Members of the AAA+ protein superfamily contribute to many diverse aspects of protein homeostasis in prokaryotic cells. As a fundamental component of numerous proteolytic machines in bacteria, AAA+ proteins play a crucial part not only in general protein quality control but also in the regulation of developmental programmes, through the controlled turnover of key proteins such as transcription factors. To manage these many, varied tasks, Hsp100/Clp and AAA+ proteases use specific adaptor proteins to enhance or expand the substrate recognition abilities of their cognate protease. Here, we review our current knowledge of the modulation of bacterial AAA+ proteases by these cellular arbitrators.

SignificanceUnderstanding the formation and structure of protective bacterial biofilms will help to design and identify antimicrobial strategies. Our experiments with the secreted major biofilm protein TasA characterize on a molecular level in vivo the transition of a folded protein into protease-resistant biofilm-stabilizing fibrils. Such conformational changes from a globular state into fibrillar structures are so far not seen for other biofilm-forming proteins. In this context, TasA can serve as a model system to study functional fibril formation from a globular state.

SummaryUsing Bacillus subtilis as a model organism, we investigated thermotolerance development by analysing cell survival and in vivo protein aggregate formation in severely heat-shocked cells primed by a mild heat shock. We observed an increased survival during severe heat stress, accompanied by a strong reduction of heat-induced cellular protein aggregates in cells lacking the ClpXP protease. We could demonstrate that the transcription factor Spx, a regulatory substrate of ClpXP, is critical for the prevention of protein aggregate formation because its regulon encodes redox chaperones, such as thioredoxin, required for protection against thiol-specific oxidative stress. Consequently B. subtilis cells grown in the absence of oxygen were more protected against severe heat shock and much less protein aggregates were detected compared to aerobically grown cells. The presented results indicate that in B. subtilis Spx and its regulon plays not only an important role for oxidative but also for heat stress response and thermotolerance development. In addition, our experiments suggest that the protection of misfolded proteins from thiol oxidation during heat shock can be critical for the prevention of cellular protein aggregation in vivo.

The chaperone-usher pathway directs the formation of adhesive surface fibres in numerous pathogenic Gram-negative bacteria. The fibres or pili consist exclusively of protein subunits that, before assembly, form transient complexes with a chaperone in the periplasm. In these chaperone:subunit complexes, the chaperone donates one b-strand to complete the imperfect immunoglobulin-like fold of the subunit. During pilus assembly, the chaperone is replaced by a polypeptide extension of another subunit in a process termed 'donor strand exchange' (DSE). Here we show that DSE occurs in a concerted reaction in which a chaperone-bound acceptor subunit is attacked by another chaperone-bound donor subunit. We provide evidence that efficient DSE requires interactions between the reacting subunits in addition to those involving the attacking donor strand. Our results indicate that the pilus assembly platforms in the outer membrane, referred to as ushers, catalyse fibre formation by increasing the effective concentrations of donor and acceptor subunits.

SummaryProtein degradation mediated by ATP-dependent proteases, such as Hsp100/Clp and related AAA+ proteins, plays an important role in cellular protein homeostasis, protein quality control and the regulation of, e.g. heat shock adaptation and other cellular differentiation processes. ClpCP with its adaptor proteins and other related proteases, such as ClpXP or ClpEP of Bacillus subtilis, are involved in general and regulatory proteolysis. To determine if proteolysis occurs at specific locations in B. subtilis cells, we analysed the subcellular distribution of the Clp system together with adaptor and general and regulatory substrate proteins, under different environmental conditions. We can demonstrate that the ATPase and the proteolytic subunit of the Clp proteases, as well as the adaptor or substrate proteins, form visible foci, representing active protease clusters localized to the polar and to the mid-cell region. These clusters could represent a compartmentalized place for protein degradation positioned at the pole close to where most of the cellular protein biosynthesis and also protein quality control are taking place, thereby spatially separating protein synthesis and degradation.

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The Hsp100/Clp protease complexes of Bacillus subtilis ClpXP and ClpCP are involved in the control of many interconnected developmental and stress response regulatory networks, including competence, redox stress response, and motility. Here we analyzed the role of regulatory proteolysis by ClpXP and ClpCP in motility development. We have demonstrated that ClpXP acts on the regulation of motility by controlling the levels of the oxidative and heat stress regulator Spx. We obtained evidence that upon oxidative stress Spx not only induces the thiol stress response, but also transiently represses the transcription of flagellar genes. Furthermore, we observed that in addition to the known impact of ClpCP via the ComK/FlgM-dependent pathway, ClpCP also affects flagellar gene expression via modulating the activity and levels of the global regulator DegU-P. This adds another layer to the intricate involvement of Clp mediated regulatory proteolysis in different gene expression programs, which may allow to integrate and coordinate different signals for a better-adjusted response to the changing environment of B. subtilis cells.

The soil-dwelling bacterium Bacillus subtilis is widely used as a model organism to study the Gram-positive branch of Bacteria. A variety of different developmental pathways, such as endospore formation, genetic competence, motility, swarming and biofilm formation, have been studied in this organism. These processes are intricately connected and regulated by networks containing e.g. alternative sigma factors, two-component systems and other regulators. Importantly, in some of these regulatory networks the activity of important regulatory factors is controlled by proteases. Furthermore, together with chaperones, the same proteases constitute the cellular protein quality control (PQC) network, which plays a crucial role in protein homeostasis and stress tolerance of this organism. In this review, we will present the current knowledge on regulatory and general proteolysis in B. subtilis and discuss its involvement in developmental pathways and cellular stress management.