1887

Abstract

The survival, physiology and gene expression profile of the phenanthrene-degrading sp. LH128 was examined after an extended period of complete nutrient starvation and compared with a non-starved population that had been harvested in exponential phase. After 6 months of starvation in an isotonic solution, only 5 % of the initial population formed culturable cells. Microscopic observation of GFP fluorescent cells, however, suggested that a larger fraction of cells (up to 80 %) were still alive and apparently had entered a viable but non-culturable (VBNC) state. The strain displayed several cellular and genetic adaptive strategies to survive long-term starvation. Flow cytometry, microscopic observation and fatty acid methyl ester (FAME) analysis showed a reduction in cell size, a change in cell shape and an increase in the degree of membrane fatty acid saturation. Transcriptome analysis showed decreased expression of genes involved in ribosomal protein biosynthesis, chromosomal replication, cell division and aromatic catabolism, increased expression of genes involved in regulation of gene expression and efflux systems, genetic translocations, and degradation of rRNA and fatty acids. Those phenotypic and transcriptomic changes were not observed after 4 h of starvation. Despite the starvation situation, the polycyclic aromatic hydrocarbon (PAH) catabolic activity was immediate upon exposure to phenanthrene. We conclude that a large fraction of cells maintain viability after an extended period of starvation apparently due to tuning the expression of a wide variety of cellular processes. Due to these survival attributes, bacteria of the genus , like strain LH128, could be considered as suitable targets for use in remediation of nutrient-poor PAH-contaminated environments.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.065870-0
2013-09-01
2024-10-03
Loading full text...

Full text loading...

/deliver/fulltext/micro/159/9/1807.html?itemId=/content/journal/micro/10.1099/mic.0.065870-0&mimeType=html&fmt=ahah

References

  1. Anderson K. L., Dunman P. M. ( 2009). Messenger RNA turnover processes in Escherichia coli, Bacillus subtilis, and emerging studies in Staphylococcus aureus . Int J Microbiol 2009:525491[PubMed] [CrossRef]
    [Google Scholar]
  2. Atkinson G. C., Tenson T., Hauryliuk V. ( 2011). The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life. PLoS ONE 6:e23479 [View Article][PubMed]
    [Google Scholar]
  3. Bastiaens L., Springael D., Wattiau P., Harms H., deWachter R., Verachtert H., Diels L. ( 2000). Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers. Appl Environ Microbiol 66:1834–1843 [View Article][PubMed]
    [Google Scholar]
  4. Betts J. C., Lukey P. T., Robb L. C., McAdam R. A., Duncan K. ( 2002). Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol Microbiol 43:717–731 [View Article][PubMed]
    [Google Scholar]
  5. Bligh E. G., Dyer W. J. ( 1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917 [View Article][PubMed]
    [Google Scholar]
  6. Brazma A., Hingamp P., Quackenbush J., Sherlock G., Spellman P., Stoeckert C., Aach J., Ansorge W., Ball C. A. & other authors ( 2001). Minimum information about a microarray experiment (MIAME) – toward standards for microarray data. Nat Genet 29:365–371 [View Article][PubMed]
    [Google Scholar]
  7. Britos L., Abeliuk E., Taverner T., Lipton M., McAdams H., Shapiro L. ( 2011). Regulatory response to carbon starvation in Caulobacter crescentus . PLoS ONE 6:e18179 [View Article][PubMed]
    [Google Scholar]
  8. Chang D. E., Smalley D. J., Conway T. ( 2002). Gene expression profiling of Escherichia coli growth transitions: an expanded stringent response model. Mol Microbiol 45:289–306 [View Article][PubMed]
    [Google Scholar]
  9. Chatterji D., Ojha A. K. ( 2001). Revisiting the stringent response, ppGpp and starvation signaling. Curr Opin Microbiol 4:160–165 [View Article][PubMed]
    [Google Scholar]
  10. Chen C., Deutscher M. P. ( 2005). Elevation of RNase R in response to multiple stress conditions. J Biol Chem 280:34393–34396 [View Article][PubMed]
    [Google Scholar]
  11. Christensen S. K., Mikkelsen M., Pedersen K., Gerdes K. ( 2001). RelE, a global inhibitor of translation, is activated during nutritional stress. Proc Natl Acad Sci U S A 98:14328–14333 [View Article][PubMed]
    [Google Scholar]
  12. Corish P., Tyler-Smith C. ( 1999). Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng 12:1035–1040 [View Article][PubMed]
    [Google Scholar]
  13. Denich T. J., Beaudette L. A., Lee H., Trevors J. T. ( 2003). Effect of selected environmental and physico-chemical factors on bacterial cytoplasmic membranes. J Microbiol Methods 52:149–182 [View Article][PubMed]
    [Google Scholar]
  14. Deutscher M. P. ( 2003). Degradation of stable RNA in bacteria. J Biol Chem 278:45041–45044 [View Article][PubMed]
    [Google Scholar]
  15. Deutscher M. P. ( 2006). Degradation of RNA in bacteria: comparison of mRNA and stable RNA. Nucleic Acids Res 34:659–666 [View Article][PubMed]
    [Google Scholar]
  16. Edgar R., Domrachev M., Lash A. E. ( 2002). Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30:207–210 [View Article][PubMed]
    [Google Scholar]
  17. Eguchi M., Nishikawa T., Macdonald K., Cavicchioli R., Gottschal J. C., Kjelleberg S. ( 1996). Responses to stress and nutrient availability by the marine ultramicrobacterium Sphingomonas sp. strain RB2256. Appl Environ Microbiol 62:1287–1294[PubMed]
    [Google Scholar]
  18. Fiala Z., Vyskocil A., Kraják V., Masín V., Emminger S., Srb V., Tejral J. ( 1999). Polycyclic aromatic hydrocarbons. I. Environmental contamination and environmental exposure. Acta Medica (Hradec Kralove) Suppl 42:Suppl.77–89 (in Czech)[PubMed]
    [Google Scholar]
  19. Fida T. T., Breugelmans P., Lavigne R., Coronado E., Johnson D. R., van der Meer J. R., Mayer A. P., Heipieper H. J., Hofkens J., Springael D. ( 2012). Exposure to solute stress affects genome-wide expression but not the polycyclic aromatic hydrocarbon-degrading activity of Sphingomonas sp. strain LH128 in biofilms. Appl Environ Microbiol 78:8311–8320 [View Article][PubMed]
    [Google Scholar]
  20. Foster P. L. ( 2007). Stress-induced mutagenesis in bacteria. Crit Rev Biochem Mol Biol 42:373–397 [View Article][PubMed]
    [Google Scholar]
  21. Givskov M., Eberl L., Møller S., Poulsen L. K., Molin S. ( 1994). Responses to nutrient starvation in Pseudomonas putida KT2442: analysis of general cross-protection, cell shape, and macromolecular content. J Bacteriol 176:7–14[PubMed]
    [Google Scholar]
  22. Golding I., Paulsson J., Zawilski S. M., Cox E. C. ( 2005). Real-time kinetics of gene activity in individual bacteria. Cell 123:1025–1036 [View Article][PubMed]
    [Google Scholar]
  23. Heipieper H. J., de Bont J. A. M. ( 1994). Adaptation of Pseudomonas putida S12 to ethanol and toluene at the level of fatty acid composition of membranes. Appl Environ Microbiol 60:4440–4444[PubMed]
    [Google Scholar]
  24. Heipieper H. J., Meinhardt F., Segura A. ( 2003). The cis-trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism. FEMS Microbiol Lett 229:1–7 [View Article][PubMed]
    [Google Scholar]
  25. Herbert K. C., Foster S. J. ( 2001). Starvation survival in Listeria monocytogenes: characterization of the response and the role of known and novel components. Microbiology 147:2275–2284[PubMed]
    [Google Scholar]
  26. Ishige T., Krause M., Bott M., Wendisch V. F., Sahm H. ( 2003). The phosphate starvation stimulon of Corynebacterium glutamicum determined by DNA microarray analyses. J Bacteriol 185:4519–4529 [View Article][PubMed]
    [Google Scholar]
  27. Ishino F., Park W., Tomioka S., Tamaki S., Takase I., Kunugita K., Matsuzawa H., Asoh S., Ohta T. & other authors ( 1986). Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein. J Biol Chem 261:7024–7031[PubMed]
    [Google Scholar]
  28. Johnsen A. R., Winding A., Karlson U., Roslev P. ( 2002). Linking of microorganisms to phenanthrene metabolism in soil by analysis of 13C-labeled cell lipids. Appl Environ Microbiol 68:6106–6113 [View Article][PubMed]
    [Google Scholar]
  29. Johnson D. R., Coronado E., Moreno-Forero S. K., Heipieper H. J., van der Meer J. R. ( 2011). Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii . BMC Microbiol 11:250 [View Article][PubMed]
    [Google Scholar]
  30. Jones M. D., Crandell D. W., Singleton D. R., Aitken M. D. ( 2011). Stable-isotope probing of the polycyclic aromatic hydrocarbon-degrading bacterial guild in a contaminated soil. Environ Microbiol 13:2623–2632 [View Article][PubMed]
    [Google Scholar]
  31. Kenyon W. J., Sayers D. G., Humphreys S., Roberts M., Spector M. P. ( 2002). The starvation-stress response of Salmonella enterica serovar Typhimurium requires σE-, but not CpxR-regulated extracytoplasmic functions. Microbiology 148:113–122[PubMed]
    [Google Scholar]
  32. Kieft T. L., Wilch E., O’connor K., Ringelberg D. B., White D. C. ( 1997). Survival and phospholipid fatty acid profiles of surface and subsurface bacteria in natural sediment microcosms. Appl Environ Microbiol 63:1531–1542[PubMed]
    [Google Scholar]
  33. Kolter R., Siegele D. A., Tormo A. ( 1993). The stationary phase of the bacterial life cycle. Annu Rev Microbiol 47:855–874 [View Article][PubMed]
    [Google Scholar]
  34. Kushner S. R. ( 2002). mRNA decay in Escherichia coli comes of age. J Bacteriol 184:4658–4665, discussion 4657 [View Article][PubMed]
    [Google Scholar]
  35. Lamoureux E. M., Brownawell B. J. ( 1999). Chemical and biological availability of sediment-sorbed hydrophobic organic contaminants. Environ Toxicol Chem 18:1733–1741 [View Article]
    [Google Scholar]
  36. Leys N. M., Ryngaert A., Bastiaens L., Verstraete W., Top E. M., Springael D. ( 2004). Occurrence and phylogenetic diversity of Sphingomonas strains in soils contaminated with polycyclic aromatic hydrocarbons. Appl Environ Microbiol 70:1944–1955 [View Article][PubMed]
    [Google Scholar]
  37. Li G.-Y., Zhang Y., Inouye M., Ikura M. ( 2009). Inhibitory mechanism of Escherichia coli RelE–RelB toxin-antitoxin module involves a helix displacement near an mRNA interferase active site. J Biol Chem 284:14628–14636 [View Article][PubMed]
    [Google Scholar]
  38. Liu M., Zhang Y., Inouye M., Woychik N. A. ( 2008). Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit. Proc Natl Acad Sci U S A 105:5885–5890 [View Article][PubMed]
    [Google Scholar]
  39. Lowder M., Unge A., Maraha N., Jansson J. K., Swiggett J., Oliver J. D. ( 2000). Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506. Appl Environ Microbiol 66:3160–3165 [View Article][PubMed]
    [Google Scholar]
  40. Manasherob R., Miller C., Kim K. S., Cohen S. N. ( 2012). Ribonuclease E modulation of the bacterial SOS response. PLoS ONE 7:e38426 [View Article][PubMed]
    [Google Scholar]
  41. Mandel M. J., Silhavy T. J. ( 2005). Starvation for different nutrients in Escherichia coli results in differential modulation of RpoS levels and stability. J Bacteriol 187:434–442 [View Article][PubMed]
    [Google Scholar]
  42. Matin A. ( 1991). The molecular basis of carbon-starvation-induced general resistance in Escherichia coli . Mol Microbiol 5:3–10 [View Article][PubMed]
    [Google Scholar]
  43. Morita R. Y. ( 1988). Bioavailability of energy and its relationship to growth and starvation survival in nature. Can J Microbiol 34:436–441 [View Article]
    [Google Scholar]
  44. Mrozik A., Piotrowska-Seget Z., Labuzek S. ( 2004). Cytoplasmatic bacterial membrane responses to environmental perturbations. Pol J Environ Stud 13:487–494
    [Google Scholar]
  45. Noirclerc-Savoye M., Morlot C., Gérard P., Vernet T., Zapun A. ( 2003). Expression and purification of FtsW and RodA from Streptococcus pneumoniae, two membrane proteins involved in cell division and cell growth, respectively. Protein Expr Purif 30:18–25 [View Article][PubMed]
    [Google Scholar]
  46. Peterson C. N., Mandel M. J., Silhavy T. J. ( 2005). Escherichia coli starvation diets: essential nutrients weigh in distinctly. J Bacteriol 187:7549–7553 [View Article][PubMed]
    [Google Scholar]
  47. Sato S., Imamura S., Ozeki Y., Kawaguchi A. ( 1992). Induction of enzymes involved in fatty acid β-oxidation in Pseudomonas fragi B-0771 cells grown in media supplemented with fatty acid. J Biochem 111:16–19[PubMed]
    [Google Scholar]
  48. Selinger D. W., Saxena R. M., Cheung K. J., Church G. M., Rosenow C. ( 2003). Global RNA half-life analysis in Escherichia coli reveals positional patterns of transcript degradation. Genome Res 13:216–223 [View Article][PubMed]
    [Google Scholar]
  49. Sjøholm O. R., Nybroe O., Aamand J., Sørensen J. ( 2010). 2,6-Dichlorobenzamide (BAM) herbicide mineralisation by Aminobacter sp. MSH1 during starvation depends on a subpopulation of intact cells maintaining vital membrane functions. Environ Pollut 158:3618–3625 [View Article][PubMed]
    [Google Scholar]
  50. Srivatsan A., Wang J. D. ( 2008). Control of bacterial transcription, translation and replication by (p)ppGpp. Curr Opin Microbiol 11:100–105 [View Article][PubMed]
    [Google Scholar]
  51. Suutari M., Laakso S. ( 1994). Microbial fatty acids and thermal adaptation. Crit Rev Microbiol 20:285–328 [View Article][PubMed]
    [Google Scholar]
  52. Takeuchi M., Hamana K., Hiraishi A. ( 2001). Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 51:1405–1417[PubMed]
    [Google Scholar]
  53. Tatusov R. L., Koonin E. V., Lipman D. J. ( 1997). A genomic perspective on protein families. Science 278:631–637 [View Article][PubMed]
    [Google Scholar]
  54. Thorne S. H., Williams H. D. ( 1997). Adaptation to nutrient starvation in Rhizobium leguminosarum bv. phaseoli: analysis of survival, stress resistance, and changes in macromolecular synthesis during entry to and exit from stationary phase. J Bacteriol 179:6894–6901[PubMed]
    [Google Scholar]
  55. Traxler M. F., Summers S. M., Nguyen H.-T., Zacharia V. M., Hightower G. A., Smith J. T., Conway T. ( 2008). The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli . Mol Microbiol 68:1128–1148 [View Article][PubMed]
    [Google Scholar]
  56. Trevors J. T. ( 2012). Can dead bacterial cells be defined and are genes expressed after cell death?. J Microbiol Methods 90:25–28 [View Article][PubMed]
    [Google Scholar]
  57. Trevors J. T., Elsas J. D., Bej A. K. ( 2012). The molecularly crowded cytoplasm of bacterial cells: dividing cells contrasted with viable but non-culturable (VBNC) bacterial cells. Curr Issues Mol Biol 15:1–6[PubMed]
    [Google Scholar]
  58. Uyttebroek M., Ortega-Calvo J. J., Breugelmans P., Springael D. ( 2006). Comparison of mineralization of solid-sorbed phenanthrene by polycyclic aromatic hydrocarbon (PAH)-degrading Mycobacterium spp. and Sphingomonas spp.. Appl Microbiol Biotechnol 72:829–836 [View Article][PubMed]
    [Google Scholar]
  59. van Overbeek L. S., Eberl L., Givskov M., Molin S., van Elsas J. D. ( 1995). Survival of, and induced stress resistance in, carbon-starved Pseudomonas fluorescens cells residing in soil. Appl Environ Microbiol 61:4202–4208[PubMed]
    [Google Scholar]
  60. Vercruysse M., Fauvart M., Jans A., Beullens S., Braeken K., Cloots L., Engelen K., Marchal K., Michiels J. ( 2011). Stress response regulators identified through genome-wide transcriptome analysis of the (p)ppGpp-dependent response in Rhizobium etli . Genome Biol 12:R17 [View Article][PubMed]
    [Google Scholar]
  61. Vestergåd M., Ekelund F., Winding A., Jacobsen C. S., Christensen S. ( 2011). Starved bacteria retain their size but lose culturability: lessons from a 5000 years old undisturbed A-horizon. Soil Biol Biochem 43:1379–1382 [View Article]
    [Google Scholar]
  62. Vorob’eva L. I. ( 2004). Stressors, stress reactions, and survival of bacteria (a review). Prikl Biokhim Mikrobiol 40:261–269[PubMed]
    [Google Scholar]
  63. Wouters K., Maes E., Spitz J. A., Roeffaers M. B. J., Wattiau P., Hofkens J., Springael D. ( 2010). A non-invasive fluorescent staining procedure allows confocal laser scanning microscopy based imaging of Mycobacterium in multispecies biofilms colonizing and degrading polycyclic aromatic hydrocarbons. J Microbiol Methods 83:317–325 [View Article][PubMed]
    [Google Scholar]
  64. Wu B., Wawrzynow A., Zylicz M., Georgopoulos C. ( 1996). Structure-function analysis of the Escherichia coli GrpE heat shock protein. EMBO J 15:4806–4816[PubMed]
    [Google Scholar]
  65. Yabuuchi E., Yamamoto H., Terakubo S., Okamura N., Naka T., Fujiwara N., Kobayashi K., Kosako Y., Hiraishi A. ( 2001). Proposal of Sphingomonas wittichii sp. nov. for strain RW1T, known as a dibenzo-p-dioxin metabolizer. Int J Syst Evol Microbiol 51:281–292[PubMed]
    [Google Scholar]
  66. Yuan Z. C., Zaheer R., Finan T. M. ( 2006). Regulation and properties of PstSCAB, a high-affinity, high-velocity phosphate transport system of Sinorhizobium meliloti . J Bacteriol 188:1089–1102 [View Article][PubMed]
    [Google Scholar]
  67. Zhang Y., Inouye M. ( 2009). The inhibitory mechanism of protein synthesis by YoeB, an Escherichia coli toxin. J Biol Chem 284:6627–6638 [View Article][PubMed]
    [Google Scholar]
  68. Zundel M. A., Basturea G. N., Deutscher M. P. ( 2009). Initiation of ribosome degradation during starvation in Escherichia coli . RNA 15:977–983 [View Article][PubMed]
    [Google Scholar]
/content/journal/micro/10.1099/mic.0.065870-0
Loading
/content/journal/micro/10.1099/mic.0.065870-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error