Toward a Mechanistic Understanding of Bacterial Rod Shape Formation and Regulation.

One of the most common bacterial shapes is a rod, yet we have a limited understanding of how this simple shape is constructed. While only six proteins are required for rod shape, we are just beginning to understand how they self-organize to build the micron-sized enveloping structures that define bacterial shape out of nanometer-sized glycan strains. Here, we detail and summarize the insights gained over the last 20 years into this complex problem that have been achieved with a wide variety of different approaches. We also explain and compare both current and past models of rod shape formation and maintenance and then highlight recent insights into how the Rod complex might be regulated.

Single-molecule dynamics show a transient lipopolysaccharide transport bridge.

Gram-negative bacteria are surrounded by two membranes. A special feature of the outer membrane is its asymmetry. It contains lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet1-3. The proper assembly of LPS in the outer membrane is required for cell viability and provides Gram-negative bacteria intrinsic resistance to many classes of antibiotics. LPS biosynthesis is completed in the inner membrane, so the LPS must be extracted, moved across the aqueous periplasm that separates the two membranes and translocated through the outer membrane where it assembles on the cell surface4. LPS transport and assembly requires seven conserved and essential LPS transport components5 (LptA-G). This system has been proposed to form a continuous protein bridge that provides a path for LPS to reach the cell surface6,7, but this model has not been validated in living cells. Here, using single-molecule tracking, we show that Lpt protein dynamics are consistent with the bridge model. Half of the inner membrane Lpt proteins exist in a bridge state, and bridges persist for 5-10 s, showing that their organization is highly dynamic. LPS facilitates Lpt bridge formation, suggesting a mechanism by which the production of LPS can be directly coupled to its transport. Finally, the bridge decay kinetics suggest that there may be two different types of bridges, whose stability differs according to the presence (long-lived) or absence (short-lived) of LPS. Together, our data support a model in which LPS is both a substrate and a structural component of dynamic Lpt bridges that promote outer membrane assembly.

Fluorogenic Probes of the Mycobacterial Membrane as Reporters of Antibiotic Action.

The genus Mycobacterium includes species such as Mycobacterium tuberculosis, which can cause deadly human diseases. These bacteria have a protective cell envelope that can be remodeled to facilitate their survival in challenging conditions. Understanding how such conditions affect membrane remodeling can facilitate antibiotic discovery and treatment. To this end, we describe an optimized fluorogenic probe, N-QTF, that reports on mycolyltransferase activity, which is vital for cell division and remodeling. N-QTF is a glycolipid probe that can reveal dynamic changes in the mycobacterial cell envelope in both fast- and slow-growing mycobacterial species. Using this probe to monitor the consequences of antibiotic treatment uncovered distinct cellular phenotypes. Even antibiotics that do not directly inhibit cell envelope biosynthesis cause conspicuous phenotypes. For instance, mycobacteria exposed to the RNA polymerase inhibitor rifampicin release fluorescent extracellular vesicles (EVs). While all mycobacteria release EVs, fluorescent EVs were detected only in the presence of RIF, indicating that exposure to the drug alters EV content. Macrophages exposed to the EVs derived from RIF-treated cells released lower levels of cytokines, suggesting the EVs moderate immune responses. These data suggest that antibiotics can alter EV content to impact immunity. Our ability to see such changes in EV constituents directly results from exploiting these chemical probes.

Tetraspanin CD82 Organizes Dectin-1 into Signaling Domains to Mediate Cellular Responses to Candida albicans.

Tetraspanins are a family of proteins possessing four transmembrane domains that help in lateral organization of plasma membrane proteins. These proteins interact with each other as well as other receptors and signaling proteins, resulting in functional complexes called "tetraspanin microdomains." Tetraspanins, including CD82, play an essential role in the pathogenesis of fungal infections. Dectin-1, a receptor for the fungal cell wall carbohydrate ß-1,3-glucan, is vital to host defense against fungal infections. The current study identifies a novel association between tetraspanin CD82 and Dectin-1 on the plasma membrane of Candida albicans-containing phagosomes independent of phagocytic ability. Deletion of CD82 in mice resulted in diminished fungicidal activity, increased C. albicans viability within macrophages, and decreased cytokine production (TNF-α, IL-1ß) at both mRNA and protein level in macrophages. Additionally, CD82 organized Dectin-1 clustering in the phagocytic cup. Deletion of CD82 modulates Dectin-1 signaling, resulting in a reduction of Src and Syk phosphorylation and reactive oxygen species production. CD82 knockout mice were more susceptible to C. albicans as compared with wild-type mice. Furthermore, patient C. albicans-induced cytokine production was influenced by two human CD82 single nucleotide polymorphisms, whereas an additional CD82 single nucleotide polymorphism increased the risk for candidemia independent of cytokine production. Together, these data demonstrate that CD82 organizes the proper assembly of Dectin-1 signaling machinery in response to C. albicans.

A central role for PBP2 in the activation of peptidoglycan polymerization by the bacterial cell elongation machinery.

Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery.

Division plane placement in pleomorphic archaea is dynamically coupled to cell shape.

One mechanism for achieving accurate placement of the cell division machinery is via Turing patterns, where nonlinear molecular interactions spontaneously produce spatiotemporal concentration gradients. The resulting patterns are dictated by cell shape. For example, the Min system of Escherichia coli shows spatiotemporal oscillation between cell poles, leaving a mid-cell zone for division. The universality of pattern-forming mechanisms in divisome placement is currently unclear. We examined the location of the division plane in two pleomorphic archaea, Haloferax volcanii and Haloarcula japonica, and showed that it correlates with the predictions of Turing patterning. Time-lapse analysis of H. volcanii shows that divisome locations after successive rounds of division are dynamically determined by daughter cell shape. For H. volcanii, we show that the location of DNA does not influence division plane location, ruling out nucleoid occlusion. Triangular cells provide a stringent test for Turing patterning, where there is a bifurcation in division plane orientation. For the two archaea examined, most triangular cells divide as predicted by a Turing mechanism; however, in some cases multiple division planes are observed resulting in cells dividing into three viable progeny. Our results suggest that the division site placement is consistent with a Turing patterning system in these archaea.

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis.

The integrity of the bacterial cell envelope is essential to sustain life by countering the high turgor pressure of the cell and providing a barrier against chemical insults. In Bacillus subtilis, synthesis of both peptidoglycan and wall teichoic acids requires a common C55 lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases. Using an optimized clustered regularly interspaced short palindromic repeat (CRISPR) system with catalytically inactive ("dead") CRISPR-associated protein 9 (dCas9)-based transcriptional repression system (CRISPR interference [CRISPRi]), we demonstrate that B. subtilis requires either of two UPP phosphatases, UppP or BcrC, for viability. We show that a third predicted lipid phosphatase (YodM), with homology to diacylglycerol pyrophosphatases, can also support growth when overexpressed. Depletion of UPP phosphatase activity leads to morphological defects consistent with a failure of cell envelope synthesis and strongly activates the σM-dependent cell envelope stress response, including bcrC, which encodes one of the two UPP phosphatases. These results highlight the utility of an optimized CRISPRi system for the investigation of synthetic lethal gene pairs, clarify the nature of the B. subtilis UPP-Pase enzymes, and provide further evidence linking the σM regulon to cell envelope homeostasis pathways. IMPORTANCE: The emergence of antibiotic resistance among bacterial pathogens is of critical concern and motivates efforts to develop new therapeutics and increase the utility of those already in use. The lipid II cycle is one of the most frequently targeted processes for antibiotics and has been intensively studied. Despite these efforts, some steps have remained poorly defined, partly due to genetic redundancy. CRISPRi provides a powerful tool to investigate the functions of essential genes and sets of genes. Here, we used an optimized CRISPRi system to demonstrate functional redundancy of two UPP phosphatases that are required for the conversion of the initially synthesized UPP lipid carrier to Und-P, the substrate for the synthesis of the initial lipid-linked precursors in peptidoglycan and wall teichoic acid synthesis.

Bacterial Filament Systems: Toward Understanding Their Emergent Behavior and Cellular Functions.

Bacteria use homologs of eukaryotic cytoskeletal filaments to conduct many different tasks, controlling cell shape, division, and DNA segregation. These filaments, combined with factors that regulate their polymerization, create emergent self-organizing machines. Here, we summarize the current understanding of the assembly of these polymers and their spatial regulation by accessory factors, framing them in the context of being dynamical systems. We highlight how comparing the in vivo dynamics of the filaments with those measured in vitro has provided insight into the regulation, emergent behavior, and cellular functions of these polymeric systems.

Cell constriction requires processive septal peptidoglycan synthase movement independent of FtsZ treadmilling in Staphylococcus aureus.

Bacterial cell division requires recruitment of peptidoglycan (PG) synthases to the division site by the tubulin homologue, FtsZ. Septal PG synthases promote septum growth. FtsZ treadmilling is proposed to drive the processive movement of septal PG synthases and septal constriction in some bacteria; however, the precise mechanisms spatio-temporally regulating PG synthase movement and activity and FtsZ treadmilling are poorly understood. Here using single-molecule imaging of division proteins in the Gram-positive pathogen Staphylococcus aureus, we showed that the septal PG synthase complex FtsW/PBP1 and its putative activator protein, DivIB, move with similar velocity around the division site. Impairing FtsZ treadmilling did not affect FtsW or DivIB velocities or septum constriction rates. Contrarily, PG synthesis inhibition decelerated or stopped directional movement of FtsW and DivIB, and septum constriction. Our findings suggest that a single population of processively moving FtsW/PBP1 associated with DivIB drives cell constriction independently of FtsZ treadmilling in S. aureus.

An exhaustive multiple knockout approach to understanding cell wall hydrolase function in Bacillus subtilis.

IMPORTANCE: In order to grow, bacterial cells must both create and break down their cell wall. The enzymes that are responsible for these processes are the target of some of our best antibiotics. Our understanding of the proteins that break down the wall- cell wall hydrolases-has been limited by redundancy among the large number of hydrolases many bacteria contain. To solve this problem, we identified 42 cell wall hydrolases in Bacillus subtilis and created a strain lacking 40 of them. We show that cells can survive using only a single cell wall hydrolase; this means that to understand the growth of B. subtilis in standard laboratory conditions, it is only necessary to study a very limited number of proteins, simplifying the problem substantially. We additionally show that the ∆40 strain is a research tool to characterize hydrolases, using it to identify three "helper" hydrolases that act in certain stress conditions.

Transcriptomic analysis of pathways associated with ITGAV/alpha(v) integrin-dependent autophagy in human B cells.

Macroautophagy/autophagy proteins have been linked with the development of immune-mediated diseases including lupus, but the mechanisms for this are unclear due to the complex roles of these proteins in multiple immune cell types. We have previously shown that a form of noncanonical autophagy induced by ITGAV/alpha(v) integrins regulates B cell activation by viral and self-antigens, in mice. Here, we investigate the involvement of this pathway in B cells from human tissues. Our data reveal that autophagy is specifically induced in the germinal center and memory B cell subpopulations of human tonsils and spleens. Transcriptomic analysis show that the induction of autophagy is related to unique aspects of activated B cells such as mitochondrial metabolism. To understand the function of ITGAV/alpha(v) integrin-dependent autophagy in human B cells, we used CRISPR-mediated knockdown of autophagy genes. Integrating data from primary B cells and knockout cells, we found that ITGAV/alpha(v)-dependent autophagy limits activation of specific pathways related to B cell responses, while promoting others. These data provide new mechanistic links for autophagy and B-cell-mediated immune dysregulation in diseases such as lupus.

Mycobacterial nucleoid-associated protein Lsr2 is required for productive mycobacteriophage infection.

Mycobacteriophages are a diverse group of viruses infecting Mycobacterium with substantial therapeutic potential. However, as this potential becomes realized, the molecular details of phage infection and mechanisms of resistance remain ill-defined. Here we use live-cell fluorescence microscopy to visualize the spatiotemporal dynamics of mycobacteriophage infection in single cells and populations, showing that infection is dependent on the host nucleoid-associated Lsr2 protein. Mycobacteriophages preferentially adsorb at Mycobacterium smegmatis sites of new cell wall synthesis and following DNA injection, Lsr2 reorganizes away from host replication foci to establish zones of phage DNA replication (ZOPR). Cells lacking Lsr2 proceed through to cell lysis when infected but fail to generate consecutive phage bursts that trigger epidemic spread of phage particles to neighbouring cells. Many mycobacteriophages code for their own Lsr2-related proteins, and although their roles are unknown, they do not rescue the loss of host Lsr2.

The structure of bacterial ParM filaments.

Bacterial ParM is a homolog of eukaryotic actin and is involved in moving plasmids so that they segregate properly during cell division. Using cryo-EM and three-dimensional reconstruction, we show that ParM filaments have a different structure from F-actin, with very different subunit-subunit interfaces. These interfaces result in the helical handedness of the ParM filament being opposite to that of F-actin. Like F-actin, ParM filaments have a variable twist, and we show that this involves domain-domain rotations within the ParM subunit. The present results yield new insights into polymorphisms within F-actin, as well as the evolution of polymer families.

Incorporation of a Chemically Diverse Set of Non-Standard Amino Acids into a Gram-Positive Organism.

The incorporation of non-standard amino acids (nsAAs) within proteins and peptides through genetic code expansion introduces novel chemical functionalities such as photo-crosslinking and bioconjugation. Given the utility of Bacillus subtilis in fundamental and applied science, we extended existing nsAA incorporation technology from Escherichia coli into B. subtilis , demonstrating incorporation of 20 unique nsAAs. The nsAAs we succeeded in incorporating within proteins conferred properties that included fluorescence, photo-crosslinking, and metal chelation. Here, we describe the reagents, equipment, and protocols to test for nsAA incorporation at a small scale (96-well plate and culture tube scales). We report specific media requirements for certain nsAAs, including two variants for different media conditions. Our protocol provides a consistent and reproducible method for incorporation of a chemically diverse set of nsAAs into a model Gram-positive organism.

The role of cell-envelope synthesis for envelope growth and cytoplasmic density in Bacillus subtilis.

All cells must increase their volumes in response to biomass growth to maintain intracellular mass density within physiologically permissive bounds. Here, we investigate the regulation of volume growth in the Gram-positive bacterium Bacillus subtilis. To increase volume, bacteria enzymatically expand their cell envelopes and insert new envelope material. First, we demonstrate that cell-volume growth is determined indirectly, by expanding their envelopes in proportion to mass growth, similarly to the Gram-negative Escherichia coli, despite their fundamentally different envelope structures. Next, we studied, which pathways might be responsible for robust surface-to-mass coupling: We found that both peptidoglycan synthesis and membrane synthesis are required for proper surface-to-mass coupling. However, surprisingly, neither pathway is solely rate-limiting, contrary to wide-spread belief, since envelope growth continues at a reduced rate upon complete inhibition of either process. To arrest cell-envelope growth completely, the simultaneous inhibition of both envelope-synthesis processes is required. Thus, we suggest that multiple envelope-synthesis pathways collectively confer an important aspect of volume regulation, the coordination between surface growth, and biomass growth.

Tuberculosis treatment failure associated with evolution of antibiotic resilience.

The widespread use of antibiotics has placed bacterial pathogens under intense pressure to evolve new survival mechanisms. Genomic analysis of 51,229 Mycobacterium tuberculosis (Mtb)clinical isolates has identified an essential transcriptional regulator, Rv1830, herein called resR for resilience regulator, as a frequent target of positive (adaptive) selection. resR mutants do not show canonical drug resistance or drug tolerance but instead shorten the post-antibiotic effect, meaning that they enable Mtb to resume growth after drug exposure substantially faster than wild-type strains. We refer to this phenotype as antibiotic resilience. ResR acts in a regulatory cascade with other transcription factors controlling cell growth and division, which are also under positive selection in clinical isolates of Mtb. Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.

Single-molecule imaging reveals that Z-ring condensation is essential for cell division in Bacillus subtilis.

Although many components of the cell division machinery in bacteria have been identified1,2, the mechanisms by which they work together to divide the cell remain poorly understood. Key among these components is the tubulin FtsZ, which forms a Z ring at the midcell. FtsZ recruits the other cell division proteins, collectively called the divisome, and the Z ring constricts as the cell divides. We applied live-cell single-molecule imaging to describe the dynamics of the divisome in detail, and to evaluate the individual roles of FtsZ-binding proteins (ZBPs), specifically FtsA and the ZBPs EzrA, SepF and ZapA, in cytokinesis. We show that the divisome comprises two subcomplexes that move differently: stationary ZBPs that transiently bind to treadmilling FtsZ filaments, and a moving complex that includes cell wall synthases. Our imaging analyses reveal that ZBPs bundle FtsZ filaments together and condense them into Z rings, and that this condensation is necessary for cytokinesis.

Understanding Beta-Lactam-Induced Lysis at the Single-Cell Level.

Mechanical rupture, or lysis, of the cytoplasmic membrane is a common cell death pathway in bacteria occurring in response to ß-lactam antibiotics. A better understanding of the cellular design principles governing the susceptibility and response of individual cells to lysis could indicate methods of potentiating ß-lactam antibiotics and clarify relevant aspects of cellular physiology. Here, we take a single-cell approach to bacterial cell lysis to examine three cellular features-turgor pressure, mechanosensitive channels, and cell shape changes-that are expected to modulate lysis. We develop a mechanical model of bacterial cell lysis and experimentally analyze the dynamics of lysis in hundreds of single Escherichia coli cells. We find that turgor pressure is the only factor, of these three cellular features, which robustly modulates lysis. We show that mechanosensitive channels do not modulate lysis due to insufficiently fast solute outflow, and that cell shape changes result in more severe cellular lesions but do not influence the dynamics of lysis. These results inform a single-cell view of bacterial cell lysis and underscore approaches of combatting antibiotic tolerance to ß-lactams aimed at targeting cellular turgor.

Designing efficient genetic code expansion in Bacillus subtilis to gain biological insights.

Bacillus subtilis is a model gram-positive bacterium, commonly used to explore questions across bacterial cell biology and for industrial uses. To enable greater understanding and control of proteins in B. subtilis, here we report broad and efficient genetic code expansion in B. subtilis by incorporating 20 distinct non-standard amino acids within proteins using 3 different families of genetic code expansion systems and two choices of codons. We use these systems to achieve click-labelling, photo-crosslinking, and translational titration. These tools allow us to demonstrate differences between E. coli and B. subtilis stop codon suppression, validate a predicted protein-protein binding interface, and begin to interrogate properties underlying bacterial cytokinesis by precisely modulating cell division dynamics in vivo. We expect that the establishment of this simple and easily accessible chemical biology system in B. subtilis will help uncover an abundance of biological insights and aid genetic code expansion in other organisms.