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The mycobacterial cell wall is profoundly regulated in response to environmental stresses, and this regulation contributes to antibiotic tolerance. The reversible phosphorylation of different cell wall regulatory proteins is a major mechanism of cell wall regulation. Eleven Serine/Threonine protein kinases (STPKs) phosphorylate many critical cell wall-related proteins in mycobacteria. PstP is the sole serine/ threonine phosphatase, but few proteins have been verified as PstP substrates. PstP is itself phosphorylated but the role of its phosphorylation in regulating its activity has been unclear. In this study we aim to discover novel substrates of PstP in Mycobacterium tuberculosis (Mtb). We show in vitro that PstP dephosphorylates two regulators of peptidoglycan in Mtb, FhaA and Wag31. We also show that a phospho-mimetic mutation of T137 on PstP negatively regulates its catalytic activity against the cell wall regulators FhaA, Wag31, CwlM, PknB and PknA, and that the corresponding mutation in Mycobacterium smegmatis (Msmeg) causes mis-regulation of peptidoglycan in vivo. We show that PstP is localized to the septum, which likely restricts its access to certain substrates. These findings on the regulation of PstP provide insight into the control of cell wall metabolism in mycobacteria.

The paper “Cell wall damage reveals spatial flexibility in peptidoglycan synthesis and a nonredundant role for RodA in mycobacteria” by Melzer et al. (E. S. Melzer, T. Kado, A. Garcia-Heredia, K. R. Gupta, et al., J Bacteriol 204:e00540-21, 2022, https://doi.org/10.1128/JB.00540-21) presents several new observations about the localization and function of cell wall enzymes in Mycobacterium smegmatis and their responses to stress. This work illustrates some important aspects of cell wall physiology in mycobacteria and also points to a new model for how peptidoglycan synthesis may be organized in pole-growing bacteria.

Cell wall peptidoglycan is a heteropolymeric mesh that protects the bacteria from internal turgor and external insults. In many rod-shaped bacteria, peptidoglycan synthesis for normal growth is achieved by two distinct pathways: the Rod complex, comprised of MreB, RodA and a cognate class B PBP, and the class A PBPs. In contrast to laterally-growing bacteria, pole-growing mycobacteria do not encode an MreB homolog and do not require SEDS protein RodA forin vitrogrowth. However, RodA contributes to survival ofMycobacterium tuberculosisin some infection models, suggesting that the protein could have a stress-dependent role in maintaining cell wall integrity. Under basal conditions, we find here that the subcellular distribution of RodA largely overlaps with that of the aPBP PonA1, and that both RodA and the aPBPs promote polar peptidoglycan assembly. Upon cell wall damage, RodA fortifiesM. smegmatisagainst lysis and, unlike aPBPs, contributes to a shift in peptidoglycan assembly from the poles to the sidewall. Neither RodA nor PonA1 relocalize; instead, the redistribution of nascent cell wall parallels that of peptidoglycan precursor synthase MurG. Our results support a model in which mycobacteria balance polar growth and cell-wide repair via spatial flexibility in precursor synthesis and extracellular insertion.ImportancePeptidoglycan synthesis is a highly successful target for antibiotics. The pathway has been extensively studied in model organisms under laboratory-optimized conditions. In natural environments, bacteria are frequently under attack. Moreover the vast majority of bacterial species are unlikely to fit a single paradigm because of differences in growth mode and/or envelope structure. Studying cell wall synthesis under non-optimal conditions and in non-standard species may improve our understanding of pathway function and suggest new inhibition strategies.Mycobacterium smegmatis,a relative of several notorious human and animal pathogens, has an unusual polar growth mode and multi-layered envelope. In this work we challengedM. smegmatiswith cell wall-damaging enzymes to characterize the roles of cell wall-building enzymes when the bacterium is under attack.

The guanosine triphosphatase (GTPase) Rab32 coordinates a cell-intrinsic host defense mechanism that restricts the replication of intravacuolar pathogens such as Salmonella. Here, we show that this mechanism requires aconitate decarboxylase 1 (IRG1), which synthesizes itaconate, a metabolite with antimicrobial activity. We find that Rab32 interacts with IRG1 on Salmonella infection and facilitates the delivery of itaconate to the Salmonella-containing vacuole. Mice defective in IRG1 rescued the virulence defect of a S. enterica serovar Typhimurium mutant specifically defective in its ability to counter the Rab32 defense mechanism. These studies provide a link between a metabolite produced in the mitochondria after stimulation of innate immune receptors and a cell-autonomous defense mechanism that restricts the replication of an intracellular bacterial pathogen.

Ribosomes require the activity of associated GTPases to synthesize proteins. Despite strong evolutionary conservation, the roles of many of these remain unknown. For example, LepA (also known as elongation factor 4) is a ribosome-associated GTPase found in bacteria, mitochondria, and chloroplasts, yet its physiological contribution to cell survival is not clear. Recently, we found that loss of lepA in Mycobacterium smegmatis (Msm) altered tolerance to rifampin, a drug that targets a non-ribosomal cellular process. To uncover the determinants of LepA-mediated drug tolerance, we characterized the whole-cell proteomes and transcriptomes of a lepA deletion mutant relative to a wild-type strain. We find that LepA is important for the steady-state abundance of an outer membrane porin, which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount of porin in the membrane, resulting in the drug tolerance phenotype of the lepA mutant. LepA control requires a sequence motif in the 5’ region of the porin transcript. Thus, LepA controls the abundance of specific proteins, likely through its activity during translation.ImportanceOur understanding of how ribosomes properly synthesis an entire cellular proteome, in all its complexity, is still evolving. Ribosomal GTPases are often highly conserved, but the roles of many are not well understood. For example, elongation factor 4, or LepA, is a ribosome-associated GTPase conserved across bacteria, mitochondria, and chloroplasts. Using whole-cell proteomics and RNA-sequencing of wild type and a lepA deletion mutant, we find that LepA improves translation of mycobacterial porins in a message-specific manner. As porins play a key role in cell wall permeability, loss of LepA produces a plethora of phenotypic changes. These findings underline the problem of building proteins into a complex cell wall, such as that of mycobacteria, and point to a solution in the use of GTPases such as LepA, that have evolved to aid in specific protein synthesis.

The mycobacterial protein LamA functions as an inhibitor of cell wall synthesis at the nascent cell pole, contributing to asymmetry in polar growth, and could represent a much-needed target for the development of anti-tuberculosis therapies. Here the authors report the discovery of a new protein (LamA) that functions as part of the 'divisome' in mycobacteria. The authors report that LamA functions as an inhibitor of cell wall synthesis at the nascent cell pole, contributing to asymmetry in polar growth. Asymmetric growth and division increases population heterogeneity, which has been linked to antibiotic tolerance and persistent infection. Here they show that removal of LamA leads to a more uniform cell population that is more effectively killed by antibiotics. Thus, LamA represents a much-needed new target for the development of anti-tuberculosis therapies. Microorganisms are often studied as populations but the behaviour of single, individual cells can have important consequences. For example, tuberculosis, caused by the bacterial pathogen Mycobacterium tuberculosis, requires months of antibiotic therapy even though the bulk of the bacterial population dies rapidly. Shorter courses lead to high rates of relapse because subpopulations of bacilli can survive despite being genetically identical to those that are easily killed1. In fact, mycobacteria create variability each time a cell divides, producing daughter cells with different sizes and growth rates2,3. The mechanism(s) that underlie this high-frequency variation and how variability relates to survival of the population are unknown. Here we show that mycobacteria actively create heterogeneity. Using a fluorescent reporter and a fluorescence-activated cell sorting (FACS)-based transposon screen, we find that deletion of lamA, a gene of previously unknown function, decreases heterogeneity in the population by decreasing asymmetric polar growth. LamA has no known homologues in other organisms, but is highly conserved across mycobacterial species. We find that LamA is a member of the mycobacterial division complex (the ‘divisome’). It inhibits growth at nascent new poles, creating asymmetry in polar growth. The kinetics of killing individual cells that lack lamA are more uniform and more rapid with rifampicin and drugs that target the cell wall. Our results show that mycobacteria encode a non-conserved protein that controls the pattern of cell growth, resulting in a population that is both heterogeneous and better able to survive antibiotic pressure.

Mycobacteria grow and divide differently compared to well-studied model bacteria. They insert new cell envelope at their poles instead of along their side walls; and, they lack obvious homologs of many well-conserved cell growth and division proteins. Furthermore, while mycobacteria share several similarities to Gram-positive bacteria, unlike these organisms they possess an outer membrane, which is abundant in long-chain fatty acids and several glycolipids and lipoglycans. A better understanding of the unique factors that make this unusual structure could lead to therapeutic targets for pathogenic mycobacteria. Here, we study a gene of previously unknown function – msmeg_0317 - predicted to be essential and associated with mycobacterial cell growth and division proteins. We find that transcriptional depletion of msmeg_0317 leads to loss of polar growth, disruption of the mycobacterial outer membrane, and cell death. Surprisingly, we also observe that depletion results in the accumulation of cell-associated lipoglycans lipomannan and lipoarabinomannan (LM/LAM), while overexpression of CwdA leads to increased shedding of LM/LAM. LM/LAM have been extensively studied in relation to infection but their role in bacterial physiology is less clear. Altogether, our data suggest that MSMEG_0317, renamed CwdA, is involved in the transport of LM/LAM to the mycobacterial outer membrane, and reveal unexpected connections between the correct localization of LM/LAM, polar growth, and the structural integrity of the mycobacterial cell envelope. IMPORTANCE Some of the most successful antibiotics target bacterial cell envelope synthesis. However, the cell envelope of mycobacteria is significantly different from that of other, more well-studied, bacteria. Its core structure consists of a covalently-linked network of peptides, carbohydrates, and fatty acids. Additionally, there are several lipids non-covalently interspersed throughout. Many of the enzymes which synthesize these lipids are known, but how they are transported remains largely unclear. Here, we discover that an essential protein, CwdA, is involved in the transport of LM/LAM, abundant lipoglycans in the mycobacterial cell envelope. Depletion of CwdA leads to the loss of polar growth and the disintegration of the mycobacterial outer membrane. These results suggest that the proteins which transport molecules across the mycobacterial cell envelope may represent an abundant source of novel drug targets for the treatment of mycobacterial infections, like tuberculosis.

Mycobacterium tuberculosis causes the world’s deadliest infectious disease. Part of the pathogen’s success is its ability to diversify itself phenotypically and survive antibiotic therapy even in the absence of genetic resistance. Here, we will highlight the physiological aspects of the pathogen that promote tolerance and describe the mechanisms by which some of these vary in a genetically identical population. A better, molecular, understanding of this phenomenon may provide the key to improved TB therapy.

International audience; Successful therapies to combat microbial diseases and cancers require incorporating ecological and evolutionary principles. Drawing upon the fields of ecology and evolutionary biology, we present a systems-based approach in which host and disease-causing factors are considered as part of a complex network of interactions, analogous to studies of "classical" ecosystems. Centering this approach around empirical examples of disease treatment, we present evidence that successful therapies invariably engage multiple interactions with other components of the host ecosystem. Many of these factors interact nonlinearly to yield synergistic benefits and curative outcomes. We argue that these synergies and nonlinear feedbacks must be leveraged to improve the study of pathogenesis in situ and to develop more effective therapies. An eco-evolutionary systems perspective has surprising and important consequences, and we use it to articulate areas of high research priority for improving treatment strategies.

In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized

Septation in bacteria requires coordinated regulation of cell wall biosynthesis and hydrolysis enzymes so that new septal cross-wall can be appropriately constructed without compromising the integrity of the existing cell wall. Bacteria with different modes of growth and different types of cell wall require different regulators to mediate cell growth and division processes. Mycobacteria have both a cell wall structure and a mode of growth that are distinct from well-studied model organisms and use several different regulatory mechanisms. Here, using Mycobacterium smegmatis , we identify and characterize homologs of the conserved cell division regulators FtsL and FtsB, and show that they appear to function similarly to their homologs in Escherichia coli . We identify a number of previously undescribed septally localized factors which could be involved in cell wall regulation. One of these, SepIVA, has a DivIVA domain, is required for mycobacterial septation, and is localized to the septum and the intracellular membrane domain. We propose that SepIVA is a regulator of cell wall precursor enzymes that contribute to construction of the septal cross-wall, similar to the putative elongation function of the other mycobacterial DivIVA homolog, Wag31. IMPORTANCE The enzymes that build bacterial cell walls are essential for cell survival but can cause cell lysis if misregulated; thus, their regulators are also essential. The number and nature of these regulators is likely to vary in bacteria that grow in different ways. The mycobacteria are a genus that have a cell wall whose composition and construction vary greatly from those of well-studied model organisms. In this work, we identify and characterize some of the proteins that regulate the mycobacterial cell wall. We find that some of these regulators appear to be functionally conserved with their structural homologs in evolutionarily distant species such as Escherichia coli , but other proteins have critical regulatory functions that may be unique to the actinomycetes.

A mutation in a microtubule-severing enzyme, katanin, causes flagella to become short due to a reduced cytoplasmic precursor pool. These results suggest that competition between flagella and cytoplasmic microtubules for a limited tubulin pool is facilitated by katanin, which is confirmed by stochastic models., Flagellar length control in Chlamydomonas reinhardtii provides a simple model system in which to investigate the general question of how cells regulate organelle size. Previous work demonstrated that Chlamydomonas cytoplasm contains a pool of flagellar precursor proteins sufficient to assemble a half-length flagellum and that assembly of full-length flagella requires synthesis of additional precursors to augment the preexisting pool. The regulatory systems that control the synthesis and regeneration of this pool are not known, although transcriptional regulation clearly plays a role. We used quantitative analysis of length distributions to identify candidate genes controlling pool regeneration and found that a mutation in the p80 regulatory subunit of katanin, encoded by the PF15 gene in Chlamydomonas, alters flagellar length by changing the kinetics of precursor pool utilization. This finding suggests a model in which flagella compete with cytoplasmic microtubules for a fixed pool of tubulin, with katanin-mediated severing allowing easier access to this pool during flagellar assembly. We tested this model using a stochastic simulation that confirms that cytoplasmic microtubules can compete with flagella for a limited tubulin pool, showing that alteration of cytoplasmic microtubule severing could be sufficient to explain the effect of the pf15 mutations on flagellar length.

Live-cell fluorescence light microscopy has emerged as an important tool in the study of cellular biology. The development of fluorescent markers in parallel with super-resolution imaging systems has pushed light microscopy into the realm of molecular visualization at the nanometer scale. Resolutions previously only attained with electron microscopes are now within the grasp of light microscopes. However, until recently, live-cell imaging approaches have eluded super-resolution microscopy, hampering it from reaching its full potential for revealing the dynamic interactions in biology occurring at the single molecule level. Here we examine recent advances in the super-resolution imaging of living cells by reviewing recent breakthroughs in single molecule localization microscopy methods such as PALM and STORM to achieve this important goal.

Structured illumination microscopy (SIM) is a method that can double the spatial resolution of wide-field fluorescence microscopy in three dimensions by using spatially structured illumination light. In this chapter, we introduce the basic principles of SIM and describe in detail several different implementations based on either a diffraction grating or liquid crystal spatial light modulators. We also describe nonlinear SIM, a method that in theory can achieve unlimited resolution. In addition, we discuss a number of key points important for high-resolution imaging.

Structured illumination microscopy (SIM) is a method that can double the spatial resolution of wide-field fluorescence microscopy in three dimensions by using spatially structured illumination light. In this chapter, we introduce the basic principles of SIM and describe in detail several different implementations based on either a diffraction grating or liquid crystal spatial light modulators. We also describe nonlinear SIM, a method that in theory can achieve unlimited resolution. In addition, we discuss a number of key points important for high-resolution imaging.

Structured illumination is a resolution-enhancement technique for widefield fluorescence microscopy that can double its spatial resolution in three dimensions by using spatially patterned excitation light. Such patterned excitation encodes conventionally unobservable high-resolution information in the form of low-resolution information, which is then computationally decoded to form a final image with extended resolution. In this chapter, we introduce the basic principles of structured illumination and describe in detail a number of key issues in its implementation as well the image reconstruction algorithms. We also discuss different forms of structured illumination and its extension, including its combination with interferometer and nonlinear structured illumination. We also present imaging results on quite a few different kinds of biological specimens.

Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it light-efficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.

Three-dimensional (3D) structured-illumination microscopy (SIM) can double the lateral and axial resolution of a wide-field fluorescence microscope but has been too slow for live imaging. Here we apply 3D SIM to living samples and record whole cells at up to 5 s per volume for >50 time points with 120-nm lateral and 360-nm axial resolution. We demonstrate the technique by imaging microtubules in S2 cells and mitochondria in HeLa cells.

Cell growth and division are required for the progression of bacterial infections. Most rod-shaped bacteria grow by inserting new cell wall along their mid-section. However, mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce new cell wall material at their poles. How mycobacteria control this different mode of growth is incompletely understood. Here we find that PonA1, a penicillin binding protein (PBP) capable of transglycosylation and transpeptidation of cell wall peptidoglycan (PG), is a major governor of polar growth in mycobacteria. PonA1 is required for growth of Mycobacterium smegmatis and is critical for M. tuberculosis during infection. In both cases, PonA1’s catalytic activities are both required for normal cell length, though loss of transglycosylase activity has a more pronounced effect than transpeptidation. Mutations that alter the amount or the activity of PonA1 result in abnormal formation of cell poles and changes in cell length. Moreover, altered PonA1 activity results in dramatic differences in antibiotic susceptibility, suggesting that a balance between the two enzymatic activities of PonA1 is critical for survival. We also find that phosphorylation of a cytoplasmic region of PonA1 is required for normal activity. Mutations in a critical phosphorylated residue affect transglycosylase activity and result in abnormal rates of cell elongation. Together, our data indicate that PonA1 is a central determinant of polar growth in mycobacteria, and its governance of cell elongation is required for robust cell fitness during both host-induced and antibiotic stress., Author Summary Bacterial infections rely on continued bacterial growth. Studying cell growth is particularly important for pathogens such as Mycobacterium tuberculosis that grow differently than model organisms. Unlike Escherichia coli or Bacillus subtilis, which grow by incorporating cell wall material along their body, mycobacteria grow from the pole. It remains unclear how mycobacteria construct and extend their poles. Our work identifies a cell wall synthase, PonA1, as a key determinant of mycobacterial polar growth. PonA1 governs polar growth through two enzymatic activities that build the cell wall’s peptidoglycan (PG); both of these activities are required for normal cell growth. Changes in the amount or activity of PonA1 leads to misplaced cell poles and inhibition of cell proliferation. PonA1 is phosphorylated, an unusual modification for PG synthases. This phosphorylation tunes the rate of cell elongation. Changing PonA1’s regulatory or enzymatic activity impacts the survival of cells in the host or when treated with antibiotics. Our work shows how mycobacterial cell pole construction and cell fitness is governed by a major cell wall synthase; these findings may have implications for other bacteria that elongate from their poles.

In most well-studied rod-shaped bacteria, peptidoglycan is primarily crosslinked by penicillin-binding proteins (PBPs). However, in mycobacteria, crosslinks formed by L,D-transpeptidases (LDTs) are highly abundant. To elucidate the role of these unusual crosslinks, we characterized Mycobacterium smegmatis cells lacking all LDTs. We find that crosslinks generate by LDTs are required for rod shape maintenance specifically at sites of aging cell wall, a byproduct of polar elongation. Asymmetric polar growth leads to a non-uniform distribution of these two types of crosslinks in a single cell. Consequently, in the absence of LDT-mediated crosslinks, PBP-catalyzed crosslinks become more important. Because of this, Mycobacterium tuberculosis (Mtb) is more rapidly killed using a combination of drugs capable of PBP- and LDT- inhibition. Thus, knowledge about the spatial and genetic relationship between drug targets can be exploited to more effectively treat this pathogen.