Proteolysis and Regulation of Bacterial Cell Growth Control

Division Of Basic Sciences - Nci

Investigators

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Abstract

For many years, our lab has investigated the role of energy-dependent proteolysis in regulation of gene expression in bacteria. The ATP-dependent cytoplasmic proteases, akin to the eukaryotic proteasome, contain ATPase domains or subunits that recognize substrates and unfold them, feeding them to the proteolytic domains. Bacteria contain multiple ATP-dependent proteases; five of them have been characterized in E. coli. Abnormal or misfolded proteins are degraded by these proteases. In addition to this quality control role, the proteases degrade proteins that are naturally unstable; for these proteins, degradation is likely to play an important biological role. Such protease substrates fall into two general classes: proteins that are always degraded, so that regulation of their abundance depends primarily on changes in synthesis, and proteins that show regulated proteolysis. In all cases, identifying how the substrate is recognized by the protease and how recognition is affected by growth conditions is important in understanding how and when regulation is carried out. Our lab showed that the Lon ATP-dependent protease regulated capsular polysaccharide synthesis and cell division by degrading the RcsA and SulA proteins, discovered and characterized the two-component Clp proteases, ClpAP and ClpXP, and investigated the roles of these proteases in vivo and in vitro. In recent years, our focus has been on the regulated degradation of the RpoS sigma factor, a subunit of RNA polymerase that directs the polymerase to specific promoters. RpoS is important for cells to switch to a stationary or stress response gene expression program; the expressed genes provide resistance to starvation, temperature extremes, and other stresses. However, RpoS and its expressed genes are detrimental when the bacteria is under optimal growth conditions. The cell regulates RpoS accumulation in a variety of ways, including at the level of translation via small RNA activators of translation, and by regulated proteolysis. We have been studying this proteolysis, one of the best examples of regulated protein turnover in E. coli. RpoS is rapidly degraded during active growth, in a process that requires the energy-dependent ClpXP protease and the adaptor protein RssB, a phosphorylatable protein that presents RpoS to the protease. RpoS becomes stable after various stress or starvation treatments; the mode of stabilization was a mystery until work from our lab led to discovery of a small, previously uncharacterized protein, now named IraP (inhibitor of RssB activity after phosphate starvation). Mutants of iraP abolish the stabilization of RpoS after phosphate starvation. IraP blocks RpoS turnover in a purified in vitro system, and directly interacts with RssB. In E. coli, phosphate starvation leads to IraP induction, due to an increase in the levels of the small molecule alarmone ppGpp; the iraP promoter has become the best example of how ppGpp positively regulates promoters. Two other small proteins also stabilize RpoS in a purified in vitro system, IraM, and IraD. These proteins are not similar in predicted structure to IraP. IraM is made in response to magnesium starvation, dependent on the PhoP and PhoQ regulators; IraD is important after DNA damage. The anti-adaptors define a new level of regulatory control, interacting with the RssB adaptor protein and blocking its ability to act; environmental signals regulate RpoS turnover by regulating expression of different anti-adaptors. In continuing collaborative studies with Sue Wickner (NCI) on the structure and function of RssB and its anti-adaptors, we use in vivo genetics and in vitro reconstitution to understand how the antiadaptors and adaptor protein work. A collaboration with A. Deaconescu (Brown University) has led to a structure of an IraD/RssB complex, providing valuable new insight into how IraD inactivates RssB and fully supporting our earlier genetic and biochemical studies. We are further defining how RssB interacts with ClpX, the ATPase subunit of the ClpXP protease. The N-terminal domain of ClpX, known to interact with some other adaptors and substrates, interacts with the RssB C-terminus. Continued dissection of this system is providing insight into how this process is balanced in the cell. Other anti-adaptors are likely to exist, based on a variety of results. A long-standing question has been how the cell recovers from stress, in particular from the antiadaptors. We have investigated this process for recovery from phosphate starvation. During this starvation, IraP is induced and stabilizes RpoS. We find that degradation of RpoS is restored rapidly after phosphate is returned to cells, and that this rapid recovery, implying active inactivation of IraP, is dependent on a feedback loop in which RpoS increases the synthesis of RssB. Another regulator of RpoS, Crl, plays a critical and unexpected role in the recovery from starvation. Crl promotes the association of RpoS with the core RNA polymerase, thus favoring expression of RpoS-dependent promoters, including the promoter for the rssB gene, encoding the adaptor. Mutational analysis of IraP demonstrates that the C-terminus of this anti-adaptor is critically necessary for rapid recovery, suggesting that it modulates the interaction of IraP with RssB. In vitro and in vivo, IraP mutant for the C-terminus is hyperactive. Therefore, the C-terminus acts as a critical negative regulator of IraP, ensuring that it is active only when required. A virtual summer student project identified a number of other likely regulators of RpoS that will be the subject of future analysis. In another aspect of RpoS regulation, H. Tabor's lab (NIDDK; deceased in 2020) had observed that cells devoid of polyamines have very low levels of RpoS. In a collaboration with them, we have confirmed and extended this work. We find that the lack of polyamines allows rapid co-translational degradation of RpoS. Ribosomal mutations that increase translational proofreading have a similar, if not as drastic, effect. In both cases, changing codon usage within the rpoS gene is sufficient to overcome much of the defect. These results suggest that previously unrecognized aspects of codon usage poise some genes, including rpoS, to be particularly sensitive to translational stress. Overall, our proteolysis studies continue to provide novel insights into regulatory mechanisms used by bacteria.