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Chapter 10 : Resuscitation of “Uncultured” Microorganisms

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Abstract:

As this chapter is about the resuscitation of uncultured or nonculturable microbes, and such microbes that are successfully resuscitated must by definition have been dormant or latent, it concentrates on the issue, particularly with reference to the actinobacteria that are the source of most of the bioactive secondary metabolites of industrial or applied interest. It shows the loss of culturability of as a decrease in the plate count at more or less constant total count. It also shows what at first sight appears to be the resuscitation of most of these previously unculturable cells (which could not form a colony in a plate count assay) as an increase in culturable (plate) count. For different cultures, the presence of appropriate concentrations of supernatant increased the culturable count of starved cells in an most-probable-number (MPN) assay by 3 to 5 orders of magnitude: the supernatant contained a resuscitation-promoting factor (Rpf) produced by culturable cells. Pheromones are substances produced by an organism that have specific effects on other organisms of the same species; although the presence of pheromones in prokaryotes was not widely recognized at the time when the existence of Rpf was proposed , this was a clear example of pheromonal activity. Given the resuscitation assay for the Rpf, the authors were able to purify it to homogeneity and thus to characterize it. If individual microbes make cell signals that can resuscitate other organisms, this raises a number of evolutionary issues.

Citation: Kell D, Mukamolova G, Finan C, Zhao H, Goodacre R, Young M, Kaprelyants A. 2004. Resuscitation of “Uncultured” Microorganisms, p 100-108. In Bull A (ed), Microbial Diversity and Bioprospecting. ASM Press, Washington, DC. doi: 10.1128/9781555817770.ch10

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Micrococcus luteus
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Gram-Positive Bacteria
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Escherichia coli
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Figures

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Figure 1

Dormancy and resuscitation in (A) Changes in the viable and total counts of grown in batch culture and subjected to starvation. Cells were grown and starved, and the total (microscopic) and viable (plate) counts were measured as described by . Time zero corresponds to the onset of the stationary phase. (B) Changes in viable and total counts and the percentage of small cells during resuscitation of a starved culture of Cells were starved for 75 days, incubated with penicillin G for 10 hours, washed, and resuscitated by the addition of growth medium as described by . Total counts, open circles; viable counts, closed circles; percentage of small cells (<0.5 μm in diameter), squares.

Citation: Kell D, Mukamolova G, Finan C, Zhao H, Goodacre R, Young M, Kaprelyants A. 2004. Resuscitation of “Uncultured” Microorganisms, p 100-108. In Bull A (ed), Microbial Diversity and Bioprospecting. ASM Press, Washington, DC. doi: 10.1128/9781555817770.ch10
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Figure 2

Schematic of the organization of the five Rpf homologs present in the genome.

Citation: Kell D, Mukamolova G, Finan C, Zhao H, Goodacre R, Young M, Kaprelyants A. 2004. Resuscitation of “Uncultured” Microorganisms, p 100-108. In Bull A (ed), Microbial Diversity and Bioprospecting. ASM Press, Washington, DC. doi: 10.1128/9781555817770.ch10
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Figure 3

Sequence and alignment of Rpf with the five Rpf-like gene products found in

Citation: Kell D, Mukamolova G, Finan C, Zhao H, Goodacre R, Young M, Kaprelyants A. 2004. Resuscitation of “Uncultured” Microorganisms, p 100-108. In Bull A (ed), Microbial Diversity and Bioprospecting. ASM Press, Washington, DC. doi: 10.1128/9781555817770.ch10
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Tables

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Table 1

Some organisms that have been shown to contain Rpf-like genes, with the number of homologs known to date

Citation: Kell D, Mukamolova G, Finan C, Zhao H, Goodacre R, Young M, Kaprelyants A. 2004. Resuscitation of “Uncultured” Microorganisms, p 100-108. In Bull A (ed), Microbial Diversity and Bioprospecting. ASM Press, Washington, DC. doi: 10.1128/9781555817770.ch10

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