Article Figures & Data
Figures
- FIG. 1.
(A) Carbazole degradation pathway via anthranilate in P. resinovorans strain CA10. (B) Genetic organization of car and ant gene clusters located on carbazole-degradative plasmid pCAR1. Black and gray pentagons represent the genes and their transcriptional directions of the ant and car gene clusters, respectively. Pentagons in ISPre1 and ISPre2 represent the transposase genes. Unknown ORFs are shown as a white pentagon. The black box located in the 5′ region of ORF9 represents the transposed 5′ portion of antA as described previously (26). ORF22 and ORF23 encoding probable AraC/XylS-type transcriptional regulators are represented by pentagons with thick lines.
- FIG. 2.
(A) Northern hybridization analysis using a 200-bp probe inside of the antA gene. Total RNA was prepared from strain CA10 cells grown on nutrient broth (lane 2), anthranilate (lane 3), and carbazole (lane 4). As a control, total RNA was similarly extracted from the cells just after starvation on CNFMM (lane 1). (B) RT-PCR amplification of the region spanning antA to antC. Total RNA extracted from the strain CA10 cells grown on anthranilate (lane 1) or carbazole (lane 2) was used as a template.
- FIG. 3.
Determination of the transcription start point of antA by primer extension. Total RNA was isolated from strain CA10 grown on anthranilate (lane 1) and carbazole (lane 2). The other lanes correspond to a sequence ladder obtained with pBCA731 as a template, and the sequence pattern is shown on the right. The arrows indicate the primer extension product and the corresponding transcription start point in the nucleotide sequence shown below. The nucleotide sequence of the Pant promoter is shown with the −35 and −10 boxes underlined.
- FIG. 4.
Deletion analysis of the 5′ region upstream of the Pant promoter. The DNA region fused with the luc gene is shown to the left. The numbers indicate the position of the 5′ termini of the transcriptional fusions relative to the transcription start point of antA (+1). Black boxes upstream of antA indicate −35 and −10 boxes of the Pant promoter, respectively. Strain CA10 was transformed by the reporter plasmids of pBRC series indicated at the middle. Luciferase activity of the cells incubated on SMM with (black bars) or without (white bars) anthranilate is shown to the right. Values and error bars represent averages and standard deviations of at least three independent experiments. RLU, relative light units.
- FIG. 5.
Expression profile of the Pant promoter. Strain CA10 harboring pBRCantA253 was inoculated on SMM containing succinate plus anthranilate (•), succinate plus catechol (▪), or succinate (▴) at time zero. Luciferase activity of the cells harvested periodically was measured. Values and error bars represent averages and standard deviations of at least three independent experiments. RLU, relative light units.
- FIG. 6.
(A) Comparison of the downstream regions of two ISPre1s on ant and car loci. The white box in the antA gene represents the deleted region in the sequence of ORF9 as described previously (26). Nearly identical regions are shown by shading. Small arrows represent the primers used in primer extension analyses. Transcription start points of two copies of Pant promoter are marked by arrows. (B) Transcription start point at the ORF9 locus determined by primer extension analysis. Total RNA was isolated from strain CA10 cells grown on anthranilate (lane 1) and carbazole (lane 2). The other lanes correspond to a sequence ladder obtained with pBCA721 as a template, and the sequence pattern is shown on the right.
- FIG. 7.
(A) Northern hybridization analysis using a 359-bp probe inside of the carAa gene. Total RNA was prepared from strain CA10 cells grown on nutrient broth (lane 2), anthranilate (lane 3), and carbazole (lane 4). As a control, total RNA was similarly extracted from the cells just after starvation on CNFMM (lane 1). (B) RT-PCR amplification of the region spanning ORF9 and carAa. Total RNA extracted from the strain CA10 cells grown on anthranilate (lane 1) or carbazole (lane 2) was used as a template.
Tables
- TABLE 1.
Bacterial strains and plasmids used in this study
Strains or plasmid Relevant characteristicsa Source or reference Strains P. resinovorans strain CA10 pCAR1 CAR+ AN+ 27 P. resinovorans strain CA10dm1 pCAR1Δ1 CAR− AN+ This study P. resinovorans strain CA10dm3 pCAR1Δ3 CAR− AN+ This study P. putida strain DS1 CAR− AN− 14 P. putida strain HS01 pCAR2 CAR+ AN+ Shintani et al., submitted Plasmids pBBR1MCS-5 GmrlacZα mob; compatible with IncP IncQ IncW 22 pBCA711 Apr, pBluescript II SK(−) containing 4.4-kb EcoRI insert of strain CA10 DNA 26 pBCA721 Apr, pBluescript II SK(−) containing 11.3-kb XhoI insert of strain CA10 DNA 26 pBCA731 Apr, pBluescript II SK(−) containing 7.5-kb EcoRV-ClaI insert of strain CA10 DNA 26 pBluescript II SK(−) Apr, lacZ Stratagene pBRCantA103 Gmr, pBBR1MCS-5 containing the region −50 to +53 from antA start codon and luc+NF This study pBRCantA113 Gmr, pBBR1MCS-5 containing the region −60 to +53 from antA start codon and luc+NF This study pBRCantA123 Gmr, pBBR1MCS-5 containing the region −70 to +53 from antA start codon and luc+NF This study pBRCantA133 Gmr, pBBR1MCS-5 containing the region −80 to +53 from antA start codon and luc+NF This study pBRCantA143 Gmr, pBBR1MCS-5 containing the region −90 to +53 from antA start codon and luc+NF This study pBRCantA153 Gmr, pBBR1MCS-5 containing the region −100 to +53 from antA start codon and luc+NF This study pBRCantA203 Gmr, pBBR1MCS-5 containing the region −150 to +53 from antA start codon and luc+NF This study pBRCantA253 Gmr, pBBR1MCS-5 containing the region −200 to +53 from antA start codon and luc+NF This study pMK Kmr, pMMB66HE inserted into DraI restriction site with 1.3-kb HincII fragment of pUC4K This study pMK22 Kmr, pMK with 1.0-kb SalI-BamHI insert containing ORF22 This study pMK23 Kmr, pMK with 1.1-kb SalI-BamHI insert containing ORF23 This study pMK2322 Kmr, pMK with 2.1-kb SalI-BamHI insert containing ORF22 and ORF23 This study pMMB66HE Apr IncQ 16 pSP-luc+NF Aprluc+NF Promega pUC4K Apr Kmr 37 pUCA1 Apr, pUC19 with 6.9-kb EcoRI insert of strain CA10 DNA 33 pUCA741 Apr, pUC119 with 8.9-kb SalI insert of strain CA10 DNA 26 ↵ a CAR+/CAR− or AN+/AN− represents ability/inability to grow on carbazole or anthranilate as a sole source of carbon, nitrogen, and energy. Apr, Gmr, and Kmr represent resistance to ampicillin, gentamicin, and kanamycin, respectively.
- TABLE 2.
Transcription from the promoter Pant in several strains
Host straina Genetic characteristics Luciferase activity (RLU/ng)b Without anthranilate With anthranilate CA10 ORF22+ ORF23+ 4.21 ± 0.34 289.63 ± 25.08 CA10dm1 ORF22+ ORF23+ 5.79 ± 0.94 279.45 ± 13.56 CA10dm3 ORF22− ORF23− 2.94 ± 0.91 220.75 ± 13.06 HS01 ORF22+ ORF23+ 0.33 ± 0.08 316.90 ± 30.94 DS1 ORF22− ORF23− 0.48 ± 0.09 0.30 ± 0.03 DS1(pMK2322) ORF22+ ORF23+ 2.78 ± 0.98 563.78 ± 7.97 DS1(pMK23) ORF22− ORF23+ 2.70 ± 0.48 546.05 ± 61.98 DS1(pMK22) ORF22+ ORF23− 0.79 ± 0.07 0.56 ± 0.06 DS1(pMK) ORF22− ORF23− 1.03 ± 0.22 0.66 ± 0.15 ↵ a P. resinovorans strains CA10, CA10dm1, and CA10dm3, and P. putida strains HS01 and DS1 were transformed by pBRCantA253. Strain DS1 was transformed by pBRCantA253 along with expression plasmids shown in parentheses. Luciferase activity was determined from the cells grown on SMM with or without anthranilate for 2 h. The values shown represent averages ± standard deviations of at least three independent experiments.
↵ b RLU, relative light units.
