GENOME ANNOUNCEMENT
Genome Sequence of Pseudomonas putida S12, a Potential Platform Strain for Industrial Production of Valuable Chemicals Fei Tao,a Yaling Shen,b Ziqi Fan,b Hongzhi Tang,a and Ping Xua State Key Laboratory of Microbial Metabolism & School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People’s Republic of China,a and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People’s Republic of Chinab
Pseudomonas putida strain S12, a well-studied solvent-tolerant bacterium, is considered a platform strain for the production of many chemicals. Here, we present a 6.28-Mb assembly of its genome sequence. We have annotated 32 coding sequences (CDSs) encoding efflux systems of organic compounds and 195 CDSs responsible for the metabolism of aromatic compounds.
B
iocatalysis is a “green” alternative of chemical catalysis for the production of valuable chemicals, especially substituted aromatic compounds (4). Currently, demand for the green production of chemicals is rapidly increasing due to the declining availability of fossil fuels and the urgency to reduce CO2 emissions (4, 13). However, such bioproduction may be hindered by the toxicity of the products of interest to the production host (12, 19). Organic-solvent-tolerant bacteria are remarkable microorganisms capable of surviving with the presence of high concentrations of organic solvents (5, 6). They provide a way to cope with the product toxicity, which is to develop solvent-tolerant microorganisms as biocatalysts (19). In fact, many researchers have successfully developed efficient solvent-tolerant whole-cell catalysts, such as solvent-tolerant biodesulfurizing strains (10, 16, 18, 20). To take advantage of solvent-tolerant bacteria in enhancing bioproduction, it is necessary to thoroughly understand the molecular mechanisms involved in adaptation to organic solvents (21). However, the solvent-tolerant mechanism of bacteria is not thoroughly known now. Therefore, it is important and urgent to clarify the solvent adaptation of bacteria. Genome sequencing is powerful for discovering molecular mechanisms, especially for seeking functional genes, and would accelerate the studies in such scientific fields (2, 17). Pseudomonas putida S12 (ATCC 700801), with its distinguished solvent-tolerant ability, is considered a good platform for bioconversion of sugars into substituted aromatic compounds (9). It has been used widely in developing many efficient biocatalysts, such as a stain for phenol production (20). Different aspects of the solvent-tolerant mechanism were also studied extensively (8, 21). However, no genomic research about it has been performed until now. Here, we present the draft genome sequence of strain S12, which was obtained using the Illumina GA system. The reads were assembled with VELVET (22), and the sequence was annotated using the RAST annotation server (1). The sequence contains 6,284,656 bases, 5,635 predicted coding sequences (CDSs), and 90 RNAs and consists of 258 large contigs (⬎200 bp in size), with a G⫹C content of 61.5%. We predicted 32 CDSs encoding efflux systems of organic compounds, which are related to the cellular resistance, including solvent tolerance and multiple-drug resistance. Gene cluster srpRSABC, an important efflux system in the solvent tolerance of the S12 strain, was also found in the genome (7, 14, 15). Moreover, 53 CDSs encoding biosynthesis of fatty acids were also annotated. There are 195 CDSs annotated for the metabolism of aromatic compounds.
November 2012 Volume 194 Number 21
Some of these CDSs are arranged as a larger gene cluster similar to the reported styrene degradation gene cluster, which is consistent with the degradation ability of the S12 strain (3, 11). Meanwhile, 180 CDSs encoding stress response were annotated, which should be further investigated for their roles in the solvent adaption. We also annotated 53 CDSs related to capsular and extracellular polysaccharides, which are corresponding to the resistance of Pseudomonas putida strains. Nucleotide sequence accession numbers. This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number ALNR00000000. The version described in this paper is the first version, ALNR01000000. ACKNOWLEDGMENTS We acknowledge Huajun Zheng and his colleagues for genome sequencing performed at Chinese National Human Genome Center at Shanghai. We also acknowledge Gerben J. Zylstra (Rutgers University, Germany) for providing us the S12 strain isolated by S. Hartmans (Agricultural University, Netherlands). The work was partially supported by the Chinese National Natural Science Foundation (31100078 and 30821005) and by the Open Funding Project of the State Key Laboratory of Bioreactor Engineering.
REFERENCES 1. Aziz RK, et al. 2008. The RAST server: rapid annotations using subsystems technology. BMC Genomics 9:75. 2. Hall N. 2007. Advanced sequencing technologies and their wider impact in microbiology. J. Exp. Biol. 210:1518 –1525. 3. Hartmans S, van der Werf MJ, de Bont JAM. 1990. Bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase. Appl. Environ. Microbiol. 56:1347–1351. 4. Hatti-Kaul R, Tornvall U, Gustafsson L, Borjesson P. 2007. Industrial biotechnology for the production of bio-based chemicals—a cradle-tograve perspective. Trends Biotechnol. 25:119 –124. 5. Inoue A, Horikoshi K. 1989. A Pseudomonas thrives in high concentrations of toluene. Nature 338:264 –266. 6. Isken S, de Bont JAM. 1998. Bacteria tolerant to organic solvents. Extremophiles 2:229 –238. 7. Kieboom J, de Bont JAM. 2001. Identification and molecular characterization of an efflux system involved in Pseudomonas putida S12 multidrug resistance. Microbiology 147:43–51.
Received 15 August 2012 Accepted 17 August 2012 Address correspondence to Ping Xu,
[email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JB.01482-12
Journal of Bacteriology
p. 5985–5986
jb.asm.org
5985
Genome Announcement
8. Kieboom J, Dennis JJ, de Bont JAM, Zylstra GJ. 1998. Identification and molecular characterization of an efflux pump involved in Pseudomonas putida S12 solvent tolerance. J. Biol. Chem. 273:85–91. 9. Koopman FW, de Winde JH, Ruijssenaars HJ. 2009. C(1) compounds as auxiliary substrate for engineered Pseudomonas putida S12. Appl. Microbiol. Biotechnol. 83:705–713. 10. Nijkamp K, van Luijk N, de Bont JAM, Wery J. 2005. The solventtolerant Pseudomonas putida S12 as host for the production of cinnamic acid from glucose. Appl. Microbiol. Biotechnol. 69:170 –177. 11. Panke S, Witholt B, Schmid A, Wubbolts MG. 1998. Towards a biocatalyst for (S)-styrene oxide production: characterization of the styrene degradation pathway of Pseudomonas sp. strain VLB120. Appl. Environ. Microbiol. 64:2032–2043. 12. Qi WW, et al. 2007. Functional expression of prokaryotic and eukaryotic genes in Escherichia coli for conversion of glucose to p-hydroxystyrene. Metab. Eng. 9:268 –276. 13. Schmid A, et al. 2001. Industrial biocatalysis today and tomorrow. Nature 409:258 –268. 14. Sun X, Dennis JJ. 2009. A novel insertion sequence derepresses efflux pump expression and preadapts Pseudomonas putida S12 for extreme solvent stress. J. Bacteriol. 191:6773– 6777. 15. Sun X, Zahir Z, Lynch KH, Dennis JJ. 2011. An antirepressor, SrpR, is
5986
jb.asm.org
16.
17. 18. 19.
20.
21.
22.
involved in transcriptional regulation of the SrpABC solvent tolerance efflux pump of Pseudomonas putida S12. J. Bacteriol. 193:2717–2725. Tao F, et al. 2011. Novel organic solvent-responsive expression vectors for biocatalysis: application for development of an organic solventtolerant biodesulfurizing strain. Bioresour. Technol. 102:9380 –9387. Tao F, et al. 2011. Genome sequence of Pseudomonas putida Idaho, a unique organic-solvent-tolerant bacterium. J. Bacteriol. 193:7011–7012. Tao F, Yu B, Xu P, Ma CQ. 2006. Biodesulfurization in biphasic systems containing organic solvents. Appl. Environ. Microbiol. 72:4604 – 4609. Verhoef S, Wierckx N, Westerhof RGM, de Winde JH, Ruijssenaars HJ. 2009. Bioproduction of p-hydroxystyrene from glucose by the solventtolerant bacterium Pseudomonas putida S12 in a two-phase water-decanol fermentation. Appl. Environ. Microbiol. 75:931–936. Wierckx NJP, Ballerstedt H, de Bont JAM, Wery J. 2005. Engineering of solvent-tolerant Pseudomonas putida S12 for bioproduction of phenol from glucose. Appl. Environ. Microbiol. 71:8221– 8227. Wijte D, van Baar BLM, Heck AJR, Altelaar AFM. 2011. Probing the proteome response to toluene exposure in the solvent tolerant Pseudomonas putida S12. J. Proteome Res. 10:394 – 403. Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18:821– 829.
Journal of Bacteriology
