Diverse nature of ClpX degradation motifs in Streptococcus mutans

Abstract

ABSTRACT Protein homeostasis is an essential process that depends on concerted effort of different proteins involved in proper protein folding, deciding the fate of misfolded proteins by either refolding them into their proper conformation or degrading the misfolded proteins. In Gram-positive bacteria, AAA+ ATPases such as ClpX, ClpC, and ClpE interact with the proteolytic ClpP and degrade misfolded proteins. Clp ATPases also maintain amounts of different proteins based on the cellular need. In streptococci, these Clp ATPases play a crucial role in various physiological processes that contribute to virulence, cell growth and division, stress tolerance, competence development, and biofilm formation. Among the Clp ATPase complexes present in low G + C Gram-positive bacteria, ClpX/P is the major proteolytic system. In contrast to numerous ClpX/P substrates identified in Escherichia coli and other bacteria, very little is known about the identity of the ClpX/P substrates in streptococci. Using a proteomic approach under late logarithmic growth condition, we screened for putative substrates that might be degraded by ClpX/P; several potential substrates were identified and verified by other methods. SpxA2 is one such candidate protein, and by Western blot, we confirmed that it indeed recognizes SpxA2. We further identified Ala-Ala-Leu, located at the C-terminal of SpxA2, as a ClpX/P degradation signal. Using a green fluorescent protein reporter system, we further confirmed several ClpX/P degradation signals. Furthermore, we found that the zinc-binding domain of ClpX is needed for substrate recognition. Our in vitro data indicate that the adaptor or other accessory factors might be needed for the ClpX/P-mediated substrate recognition or degradation. Additional investigations are needed to determine the identity of these factors in the cell. IMPORTANCE Cytoplasmic Clp-related proteases play a major role in maintaining cellular proteome in bacteria. ClpX/P is one such proteolytic complex that is important for conserving protein homeostasis. In this study, we investigated the role of ClpX/P in Streptococcus mutans , an important oral pathogen. We identified several putative substrates whose cellular levels are regulated by ClpX/P in S. mutans and subsequently discovered several recognition motifs that are critical for degradation. Our study is the first comprehensive analysis of determining ClpX/P motifs in streptococci. We believe that identifying the substrates that are regulated by ClpX/P will enhance our understanding about virulence regulation in this important group of pathogens.