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.

Wastewater treatment has made great strides in the last century. A century ago, gastrointestinal distress was commonplace due to improper sanitation and water treatment. However, the processes used today are still not fully understood, and in many ways are treated as “black box” processes (Forster et al 2002). This is partly due to our lack of understanding about the growth response of microbial populations present in these systems under varying environmental and operational conditions. The situation of operating these crucial engineered systems while not fully understanding them is very much the status quo. Traditional methods of measuring microbial growth are suited for pure cultures, or may only reveal composite growth rates for an environmental sample. Typically, growth rate information is obtained by letting cells grow suspended in a nutritive media and taking a time series of spectrophotometer measurements of the suspension’s turbidity. But this method only measures the bulk growth rate, and does not provide independent simultaneous measures of growth rate for each constituent species in the mixture. Presently we can identify and enumerate distinct microbial populations, as well as determine function (Amann et al 1998), but we would further benefit from knowing how fast microbes are growing under a variety of operational conditions. To date, developing a valid technique has been attempted (Pollard 1998) with radioactively-labeled DNA and DNA hybridization, but in addition to the restrictions on using radioactive material, suffers because not all bacteria incorporate the thymidine used in the assay. Other techniques, such as in situ identification of uncultured bacteria and microbial community composition that target ribosomal RNA (rRNA) have been useful (Amann 2000) for studying microbial communities in wastewater systems. A relatively new method, FISH with microautoradiography (FISH-MAR), identifies which individual cells are consuming a particular compound (Lee et al 1999), as well as the substrate uptake rate (Nielsen et al 2003). However, the substrate uptake rate has not been correlated with growth rate. While a powerful method appropriate for certain applications, FISH-MAR suffers from a few limitations. It is a notably difficult method to master, involving several days of work, and is limited to laboratories authorized to work with radioisotopes. A faster, simpler method would be beneficial for wastewater treatment facilities. Current mathematical models, for instance ASM3 for the activated sludge process (Gujer et al 1999) and ADM1 for the anaerobic digestion process [6], would benefit from accurate measured specific growth rates of distinct microbial populations in full-scale wastewater treatment systems. As environmental engineers, complete and accurate information on distinct microbial populations and their specific growth rates will lead to optimal designs and operation of bioprocesses.

A new molecular biology method has been developed to measure the specific growth rate of a distinct microbial population and has the potential to be used for a mixed environmental sample. This new method has been named RT- RiboSyn, based upon the use of reverse transcription and primer extension (RT&PE) to measure the rate of ribosome synthesis (RiboSyn). Figure 1 illustrates the basic processes involved in cell division and ribosome synthesis.

During log growth, each cell is actively synthesizing new ribosomes to be divided into two new daughter cells (Figure 1A). For a constant specific growth rate, the average number of ribosomes per cell remains constant. Thus, the specific rate of ribosome synthesis is the same as the specific growth rate of the cell. Ribosome synthesis starts with expression, which generates a polycistronic rRNA transcript that contains the three rRNAs. RNAse III cleaves this transcript of the rrn operon enzymatically into 5S, 16S, and 23S segments, and each of these “precursor” segments has extraneous RNA from the 5’ and 3’ ends removed to form “mature” rRNA for assembly into ribosomes. Ribosomes contain the mature rRNA and ribosomal proteins. The precursor rRNA represents new ribosomes, while the mature rRNA represents the extant, functional ribosomes (Figure 1B).

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