RT-RiboSyn – A New Method to Measure the Specific Growth Rates of Distinct Microbial Populations in Engineered Systems

A molecular biology based method called RT-RiboSyn has been developed to measure the specific growth rate of microbial populations. This method analyzes culture samples that have been exposed to chloramphenicol for defined times. Chloramphenicol disrupts ribosome synthesis, which causes a buildup of the level of precursor 16S rRNA within the cells. Specific microbial populations can be targeted, because of signature sequences present in both precursor and mature 16S rRNA. The method measures the rate of increase of the precursor 16S rRNA within the cells, which is used to measure the specific growth rate of a specific microbial population. This link between the specific rate of ribosome synthesis and specific growth rate for a cell is true for log growth, and also stationary phase where the specific growth rate is zero. RT-RiboSyn, is an ex situ method that utilizes a reverse transcription and primer extension (RT&PE) method to analyze the RNA extracted from a series of samples treated with chloramphenicol. A single fluorescently labeled primer that is specific for a microbial population and targets an interior region of both pre16S and 16S rRNA is employed. The pre16S/16S rRNA can be determined by separating the RT&PE products, which have different lengths, and measuring the fluorescent intensity of each. A pure culture of Acinetobacter calcoaceticus was exposed to chloramphenicol during log growth and stationary phases at two different temperatures, and a time series of samples were taken. Specific growth rates calculated with RT-RiboSyn for the log phase samples were within 16% of the specific growth rate determined from the spectrophotometer using a Eub338 probe. Further testing using the Acin0659 probe yielded specific growth rates that were within 22.5% of those calculated from spectrophotometer readings. Specific growth rates determined with RT-RiboSyn for stationary phase samples were 81% lower than rates calculated by spectrophotometer, however both rates were very low. The method has the potential to identify members of a microbial population (species or strain) that are growing rapidly relative to the other members present. This method is useful for determining the growth rates of microbial populations in natural and engineered systems, possibly allowing for the optimization of these systems.