In the 1960s-1980s, determination of bacterial growth rates was an important tool in microbial genetics, biochemistry, molecular biology, and microbial physiology. The exciting technical developments of the 1990s and the 2000s eclipsed that tool; as a result, many investigators today lack experience with growth rate measurements. Recently, investigators in a number of areas have started to use measurements of bacterial growth rates for a variety of purposes. Those measurements have been greatly facilitated by the availability of microwell plate readers that permit the simultaneous measurements on up to 384 different cultures. Only the exponential (logarithmic) portions of the resulting growth curves are useful for determining growth rates, and manual determination of that portion and calculation of growth rates can be tedious for high-throughput purposes. Here, we introduce the program GrowthRates that uses plate reader output files to automatically determine the exponential portion of the curve and to automatically calculate the growth rate, the maximum culture density, and the duration of the growth lag phase. GrowthRates is freely available for Macintosh, Windows, and Linux. We discuss the effects of culture volume, the classical bacterial growth curve, and the differences between determinations in rich media and minimal (mineral salts) media. This protocol covers calibration of the plate reader, growth of culture inocula for both rich and minimal media, and experimental setup. As a guide to reliability, we report typical day-to-day variation in growth rates and variation within experiments with respect to position of wells within the plates.
Most phenotypes are determined by molecular systems composed of specifically interacting molecules. However, unlike for individual components, little is known about the distributions of mutational effects of molecular systems as a whole. We ask how the distribution of mutational effects of a transcriptional regulatory system differs from the distributions of its components, by first independently, and then simultaneously, mutating a transcription factor and the associated promoter it represses. We find that the system distribution exhibits increased phenotypic variation compared to individual component distributions - an effect arising from intermolecular epistasis between the transcription factor and its DNA-binding site. In large part, this epistasis can be qualitatively attributed to the structure of the transcriptional regulatory system and could therefore be a common feature in prokaryotes. Counter-intuitively, intermolecular epistasis can alleviate the constraints of individual components, thereby increasing phenotypic variation that selection could act on and facilitating adaptive evolution.
Background Horizontal gene transfer, the acquisition of genes across species boundaries, is a major source of novel phenotypes that enables microbes to rapidly adapt to new environments. How the transferred gene alters the growth – fitness – of the new host affects the success of the horizontal gene transfer event and how rapidly the gene spreads in the population. Several selective barriers – factors that impact the fitness effect of the transferred gene – have been suggested to impede the likelihood of horizontal transmission, however experimental evidence is scarce. The objective of this study was to determine the fitness effects of orthologous genes transferred from Salmonella enterica serovar Typhimurium to Escherichia coli to identify the selective barriers using highly precise experimental measurements. Results We found that most gene transfers result in strong fitness costs. Previously identified evolutionary barriers — gene function and the number of protein-protein interactions — did not predict the fitness effects of transferred genes. In contrast, dosage sensitivity, gene length, and the intrinsic protein disorder significantly impact the likelihood of a successful horizontal transfer. Conclusion While computational approaches have been successful in describing long-term barriers to horizontal gene transfer, our experimental results identified previously underappreciated barriers that determine the fitness effects of newly transferred genes, and hence their short-term eco-evolutionary dynamics.
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