Transcriptional heterogeneity of catabolic genes on the plasmid pCAR1 causes host-specific carbazole degradation

Abstract

To elucidate why plasmid-borne catabolic ability differs among host bacteria, we assessed the expression dynamics of the P_ant promoter on the carbazole-degradative conjugative plasmid pCAR1 in Pseudomonas putida KT2440(pCAR1) (hereafter, KTPC) and Pseudomonas resinovorans CA10. The P_ant promoter regulates the transcription of both the car and ant operons, which are responsible for converting carbazole into anthranilate and anthranilate into catechol, respectively. In the presence of anthranilate, transcription of the P_ant promoter is induced by the AraC/XylS family regulator AntR, encoded on pCAR1. A reporter cassette containing the P_ant promoter followed by gfp was inserted into the chromosomes of KTPC and CA10. After adding anthranilate, GFP expression in the population of CA10 showed an unimodal distribution, whereas a small population with low GFP fluorescence intensity appeared for KTPC. CA10 has a gene, antR_CA, that encodes an iso-functional homolog of AntR on its chromosome. When antR_CA was disrupted, a small population with low GFP fluorescence intensity appeared. In contrast, overexpression of pCAR1-encoded AntR in KTPC resulted in unimodal expression under the P_ant promoter. These results suggest that the expression of pCAR1-encoded AntR is insufficient to ameliorate the stochastic expression of the P_ant promoter. Raman spectra of single cells collected using deuterium-labeled carbazole showed that the C-D Raman signal exhibited greater variability for KTPC than CA10. These results indicate that heterogeneity at the transcriptional level of the P_ant promoter due to insufficient AntR availability causes fluctuations in the pCAR1-borne carbazole-degrading capacity of host bacterial cells.IMPORTANCEHorizontally acquired genes increase the competitiveness of host bacteria under selective conditions, although unregulated expression of foreign genes may impose fitness costs. The "appropriate" host for a plasmid is empirically known to maximize the expression of plasmid-borne traits. In the case of pCAR1-harboring Pseudomonas strains, P. resinovorans CA10 exhibits strong carbazole-degrading capacity, whereas P. putida KT2440 harboring pCAR1 exhibits low degradation capacity. Our results suggest that a chromosomally encoded transcription factor affects transcriptional and metabolic fluctuations in host cells, resulting in different carbazole-degrading capacities as a population. This study may provide a clue for determining appropriate hosts for a plasmid and for regulating the expression of plasmid-borne traits, such as the degradation of xenobiotics and antibiotic resistance.

Keywords: Pseudomonas; Raman spectroscopy; flow cytometry; heterogeneity; plasmids.

Fig 1

Carbazole catabolic pathway and the car and ant genes on pCAR1 involved in carbazole degradation. The carbazole catabolic pathway in Pseudomonas strains harboring pCAR1 is shown, along with the names of the enzymes involved and genetic organization of the car and ant operons on pCAR1 (10). Transcription from the P_ant promoter is induced by AntR in the presence of anthranilate, whereas P_carAa is a constitutive promoter.

Fig 2

Expression dynamics of the P_ant promoter in three Pseudomonas strains harboring the plasmid pCAR1. CA10R::P_ant-gfp (A), KTPC::P_ant-gfp (B), and PFPC-Km::P_ant-gfp (C) were exposed to 0.01% anthranilate, and GFP fluorescence intensity of each cell was measured using flow cytometry; 100,000 cells were analyzed for each sample. The upper panels show the distribution of GFP fluorescence intensity in cells incubated for 0, 1, 2, and 3 h. CA10R::gfp (A), KTPC::gfp (B), and PFPC-Km::gfp (C) were used as negative controls for each panel, and the results are shown as histograms with gray shading. The lower panels show the distribution of GFP fluorescence intensity in cells incubated for 3 h after adding anthranilate. The ratio of bacterial cells exhibiting GFP fluorescence intensities lower and higher than the maximum intensity of the negative control is shown in each panel.

Fig 3

Expression dynamics of the P_ant promoter in KTPC::P_ant-gfp and PFPC-Km::P_ant-gfp after sorting populations with low and high GFP fluorescence intensities. After 3 h of incubation in the presence of anthranilate, the cells with GFP fluorescence intensities lower and higher than the maximum intensity of the negative controls (lower panels in Fig. 2) were collected and grown separately in LB. The resultant cultures were analyzed using flow cytometry after adding 0.01% anthranilate. The results for KTPC::P_ant-gfp (A) and PFPC-Km::P_ant-gfp (B) are shown. “Low” and “High” indicate that cells with lower and higher GFP fluorescence intensities, respectively, were used to prepare the cultures subsequently subjected to flow cytometry. The results for the negative controls [KTPC::gfp (A) and PFPC-Km::gfp (B)] are shown as histograms with gray shading.

Fig 4

Expression dynamics of the P_ant promoter in CA10RΔantR_CA::P_ant-gfp. The GFP fluorescence intensity of each cell was measured using flow cytometry after exposure to 0.01% anthranilate. The upper panel shows the distributions of GFP fluorescence intensity after 0, 1, 2, and 3 h of incubation. The result for the negative control (CA10R::gfp) is shown as a histogram with gray shading. The lower panel shows the distribution of GFP fluorescence intensity of cells incubated for 3 h after adding anthranilate. The ratio of bacterial cells exhibiting GFP fluorescence intensities lower and higher than the maximum intensity of the negative control is shown.

Fig 5

Expression dynamics of the P_ant promoter in KTPC::P_ant-gfp(pBRKantR). KTPC::P_ant-gfp(pBRKantR) (A) and the vector control KTPC::P_ant-gfp(pBBR1MCS-2) (B) were exposed to 0.01% anthranilate, and the GFP fluorescence intensity of each cell was measured using flow cytometry. The upper panels show the distributions of GFP fluorescence intensity in cells incubated for 0, 1, 2, and 3 h. KTPC::gfp(pBBR1MCS-2) was used as the negative control for both analyses and the results are shown as histograms with gray shading. The lower panels show the distributions of GFP fluorescence intensity of cells incubated for 3 h after adding anthranilate. The ratio of bacterial cells exhibiting GFP fluorescence intensities lower and higher than the maximum intensity of the negative control is shown on each panel.

Fig 6

Carbazole-degrading capacities of CA10R and KTPC assessed at the single-cell level using Raman microspectroscopy. (A, B) Growth curves of CA10R (A) and KTPC (B) in CNF buffer supplemented with 0.1% (wt/vol) CAR-d₈ as the sole source of carbon and nitrogen. Data represent mean ± SEM (n = 3). (C, D) Raman spectra of single CA10R (C) and KTPC (D) cells measured after 8, 12, 16, 20, and 24 h of incubation in medium containing CAR-d₈. Each spectrum is the average of 30 spectra obtained from 30 bacterial cells and spectra are shown with vertical offset for clarity. The shaded area represents the 1σ error envelope. All of the averaged spectra have been normalized to the area of the C–H stretching band at 2935 cm^–1. (E, F) Box plots of relative C–D stretching band intensity, C–D/(C–H + C–D), at various incubation times for CA10R (E) and KTPC (F). The ratio was calculated by dividing the area intensity of the C–D stretching band by the sum of the C–H and C–D stretching band intensities.