The degradation of RcsA by ClpYQ(HslUV) protease in Escherichia coli

Abstract

In Escherichia coli, RcsA, a positive activator for transcription of cps (capsular polysaccharide synthesis) genes, is degraded by the Lon protease. In lon mutant, the accumulation of RcsA leads to overexpression of capsular polysaccharide. In a previous study, overproduction of ClpYQ(HslUV) protease represses the expression of cpsB∷lacZ, but there has been no direct observation demonstrating that ClpYQ degrades RcsA. By means of a MBP-RcsA fusion protein, we showed that RcsA activated cpsB∷lacZ expression and could be rapidly degraded by Lon protease in SG22622 (lon(+)). Subsequently, the comparative half-life experiments performed in the bacterial strains SG22623 (lon) and AC3112 (lon clpY clpQ) indicated that the RcsA turnover rate in AC3112 was relatively slow and RcsA was stable at 30°C or 41°C. In addition, ClpY could interact with RscA in an in vitro pull-down assay, and the more rapid degradation of RcsA was observed in the presence of ClpYQ protease at 41°C. Thus, we conclude that RcsA is indeed proteolized by ClpYQ protease.

New Insights into the Non-orthodox Two Component Rcs Phosphorelay System

“…The expression of matA in the mat ( ecp ) operon encoding fimbrial adhesin is induced at acidic pH, low temperature, or high acetate concentration ( Lehti et al, 2013 ). Finally, RcsA is unstable at 37°C and can be degraded by the ClpYQ and Lon proteases ( Allen et al, 1987 ; Stout et al, 1991 ; Chang et al, 2016 ). The regulatory roles of RcsB heterodimers are largely controlled by the availability and regulation of the auxiliary regulators.…”

Escherichia coli Proteome Microarrays Identified the Substrates of ClpYQ Protease

“…The restrictive enzymes EcoRI and HindIII were used to cleavage the gene from pB42AD-ybaB plasmid, and the gene was then ligated to pMal-c2x (NEB®) and pTH18kr-mbp vectors (35), which were prepared using the same restrictive enzymes, to construct pMal-c2x-ybaB, and pTH18kr-mbp-ybaB. pET21a-(6x his tag)-clpY and pET21a-clpQ-(6x his-tag), the expression vectors used for protein purification of His(6x)-ClpY and ClpQ-His(6x) were constructed by Hsieh et, al (27).…”

“…The SG22623 was constructed from wild type, which was knocking out lon gene. The AC3112 was constructed from SG22623 by further knocking out the clpQ and clpY gene (35). These 2 KO strains will help us to investigate the in vivo degradation of YbaB.…”

“…It suggested the substrate of ClpYQ protease may be degraded by the Lon protease when E. coli suffering from ClpYQ functionless mutation (36). Thus, both strains SG22623 and AC3112 were considered as ATP-dependent protease deficient strains without the Lon protease, and the difference in the proteomic profiles between these two strains would be derived by the expression of ClpYQ protease in SG22623 (35). Samples of ybaB were cloned into pTH18kr-mbp plasmids and then transformed into SG22623 and AC3112 strains.…”

Characterization of the Sinorhizobium meliloti HslUV and ClpXP Protease Systems in Free-Living and Symbiotic States

“…Bacterial HslUV and ClpXP1 complexes are orthologous to eukaryotic 26S proteasomes and function similarly to maintain the integrity of the cell's proteome by degrading damaged or misfolded protein. Furthermore, work in multiple bacterial systems has shown that both protease systems carry out targeted degradation of regulatory proteins and are involved in many cellular processes, including the SOS response (11), cell cycle regulation (12), heat stress (13,14), polysaccharide biosynthesis (15), pilus formation and DNA transfer (16), RNA metabolism (17), and SNF (12). Furthermore, exposing S. meliloti to conditions found within root nodules, including incubation with legume host-derived antimicrobial peptide NCR335 (2) or shifting growth media to acidic pH (18), results in increased expression of clpXP1 and hslUV.…”