Detection of pH Levels

Why use a pH-inducible promoter?

In order to execute the alkalinity response only when it is needed,, it was first necessary to identify pH-inducible promoters that are active at or near pH 5.5 (citation needed). At pH 5.5 or below, the rate of demineralization of the tooth exceeds that of the re-mineralization process provided by saliva (citation needed). This results in erosion of the hard tissues of the tooth (citation needed). Thus, a promoter induced at pH 5.5 provides useful transcriptional control over genes that might prevent the progression of tooth decay.

Identifying a pH-inducible promoter

The E. coli genome contains genes which demonstrate elevated levels of transcription at or near pH 5.5. Tucker, et al. (http://jb.asm.org/content/184/23/6551.full) performed a comprehensive study that identified acid-inducible genes contained within the E. coli genome. The team compared the expression levels of genes in cells grown at pH 5.5 to cells grown in pH 7.4. They found that the transcription of the asr and gadA genes were considerably induced at pH 5.5. In fact, the asr gene was the most significantly pH-induced gene identified. Our team proceeded with isolating the promoters of the asr and gadA genes to be used as the pH-inducible promoters driving elevated levels of gene expression within our dual-state promoter.

GadA is a crucial component in the acid stress response of E. coli

On the other hand, the gadA gene is better-characterized. The gadA gene is a part of the gad system, which is an acid-inducible glutamate decarboxylase-based acid resistance system that enables the survival of E. coli under acid stress conditions. (http://jb.asm.org/content/188/23/8118.full). GadA is a gene encoding a glutamate decarboxylase. (http://jb.asm.org/content/185/15/4644.full, http://jb.asm.org/content/188/23/8118.full) The process of decarboxylating glutamate consumes protons that leach into the cell under acid stress. (http://jb.asm.org/content/188/23/8118.full) In this manner, the gad system manages protons that would otherwise drop the cellular pH below levels at which E. coli could survive.

The function of the gad system explains why these genes experience high transcription levels at low pH. Despite this functional explanation, modes of transcriptional control over the gad system are rather complex. Transcriptional factors RpoS, cyclic AMP receptor protein, HN-S and EvgA all play a role in transcriptional regulation. INCOMPLETE SECTION -Do more research, since this section is complicated.

Identification of gadA and asr promoter regions

Transcriptional regulation of asr promoter

As of yet, the function of the asr gene, or “acid-shock RNA” gene, and the mechanism responsible for its induction are still unclear. However, Iien et al. have taken significant steps toward characterizing the gene. They propose that asr encodes a periplasmic or outer-membrane protein. Knockout experiments illustrated that the PhoBR operon plays a significant role in activating the asr gene. They demonstrated through mobility shift electrophoresis that the PhoB protein binds to the promoter region of asr. By analyzing the sequence of the asr promoter region, they revealed that it contains a sequence similar to that of the Pho box, which is a consensus sequence known to bind the PhoB protein. The Pho box can be found in the promoter regions of other PhoB-regulated genes. (http://jb.asm.org/content/181/7/2084.long)This evidence suggests thatthe regulatory protein PhoB indeed exerts some transcriptional control over the asr gene.