Research scientists from INRA, AgroParisTech, CNRS, INSERM and the University of Montpellier have succeeded in observing the expression of bacterial genes with an unprecedented accuracy. Using fluorescence-based techniques and microscopy, the scientists were able to count the number of synthesized proteins to within one molecule, and within each individual bacteria of a population. By observing an early stage in the gene expression, they also succeeded in associating the fluctuations in gene expression from one cell to another with the molecular mechanisms regulating the activity of the genes being studied. This advance could be a step forward towards predicting the type of mechanism regulating the expression of a gene, based on the fluctuation profile of its expression. It also opens up an interesting perspective for synthetic biology since it helps to better understand the random part of gene expression in synthetic constructions. These results were published on December 22nd, 2011 in the on-line version of the PNAS.

The level of expression of most genes in a cell depends on the environment in which the cell is placed. Numerous gene expression-regulating mechanisms adjust the expression of each gene to the current environment, thus allowing the gene to adapt to this environment. However, even in a stable environment, a given gene is not always expressed at the same level in each cell of a population. This is because the gene expression mechanism is a largely stochastic process involving much noise. This means that it is not a regular, continuous, entirely determined process, but rather a partly random process. At the scale of a single cell, only a small number of molecules is involved: one single copy of the gene, a few gene regulating molecules, a few molecules to transcribe this gene into messenger NRA, plus a few molecules to convert this messenger NRA into protein, etc. Therefore in certain cases, the stochastic quality of gene expression can induce a phenotypic heterogeneity within a population that is otherwise perfectly identical from a genetic point of view: schematically speaking, one sub-population turns 'green’ while another turns 'red’ and yet they are genetically identical and placed in the same environment.



© CNRS/CBS

Different levels of expression within a same gene in bacteria of a same population, shown by different colors.

A team of microbiologists from INRA and AgroParisTech, working with a team of biophysicists from the CNRS, INSERM, the University of Montpellier and a mathematician from the CNRS worked together to develop a new method of measuring versus time the expression of a given gene, albeit weak, in each bacterial cell of a population. And they did this without destroying the cells and by counting directly and giving an absolute number of molecules produced. They concentrated on the first stage of expression, the transcription of the gene into messenger NRA, with the aim of determining the degree and characteristics of the random process inherent in each precise stage. They studied a small group of cells involved in the breakdown and synthesis of glucose during a precise environmental change in the model bacteria Bacillus subtilis after studying the molecular gene regulating mechanisms. They built a mathematical model based on previous experience from these mechanisms in order to analyze and interpret their impact on the random character of gene expression in the genes studied, both in basal and induced states.

Recent work has demonstrated that gene expression takes place in ‘bursts’, separated by periods of inactivity. The frequency and strength of these bursts characterize the expression of a given gene at cell scale and help to better understand how the cells of this gene adapt to change. It is particularly important to identify these characteristics when gene expression is at basal level, in other words, when current conditions do not require gene expression - because stochasticity is most marked at this point (in these conditions, the number of molecules involved in gene expression is lower). This allows us to understand how natural selection has best “prepared” the cell population to adapt to an environmental condition in which the given gene needs to be expressed. We can use the metaphor of “basal respiration” of a basal gene in each cell to better understand how the gene is subsequently induced at population level, when an environmental change occurs. More generally, the research scientists were able to associate the characteristics of the specific molecular mechanisms regulating the expression of each gene studied, with the stochastic characteristics of expression from one cell to another.



This work led to the development of a powerful method of exploring the random part of gene expression at bacterial cell level (that can also be used for eukaryotic cells). Using this type of measurement helps to refine modeling of gene expression and hence to both understand and to more accurately predict gene behavior depending on environmental conditions. Moreover, from a synthetic biology point of view, it is important to be able to find the association between a given gene expression-regulating mechanism and a variation profile of this expression between the individual cells of a same clonal population, in other words, cells that carry exactly the same genetic data.