Assembly of an atrazine catabolic operon and its introduction to Gram-negative hosts for robust and stable degradation of triazine herbicides

ABSTRACT In 1995, Pseudomonas sp. ADP, capable of metabolizing atrazine, was isolated from contaminated soil. Genes responsible for atrazine mineralization were found scattered in the 108.8 kb pADP-1 plasmid carried by this strain, some of them flanked by insertion sequences rendering them unstable. The goal of this work was to construct a transcriptional unit containing the atz operon in an easy to transfer manner, to be introduced and inherited stably by Gram-negative bacteria. atz genes were PCR amplified, joined into an operon and inserted onto the mobilizable plasmid pBAMD1–2. Primers were designed to add efficient transcription and translation signals. Plasmid bearing the atz operon was transferred to different Gram-negative strains by conjugation, which resulted in Tn5 transposase-mediated chromosomal insertion of the atz operon. To test the operon activity, atrazine degradation by transposants was assessed both colorimetrically and by high-performance liquid chromatography (HPLC). Transposants mineralized atrazine more efficiently than wild-type Pseudomonas sp. ADP and did not accumulate cyanuric acid. Atrazine degradation was not repressed by simple nitrogen sources. Genes conferring atrazine-mineralizing capacities were stable and had little or null effect on the fitness of different transposants. Introduction of catabolic operons in a stable fashion could be used to develop bacteria with better degrading capabilities useful in bioremediation.

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Gene Sequence and Properties of ans-Triazine Ring-Cleavage Enzyme from Pseudomonassp. Strain NRRLB-12227

Applied and Environmental Microbiology ◽

10.1128/aem.65.8.3512-3517.1999 ◽

1999 ◽

Vol 65 (8) ◽

pp. 3512-3517 ◽

Cited By ~ 61

Author(s):

Jeffrey S. Karns

Keyword(s):

High Performance ◽

Cyanuric Acid ◽

Nitrogen Sources ◽

Complete Removal ◽

Competitive Inhibitor ◽

Ring Structure ◽

Ring Cleavage ◽

Triazine Ring ◽

Cloned Gene ◽

Acid Amidohydrolase

ABSTRACT Pesticides based on the s-triazine ring structure are widely used in cultivation of food crops. Cleavage of thes-triazine ring is an important step in the mineralization of s-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-s-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO2 and ammonia. The trzD gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 fromPseudomonas sp. strain NRRLB-12227, a strain that is capable of utilizing s-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts ofEscherichia coli containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparentKm of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other s-triazine or hydroxypyrimidine compound, although barbituric acid (2,4,6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of trzD nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein.

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The atzABC Genes Encoding Atrazine Catabolism Are Located on a Self-Transmissible Plasmid in Pseudomonas sp. Strain ADP

Applied and Environmental Microbiology ◽

10.1128/aem.64.6.2323-2326.1998 ◽

1998 ◽

Vol 64 (6) ◽

pp. 2323-2326 ◽

Cited By ~ 112

Author(s):

Mervyn L. de Souza ◽

Lawrence P. Wackett ◽

Michael J. Sadowsky

Keyword(s):

Escherichia Coli ◽

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

Soil Bacteria ◽

High Performance ◽

Southern Hybridization ◽

Cyanuric Acid ◽

Pseudomonas Sp ◽

Transmissible Plasmid ◽

Genes Encoding

ABSTRACT Pseudomonas sp. strain ADP initiates atrazine catabolism via three enzymatic steps, encoded by atzA, -B, and -C, which yield cyanuric acid, a nitrogen source for many bacteria. In-well lysis, Southern hybridization, and plasmid transfer studies indicated that theatzA, -B, and -C genes are localized on a 96-kb self-transmissible plasmid, pADP-1, inPseudomonas sp. strain ADP. High-performance liquid chromatography analyses showed that cyanuric acid degradation was not encoded by pADP-1. pADP-1 was transferred to Escherichia coli strains at a frequency of 4.7 × 10−2. This suggests a potential molecular mechanism for the dispersion of theatzABC genes to other soil bacteria.

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Biological granulated activated carbon fluidized bed reactor for atrazine remediation

Water Science & Technology ◽

10.2166/wst.2004.0845 ◽

2004 ◽

Vol 49 (11-12) ◽

pp. 215-222 ◽

Cited By ~ 11

Author(s):

M. Herzberg ◽

C.G. Dosoretz ◽

S. Tarre ◽

M. Beliavski ◽

M. Green

Keyword(s):

Fluidized Bed ◽

Fluidized Bed Reactor ◽

Batch Adsorption ◽

Pseudomonas Sp ◽

Partial Penetration ◽

Biofilm Carrier ◽

Degradation Rates ◽

Degrading Bacteria ◽

Atrazine Degradation ◽

Granulated Activated Carbon

To show that an adsorbing biofilm carrier (GAC) can be advantageous for atrazine bioremediation over a non-adsorbing carrier, fluidized bed (FB) reactors were operated under atrazine limiting concentrations using Pseudomonas sp. strain ADP as the atrazine degrading bacteria. The following interrelated subjects were investigated: 1) atrazine adsorption to GAC under conditions of atrazine partial penetration in the biofilm, 2) differences in atrazine degradation rates and 3) stability of atrazine biodegradation under non-sterile anoxic conditions in the GAC reactor versus a reactor with a non-adsorbing biofilm carrier. Results from batch adsorption tests together with modeling best described the biofilm as patchy in nature with covered and non-biofilm covered areas. Under conditions of atrazine partial penetration in the biofilm, atrazine adsorption occurs in the non-covered areas and is consequently desorbed at the base of the biofilm substantially increasing the active biofilm surface area. The double flux of atrazine to the biofilm in the GAC reactor results in lower effluent atrazine concentrations as compared to a FB reactor with a non-adsorbing carrier. Moreover, under non-sterile denitrification conditions, atrazine degradation stability was found to be much higher (several months) using GAC as a biofilm carrier while non-adsorbing carrier reactors showed sharp deterioration within 30 days due to contamination of non-atrazine degrading bacteria.

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Heavy Vacuum Gas Oil Upregulates the Rhamnosyltransferases and Quorum Sensing Cascades of Rhamnolipids Biosynthesis in Pseudomonas sp. AK6U

Molecules ◽

10.3390/molecules26144122 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4122

Author(s):

Sarah A. Alkhalaf ◽

Ahmed R. Ramadan ◽

Christian Obuekwe ◽

Ashraf M. El Nayal ◽

Nasser Abotalib ◽

...

Keyword(s):

Quorum Sensing ◽

High Performance ◽

Lower Surface ◽

Comparative Approach ◽

Vacuum Gas Oil ◽

Pseudomonas Sp ◽

Gas Oil ◽

Expression Of Genes ◽

Regulation Of Biosynthesis ◽

Heavy Vacuum Gas Oil

We followed a comparative approach to investigate how heavy vacuum gas oil (HVGO) affects the expression of genes involved in biosurfactants biosynthesis and the composition of the rhamnolipid congeners in Pseudomonas sp. AK6U. HVGO stimulated biosurfactants production as indicated by the lower surface tension (26 mN/m) and higher yield (7.8 g/L) compared to a glucose culture (49.7 mN/m, 0.305 g/L). Quantitative real-time PCR showed that the biosurfactants production genes rhlA and rhlB were strongly upregulated in the HVGO culture during the early and late exponential growth phases. To the contrary, the rhamnose biosynthesis genes algC, rmlA and rmlC were downregulated in the HVGO culture. Genes of the quorum sensing systems which regulate biosurfactants biosynthesis exhibited a hierarchical expression profile. The lasI gene was strongly upregulated (20-fold) in the HVGO culture during the early log phase, whereas both rhlI and pqsE were upregulated during the late log phase. Rhamnolipid congener analysis using high-performance liquid chromatography-mass spectrometry revealed a much higher proportion (up to 69%) of the high-molecularweight homologue Rha–Rha–C10–C10 in the HVGO culture. The results shed light on the temporal and carbon source-mediated shifts in rhamonlipids’ composition and regulation of biosynthesis which can be potentially exploited to produce different rhamnolipid formulations tailored for specific applications.

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Water ingestion during swimming activities in a pool: A pilot study

Journal of Water and Health ◽

10.2166/wh.2006.0026 ◽

2006 ◽

Vol 4 (4) ◽

pp. 425-430 ◽

Cited By ~ 142

Author(s):

Alfred P. Dufour ◽

Otis Evans ◽

Thomas D. Behymer ◽

Ricardo Cantú

Keyword(s):

High Performance ◽

Cyanuric Acid ◽

The Body ◽

Swimming Activity ◽

Swimming Pool ◽

Urinary Proteins ◽

Analytical Methodology ◽

Water Ingestion ◽

Pool Water ◽

Swimming Pool Water

Chloroisocyanurates are commonly added to outdoor swimming pools to stabilize chlorine disinfectants. The chloroisocyanurates decompose slowly to release chlorine and cyanuric acid. Studies conducted to determine if the chloroisocyanurates might be toxic to swimmers showed that they were not and that ingested cyanuric acid passed through the body unmetabolized. This fact was used to determine the amount of water swallowed during swimming activity. Fifty-three recreational swimmers, using a community swimming pool disinfected with cyanuric acid stabilized chlorine, participated in the study. The participants did not swim on the day before or after the test swim. The swimmers were asked to actively swim for at least 45 minutes and to collect their urine for the next 24 hours. Cyanuric acid was measured in pool water using high performance liquid chromatography and porous graphitic carbon columns with UV detection. The urine sample assay required a clean-up procedure to remove urinary proteins and interfering substances. Results of the study indicate that non-adults ingest about twice as much water as adults during swimming activity. The average amount of water swallowed by non-adults and adults was 37 ml and 16 ml, respectively. The design for this study and the analytical methodology used to assay cyanuric acid in swimming pool water and human urine were effective for measuring the volume of water swallowed during swimming activity.

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Nitrogen Control of Atrazine Utilization in Pseudomonas sp. Strain ADP

Applied and Environmental Microbiology ◽

10.1128/aem.69.12.6987-6993.2003 ◽

2003 ◽

Vol 69 (12) ◽

pp. 6987-6993 ◽

Cited By ~ 56

Author(s):

Vicente García-González ◽

Fernando Govantes ◽

Liz J. Shaw ◽

Richard G. Burns ◽

Eduardo Santero

Keyword(s):

Wild Type Strain ◽

Nitrogen Sources ◽

Degradation Pathway ◽

Strong Impact ◽

Soil Microcosms ◽

Degradation Rates ◽

Herbicide Atrazine ◽

Mutant Derivative ◽

Nitrogen Repression ◽

Atrazine Degradation

ABSTRACT Pseudomonas sp. strain ADP uses the herbicide atrazine as the sole nitrogen source. We have devised a simple atrazine degradation assay to determine the effect of other nitrogen sources on the atrazine degradation pathway. The atrazine degradation rate was greatly decreased in cells grown on nitrogen sources that support rapid growth of Pseudomonas sp. strain ADP compared to cells cultivated on growth-limiting nitrogen sources. The presence of atrazine in addition to the nitrogen sources did not stimulate degradation. High degradation rates obtained in the presence of ammonium plus the glutamine synthetase inhibitor MSX and also with an Nas− mutant derivative grown on nitrate suggest that nitrogen regulation operates by sensing intracellular levels of some key nitrogen-containing metabolite. Nitrate amendment in soil microcosms resulted in decreased atrazine mineralization by the wild-type strain but not by the Nas− mutant. This suggests that, although nitrogen repression of the atrazine catabolic pathway may have a strong impact on atrazine biodegradation in nitrogen-fertilized soils, the use of selected mutant variants may contribute to overcoming this limitation.

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Identification of a Suitable Solid Carrier as a Matrix for the Permeable Reactive Barrier of Laboratory Scale Model Aquifer for Atrazine Degradation

Tribhuvan University Journal ◽

10.3126/tuj.v25i1.3754 ◽

1970 ◽

Vol 25 (1) ◽

pp. 119-128 ◽

Cited By ~ 1

Author(s):

Binita K Shrestha ◽

Pawan Raj Shakya ◽

MIM Soares

Keyword(s):

Laboratory Scale ◽

Permeable Reactive Barrier ◽

Scale Model ◽

Pseudomonas Sp ◽

Reactive Barrier ◽

Solid Carrier ◽

Different Types ◽

Simultaneous Loss ◽

Static Conditions ◽

Atrazine Degradation

Three different types of physical substrata such as sand, granualar activated carbon (GAC) and plastic macaroni beads were selected to identify a suitable solid carrier as a course matrix for the permeable reactive barrier (PRB) in a sand filled two-dimensional laboratory-scale model aquifer. An adhesion experiment was performed and tested with Pseudomonas sp ADP (PASP) under agitated as well as static conditions. In static conditions, adhesion to GAC was the highest (80%) followed by that to beads (60%). No adhesion to sand was observed under static or agitated conditions. Since atrazine is adsorbed by GAC, its bioavailability for degradation by PADP may be affected. To test this, simultaneous loss of atrazine and release of chloride (dechlorination, the first step in the pathway of atrazine degradation by PADP) were determined in the presence and absence of GAC. Although cells remained viable, no significant chloride release was detected. Thus, plastic macaroni beads were identified and selected as the most suitable support for PADP in the biological permeable reactive barrier (BPRB) of the model aquifer.Key words: Suitable Solid; Laboratory Scale; PADPTribhuvan University JournalVol. XXV, No. 1, 2005Page:119-128Uploaded date: 26, September, 2010

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Complete Genome Sequence of Pseudomonas sp. Strain phDV1, an Isolate Capable of Efficient Degradation of Aromatic Hydrocarbons

Microbiology Resource Announcements ◽

10.1128/mra.01171-18 ◽

2019 ◽

Vol 8 (2) ◽

Author(s):

Hao Xie ◽

Giannis Valsamidis ◽

Eirini Mathioudaki ◽

Georgios Tsiotis

Keyword(s):

Genome Sequence ◽

Aromatic Hydrocarbons ◽

Complete Genome Sequence ◽

Complete Genome ◽

Pseudomonas Sp ◽

Negative Bacterium ◽

Gram Negative ◽

Content Type

Pseudomonas sp. strain phDV1 is a Gram-negative bacterium capable of degrading aromatic hydrocarbons. Here, we present the complete genome sequence of this strain, which consists of 4,727,682 bp, with a 62.3% G+C content and 4,574 genes.

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THE ISOLATION AND CHARACTERIZATION OF METHANOMONAS METHANOOXIDANS BROWN AND STRAWINSKI

Canadian Journal of Microbiology ◽

10.1139/m64-100 ◽

1964 ◽

Vol 10 (5) ◽

pp. 791-799 ◽

Cited By ~ 41

Author(s):

L. R. Brown ◽

R. J. Strawinski ◽

C. S. McCleskey

Keyword(s):

Formic Acid ◽

Inorganic Nitrogen ◽

Nitrogen Sources ◽

Energy Sources ◽

Resting Cells ◽

Gram Negative ◽

Oxidation Of Methane ◽

Isolation And Characterization ◽

Trapping Agent

Procedures for the isolation and characterization of Metkanomonas methanooxidans Brown and Strawinski are described. Isolates from varied sources are alike in cellular morphology, inasmuch as they form only microcolonies, and in their dependence on methane or methanol as carbon and energy sources for growth. Both organic and inorganic nitrogen sources are used. The organism is a Gram negative non-sporeforming rod, 1.5 to 3.0 μ by 1.0 μ in size, and motile by means of a single polar flagellum. In growing cultures the oxygen/methane ratio was approximately 1.1 and in resting cells 1.7. The R.Q. for methane with resting cells was 0.43. Resting cells were unable to oxidize organic compounds other than methane, methanol, formaldehyde, and formate. Formic acid was detected in test solutions after cell suspensions had metabolized methane, methanol, and formaldehyde. Using sodium sulphite as trapping agent for formaldehyde, it was found that 60 to 70% of the methane or methanol consumed was converted to formaldehyde. In the presence of iodoacetate, 70% of the methane consumed was present terminally as methanol. Thus it was shown that methanol, formaldehyde, and formic acid are sequential intermediates in the oxidation of methane by these organisms.

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DUF3380 Domain from a Salmonella Phage Endolysin Shows PotentN-Acetylmuramidase Activity

Applied and Environmental Microbiology ◽

10.1128/aem.00446-16 ◽

2016 ◽

Vol 82 (16) ◽

pp. 4975-4981 ◽

Cited By ~ 22

Author(s):

Lorena Rodríguez-Rubio ◽

Hans Gerstmans ◽

Simon Thorpe ◽

Stéphane Mesnage ◽

Rob Lavigne ◽

...

Keyword(s):

Enzymatic Activity ◽

Thermal Resistance ◽

High Performance ◽

Antimicrobial Agents ◽

Biochemical Characterization ◽

Specific Activity ◽

Three Dimensional ◽

Dimensional Structure ◽

Gram Negative ◽

Content Type

ABSTRACTBacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novelSalmonellabacteriophage endolysin, Gp110, which comprises an uncharacterizeddomain ofunknownfunction (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/μM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 hasN-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond betweenN-acetylmuramic acid andN-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents.IMPORTANCEWe report the functional and biochemical characterization of theSalmonellaphage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 hasN-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

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