Caulobacter crescentus folding machines at the interface of ...

Caulobacter crescentus foldingmachines at the interface ofinheritance, cell division, andenergy metabolism Kristen Schroeder

Kristen Schroeder    Caulobacter crescentus folding m

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Doctoral Thesis in Molecular Bioscience at Stockholm University, Sweden 2021

Department of Molecular Biosciences,The Wenner-Gren Institute

ISBN 978-91-7911-498-5

Kristen SchroederB.Sc. Honours degree in 2010 fromUniversity of Saskatchewan. M.Sc.degree in 2013 from the WesternCollege of Veterinary Medicine,University of Saskatchewan. StartedPhD studies at the IMPRS Marburg in2015 and continued at StockholmUniversity in 2016.

From bacteria to humans, all organisms need to accomplish certaintasks, such as finding and processing nutrients for energy, growing, andsensing and responding to their environment. One process almost alllife has in common is the stress response, where molecular chaperonesfunction to repair and remove damage caused to cells by stress. Sincethis stress response is so widely important to life, we study how itworks in bacteria to gain a better understanding of its function inprotecting tasks such as nutrition and growth during stress, and also tounderstand what happens when stress becomes too intense for anorganism to handle.     Collectively, the work of this thesis furthers understanding ofbacterial aging, as well as how chaperones of the stress responseprotect the critical tasks of division, energy generation, andmetabolism. Together, the findings of this research shed light into howfundamental processes of biology are protected by chaperones of thestress response.

Caulobacter crescentus folding machines at theinterface of inheritance, cell division, and energymetabolismKristen Schroeder

Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience atStockholm University to be publicly defended on Thursday 10 June 2021 at 10.00 in ViviTäckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, online via Zoom (public link isavailable at the department website).

AbstractAll living cells must perform essential biological processes while monitoring and responding to environmental cues.Bacteria are accessible experimental systems in which to study the function of conserved central processes. One of the mosthighly conserved systems between different organisms is the proteostasis network; a group of chaperones and proteasesthat work collectively to repair and remove damaged proteins that accumulate in living systems. In the work of this thesis,we investigate how folding machines of the proteostasis network are integrated with central biological processes in themodel organism Caulobacter crescentus, and examine how these relationships change during stress.

Asymmetrically-dividing C. crescentus has previously been described to undergo aging, with the accumulation ofprotein aggregates in the larger stalked cell proposed to drive replicative decline in this organism. In study I, we establishC. crescentus as a model for monitoring the dynamic cellular response to protein aggregation. Using this system, wedemonstrate that protein aggregates are shared during division, and do not preferentially collect in one cell type.

The ubiquitous GroESL folding machine, which provides a specialized environment for folding specific proteins, hasbeen previously linked to the C. crescentus cell cycle through an unknown mechanism. In study II, we discover thatGroESL folding is required to support division both in optimal conditions and during mild stress. Specifically, we find thatGroESL supports the function of proteins that interact with the highly conserved bacterial division scaffold FtsZ, as wellas proteins that direct synthesis of the peptidoglycan cell envelope layer.

In study III we investigate the functional link between GroESL folding and energy metabolism, and find that thechaperonin has a conserved role in folding respiratory and metabolic proteins, thereby supporting the central pathways theseproteins function in. Furthermore, we find that GroESL protects several of these proteins from aggregation during stress.

Taken together, the work of this thesis addresses current models of prokaryotic damage segregation and aging, expandson how chaperonin folding is integrated into the essential process of division, and demonstrates a functional role for proteinfolding in protecting energy metabolism during stress. The findings of this research thereby provide novel insight into howfundamental biological processes interface with protein folding machines.

Keywords: chaperones, DnaK, chaperonin, GroESL, protein quality control, protein folding, stress response, cellularaging, cell division, bacterial respiration, Caulobacter crescentus.

Stockholm 2021http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-192428

ISBN 978-91-7911-498-5ISBN 978-91-7911-499-2

Department of Molecular Biosciences, The Wenner-Gren Institute

Stockholm University, 106 91 Stockholm

CAULOBACTER CRESCENTUS FOLDING MACHINES AT THEINTERFACE OF INHERITANCE, CELL DIVISION, AND ENERGYMETABOLISM 

Kristen Schroeder

Caulobacter crescentus foldingmachines at the interface ofinheritance, cell division, andenergy metabolism 

Kristen Schroeder

©Kristen Schroeder, Stockholm University 2021 ISBN print 978-91-7911-498-5ISBN PDF 978-91-7911-499-2 Cover micrographs are assorted Caulobacter experiments performed during my PhD. Printed in Sweden by Universitetsservice US-AB, Stockholm 2021

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SUMMARY

All living cells must perform essential biological processes while monitoring and responding to environmental cues. Bacteria are accessible experimental systems in which to study the function of conserved central processes. One of the most highly conserved systems between different organisms is the proteostasis network; a group of chaperones and proteases that work collec-tively to repair and remove damaged proteins that accumulate in living sys-tems. In the work of this thesis, we investigate how folding machines of the proteostasis network are integrated with central biological processes in the model organism Caulobacter crescentus, and examine how these relation-ships change during stress.

Asymmetrically-dividing C. crescentus has previously been described to undergo aging, with the accumulation of protein aggregates in the larger stalked cell proposed to drive replicative decline in this organism. In study I, we establish C. crescentus as a model for monitoring the dynamic cellular response to protein aggregation. Using this system, we demonstrate that pro-tein aggregates are shared during division, and do not preferentially collect in one cell type.

The ubiquitous GroESL folding machine, which provides a specialized environment for folding specific proteins, has been previously linked to the C. crescentus cell cycle through an unknown mechanism. In study II, we discover that GroESL folding is required to support division both in optimal conditions and during mild stress. Specifically, we find that GroESL sup-ports the function of proteins that interact with the highly conserved bacterial division scaffold FtsZ, as well as proteins that direct synthesis of the pepti-doglycan cell envelope layer.

In study III we investigate the functional link between GroESL folding and energy metabolism, and find that the chaperonin has a conserved role in folding respiratory and metabolic proteins, thereby supporting the central pathways these proteins function in. Furthermore, we find that GroESL pro-tects several of these proteins from aggregation during stress.

Taken together, the work of this thesis addresses current models of pro-karyotic damage segregation and aging, expands on how chaperonin folding is integrated into the essential process of division, and demonstrates a func-tional role for protein folding in protecting energy metabolism during stress. The findings of this research thereby provide novel insight into how funda-mental biological processes interface with protein folding machines.

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POPULÄRVETENSKAPLIG SAMMANFATTNING

Från bakterier till människor, alla organismer behöver uträtta vissa saker i livet, såsom att hitta och tillvarata näring för energi, att växa och att intera-gera med sin omgivning. Något som nästan allt liv har gemensamt är stress-responsen där så kallade molekylära chaperoner reparerar skador orsakade på cellen av stress. Eftersom denna stressrespons är av sådan stor vikt för liv, studerar vi hur det fungerar i bakterier, för att skapa oss en bättre förståelse i hur det skyddar processer såsom näringsintag och tillväxt, samt vad som händer när stressen blir för intensiv för en organism att hantera. I studie I, adresserar vi hur skador som inte kan bli lagade av stressrespon-sen ärvs vidare i bakterien Caulobacter crescentus. Tidigare antog man att denna bakterie åldras på grund av ackumulerad skada, likt enklare eukaryota organismer såsom jäst. När C. crescentus delar sig fördelas sådana skador jämnt mellan de två nya cellerna, vilket indikerar att denna bakterie inte åldras på samma sätt som jäst. I studie II av denna avhandling tittar vi på funktionen av GroESL – en spe-cifik typ av chaperon som är en del av stressresponsen och som dessutom tycks ha en okaraktäriserad roll där den hjälper C. crescentus att dela sig. Vi upptäckte att GroESL försvarar funktionen av flera proteiner som assisterar C. crescentus cytoskelett under celldelningen, samt funktionen av flertalet proteiner involverade i att bygga upp den bakteriella cellväggen. I studie III fortsätter vi våra studier av GroESL, men med fokus på dess okaraktäriserade roll i att stötta ämnesomsättning och energiutvinning vilken vi identifierade i studie II, något som också har identifierats i andra bakte-rier. Vi upptäckte att GroESL-chaperonen skyddar vissa delar av det bakteri-ella andningssystemet mot stress och utöver det främjar dess funktion när stress är frånvarande. Tillsammans kan arbetet i denna avhandling utöka vår förståelse för hur bakterier åldras samt hur chaperoner ur stressresponsen försvarar kritiska processer såsom celldelning, energitillverkning och ämnesomsättning. Till-sammans belyser upptäckterna av denna forskning hur fundamentala proces-ser i biologin skyddas av chaperoner ur stressresponsen.

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POPULAR SUMMARY

From bacteria to humans, all organisms need to accomplish certain tasks in their lives, such as finding and processing nutrients for energy, growing, and sensing and responding to their environment. One process almost all life has in common is the stress response, where molecular chaperones function to repair and remove damage caused to cells during stress. Since this stress response is so widely important to life, we study how it works in bacteria to gain a better understanding of its function in protecting tasks such as nutri-tion and growth during stress, and also to understand what happens when stress becomes too intense for an organism to cope.

In study I, we address how damage that cannot be fixed by the stress re-sponse is inherited in the bacterium Caulobacter crescentus. It was suggest-ed that this bacteria ages due to accumulation of damage, in a manner similar to simple eukaryotic organisms such as yeast. In C. crescentus, damage that the stress response cannot fix is shared when it divides into two cells, indi-cating that this bacteria does not age in the same way as yeast.

In study II of this thesis, we look at the function of GroESL; a specific type of chaperone that is part of the stress response and additionally per-forms an uncharacterized role in helping C. crescentus to divide. We find that GroESL protects the function of several proteins that assist the C. cres-centus cytoskeleton during division, and also the function of several proteins involved in building the bacterial cell wall.

In study III, we further investigate GroESL for an uncharacterized role in supporting metabolism and energy generation we identified in study II, that has also been identified in other bacteria. We find that the GroESL chaper-one protects certain parts of the bacterial respiratory system from stress, and additionally helps them to function when stress is absent.

Collectively, the work of this thesis furthers our understanding of bacteri-al aging, as well as how chaperones of the stress response protect critical tasks such as division, energy generation, and metabolism. Together, the findings of this research shed light into how fundamental processes of biolo-gy are protected by chaperones of the stress response.

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LIST OF PUBLICATIONS

This thesis is based on the following studies:

I Schramm, F.D.*, Schroeder, K.*, Alvelid, J., Testa, I., & Jonas, K. (2019). Growth-driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells in Caulobacter crescentus. Molecular Microbiology, 111(6); 1430-1448.

II Schroeder, K., Heinrich, K., Neuwirth, I., & Jonas, K. (2021). The chaperonin GroESL facilitates Caulobacter crescentus cell division by supporting the functions of the Z-ring regulators FtsA and FzlA. mBio, 12(3); e03564-20.

III Schroeder, K., Furniss, R.C.D., & Jonas, K. Chaperonin folding protects energy metabolism via TCA cycle and respiratory chain proteins in the obligate aerobe Caulobacter crescentus. Manuscript.

* equal contribution.

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RELATED PUBLICATIONS

The following manuscripts are not included in this thesis:

I Wassing, G.M., Lidberg, K., Sigurlásdóttir, S., Frey, J., Schroeder, K., Ilehag, N., Lindås, A-C., Jonas, K., & Jonsson, A-B. Meningococcal DNA binds to the human beta-defensin 2 and blocks its lethal effect against the bacteria. Submitted.

II Li, F., Bähre, H., Mohanty, S., Brauner, A., Schroeder, K., Jonas, K., Rohde, M., & Römling, U. Morphological and physiological effects of a single amino acid substitution in the patatin-like phospholipase CapV in Escherichia coli. Manuscript.

The following reviews have been published during the course of doctoral study:

I Schramm, F.D., Schroeder, K., & Jonas, K. (2020). Protein

aggregation in bacteria. FEMS Microbiology Reviews, 44(1); 54-72.

II Schroeder, K., & Jonas, K. (2021). The protein quality control network in Caulobacter crescentus. Frontiers in Molecular Biosciences, 8; 682967.

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ABBREVIATIONS

ATP adenosine triphosphate NADH nicotinamide adenine CIRCE controlling inverted dinucleotide repeat of chaperonin PBP penicillin-binding expression protein CO carbon monoxide PG peptidoglycan CTC C-terminal conserved PMF proton motive force DNA deoxyribonucleic acid ROS reactive oxygen species GlcNAc N-acetylglucosamine SEDS shape, elongation, GTP guanosine triphosphate division, and sporulation HSP heat shock protein TCA tricarboxylic acid LPS lipopolysaccharide UDP uridine diphosphate

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CONTENTS

SUMMARY ...................................................................................................... i

POPULÄRVETENSKAPLIG SAMMANFATTNING ......................................... ii

POPULAR SUMMARY .................................................................................. iii

LIST OF PUBLICATIONS .............................................................................. iv

RELATED PUBLICATIONS ........................................................................... v

ABBREVIATIONS .......................................................................................... vi

PREFACE ...................................................................................................... 1 The organization of molecular life ................................................................................... 1

INTRODUCTION ........................................................................................... 2 1. Essential life processes .............................................................................................. 2

1.1. DNA replication and chromosome segregation .................................................. 2 1.2. Synthesis of the cell envelope ............................................................................ 3 1.3. Bacterial cell division .......................................................................................... 6 1.4. Respiration ......................................................................................................... 8 1.5. Regulation of life cycle ..................................................................................... 10

2. The model organism Caulobacter crescentus .......................................................... 11 3. Phenotypic responses to stress ................................................................................ 12 4. Protein folding ........................................................................................................... 13 5. Threats to native protein folding ............................................................................... 14 6. The proteostasis network ......................................................................................... 15

6.1. DnaKJ/E ........................................................................................................... 15 6.2. GroESL ............................................................................................................ 18 6.3. Proteases ......................................................................................................... 20 6.4. ClpB ................................................................................................................. 21 6.5. Holdases and ATP-independent chaperones .................................................. 21

7. Protein aggregation and lifespan .............................................................................. 22 8. The function of protein folding .................................................................................. 23

AIMS OF THIS THESIS ............................................................................... 25

MAIN FINDINGS .......................................................................................... 26 STUDY I ....................................................................................................................... 26

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STUDY II ...................................................................................................................... 28 STUDY III ..................................................................................................................... 31

FUTURE PERSPECTIVES .......................................................................... 33

ACKNOWLEDGEMENTS ............................................................................ 36

REFERENCES ............................................................................................ 39

1

PREFACE

The organization of molecular life All life forms coordinate a myriad of molecular-scale processes to perform tasks that range from nutrition and reproduction, to environmental sensing and stress responses. In 1972 Dr. Jacques Monod said; "Tout ce qui est vrai pour le Colibacille est vrai pour l'éléphant", conveying that living organisms from the microscopic bacterium Escherichia coli to large animals such as elephants must operate similar processes and navigate similar challenges as an integral part of life. The reductionist philosophy has limits, as even the most highly conserved processes are adapted to the individual needs of dis-crete organisms, however the cellular units of life do generally share con-served practices such as oxidizing nutrients, sensing environmental cues, and generating energy. As such, investigating biological processes in accessible and relatively simple prokaryotic systems has been employed as a powerful method of understanding the molecular activities that operate in bacteria, eukaryotes, and even humans.

While understanding the molecular mechanisms driving life has led to advances in medicine and industry, prokaryotes also make up the largest fraction of biomass on earth—with an estimated number of bacteria ranging over 5x1030. This diverse community of terrestrial prokaryotes contributes irreplaceable functions to the world; from providing cobalamin for the global food web, to fixing atmospheric nitrogen, and more recently to remediating industrial toxic wastes. With this in mind, understanding how bacteria func-tion is not only important as a window into the function of central biological processes, but also to understanding the world we live in.

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INTRODUCTION

1. Essential life processes Taking a simple view of the essential processes of life, all cells must be able to replicate the genetic material, synthesize biomass, divide, and organize these events spatially and temporally, all while continuously sensing and responding to their environment. In the following sections these essential processes are described, with a focus on the general concepts established using prokaryotic model organisms, and in particular on how these processes are directed and regulated by functions of the proteome. As the essential processes of bacteria are modulated by external cues, how environmental sensing and responding is integrated with these processes, and how it might be disrupted by stress is discussed alongside.

1.1. DNA replication and chromosome segregation Duplication of the genome is a highly controlled step in replication, as dam-age to the genetic material, occurring for example as the result of UV radia-tion or presence of genotoxic compounds, can result in fitness defects or cell death. In bacteria, DNA replication begins by interaction of the highly con-served replication initiation protein DnaA with the chromosomal origin of replication (1). The essential activity of DnaA in replication initiation means that many internal regulatory systems target this interaction in order to mod-ulate chromosome duplication in accordance with environmental conditions (2,3). Once replication is initiated, DNA synthesis proceeds bidirectionally from the origin, and replication terminates with decatenation and segregation of the duplicated chromosomes to each progeny cell. The fidelity of this process is ensured through proofreading and rescue mechanisms, and also by the SOS response (4), which is dedicated to sensing and resolving genotoxic stress.

The circular chromosomes and plasmids of bacteria are tightly wound and packed into the area of the cell known as the nucleoid, which varies in its structure from occupying the entire interior of the cell as in Caulobacter crescentus (hereafter Caulobacter) (5), or multiple small areas of the cytosol as in Mycobacterial species (6,7). The packing and organization of the nu-cleoid is dynamically coordinated with the presence of other intracellular structures, such as polyphosphate granules and polyribosomes (8,9), which

3

can be illustrated through increases or decreases in nucleoid compression that occur in concert with the translational activity of the cell (9–11). The nucleoid typically occupies a large fraction of the cytoplasmic volume, and is thought to act as an organizational scaffold, as activity of nucleoid-associated proteins or even the chromosome itself can impose limits on the positioning of other large intracellular structures or life cycle events (12,13).

1.2. Synthesis of the cell envelope To support the generation of a new chromosome and the goal of creating a new cell by dividing, bacteria add biomass to increase both metabolic capac-ity and volume during the pre-divisional period. To support the increase in volume, the bacterial envelope must coordinately increase in surface area to maintain cellular dimensions (14,15). Though exceptions exist (16,17), the envelope of bacteria typically consists of either one or more lipid membranes as well as a rigid, yet flexible, structural layer of PG. Collectively, these layers provide a selective barrier between intracellular and extracellular en-vironments, and perform functions such as maintaining bacterial shape, regu-lating macromolecule and ion transport, communicating environmental in-formation, and spatially organizing processes spanning this compartment. In order to create the platform for these processes, bacteria utilize de novo syn-thesis of PG, and can modify products of the fatty acid synthetic pathway or sometimes use existing phospholipids for creation of membrane lipids (18,19). Curiously, while they are intimately connected and must collective-ly cover the surface area of the cell, the processes of PG synthesis and mem-brane synthesis may be uncoupled, which can lead to the development of PG-free morphotypes in some species (16).

The bacterial cytoplasmic membrane is a lipid bilayer that hosts a wide variety of integral, peripheral, and membrane-associated proteins. The bi-layer itself is shaped through properties of the lipid head and fatty acid groups, as well as the phase of the bilayer (18,20). This global shaping of the inner membrane is used to organize intracellular processes, such as directing synthesis of the structural PG layer (discussed below) to correct inward-bending portions of the lateral walls of rod-shaped bacteria, or positioning of chemosensory arrays at the highly curved poles (21–25). Reminiscent of the lipid rafts present in eukaryotic membranes (26), the bacterial cytoplasmic membrane is further sub-organized into functional membrane microdomains that restrict the free lateral diffusion of membrane proteins (27–29). Micro-domains are segregated based on fluidity, which is maintained in the existing membrane through the activity of cytoplasmic cytoskeletal structure (30–32). The fluidity and packing of the lipid bilayer is in turn regulated by the fatty acids attached to the lipid head group (18,20), and while this process is mod-ulated differently in different organisms (33), the membrane must be main-tained within a certain range of fluidity to support to function of essential

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processes (18,34). As the membrane is sensitive to changes in temperature, osmolarity, pH, and salinity, fatty acid and phospholipid synthesis must be tuned to respond with dynamic changes in membrane fluidity and thickness to sustain membrane homeostasis (35,36). Many connections have been made between membrane homeostasis and processes such as the generation of cell shape by PG and cell division (discussed below) (37), and not only due to the large number of integral membrane proteins involved. Reversible interactions, such as that between membrane phospholipids and terminal amphipathic helices that insert into the membrane, are frequently present in membrane-associated proteins (37), and these can be influenced through environmentally-induced changes in membrane properties (37–39). The PG layer lies on the periplasmic side of the cytoplasmic membrane, where it functions to provide structure and shape to the boundaries of the cell. The importance of keeping this layer intact is reflected in the dramatic phenotypes of PG-targeting antibiotics, where the intracellular contents of bacteria escape through defective or weak PG, however PG must also rou-tinely be broken over nearly the whole surface area of the cell to allow for growth. To grow the PG layer, the membrane-associated precursor lipid II is first synthesized from UDP-GlcNAc by a series of six cytoplasmic enzymes (MurA-MurF), whose activities have been well characterized (40). Once the lipid II precursor is constructed and attached to the membrane, it is trans-ported into the periplasm through the action of lipid II flippases (41). Upon reaching the periplasm, lipid II is knitted into the existing PG layer through the activity of SEDS family proteins acting in partnership with PBPs (42,43). To organize and concentrate these steps of PG synthesis, coordinated clus-ters of synthetic and lytic proteins form large multiprotein complexes di-rected either towards overall PG-layer growth (elongasome), or division (divisome). While the inter-protein interactions and regulation governing assembly and regulation of these complexes is an area of ongoing investiga-tion, the bacterial actin homologue MreB and the glycosyltransferase MurG have been reported to provide scaffolding functions for PG biosynthesis (44–46). MreB is a cytoskeletal protein that organizes PG synthesis performed by the elongasome, and although MreB is a cytoplasmic protein, it communi-cates with PG synthetic enzymes active in the periplasm and coordinates microdomain structure of the inner membrane (32,37,46). Through the polymerization of MreB into filaments that move circumferentially around the inner membrane surface (46), the PG layer is evenly remodelled to make room for the additional volume of the growing bacterial cell.

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Figure 1. Steps of PG biosynthesis in Caulobacter. Six cytoplasmic enzymes (MurA-MurF) create the PG precursor lipid II, which is anchored onto the membrane through the action of the integral membrane protein MraY, and flipped to the periplasmic leaflet of the membrane through the action of MurJ. Once in the periplasm, space for the lipid II precursor is created through the activity of PG hydrolases (SdpA, SdpB, AmiC, LpdF), which may be active as a part of the elongasome or divisome. Lipid II is attached into the growing PG layer through the activity of SEDS protein-PBP pairs, which are specific to the elongasome (RodA-PBP) or divisome (FtsW-FtsI). Model is construct-ed based on (41–43,47,48). Membrane and PG created with Biorender (Bio-render.com).

The integral or peripheral membrane proteins and periplasmic proteins

supporting the envelope are all translated in the cytoplasm, therefore they must undergo insertion into the membrane, translocation over the membrane into the periplasm, or even further transport into the protein-rich outer mem-brane present in Gram-negative bacteria. This is accomplished through the activity of specialized protein transport complexes, which include the Sec and Tat pathways (49,50). Proteins may be transported co-translationally during their synthesis through the Sec pathway, which inserts many cyto-plasmic membrane proteins, recognized as such by either the YidC or SecB chaperone proteins. Additionally, proteins may be translocated through the Tat pathway, which transfers proteins over the membrane after their transla-tion is complete. Since ATP is not present in the periplasm, biogenesis of periplasmic and outer membrane components is intricately connected to the cytosol through periplasmic bridges or uses strategies dependent on thermo-dynamic favourability (51). Even while the periplasmic and cytoplasmic

MurF

MurDMurC

MurB

MurAMurE

MurG+

DapA +

MurJRodAFtsW

PBPsFtsIMraY

SdpASdpB

AmiC LpdF

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compartments are separated, information must be continually shared across the membrane in order to coordinate environmental sensing, nutritional sta-tus, and division (14,15,52).

1.3. Bacterial cell division When bacteria have fulfilled the prerequisites for division, which may in-clude growing to a sufficient size (14,53,54), or receiving regulatory cues from the bacterial cell cycle (55,56), the focus of cell envelope synthesis shifts towards creating two new cell poles as a part of division. For this, the specialized proteins of the divisome condense at the incipient division site, followed by their regulated activation to synthesize and remodel the cell envelope (57–60). Building cell poles is both a sensitive and resource-intensive process (14), therefore the process of cell division is connected with the nutritional and stress status of the cell (61–63). While most essential proteins of the divisome have been identified in model bacterial organisms (64), the interactions between these proteins and how they influence division are still being uncovered.

Figure 2. The divisome complex of Caulobacter. Polymers of the GTPase FtsZ (Z) form a discontinuous ring structure at the incipient division site (Z-ring). FtsZ-interacting proteins ZapA, FzlA, KidO, and ZauP regulate FtsZ polymer structure. The Z-ring is attached to the membrane through the activi-ty of the membrane anchors FtsA, FzlC, and FtsEX. Subcomplexes consisting of FtsW/I/N and FtsQ/L/B assemble at the Z-ring and perform septal PG syn-thesis and unknown functions in divisome stability, respectively. MurG and MreB (B) are also recruited to the Z-ring to assist in synthesis of the PG layer. DipM and FtsEX stimulate PG hydrolysis, via SdpA, SdpB, and AmiC

ZapA

FtsAFzlC

ZZZZZauPFzlA

KidO

FtsX

FtsEFtsK

FtsQ

FtsL FtsB

FtsW

BBBBMurG

Pal

FtsN FtsI

TolQ

TolR

TolA

SdpASdpB

DipM

AmiC

LpdF

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(DipM) and LpdF (FtsX) hydrolases. FtsK performs a role in ensuring chro-mosome segregation. The Tol-Pal complex connects division activities with constriction of the outer membrane. Model is constructed based on (47,48,59,65–67). Cell envelope and DNA images created with Biorender (Biorender.com).

The cytoskeletal protein FtsZ is a tubulin homologue that is the first pro-tein to arrive at the incipient division site (64,68,69), and is the most widely conserved bacterial division protein—able even to induce mitochondrial fission in human cells (70,71). FtsZ is most often organized as a CTC do-main, a flexible linker, and a GTPase domain, which regulates the ability of FtsZ to polymerize into dynamic filaments that form a discontinuous ring-like structure called the Z-ring (69,72,73). Reflecting its importance, loss of FtsZ function is associated with a complete inability to form the divisome complex in many species, as it coordinates the placement and activity of division-linked membrane anchors, cytoskeletal elements, PG synthases and hydrolases, as well as proteins that connect chromosome segregation and septation initiation with division (57,64,69). As the central coordinator of division, FtsZ is heavily regulated by nutritional and stress signalling (74), and several different systems have been described to restrict FtsZ polymeri-zation to the correct time and place (64).

Bacteria use a set of FtsZ-interacting proteins to regulate the polymeriza-tion and dynamics of the Z-ring, with different species possessing various combinations of conserved and less conserved interactors. Membrane-anchoring proteins interact with the CTC domain of FtsZ to regulate attach-ment of the Z-ring onto the cytoplasmic membrane. The actin homologue FtsA is one such FtsZ membrane anchor (75–78), that is essential in many, though not all (79), bacteria. FtsA localizes to the cytoplasmic membrane through an amphipathic helix (39,75), tuning both the membrane and ener-getic status of the cell to division. Other anchoring proteins may be used in addition to FtsA, such as ZipA in E. coli (80), or FzlC in Caulobacter (81,82), or the function of anchoring the Z-ring to the membrane may be performed by other proteins, such as SepF in Bacillus subtilis and FtsW in Mycobacteria (83,84). Apart from regulating the location of FtsZ, many division proteins regulate FtsZ polymer structure via interaction with its GTPase domain. In α-proteobacteria, the division protein FzlA interacts with the FtsZ GTPase domain, introducing curvature into FtsZ polymers as well as influencing their dynamics (82,85,86). As for several FtsZ-interacting proteins that connect other envelope remodelling processes with FtsZ, FzlA has a role in activating constriction through an interaction with PBP syn-thases (86).

With the Z-ring in place to coordinate activity of the divisome, divi-sion-specific PG synthetic and lytic enzymes are recruited and activated to extend the PG layer. Several proteins are thought to act as regulatory hubs or

8

scaffolds for specific aspects of this process, such as the PG synthetic protein MurG and the periplasmic LytM-domain protein DipM (45,47,48). These proteins assemble to the divisome without a direct FtsZ interaction, but are nevertheless dependent on the presence of the Z-ring (59,65,87). As divi-some assembly progresses, the directional movement of FtsZ polymers by subunit assembly and disassembly, or treadmilling, about the division plane is observed (88,89), and in some organisms this movement directly carries the division-specific PG synthases FtsI and FtsW around the cell circumfer-ence to build the new cell poles (88–90). As the new poles are completed, the processes of PG hydrolysis and membrane fusion take over in the final phase of division. Here, a suite of functionally redundant lytic enzymes are activated by periplasmic regulators such as LpdF and DipM to mediate the final separation of a cell from its sibling (47,48). This process of cell separa-tion is still coordinated with the constricting Z-ring; for example, the ABC transporter complex FtsEX interacts both with FtsZ and PG hydrolytic fac-tors to recruit and activate their activity (91–95). Lastly, as membrane fusion and cell separation complete, FtsZ polymers and the divisome are disassem-bled and, if environmental conditions permit, cells return focus to biosynthe-sis in preparation for subsequent division events

1.4. Respiration In order to power the processes of growth and division, cells must be able to uptake and catabolize nutrients into high energy compounds, such as ATP. The oxidation of nutrients to generate high energy compounds is most often accomplished using oxidative phosphorylation. Here, a group of membrane-localized complexes shuttle electrons to a terminal electron acceptor, gener-ating a PMF that can be used to power the membrane-spanning ATP syn-thase (96). In mitochondria and chloroplasts, this process occurs via a rela-tively standard aerobic pathway, where electrons enter the respiratory chain via NADH dehydrogenase (Complex I) and succinate dehydrogenase (Com-plex II), which oxidize metabolites (NADH or succinate, respectively) gen-erated by the TCA cycle, generating reduced quinone in the process (96). Electrons may also enter the respiratory chain via electron transfer flavopro-teins, where quinone is also reduced (96). Cytochrome c oxidoreductase (Complex III) oxidizes the reduced quinone, passing the electrons onwards to cytochrome acceptors. The terminal cytochrome c oxidase (Complex IV) then oxidizes cytochromes, finally passing the electrons to acceptor oxygen molecules. With the exception of Complex II, the respiratory complexes pump protons across the membrane during these reactions, generating a pro-ton gradient that is later used to drive function of the ATP synthase.

9

Figure 3. The aerobic respiratory chain components and major reactions in Caulobacter, predicted based on homology. Metabolites from the TCA cycle are oxidized through the activity of NADH dehydrogenase (nuoA-N, analo-gous to Complex I of mitochondria) and succinate dehydrogenase (sdhA-D, analogous to Complex II). NADH dehydrogenase and succinate dehydrogen-ase reduce the quinone pool, and NADH dehydrogenase additionally pumps protons across the inner membrane as a part of its activity. Homologues of mi-tochondrial ubiquinol-cytochrome c reductase (CCNA_00505-507, Complex III) oxidize reduced quinone. Caulobacter possesses four terminal oxidases, two of which have low oxygen affinity (qoxA-D, coxAB), and two of which exhibit high affinity for oxygen and are used in hypoxic conditions (ccoN-Q, cydAB) (97). Lastly, the ATP synthase uses the proton gradient generated by the respiratory complexes to power synthesis of ATP. IM, inner membrane; OM, outer membrane; Q, quinone.

While the general phenomenon of coupling electron transport to the gen-

eration of a proton gradient is the same, prokaryotes have highly branched oxidative phosphorylation pathways that can use a wide variety of com-pounds to donate electrons (96). Both Mycobacteria and Caulobacter, which are strictly aerobic organisms, switch between terminal oxidases with high and low affinity for oxygen depending on the environmental challenge (97,98). For these organisms, expression of a high affinity terminal oxidase can aid in survival of hypoxic environments, and in the case of Mycobacte-ria, the presence of the toxic gas CO (98). For the metabolically flexible E. coli, which can respire aerobically, anaerobically or by utilizing fermenta-tion, five discrete NADH dehydrogenase complexes alone can be deployed to tailor respiration to its environment (99). Adding further complexity to prokaryotic respiration, bacteria are capable of moving electrons between a wide variety of redox pairs, offering further robustness to environmental challenges (100).

The flexibility of branched respiratory pathways may permit a diversity of routes to generating energy, however environmental conditions often change

QQH2Q

sdhB

sdhA

sdhCsdhD

nuoFnuoEnuoG

nuoI

nuoC

nuoB

nuoD

nuoA

nuoLnuoMnuoNnuoKnuoJnuoH CCNA_00505

CCNA_00507CCNA_00506

Complex I Complex II

Complex III

Complex IVNADH NAD+ + H+

succinate fumarate

H+ H+

coxABCqoxABCD

ccoNOPQcydAB

H+

IM

OM

TCA cycle

TCA cycleATP Synthase

Pi + ADP ATP + H2OH+

Q QH2 QH2

H2OH+ + O2

10

swiftly and slow transcriptional responses may be insufficient to ensure an adequate energy supply is present to power survival processes. Dynamic relocalization of existing complexes may aid in quickly rewiring respiration, or regulation of respiratory complex assembly may control activity or effi-ciency (101). In E. coli, respiratory complexes have been found to cluster together by type, rather than as sites containing the full electron transport chain as occurs in mitochondria (102), and free diffusion of quinone is thought to shuttle electrons between these respiratory complex zones (102). As an example of dynamic localization affecting activity, movement of the E. coli nitrate reductase complex from distributed clusters to pole-associated patches facilitates increased efficiency of the respiratory chain during ni-trate-respiring conditions (103).

The product of respiratory complex function, PMF, is not only an essen-tial driver of ATP synthesis, but is also intertwined with the regulation of central metabolism, envelope homeostasis, and cell division. Generation of PMF is influenced by TCA cycle activity, where increased levels of acetyl-coA result in increased respiratory activity. This activity must be balanced, however, as the electron transport chain is the main source of endogenous ROS, which can threaten protein folding and damage the chromosome (104). Changes in fatty acid availability and membrane fluidity also affect PMF and must be balanced with respiration (33,105), as quinone diffusion is impaired in low fluidity membranes, and protons are able to leak across highly fluid membranes. The fluidity and organization of the membrane may furthermore affect the ability to redistribute respiratory complexes and modulate their activity (99). Finally, PMF is known to affect localization of weak amphi-pathic helices, such as that present on the membrane anchors of FtsA and MinD (39). The mechanisms by which PMF is integrated with metabolism, redox homeostasis, cell envelope function, and events of the cell cycle such as division are complex, and the full picture of energy metabolism homeo-stasis has not yet been uncovered.

1.5. Regulation of life cycle Each of the essential life processes described in the previous sections must be functional to support life, however they must also be temporally coordi-nated at the correct time and condition in order for an organism to success-fully replicate. In contrast to the strict and conserved checkpoints of the eu-karyotic cell cycle, bacteria employ a more varied approach to life cycle regulation that is thought to depend on environmental niche and lifestyle. Most bacteria regulate events such as DNA replication and cell division ac-cording to environmental cues, however this is accomplished in different ways by different organisms. In E. coli and B. subtilis, DNA replication and division are coupled through size (14,53,54,106), and in B. subtilis the con-trol of a sporulation program that aids in survival of harsh conditions is addi-

11

tionally integrated. Other organisms, such as Caulobacter and Mycobacteria, utilize oscillating patterns of gene transcription to drive progression of a bacterial cell cycle (107,108). As discussed below, Caulobacter additionally makes use of regulated proteolysis to refine the oscillations of cell differenti-ation and development factors that drive progression of its asymmetric cell cycle (109,110). In all cases, life cycle regulation is integrated with stress responses in order to protect sensitive essential processes during unfavoura-ble environmental conditions.

2. The model organism Caulobacter crescentus Caulobacter species are aerobic, chemorganotrophic α-proteobacteria that can be found in freshwater and marine environments, as well as in soil (111,112). Caulobacter crescentus was developed as a model of prokaryotic developmental biology due to its asymmetric cell cycle, which features cell type differentiation. Each Caulobacter division event results in the produc-tion of a stalked cell type and a swarmer cell type, which are morphological-ly and functionally distinct. The swarmer cell is a non-replicative and motile cell, possessing chemosensory arrays that guide it to nutrient-filled environ-ments where it ejects its polar flagella and elaborates a stalk as part of its terminal differentiation into a stalked cell (113). The stalked cell is a station-ary, replicative cell that is attached to surfaces (or other Caulobacter, form-ing rosettes (114)) through a holdfast polysaccharide present at the distal end of its polar stalk (115,116). The stalked cell type initiates DNA replication and divides to produce swarmer cells, and as this ability to produce addition-al swarmer cells declines over successive generations (117), it has been pro-posed to experience replicative aging in a manner similar to the mother cell of yeast (118,119). In order to faithfully produce the two morphotypes, Cau-lobacter utilizes a tightly regulated cell cycle where environmental and in-ternal cues are integrated with the organization of cells in time and space. Five major cell cycle regulators are known to drive this program; the master regulator CtrA (107), the DNA replication initiator DnaA (120), the DNA methyltransferase CcrM (121), the swarmer cell regulator SciP (122), and transcriptional activator GcrA (123). The abundance and activity of several of these regulators is further modulated by proteolytic degradation (2,124–126), in a configuration reminiscent of the cyclin-CDK complexes that drive the eukaryotic cell cycle. Further oscillations in the transcriptome (107), proteome (127), and metabolome (128) have been defined to occur through-out the cell cycle, linked to the events driven by the master regulators.

12

Figure 4. The Caulobacter cell cycle is driven through the activity of five ma-jor regulators. Caulobacter exhibits an asymmetric cell cycle where one swarmer cell and one stalked cell are created during each division event. The motile, non-replicative swarmer cell is induced by nutritional cues to differen-tiate into a stationary, replicative stalked cell. The stalked cell initiates DNA replication, completes chromosome duplication, and finally divides again into a stalked and swarmer cell. The master regulator CtrA is an inhibitor of DNA replication that is degraded during the swarmer-to-stalked cell transition. DnaA is the DNA replication initiator protein that binds the chromosomal origin of replication when CtrA is not present to repress DNA replication. The CcrM methyltransferase activates transcription of cell-cycle regulated genes during chromosome duplication through epigenetic regulation. GcrA interacts with the housekeeping sigma factor σ73 to activate transcription of a subset of genes. SciP is an inhibitor of CtrA-directed gene expression that prevents di-vision-related genes from being expressed in the swarmer cell. Timing of these functions of the major regulators in cell cycle progression is illustrated by coloured bands. Model is constructed from (113), Caulobacter cell cycle diagram is adapted from (129).

3. Phenotypic responses to stress Stress can be caused by the presence or absence of many different environ-mental factors, and while the mechanisms by which stress impinges on an organism vary widely, similar phenotypic responses can be observed in re-sponse. One prominent example is stress-induced deviation from normal cell shape parameters, which occurs when the sensitive processes of division or the cell cycle are halted while cell growth continues. Diverse bacteria under-go a filamentation response during host infection, heat exposure, UV light

swar

mer

-to-s

talk

ed

tran

sitio

n

swarmer

stalkedpre-

divisional

divi

sion

CtrADnaACcrMGcrASciP

13

exposure, osmotic changes, antibiotic pressure, and biofilm participation, including Caulobacter (130,131). This filamentation may stem from the direct loss or malfunction of one or more essential proteins as a result of the stress, or indirectly when stress-sensing mechanisms intentionally target the function of essential proteins in the interests of survival. For example, geno-toxic stress induces the synthesis of SidA and DidA in Caulobacter, which inhibit the division PG synthases FtsW and FtsI (61,62). Delaying cell divi-sion in response to internal problems, in this case genotoxic stress, may give the affected cell time to repair or prevent further damage. Filamentation may also be incorporated into other survival responses, such as preventing inges-tion by host cells, or surviving in resource-limited environments (130).

Bacteria exposed to stress may also arrest both division and growth. In this response, expensive growth processes are shut down with the aim of reducing metabolic costs and surviving until conditions improve. Growth arrest can be observed in response to oxidative, pH, and starvation stress, as well as in response to high intensity heat or antibiotic stress (132,133). In the case of growth arrest from high intensity heat stress, the energetic cost of producing HSPs and operating the protective response are thought to drain available resources away from translation, resulting in fewer resources for building biomass (133). Some stresses may also directly target the ribosome and shut down the ability to translate proteins required for growth, as is the mechanism of several antibiotic classes. In the case of oxidative stress, growth capacity is restricted through a drastic decrease in ATP levels, while oxidation of amino acid residues can irreparably damage susceptible proteins (134–136). As stress threatening the proteome is a frequent danger to many organisms, a highly conserved response exists to repair protein damage and promote survival. In the following sections, how individual protein structure and function is affected by stress, and how this is repaired and safeguarded by a conserved protective response are discussed.

4. Protein folding To carry out the specific functions of the molecular pathways that support essential processes, bacteria translate several thousand discrete protein spe-cies. As the polypeptide chain is translated and moves towards the ribosome exit tunnel, interactions between amino acids begin to induce the develop-ment of secondary folded protein structure in a thermodynamically favoured process (137). This higher order structure develops through features such as hydrophobic residues and disulfide bonds, and correct orientation of these features facilitates the solubility of a protein in the crowded, hydrophilic intracellular environment. Hydrophobic resides typically become located in the interior of the folded protein structure, with hydrophilic resides on the exterior. Modifications of amino acids, such as redox-sensitive sulfur-

14

containing amino acids, can also influence the conformation of a protein (135,138). The sum of interactions occurring within a protein species and between its environment as it exits the ribosome provoke instantaneous fold-ing from an unfolded, energetically expensive conformation to one that pop-ulates a more stable energy state in dilute environments (139–141). As the interior of a cell is crowded with other proteins, the ribosome-associated chaperone trigger factor is present in bacteria and chloroplasts to help stabi-lize the nascent chain during translation and prevent aberrant interactions or misfolding (142). In this way, many small proteins are able to achieve their native conformation in vivo rapidly and without further assistance. These proteins are then free to perform their cellular function until they are re-moved by proteolysis at the end of their lifespan or after sustaining damage.

5. Threats to native protein folding While small basic proteins readily take on their native conformation in the environment of the ribosome and with the assistance of trigger factor, larger proteins or those with complex folds may form stable, partially-folded states upon translation (139,140). These folding intermediates may require time scales that are incompatible with the requirements of biological processes to fully fold into their final conformation (141,143). Furthermore, environmen-tal changes may affect the ribosome or protein folding parameters, and the appearance of proteotoxic stress may additionally threaten or damage sus-ceptible segments of the proteome (144–146). If the ribosome becomes ob-structed or impaired, such as when bacteria are exposed to the antibiotic kanamycin (147), mistranslated protein species can be created that fold into aberrant structures. These aberrant proteins may reduce the effectivity of translation, as resources are used to create non-functional species, and may exhibit inappropriate activity that disrupts normal cellular signalling or met-abolic processes, such as by fostering creation of ROS (139,147,148). While misfolded protein species may be created through translation errors during biogenesis, mature proteins can also be structurally damaged and induced to misfold, for example through oxidation of cysteine and methionine residues (135,138). Less permanent threats to protein folding include changes in tem-perature, crowding, ionic strength, or the presence of compounds such as heavy metals (137,144,149), all of which may provoke unfolding of the na-tive structure of susceptible mature proteins, or of nascent chains. The clas-sical experimental unfolding stress is heat shock, where increases in temper-ature denature intracellular proteins in bulk (145), however declining levels of the hydrotrope ATP during entry into stationary phase, or during oxida-tive stress, has also been examined for its effect on intracellular crowding and protein folding (150–152), as well as mistranslation and protein misfold-ing due to antibiotic treatment (147,153).

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As a consequence of protein unfolding or misfolding, hydrophobic resi-dues normally buried in the interior of a protein may become exposed and interact with hydrophobic patches of other unfolded proteins in an attempt to be shielded from the hydrophilic cytoplasm. Through these interactions, unfolded proteins can form stable insoluble arrays, called aggregates, that deprive the cell of the function of the constituent proteins. Protein aggregates start as small clusters, but can grow so large they become apparent at a tissue scale (148). Accumulation of protein aggregates is associated with the de-velopment of pathologies ranging from growth defects in unicellular organ-isms to neurological degeneration in multicellular eukaryotes, indicating that control and repair of aggregated proteins is critical to survival (148,153–157).

6. The proteostasis network Proteostasis networks consist of a group of highly conserved chaperones, proteases, disaggregases, holdases, and accessory proteins that collectively function to curate and protect the proteome of an organism against protein aggregation. The major conserved proteins of bacterial proteostasis networks are introduced in the following sections, with a focus on protein folding pathways that are active in the Caulobacter cytosol. While the contribution of proteases to the Caulobacter cell cycle has undergone intensive study (109,110,124–126), less is known of the contribution of its two ATP-dependent chaperones; DnaKJ/E and GroESL.

6.1. DnaKJ/E The chaperone DnaK (Hsp70) functions together with its DnaJ (Hsp40) co-chaperonin, and together these proteins recognize the hydrophobic residues exposed in unfolded or misfolded proteins. While DnaKJ/E is expressed during optimal conditions and can generally assist folding intermediates into their native conformation during biogenesis, the chaperone performs its most critical role in stabilizing and refolding proteins that unfold during stress (158,159). The folding mechanism of this chaperone occurs through DnaKJ binding onto hydrophobic patches as a molecular clamp, after which ATP hydrolysis stabilizes the chaperone-client complex. Stable DnaKJ binding serves to protect the client protein from interacting with other unfolded pro-teins, supporting refolding over non-productive aggregation. Finally, the GrpE nucleotide exchange factor regenerates ATP on DnaK, preparing it for subsequent rounds of folding (160). Some organisms possess multiple DnaK-like proteins, as well as multiple DnaJ-like proteins that specify DnaK towards different functions. For example, E. coli possesses six J-domain proteins, and while the function of some is unknown, CbpA specifies DnaK

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Figure 5. The proteostasis network of Caulobacter during optimal conditions. During optimal conditions, the proteostasis network of Caulobacter provides functions in development, cell cycle progression, and stress sensing. Hypothe-sized or known client proteins of the protein folding machines are indicated in circles, and by question marks where additional unknown substrates exist. Functional categories of known substrates are as follows: blue, division; or-ange, development; maroon, stress regulation; green, PG synthesis. Refolding or holding cycles are shown with gray circular arrows, and degradation with dashed lines. Blue dashed arrows indicate points of collaboration between proteostasis network proteins. Membrane image created with Biorender (Bio-render.com). Figure is modified from (129).

activity in curli fiber production and organization of biofilms, while DjlA specifies an activity in capsule synthesis (161,162). Whether some J-domain proteins may specify DnaK activity to different stresses or client pools, as occurs in eukaryotes (163), remains to be determined in bacteria.

While E. coli only strictly requires DnaKJ/E for survival during stress (158), it is typically expressed in optimal conditions, and organisms such as Caulobacter and Mycobacteria smegmatis have been found to require this chaperone at all temperatures (7,159). In the absence of stress in proteobac-teria DnaKJ/E has a conserved role in targeting the heat shock sigma factor σ32 for degradation by the membrane-bound protease FtsH (164,165), and while E. coli is able to survive without this interaction (158), Caulobacter exhibits a stronger requirement for dampening the σ32 regulon during opti-mal conditions (159). In other organisms however, the folding activity of DnaKJ/E is required during protein biogenesis in the absence of stress, as in

FtsH

DnaJ

σ32HfiA

GroEL

FzlAFtsA

HrcA

CnoX

SS SSHH

SecB

ClpX

ClpP

ClpP

Lon

ClpA

Protein folding Proteolysis

σ32

?

? Lon

GroES

DnaK

?

DapA

MurA

KidO

Trigger factor

SciP, CcrM, DnaA

FtsA, SdhA

FtsZ, SdhA

17

M. smegmatis, where DnaKJ/E is required at all temperatures due to the de-pendency of the essential, multimodular lipid synthase FASI on the chaper-one (7). As DnaKJ/E is highly stress-responsive, integration of chaperone availability into stress-sensing regulatory circuits has also been described. For example, DnaKJ/E performs a role in Caulobacter development through its interaction with the surface attachment inhibitor HfiA (166). In optimal conditions, a portion of HfiA interacts with DnaKJ/E and is maintained in a stable, active form that inhibits holdfast synthesis, however increases in the amount of DnaKJ/E during unfolding stress is thought to increase the amount of stable HfiA, promoting dispersal away from environments with proteotoxic attributes (166).

When proteotoxic stress is encountered, DnaKJ/E is titrated away from σ32 by the increasing presence of unfolded proteins (164,167). Accumulating σ32 is then able to induce expression of HSPs, and, in combination with sim-ultaneous stress-induced unfolding of the housekeeping sigma factor σ70 (σ73 in Caulobacter) (167,168), redirect transcription and translation to the pro-tective heat shock response. As DnaKJ/E performs such a central role in deploying the response to unfolding stress, mechanisms to halt essential processes during stress are linked into the shift in DnaKJ/E function. This is illustrated by DnaKJ/E regulation of DnaA stability via induction of the pro-tease Lon during stress in Caulobacter (2). The shift between DnaKJ/E func-tioning as a σ32 regulator and a folding catalyst is also accompanied by dy-namic changes in its subcellular localization, as the chaperone reorganizes from a diffuse pattern to become concentrated at foci of protein aggregation. These foci are located at the poles in E. coli (169), as multiple foci in Myco-bacteria (7,153), and in study I we show that DnaKJ/E-decorated foci of protein aggregation are distributed throughout the cell volume in Caulobac-ter (170). DnaKJ/E is capable of interacting broadly with the proteome to promote refolding, and the profile of aggregated proteins that depend on DnaKJ/E folding is similar between E. coli and Caulobacter (145,159). Through its general refolding activity, DnaKJ/E is the major chaperone re-sponsible for restoring the proteome into a folded state, preventing irreversi-ble aggregation, and ensuring survival of adverse conditions.

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Figure 6. The proteostasis network of Caulobacter during stress. (A) During proteotoxic stress conditions such as heat shock, proteins unfold and form ag-gregate structures (grey squiggles). Hypothesized or known client proteins with a relationship to unfolding stress are indicated in circles. DnaKJ/E, ClpB and GroESL participate in protein refolding and disaggregation. The small heat shock proteins (sHSP1 and sHSP2) stabilize unfolded proteins. The pro-teases ClpAP and Lon participate in degradation of unfolded proteins and provide roles in halting essential processes. The contributions of the FtsH and HslUV proteases are unknown. (B) During oxidative stress, the specialized holdase CnoX is upregulated and activated to help stabilize unfolded proteins. When reducing conditions are restored, these substrates are passed to GroESL and DnaKJ/E for refolding. Membrane image created with Biorender (Bioren-der.com). Figure is modified from (129).

6.2. GroESL Most bacteria require the folding contributions of at least one type I chap-eronin homologue, which functions as two heptameric rings of the GroEL (Cpn60, Hsp60) subunit, and a heptamer of the GroES (Cpn10, Hsp10) sub-

ClpBDnaK

sHSP1 sHSP2

ClpA

ClpP

FtsH

Lon

DNA replication

DnaA

DnaJσ73

σ32

GroES

GroEL

O2-

O2-O2

-CnoX

GroESL

DnaKJ

SSHH

Stress release

HH

HH

HslVHslU

?

B

A

FtsA

mdhNuoC

FabA

19

unit (141). The GroEL heptamers are stacked as two concentric rings, each forming a central cavity lined with hydrophobic residues. Through the resi-dues present on the apical domains, the GroEL rings can alternately interact with the exposed hydrophobic residues present in unfolded client proteins (171). Interaction with a client protein and the binding of ATP causes a con-formational shift that pulls the client down into the GroEL ring cavity, and binding of the GroES co-chaperone fully encapsulates it (141,171). Once encapsulated, a series of conformational changes in GroEL switch the interi-or of the ring cavity from a hydrophobic to hydrophilic environment, pro-moting burial of hydrophobic residues exposed on the client protein. Finally, hydrolysis of ATP induces disassembly of the GroESL-client complex, completing one folding cycle (141,171). This mechanism of capturing sub-strates is currently thought to provide kinetic assistance to folding proteins, speeding spontaneous folding to biologically relevant timescales by provid-ing an environment where dwelling as a folding intermediate is avoided (141,171). Due to size limitations of the GroESL chamber, client proteins of less than 60 kDa are preferred by the chaperonin, however a small number of larger proteins may use GroESL for folding without being fully encapsulated (172,173). Although it remains difficult to accurately predict if a protein will require GroESL for folding into its native form, specific physicochemical signatures have been identified as being enriched in GroESL substrates (174–177). For example, the presence of the α/β TIM barrel fold structure in aldolases is highly associated with GroESL dependency (173,178–180), however some proteins featuring this fold do not require GroESL for fold-ing, and furthermore, exogenous proteins without this fold can be mutated to have an absolute chaperonin requirement (181,182).

The GroESL chaperonin is capable of interacting with a larger proportion of the proteome than strictly depends on its folding environment, and has been described to be responsible for folding approximately 5-10% of ex-pressed proteins in E. coli, some of which are shared with DnaKJ/E (173,183). In the course of identifying which E. coli proteins have a strict dependency on the chaperonin environment for folding, 57 proteins were identified of which six are considered essential (173,178), although addition-al genetic or metabolic intervention may be able to bypass the function of some of these (184). Several of the proteins identified as obligate substrates of E. coli GroESL play crucial roles in fatty acid synthesis (FabF), in regula-tion of FtsZ activity (FtsE), and in oxidative phosphorylation (SdhA, NuoC) (173). The requirement of several essential proteins for GroESL folding means that GroESL is also essential for viability during both stress and op-timal conditions, and has been found to be essential in nearly all examined bacteria (185), as well as in mitochondria and chloroplasts (186,187). Re-flecting its utility, the chaperonin has undergone duplication in several spe-cies (185), with additional groESL genes becoming tailored to specific pro-cesses. For example, the Mycobacterial groESL2 is specialized to fold the

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type II fatty acid synthase KasA (188), and the five GroESL paralogues of Sinorhizobium meliloti perform overlapping roles in nitrogen fixation, stress response, and housekeeping (185,189). While few GroESL client proteins have been characterized in species other than E. coli, the chaperonin has been linked phenotypically to cell envelope synthesis and division in several organisms (188,190–192). In study II, we describe multiple points of inter-action between the cell envelope and division and GroESL in Caulobacter (193). Interestingly, some of the members of the Mollicute class of bacteria lack a chaperonin homologue (194,195), indicating that while chaperonin folding is utilized by the vast majority of organisms, it is not an absolute requirement for life.

To accomplish regulation of GroESL induction (sometimes together with DnaKJ/E), a regulatory circuit consisting of the CIRCE element and the HrcA transcriptional repressor are widely used outside of E. coli. In B. sub-tilis, Chlamydial species, Mycobacteria and Caulobacter, HrcA is an aggre-gation-prone protein dependent on GroESL folding to take on a stable, fold-ed structure that is competent to bind the CIRCE operator (196–198). In organisms excepting Caulobacter, this interaction changes during proteotox-ic stress, when HrcA is destabilized and GroESL is occupied by unfolding proteins, thereby allowing derepression of the chaperonin until declining levels of unfolded proteins allow for folded HrcA to accumulate again (199). In contrast to this negative regulation by CIRCE-HrcA, Caulobacter utilizes this regulatory circuit to imprint cell cycle regulation on expression of GroESL (200–202). However, as high levels of the chaperonin can be stably detected in synchronized cultures, the purpose of this cell cycle regulation has remained enigmatic. Nevertheless, upregulation of GroESL is critically required to withstand proteotoxic stress in Caulobacter and other organisms, with the groESL operon strongly induced in response to heat as well as etha-nol and salt stress (132,203). The upregulation of the chaperonin has addi-tionally been linked to tolerance of oxidative stress (191,204), and recently a mechanism has been described where GroESL collaborates with the oxida-tive stress-activated chaperedoxin CnoX (135,205). Here, CnoX stabilizes proteins that have been unfolded through oxidative stress, and transfers these to the chaperonin for refolding when reducing conditions are restored (135). Which proteins depend on GroESL refolding during stress has not been yet well characterized, however in study III we uncover that a subset of Cau-lobacter proteins that interact with GroESL during their biogenesis also re-quire its protection from proteotoxic stress.

6.3. Proteases In addition to protein folding machines, proteases make up an important class of proteostasis network proteins. While the aims of this thesis are di-rected towards understanding the role of protein folding machines, cyto-

21

plasmic proteases collaborate with chaperones to preserve the integrity of the proteome during both optimal and stress conditions. Highly conserved prote-ases contain a chaperone subunit or domain, which provides substrate recog-nition and unfolds substrate proteins. Bacterial unfoldase subunits include ClpA, ClpC, ClpE, and ClpX, all of which can associate with the ClpP pep-tidase subunit, as well as HslU, which associates exclusively with the HslV peptidase subunit (146). Proteases may also be comprised of a single poly-peptide that forms both unfoldase and peptidase domains, as is the case for the protease Lon and the membrane-associated protease FtsH. Most proteas-es are active during optimal conditions, where they contribute roles in regu-lating essential processes and stress sensing. For example, FtsH regulates lipid A biosynthesis in E. coli through precise control of the LPS biosynthet-ic protein LpxC (206), and ClpCP degrades the PG biosynthetic protein Mu-rAA in B. subtilis upon entry into stationary phase (207). The activity of proteases during optimal conditions has been a topic of particularly intensive study in Caulobacter, as approximately 5% of the proteome is rapidly turned over during the cell cycle (127), and ClpXP and Lon have been found to perform integral roles in promoting cell differentiation and progression of the transcriptional cell cycle circuit (109,110). Most proteases are part of the stress-responsive σ32 regulon, and therefore their expression is induced by stress. During stress, proteases function to quickly alter activity of essential processes, for example when Lon degrades the DNA replication initiator DnaA (2), as well as to clear damaged proteins from the cell.

6.4. ClpB The ClpB (Hsp104) disaggregase is related to the unfoldase domains of pro-teases, however instead of associating with a peptidase subunit, it assists in protein refolding through collaboration with the DnaKJ/E chaperone (208–211). Expression of the hexameric ClpB ring is strongly induced by stress, during which it performs an important function in pulling unfolded proteins away from protein aggregates (146,168,212). Reflecting its importance in disaggregating proteins, ClpB mutants are impaired in stress survival in many organisms (153,168,213,214). Using an approach similar to that used previously in E. coli (156), in study I, we utilize the inability of ∆clpB mu-tants to dissolve protein aggregates as a tool to determine the aggregate in-heritance pattern in Caulobacter.

6.5. Holdases and ATP-independent chaperones In addition to the major conserved chaperones and proteases, a suite of holdases and other proteins may cooperate to direct the activity of generalist proteostasis machines towards specific goals, or may be dedicated towards

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specific cellular tasks. These proteins include the stress-responsive small heat shock proteins, classified according to the presence of an alpha-crystallin domain, process-specific holdases such as the protein transport chaperone SecB, and chaperedoxins such as CnoX and Hsp33 (49,134,135,215). A shared function between these different holdases is to bind unfolded proteins and maintain them in a folding-competent state—either in optimal conditions during biogenesis and transport, or during stress conditions that provoke protein unfolding. Collaboration between holdases, chaperones, and proteases is also observed, with one example being how the small heat shock proteins assist DnaKJ/E-ClpB in remediating protein ag-gregation (216,217).

7. Protein aggregation and lifespan While the proteostasis network of bacteria is designed to effectively protect and repair proteins functioning in essential processes, damage accumulates and the reproductive capacity of prokaryotes is not infinite (218). As in eu-karyotes, accumulation of cellular damage throughout the lifespan of a cell affects the ability of prokaryotes to continue replicative processes (118,219,219–223). Unequal distribution of cellular damage during division is linked to the replicative decline of damage-inheriting cells (223–226), and aggregated protein has been proposed to be an heritable aging factor driving this process in prokaryotes (118,119). Therefore, much investigation has been performed into how prokaryotes share aggregated protein across gener-ations, and what the consequences of inheriting or escaping inheritance of these proposed aging factors are.

What has become evident is that the biophysical properties of the cyto-plasm and intensity of stress both affect how aggregated protein is shared by actively dividing bacteria. In E. coli and B. subtilis protein aggregates typi-cally form at the poles and midcell, in the space unoccupied by the nucleoid (10,118,156,169,227–230). In Caulobacter and Mycobacteria however, dif-ferences in nucleoid structure (discussed above and in study I) give rise to the development of multiple foci of protein aggregation (7,153,170). The organization of the cytoplasm can further impact how protein aggregates are managed, as while Caulobacter aggregates were determined to be relatively static in study I, Mycobacterial aggregates collect at the pole (153,170). During subsequent division, these different subcellular organizations of ag-gregates give rise to markedly different patterns of inheritance (146). The intensity of stress also frequently affects the number, size, and stability of protein aggregates that develop, with consequences for how these aggregates are managed during survival. Studies modelling the effects of accumulating damage in bacterial populations suggest that during high intensity stress, repair processes become inefficient and sequestration of damage or protein

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aggregates within one segment of the population confers a growth advantage on the damage-evading segment (219,220,231). This model is consistent with early experimental reports indicating that retention of protein aggre-gates is associated with reduced growth rate (118,156), and supported by the clear consequences of aggregate retention in Mycobacteria (153), however other reports have indicated that inheritance of aggregate-associated HSPs confers an advantage in resistance against future stresses (232). In study I we determine the aggregate inheritance pattern in Caulobacter, demonstrat-ing that protein aggregates are maintained in stalked and swarmer cell popu-lations in stable ratios over several divisions, and found no population growth defect in aggregate-retaining cells (170). While these results refute protein aggregates as the aging factor accumulating in the Caulobacter stalked cell, the age of the envelope compartment present at the cell poles has also been associated with replicative decline (118,156,219,221,233), and may underlie the observed replicative decline of the stalked cell.

8. The function of protein folding Chaperones are capable of binding proteins that are both reversibly and irre-versibly denatured in vitro, however in vivo this class of proteins acts to pro-tect proteins before irreversible damage occurs (234). A global picture of how the proteostasis network maintains the proteome and resists overwhelm-ing aggregation has been modeled (234–236), but the contribution of indi-vidual client proteins as they flow through cycles of synthesis, folding, re-folding, and degradation depends on a myriad of mechanisms, taking into account factors from propensity to misfold to environmental niche. As demonstrated by the examples of HfiA, σ73, and HrcA, the stability of a pro-tein, mediated through its folding state, offers an opportunity for modulation of its function with the availability of protein folding machines and the stress state of the cell. For some proteins, this regulation through folding may be advantageous, however for other proteins, such as respiratory chain complex components or cytoskeletal elements, it is of crucial importance that their functions are protected during stress. Much remains to be discovered on how proteins of essential processes interact with the folding machines of the pro-teostasis network during their biogenesis, and the mechanisms of how they are defended from stress-induced damage. This is particularly true for the chaperonin GroESL, where folding of client proteins has been studied pri-marily in vitro or in isolation from the biological pathway they participate in. Furthermore, as chaperonins in particular are associated with providing a variety of moonlighting functions (237), studying their conserved and spe-cialized folding functions in organisms other than E. coli is important to understanding the central biological functions of prokaryotic GroESL. In order to better understand this process, the overarching aim of the work con-

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tained herein was to uncover how protein folding is integrated with essential processes in the model organism Caulobacter, and how these relationships change during stress.

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AIMS OF THIS THESIS

The aim of this thesis is to further understanding of how the proteostasis network and specifically protein folding is integrated with essential bacterial processes, with a special focus on processes operating in the cytoplasm to support the cell envelope.

This thesis is divided to address the following specific aims:

I To determine how protein aggregate inheritance is managed during division in asymmetrically-dividing Caulobacter.

II To identify how the chaperonin GroESL is integrated into the Caulobacter cell cycle.

III To understand the functional contributions of GroESL-mediated folding to energy metabolism in the aerobic microorganism Caulobacter crescentus.

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MAIN FINDINGS

STUDY I

Growth-driven displacement of protein aggregates along the cell length ensures partitioning to both daughter cells in Caulobacter crescentus.

Prior to this study, protein aggregation inheritance had been investigated in asymmetrically dividing yeast and in symmetrically dividing rod-shaped bacteria (118,153,156,222–224,238). Previous work in the asymmetrically dividing Caulobacter demonstrated replicative decline of the stalked cell type (119), similar to what is observed in yeast. As retention of protein ag-gregates in the mother cell is proposed to underpin aging of this cell type, we investigated the inheritance of protein aggregates in Caulobacter. In this study, we established Caulobacter as a system for studying protein aggrega-tion, characterized the dynamics of protein aggregation and resolution pro-voked by stress of different intensities, and discovered how persistent aggre-gates are distributed during division.

The Caulobacter proteostasis network as a model of protein aggregation. In this study we created and validated a set of tools to study aggregate for-mation and resolution in Caulobacter. Using fluorescent fusions to the pro-teostasis network proteins DnaK and ClpB, as well as to a set of endogenous proteins we identified as aggregation-prone, we determined that protein ag-gregates form as multiple clusters distributed throughout the cell volume. Using genetic knockouts of small heat shock proteins, ClpB, and the prote-ase Lon, we further determined that the concerted activity of DnaK and ClpB is required to resolve protein aggregates once they have formed. In the ab-sence of ClpB, protein aggregates were highly persistent as discrete punctate foci.

Strategies of aggregate management during division depend on stress intensity. Using time lapse microscopy to assess protein aggregation dynamics during sustained stress and recovery, we determined that two modes of aggregate management exist. During stress of low to medium intensity, protein aggre-

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gates are rapidly dissolved through the action of the proteostasis network, resulting only in mild effects on growth and cell shape. During high intensity stress, strong effects on growth and cell shape were observed, protein aggre-gates persisted for longer time scales, and these more persistent aggregates were associated with greater defects in recovery. We determined that the persistent aggregates formed by high intensity stress were managed through dilution in the growing culture.

Caulobacter partitions persistent aggregates to both daughter cells. Using a ClpB mutant (where all aggregates are managed through dilution), we determined that protein aggregates do not collect in a specific cellular location over time, but are instead displaced, or spread apart, in proportion to the growth of the cell and its descendants. Using this method of aggregate partitioning, protein aggregates only very rarely became closer to the stalked pole, and placement of the division plane in the pre-divisional cell deter-mined the likelihood of inheriting an aggregate. The fraction of aggregates existing at the pole, as well as the fraction of aggregates inherited by a stalked cell was stable across several generations. This describes a new mode of microbial aggregate inheritance, where insoluble protein deposits are distributed through a combination of growth and division.

Figure 7. Model of persistent aggregate inheritance in Caulobacter. As Cau-lobacter grows, aggregates formed in response to stress are displaced in pro-portion to the elongation of the cell, while maintaining their relative position. Aggregates forming at the poles are not displaced from this region, as the poles are not areas of active growth in Caulobacter (87). Positioning of the division plane governs the distribution of aggregates between swarmer and stalked cells, and cell elongation in post-division or post-differentiation stalked cells drives further displacement before subsequent divisions. Num-bers represent the relative position between the old (0) pole and the new pole (1). Figure is reproduced from (170).

protein aggregatedirection of cell elongation

00.250.50.75

1

00.250.50.75

1

00.250.50.75

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STUDY II The chaperonin GroESL facilitates Caulobacter crescentus cell division by supporting the functions of the Z-ring regulators FtsA and FzlA.

Previous work in Caulobacter linked the major ATP-dependent chaperones to the cell cycle through DnaKJ/E regulation of DNA replication (2), and through GroESL by an unknown mechanism (191). Therefore, in this study we investigated the involvement of Caulobacter GroESL in cell cycle pro-gression, identifying several proteins involved in division and PG biosynthe-sis to have an interaction with the chaperonin. We further investigated the function of the Z-ring when GroESL levels were insufficient, and character-ized FtsZ-interacting proteins as mediating a link between protein biosynthe-sis and cell division.

GroESL is integrated into the cell cycle at the point of cell division. Similarly to E. coli, we expected that Caulobacter GroESL would be respon-sible for folding several essential proteins, therefore we first assessed at what point depletion of GroESL arrests the cell cycle. We demonstrated that in-sufficient GroESL does not interfere with DNA replication, and that the transcriptional circuit of cell cycle regulation in Caulobacter is only mildly affected when GroESL levels are reduced. These results indicated that when GroESL folding is insufficient, a problem specific to division or cell septa-tion occurs.

GroESL assists cell envelope and division proteins in becoming soluble. Using a proteomics approach, we identified proteins with decreased solubili-ty specifically when GroESL folding was insufficient for survival. Through this experiment, we identified that several proteins of the PG biosynthetic pathway and related metabolism (DapA, MurG, MurA), as well as several proteins of Caulobacter division (KidO, FtsA, FzlA) are assisted in their solubility by GroESL. Additionally, we found that the proteins DapA, Mu-rA, and KidO are degraded when GroESL is insufficient, indicating chap-eronin collaboration with one or more proteases.

GroESL folding supports several points in PG biosynthesis. Classical experiments performed in E. coli show that an interaction between DapA and GroESL is required to support PG biosynthesis (239), therefore we determined if a similar requirement existed in Caulobacter. Surprisingly, neither providing the DapA reaction product nor increasing the levels of MurG or MurA improved the PG synthesis defect of insufficient GroESL. We observed altered localization of MurG, supporting the possibility that it becomes aggregated when GroESL levels are insufficient. As supplementing

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the identified PG biosynthetic proteins individually did not improve the PG defect, we conclude GroESL folding supports multiple points in the PG bio-synthetic pathway.

Increased FtsA and FzlA can specifically compensate for the division defect of GroESL depletion. We found that constriction, mediated by the dynamics of the Z-ring, was slowed with even a slight decline in GroESL levels, suggesting that one or more FtsZ-interacting proteins interacts with GroESL. We demonstrated that increasing levels of the constriction regulator FzlA or the FtsZ membrane anchor FtsA, both identified in our proteomics screen, could compensate for the cell division defect imposed by insufficient GroESL folding. While providing extra amounts of these proteins did not restore the biomass defect of GroESL depletion, filamentation was significantly delayed and cells com-pleted more division events. We found that previously described suppressor mutations obviating the need for FzlA were unable to improve the cell divi-sion defect, suggesting FtsA may have a primary interaction with GroESL. The GroESL-FtsA interaction is required for division during mild stress. Upregulation of GroESL is required to withstand proteotoxic stress, however the proteins that it supports during this condition have not been described. We therefore assessed if increasing levels of FtsA could improve the cell division defect observed when GroESL is restricted during mild heat stress, and demonstrated that this increased cell division events. As wild type Cau-lobacter is similarly observed to halt cell division during elevated tempera-ture (observed in (132) and study I), the GroESL-FtsA interaction may be restricted to specific conditions where it is safe to divide.

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Figure 8. GroESL supports the function of several division proteins and PG biosynthetic enzymes during Caulobacter division. The chaperonin GroESL assists client proteins from stable folding intermediates into the native con-formation (star). GroESL folding supports the solubility of FtsA, FzlA, and KidO in the divisome, and MurG, DapA, and MurA in the PG biosynthetic pathway. Proteins indicated in red are able to temporarily rescue the division defect of insufficient GroESL folding if provided in excess. Proteins indicated in purple are degraded if synthesized in the absence of sufficient GroESL folding. Proteins indicated in green exhibit altered localization when GroESL folding is insufficient. Proteins indicated in grey do not exhibit a relationship between GroESL folding and their solubility. Dashed lines indicate interac-tions that may proceed through unidentified intermediates. OM, outer mem-brane; IM, inner membrane; Z, FtsZ. Membrane and PG images created with Biorender (Biorender.com). Figure is reproduced from (193).

PG synthesisCell division

GroESL

FtsA FtsA FtsA MurG

MraYMurF

MurEMurD

MurCMurB

MurA

KidO

ZZZZ

DapA

Lipid IIIM

OM

PG

FzlA

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STUDY III Chaperonin folding protects energy metabolism via TCA cycle and res-piratory chain proteins in the obligate aerobe Caulobacter crescentus. Subunits of several respiratory complexes have an obligate requirement for GroESL folding in E. coli (173,178), suggesting that chaperonin folding is intricately connected with energy metabolism. In study II we identified that Caulobacter homologues of GroESL-dependent NADH dehydrogenase and succinate dehydrogenase subunits also exhibited a relationship with GroESL for their solubility. As the obligate aerobe Caulobacter has a more limited repertoire of respiratory complexes than E. coli, we investigated the func-tional relationship between GroESL folding and respiration in this organism. Furthermore, since metabolism and lipid biosynthesis are intimately con-nected with the function of respiration, we additionally assessed the function of identified conserved GroESL client homologues in fatty acid biosynthesis and the TCA cycle.

GroESL client proteins that function in energy metabolism are sensitive to proteotoxic stress. We firstly utilized proteomics to identify proteins enriched in the insoluble fraction when GroESL levels were restricted in the presence of mild proteo-toxic stress. We identified the NADH dehydrogenase subunit NuoC, the fatty acid biosynthetic protein FabA, and the TCA cycle protein mdh all to become more insoluble specifically when GroESL levels were unable to be raised to cope with the increased folding demand. This indicates a role for the chaperonin not only in the biogenesis of these proteins, but also in pro-tecting them from stress.

GroESL is required to support fatty acid biosynthesis, membrane potential, and carbon catabolism. To determine if the solubility interaction between the energy metabolism proteins was reflected in a functional relationship, we determined the effects of reduced GroESL folding on pathways containing the identified candi-dates. Insufficient GroESL folding resulted in destabilization of fatty acid biosynthesis and hypersensitivity to fatty acid-targeting antibiotics, as well as a reduction in membrane potential and in carbon catabolism. Collectively, this supports a relevant role for GroESL folding in the biogenesis of energy metabolism proteins.

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Specific metabolic supplementation can partially compensate for the functional deficits of insufficient GroESL folding. As the pathways of fatty acid synthesis, respiration, and carbon catabolism are linked, we attempted to separate the specific contributions of each path-way to the functional defects of insufficient GroESL. However, supplemen-tation with unsaturated fatty acids or TCA cycle intermediates did not restore biomass potential. Fatty acid supplementation exacerbated the division de-fects investigated in study II, however growth in excess TCA cycle inter-mediates increased the number of cell division events when GroESL was insufficient. Furthermore, the specific addition of fumarate was able to re-store membrane potential. These data indicate that the interplay between GroESL and its client proteins is highly complex, but that GroESL folding capacity is required to support energy metabolism.

Figure 9. Energy metabolism proteins that require GroESL for solubility in Caulobacter in optimal and stress conditions. GroESL folding supports the solubility of a cluster of proteins involved in Caulobacter energy metabolism, including the TCA cycle, oxidative phosphorylation, and fatty acid biosynthe-sis. Proteins indicated in red exhibit decreased solubility when GroESL fold-ing is insufficient in optimal conditions. Proteins indicated in yellow exhibit decreased solubility when GroESL folding is insufficient in optimal condi-tions as well as during mild stress. Proteins indicated in blue have not been identified to have a relationship between their solubility and GroESL folding. Green arrows indicate reactions of the TCA cycle. OM, outer membrane; IM, inner membrane.

Respiratory Complex II sdhB

sucC

TCA Cycle

mdh

fumC

sucDsdhA

malate

fumarate

succinate

succinyl-CoA

oxaloacetate

sdhCsdhD

nuoFnuoEnuoG

nuoInuoC

nuoBnuoD

nuoA

nuoLnuoMnuoN

nuoKnuoJ

nuoH

IMOM

Respiratory Complex I

Oxidative Phosphorylation

fabAfabFacetyl-coA

Fatty Acid Biosynthesis

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FUTURE PERSPECTIVES

In study I we showed that protein aggregate inheritance is governed by the intracellular environment and positioning of the division plane in Caulobac-ter. As the experiments of this study were conducted using mixed popula-tions, one open question is if the response to protein aggregation is different between the swarmer and stalked cell. The two cell types are different in protein complements (127), and have been demonstrated to deploy different protein-based mechanisms for escaping or withstanding stress (240). While addressing the impact of proteotoxic stress on the stalked and swarmer cell types is a technically demanding investigation, understanding if cell type-specific sensitivity to stress exists in prokaryotes could open the door for future investigations into the regulation of bacterial differentiation. Further-more, protein aggregate management in bacteria with even more complex cell types—such as that of the spore-forming Streptomycetes—would be an interesting study to further address strategies used by specialized cell types to manage protein aggregation.

Another remaining open question from this study centers on how protein aggregates are resolved during mistranslation stress. During heat stress, ClpB is highly induced and collaborates with DnaKJ/E to dissolve protein aggregates, however during mistranslation stress we did not observe robust induction of the σ32-dependent heat shock response, and by extension ClpB. If one or more proteases are responsible for aggregate resolution in this con-dition, or if another mechanism exists to resolve misfolded proteins caused through mistranslation stress remains an open avenue for investigation.

Lastly, the findings of this study did not support accumulation of protein aggregates in the stalked cell as a mechanism that could explain replicative decline in this morphotype (117). Inheritance of the oldest pre-existing por-tion of a cell has been associated with replicative decline in E. coli, and fur-thermore inheritance of older, spatially-restricted membrane components imposes a replicative cost to this organism (118,156,219,221,233). Since the stalked cell possesses many polar factors involved in signalling and morpho-genesis, and organizes these into a phase-separated polar domain (150,241), it would be interesting to test if the composition or phase properties of this microdomain decay as the stalked cell ages.

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In study II, we identified functional relationships between GroESL and a set of proteins acting in cell envelope biogenesis and cell division. During the course of this study we identified several PG biosynthetic enzymes to have a relationship with GroESL folding, but were unable to find a condition that could improve PG biosynthesis when GroESL levels were insufficient. Sup-plementing later stages of this pathway, or boosting levels of multiple PG pathway enzymes may reveal how PG biosynthesis is linked with cytosolic proteostasis in Caulobacter. However, as accumulation of the PG biosyn-thetic proteins DapA and MurA was prevented during periods of substantial-ly reduced GroESL activity, collaboration between chaperonin availability and a yet unknown protease may regulate the abundance of these proteins. Identification of the proteases responsible for PG biosynthetic enzyme deg-radation is a clear next step in understanding how cell envelope synthesis is regulated by the proteostasis network.

As in vitro analysis of the interaction between GroESL and the candidate client proteins we identified has not been performed, assessment of folding interactions, particularly between FtsA and GroESL, will be important to address. While eukaryotic actin proteins exhibit chaperonin dependency (242,243), E. coli GroESL is not competent to fold actin (242). If FtsA is indeed a bona fide Caulobacter GroESL substrate, investigating the folding mechanism and comparing this to both mitochondrial actin and E. coli FtsA folding by their respective native chaperonins could shed light on the evolu-tionary path of the chaperonin-actin protein relationship.

During this study we also identified that division is supported by the GroESL-FtsA interaction during mild heat stress. It remains unclear if a re-duction in FtsA function and subsequent block in division is perhaps benefi-cial to surviving stresses, as has been hypothesized for filamentation strate-gies (130). Notably, FtsA is degraded in Caulobacter by the protease ClpAP (244), suggesting it may exhibit complex regulation during stress in this organism. Experiments to determine how the folding and degradation of FtsA occurs during stress would clarify how FtsA function is regulated by environmental changes. In study III we address the functional link between GroESL and proteins involved in energy metabolism that are known to depend on the chaperonin in other organisms. As the processes of carbon metabolism, oxidative phos-phorylation, and membrane homeostasis are intertwined, and further can vary widely by organism, investigation in Caulobacter may yield insight into how these processes function in the α-proteobacteria, and how regulation is conserved with other prokaryotes. More investigation is being performed into the respiratory activities of bacteria other than E. coli (98,100), and study of how chaperonin folding is integrated into respiration of an obligate aerobe adds a needed perspective to this discussion. Activity of other proteo-stasis network proteins has been linked to respiration in Caulobacter (245),

35

and genetic regulation of certain respiratory components during hypoxia has been described (97), however uncovering the full network of respiratory complexes and any existing links with the proteostasis network is a prerequi-site for continuing to develop the Caulobacter model.

GroESL has been linked to stabilizing the membrane as a lipochaperonin (246), and is highly expressed following membrane stresses in diverse bacte-ria (132,203). Links between GroESL and protein transport and cytoplasmic membrane function are not yet well understood, and given the large number of antimicrobial agents that target this compartment, it is important to re-solve how the cytosolic chaperonin is specialized to promote survival of membrane targeting stresses. Specifically, determining how the function of stress-sensitive GroESL candidate client proteins identified in this study is protected during membrane-targeting stress will be important to understand-ing how bacteria resist this common antimicrobial strategy at the proteome level.

Finally, as we identify certain subunits of NADH dehydrogenase and suc-cinate dehydrogenase to have a conserved interaction with Caulobacter GroESL, and further identify subunits of these complexes to be aggregation-prone proteins (see study I for SdhA aggregation), an intriguing next step would be to monitor assembly of these complexes during optimal and stress conditions, as well as when chaperonin availability is reduced. Connecting observation of complex assembly or aggregation of these subunits at super-resolution with functional assessment would yield insight into how protein folding or complex assembly is regulated during stress.

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ACKNOWLEDGEMENTS

Well, here we are. There are so many people who helped me get to this point, I wish I had the time and space here to thank each one of you for how you’ve shaped this journey. Firstly I would like to thank my supervisor Dr. Kristina Jonas for giving me the opportunity to do a PhD in your lab. I have grown a lot as a scientist in this environment, and I appreciate the independence I had during this time to develop projects based on my own interests. Thank you for always sup-porting me going after an interesting hypothesis or result, and for reminding me that phenotypes are never black and white. I would also like to thank all the senior scientists who have been part of my committees over the years, including my co-supervisor Dr. Stefan Åström, Dr. Claes Andréasson, Dr. Sabrina Büttner, and Dr. Ann-Beth Jonsson. I would also like to thank my mentors from the University of Saskatchewan, and in particular Dr. Vikram Misra and Dr. Joyce Wilson for supporting my journey to holding a PhD. In addition, I would like to thank Dr. Benja-min Rosser, Dr. William Kulyk, Dr. Patrick Krone, and everyone from the ACB department for letting me moonlight in the world of anatomy re-search and being great mentors and colleagues. Finally, I would like to thank Dr. Barry Ziola for picking me out of class one day and giving me the op-portunity to work in a research lab. It's been an incredible journey since then.

To all the members of the Jonas Lab, past and present, thank you for your help in keeping experiments running and for your thoughtful input during journal clubs and lab meetings. In particular, I would like to thank Deike, for modelling how to tackle scientific challenges with rigor and for being a nice desk neighbour; Michele, for always giving insightful advice and for ampli-fying my scientific voice; and to Chris for collaborating on the energy me-tabolism project and for being a kind human always up for discussing some random result I unearthed in an old notebook. Thank you to Blanca, for bringing a sense of community and friendship even during social distancing; to Matthias for always having time for a chat over the fence and for the fun of naming the incubators and hunting for more flasks during teaching; to Aswathy for your sense of humour and for letting me spam you with science

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memes; and to Joel for also being very enthusiastic about microscopy and journal club papers. Thank you all for being supportive people to be in the lab with, especially during the pandemic this past year. I would also like to thank David L., Kristina H., and Fred for your help with getting started with Caulobacter in Marburg all those years ago, and for your work in our collaborations. To my student Ines, thank you for being a star with your part of the GroESL project, and for being patient while I was running a hundred experiments. Lastly, to all the students and interns who came through the lab during my time here, thank you for sharing your excitement for science and creating a nice atmosphere to work in. I would also like to thank my collaborators, especially Jonatan for your patience through the STED troubleshooting, but also Dr. Ilaria Testa and everyone from the Testa Lab for being so helpful. Thank you to Dr. Erin Goley for providing so much experimental material, and then providing ad-vice on what I found with it. And thank you to Gabriella, Fengyang, and to everyone who came by to look at cool stuff under the microscope with me.

I would also like to thank the scientific communities I’ve been fortunate to be a part of during my PhD. Firstly, thank you to all of the ever-changing community of Gamma 5 and in particular the Hudson and PEOZ groups, who created the working environment. In particular, thank you to Markus, Johannes, Ivana, Danuta, Linnea, Lotta, Natalie, Björn, Antonino, Pa-tricia, Sissi, David H., Karen, and Jan, for all the lunchtime conversations, and especially for the inception of ‘Science! The Musical’. Thank you also to Nick and Tobi for keeping me company in the lab during the early pan-demic times. Thank you to the community at MBW, and especially to Gelana and Birgit-ta from administration for helping me with paperwork and poster printing. Thank you to Anna for helping me figure out how to normalize the insoluble fraction. To my teaching pals, thank you for the great times, especially to Sara, for showing me how everything worked as a PhD student at SU, to Gabriella for the yogurt-making and PG diagrams and for being a ray of sunshine, and to Franzi for attempting to integrate us SciLifeLab outliers into the MBW social circle. I would also like to thank the IMPRS-Marburg, especially to Manuel, Shankar, Carolina, Bob, Janina, Sofia, Francisco, Tarryn and Hanna, and also Wiebke for being excellent humans and excellent researchers, for sharing the experience of moving to rural Germany with me, and for making my time there so memorable and so so so hard to leave. Finally, thank you to the CauloSlackers community for all the suggestions and for being such a welcoming and friendly community of scientists.

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I would also like to thank all of my friends and the people who have sup-ported me during these last five years. Thank you to Hanna and Tarryn for your unwavering support and wisdom, for always being there to talk about everything from big life decisions to very small glassware, and for the count-less thousands of heart stickers. Thank you to my people back in Canada and across the world; Kristen and Matthew, Angie, Karen, Nykki and Regan, Candice, Kathee, Rachelle, Melanie, Janna, and Stacey and everyone who has supported me. Thank you to my sister Nicole for being chill about get-ting a wall of texts at 4am mountain time, and to my parents for helping me coordinate moving to a different continent. Thank you to my people here in Sweden; to the Strahd group (Kirsti, Frej, Johannes, Sophia, and Oliver), and the Alien group (Johannes, Fabian, Chester, and Oliver) for the adventures. Thank you to Malin, Sophia, Mia, Catta, Jessica, and Anna for adopting me into your deep-thinking, deep-feeling group, and for all the laughter over the years. I would also like to thank all of the people in all of the dance and aerial arts studios I’ve walked into over the years who have helped me tame my stress-encumbered SNS, forget about science problems, and fly. You were instru-mental in me surviving my PhD, yet are so numerous it would take another book to list you all. Thank you to Kevin and my writing group for giving me feedback on the frankly exhausting number of adjectives and the sheer paucity of periods I’m prone to. It has been invaluable training for the Spring of Writing Every-thing. Now that I'm done with this book, maybe I'll write about Aoife and Scayhach :) And last but most certainly not least, thank you to Henrik for being there through all of it. I truly would not be here writing the acknowledgements section of this thesis without your support. Thank you for always resolutely insisting that I am an amazing scientist, and for suffering through countless midnight practice presentations about chaperonins. ♥

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