Journal
MICROBIOLOGY
Volume 78, Issue 5, Pages 585-592Publisher
MAIK NAUKA/INTERPERIODICA/SPRINGER
DOI: 10.1134/S0026261709050099
Keywords
nonculturable cells of Erwinia carotovora; autoregulators; ultrastructure; resuscitation; quantitative PCR analysis
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Funding
- Russian Foundation for Basic Research [08-04-01518-a, 09-0400915-a]
- President of the Russian Federation [NSh-5492.2008.4]
- Presidium of the Russian Academy of Sciences
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Reversible transition of the phytopathogenic gram-negative bacterium Erwinia carotovora, subsp. atroseptica, strain SCRI1043, to a dormant state was demonstrated; it was associated with a complete loss of cell ability to form colonies on the standard medium, i.e., with acquiring non-culturability. Entering of Erwinia cells to a nonculturable state occurred after lono-term incubation (100-150 days) of the stationary-phase cell suspensions in either a fresh complete medium or in the carbon-free mineral medium or treatment with a chemical analogue of microbial anabiosis autoinducers (4 x 10(-4) M of C-12-alkylhydroxybenzene, AHB). However, confocal laser microscopy of the cells stained with the Live/Dead BacLight kit revealed that the majority of E. carotovora cells (90%) from long-incubated suspensions retained membrane integrity. In these suspensions, round cells of smaller size prevailed, with the envelope, containing an electron-dense outer layer and an underlying layer of lower density; the cytoplasm was coarse-granulated. Detection of nonculturable E. carotovora cells by quantitative real-time PCR analysis (Q-PCR) with specific primers by using standard procedures of sample preparation was shown to be inefficient. A special procedure including cell washing from the incubation medium in the absence of growth stimulation was developed, which promoted recovery of the colony-forming ability of the cells (up to 10% of the initial CFU number) and improved cell detection by Q-PCR from the number of genomic copies. The results provided further insight into the ways of long-term survival of phytopathogenic bacteria under environmental changes and carbon starvation.
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