ABSTRACT

To elucidate why plasmid-borne catabolic ability differs among host bacteria, we assessed the expression dynamics of the Pant promoter on the carbazole-degradative conjugative plasmid pCAR1 in Pseudomonas putida KT2440(pCAR1) (hereafter, KTPC) and Pseudomonas resinovorans CA10. The Pant 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 Pant promoter is induced by the AraC/XylS family regulator AntR, encoded on pCAR1. A reporter cassette containing the Pant 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, antRCA, that encodes an iso-functional homolog of AntR on its chromosome. When antRCA 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 Pant promoter. These results suggest that the expression of pCAR1-encoded AntR is insufficient to ameliorate the stochastic expression of the Pant 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 Pant promoter due to insufficient AntR availability causes fluctuations in the pCAR1-borne carbazole-degrading capacity of host bacterial cells.

IMPORTANCE

Horizontally 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.

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REFERENCES

1.
Nojiri H, Shintani M, Omori T. 2004. Divergence of mobile genetic elements involved in the distribution of xenobiotic-catabolic capacity. Appl Microbiol Biotechnol 64:154–174.
2.
Segura A, Molina L, Ramos JL. 2014. Plasmid-mediated tolerance toward environmental pollutants. Microbiol Spectr 2.
3.
Ramos JL, Marqués S, Timmis KN. 1997. Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. Annu Rev Microbiol 51:341–373.
4.
Nojiri H. 2013. Impact of catabolic plasmids on host cell physiology. Curr Opin Biotechnol 24:423–430.
5.
Vial L, Hommais F. 2020. Plasmid-chromosome cross-talks. Environ Microbiol 22:540–556.
6.
Miyakoshi M, Nishida H, Shintani M, Yamane H, Nojiri H. 2009. High-resolution mapping of plasmid transcriptomes in different host bacteria. BMC Genomics 10:12.
7.
Takahashi Y, Shintani M, Li L, Yamane H, Nojiri H. 2009. Carbazole-degradative IncP-7 plasmid pCAR1.2 is structurally unstable in Pseudomonas fluorescens Pf0-1, which accumulates catechol, the intermediate of the carbazole degradation pathway. Appl Environ Microbiol 75:3920–3929.
8.
Shintani M, Tokumaru H, Takahashi Y, Miyakoshi M, Yamane H, Nishida H, Nojiri H. 2011. Alterations of RNA maps of IncP-7 plasmid PCAR1 in various Pseudomonas bacteria. Plasmid 66:85–92.
9.
Takahashi Y, Shintani M, Takase N, Kazo Y, Kawamura F, Hara H, Nishida H, Okada K, Yamane H, Nojiri H. 2015. Modulation of primary cell function of host Pseudomonas bacteria by the conjugative plasmid pCAR1. Environ Microbiol 17:134–155.
10.
Nojiri H. 2012. Structural and molecular genetic analyses of the bacterial carbazole degradation system. Biosci Biotechnol Biochem 76:1–18.
11.
Nojiri H, Sekiguchi H, Maeda K, Urata M, Nakai S, Yoshida T, Habe H, Omori T. 2001. Genetic characterization and evolutionary implications of a car gene cluster in the carbazole degrader Pseudomonas sp. strain CA10. J Bacteriol 183:3663–3679.
12.
Ouchiyama N, Zhang Y, Omori T, Kodama T. 1993. Biodegradation of carbazole by Pseudomonas spp. CA06 and CA10. Biosci Biotechnol Biochem 57:455–460.
13.
Urata M, Miyakoshi M, Kai S, Maeda K, Habe H, Omori T, Yamane H, Nojiri H. 2004. Transcriptional regulation of the ant operon, encoding two-component anthranilate 1,2-dioxygenase, on the carbazole-degradative plasmid pCAR1 of Pseudomonas resinovorans strain CA10. J Bacteriol 186:6815–6823.
14.
Miyakoshi M, Urata M, Habe H, Omori T, Yamane H, Nojiri H. 2006. Differentiation of carbazole catabolic operons by replacement of the regulated promoter via transposition of an insertion sequence. J Biol Chem 281:8450–8457.
15.
Engl C. 2019. Noise in bacterial gene expression. Biochem Soc Trans 47:209–217.
16.
Silva-Rocha R, de Lorenzo V. 2012. Stochasticity of TOL plasmid catabolic promoters sets a bimodal expression regime in Pseudomonas putida mt-2 exposed to M-xylene. Mol Microbiol 86:199–211.
17.
Guantes R, Benedetti I, Silva-Rocha R, de Lorenzo V. 2016. Transcription factor levels enable metabolic diversification of single cells of environmental bacteria. ISME J 10:1122–1133.
18.
Simon R, Priefer U, Pühler A. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nat Biotechnol 1:784–791.
19.
Miyakoshi M, Shintani M, Terabayashi T, Kai S, Yamane H, Nojiri H. 2007. Transcriptome analysis of Pseudomonas putida KT2440 harboring the completely sequenced IncP-7 plasmid pCAR1. J Bacteriol 189:6849–6860.
20.
Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM. 1995. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176.
21.
Maeda K, Nojiri H, Shintani M, Yoshida T, Habe H, Omori T. 2003. Complete nucleotide sequence of carbazole/dioxin-degrading plasmid pCAR1 in Pseudomonas resinovorans strain CA10 indicates its mosaicity and the presence of large catabolic transposon Tn4676. J Mol Biol 326:21–33.
22.
Shintani M, Yano H, Habe H, Omori T, Yamane H, Tsuda M, Nojiri H. 2006. Characterization of the replication, maintenance, and transfer features of the IncP-7 plasmid pCAR1, which carries genes involved in carbazole and dioxin degradation. Appl Environ Microbiol 72:3206–3216.
23.
Takahashi Y, Shintani M, Yamane H, Nojiri H. 2009. The complete nucleotide sequence of pCAR2: pCAR2 and pCAR1 were structurally identical IncP-7 carbazole degradative plasmids. Biosci Biotechnol Biochem 73:744–746.
24.
Shintani M, Hosoyama A, Ohji S, Tsuchikane K, Takarada H, Yamazoe A, Fujita N, Nojiri H. 2013. Complete genome sequence of the carbazole degrader Pseudomonas resinovorans strain CA10. Genome Announc 1:e00488-13.
25.
Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A. 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73.
26.
Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP. 1998. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86.
27.
Figurski DH, Helinski DR. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76:1648–1652.
28.
Martínez-García E, Fraile S, Algar E, Aparicio T, Velázquez E, Calles B, Tas H, Blázquez B, Martín B, Prieto C, Sánchez-Sampedro L, Nørholm MHH, Volke DC, Wirth NT, Dvořák P, Alejaldre L, Grozinger L, Crowther M, Goñi-Moreno A, Nikel PI, Nogales J, de Lorenzo V. 2023. SEVA 4.0: an update of the standard European vector architecture database for advanced analysis and programming of bacterial phenotypes. Nucleic Acids Research 51:D1558–D1567.
29.
Choi KH, Gaynor JB, White KG, Lopez C, Bosio CM, Karkhoff-Schweizer RR, Schweizer HP. 2005. A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2:443–448.
30.
Li M, Huang WE, Gibson CM, Fowler PW, Jousset A. 2013. Stable isotope probing and Raman spectroscopy for monitoring carbon flow in a food chain and revealing metabolic pathway. Anal Chem 85:1642–1649.
31.
Berry D, Mader E, Lee TK, Woebken D, Wang Y, Zhu D, Palatinszky M, Schintlmeister A, Schmid MC, Hanson BT, Shterzer N, Mizrahi I, Rauch I, Decker T, Bocklitz T, Popp J, Gibson CM, Fowler PW, Huang WE, Wagner M. 2015. Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells. Proc Natl Acad Sci U S A 112:E194–E203.
32.
Noothalapati Venkata HN, Shigeto S. 2012. Stable isotope-labeled Raman imaging reveals dynamic proteome localization to lipid droplets in single fission yeast cells. Chem Biol 19:1373–1380.
33.
Yasuda M, Takeshita N, Shigeto S. 2021. Deuterium-labeled raman tracking of glucose accumulation and protein metabolic dynamics in Aspergillus nidulans hyphal tips. Sci Rep 11:1279.
34.
Baltrus DA. 2013. Exploring the costs of horizontal gene transfer. Trends Ecol Evol 28:489–495.
35.
Shintani M, Yoshida T, Habe H, Omori T, Nojiri H. 2005. 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. Appl Microbiol Biotechnol 67:370–382.
36.
Shintani M, Nojiri H, Yoshida T, Habe H, Omori T. 2003. Carbazole/dioxin-degrading car gene cluster is located on the chromosome of Pseudomonas stutzeri strain OM1 in a form different from the simple transposition of Tn4676. Biotechnol Lett 25:1255–1261.
37.
Yao Y, Maddamsetti R, Weiss A, Ha Y, Wang T, Wang S, You L. 2022. Intra- and interpopulation transposition of mobile genetic elements driven by antibiotic selection. Nat Ecol Evol 6:555–564.
38.
Tropel D, van der Meer JR. 2004. Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiol Mol Biol Rev 68:474–500.
39.
Cortés-Avalos D, Martínez-Pérez N, Ortiz-Moncada MA, Juárez-González A, Baños-Vargas AA, Estrada-de Los Santos P, Pérez-Rueda E, Ibarra JA. 2021. An update of the unceasingly growing and diverse AraC/XylS family of transcriptional activators. FEMS Microbiol Rev 45:fuab020.
40.
Nikel PI, Silva-Rocha R, Benedetti I, de Lorenzo V. 2014. The private life of environmental bacteria: pollutant biodegradation at the single cell level. Environ Microbiol 16:628–642.
41.
Wang Y, Li J, Liu A. 2017. Oxygen activation by mononuclear nonheme iron dioxygenases involved in the degradation of aromatics. J Biol Inorg Chem 22:395–405.
42.
San Millan A, MacLean RC. 2017. Fitness costs of plasmids: a limit to plasmid transmission. Microbiol Spectr 5.
43.
San Millan A, Toll-Riera M, Qi Q, MacLean RC. 2015. Interactions between horizontally acquired genes create a fitness cost in Pseudomonas aeruginosa. Nat Commun 6:6845.
44.
Kawano H, Suzuki-Minakuchi C, Sugiyama D, Watanabe N, Takahashi Y, Okada K, Nojiri H. 2020. A novel small RNA on the Pseudomonas putida KT2440 chromosome is involved in the fitness cost imposed by IncP-1 plasmid Rp4. Front Microbiol 11:1328.
45.
Sambrook J, Russell D. 2001. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
46.
Shintani M, Habe H, Tsuda M, Omori T, Yamane H, Nojiri H. 2005. Recipient range of IncP-7 conjugative plasmid pCAR2 from Pseudomonas putida HS01 is broader than from other Pseudomonas strains. Biotechnol Lett 27:1847–1853.
47.
Choi KH, Schweizer HP. 2005. An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants. BMC Microbiol 5:30.
48.
Suzuki-Minakuchi C, Hirotani R, Shintani M, Takeda T, Takahashi Y, Matsui K, Vasileva D, Yun C-S, Okada K, Yamane H, Nojiri H. 2015. Effects of three different nucleoid-associated proteins encoded on IncP-7 plasmid PCAR1 on host Pseudomonas putida KT2440. Appl Environ Microbiol 81:2869–2880.
49.
Nakamura T, Suzuki-Minakuchi C, Kawano H, Kanesaki Y, Kawasaki S, Okada K, Nojiri H. 2020. H-NS family proteins drastically change their targets in response to the horizontal transfer of the catabolic plasmid pCAR1. Front Microbiol 11:1099.
50.
Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG. 2012. Primer3--new capabilities and interfaces. Nucleic Acids Res 40:e115.
51.
Horiue H, Sasaki M, Yoshikawa Y, Toyofuku M, Shigeto S. 2020. Raman spectroscopic signatures of carotenoids and polyenes enable label-free visualization of microbial distributions within pink biofilms. Sci Rep 10:7704.
52.
Huang C-K, Ando M, Hamaguchi H, Shigeto S. 2012. Disentangling dynamic changes of multiple cellular components during the yeast cell cycle by in vivo multivariate raman imaging. Anal Chem 84:5661–5668.
53.
Huang C-K, Hamaguchi H, Shigeto S. 2011. In vivo multimode raman imaging reveals concerted molecular composition and distribution changes during yeast cell cycle. Chem Commun (Camb) 47:9423–9425.

Information & Contributors

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Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 90Number 221 February 2024
eLocator: e01247-23
Editor: Isaac Cann, University of Illinois Urbana-Champaign, Champaign, Illinois, USA
PubMed: 38289097

History

Received: 20 July 2023
Accepted: 21 December 2023
Published online: 30 January 2024

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Keywords

  1. flow cytometry
  2. heterogeneity
  3. plasmids
  4. Pseudomonas
  5. Raman spectroscopy

Contributors

Authors

Agro-Biotechnology Research Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan
Author Contributions: Conceptualization, Funding acquisition, Project administration, and Writing – original draft.
Natsumi Yamamoto
Agro-Biotechnology Research Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
Author Contributions: Data curation, Formal analysis, and Writing – original draft.
Saki Takahira
Agro-Biotechnology Research Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
Author Contributions: Data curation, Formal analysis, and Writing – original draft.
Masataka Yamaguchi
Department of Chemistry, Graduate School of Science and Technology, Kwansei Gakuin University, Hyogo, Japan
Author Contributions: Data curation and Formal analysis.
Yutaro Takeda
Agro-Biotechnology Research Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
Author Contributions: Data curation and Formal analysis.
Kazunori Okada
Agro-Biotechnology Research Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
Author Contributions: Supervision and Writing – review and editing.
Shinsuke Shigeto
Department of Chemistry, Graduate School of Science and Technology, Kwansei Gakuin University, Hyogo, Japan
Author Contributions: Formal analysis, Funding acquisition, Supervision, and Writing – review and editing.
Agro-Biotechnology Research Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, Tokyo, Japan
Author Contributions: Conceptualization, Funding acquisition, Project administration, Supervision, and Writing – review and editing.

Editor

Isaac Cann
Editor
University of Illinois Urbana-Champaign, Champaign, Illinois, USA

Notes

The authors declare no conflict of interest.

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