A Simple Screen to Identify Promoters Conferring High Levels of Phenotypic Noise

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Abstract
Genetically identical populations of unicellular organisms often show marked variation in some phenotypic traits. To investigate the molecular causes and possible biological functions of this phenotypic noise, it would be useful to have a method to identify genes whose expression varies stochastically on a certain time scale. Here, we developed such a method and used it for identifying genes with high levels of phenotypic noise in Salmonella enterica ssp. I serovar Typhimurium (S. Typhimurium). We created a genomic plasmid library fused to a green fluorescent protein (GFP) reporter and subjected replicate populations harboring this library to fluctuating selection for GFP expression using fluorescent-activated cell sorting (FACS). After seven rounds of fluctuating selection, the populations were strongly enriched for promoters that showed a high amount of noise in gene expression. Our results indicate that the activity of some promoters of S. Typhimurium varies on such a short time scale that these promoters can absorb rapid fluctuations in the direction of selection, as imposed during our experiment. The genomic fragments that conferred the highest levels of phenotypic variation were promoters controlling the synthesis of flagella, which are associated with virulence and host–pathogen interactions. This confirms earlier reports that phenotypic noise may play a role in pathogenesis and indicates that these promoters have among the highest levels of noise in the S. Typhimurium genome. This approach can be applied to many other bacterial and eukaryotic systems as a simple method for identifying genes with noisy expression. According to the conventional view, the characteristics of an organism are determined by nature and nurture—by its genes and by the environment it lives in. Consequently, one would expect that two organisms that share the same genes and live in the same environment have identical characteristics. Recently it has become clear that this expectation is often not borne out; clonal families of simple organisms living under constant conditions often show variation in biological traits and sometimes even have markedly different properties and do different things. In order to investigate molecular causes and possible biological functions of such phenotypic noise, it would be very valuable to have a simple and fast method for identifying biological traits that are particularly noisy. Here, we developed such a method, and screened for noisy traits in the bacterial pathogen Salmonella typhimurium that vary at a time scale of one day. We found that traits involved in interaction with the host are particularly noisy, suggesting that phenotypic noise might be important in pathogenesis. This method can be readily adopted for other organisms and might contribute to elucidating the role of noise in biology.