Steroid Degradation in Comamonas testosteroni TA441: Identification of Metabolites and the Genes Involved in the Reactions Necessary before D-Ring Cleavage

ABSTRACT Bacterial steroid degradation has been studied mainly with Rhodococcus equi (Nocardia restrictus) and Comamonas testosteroni as representative steroid degradation bacteria for more than 50 years. The primary purpose was to obtain materials for steroid drugs, but recent studies showed that many genera of bacteria (Mycobacterium, Rhodococcus, Pseudomonas, etc.) degrade steroids and that steroid-degrading bacteria are globally distributed and found particularly in wastewater treatment plants, the soil, plant rhizospheres, and the marine environment. The role of bacterial steroid degradation in the environment is, however, yet to be revealed. To uncover the whole steroid degradation process in a representative steroid-degrading bacterium, C. testosteroni, to provide basic information for further studies on the role of bacterial steroid degradation, we elucidated the two indispensable oxidative reactions and hydration before D-ring cleavage in C. testosteroni TA441. In bacterial oxidative steroid degradation, A- and B-rings of steroids are cleaved to produce 2-hydroxyhexa-2,4-dienoic acid and 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. The latter compound was revealed to be degraded to the coenzyme A (CoA) ester of 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid, which is converted to the CoA ester of 9,17-dioxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid by ORF31-encoded hydroxylacyl dehydrogenase (ScdG), followed by conversion to the CoA ester of 9,17-dioxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid by ORF4-encoded acyl-CoA dehydrogenase (ScdK). Then, a water molecule is added by the ORF5-encoded enoyl-CoA hydratase (ScdY), which leads to the cleavage of the D-ring. The conversion by ScdG is presumed to be a reversible reaction. The elucidated pathway in C. testosteroni TA441 is different from the corresponding pathways in Mycobacterium tuberculosis H37Rv. IMPORTANCE Studies on representative steroid degradation bacteria Rhodococcus equi (Nocardia restrictus) and Comamonas testosteroni were initiated more than 50 years ago primarily to obtain materials for steroid drugs. A recent study showed that steroid-degrading bacteria are globally distributed and found particularly in wastewater treatment plants, the soil, plant rhizospheres, and the marine environment, but the role of bacterial steroid degradation in the environment is yet to be revealed. This study aimed to uncover the whole steroid degradation process in C. testosteroni TA441, in which major enzymes for steroidal A- and B-ring cleavage were elucidated, to provide basic information for further studies on bacterial steroid degradation. C. testosteroni is suitable for exploring the degradation pathway because the involvement of degradation-related genes can be determined by gene disruption. We elucidated the two indispensable oxidative reactions and hydration before D-ring cleavage, which appeared to differ from those present in Mycobacterium tuberculosis H37Rv.

Download Full-text

Steroid Degradation in Comamonas testosteroni TA441: Identification of the Entire β-Oxidation Cycle of the Cleaved B Ring

Applied and Environmental Microbiology ◽

10.1128/aem.01204-19 ◽

2019 ◽

Vol 85 (20) ◽

Cited By ~ 4

Author(s):

Masae Horinouchi ◽

Hiroyuki Koshino ◽

Michal Malon ◽

Hiroshi Hirota ◽

Toshiaki Hayashi

Keyword(s):

Gene Clusters ◽

Degradation Process ◽

Degradation Pathway ◽

Ring Cleavage ◽

Comamonas Testosteroni ◽

Content Type ◽

Coa Transferase ◽

Degrading Bacteria ◽

Globally Distributed

ABSTRACT Comamonas testosteroni TA441 degrades steroids via aromatization of the A ring, followed by degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, mainly by β-oxidation. In this study, we revealed that 7β,9α-dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-coenzyme A (CoA) ester is dehydrogenated by (3S)-3-hydroxylacyl CoA-dehydrogenase, encoded by scdE (ORF27), and then the resultant 9α-hydroxy-7,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is converted by 3-ketoacyl-CoA transferase, encoded by scdF (ORF23). With these results, the whole cycle of β-oxidation on the side chain at C-8 of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid is clarified; 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is dehydrogenated at C-6 by ScdC1C2, followed by hydration by ScdD. 7β,9α-Dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-CoA ester then is dehydrogenated by ScdE to be converted to 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid-CoA ester and acetyl-CoA by ScdF. ScdF is an ortholog of FadA6 in Mycobacterium tuberculosis H37Rv, which was reported as a 3-ketoacyl-CoA transferase involved in C ring cleavage. We also obtained results suggesting that ScdF is also involved in C ring cleavage, but further investigation is required for confirmation. ORF25 and ORF26, located between scdF and scdE, encode enzymes belonging to the amidase superfamily. Disrupting either ORF25 or ORF26 did not affect steroid degradation. Among the bacteria having gene clusters similar to those of tesB to tesR, some have both ORF25- and ORF26-like proteins or only an ORF26-like protein, but others do not have either ORF25- or ORF26-like proteins. ORF25 and ORF26 are not crucial for steroid degradation, yet they might provide clues to elucidate the evolution of bacterial steroid degradation clusters. IMPORTANCE Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in relation to human health is attracting attention. The overall aerobic degradation of the four basic steroidal rings has been proposed; however, there is still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in Comamonas testosteroni TA441 as a model of steroid-degrading bacteria. C. testosteroni is one of the most studied representative steroid-degrading bacteria and is suitable for exploring the degradation pathway, because the involvement of degradation-related genes can be determined by gene disruption. Here, we elucidated the entire β-oxidation cycle of the cleaved B ring. This cycle is essential for the following C and D ring cleavage.

Download Full-text

Identification of the CoA-ester intermediates and genes involved in the cleavage and degradation of the steroidal C-ring by Comamonas testosteroni TA441

Applied and Environmental Microbiology ◽

10.1128/aem.01102-21 ◽

2021 ◽

Author(s):

Masae Horinouchi ◽

Toshiaki Hayashi

Keyword(s):

Degradation Process ◽

Intermediate Compound ◽

Degradation Pathway ◽

Hydroxyl Group ◽

Ring Cleavage ◽

Comamonas Testosteroni ◽

Dioic Acid ◽

Coa Transferase ◽

Degrading Bacteria ◽

Globally Distributed

Comamonas testosteroni TA441 degrades steroids aerobically via aromatization of the A-ring accompanied by B-ring cleavage, followed by D- and C-ring cleavage. We previously revealed major enzymes and intermediate compounds in A,B-ring cleavage, β-oxidation cycle of the cleaved B-ring, and partial C,D-ring cleavage process. Here, we elucidated the C-ring cleavage and the β-oxidation cycle that follows. ScdL1L2, a 3-ketoacid Coenzyme A (CoA) transferase which belongs to the SugarP_isomerase superfamily, was thought to cleave the C-ring of 9-oxo-1,2,3,4,5,6,10,19-octanor-13,17-secoandrost-8(14)-ene-7,17-dioic acid-CoA ester, the key intermediate compound in the degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid (3aα- H -4α [3′-propionic acid]-7aβ-methylhexahydro-1,5-indanedione; HIP)-CoA ester in the previous study; however, this study suggested that ScdL1L2 is the isomerase of the derivative with a hydroxyl group at C-14 which cleaves C ring. The subsequent ring-cleaved product was indicated to be converted to 4-methyl-5-oxo-octane-1,8-dioic acid-CoA ester mainly by ORF33-encoded CoA-transferase (named ScdJ), followed by dehydrogenation by ORF21 and 22-encoded acyl-CoA dehydrogenase (named ScdM1M2). Then a water molecule is added by ScdN for further degradation by β-oxidation. ScdN is considered to catalyze the last reaction in C,D-ring degradation by the enzymes encoded in the steroid degradation gene cluster tesB to tesR . IMPORTANCE Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in human is attracting attention. The overall degradation of steroidal four rings is proposed, however there are still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in C. testosteroni , which is one of the most studied representative steroid degrading bacteria and is suitable for exploring the degradation pathway because the involvement of degradation-related genes can be determined by gene disruption.

Download Full-text

Estrogen Degraders and Estrogen Degradation Pathway Identified in an Activated Sludge

Applied and Environmental Microbiology ◽

10.1128/aem.00001-18 ◽

2018 ◽

Vol 84 (10) ◽

Cited By ~ 25

Author(s):

Yi-Lung Chen ◽

Han-Yi Fu ◽

Tzong-Huei Lee ◽

Chao-Jen Shih ◽

Lina Huang ◽

...

Keyword(s):

Wastewater Treatment ◽

Activated Sludge ◽

Wastewater Treatment Plants ◽

Sewage Treatment Plant ◽

Degradation Pathway ◽

Bacterial Degradation ◽

Ring Cleavage ◽

Main Process ◽

Content Type ◽

Degrading Bacteria

ABSTRACTThe environmental release and fate of estrogens are becoming an increasing public concern. Bacterial degradation has been considered the main process for eliminating estrogens from wastewater treatment plants. Various bacterial isolates are reportedly capable of aerobic estrogen degradation, and several estrogen degradation pathways have been proposed in proteobacteria and actinobacteria. However, the ecophysiological relevance of estrogen-degrading bacteria in the environment is unclear. In this study, we investigated the estrogen degradation pathway and corresponding degraders in activated sludge collected from the Dihua Sewage Treatment Plant, Taipei, Taiwan. Cultivation-dependent and cultivation-independent methods were used to assess estrogen biodegradation in the collected activated sludge. Estrogen metabolite profile analysis revealed the production of pyridinestrone acid and two A/B-ring cleavage products in activated sludge incubated with estrone (1 mM), which are characteristic of the 4,5-secopathway. PCR-based functional assays detected sequences closely related to alphaproteobacterialoecC, a key gene of the 4,5-secopathway. Metagenomic analysis suggested thatNovosphingobiumspp. are major estrogen degraders in estrone-amended activated sludge.Novosphingobiumsp. strain SLCC, an estrone-degrading alphaproteobacterium, was isolated from the examined activated sludge. The general physiology and metabolism of this strain were characterized. Pyridinestrone acid and the A/B-ring cleavage products were detected in estrone-grown strain SLCC cultures. The production of pyridinestrone acid was also observed during the aerobic incubation of strain SLCC with 3.7 nM (1 μg/liter) estrone. This concentration is close to that detected in many natural and engineered aquatic ecosystems. The presented data suggest the ecophysiological relevance ofNovosphingobiumspp. in activated sludge.IMPORTANCEEstrogens, which persistently contaminate surface water worldwide, have been classified as endocrine disruptors and human carcinogens. We contribute new knowledge on the major estrogen biodegradation pathway and estrogen degraders in wastewater treatment plants. This study considerably advances the understanding of environmental estrogen biodegradation, which is instrumental for the efficient elimination of these hazardous pollutants. Moreover, this study substantially improves the understanding of microbial estrogen degradation in the environment.

Download Full-text

The Role of the Reactive Species Involved in the Photocatalytic Degradation of HDPE Microplastics Using C,N-TiO2 Powders

Polymers ◽

10.3390/polym13070999 ◽

2021 ◽

Vol 13 (7) ◽

pp. 999 ◽

Cited By ~ 1

Author(s):

Aranza Denisse Vital-Grappin ◽

Maria Camila Ariza-Tarazona ◽

Valeria Montserrat Luna-Hernández ◽

Juan Francisco Villarreal-Chiu ◽

Juan Manuel Hernández-López ◽

...

Keyword(s):

Wastewater Treatment Plants ◽

Reaction System ◽

Terrestrial Ecosystems ◽

Degradation Process ◽

Reactive Species ◽

Hazardous Substances ◽

Wide Range ◽

Degradation Behaviors ◽

Environmentally Friendly Process

Microplastics (MPs) are distributed in a wide range of aquatic and terrestrial ecosystems throughout the planet. They are known to adsorb hazardous substances and can transfer them across the trophic web. To eliminate MPs pollution in an environmentally friendly process, we propose using a photocatalytic process that can easily be implemented in wastewater treatment plants (WWTPs). As photocatalysis involves the formation of reactive species such as holes (h+), electrons (e−), hydroxyl (OH●), and superoxide ion (O2●−) radicals, it is imperative to determine the role of those species in the degradation process to design an effective photocatalytic system. However, for MPs, this information is limited in the literature. Therefore, we present such reactive species’ role in the degradation of high-density polyethylene (HDPE) MPs using C,N-TiO2. Tert-butanol, isopropyl alcohol (IPA), Tiron, and Cu(NO3)2 were confirmed as adequate OH●, h+, O2●− and e− scavengers. These results revealed for the first time that the formation of free OH● through the pathways involving the photogenerated e− plays an essential role in the MPs’ degradation. Furthermore, the degradation behaviors observed when h+ and O2●− were removed from the reaction system suggest that these species can also perform the initiating step of degradation.

Download Full-text

Chitin Heterodisaccharide, Released from Chitin by Chitinase and Chitin Oligosaccharide Deacetylase, Enhances the Chitin-Metabolizing Ability of Vibrio parahaemolyticus

Journal of Bacteriology ◽

10.1128/jb.00270-19 ◽

2019 ◽

Vol 201 (20) ◽

Cited By ~ 1

Author(s):

Takako Hirano ◽

Manabu Okubo ◽

Hironobu Tsuda ◽

Masahiro Yokoyama ◽

Wataru Hakamata ◽

...

Keyword(s):

Vibrio Parahaemolyticus ◽

Pcr Analysis ◽

Content Type ◽

Chitin Degradation ◽

Expression Of Genes ◽

Peptide Mass ◽

Degrading Bacteria ◽

Genes Encoding ◽

Chitinase Production

ABSTRACT Vibrio parahaemolyticus RIMD2210633 secretes both chitinase and chitin oligosaccharide deacetylase and produces β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) from chitin. Previously, we reported that GlcNAc-GlcN induces chitinase production by several strains of Vibrio harboring chitin oligosaccharide deacetylase genes (T. Hirano, K. Kadokura, T. Ikegami, Y. Shigeta, et al., Glycobiology 19:1046–1053, 2009). The metabolism of chitin by Vibrio was speculated on the basis of the findings of previous studies, and the role of chitin oligosaccharide produced from chitin has been well studied. However, the role of GlcNAc-GlcN in the Vibrio chitin degradation system, with the exception of the above-mentioned function as an inducer of chitinase production, remains unclear. N,N′-Diacetylchitobiose, a homodisaccharide produced from chitin, is known to induce the expression of genes encoding several proteins involved in chitin metabolism in Vibrio strains (K. L. Meibom, X. B. Li, A. Nielsen, C. Wu, et al., Proc Natl Acad Sci U S A 101:2524–2529, 2004). We therefore hypothesized that GlcNAc-GlcN also affects the expression of enzymes involved in chitin metabolism in the same manner. In this study, we examined the induction of protein expression by several sugars released from chitin using peptide mass fingerprinting and confirmed the expression of genes encoding enzymes involved in chitin metabolism using real-time quantitative PCR analysis. We then confirmed that GlcNAc-GlcN induces the expression of genes encoding many soluble enzymes involved in chitin degradation in Vibrio parahaemolyticus. Here, we demonstrate that GlcNAc-GlcN enhances the chitin-metabolizing ability of V. parahaemolyticus. IMPORTANCE We demonstrate that β-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) enhances the chitin-metabolizing ability of V. parahaemolyticus. Members of the genus Vibrio are chitin-degrading bacteria, and some species of this genus are associated with diseases affecting fish and animals, including humans (F. L. Thompson, T. Iida, and J. Swings, Microbiol Mol Biol Rev 68:403–431, 2004; M. Y. Ina-Salwany, N. Al-Saari, A. Mohamad, F.-A. Mursidi, et al., J Aquat Anim Health 31:3–22, 2019). Studies on Vibrio are considered important, as they may facilitate the development of solutions related to health, food, and aquaculture problems attributed to this genus. This report enhances the current understanding of chitin degradation by Vibrio bacteria.

Download Full-text

Proteogenomic Elucidation of the Initial Steps in the Benzene Degradation Pathway of a Novel Halophile, Arhodomonas sp. Strain Rozel, Isolated from a Hypersaline Environment

Applied and Environmental Microbiology ◽

10.1128/aem.01327-12 ◽

2012 ◽

Vol 78 (20) ◽

pp. 7309-7316 ◽

Cited By ~ 25

Author(s):

Sonal Dalvi ◽

Sei Azetsu ◽

Marianna A. Patrauchan ◽

Deniz F. Aktas ◽

Babu Z. Fathepure

Keyword(s):

Mass Spectrometry ◽

Tricarboxylic Acid Cycle ◽

Draft Genome ◽

Degradation Pathway ◽

Phenol Hydroxylase ◽

Ring Cleavage ◽

Content Type ◽

Benzene Degradation ◽

Chromatography Mass Spectrometry

ABSTRACTLately, there has been a special interest in understanding the role of halophilic and halotolerant organisms for their ability to degrade hydrocarbons. The focus of this study was to investigate the genes and enzymes involved in the initial steps of the benzene degradation pathway in halophiles. The extremely halophilic bacteriaArhodomonassp. strain Seminole andArhodomonassp. strain Rozel, which degrade benzene and toluene as the sole carbon source at high salinity (0.5 to 4 M NaCl), were isolated from enrichments developed from contaminated hypersaline environments. To obtain insights into the physiology of this novel group of organisms, a draft genome sequence of the Seminole strain was obtained. A cluster of 13 genes predicted to be functional in the hydrocarbon degradation pathway was identified from the sequence. Two-dimensional (2D) gel electrophoresis and liquid chromatography-mass spectrometry were used to corroborate the role of the predicted open reading frames (ORFs). ORFs 1080 and 1082 were identified as components of a multicomponent phenol hydroxylase complex, and ORF 1086 was identified as catechol 2,3-dioxygenase (2,3-CAT). Based on this analysis, it was hypothesized that benzene is converted to phenol and then to catechol by phenol hydroxylase components. The resulting catechol undergoes ring cleavage via the meta pathway by 2,3-CAT to form 2-hydroxymuconic semialdehyde, which enters the tricarboxylic acid cycle. To substantiate these findings, the Rozel strain was grown on deuterated benzene, and gas chromatography-mass spectrometry detected deuterated phenol as the initial intermediate of benzene degradation. These studies establish the initial steps of the benzene degradation pathway in halophiles.

Download Full-text

Metagenomes Reveal Global Distribution of Bacterial Steroid Catabolism in Natural, Engineered, and Host Environments

mBio ◽

10.1128/mbio.02345-17 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 26

Author(s):

Johannes Holert ◽

Erick Cardenas ◽

Lee H. Bergstrand ◽

Elena Zaikova ◽

Aria S. Hahn ◽

...

Keyword(s):

Wastewater Treatment ◽

Marine Environment ◽

Wastewater Treatment Plants ◽

Marine Sponges ◽

Model Organisms ◽

Ecological Significance ◽

Data Set ◽

Degrading Bacteria ◽

Globally Distributed ◽

Biochemical Diversity

ABSTRACT Steroids are abundant growth substrates for bacteria in natural, engineered, and host-associated environments. This study analyzed the distribution of the aerobic 9,10-seco steroid degradation pathway in 346 publically available metagenomes from diverse environments. Our results show that steroid-degrading bacteria are globally distributed and prevalent in particular environments, such as wastewater treatment plants, soil, plant rhizospheres, and the marine environment, including marine sponges. Genomic signature-based sequence binning recovered 45 metagenome-assembled genomes containing a majority of 9,10-seco pathway genes. Only Actinobacteria and Proteobacteria were identified as steroid degraders, but we identified several alpha- and gammaproteobacterial lineages not previously known to degrade steroids. Actino- and proteobacterial steroid degraders coexisted in wastewater, while soil and rhizosphere samples contained mostly actinobacterial ones. Actinobacterial steroid degraders were found in deep ocean samples, while mostly alpha- and gammaproteobacterial ones were found in other marine samples, including sponges. Isolation of steroid-degrading bacteria from sponges confirmed their presence. Phylogenetic analysis of key steroid degradation proteins suggested their biochemical novelty in genomes from sponges and other environments. This study shows that the ecological significance as well as taxonomic and biochemical diversity of bacterial steroid degradation has so far been largely underestimated, especially in the marine environment. IMPORTANCE Microbial steroid degradation is a critical process for biomass decomposition in natural environments, for removal of important pollutants during wastewater treatment, and for pathogenesis of bacteria associated with tuberculosis and other bacteria. To date, microbial steroid degradation was mainly studied in a few model organisms, while the ecological significance of steroid degradation remained largely unexplored. This study provides the first analysis of aerobic steroid degradation in diverse natural, engineered, and host-associated environments via bioinformatic analysis of an extensive metagenome data set. We found that steroid-degrading bacteria are globally distributed and prevalent in wastewater treatment plants, soil, plant rhizospheres, and the marine environment, especially in marine sponges. We show that the ecological significance as well as the taxonomic and biochemical diversity of bacterial steroid degradation has been largely underestimated. This study greatly expands our ecological and evolutionary understanding of microbial steroid degradation.

Download Full-text

The role of governance indicators to minimize the carbon emission: a study of Saudi Arabia

Management of Environmental Quality An International Journal ◽

10.1108/meq-11-2020-0275 ◽

2021 ◽

Vol ahead-of-print (ahead-of-print) ◽

Author(s):

Suleman Sarwar ◽

Majid Ibrahim Alsaggaf

Keyword(s):

Saudi Arabia ◽

Carbon Emission ◽

Environmental Behavior ◽

Degradation Process ◽

Content Type ◽

Governance Indicators ◽

Regulatory Quality ◽

Governance Effectiveness ◽

The Impact

PurposeThe main purpose of the study is to examine the impact of governance indicators, to control carbon emission, through the environmental awareness and pro-environmental behavior. Previously, researchers have attempted to explore the factors of carbon emission and report the number of solutions. Despite that, the environmental degradation process is surging.Design/methodology/approachThe study uses quantile regressions (QR) techniques by using the yearly data of Saudi Arabia for the period of 1970–2018. QR reports the results at different quantiles which is useful for conclusion.FindingsThe empirical results have confirmed the significant and negative coefficients of governance indicators, mentioning that governance effectiveness and regulatory quality leads to reduction of carbon emission, in the case of Saudi Arabia.Practical implicationsThe Saudi Government has to improve governance effectiveness to eradicate environmental hazards. However, it is necessary to enhance the quality of regulations regarding formation as well as the implementation of policies to confirm that firms and public follow the pro-environmental behavior.Originality/valueThe study is the pioneer, which addresses the governance parameters, governance effectiveness and regulatory quality to minimize the carbon emission for Saudi Arabia.

Download Full-text

Role of IncP-1β Plasmids pWDL7::rfpand pNB8c in Chloroaniline Catabolism as Determined by Genomic and Functional Analyses

Applied and Environmental Microbiology ◽

10.1128/aem.07480-11 ◽

2011 ◽

Vol 78 (3) ◽

pp. 828-838 ◽

Cited By ~ 33

Author(s):

J. E. Król ◽

J. T. Penrod ◽

H. McCaslin ◽

L. M. Rogers ◽

H. Yano ◽

...

Keyword(s):

Single Copy ◽

Degradation Pathway ◽

Bacterial Degradation ◽

Ring Cleavage ◽

Close Relative ◽

Broad Host Range ◽

Content Type ◽

Aromatic Ring Cleavage ◽

Functional Analyses

ABSTRACTBroad-host-range catabolic plasmids play an important role in bacterial degradation of man-made compounds. To gain insight into the role of these plasmids in chloroaniline degradation, we determined the first complete nucleotide sequences of an IncP-1 chloroaniline degradation plasmid, pWDL7::rfpand its close relative pNB8c, as well as the expression pattern, function, and bioaugmentation potential of the putative 3-chloroaniline (3-CA) oxidation genes. Based on phylogenetic analysis of backbone proteins, both plasmids are members of a distinct clade within the IncP-1β subgroup. The plasmids are almost identical, but whereas pWDL7::rfpcarries a duplicate inverted catabolic transposon, Tn6063, containing a putative 3-CA oxidation gene cluster,dcaQTA1A2BR, pNB8c contains only a single copy of the transposon. No genes for an aromatic ring cleavage pathway were detected on either plasmid, suggesting that only the upper 3-CA degradation pathway was present. ThedcaA1A2Bgene products expressed from a high-copy-number vector were shown to convert 3-CA to 4-chlorocatechol inEscherichia coli. Slight differences in thedcapromoter region between the plasmids and lack of induction of transcription of the pNB8cdcagenes by 3-CA may explain previous findings that pNB8C does not confer 3-CA transformation. Bioaugmentation of activated sludge with pWDL7::rfpaccelerated removal of 3-CA, but only in the presence of an additional carbon source. Successful bioaugmentation requires complementation of the upper pathway genes with chlorocatechol cleavage genes in indigenous bacteria. The genome sequences of these plasmids thus help explain the molecular basis of their catabolic activities.

Download Full-text

Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine and Its Mononitroso Derivative Hexahydro-1-Nitroso-3,5-Dinitro-1,3,5-Triazine by Klebsiella pneumoniae Strain SCZ-1 Isolated from an Anaerobic Sludge

Applied and Environmental Microbiology ◽

10.1128/aem.68.11.5336-5341.2002 ◽

2002 ◽

Vol 68 (11) ◽

pp. 5336-5341 ◽

Cited By ~ 73

Author(s):

Jian-Shen Zhao ◽

Annamaria Halasz ◽

Louise Paquet ◽

Chantale Beaulieu ◽

Jalal Hawari

Keyword(s):

Klebsiella Pneumoniae ◽

Removal Rate ◽

Dry Weight ◽

Degradation Process ◽

Degradation Pathway ◽

Ring Cleavage ◽

Anaerobic Sludge ◽

Initial Reaction ◽

Total N

ABSTRACT In previous work, we found that an anaerobic sludge efficiently degraded hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), but the role of isolates in the degradation process was unknown. Recently, we isolated a facultatively anaerobic bacterium, identified as Klebsiella pneumoniae strain SCZ-1, using MIDI and the 16S rRNA method from this sludge and employed it to degrade RDX. Strain SCZ-1 degraded RDX to formaldehyde (HCHO), methanol (CH3OH) (12% of total C), carbon dioxide (CO2) (72% of total C), and nitrous oxide (N2O) (60% of total N) through intermediary formation of methylenedinitramine (O2NNHCH2NHNO2). Likewise, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) was degraded to HCHO, CH3OH, and N2O (16.5%) with a removal rate (0.39 μmol · h−1 · g [dry weight] of cells−1) similar to that of RDX (0.41 μmol · h−1 · g [dry weight] of cells−1) (biomass, 0.91 g [dry weight] of cells · liter−1). These findings suggested the possible involvement of a common initial reaction, possibly denitration, followed by ring cleavage and decomposition in water. The trace amounts of MNX detected during RDX degradation and the trace amounts of hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine detected during MNX degradation suggested that another minor degradation pathway was also present that reduced —NO2 groups to the corresponding —NO groups.

Download Full-text