The Escherichia coli flagella operons show a precise temporal pattern of expression. In the past, this expression pattern was studied using lacZ fusion constructs and DNA microarrays, both of which require bacterial cell lysis. Recently, Kalir and colleagues reported on a system they developed that allows researchers to monitor gene expression in live bacteria. This approach, which involves the use of green fluorescent protein (GFP) reporter gene constructs, can be used for real-time analysis of gene expression in bacteria. The authors used the technique to do time-course studies of the flagellar operons. Their system, however, could be extended to study other prokaryotic or eukaryotic genes.

The bacterial flagellum is composed of three components: a basal body, a hook, and a filament. The basal body contains a set of rings that are positioned in the cytoplasm, plasma membrane, and cell wall, respectively. The plasma membrane ring is believed to act as a motor; its rotation leads to the propeller-like motion of the flagellum. The other rings act to keep the basal body in place. The basal body's rings are attached through the hook (a bolt-like structure) to the filament. The filament is the "whip" of the flagellum and is responsible for its movement. This movement allows the bacteria to move towards nutrients in a process known as chemotaxis. Chemotaxis involves not only intact, motile flagella, but also sensory receptors and a signal transduction pathway.

Interestingly, the 14 operons (functionally related gene(s) under the control of a common promoter) that code for the flagella components and chemotaxis proteins show a precise and timely order of expression. Based on their order of expression, these operons have been grouped into 3 classes. The group 1 operon codes for a transcriptional activator for the group 2 operons. The group 2 operons code for the basal body and hook proteins, as well a transcriptional activator for the group 3 operons. The group 3 operons code for the filament proteins and the chemotaxis signal transduction system. This order of expression is logical. For example, there is no reason for the bacteria to express the filament protein without a docking hook and basal body. Also, expression of the signal transduction proteins would be pointless without intact flagella being present.

Previously, temporal expression of the flagellar operons was studied using DNA microarrays and lacZ fusion constructs. Both of these approaches require cell lysis and thus do not allow for real-time examination of gene expression levels. Furthermore, these techniques are associated with relatively large sample-to-sample variability; variations in detected expression levels between experiments are typically two-fold or more.

The new assay developed by Kalir and colleagues, and described in the June 15th issue of Science, monitors gene expression in live bacteria using GFP. Because there is no need for bacterial lysis, the average error between repeat experiments is less than 10%, making the assay more precise than previous experimental approaches. The technique also allows for real-time analysis of gene expression levels, which in turn, gives a more accurate picture of the timing of gene transcription.

Kalir and colleagues used this expression kinetics assay to monitor the temporal expression of the E. coli flagella operons. The authors cloned the promoters of the 14 flagella operons upstream of the reporter gene, GFP. Next, separate bacterial populations were transformed with these constructs, grown in rich media, and then diluted into minimal media in a 96-well plate. The authors suggested that this dilution could have been responsible for the observed synchronization of flagella biosynthesis in the bacteria (growing in the different wells). GFP expression levels in the bacteria were then monitored every six minutes. By detecting reporter gene activity over time, the researchers were able to monitor the promoter activity of the flagellar operons. The activity of the various flagellar promoters was then plotted over time, using a computerized analysis algorithm.

Using this approach, the authors were able to get a more detailed picture of the temporal expression of flagellar operons. They found that the temporal pattern of flagella operon expression was more complicated and detailed than previously realized. For example, the authors showed that the proteins comprising the cytoplasmic ring, inner membrane ring, and cell wall rings of the basal body are expressed sequentially. In other words, the genes are expressed in order of protein location within the flagellum, such that proteins on the cytoplasmic side are synthesized prior to those found on the extracellular side.

Although the mechanism behind this temporal expression is not clear, the authors hypothesized an 'affinity model' to explain the process. According to this model promoters within each of the three classes of operons have different affinities for their respective transcription factors. At low levels of transcriptional activator, only class 2 (or class 3) operons with high-affinity promoters get expressed. As the levels of the appropriate activator increase, the operons with lower affinity promoters get turned on as well.

While the expression kinetics assay was used to examine the temporal expression of E. coli flagella operons, it could easily be extended to other prokaryotic operons and certain eukaryotic genes. One interesting possibility is to use this technique for time course studies of sporulation genes, which also show a fascinating temporal expression pattern.

References

Kalir, S., McClure, J., Pabbaraju, K., Southward, C., Ronen, M., Leibler, S., Surette, M. G., and Alon, U. (2001) Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria Science 292: 2080-2083.