Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation

ABSTRACTRhodococcus erythropolisBG43 is able to degrade thePseudomonas aeruginosaquorum sensing signal molecules PQS (Pseudomonasquinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resultingN-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters,aqdA1B1C1andaqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. AnaqdRgene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to theaqdA2B2C2cluster. Expression ofaqdA2B2C2inEscherichia coliconferred the ability to degrade HHQ and PQS to anthranilic acid; however, forE. colitransformed withaqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to formN-octanoylanthranilic acid and carbon monoxide and revealed apparentKmandkcatvalues for PQS of ∼27 μM and 21 s−1, respectively. Heterologous expression of the PQS dioxygenase geneaqdC1oraqdC2inP. aeruginosaPAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases.

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Interference with Pseudomonas aeruginosa Quorum Sensing and Virulence by the Mycobacterial Pseudomonas Quinolone Signal Dioxygenase AqdC in Combination with the N-Acylhomoserine Lactone Lactonase QsdA

Infection and Immunity ◽

10.1128/iai.00278-19 ◽

2019 ◽

Vol 87 (10) ◽

Cited By ~ 4

Author(s):

Franziska S. Birmes ◽

Ruth Säring ◽

Miriam C. Hauke ◽

Niklas H. Ritzmann ◽

Steffen L. Drees ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Virulence Factors ◽

Rhodococcus Erythropolis ◽

Quorum Quenching ◽

Signal Molecules ◽

Content Type ◽

Acylhomoserine Lactone ◽

Regulatory Effects ◽

Epithelial Lung Cells

ABSTRACT The nosocomial pathogen Pseudomonas aeruginosa regulates its virulence via a complex quorum sensing network, which, besides N-acylhomoserine lactones, includes the alkylquinolone signal molecules 2-heptyl-3-hydroxy-4(1H)-quinolone (Pseudomonas quinolone signal [PQS]) and 2-heptyl-4(1H)-quinolone (HHQ). Mycobacteroides abscessus subsp. abscessus, an emerging pathogen, is capable of degrading the PQS and also HHQ. Here, we show that although M. abscessus subsp. abscessus reduced PQS levels in coculture with P. aeruginosa PAO1, this did not suffice for quenching the production of the virulence factors pyocyanin, pyoverdine, and rhamnolipids. However, the levels of these virulence factors were reduced in cocultures of P. aeruginosa PAO1 with recombinant M. abscessus subsp. massiliense overexpressing the PQS dioxygenase gene aqdC of M. abscessus subsp. abscessus, corroborating the potential of AqdC as a quorum quenching enzyme. When added extracellularly to P. aeruginosa cultures, AqdC quenched alkylquinolone and pyocyanin production but induced an increase in elastase levels. When supplementing P. aeruginosa cultures with QsdA, an enzyme from Rhodococcus erythropolis which inactivates N-acylhomoserine lactone signals, rhamnolipid and elastase levels were quenched, but HHQ and pyocyanin synthesis was promoted. Thus, single quorum quenching enzymes, targeting individual circuits within a complex quorum sensing network, may also elicit undesirable regulatory effects. Supernatants of P. aeruginosa cultures grown in the presence of AqdC, QsdA, or both enzymes were less cytotoxic to human epithelial lung cells than supernatants of untreated cultures. Furthermore, the combination of both aqdC and qsdA in P. aeruginosa resulted in a decline of Caenorhabditis elegans mortality under P. aeruginosa exposure.

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SfnR2 Regulates Dimethyl Sulfide-Related Utilization inPseudomonas aeruginosaPAO1

Journal of Bacteriology ◽

10.1128/jb.00606-18 ◽

2018 ◽

Vol 201 (4) ◽

Cited By ~ 1

Author(s):

Benjamin R. Lundgren ◽

Zaara Sarwar ◽

Kyle S. Feldman ◽

Joseph M. Shoytush ◽

Christopher T. Nomura

Keyword(s):

Pseudomonas Aeruginosa ◽

Target Genes ◽

Dimethyl Sulfide ◽

Sulfur Compound ◽

Main Function ◽

Gene Encoding ◽

Content Type ◽

First Results ◽

Terrestrial Environments ◽

Initial Activation

ABSTRACTDimethyl sulfide (DMS) is a volatile sulfur compound produced mainly from the degradation of dimethylsulfoniopropionate (DMSP) in marine environments. DMS undergoes oxidation to form dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO2), and methanesulfonate (MSA), all of which occur in terrestrial environments and are accessible for consumption by various microorganisms. The purpose of the present study was to determine how the enhancer-binding proteins SfnR1 and SfnR2 contribute to the utilization of DMS and its derivatives inPseudomonas aeruginosaPAO1. First, results from cell growth experiments showed that deletion of eithersfnR2orsfnG, a gene encoding a DMSO2-monooxygenase, significantly inhibits the ability ofP. aeruginosaPAO1 to use DMSP, DMS, DMSO, and DMSO2as sulfur sources. Deletion of thesfnR1ormsuEDCgenes, which encode a MSA desulfurization pathway, did not abolish the growth ofP. aeruginosaPAO1 on any sulfur compound tested. Second, data collected from β-galactosidase assays revealed that themsuEDC-sfnR1operon and thesfnGgene are induced in response to sulfur limitation or nonpreferred sulfur sources, such as DMSP, DMS, and DMSO, etc. Importantly, SfnR2 (and not SfnR1) is essential for this induction. Expression ofsfnR2is induced under sulfur limitation but independently of SfnR1 or SfnR2. Finally, the results of this study suggest that the main function of SfnR2 is to direct the initial activation of themsuEDC-sfnR1operon in response to sulfur limitation or nonpreferred sulfur sources. Once expressed, SfnR1 contributes to the expression ofmsuEDC-sfnR1,sfnG, and other target genes involved in DMS-related metabolism inP. aeruginosaPAO1.IMPORTANCEDimethyl sulfide (DMS) is an important environmental source of sulfur, carbon, and/or energy for microorganisms. For various bacteria, includingPseudomonas,Xanthomonas, andAzotobacter, DMS utilization is thought to be controlled by the transcriptional regulator SfnR. Adding more complexity, some bacteria, such asAcinetobacter baumannii,Enterobacter cloacae, andPseudomonas aeruginosa, possess two, nonidentical SfnR proteins. In this study, we demonstrate that SfnR2 and not SfnR1 is the principal regulator of DMS metabolism inP. aeruginosaPAO1. Results suggest that SfnR1 has a supportive but nonessential role in the positive regulation of genes required for DMS utilization. This study not only enhances our understanding of SfnR regulation but, importantly, also provides a framework for addressing gene regulation through dual SfnR proteins in other bacteria.

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Novel Reporter for Identification of Interference with Acyl Homoserine Lactone and Autoinducer-2 Quorum Sensing

Applied and Environmental Microbiology ◽

10.1128/aem.03290-14 ◽

2014 ◽

Vol 81 (4) ◽

pp. 1477-1489 ◽

Cited By ~ 28

Author(s):

Nancy Weiland-Bräuer ◽

Nicole Pinnow ◽

Ruth A. Schmitz

Keyword(s):

Quorum Sensing ◽

Vibrio Fischeri ◽

Quorum Quenching ◽

Homoserine Lactone ◽

Signal Molecules ◽

Lethal Gene ◽

Reporter Strain ◽

Content Type ◽

E Coli ◽

Autoinducer 2

ABSTRACTTwo reporter strains were established to identify novel biomolecules interfering with bacterial communication (quorum sensing [QS]). The basic design of theseEscherichia coli-based systems comprises a gene encoding a lethal protein fused to promoters induced in the presence of QS signal molecules. Consequently, theseE. colistrains are unable to grow in the presence of the respective QS signal molecules unless a nontoxic QS-interfering compound is present. The first reporter strain designed to detect autoinducer-2 (AI-2)-interfering activities (AI2-QQ.1) contained theE. coliccdBlethal gene under the control of theE. colilsrApromoter. The second reporter strain (AI1-QQ.1) contained theVibrio fischeriluxIpromoter fused to theccdBgene to detect interference with acyl-homoserine lactones. Bacteria isolated from the surfaces of several marine eukarya were screened for quorum-quenching (QQ) activities using the established reporter systems AI1-QQ.1 and AI2-QQ.1. Out of 34 isolates, two interfered with acylated homoserine lactone (AHL) signaling, five interfered with AI-2 QS signaling, and 10 were demonstrated to interfere with both signal molecules. Open reading frames (ORFs) conferring QQ activity were identified for three selected isolates (Photobacteriumsp.,Pseudoalteromonassp., andVibrio parahaemolyticus). Evaluation of the respective heterologously expressed and purified QQ proteins confirmed their ability to interfere with the AHL and AI-2 signaling processes.

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The Widespread Multidrug-Resistant Serotype O12 Pseudomonas aeruginosa Clone Emerged through Concomitant Horizontal Transfer of Serotype Antigen and Antibiotic Resistance Gene Clusters

mBio ◽

10.1128/mbio.01396-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 21

Author(s):

Sandra Wingaard Thrane ◽

Véronique L. Taylor ◽

Luca Freschi ◽

Irena Kukavica-Ibrulj ◽

Brian Boyle ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Horizontal Transfer ◽

Core Genome ◽

Gene Clusters ◽

Multidrug Resistant ◽

Clinical Settings ◽

Content Type ◽

The Past ◽

Antibiotic Resistance Determinant

ABSTRACTThe O-specific antigen (OSA) inPseudomonas aeruginosalipopolysaccharide is highly varied by sugar identity, side chains, and bond between O-repeats. These differences classifiedP. aeruginosainto 20 distinct serotypes. In the past few decades, O12 has emerged as the predominant serotype in clinical settings and outbreaks. These serotype O12 isolates exhibit high levels of resistance to various classes of antibiotics. Here, we explore how theP. aeruginosaOSA biosynthesis gene clusters evolve in the population by investigating the association between the phylogenetic relationships among 83P. aeruginosastrains and their serotypes. While most serotypes were closely linked to the core genome phylogeny, we observed horizontal exchange of OSA biosynthesis genes among phylogenetically distinctP. aeruginosastrains. Specifically, we identified a “serotype island” ranging from 62 kb to 185 kb containing theP. aeruginosaO12 OSA gene cluster, an antibiotic resistance determinant (gyrAC248T), and other genes that have been transferred betweenP. aeruginosastrains with distinct core genome architectures. We showed that these genes were likely acquired from an O12 serotype strain that is closely related toP. aeruginosaPA7. Acquisition and recombination of the “serotype island” resulted in displacement of the native OSA gene cluster and expression of the O12 serotype in the recipients. Serotype switching by recombination has apparently occurred multiple times involving bacteria of various genomic backgrounds. In conclusion, serotype switching in combination with acquisition of an antibiotic resistance determinant most likely contributed to the dissemination of the O12 serotype in clinical settings.IMPORTANCEInfection rates in hospital settings by multidrug-resistant (MDR)Pseudomonas aeruginosaclones have increased during the past decades, and serotype O12 is predominant among these epidemic strains. It is not known why the MDR phenotype is associated with serotype O12 and how this clone type has emerged. This study shows that evolution of MDR O12 strains involved a switch from an ancestral O4 serotype to O12. Serotype switching was the result of horizontal transfer and genetic recombination of lipopolysaccharide (LPS) biosynthesis genes originating from an MDR taxonomic outlierP. aeruginosastrain. Moreover, the recombination event also resulted in acquisition of antibiotic resistance genes. These results impact on our understanding of MDR outbreak strain and serotype evolution and can potentially assist in better monitoring and prevention.

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Complete genome sequence of Rhodococcus erythropolis BG43 (DSM 46869), a degrader of Pseudomonas aeruginosa quorum sensing signal molecules

Journal of Biotechnology ◽

10.1016/j.jbiotec.2015.07.014 ◽

2015 ◽

Vol 211 ◽

pp. 99-100 ◽

Cited By ~ 4

Author(s):

Christian Rückert ◽

Franziska S. Birmes ◽

Christine Müller ◽

Heiko Niewerth ◽

Anika Winkler ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Rhodococcus Erythropolis ◽

Signal Molecules

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Conversion of the Pseudomonas aeruginosa Quinolone Signal and Related Alkylhydroxyquinolines by Rhodococcus sp. Strain BG43

Applied and Environmental Microbiology ◽

10.1128/aem.02342-14 ◽

2014 ◽

Vol 80 (23) ◽

pp. 7266-7274 ◽

Cited By ~ 14

Author(s):

Christine Müller ◽

Franziska S. Birmes ◽

Heiko Niewerth ◽

Susanne Fetzner

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Rhodococcus Erythropolis ◽

Opportunistic Pathogen ◽

Biosynthetic Enzyme ◽

Signal Molecule ◽

Content Type ◽

Cell Extracts ◽

Specific Pathway ◽

Rhodococcus Sp

ABSTRACTA bacterial strain, which based on the sequences of its 16S rRNA,gyrB,catA, andqsdAgenes, was identified as aRhodococcussp. closely related toRhodococcus erythropolis, was isolated from soil by enrichment on thePseudomonasquinolone signal [PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone], a quorum sensing signal employed by the opportunistic pathogenPseudomonas aeruginosa. The isolate, termedRhodococcussp. strain BG43, cometabolically degraded PQS and its biosynthetic precursor 2-heptyl-4(1H)-quinolone (HHQ) to anthranilic acid. HHQ degradation was accompanied by transient formation of PQS, and HHQ hydroxylation by cell extracts required NADH, indicating that strain BG43 has a HHQ monooxygenase isofunctional to the biosynthetic enzyme PqsH ofP. aeruginosa. The enzymes catalyzing HHQ hydroxylation and PQS degradation were inducible by PQS, suggesting a specific pathway. Remarkably,Rhodococcussp. BG43 is also capable of transforming 2-heptyl-4-hydroxyquinoline-N-oxide to PQS. It thus converts an antibacterial secondary metabolite ofP. aeruginosato a quorum sensing signal molecule.

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AidC, a NovelN-Acylhomoserine Lactonase from the Potato Root-Associated Cytophaga-Flavobacteria-Bacteroides (CFB) Group Bacterium Chryseobacterium sp. Strain StRB126

Applied and Environmental Microbiology ◽

10.1128/aem.02188-12 ◽

2012 ◽

Vol 78 (22) ◽

pp. 7985-7992 ◽

Cited By ~ 39

Author(s):

Wen-Zhao Wang ◽

Tomohiro Morohoshi ◽

Nobutaka Someya ◽

Tsukasa Ikeda

Keyword(s):

High Performance ◽

Genomic Library ◽

Root Surface ◽

Signal Molecules ◽

Gram Negative Bacteria ◽

Potato Root ◽

Gene Encoding ◽

Content Type ◽

Degrading Enzymes

ABSTRACTN-Acylhomoserine lactones (AHLs) are used as quorum-sensing (QS) signal molecules by many Gram-negative bacteria. We have reported thatChryseobacteriumsp. strain StRB126, which was isolated from the root surface of potato, has AHL-degrading activity. In this study, we cloned and characterized theaidCgene from the genomic library of StRB126. AidC has AHL-degrading activity and shows homology to several metallo-β-lactamase proteins fromBacteroidetes, although not to any known AHL-degrading enzymes. Purified AidC, as a maltose-binding fusion protein, showed high degrading activity against all tested AHLs, whether short- or long-chain forms, with or without substitution at carbon 3. High-performance liquid chromatography (HPLC) analysis revealed that AidC functions as an AHL lactonase catalyzing AHL ring opening by hydrolyzing lactones. An assay to determine the effects of covalent and ionic bonding showed that Zn2+is important to AidC activity bothin vitroandin vivo. In addition, theaidCgene could also be PCR amplified from several otherChryseobacteriumstrains. In conclusion, this study indicated that theaidCgene, encoding a novel AHL lactonase, may be widespread throughout the genusChryseobacterium. Our results extend the diversity and known bacterial hosts of AHL-degrading enzymes.

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Efficient Biostimulation of Native and Introduced Quorum-Quenching Rhodococcus erythropolis Populations Is Revealed by a Combination of Analytical Chemistry, Microbiology, and Pyrosequencing

Applied and Environmental Microbiology ◽

10.1128/aem.06159-11 ◽

2011 ◽

Vol 78 (2) ◽

pp. 481-492 ◽

Cited By ~ 44

Author(s):

Amélie Cirou ◽

Samuel Mondy ◽

Shu An ◽

Amélie Charrier ◽

Amélie Sarrazin ◽

...

Keyword(s):

Analytical Chemistry ◽

Root Colonization ◽

Plant Root ◽

Rhodococcus Erythropolis ◽

Quorum Quenching ◽

Content Type ◽

Potato Plants ◽

Molecular Approaches ◽

Degrading Bacteria ◽

Integrative Study

ABSTRACTDegradation of the quorum-sensing (QS) signals known asN-acylhomoserine lactones (AHL) by soil bacteria may be useful as a beneficial trait for protecting crops, such as potato plants, against the worldwide pathogenPectobacterium. In this work, analytical chemistry and microbial and molecular approaches were combined to explore and compare biostimulation of native and introduced AHL-degradingRhodococcus erythropolispopulations in the rhizosphere of potato plants cultivated in farm greenhouses under hydroponic conditions. We first identified gamma-heptalactone (GHL) as a novel biostimulating agent that efficiently promotes plant root colonization by AHL-degradingR. erythropolispopulation. We also characterized an AHL-degrading biocontrolR. erythropolisisolate, R138, which was introduced in the potato rhizosphere. Moreover, root colonization by AHL-degrading bacteria receiving different combinations of GHL and R138 treatments was compared by using a cultivation-based approach (percentage of AHL-degrading bacteria), pyrosequencing of PCR-amplifiedrrsloci (total bacterial community), and quantitative PCR (qPCR) of theqsdAgene, which encodes an AHL lactonase inR. erythropolis. Higher densities of the AHL-degradingR. erythropolispopulation in the rhizosphere were observed when GHL treatment was associated with biocontrol strain R138. Under this condition, the introducedR. erythropolispopulation displaced the nativeR. erythropolispopulation. Finally, chemical analyses revealed that GHL, gamma-caprolactone (GCL), and their by-products, gamma-hydroxyheptanoic acid and gamma-hydroxycaproic acid, rapidly disappeared from the rhizosphere and did not accumulate in plant tissues. This integrative study highlights biostimulation as a potential innovative approach for improving root colonization by beneficial bacteria.

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Discovery and Biosynthesis of the Antibiotic Bicyclomycin in Distantly Related Bacterial Classes

Applied and Environmental Microbiology ◽

10.1128/aem.02828-17 ◽

2018 ◽

Vol 84 (9) ◽

Cited By ~ 12

Author(s):

Natalia M. Vior ◽

Rodney Lacret ◽

Govind Chandra ◽

Siobhán Dorai-Raj ◽

Martin Trick ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Gene Cluster ◽

Gene Clusters ◽

Opportunistic Pathogen ◽

Biosynthetic Gene Cluster ◽

Wide Distribution ◽

Biosynthetic Gene ◽

P450 Monooxygenase ◽

Content Type ◽

Transcription Termination Factor

ABSTRACTBicyclomycin (BCM) is a clinically promising antibiotic that is biosynthesized byStreptomyces cinnamoneusDSM 41675. BCM is structurally characterized by a core cyclo(l-Ile-l-Leu) 2,5-diketopiperazine (DKP) that is extensively oxidized. Here, we identify the BCM biosynthetic gene cluster, which shows that the core of BCM is biosynthesized by a cyclodipeptide synthase, and the oxidative modifications are introduced by five 2-oxoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase. The discovery of the gene cluster enabled the identification of BCM pathways encoded by the genomes of hundreds ofPseudomonas aeruginosaisolates distributed globally, and heterologous expression of the pathway fromP. aeruginosaSCV20265 demonstrated that the product is chemically identical to BCM produced byS. cinnamoneus. Overall, putative BCM gene clusters have been found in at least seven genera spanningActinobacteriaandProteobacteria(Alphaproteobacteria,Betaproteobacteria, andGammaproteobacteria). This represents a rare example of horizontal gene transfer of an intact biosynthetic gene cluster across such distantly related bacteria, and we show that these gene clusters are almost always associated with mobile genetic elements.IMPORTANCEBicyclomycin is the only natural product antibiotic that selectively inhibits the transcription termination factor Rho. This mechanism of action, combined with its proven biological safety and its activity against clinically relevant Gram-negative bacterial pathogens, makes it a very promising antibiotic candidate. Here, we report the identification of the bicyclomycin biosynthetic gene cluster in the known bicyclomycin-producing organismStreptomyces cinnamoneus, which will enable the engineered production of new bicyclomycin derivatives. The identification of this gene cluster also led to the discovery of hundreds of bicyclomycin pathways encoded in highly diverse bacteria, including in the opportunistic pathogenPseudomonas aeruginosa. This wide distribution of a complex biosynthetic pathway is very unusual and provides an insight into how a pathway for an antibiotic can be transferred between diverse bacteria.

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Biodegradation of the Organic Disulfide 4,4′-Dithiodibutyric Acid by Rhodococcus spp.

Applied and Environmental Microbiology ◽

10.1128/aem.02059-15 ◽

2015 ◽

Vol 81 (24) ◽

pp. 8294-8306 ◽

Cited By ~ 16

Author(s):

Heba Khairy ◽

Jan Hendrik Wübbeler ◽

Alexander Steinbüchel

Keyword(s):

Carbon Source ◽

Sole Carbon Source ◽

Rhodococcus Erythropolis ◽

Model Organism ◽

Hypothetical Protein ◽

Gene Encoding ◽

Content Type ◽

Gene Coding ◽

Initial Cleavage ◽

Genes Encoding

ABSTRACTFourRhodococcusspp. exhibited the ability to use 4,4′-dithiodibutyric acid (DTDB) as a sole carbon source for growth. The most important step for the production of a novel polythioester (PTE) using DTDB as a precursor substrate is the initial cleavage of DTDB. Thus, identification of the enzyme responsible for this step was mandatory. BecauseRhodococcus erythropolisstrain MI2 serves as a model organism for elucidation of the biodegradation of DTDB, it was used to identify the genes encoding the enzymes involved in DTDB utilization. To identify these genes, transposon mutagenesis ofR. erythropolisMI2 was carried out using transposon pTNR-TA. Among 3,261 mutants screened, 8 showed no growth with DTDB as the sole carbon source. In five mutants, the insertion locus was mapped either within a gene coding for a polysaccharide deacetyltransferase, a putative ATPase, or an acetyl coenzyme A transferase, 1 bp upstream of a gene coding for a putative methylase, or 176 bp downstream of a gene coding for a putative kinase. In another mutant, the insertion was localized between genes encoding a putative transcriptional regulator of the TetR family (noxR) and an NADH:flavin oxidoreductase (nox). Moreover, in two other mutants, the insertion loci were mapped within a gene encoding a hypothetical protein in the vicinity ofnoxRandnox. The interruption mutant generated,R. erythropolisMI2noxΩtsr, was unable to grow with DTDB as the sole carbon source. Subsequently,noxwas overexpressed and purified, and its activity with DTDB was measured. The specific enzyme activity of Nox amounted to 1.2 ± 0.15 U/mg. Therefore, we propose that Nox is responsible for the initial cleavage of DTDB into 2 molecules of 4-mercaptobutyric acid (4MB).

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