1887

Abstract

strain UT26, whose γ-hexachlorocyclohexane-degrading ability has been studied in detail, is a typical aerobic and heterotrophic bacterium that needs organic carbon sources for its growth, and cannot grow on a minimal salt agar medium prepared without adding any organic carbon sources. Here, we isolated a mutant of UT26 with the ability to grow to visible state on such an oligotrophic medium from a transposon-induced mutant library. This high-yield growth under oligotrophic conditions (HYGO) phenotype was CO-dependent and accompanied with CO incorporation. In the HYGO mutant, a transposon was inserted just upstream of the putative Zn-dependent alcohol dehydrogenase (ADH) gene () so that the gene was constitutively expressed, probably by the transposon-derived promoter. The -deletion mutant (UT26DAX) harbouring a plasmid carrying the gene under the control of a constitutive promoter exhibited the HYGO phenotype. Moreover, the HYGO mutants spontaneously emerged among the UT26-derived hypermutator strain cells, and was highly expressed in these HYGO mutants, while no HYGO mutant appeared among UT26DAX-derived hypermutator strain cells, indicating the necessity of for the HYGO phenotype. His-tagged AdhX that was expressed in and purified to homogeneity showed ADH activity towards methanol and other alcohols. Mutagenesis analysis of the gene indicated a correlation between the ADH activity and the HYGO phenotype. These results demonstrated that the constitutive expression of an -encoding protein with ADH activity in UT26 leads to the CO-dependent HYGO phenotype. Identical or nearly identical orthologues were found in other sphingomonad strains, and most of them were located on plasmids, suggesting that the -mediated HYGO phenotype may be an important adaptation strategy to oligotrophic environments among sphingomonads.

Funding
This study was supported by the:
  • Yuji Nagata , Institute for Fermentation, Osaka (IFO)
  • Masataka Tsuda , Grants-in-Aid for Scientific Research (B) from JSPS , (Award 17H03781)
  • Yuji Nagata , Grants-in-Aid for Scientific Research (B) from JSPS , (Award 19H02865)
  • Yuji Nagata , Society of the Friendly Sons of St. Patrick for the Relief of Emigrants from Ireland , (Award no. 24658068, 26660054, and 16K14877)
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2020-04-20
2020-06-04
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References

  1. Nagata Y, Endo R, Ito M, Ohtsubo Y, Tsuda M. Aerobic degradation of lindane (γ-hexachlorocyclohexane) in bacteria and its biochemical and molecular basis. Appl Microbiol Biotechnol 2007; 76: 741 752 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  2. Nagata Y, Natsui S, Endo R, Ohtsubo Y, Ichikawa N et al. Genomic organization and genomic structural rearrangements of Sphingobium japonicum UT26, an archetypal γ-hexachlorocyclohexane-degrading bacterium. Enzyme Microb Technol 2011; 49: 499 508 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  3. Endo R, Kamakura M, Miyauchi K, Fukuda M, Ohtsubo Y et al. Identification and characterization of genes involved in the downstream degradation pathway of gamma-hexachlorocyclohexane in Sphingomonas paucimobilis UT26. J Bacteriol 2005; 187: 847 853 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  4. Endo R, Ohtsubo Y, Tsuda M, Nagata Y. Identification and characterization of genes encoding a putative ABC-type transporter essential for utilization of gamma-hexachlorocyclohexane in Sphingobium japonicum UT26. J Bacteriol 2007; 189: 3712 3720 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  5. Boutte CC, Crosson S. Bacterial lifestyle shapes stringent response activation. Trends Microbiol 2013; 21: 174 180 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  6. Kuznetsov SI, Dubinina GA, Lapteva NA. Biology of oligotrophic bacteria. Annu Rev Microbiol 1979; 33: 377 387 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  7. Schut F, Prins RA, Gottschal JC. Oligotrophy and pelagic marine bacteria: facts and fiction. Aquat Microb Ecol 1997; 12: 177 202 [CrossRef]
    [Google Scholar]
  8. Yoshida N, Inaba S, Takagi H. Utilization of atmospheric ammonia by an extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4. J Biosci Bioeng 2014; 117: 28 32 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  9. Yoshida N, Ohhata N, Yoshino Y, Katsuragi T, Tani Y et al. Screening of carbon dioxide-requiring extreme oligotrophs from soil. Biosci Biotechnol Biochem 2007; 71: 2830 2832 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  10. Ohhata N, Yoshida N, Egami H, Katsuragi T, Tani Y et al. An extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4, isolated from crude oil. J Bacteriol 2007; 189: 6824 6831 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  11. Yano T, Yoshida N, Yu F, Wakamatsu M, Takagi H. The glyoxylate shunt is essential for CO2-requiring oligotrophic growth of Rhodococcus erythropolis N9T-4. Appl Microbiol Biotechnol 2015; 99: 5627 5637 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  12. Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning : a Laboratory Manual Cold Spring Harbor, N Y: Cold Spring Harbor Laboratory; 1982
    [Google Scholar]
  13. Imai R, Nagata Y, Senoo K, Wada H, Fukuda M et al. Dehydrochlorination of γ-hexachlorocyclohexane (γ-BHC) by γ-BHC-assimilating Pseudomonas paucimobilis . Agric Biol Chem 1989; 53: 2015 2017
    [Google Scholar]
  14. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: a Laboratory Manual , 2nd ed. Cold Spring Harbor, N.Y., USA: Cold Spring Harbor laboratory Press; 1989
    [Google Scholar]
  15. Ohtsubo Y, Ikeda-Ohtsubo W, Nagata Y, Tsuda M. GenomeMatcher: a graphical user interface for DNA sequence comparison. BMC Bioinformatics 2008; 9: 376 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  16. Dennis JJ, Zylstra GJ. Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. Appl Environ Microbiol 1998; 64: 2710 2715 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  17. Nishiyama E, Ohtsubo Y, Nagata Y, Tsuda M. Identification of Burkholderia multivorans ATCC 17616 genes induced in soil environment by in vivo expression technology. Environ Microbiol 2010; 12: 2539 2558 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  18. Guzmán C, Bagga M, Kaur A, Westermarck J, Abankwa D. ColonyArea: an ImageJ plugin to automatically quantify colony formation in clonogenic assays. PLoS One 2014; 9: e92444 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  19. Kahm M, Hasenbrink G, Lichtenberg-Fraté H, Ludwig J, Kschischo M. Grofit: Fitting biological growth curves with R. J Stat Softw 2010; 33: 36093 [CrossRef]
    [Google Scholar]
  20. Kaczmarczyk A, Vorholt JA, Francez-Charlot A. Markerless gene deletion system for sphingomonads. Appl Environ Microbiol 2012; 78: 3774 3777 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  21. Itakura M, Tabata K, Eda S, Mitsui H, Murakami K et al. Generation of Bradyrhizobium japonicum mutants with increased N2O reductase activity by selection after introduction of a mutated dnaQ gene. Appl Environ Microbiol 2008; 74: 7258 7264 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  22. Kovach ME, Phillips RW, Elzer PH, Roop RM, Peterson KM. pBBR1MCS: a broad-host-range cloning vector. Biotechniques 1994; 16: 800 802 [PubMed] [PubMed]
    [Google Scholar]
  23. Ito M, Prokop Z, Klvana M, Otsubo Y, Tsuda M et al. Degradation of β-hexachlorocyclohexane by haloalkane dehalogenase LinB from gamma-hexachlorocyclohexane-utilizing bacterium Sphingobium sp. MI1205. Arch Microbiol 2007; 188: 313 325 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  24. Robinson GA, Bailey CJ, Dowds BC. Gene structure and amino acid sequences of alcohol dehydrogenases of Bacillus stearothermophilus . Biochim Biophys Acta 1994; 1218: 432 434 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  25. Willis LB, Walker GC. Identification of the Rhizobium meliloti alcohol dehydrogenase gene (adhA) and heterologous expression in Alcaligenes eutrophus . Biochim Biophys Acta 1998; 1384: 197 203 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  26. Bar-Even A, Noor E, Milo R. A survey of carbon fixation pathways through a quantitative lens. J Exp Bot 2012; 63: 2325 2342 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  27. Hügler M, Sievert SM. Beyond the Calvin cycle: autotrophic carbon fixation in the ocean. Ann Rev Mar Sci 2011; 3: 261 289 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  28. Seto M, Alexander M. Effect of bacterial density and substrate concentration on yield coefficients. Appl Environ Microbiol 1985; 50: 1132 1136 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  29. Tarao M, Seto M. Estimation of the yield coefficient of Pseudomonas sp. strain DP-4 with a low substrate (2,4-dichlorophenol [DCP]) concentration in a mineral medium from which uncharacterized organic compounds were eliminated by a non-DCP-degrading organism. Appl Environ Microbiol 2000; 66: 566 570 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  30. Tabata M, Ohhata S, Kawasumi T, Nikawadori Y, Kishida K et al. Complete genome sequence of a γ-hexachlorocyclohexane degrader, Sphingobium sp. strain TKS, isolated from a γ-hexachlorocyclohexane-degrading microbial community. Genome Announc 2016; 4: e00247 16 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  31. Tabata M, Ohhata S, Nikawadori Y, Sato T, Kishida K et al. Complete genome sequence of a γ-hexachlorocyclohexane-degrading bacterium, Sphingobium sp. strain MI1205. Genome Announc 2016; 4: e00246 16 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  32. Tabata M, Ohtsubo Y, Ohhata S, Tsuda M, Nagata Y. Complete genome sequence of the γ-hexachlorocyclohexane-degrading bacterium Sphingomonas sp. strain MM-1. Genome Announc 2013; 1: e00247 13 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  33. Nagata Y, Senbongi J, Ishibashi Y, Sudo R, Miyakoshi M et al. Identification of Burkholderia multivorans ATCC 17616 genetic determinants for fitness in soil by using signature-tagged mutagenesis. Microbiology 2014; 160: 883 891 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  34. Watsuji T-O, Kato T, Ueda K, Beppu T. CO2 supply induces the growth of Symbiobacterium thermophilum, a syntrophic bacterium. Biosci Biotechnol Biochem 2006; 70: 753 756 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  35. Yoshida N, Hayasaki T, Takagi H. Gene expression analysis of methylotrophic oxidoreductases involved in the oligotrophic growth of Rhodococcus erythropolis N9T-4. Biosci Biotechnol Biochem 2011; 75: 123 127 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  36. Lal R, Dogra C, Malhotra S, Sharma P, Pal R, Diversity PR. Diversity, distribution and divergence of Lin genes in hexachlorocyclohexane-degrading sphingomonads. Trends Biotechnol 2006; 24: 121 130 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  37. Stolz A. Molecular characteristics of xenobiotic-degrading sphingomonads. Appl Microbiol Biotechnol 2009; 81: 793 811 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  38. Stolz A. Degradative plasmids from sphingomonads. FEMS Microbiol Lett 2014; 350: 9 19 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  39. Tabata M, Ohhata S, Nikawadori Y, Kishida K, Sato T et al. Comparison of the complete genome sequences of four γ-hexachlorocyclohexane-degrading bacterial strains: insights into the evolution of bacteria able to degrade a recalcitrant man-made pesticide. DNA Res 2016; 23: 581 599 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  40. Copley SD, Rokicki J, Turner P, Daligault H, Nolan M et al. The whole genome sequence of Sphingobium chlorophenolicum L-1: insights into the evolution of the pentachlorophenol degradation pathway. Genome Biol Evol 2012; 4: 184 198 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  41. Miller TR, Delcher AL, Salzberg SL, Saunders E, Detter JC et al. Genome sequence of the dioxin-mineralizing bacterium Sphingomonas wittichii RW1. J Bacteriol 2010; 192: 6101 6102 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  42. Masai E, Katayama Y, Fukuda M. Genetic and biochemical investigations on bacterial catabolic pathways for lignin-derived aromatic compounds. Biosci Biotechnol Biochem 2007; 71: 1 15 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  43. Parthasarathy S, Azam S, Lakshman Sagar A, Narasimha Rao V, Gudla R et al. Genome-guided insights reveal organophosphate-degrading Brevundimonas diminuta as Sphingopyxis wildii and define its versatile metabolic capabilities and environmental adaptations. Genome Biol Evol 2017; 9: 77 81 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  44. Santini JM, Sly LI, Schnagl RD, Macy JM. A new chemolithoautotrophic arsenite-oxidizing bacterium isolated from a gold mine: phylogenetic, physiological, and preliminary biochemical studies. Appl Environ Microbiol 2000; 66: 92 97 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  45. White DC, Sutton SD, Ringelberg DB. The genus Sphingomonas: physiology and ecology. Curr Opin Biotechnol 1996; 7: 301 306 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  46. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA et al. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 1995; 166: 175 176 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
  47. Nagata Y, Futamura A, Miyauchi K, Takagi M. Two different types of dehalogenases, LinA and LinB, involved in the γ-HCH degradation in Sphingomonas paucimobilis UT26 are localized in the periplasmic space without molecular processing. J Bacteriol 1999; 181: 5409 5413 [CrossRef] [PubMed] [PubMed]
    [Google Scholar]
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