Date of Award

2012

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Immunology

Abstract

While reversible acetylation of proteins has been well studied in eukaryotes and is now recognized in bacteria, global protein acetylation in bacteria is a recently appreciated phenomenon. Protein acetylation is known to affect almost every aspect of cellular physiology in eukaryotes and there is proteomic evidence that this may also hold true in bacteria. In eukaryotes, lysines are acetylated by acetyltransferases that use acetyl-CoA as the acetyl group source, and de-acetylated by deacetylases. In bacteria, this reversible process uses enzymes homologous to those used by eukaryotes.

Our lab has recently found that acetylation of RNA polymerase (RNAP) can activate transcription at the CpxR-dependent promoter cpxP independent of activation by its cognate sensor kinase CpxA [1]. The RNAP acetylation is detected when cells grow in mixed amino acids (e.g. tryptone broth (TB)) supplemented with a glycolytic carbon source such as 0.4% glucose. In E. coli, this growth condition results in a global increase in protein acetylation [1]. CpxA-independent activation of cpxP in response to glucose requires AcCoA as the acetyl group donor, the acetyltransferase YfiQ, and K298 on the α-CTD of RNAP, which is acetylated in a YfiQ-dependent manner. This activation is reversed by the deacetylase CobB. These data support the hypothesis that glucose-induced CpxA-independent activation of cpxP transcription involves acetylation of K298 [1]. This effect of acetylation on cpxP transcription was discovered using a cpxP-lacZ-promoter fusion inserted in single copy into the chromosome [1, 2] and confirmed using a cpxP-lux reporter fusion carried by a low-copy plasmid (Lima, Patel and Wolfe, unpublished data).

I performed a screen of CpxR-regulated promoters each carried on the same low copy plasmid [3], testing each promoter's response to glucose. These plasmids were originally characterized in a constitutive kinase mutant of CpxA known as CpxA*[4, 5]. These mutants accumulate high levels of P-CpxR and the response to CpxA* depends entirely on CpxR phosphorylation [3-5]. This screen identified two categories of CpxR-regulated promoters: (1) those that are activated by CpxA* and (2) those that are repressed by CpxA* [3]. When I investigated the response to glucose by these promoters, I found that the response to glucose did not necessarily correlate with the response to CpxA*. This suggests that glucose can control CpxR-regulated promoters by a mechanism distinct from the conventional phosphorylation cascade.

To generalize glucose-induced control of CpxR-regulated promoters, we further characterized the glucose response of the CpxR-regulated and OmpR-dependent promoters ompC and ompF. The EnvZ-OmpR two-component system is one of the most extensively studied two-component systems in bacteria. Like CpxA, EnvZ is a sensor kinase that responds to changes in the extracellular environment, most prominently osmolarity and pH. Also like CpxA, it can act either as a net kinase or a net phosphatase for its cognate response regulator, OmpR. While the role of phosphorylation in the regulation of OmpR-dependent promoters is well known, the role of acetylation in this pathway has not been studied. The cpxP promoter responds similarly to both glucose and the CobB inhibitor, NAM, through an acetylation mechanism. When we saw that ompC and ompF responded similarly to glucose and NAM, we had compelling evidence that suggested that acetylation might influence OmpR-dependent transcription. If glucose was affecting ompC and ompF transcription through an acetylation-dependent mechanism, then we hypothesized that it might be through a mechanism similar to the way glucose affects transcription at the cpxP promoter.

If glucose affected transcription at the ompC and ompF promoters using the mechanism used at the cpxP promoter, then we proposed that it would require AcCoA as an acetyl group donor, the acetyltransferase YfiQ, K298 on the α-CTD of RNAP and be sensitive to CobB. We investigated the role of each of these components in ompC and ompF transcription and found that glucose does indeed affect transcription at these promoters, but it does so using a mechanism distinct from glucose-induced cpxP transcription. Therefore, the role of acetylation at the ompC and ompF promoters remains to be determined.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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