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1.
Mervyn L. de Souza Jennifer Seffernick Betsy Martinez Michael J. Sadowsky Lawrence P. Wackett 《Journal of bacteriology》1998,180(7):1951-1954
Pseudomonas strain ADP metabolizes the herbicide atrazine via three enzymatic steps, encoded by the genes atzABC, to yield cyanuric acid, a nitrogen source for many bacteria. Here, we show that five geographically distinct atrazine-degrading bacteria contain genes homologous to atzA, -B, and -C. The sequence identities of the atz genes from different atrazine-degrading bacteria were greater than 99% in all pairwise comparisons. This differs from bacterial genes involved in the catabolism of other chlorinated compounds, for which the average sequence identity in pairwise comparisons of the known members of a class ranged from 25 to 56%. Our results indicate that globally distributed atrazine-catabolic genes are highly conserved in diverse genera of bacteria.Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)- 1,3,5-triazine] is a herbicide used for controlling broad-leaf and grassy weeds and is relatively persistent in soils (51). Atrazine and other s-triazine compounds have been detected in ground and surface waters at levels exceeding the Environmental Protection Agency’s maximum contaminant level of 3 ppb (30).Microbial populations exposed to synthetic chlorinated compounds, such as atrazine, often respond by producing enzymes that degrade these molecules. Most of our current understanding of the genes and enzymes involved in atrazine degradation derives from studies using Pseudomonas strain ADP, in which the first three enzymatic steps in atrazine degradation have been defined (6, 14, 15, 48). The genes atz A, -B, and -C, which encode these enzymes, have been cloned and sequenced. Atrazine chlorohydrolase (AtzA), hydroxyatrazine ethylaminohydrolase (AtzB), and N-isopropylammelide isopropylaminohydrolase (AtzC) sequentially convert atrazine to cyanuric acid (6, 14, 15, 48) (Fig. (Fig.1).1). Cyanuric acid and related compounds are catabolized by many soil bacteria (10, 11, 17, 24, 26, 61), and by Pseudomonas sp. ADP, to carbon dioxide and ammonia (35). This provides the evolutionary pressure for the atzA, -B, and -C genes to permit bacterial growth on the more than one billion pounds of atrazine that have been applied to soils globally (20). Here we used a knowledge of the atzA, -B, and -C gene sequences to investigate the presence of homologous genes in other atrazine-degrading bacteria. In this study, we report that five atrazine-degrading microorganisms, which were recently isolated from geographically separated sites exposed to atrazine, contained nearly identical atzA, -B, and -C genes. Open in a separate windowFIG. 1Pathway for atrazine catabolism to cyanuric acid in Pseudomonas sp. strain ADP.
Open in a separate windowaIsolate identity based on 16S rRNA sequence analysis. bND, not determined.
Open in a separate windowaDNA sequences obtained from each strain by using the ataA, -B, and -C primers were compared with the atzABC gene sequences from Pseudomonas strain ADP. A review of the literature on other bacterial catabolic pathways indicated a much greater degree of divergence when genes encoding enzymes for the catabolism of other commercially relevant chlorinated compounds were compared (Table (Table3).3). As with atrazine, multiple bacterial strains that catabolize 1,2-dichloroethane, chloroacetic acid, 2,4-dichlorophenoxyacetate, dichloromethane, and 4-chlorobenzoate have been isolated. A comparison of the gene sequences encoding the initiating reactions in the catabolism of each of those compounds revealed that sequence divergence was comparatively high. In pairwise comparisons within each gene class, the average sequence identities ranged from 25 to 56% (divergence was 46 to 75%). With the atzABC genes, by contrast, there is at most a 1% sequence difference within the sequenced gene region (Table (Table2).2). Moreover, the atzB sequences were completely identical, and the atzC genes diverged by only 1 bp in one of the five strains tested. This suggests that the atz genes recently arose from a single origin and have become distributed globally. Similarly, identical parathion hydrolase genes were isolated from two bacteria representing different genera and global locations (40, 52, 53).
Open in a separate windowaAll possible pairwise alignments of translated gene sequences were made. The average percent identity is the mean of the percent identity values for all pairwise alignments ± standard error of the mean. bIncludes full protein sequences as well as partial protein sequences of ≥100 amino acids. cSequence identity within a 0.5-kb PCR product for atzA and -B and within a 0.6-kb PCR product for atzC. Six sequences were analyzed for atzA, and five were analyzed for atzB and -C. The data presented here provide further support for previous studies suggesting that hydroxyatrazine in the environment derives from biological processes (36), and not solely from abiotic reactions (2, 9). The present data, and a recent report by Bouquard et al. (8), indicate that the gene encoding atrazine chlorohydrolase is widespread in the United States and Europe.Our observations argue for a single, recent evolutionary origin of the atz genes and their subsequent global distribution. We have recently localized the atzA, -B, and -C genes to a large, self-transmissible plasmid in Pseudomonas strain ADP (12), and possible mechanisms of transfer of the atzABC genes are currently under investigation. 相似文献
Atrazine-catabolizing bacteria used in this study.
Until recently, attempts at isolating bacteria (18) or fungi (27) that completely degrade atrazine to carbon dioxide, ammonia, and chloride were unsuccessful. While several microorganisms were shown to dealkylate atrazine, they were unable to displace the chlorine atom (41, 54). Since 1994, several research groups have independently isolated atrazine-degrading bacteria that displaced the chlorine atom and mineralized atrazine (3, 7, 13, 35, 39, 46). Six of these bacterial cultures, listed in Table Table1,1, were studied here, and the Clavibacter strain had been investigated previously (13).TABLE 1
Recently isolated atrazine-catabolizing bacteriaGenus | Strain | Location where isolated | Yr reported (reference) |
---|---|---|---|
Pseudomonasa | ADP | Agricultural-chemical dealership site, Little Falls, Minn. | 1995 (35) |
Ralstoniaa | M91-3 | Agricultural soil, Ohio | 1995 (46, 55) |
Mixed culture | Basel, Switzerland | 1995 (57) | |
Clavibacter | Agricultural soil, Riverside, Calif. | 1996 (13) | |
Agrobacterium | J14a | Agricultural soil, Nebraska | 1996 (39) |
NDb | 38/38 | Atrazine-contaminated soil, Indiana | 1996 (3) |
Alcaligenesa | SG1 | Industrial settling pond, San Gabriel, La. | 1997 (7) |
Detection of atzA, -B, and -C homologs in atrazine-degrading microorganisms by PCR analysis.
Recently isolated atrazine-degrading bacteria were screened for the presence of DNA homologous to the Pseudomonas strain ADP atzABC genes, which encode enzymes transforming atrazine to cyanuric acid (Fig. (Fig.1).1). Total genomic DNA was isolated from each of these bacteria as described elsewhere (49), and the PCR technique was used to amplify sequences internal to the atzA, -B, and -C genes as described elsewhere (13). Custom primers were designed specifically for atzA (5′CCATGTGAACCAGATCCT3′ and 5′TGAAGCGTCCACATTACC3′), atzB (5′TCACCGGGGATGTCGCGGGC3′ and 5′CTCTCCCGCATGGCATCGGG3′), and atzC (5′GCTCACATGCAGGTACTCCA3′ and 5′GTACCATATCACCGTTTGCCA3′) by using the Primer Designer package, version 2.01 (Scientific and Educational Software, State Line, Pa.), and were synthesized by Gibco BRL (Gaithersburg, Md.). PCR fragments were amplified by using Taq DNA polymerase (Gibco BRL) (22) and were separated from primers on a 1.0% agarose gel. The results of these studies (Fig. (Fig.2)2) indicated that PCR amplification consistently produced DNA fragments of 0.5 kb for all organisms when the atzA or -B primers were used and fragments of 0.6 kb when the atzC primers were used. Open in a separate windowFIG. 2PCR analysis with primers designed to amplify internal regions of atzA (lanes 1 to 5), atzB (lanes 6 to 10), and atzC (lanes 11 to 15). The atrazine-degrading bacteria analyzed were Pseudomonas strain ADP (35) (lanes 1, 6, and 11), Alcaligenes strain SGI (7) (lanes 2, 7, and 12), Ralstonia strain M91-3 (46) (lanes 3, 8, and 13), Agrobacterium strain J14a (39) (lanes 4, 9, and 14), and isolate 38/38 (3) (lanes 5, 10, and 15). Values to the right of the gel are sizes (in kilobase pairs).Southern hybridization analyses were performed on the PCR-amplified DNA as described elsewhere (49) to confirm the presence of homologous DNA. We used a 0.6-kb ApaI/PstI fragment from pMD4 (15), a 1.5-kb BglII fragment from pATZB-2 (6), and a 2.0-kb EcoRI/AvaI fragment from pTD2.5 (48) as probes for atzA, -B, and -C genes, respectively. DNA probes were labeled with [α-32P]dCTP by using the Rediprime Random Primer Labeling Kit (Amersham Life Science, Arlington Heights, Ill.) according to the manufacturer’s instructions. Southern hybridization analyses, performed under stringent conditions, confirmed that each strain contained DNA homologous to atzA, -B, and -C (data not shown). With strain M91-3 and isolate 38/38, however, in addition to the expected 0.5-kb atzB PCR product (Fig. (Fig.2,2, lanes 8 and 10), a 1.2-kb fragment was also obtained. However, no hybridization to this fragment was seen with the atzB probe. Similar investigations showed that a mixed culture obtained from Switzerland (Table (Table1),1), capable of degrading atrazine, also contained DNA homologous to all three atz genes (12).As a negative control, bacteria known not to degrade atrazine were analyzed. PCR analyses were carried out with genomic DNA from the following randomly chosen laboratory strains: Rhodococcus chlorophenolicus (1), Flavobacterium sp. (47), Streptomyces coelicolor M145 (21), Amycolatopsis mediterranei (19), Agrobacterium strain A136 and strain A348 (A136/pTiA6NC) (60), Arthrobacter globiformis MN1 (45), Bradyrhizobium japonicum (33), Rhizobium sp. strain NGR 234 (44), Pseudomonas NRRLB12228, and Klebsiella pneumoniae 99 (16). None of these strains contained DNA that was amplified by PCR using the primers designed to identify the atzA, -B, or -C gene (data not shown).DNA sequences of atzA, -B, and -C homologs in atrazine-degrading microorganisms.
DNAs amplified from the five strains in Table Table11 with the atzA, -B, and -C primers were purified from gel slices by using the GeneClean II System (Bio 101, Inc., Vista, Calif.) and sequenced with a PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit (Perkin-Elmer Corp., Norwalk, Conn.) and an ABI model 373A DNA sequencer (Applied Biosystems, Foster City, Calif.). The GCG sequence analysis software package (Genetics Computer Group, Inc., Madison, Wis.) was used for all DNA and protein sequence comparisons and alignments. Table Table22 summarizes these data. The PCR-amplified genes were ≥99% identical to the Pseudomonas strain ADP atzA, -B, and -C genes in all pairwise comparisons of DNA sequences. This remarkable sequence identity suggested that each atz gene in the different genera was derived from a common ancestor and that they have diverged evolutionarily only to a limited extent.TABLE 2
Sequence identities of atzABC homologs from different atrazine-degrading bacteriaStrain | % DNA sequence identitya
| ||
---|---|---|---|
atzA | atzB | atzC | |
Pseudomonas ADP | 100 | 100 | 100 |
Alcaligenes SG1 | 99.2 | 100 | 100 |
Ralstonia M91-3 | 99.0 | 100 | 100 |
Agrobacterium J14a | 99.1 | 100 | 100 |
Isolate 38/38 | 99.3 | 100 | 99.8 |
TABLE 3
Sequence comparisons of isofunctional bacterial enzymes that catabolize chlorinated compoundsGene | Enzyme | Average % protein sequence identitya (no. of pairwise comparisons) | References |
---|---|---|---|
dhlA, dhaA | Haloalkane dehalogenase | 25.0 (1) | 23, 31 |
dehC, hadL, dehH, dehH1, dehH2, dhlB, dehCI, dehCII | 2-Haloacid dehalogenase | 36.6 ± 3.9 (36) | 5, 25, 28, 29, 42, 43, 50, 59 |
tfdA | 2,4-Dichlorophenoxyacetate monooxygenase | 43.2 ± 4.6 (21)b | 34, 37, 38, 56, 58 |
dcmA | Dichloromethane dehalogenase | 56.0 (1) | 4, 32 |
atzA | Atrazine chlorohydrolase | 98.6 ± 0.12 (15)c | This study |
atzB | Hydroxyatrazine ethylaminohydrolase | 100 (10)c | This study |
atzC | N-Isopropylammelide isopropylaminohydrolase | 99.0 ± 0.43 (10)c | This study |
2.
Vicente García-Gonzlez Fernando Govantes Liz J. Shaw Richard G. Burns Eduardo Santero 《Applied microbiology》2003,69(12):6987-6993
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. 相似文献
3.
4.
5.
In laboratory settings, the ability of bacteria and fungi to degrade many environmental contaminants is well proven. However, the potential of microbial inoculants in soil remediation has not often been realized because catabolically competent strains rarely survive and proliferate in soil, and even if they do, they usually fail to express their desired catabolic potential. One method to address the survival problem is formulating the microorganisms with physical and chemical support systems. This study investigates the survival of Pseudomonas sp. strain ADP in sterile soil and its retention of atrazine-degrading functionality. Assessment was conducted with free and zeolite-immobilized bacteria incorporated into the soil. Pseudomonas sp. strain ADP remained viable for at least 10 weeks when stored at 15°C in sterile soil. Cell numbers increased for both free and zeolite-immobilized bacteria during this period, except for free cells when grown in Miller's Luria-Bertani medium, which exhibited constant cell numbers over the 10 weeks. Only the zeolite-immobilized cell retained full functionality to degrade atrazine after 10 weeks in sterile soil regardless of the medium used to culture Pseudomonas sp. strain ADP. Functionality was diminished in free-cell inoculations except when using an improved culture medium. Survival of zeolite-immobilized Pseudomonas sp. strain ADP separated from the soil matrix after 10 weeks’ incubation was significantly (p < .05) greater than in soil inoculated with free cells or in the soil fraction inoculated by release from zeolite-immobilized Pseudomonas sp. strain ADP. 相似文献
6.
Seffernick JL McTavish H Osborne JP de Souza ML Sadowsky MJ Wackett LP 《Biochemistry》2002,41(48):14430-14437
Atrazine chlorohydrolase (AtzA) from Pseudomonas sp. ADP initiates the metabolism of the herbicide atrazine by catalyzing a hydrolytic dechlorination reaction to produce hydroxyatrazine. Sequence analysis revealed AtzA to be homologous to metalloenzymes within the amidohydrolase protein superfamily. AtzA activity was experimentally shown to depend on an enzyme-bound, divalent transition-metal ion. Loss of activity obtained by incubating AtzA with the chelator 1,10-phenanthroline or oxalic acid was reversible upon addition of Fe(II), Mn(II), or Co(II) salts. Experimental evidence suggests a 1:1 metal to subunit stoichiometry, with the native metal being Fe(II). Our data show that the inhibitory effects of metals such as Zn(II) and Cu(II) are not the result of displacing the active site metal. Taken together, these data indicate that AtzA is a functional metalloenzyme, making this the first report, to our knowledge, of a metal-dependent dechlorinating enzyme that proceeds via a hydrolytic mechanism. 相似文献
7.
Genes for Chlorogenate and Hydroxycinnamate Catabolism (hca) Are Linked to Functionally Related Genes in the dca-pca-qui-pob-hca Chromosomal Cluster of Acinetobacter sp. Strain ADP1
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Michael A. Smith Valerie B. Weaver David M. Young L. Nicholas Ornston 《Applied microbiology》2003,69(1):524-532
8.
N Shapir R T Mandelbaum H Gottlieb 《Journal of industrial microbiology & biotechnology》1998,20(3-4):153-159
Wastewater from atrazine manufacturing plants contains large amounts of residual atrazine and atrazine synthesis products,
which must be removed before disposal. One of the obstacles to biological treatment of these wastewaters is their high salt
content, eg, up to 4% NaCl (w/v). To enable biological treatment, bacteria capable of atrazine mineralization must be adapted
to high-salinity conditions. A recently isolated atrazine-degrading bacterium, Pseudomonas sp strain ADP, originally isolated from contaminated soils was adapted to biodegradation of atrazine at salt concentrations
relevant to atrazine manufacturing wastewater. The adaptation mechanism was based on the ability of the bacterium to produce
trehalose as its main osmolyte. Trehalose accumulation was confirmed by natural-abundance 1H NMR spectral analysis. The bacterium synthesized trehalose de novo in the cells, but could not utilize trehalose added to the growth medium. Interestingly, the bacterium could not produce
glycine betaine (a common compatible solute), but addition of 1 mM of glycine betaine to the medium induced salt tolerance.
Osmoregulated Pseudomonas sp strain ADP, feeding on citrate decreased the concentration of atrazine in non-sterile authentic wastewater from 25 ppm
to below 1 ppm in less than 2 days. The results of our study suggest that salt-adapted Pseudomonas sp strain ADP can be used for atrazine degradation in salt-containing wastewater.
Received 26 August 1997/ Accepted in revised form 06 December 1997 相似文献
9.
Simultaneous Degradation of Atrazine and Phenol by Pseudomonas sp. Strain ADP: Effects of Toxicity and Adaptation
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Grit Neumann Riho Teras Liis Monson Maia Kivisaar Frieder Schauer Hermann J. Heipieper 《Applied microbiology》2004,70(4):1907-1912
The strain Pseudomonas sp. strain ADP is able to degrade atrazine as a sole nitrogen source and therefore needs a single source for both carbon and energy for growth. In addition to the typical C source for Pseudomonas, Na2-succinate, the strain can also grow with phenol as a carbon source. Phenol is oxidized to catechol by a multicomponent phenol hydroxylase. Catechol is degraded via the ortho pathway using catechol 1,2-dioxygenase. It was possible to stimulate the strain in order to degrade very high concentrations of phenol (1,000 mg/liter) and atrazine (150 mg/liter) simultaneously. With cyanuric acid, the major intermediate of atrazine degradation, as an N source, both the growth rate and the phenol degradation rate were similar to those measured with ammonia as an N source. With atrazine as an N source, the growth rate and the phenol degradation rate were reduced to ~35% of those obtained for cyanuric acid. This presents clear evidence that although the first three enzymes of the atrazine degradation pathway are constitutively present, either these enzymes or the uptake of atrazine is the bottleneck that diminishes the growth rate of Pseudomonas sp. strain ADP with atrazine as an N source. Whereas atrazine and cyanuric acid showed no significant toxic effect on the cells, phenol reduces growth and activates or induces typical membrane-adaptive responses known for the genus Pseudomonas. Therefore Pseudomonas sp. strain ADP is an ideal bacterium for the investigation of the regulatory interactions among several catabolic genes and stress response mechanisms during the simultaneous degradation of toxic phenolic compounds and a xenobiotic N source such as atrazine. 相似文献
10.
11.
Identification and Sequencing of β-Myrcene Catabolism Genes from Pseudomonas sp. Strain M1
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Sandra Iurescia Andrea M. Marconi Daniela Tofani Augusto Gambacorta Annalisa Patern Chiara Devirgiliis Mariët J. van der Werf Elisabetta Zennaro 《Applied microbiology》1999,65(7):2871-2876
The M1 strain, able to grow on β-myrcene as the sole carbon and energy source, was isolated by an enrichment culture and identified as a Pseudomonas sp. One β-myrcene-negative mutant, called N22, obtained by transposon mutagenesis, accumulated (E)-2-methyl-6-methylen-2,7-octadien-1-ol (or myrcen-8-ol) as a unique β-myrcene biotransformation product. This compound was identified by gas chromatography-mass spectrometry. We cloned and sequenced the DNA regions flanking the transposon and used these fragments to identify the M1 genomic library clones containing the wild-type copy of the interrupted gene. One of the selected cosmids, containing a 22-kb genomic insert, was able to complement the N22 mutant for growth on β-myrcene. A 5,370-bp-long sequence spanning the region interrupted by the transposon in the mutant was determined. We identified four open reading frames, named myrA, myrB, myrC, and myrD, which can potentially code for an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-coenzyme A (CoA) synthetase, and an enoyl-CoA hydratase, respectively. myrA, myrB, and myrC are likely organized in an operon, since they are separated by only 19 and 36 nucleotides (nt), respectively, and no promoter-like sequences have been found in these regions. The myrD gene starts 224 nt upstream of myrA and is divergently transcribed. The myrB sequence was found to be completely identical to the one flanking the transposon in the mutant. Therefore, we could ascertain that the transposon had been inserted inside the myrB gene, in complete agreement with the accumulation of (E)-2-methyl-6-methylen-2,7-octadien-1-ol by the mutant. Based on sequence and biotransformation data, we propose a pathway for β-myrcene catabolism in Pseudomonas sp. strain M1. 相似文献
12.
13.
ntn Genes Determining the Early Steps in the Divergent Catabolism of 4-Nitrotoluene and Toluene in Pseudomonas sp. Strain TW3 总被引:2,自引:0,他引:2
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Pseudomonas sp. strain TW3 is able to oxidatively metabolize 4-nitrotoluene and toluene via a route analogous to the upper pathway of the TOL plasmids. We report the sequence and organization of five genes, ntnWCMAB*, which are very similar to and in the same order as the xyl operon of TOL plasmid pWW0 and present evidence that they encode enzymes which are expressed during growth on both 4-nitrotoluene and toluene and are responsible for their oxidation to 4-nitrobenzoate and benzoate, respectively. These genes encode an alcohol dehydrogenase homolog (ntnW), an NAD+-linked benzaldehyde dehydrogenase (ntnC), a two-gene toluene monooxygenase (ntnMA), and part of a benzyl alcohol dehydrogenase (ntnB*), which have 84 to 99% identity at the nucleotide and amino acid levels with the corresponding xylWCMAB genes. The xylB homolog on the TW3 genome (ntnB*) appears to be a pseudogene and is interrupted by a piece of DNA which destroys its functional open reading frame, implicating an additional and as-yet-unidentified benzyl alcohol dehydrogenase gene in this pathway. This conforms with the observation that the benzyl alcohol dehydrogenase expressed during growth on 4-nitrotoluene and toluene differs significantly from the XylB protein, requiring assay via dye-linked electron transfer rather than through a nicotinamide cofactor. The further catabolism of 4-nitrobenzoate and benzoate diverges in that the former enters the hydroxylaminobenzoate pathway as previously reported, while the latter is further metabolized via the β-ketoadipate pathway. 相似文献
14.
Biochemical and Genetic Analyses of Ferulic Acid Catabolism in Pseudomonas sp. Strain HR199 总被引:2,自引:0,他引:2
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The gene loci fcs, encoding feruloyl coenzyme A (feruloyl-CoA) synthetase, ech, encoding enoyl-CoA hydratase/aldolase, and aat, encoding β-ketothiolase, which are involved in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199 (DSM7063), were localized on a DNA region covered by two EcoRI fragments (E230 and E94), which were recently cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The nucleotide sequences of parts of fragments E230 and E94 were determined, revealing the arrangement of the aforementioned genes. To confirm the function of the structural genes fcs and ech, they were cloned and expressed in Escherichia coli. Recombinant strains harboring both genes were able to transform ferulic acid to vanillin. The feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities of the fcs and ech gene products, respectively, were confirmed by photometric assays and by high-pressure liquid chromatography analysis. To prove the essential involvement of the fcs, ech, and aat genes in the catabolism of ferulic acid and eugenol in Pseudomonas sp. strain HR199, these genes were inactivated separately by the insertion of omega elements. The corresponding mutants Pseudomonas sp. strain HRfcsΩGm and Pseudomonas sp. strain HRechΩKm were not able to grow on ferulic acid or on eugenol, whereas the mutant Pseudomonas sp. strain HRaatΩKm exhibited a ferulic acid- and eugenol-positive phenotype like the wild type. In conclusion, the degradation pathway of eugenol via ferulic acid and the necessity of the activation of ferulic acid to the corresponding CoA ester was confirmed. The aat gene product was shown not to be involved in this catabolism, thus excluding a β-oxidation analogous degradation pathway for ferulic acid. Moreover, the function of the ech gene product as an enoyl-CoA hydratase/aldolase suggests that ferulic acid degradation in Pseudomonas sp. strain HR199 proceeds via a similar pathway to that recently described for Pseudomonas fluorescens AN103. 相似文献
15.
Asymbiotic Acetylene Reduction by a Fast-Growing Cowpea Rhizobium Strain with Nitrogenase Structural Genes Located on a Symbiotic Plasmid 总被引:3,自引:1,他引:2
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A procedure was designed which enabled the detection of ex planta nitrogenase activity in the fast-growing cowpea Rhizobium strain IHP100. Nitrogenase activity in agar culture under air occurred at a rate similar to that found for Bradyrhizobium strain CB756 but lower than that for Rhizobium strain ORS571. Hybridization studies showed that both nod and nif genes were located on a 410-kilobase Sym plasmid in strain IHP100. 相似文献
16.
Hydroxycinnamate (hca) Catabolic Genes from Acinetobacter sp. Strain ADP1 Are Repressed by HcaR and Are Induced by Hydroxycinnamoyl-Coenzyme A Thioesters
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Hydroxycinnamates are plant products catabolized through the diphenol protocatechuate in the naturally transformable bacterium Acinetobacter sp. strain ADP1. Genes for protocatechuate catabolism are central to the dca-pca-qui-pob-hca chromosomal island, for which gene designations corresponding to catabolic function are dca (dicarboxylic acid), pca (protocatechuate), qui (quinate), pob (p-hydroxybenzoate), and hca (hydroxycinnamate). Acinetobacter hcaC had been cloned and shown to encode a hydroxycinnamate:coenzyme A (CoA) SH ligase that acts upon caffeate, p-coumarate, and ferulate, but genes for conversion of hydroxycinnamoyl-CoA to protocatechuate had not been characterized. In this investigation, DNA from pobS to an XbaI site 5.3 kb beyond hcaC was captured in the plasmid pZR8200 by a strategy that involved in vivo integration of a cloning vector near the hca region of the chromosome. pZR8200 enabled Escherichia coli to convert p-coumarate to protocatechuate in vivo. Sequence analysis of the newly cloned DNA identified five open reading frames designated hcaA, hcaB, hcaK, hcaR, and ORF1. An Acinetobacter strain with a knockout of HcaA, a homolog of hydroxycinnamoyl-CoA hydratase/lyases, was unable to grow at the expense of hydroxycinnamates, whereas a strain mutated in HcaB, homologous to aldehyde dehydrogenases, grew poorly with ferulate and caffeate but well with p-coumarate. A chromosomal fusion of lacZ to the hcaE gene was used to monitor expression of the hcaABCDE promoter. LacZ was induced over 100-fold by growth in the presence of caffeate, p-coumarate, or ferulate. The protein deduced to be encoded by hcaR shares 28% identity with the aligned E. coli repressor, MarR. A knockout of hcaR produced a constitutive phenotype, as assessed in the hcaE::lacZ-Kmr genetic background, revealing HcaR to be a repressor as well. Expression of hcaE::lacZ in strains with knockouts in hcaA, hcaB, or hcaC revealed unambiguously that hydroxycinnamoyl-CoA thioesters relieve repression of the hcaABCDE genes by HcaR. 相似文献
17.
18.
Genes Involved in Cyclic Lipopeptide Production Are Important for Seed and Straw Colonization by Pseudomonas sp. Strain DSS73 总被引:1,自引:0,他引:1
Tommy H. Nielsen Ole Nybroe Birgit Koch Michael Hansen Jan S?rensen 《Applied microbiology》2005,71(7):4112-4116
Survival in natural bulk soil and colonization of sugar beet seeds and barley straw residues were determined for Pseudomonas sp. strain DSS73 and Tn5 mutants in amsY (encoding a peptide synthetase involved in production of the cyclic lipopeptide amphisin) and gacS (encoding the sensory kinase of the two-component GacA/GacS regulatory system). No differences in survival or growth in response to carbon amendment (citrate) were observed in bulk soil. However, both mutants were impaired in their colonization of sugar beet seeds and barley straw residues by an inoculum established in the bulk soil. The two mutants had comparable colonization phenotypes, suggesting that amphisin production is more important for colonization than other gacS-controlled traits. 相似文献
19.
Atrazine chlorohydrolase from Pseudomonas sp. strain ADP: gene sequence, enzyme purification, and protein characterization. 总被引:13,自引:1,他引:12
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Pseudomonas sp. strain ADP metabolizes atrazine to carbon dioxide and ammonia via the intermediate hydroxyatrazine. The genetic potential to produce hydroxyatrazine was previously attributed to a 1.9-kb AvaI DNA fragment from strain ADP (M. L. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373-3378, 1995). In this study, sequence analysis of the 1.9-kb AvaI fragment indicated that a single open reading frame, atzA, encoded an activity transforming atrazine to hydroxyatrazine. The open reading frame for the chlorohydrolase was determined by sequencing to be 1,419 nucleotides and encodes a 473-amino-acid protein with a predicted subunit molecular weight of 52,421. The deduced amino acid sequence matched the first 10 amino acids determined by protein microsequencing. The protein AtzA was purified to homogeneity by ammonium sulfate precipitation and anion-exchange chromatography. The subunit and holoenzyme molecular weights were 60,000 and 245,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively. The purified enzyme in H2(18)O yielded [18O]hydroxyatrazine, indicating that AtzA is a chlorohydrolase and not an oxygenase. The most related protein sequence in GenBank was that of TrzA, 41% identity, from Rhodococcus corallinus NRRL B-15444R. TrzA catalyzes the deamination of melamine and the dechlorination of deethylatrazine and desisopropylatrazine but is not active with atrazine. AtzA catalyzes the dechlorination of atrazine, simazine, and desethylatrazine but is not active with melamine, terbutylazine, or desethyldesisopropylatrazine. Our results indicate that AtzA is a novel atrazine-dechlorinating enzyme with fairly restricted substrate specificity and contributes to the microbial hydrolysis of atrazine to hydroxyatrazine in soils and groundwater. 相似文献
20.
Arthrobacter aurescens TC1 Atrazine Catabolism Genes trzN, atzB, and atzC Are Linked on a 160-Kilobase Region and Are Functional in Escherichia coli 总被引:1,自引:0,他引:1
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Kannika Sajjaphan Nir Shapir Lawrence P. Wackett Michael Palmer Barbara Blackmon Jeff Tomkins Michael J. Sadowsky 《Applied microbiology》2004,70(7):4402-4407
Arthrobacter aurescens strain TC1 metabolizes atrazine to cyanuric acid via TrzN, AtzB, and AtzC. The complete sequence of a 160-kb bacterial artificial chromosome clone indicated that trzN, atzB, and atzC are linked on the A. aurescens genome. TrzN, AtzB, and AtzC were shown to be functional in Escherichia coli. Hybridization studies localized trzN, atzB, and atzC to a 380-kb plasmid in A. aurescens strain TC1. 相似文献