Strain CA10 Research Articles

Synergy Between Polyvinylpyrrolidone-Coated Silver Nanoparticles and Azole Antifungal Against Drug-Resistant Candida albicans.

In the clinical practice, resistance of Candida albicans to antifungal agents has frequently emerged. Silver-nanoparticles (Ag-NPs) have been demonstrated to have the antifungal property. We investigated the potential for synergy between polyvinylpyrrolidone (PVP)-coated Ag-NPs and azole antifungal, such as fluconazole or voriconazole, against drug-resistant C. albicans strain CA10. When antifungal agent was examined alone, fluconazole and voriconazole did not kill drug-resistant C. albicans, and PVP-coated Ag-NPs had only the moderate killing ability. In contrast, the combinational treatment of PVP-coated Ag-NPs with fluconazole or voriconazole was effective in being against the drug-resistant C. albicans. After the combinational treatment, we detected the disruption of cell membrane integrity, the tendency of PVP-coated Ag-NPs to adhere to cell membrane, and the inhibition of budding process. Moreover, after the combinational treatment, the defects in ergosterol signaling and efflux pump functions were detected. Our results suggest that the combinational use of engineered nanomaterials (ENMs), such as PVP-coated Ag-NPs, with the conventional antifungal may be a viable strategy to combat drug-resistant fungal infection.

Expression, purification and characterization of meta-cleavage enzyme CarBaBb from Novosphiongobium sp. KA1

The meta-cleavage enzyme CarBaBb of carbazole-degrader Novosphiongobium sp. KA1 were cloned, expressed and purified to homogeneity in Escherichia coli strain. The enzyme was cloned with 6x histidine residues attached at the C-terminal of large subunit CarBb for purification using affinity chromatography method prior to gel filtration chromatography. The CarBaBb, a two-subunit meta-cleavage enzyme, approximately 30 kda for CarBb dan 10 kDa for CarBa, was found to be α2β2-heterotetrameric (Mr 80,000), showed highest activity at pH 8.5 and temperature 30°C. CarBaBb showed highest catalytic activity towards 2,3-dihydroxybiphenyl with kcat/Km 4.1 M-1s-1, and overall higher catalytic activities towards biphenyl-type substrates in comparison to catechol-type substrates. Based on the similarities, this meta-cleavage enzyme from Novosphiongobium sp. KA1 would also be a good candidate for protein crystallization and structural studies apart from CarBaBb from strain P. resinovorans strain CA10.

Erratum: The Effect of Plagiochin E Alone and in Combination with Fluconazole on the Ergosterol Biosynthesis of Candida albicans [Biol. Pharm. Bull. 32(1): 36-40 (2009)

Correction: In the manuscript described above, we have described the effect of plagiochin E alone and in combination with fluconazole on the ergosterol biosynthesis of Candida albicans. Now we found a fatal error occurred during the sterol content calculation according to Fig. 3A. Based on this wrong datum, the calculation of relative sterol composition of C. albicans CA10 has provided wrong results in Table 2. According to the correct data, for plagiochin E on the resistant strain CA10, ergosterol percentage was reduced, but, lanosterol and 14α-methylfecosterol were accumulated. So when FLC-resistant strain was treated by PLE, the accumulation of 14α-methylated sterols (lanosterol and 14α-methylfecosterol) and the down regulation of ERG11 may indicate the other mechanism for resistance reverse.We therefore deleted this publication of Biol. Pharm. Bull.Ling Mei SUNSchool of Pharmaceutical Sciences, Shandong University, ChinaFebruary 3, 2009

Crystal structure of the ferredoxin component of carbazole 1,9a‐dioxygenase of Pseudomonas resinovorans strain CA10, a novel Rieske non‐heme iron oxygenase system

The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 catalyzes the dioxygenation of carbazole; the 9aC carbon bonds to a nitrogen atom and its adjacent 1C carbon as the initial reaction in the mineralization pathway. The CARDO system is composed of ferredoxin reductase (CarAd), ferredoxin (CarAc), and terminal oxygenase (CarAa). CarAc acts as a mediator in the electron transfer from CarAd to CarAa. To understand the structural basis of the protein-protein interactions during electron transport in the CARDO system, the crystal structure of CarAc was determined at 1.9 A resolution by molecular replacement using the structure of BphF, the biphenyl 2,3-dioxygenase ferredoxin from Burkholderia cepacia strain LB400 as a search model. CarAc is composed of three beta-sheets, and the structure can be divided into two domains, a cluster-binding domain and a basal domain. The Rieske [2Fe-2S] cluster is located at the tip of the cluster-binding domain, where it is exposed to solvent. While the overall folding of CarAc and BphF is strongly conserved, the properties of their surfaces are very different from each other. The structure of the cluster-binding domain of CarAc is more compact and protruding than that of BphF, and the distribution of electric charge on its molecular surface is very different. Such differences are thought to explain why these ferredoxins can act as electron mediators in respective electron transport chains composed of different-featured components.

Large plasmid pCAR2 and class II transposon Tn4676 are functional mobile genetic elements to distribute the carbazole/dioxin-degradative car gene cluster in different bacteria

The carbazole-catabolic plasmid pCAR1 isolated from Pseudomonas resinovorans strain CA10 was sequenced in its entirety; and it was found that pCAR1 carries the class II transposon Tn4676 containing carbazole-degradative genes. In this study, a new plasmid designated pCAR2 was isolated from P. putida strain HS01 that was a transconjugant from mating between the carbazole-degrader Pseudomonas sp. strain K23 and P. putida strain DS1. Southern hybridization and nucleotide sequence analysis of pCAR1 and pCAR2 revealed that the whole backbone structure was very similar in each. Plasmid pCAR2 was self-transmissible, because it was transferred from strain HS01 to P. fluorescens strain IAM12022 at the frequency of 2 x 10(-7) per recipient cell. After the serial transfer of strain HS01 on rich medium, we detected the transposition of Tn4676 from pCAR2 to the HS01 chromosome. The chromosome-located copy of Tn4676 was flanked by a 6-bp target duplication, 5'-AACATC-3'. These results experimentally demonstrated the transferability of pCAR2 and the functionality of Tn4676 on pCAR2. It was clearly shown that plasmid pCAR2 and transposon Tn4676 are active mobile genetic elements that can mediate the horizontal transfer of genes for the catabolism of carbazole.

Divergent structures of carbazole degradative car operons isolated from gram-negative bacteria.

Southern hybridization analysis of the genomes from the newly-isolated 10 carbazole (CAR)-utilizing bacteria revealed that 8 of the isolates carried gene clusters homologous to the CAR-catabolic car operon of Pseudomonas resinovorans strain CA10. Sequencing analysis showed that two car operons and the neighboring regions of Pseudomonas sp. strain K23 are nearly identical to that of strain CA10. In contrast to strains CA10 and K23, carEF genes did not exist downstream of the car gene cluster of Janthinobacterium sp. strain J3. In the car gene clusters, strains CA10, K23 and J3 have Rieske-type ferredoxin as a component of carbazole dioxygenase, although Sphingomonas sp. strain KA1 possesses a putidaredoxin-type ferredoxin. We confirmed that this putidaredoxin-type ferredoxin CarAc can function as an electron mediator to CarAa of strain KA1. In the upstream regions of the carJ3 and carKA1 gene clusters, ORFs whose deduced amino acid sequences showed homology to GntR-family transcriptional regulators were identified.

Crystal structure of a histidine-tagged serine hydrolase involved in the carbazole degradation (CarC enzyme)

2-Hydroxy-6-oxo-6-(2 ′-aminophenyl)-hexa-2,4-dienoate hydrolases (CarC enzymes) from two carbazole-degrading bacteria were purified using recombinant Escherichia coli strains with the histidine (His)-tagged purification system. The His-tagged CarC (ht-CarC) enzymes from Pseudomonas resinovorans strain CA10 (ht-CarC CA10) and Janthinobacterium sp. strain J3 (ht-CarC J3) exhibited hydrolase activity toward 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate as the purified native CarC CA10 did. ht-CarC J3 was crystallized in the space group I422 with cell dimensions of a= b=130.3 Å, c=84.5 Å in the hexagonal setting, and the crystal structure of ht-CarC J3 was determined at 1.86 Å resolution. The final refined model of ht-CarC J3 yields an R-factor of 21.6%, although the electron-density corresponding to Ile146 to Asn155 was ambiguous in the final model. We compared the known structures of BphD from Rhodococcus sp. strain RHA1 and CumD from Pseudomonas fluorescens strain IP01. The backbone conformation of ht-CarC J3 was better superimposed with CumD than with BphD RHA1. The side-chain directions of Arg185 and Trp262 residues in the substrate binding pockets of these enzymes were different among these proteins, suggesting that these residues may take a conformational change during the catalytic cycles.

Complete Nucleotide Sequence of Carbazole/Dioxin-degrading Plasmid pCAR1 in Pseudomonas resinovorans Strain CA10 Indicates its Mosaicity and the Presence of Large Catabolic Transposon Tn 4676

The car and ant operons originally isolated from Pseudomonas resinovorans strain CA10 contain the genes encoding the carbazole/dioxin-degrading enzymes and anthranilate 1,2-dioxygenase, respectively, and are located on the plasmid pCAR1. The entire nucleotide sequence of pCAR1 was determined to elucidate the mechanism by which the car operon may have been assembled and distributed in nature. pCAR1 is a 199,035-bp circular plasmid, and carries 190 open reading frames. Although the incompatibility group of pCAR1 is unclear, its potential origin for replication, OriP, and Rep and Par proteins appeared to be closely related to those of plasmid pL6.5 isolated from Pseudomonas fluorescens. The potential tellurite-resistance klaABC genes identified in the neighboring region of repA gene were also related to those in IncP plasmid originally identified from pseudomonads. On the other hand, we found genes encoding proteins that showed low but significant homology (20–45% identity) with Trh and Tra proteins from Enterobacteriaceae, which are potentially involved in conjugative transfer of plasmids or genomic island, suggesting that pCAR1 is also a conjugative plasmid. In pCAR1, we found tnpAcCST genes that encoded the proteins showing >70% length-wise identities with those are encoded by the toluene/xylene-degrading transposon Tn 4651 of TOL plasmid pWW0. Both car and ant degradative operons were found within a 72.8-kb Tn 4676 sequence defined by flanking tnpAcC and tnpST genes and bordered by a 46-bp inverted repeat (IR). Within Tn 4676 and its flanking region, we found the remnants of numerous mobile genetic elements, such as the duplicated transposase genes that are highly homologous to tnpR of Tn 4653 and the multiple candidates of IRs for Tn 4676 and Tn 4653-like element. We also found distinct regions with high and low G+C contents within Tn 4676, which contain an ant operon and car operon, respectively. These results suggested that multiple step assembly could have taken place before the current structure of Tn 4676 had been captured.

Expression, purification, and characterization of 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase from carbazole-degrader Pseudomonas resinovorans strain CA10.

The two-subunit meta-cleavage enzyme, 2'-aminobiphenyl-2,3-diol 1,2-dioxygenase (CarBaBb), from the carbazole degrader Pseudomonas resinovorans strain CA10 was purified to homogeneity from an Escherichia coli strain carrying the expression vector pUCA503, in which two copies of the carBaBb genes are tandemly linked. SDS-PAGE and gel filtration showed that CarB was a alpha2beta2-heterotetrameric enzyme with subunit molecular masses of approximately 10,000 for CarBa and 29,000 for CarBb. The optimum pH for activity was 8.5 and that of temperature was 35 degrees C. The CarB enzyme had a Km of 14 microM and a kcat/Km of 0.25 microM(-1) s(-1) for 2'-aminobiphenyl-2,3-diol, and the catalytic activities for biphenyl-type catecholic substrates were higher than those for monoaromatic catechol derivatives. The enzyme was originally isolated as a meta-cleavage enzyme for 2'-aminobiphenyl-2,3-diol involved in carbazole degradation, but the enzyme was highly specific for 2,3-dihydroxybiphenyl.

Purification and Characterization ofmeta-Cleavage Compound Hydrolase from a Carbazole DegraderPseudomonas resinovoransStrain CA10

2-Hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4dienoic acid [6-(2'-aminophenyl)-HODA] hydrolase, involved in carbazole degradation by Pseudomonas resinovorans strain CA10, was purified to near homogeneity from an overexpressing Escherichia coli strain. The enzyme was dimeric, and its optimum pH was 7.0-7.5. Phylogenetic analysis showed the close relationship of this enzyme to other hydrolases involved in the degradation of monocyclic aromatic compounds, and this enzyme was specific for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (6-phenyl-HODA), having little activity toward 2-hydroxy-6-oxohepta-2,4-dienoic acid and 2-hydroxymuconic semialdehyde. The enzyme had a Km of 2.51 microM and k(cat) of 2.14 (s(-1)) for 6-phenyl-HODA (50 mM sodium phosphate, pH 7.5, 25 degrees C). The effect of the presence of an amino group or hydroxyl group at the 2'-position of phenyl moiety of 6-phenyl-HODA on the enzyme activity was found to be small; the activity decreased only in the order of 6-(2'-aminophenyl)-HODA (2.44 U/mg) > 6-phenyl-HODA (1.99 U / mg) > 2-hydroxy-6-oxo-6-(2'-hydroxyphenyl)-hexa-2,4-dienoic acid (1.05 U/mg). The effects of 2'-substitution on the activity were in accordance with the predicted reactivity based on the calculated lowest unoccupied molecular orbital energy for these substrates.

Purification and Characterization of Carbazole 1,9a-Dioxygenase, a Three-Component Dioxygenase System of Pseudomonas resinovorans Strain CA10

The carbazole 1,9a-dioxygenase (CARDO) system of Pseudomonas resinovorans strain CA10 consists of terminal oxygenase (CarAa), ferredoxin (CarAc), and ferredoxin reductase (CarAd). Each component of CARDO was expressed in Escherichia coli strain BL21(DE3) as a native form (CarAa) or a His-tagged form (CarAc and CarAd) and was purified to apparent homogeneity. CarAa was found to be trimeric and to have one Rieske type [2Fe-2S] cluster and one mononuclear iron center in each monomer. Both His-tagged proteins were found to be monomeric and to contain the prosthetic groups predicted from the deduced amino acid sequence (His-tagged CarAd, one FAD and one [2Fe-2S] cluster per monomer protein; His-tagged CarAc, one Rieske type [2Fe-2S] cluster per monomer protein). Both NADH and NADPH were effective as electron donors for His-tagged CarAd. However, since the k(cat)/K(m) for NADH is 22.3-fold higher than that for NADPH in the 2,6-dichlorophenolindophenol reductase assay, NADH was supposed to be the physiological electron donor of CarAd. In the presence of NADH, His-tagged CarAc was reduced by His-tagged CarAd. Similarly, CarAa was reduced by His-tagged CarAc, His-tagged CarAd, and NADH. The three purified proteins could reconstitute the CARDO activity in vitro. In the reconstituted CARDO system, His-tagged CarAc seemed to be indispensable for electron transport, while His-tagged CarAd could be replaced by some unrelated reductases.

Enhanced degradation of carbazole and 2,3-dichlorodibenzo-p-dioxin in soils by Pseudomonas resinovorans strain CA10.

We studied the degradation of carbazole (CAR) and 2,3-dichlorodibenzo-p-dioxin (2,3-DCDD) in soils inoculated with carbazole- and dioxin-degrader Pseudomonas resinovorans strain CA10. By using Tn5-based transposon delivery systems, this bacterium was chromosomally marked with a tandem green fluorescent protein (gfp) gene. Real-time competitive PCR and direct counting using the (gfp) marker were employed to monitor the total number of carbazole 1,9a-dioxygenase gene (carAa) and survival of CA10 cells in the soil and soil slurry microcosms. Bioaugmentation studies indicated that the survival of the marked CA10 cells in soil microcosms was strongly influenced by pH and organic matter. While the number of the marked CA10 cells decreased rapidly in pH 6 with low organic matter, a high cell density was maintained in pH 7.3 with 2.5% organic matters up to 21 days after inoculation. In pH 7.3 soil, the period needed for complete degradation of CAR (100 microg kg(-1)) was markedly shortened from 21 to 7 days by the inoculation with the CA10 cells. Single inoculation of CA10 cells into the soil slurry system of 2,3-DCDD-contaminated soil enhanced the degradation of 2,3-DCDD from 25.0% to 37.0%. In this system, the population density of CA10 cells and the total number of carAa gene were maintained up to 14 days after inoculation. By repeated inoculation (every 2 days) with CA10 cells each at a density of 10(9) CFU g(-1) of soil, almost all of the 2,3-DCDD (1 microg kg(-1)) was degraded within 14 days. Results of these experiments suggest that P. resinovorans strain CA10 may be an important resource for bioremediation of CAR and chlorinated dibenzo-p-dioxin in contaminated soils.

Crystallization and preliminary crystallographic analysis of the terminal oxygenase component of carbazole 1,9a-dioxygenase of Pseudomonas resinovorans strain CA10.

The terminal oxygenase component (CarAa) of carbazole 1,9a-dioxygenase from Pseudomonas resinovorans strain CA10 was crystallized at 293 K using the sitting-drop vapour-diffusion method under the following conditions: 0.1 M sodium citrate pH 5.6 in the presence of 0.5 M ammonium sulfate and 1.0 M lithium sulfate. By using additive reagents with the crystallizing condition, improved diffraction was obtained from the crystals. Preliminary X-ray diffraction analysis indicated that CarAa crystals are hexagonal and belong to space group P6(2) or P6(4), with unit-cell parameters a = b = 244.5, c = 65.7 A, alpha = beta = 90.0, gamma = 120.0 degrees. Diffraction data were collected to 3.0 A resolution. The V(M) value is 2.16 A(3) Da(-1), which indicates a solvent content of 43.0%. This is the first report of crystallization of the terminal oxygenase component of an angular-type dioxygenase.

Sphingomonas sp. strain KA1, carrying a carbazole dioxygenase gene homologue, degrades chlorinated dibenzo-p-dioxins in soil.

Hybridization analysis showed that a newly isolated carbazole (CAR)-degrading bacterium Sphingomonas sp. strain KA1 did not possess the gene encoding the terminal oxygenase component (carAa) of CAR 1,9a-dioxygenase at high homology (more than 90% identity) to that of another CAR-degrader, Pseudomonas resinovorans strain CA10. However, PCR experiments using the primers for amplifying the internal fragment of the carAa gene (810 bp for strain CA10) showed that a PCR product of unexpected size (1100 bp) was amplified. Sequence analysis revealed that this DNA region contained the portion of two possible ORFs, which showed moderate homology to CarAa and CarBa from strain CA10 (61% and 40% identities at the amino acid level, respectively). Inoculation of strain KA1 into dioxin-contaminated model soil resulted in 96% and 70% degradation of 2-mono- and 2,3-dichlorinated dibenzo-p-dioxin, respectively, after 7-day incubation.

Sphingomonas sp. strain KA1, carrying a carbazole dioxygenase gene homologue, degrades chlorinated dibenzo- p-dioxins in soil

Hybridization analysis showed that a newly isolated carbazole (CAR)-degrading bacterium Sphingomonas sp. strain KA1 did not possess the gene encoding the terminal oxygenase component ( carAa) of CAR 1,9a-dioxygenase at high homology (more than 90% identity) to that of another CAR-degrader, Pseudomonas resinovorans strain CA10. However, PCR experiments using the primers for amplifying the internal fragment of the carAa gene (810 bp for strain CA10) showed that a PCR product of unexpected size (1100 bp) was amplified. Sequence analysis revealed that this DNA region contained the portion of two possible ORFs, which showed moderate homology to CarAa and CarBa from strain CA10 (61% and 40% identities at the amino acid level, respectively). Inoculation of strain KA1 into dioxin-contaminated model soil resulted in 96% and 70% degradation of 2-mono- and 2,3-dichlorinated dibenzo- p-dioxin, respectively, after 7-day incubation.

Organization and transcriptional characterization of catechol degradation genes involved in carbazole degradation by Pseudomonas resinovorans strain CA10.

Pseudomonas resinovorans strain CA10 assimilates catechol, which is an intermediate of carbazole degradation, by ortho cleavage pathway enzymes encoded by the catR, catBCA operon. Cat proteins of strain CA10 were very similar to those of P. putida, although the relatedness in non-coding regions was not high. It was found that catBCA genes were induced in carbazole-grown cells as a single transcriptional unit.

Detailed comparison between the substrate specificities of two angular dioxygenases, dibenzofuran 4,4a-dioxygenase from Terrabacter sp. and carbazole 1,9a-dioxygenase from Pseudomonas resinovorans

The preferred substrates in angular dioxygenation, monooxygenation, and lateral dioxygenation by dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 and carbazole 1,9a-dioxygenase (CARDO) from Pseudomonas resinovorans strain CA10 are shown to be distinctly different. The preferred oxygenation reactions suggest that DFDO evolved from a polycyclic aromatic hydrocarbon dioxygenase and that its most preferred substrates were fluorene and 9-fluorenone. The angular dioxygenases involved in the degradation pathway of dibenzofuran (dioxin) and fluorene are closely related in function, while CARDO is a novel enzyme not only phylogenetically but also functionally.

Quantification of the carbazole 1,9a-dioxygenase gene by real-time competitive PCR combined with co-extraction of internal standards

The fluorogenic probe assay, competitive polymerase chain reaction (PCR) and co-extraction with internal standard cells were combined to develop a rapid, sensitive, and accurate quantification method for the copy number of a target carbazole 1,9a-dioxygenase gene ( carAa) and the cell number of Pseudomonas sp. strain CA10. The internal standard DNA was modified by replacement of a 20-bp long region with one for binding a specific probe in fluorogenic PCR (TaqMan). The resultant DNA fragment was similar to the corresponding region of the intact carAa gene in terms of G+C content. When used as a competitor in the PCR reaction, the internal standard DNA was distinguishable from the target carAa gene by two specific fluorogenic probes with different fluorescence labels, and was automatically detected in a single tube using the ABI7700 sequence detection system. To minimize variations in the efficiency of cell lysis and DNA extraction between the samples, the co-extraction method was combined. A mini-transposon was used to introduce competitor DNA into the genome of other pseudomonads, and the resultant construct was used as the standard cell. After the addition of a fixed amount of the internal standard cells to soil samples, total DNA was extracted (co-extraction). Using this method, the copy number of the carAa gene and the cell number of strain CA10 in soil samples could be quantified rapidly.

Degradation of chlorinated dibenzofurans and dibenzo-p-dioxins by two types of bacteria having angular dioxygenases with different features.

Two kinds of bacteria having different-structured angular dioxygenases-a dibenzofuran (DF)-utilizing bacterium, Terrabacter sp. strain DBF63, and a carbazole (CAR)-utilizing bacterium, Pseudomonas sp. strain CA10-were investigated for their ability to degrade some chlorinated dibenzofurans (CDFs) and chlorinated dibenzo-p-dioxins (CDDs) (or, together, CDF/Ds) using either wild-type strains or recombinant Escherichia coli strains. First, it was shown that CAR 1,9a-dioxygenase (CARDO) catalyzed angular dioxygenation of all mono- to triCDF/Ds investigated in this study, but DF 4,4a-dioxygenase (DFDO) did not degrade 2,7-diCDD. Secondly, degradation of CDF/Ds by the sets of three enzymes (angular dioxygenase, extradiol dioxygenase, and meta-cleavage compound hydrolase) was examined, showing that these enzymes in both strains were able to convert 2-CDF to 5-chlorosalicylic acid but not other tested substrates to the corresponding chlorosalicylic acid (CSA) or chlorocatechol (CC). Finally, we tested the potential of both wild-type strains for cooxidation of CDF/Ds and demonstrated that both strains degraded 2-CDF, 2-CDD, and 2,3-diCDD to the corresponding CSA and CC. We investigated the sites for the attack of angular dioxygenases in each CDF/D congener, suggesting the possibility that the angular dioxygenation of 2-CDF, 2-CDD, 2,3-diCDD, and 1,2,3-triCDD (10 ppm each) by both DFDO and CARDO occurred mainly on the nonsubstituted aromatic nuclei.

Quantification of the carbazole 1,9a-dioxygenase gene by real-time competitive PCR combined with co-extraction of internal standards.

The fluorogenic probe assay, competitive polymerase chain reaction (PCR) and co-extraction with internal standard cells were combined to develop a rapid, sensitive, and accurate quantification method for the copy number of a target carbazole 1,9a-dioxygenase gene (carAa) and the cell number of Pseudomonas sp. strain CA10. The internal standard DNA was modified by replacement of a 20-bp long region with one for binding a specific probe in fluorogenic PCR (TaqMan). The resultant DNA fragment was similar to the corresponding region of the intact carAa gene in terms of G+C content. When used as a competitor in the PCR reaction, the internal standard DNA was distinguishable from the target carAa gene by two specific fluorogenic probes with different fluorescence labels, and was automatically detected in a single tube using the ABI7700 sequence detection system. To minimize variations in the efficiency of cell lysis and DNA extraction between the samples, the co-extraction method was combined. A mini-transposon was used to introduce competitor DNA into the genome of other pseudomonads, and the resultant construct was used as the standard cell. After the addition of a fixed amount of the internal standard cells to soil samples, total DNA was extracted (co-extraction). Using this method, the copy number of the carAa gene and the cell number of strain CA10 in soil samples could be quantified rapidly.