Threonine Phosphorylation Fine-Tunes the Regulatory Activity of Histone-Like Nucleoid Structuring Protein in Salmonella Transcription.

Lizhi Hu,Dezhi Yang,Wei Kong,Qiangqiang Han,Lin Guo,Yixin Shi

doi:10.3389/fmicb.2019.01515

Abstract

Histone-like nucleoid structuring protein (H-NS) in enterobacteria plays an important role in facilitating chromosome organization and functions as a crucial transcriptional regulator for global gene regulation. Here, we presented an observation that H-NS of Salmonella enterica serovar Typhimurium could undergo protein phosphorylation at threonine 13 residue (T13). Analysis of the H-NS wild-type protein and its T13E phosphomimetic substitute suggested that T13 phosphorylation lead to alterations of H-NS structure, thus reducing its dimerization to weaken its DNA binding affinity. Proteomic analysis revealed that H-NS phosphorylation exerts regulatory effects on a wide range of genetic loci including the PhoP/PhoQ-regulated genes. In this study, we investigated an effect of T13 phosphorylation of H-NS that rendered transcription upregulation of the PhoP/PhoQ-activated genes. A lower promoter binding of the T13 phosphorylated H-NS protein was correlated with a stronger interaction of the PhoP protein, i.e., a transcription activator and also a competitor of H-NS, to the PhoP/PhoQ-dependent promoters. Unlike depletion of H-NS which dramatically activated the PhoP/PhoQ-dependent transcription even in a PhoP/PhoQ-repressing condition, mimicking of H-NS phosphorylation caused a moderate upregulation. Wild-type H-NS protein produced heterogeneously could rescue the phenotype of T13E mutant and fully restored the PhoP/PhoQ-dependent transcription enhanced by T13 phosphorylation of H-NS to wild-type levels. Therefore, our findings uncover a strategy in S. typhimurium to fine-tune the regulatory activity of H-NS through specific protein phosphorylation and highlight a regulatory mechanism for the PhoP/PhoQ-dependent transcription via this post-translational modification.

Highlights

Bacterial nucleoid-associated proteins play an essential role in chromosome organization
In Salmonella enterica, Histone-like nucleoid structuring protein (H-NS) plays a pivotal role in silencing horizontally acquired virulence genes that are grouped in clusters, referred to as Salmonella pathogenicity islands (SPIs), whose promoter sequences contain higher AT base contents than the resident genome (O’Byrne and Dorman, 1994; Kutsukake, 1997; Lucchini et al, 2006)
Phosphorylation should take place at threonine 13, but not at threonine 22 or threonine 25, because of the presence of two ions with m/z (z = +1) of y11+ (1379.6970) and y12+ (1539.72693), respectively (Figure 1A). This threonine phosphorylation (i.e., threonine 13 residue (T13) phosphorylation) in H-NS was further confirmed by an immunoprecipitation and immunoblot analysis using an hns-HA strain constructed in our previous study (Kong et al, 2008), which produced an H-NS protein carrying a C-terminal HA-tag

Summary

INTRODUCTION

Bacterial nucleoid-associated proteins play an essential role in chromosome organization. H−NS-elicited repression is relieved by a wide range of transcriptional regulators that act on H−NS−bound promoters, removing this silencer or altering the H-NS-DNA structures Among these regulators, the PhoP/PhoQ two-component system governs more than 40 genetic loci which are essential for S. According to our previous studies, transcription of many horizontally acquired genetic loci such as the ugtL and pagC genes are governed by a PhoP/PhoQ- and SlyA-dependent feedforward regulatory loop (Shi et al, 2004b; Zhao et al, 2008). Both ugtL and pagC genes are activated when the PhoP and SlyA regulators simultaneously bind to their promoter regions to release the H-NS protein. This study provides an example in which protein phosphorylation can modulate the function of H-NS through structural alternations

MATERIALS AND METHODS

References or source

RESULTS AND DISCUSSION