The Bacterial Two-Hybrid System Uncovers the Involvement of Acetylation in Regulating of Lrp Activity in Salmonella Typhimurium.

Ran Qin,Qiufen Zhang,Zhongli Cui,Jie Ren,Yu-Feng Yao,Yu Sang,Shuxian Li

doi:10.3389/fmicb.2016.01864

Abstract

N𝜀-lysine acetylation is an abundant and important Post-translational modification in bacteria. We used the bacterial two-hybrid system to screen the genome library of the Salmonella Typhimurium to identify potential proteins involved in acetyltransferase Pat – or deacetylase CobB-mediated acetylation. Then, the in vitro (de)acetylation assays were used to validate the potential targets, such as STM14_1074, NrdF, RhaR. Lrp, a leucine-responsive regulatory protein and global regulator, was shown to interact with Pat. We further demonstrate that Lrp could be acetylated by Pat and deacetylated by NAD+-dependent CobB in vitro. Specifically, the conserved lysine residue 36 (K36) in helix-turn-helix (HTH) DNA-binding domain of Lrp was acetylated. Acetylation of K36 impaired the function of Lrp through altering the affinity with the target promoter. The mutation of K36 in chromosome mimicking acetylation enhanced the transcriptional level of itself and attenuated the mRNA levels of Lrp-regulated genes including fimA, which was confirmed by yeast agglutination assay. These findings demonstrate that the acetylation regulates the DNA-binding activity of Lrp, suggesting that acetylation modification of transcription factors is a conserved regulatory manner to modulate gene expression in bacteria and eukaryotes.

Highlights

Nε-lysineacetylation, as a key post-translational modification, plays critical roles in various biological processes, including transcriptional regulation, aging, and metabolism (Grunstein, 1997; Cohen et al, 2004; Lu et al, 2011)
We found Lrp K36 is highly conserved in bacteria (Figure 4A), and this residue was conserved in several other DNA binding proteins (Figure 4B)
Electrophoretic Mobility Shift Assay (EMSA) results showed that both the wild type Lrp and K36R had the similar and strong DNA-binding affinity to the fimZ promoter, while K36Q possessed a significantly weaker ability to bind the fimZ promoter (Figure 4C). These findings indicate that K36 residue is indispensable for DNAbinding ability of Lrp, and suggest that the acetylation of K36 is associated with DNA-binding ability of Lrp

Summary

Introduction

Nε-lysine (de)acetylation, as a key post-translational modification, plays critical roles in various biological processes, including transcriptional regulation, aging, and metabolism (Grunstein, 1997; Cohen et al, 2004; Lu et al, 2011). There are two distinct mechanisms in bacteria to regulate the protein acetylation. Acetylation Regulates Lrp Activity group from acetyl-CoA (Ac-CoA) to a deprotonated lysine. The other is non-enzymatic, whereby acetyl phosphate (AcP) serves as the acetyl donor to a deprotonated lysine (Hentchel and Escalante-Semerena, 2015; Wolfe, 2016). Nicotinamide adenine dinucleotide (NAD+)-dependent CobB (NAD+-dependent protein deacylase) has been known as the only lysine deacetylase (KDAC) for a long time (Starai et al, 2002). Current study uncovers that YcgC, representing a new protein deacetylase family, exhibits KDAC activity. YcgC targets a distinct set of substrates from CobB (Tu et al, 2015)

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Conclusion