Evaluation of Two Culture-Independent Methods for Quantification of the Fraction of K-and r-Strategists within Water Microbial Communities
Master thesis
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http://hdl.handle.net/11250/2351608Utgivelsesdato
2015Metadata
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Sammendrag
The r/K concept is a theoretical concept that can serve as a tool in evaluating the risk of invasion by pathogenic bacterial species in rearing of marine fish larvae. In rearing of marine fish larvae, a dominance of opportunistic, fast-growing r-strategists can result in poor survival of the fish. Dominance of slow-growing K-strategists have been shown to increase the survival and growth. The r-strategists generally exhibits many copies of the 16S rRNA gene in their genomes, whereas the K-strategists have one or a few. Moreover, the maximum specific growth rate also correlates with the maximum RNA content of a cell. Fast-growing r-strategists thus have a higher maximum RNA content per cell, than the slow-growing K-strategists.
The aim of this study was to test two methods based upon this fact, as possible methods for determining the fraction of r-or K-strategists in seawater communities. One quantitative real-time PCR (rtPCR) method was used to quantify the number of genomic 16S rRNA genes. Maximum cellular RNA content was determined by a flow cytometry analysis of single cells after a nutrient pulse, to induce the cells to grow at maximum specific growth rate. The methods were established in pure cultures and then in intact microbial communities where a shift from K- to r-selection was established, with gradually a succession towards K-selection.
The study in pure bacterial strains confirmed the hypothesis of a correlation between maximum specific growth rate and 16S rRNA gene copy numbers per genome. Moreover, the maximum RNA content of bacterial cells correlated with maximum specific growth rate. In addition, the fast-growing r-strategist was found to divide within 8 hours incubation with nutrients at 15°C, whereas slow-growing K-strategist did not. The r-selected communities showed considerably higher maximum RNA content and higher 16S rRNA gene copy numbers per genome, than K-selected communities.
Of the two methods evaluated, the flow cytometry method was best suited to determine the fraction of r- or K-strategists within seawater microbial communities, e.g. rearing water for marine fish larvae. However, the method is based on a nutrient pulse to achieve maximum cellular RNA content, which is a disadvantage, because the optimal growth conditions for uncultivable cells are not identified. The rtPCR method lacked a precise normalization, and an accurate estimate of the 16S rRNA gene copy number per genome was thus not achieved. Ideal normalization could be achieved by using a single-copy protein gene and designing universal bacterial PCR primers targeting the gene. If a successful normalization by a single-copy gene is achieved, the rtPCR method, or possibly the proposed digital droplet PCR (ddPCR) method could be alternative methods to determine the fraction of r- or K-strategists in microbial communities. A method like this relies on analysis on DNA level, and would be cultivation independent, as opposed to the flow cytometry method.