cluster is tightly repressed. However, mutant strains that can degrade phenol arise by spontaneous mutations of a repressor gene during incubation with phenol. Here, we report the draft genome sequence of strain TA441.

scholarly journals Decarboxylating and Nondecarboxylating Glutaryl-Coenzyme A Dehydrogenases in the Aromatic Metabolism of Obligately Anaerobic Bacteria

2009 ◽  
Vol 191 (13) ◽  
pp. 4401-4409 ◽  
Author(s):  
Simon Wischgoll ◽  
Martin Taubert ◽  
Franziska Peters ◽  
Nico Jehmlich ◽  
Martin von Bergen ◽  
...  
Keyword(s):  
Coenzyme A ◽  
Anaerobic Bacteria ◽  
Oxidative Decarboxylation ◽  
Kinetic Properties ◽  
Strictly Anaerobic ◽  
Amino Acid Residues ◽  
Geobacter Metallireducens ◽  
Sulfate Reducing ◽  
Reverse Transcription Pcr ◽  
Gene Coding

ABSTRACT In anaerobic bacteria using aromatic growth substrates, glutaryl-coenzyme A (CoA) dehydrogenases (GDHs) are involved in the catabolism of the central intermediate benzoyl-CoA to three acetyl-CoAs and CO2. In this work, we studied GDHs from the strictly anaerobic, aromatic compound-degrading organisms Geobacter metallireducens (GDHGeo) (Fe[III] reducing) and Desulfococcus multivorans (GDHDes) (sulfate reducing). GDHGeo was purified from cells grown on benzoate and after the heterologous expression of the benzoate-induced bamM gene. The gene coding for GDHDes was identified after screening of a cosmid gene library. Reverse transcription-PCR revealed that its expression was induced by benzoate; the product was heterologously expressed and isolated. Both wild-type and recombinant GDHGeo catalyzed the oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA at similar rates. In contrast, recombinant GDHDes catalyzed only the dehydrogenation to glutaconyl-CoA. The latter compound was decarboxylated subsequently to crotonyl-CoA by the addition of membrane extracts from cells grown on benzoate in the presence of 20 mM NaCl. All GDH enzymes were purified as homotetramers of a 43- to 44-kDa subunit and contained 0.6 to 0.7 flavin adenine dinucleotides (FADs)/monomer. The kinetic properties for glutaryl-CoA conversion were as follows: for GDHGeo, the Km was 30 ± 2 μM and the V max was 3.2 ± 0.2 μmol min−1 mg−1, and for GDHDes, the Km was 52 ± 5 μM and the V max was 11 ± 1 μmol min−1 mg−1. GDHDes but not GDHGeo was inhibited by glutaconyl-CoA. Highly conserved amino acid residues that were proposed to be specifically involved in the decarboxylation of the intermediate glutaconyl-CoA were identified in GDHGeo but are missing in GDHDes. The differential use of energy-yielding/energy-demanding enzymatic processes in anaerobic bacteria that degrade aromatic compounds is discussed in view of phylogenetic relationships and constraints of overall energy metabolism.

scholarly journals Clostridium acidurici Electron-Bifurcating Formate Dehydrogenase

2013 ◽  
Vol 79 (19) ◽  
pp. 6176-6179 ◽  
Author(s):  
Shuning Wang ◽  
Haiyan Huang ◽  
Jörg Kahnt ◽  
Rudolf K. Thauer
Keyword(s):  
Uric Acid ◽  
Gene Cluster ◽  
Formate Dehydrogenase ◽  
Specific Activity ◽  
Enzyme Complex ◽  
Content Type ◽  
Cell Extracts

ABSTRACTCell extracts of uric acid-grownClostridium aciduricicatalyzed the coupled reduction of NAD+and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene clusterhylCBA-fdhF2.

scholarly journals Cyclohexa-1,5-Diene-1-Carbonyl-Coenzyme A (CoA) Hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a Common Benzoyl-CoA Degradation Pathway in Facultative and Strict Anaerobes

2006 ◽  
Vol 189 (3) ◽  
pp. 1055-1060 ◽  
Author(s):  
Franziska Peters ◽  
Yoshifumi Shinoda ◽  
Michael J. McInerney ◽  
Matthias Boll
Keyword(s):  
Coenzyme A ◽  
Anaerobic Bacteria ◽  
Degradation Pathway ◽  
Ring Cleavage ◽  
Strictly Anaerobic ◽  
Significant Activity ◽  
Geobacter Metallireducens ◽  
Sulfate Reducing ◽  
Coa Reductase ◽  
Syntrophus Aciditrophicus

ABSTRACT In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of β-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km , 80 and 35 μM; V max, 350 and 550 μmol min−1 mg−1, respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.

scholarly journals Degradation of Phenol via Meta Cleavage Pathway by Pseudomonas fluorescens PU1

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Md. Mahiudddin ◽  
A. N. M. Fakhruddin ◽  
Abdullah-Al-Mahin
Keyword(s):  
Phenolic Compounds ◽  
Pseudomonas Fluorescens ◽  
Phenol Degradation ◽  
Carbon Sources ◽  
Soil Microflora ◽  
Ring Cleavage ◽  
Cell Extracts ◽  
Polluted Sites ◽  
Important Means ◽  
Biodegradation Efficiency

Degradation of phenolics by members of soil microflora is an important means by which these substances are removed from the environment thus reducing environmental pollution. Biodegradation by microorganisms offers unique opportunities to destroy or render phenolic compounds. A bacterium, PU1, identified as Pseudomonas fluorescens PU1, was investigated for its ability to grow on and degrade phenols as sole carbon sources in aerobic shaking batch culture. The organism degraded up to 1000 ppm of phenol using meta cleavage pathway. The pathways for phenol degradation were proposed by the identification of metabolites and assay of ring cleavage enzymes in cell extracts. Phenol was degraded via catechol with subsequent metaring cleavage. Cell growth increased as the phenol concentrations increased up to 1000 ppm phenol. The biodegradation efficiency, degradation extent, and metabolic pathway of phenol were determined to provide useful clues for further application of this isolate in the engineered bioremediation systems. The paper's results suggest that Pseudomonas fluorescens PU1 strain could be a good candidate for remediation of phenol contaminants from heavily polluted sites.

scholarly journals Identification and Characterization of Epoxide Carboxylase Activity in Cell Extracts of Nocardia corallina B276

1998 ◽  
Vol 180 (8) ◽  
pp. 2072-2078 ◽  
Author(s):  
Jeffrey R. Allen ◽  
Scott A. Ensign
Keyword(s):  
Enzyme System ◽  
Time Dependent ◽  
High Level Expression ◽  
Carboxylase Activity ◽  
Cell Extracts ◽  
Keto Acids ◽  
Nocardia Corallina ◽  
High Level ◽  
Identification And Characterization

ABSTRACT The metabolism of aliphatic epoxides (epoxyalkanes) by the alkene-utilizing actinomycete Nocardia corallina B276 was investigated. Suspensions of N. corallina cells grown with propylene as the carbon source readily degraded propylene and epoxypropane, while suspensions of glucose-grown cells did not. The addition of propylene and epoxypropane to glucose-grown cells resulted in a time-dependent increase in propylene- and epoxypropane-degrading activities that was prevented by the addition of rifampin and chloramphenicol. The expression of alkene- and epoxide-degrading activities was correlated with the high-level expression of several polypeptides not present in extracts of glucose-grown cells. Epoxypropane and epoxybutane degradation by propylene-grown cell suspensions of N. corallina was stimulated by the addition of CO2 and inhibited by the depletion of CO2. Cell extracts catalyzed the carboxylation of epoxypropane to form acetoacetate in a reaction that was dependent on the addition of CO2, NAD+, and a reductant (NADPH or dithiothreitol). In the absence of CO2, epoxypropane was isomerized by cell extracts to form acetone at a rate approximately 10-fold lower than the rate of epoxypropane carboxylation. Methylepoxypropane was found to be a time-dependent, irreversible inactivator of epoxyalkane-degrading activity. These properties demonstrate that epoxyalkane metabolism in N. corallinaoccurs by a carboxylation reaction forming β-keto acids as products and provide evidence for the involvement in this reaction of an epoxide carboxylase with properties and cofactor requirements similar to those of the four-component epoxide carboxylase enzyme system of the gram-negative bacterium Xanthobacter strain Py2 (J. R. Allen and S. A. Ensign, J. Biol. Chem. 272:32121–32128, 1997). The addition of epoxide carboxylase component I fromXanthobacter strain Py2 to methylepoxypropane-inactivatedN. corallina extracts restored epoxide carboxylase activity, and the addition of epoxide carboxylase component II fromXanthobacter Py2 to active N. corallinaextracts stimulated epoxide isomerase rates to the same levels observed with the purified Xanthobacter system. Antibodies raised against Xanthobacter strain Py2 epoxide carboxylase component I cross-reacted with a polypeptide in propylene-grownN. corallina extracts with the same molecular weight as component I but did not cross-react with glucose-grown extracts. Together, these results suggest a common pathway of epoxyalkane metabolism for phylogenetically distinct bacteria that involves CO2 fixation and the activity of a multicomponent epoxide carboxylase enzyme system.

scholarly journals Genes, Enzymes, and Regulation of para-Cresol Metabolism in Geobacter metallireducens

2007 ◽  
Vol 189 (13) ◽  
pp. 4729-4738 ◽  
Author(s):  
Franziska Peters ◽  
Dimitri Heintz ◽  
Jörg Johannes ◽  
Alain van Dorsselaer ◽  
Matthias Boll
Keyword(s):  
Alcohol Oxidation ◽  
Regulatory Elements ◽  
Denitrifying Bacteria ◽  
Regulation Of Transcription ◽  
Strictly Anaerobic ◽  
Geobacter Metallireducens ◽  
Α Subunit ◽  
Proteomic Approach ◽  
Activity Measurements ◽  
Hydroxybenzyl Alcohol

ABSTRACT In aerobic and facultatively anaerobic bacteria, the degradation of para-cresol (p-cresol) involves the initial hydroxylation to p-hydroxybenzyl alcohol by water catalyzed by the soluble, periplasmatic flavocytochrome p-cresol methylhydroxylase (PCMH; α2β2 composition). In denitrifying bacteria the further metabolism proceeds via oxidation to p-hydroxybenzoate, the formation of p-hydroxybenzoyl-coenzyme A (CoA), and the subsequent dehydroxylation of the latter to benzoyl-CoA by reduction. In contrast, the strictly anaerobic Desulfobacterium cetonicum degrades p-cresol by addition to fumarate, yielding p-hydroxybenzylsuccinate. In this work, in vitro enzyme activity measurements revealed that the obligately anaerobic Geobacter metallireducens uses the p-cresol degradation pathway of denitrifying bacteria. Surprisingly, PCMH, which is supposed to catalyze both p-cresol hydroxylation and p-hydroxybenzyl alcohol oxidation to the corresponding aldehyde, was located in the membrane fraction. The α subunit of the enzyme was present in two isoforms, suggesting an αα′β2 composition. We propose that the unusual asymmetric architecture and the membrane association of PCMH might be important for alternative electron transfer routes to either cytochrome c (in the case of p-cresol oxidation) or to menaquinone (in the case of p-hydroxybenzyl alcohol oxidation). Unusual properties of further enzymes of p-cresol metabolism, p-hydroxybenzoate-CoA ligase, and p-hydroxybenzoyl-CoA reductase were identified and are discussed. A proteomic approach identified a gene cluster comprising most of the putative structural genes for enzymes involved in p-cresol metabolism (pcm genes). Reverse transcription-PCR studies revealed a different regulation of transcription of pcm genes and the corresponding enzyme activities, suggesting the presence of posttranscriptional regulatory elements.

scholarly journals Transcription attenuation-derived small RNA rnTrpL regulates tryptophan biosynthesis gene expression in trans

2019 ◽  
Vol 47 (12) ◽  
pp. 6396-6410 ◽  
Author(s):  
Hendrik Melior ◽  
Siqi Li ◽  
Ramakanth Madhugiri ◽  
Maximilian Stötzel ◽  
Saina Azarderakhsh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Posttranscriptional Regulation ◽  
Sinorhizobium Meliloti ◽  
Regulation Mechanism ◽  
Base Pairs ◽  
Bacterial Gene ◽  
Bacterial Gene Expression ◽  
In Trans ◽  
Transcription Attenuation ◽  
In Cis

Abstract Ribosome-mediated transcription attenuation is a basic posttranscriptional regulation mechanism in bacteria. Liberated attenuator RNAs arising in this process are generally considered nonfunctional. In Sinorhizobium meliloti, the tryptophan (Trp) biosynthesis genes are organized into three operons, trpE(G), ppiD-trpDC-moaC-moeA, and trpFBA-accD-folC, of which only the first one, trpE(G), contains a short ORF (trpL) in the 5′-UTR and is regulated by transcription attenuation. Under conditions of Trp sufficiency, transcription is terminated between trpL and trpE(G), and a small attenuator RNA, rnTrpL, is produced. Here, we show that rnTrpL base-pairs with trpD and destabilizes the polycistronic trpDC mRNA, indicating rnTrpL-mediated downregulation of the trpDC operon in trans. Although all three trp operons are regulated in response to Trp availability, only in the two operons trpE(G) and trpDC the Trp-mediated regulation is controlled by rnTrpL. Together, our data show that the trp attenuator coordinates trpE(G) and trpDC expression posttranscriptionally by two fundamentally different mechanisms: ribosome-mediated transcription attenuation in cis and base-pairing in trans. Also, we present evidence that rnTrpL-mediated regulation of trpDC genes expression in trans is conserved in Agrobacterium and Bradyrhizobium, suggesting that the small attenuator RNAs may have additional conserved functions in the control of bacterial gene expression.

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