Abstract

The most widely used expression systems in bacteria are based on the Escherichia coli lac promoter. Furthermore, elements of the lac operon are used today in systems and synthetic biology. In most cases, the free inducers IPTG or TMG are used. Here we report a systematic comparison of lac promoter induction by TMG and IPTG that focuses on aspects of inducer uptake, population heterogeneity, and a potential influence of the transacetylase, LacA.

We provide induction curves in E. coli LJ110 and in isogenic lacY and lacA mutant strains and show that both inducers are substrates of lactose permease at low inducer concentrations but can also enter cells independently of lactose permease if it is present at high concentrations. higher. Using a GFP reporter strain, we compared the induction of TMG and IPTG at the single-cell level and showed that bimodal induction with IPTG occurred at approximately ten-fold lower concentrations than with TMG.

Furthermore, we observed that the induction of the lac operon is influenced by the transacetylase, LacA. By comparing two Plac-GFP reporter strains with and without a lacA deletion, we were able to show that in the lacA+ strain the fluorescence level decreased after a few hours, while the fluorescence increased even more in the lacA− strain. The results indicate that through the activity of LacA, the concentration of IPTG can be reduced below a threshold concentration of induction, an influence that must be taken into account if low amounts of inducer are used.

What causes the lac operon to become activated?

E. coli bacteria can break down lactose, but it’s not their favourite fuel. If glucose is present, they would prefer to use it. Glucose requires fewer steps and less energy to break down than lactose. However, if lactose is the only sugar available, E. coli will go ahead and use it for energy. To use lactose, bacteria must express the lac operon genes, which encode key enzymes for lactose absorption and metabolism. To be as efficient as possible, E. coli should express the lac operon only when two conditions are met:

• Lactose is available and
• Glucose is not available

How are lactose and glucose levels detected, and how do changes in levels affect transcription of the lac operon? Two regulatory proteins are involved:

• One, the lac repressor, acts as a lactose sensor.
• The other catabolite activating protein (CAP) acts as a glucose sensor.

These proteins bind to the DNA of the lac operon and regulate its transcription as a function of lactose and glucose levels. Let’s take a look at how this works.

Structure of the lac operon

The lac operon induction contains three genes: lacZ, lacY, and lacA. These genes are transcribed as a single mRNA, under the control of a promoter. Genes in the lac operon specify proteins that help the cell use lactose. lacZ encodes an enzyme that splits lactose into monosaccharides (single-unit sugars) that can be incorporated by glycolysis. Similarly, lacY encodes a membrane-embedded transporter that helps bring lactose into the cell. In addition to the three genes, the lac operon also contains several regulatory DNA sequences. These are regions of DNA to which particular regulatory proteins can bind, controlling the transcription of the operon.

• The promoter is the binding site for RNA polymerase, the enzyme that carries out transcription.
• The operator is a negative regulatory site bound by the lac repressor protein. The operator overlaps with the promoter, and when the lac repressor binds, RNA polymerase cannot bind to the promoter and start transcription.
• The CAP binding site is a positive regulatory site that binds catabolite-activating protein (CAP). When CAP binds to this site, it promotes transcription by helping RNA polymerase bind to the promoter.

The lac repressor

The lac repressor is a protein that represses (inhibits) transcription of the lac operon. It does this by binding to the operator, which partially overlaps with the promoter. When it binds, the lac repressor gets in the way of the RNA polymerase and prevents it from transcribing the operon. When lactose is not available, the lac repressor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, the lac repressor loses its ability to bind DNA. It floats outside the operator, clearing the way for RNA polymerase to transcribe the operon.

This change in the lac repressor is caused by the small molecule allolactose, an isomer (rearranged version) of lactose. When lactose is available, some molecules will be converted to allolactose inside the cell. Allolactose binds to the lac repressor and causes it to change shape so that it can no longer bind to DNA. Allolactose is an example of an inducer, a small molecule that triggers the expression of a gene or operon. The lac operon is considered an inducible operon because it is usually turned off (repressed) but can be turned on in the presence of the inducer allolactose.