Cell Poles Research Articles

Extrachromosomal Nucleolus-Like Compartmentalization by a Plasmid-Borne Ribosomal RNA Operon and Its Role in Nucleoid Compaction.

In the fast-growing Escherichia coli cells, RNA polymerase (RNAP) molecules are concentrated and form foci at clusters of ribosomal RNA (rRNA) operons resembling eukaryotic nucleolus. The bacterial nucleolus-like organization, spatially compartmentalized at the surface of the compact bacterial chromosome (nucleoid), serves as transcription factories for rRNA synthesis and ribosome biogenesis, which influences the organization of the nucleoid. Unlike wild type that has seven rRNA operons in the genome in a mutant that has six (Δ6rrn) rRNA operons deleted in the genome, there are no apparent transcription foci and the nucleoid becomes uncompacted, indicating that formation of RNAP foci requires multiple copies of rRNA operons clustered in space and is critical for nucleoid compaction. It has not been determined, however, whether a multicopy plasmid-borne rRNA operon (prrnB) could substitute the multiple chromosomal rRNA operons for the organization of the bacterial nucleolus-like structure in the mutants of Δ6rrn and Δ7rrn that has all seven rRNA operons deleted in the genome. We hypothesized that extrachromosomal nucleolus-like structures are similarly organized and functional in trans from prrnB in these mutants. In this report, using multicolor images of three-dimensional superresolution Structured Illumination Microscopy (3D-SIM), we determined the distributions of both RNAP and NusB that are a transcription factor involved in rRNA synthesis and ribosome biogenesis, prrnB clustering, and nucleoid structure in these two mutants in response to environmental cues. Our results found that the extrachromosomal nucleolus-like organization tends to be spatially located at the poles of the mutant cells. In addition, formation of RNAP foci at the extrachromosomal nucleolus-like structure condenses the nucleoid, supporting the idea that active transcription at the nucleolus-like organization is a driving force in nucleoid compaction.

Constriction Rate Modulation Can Drive Cell Size Control and Homeostasis in C. crescentus

SummaryRod-shaped bacteria typically grow first via sporadic and dispersed elongation along their lateral walls and then via a combination of zonal elongation and constriction at the division site to form the poles of daughter cells. Although constriction comprises up to half of the cell cycle, its impact on cell size control and homeostasis has rarely been considered. To reveal the roles of cell elongation and constriction in bacterial size regulation during cell division, we captured the shape dynamics of Caulobacter crescentus with time-lapse structured illumination microscopy and used molecular markers as cell-cycle landmarks. We perturbed the constriction rate using a hyperconstriction mutant or fosfomycin ([(2R,3S)-3-methyloxiran-2-yl]phosphonic acid) inhibition. We report that the constriction rate contributes to both size control and homeostasis, by determining elongation during constriction and by compensating for variation in pre-constriction elongation on a single-cell basis.

A unique cytoplasmic ATPase complex defines the Legionella pneumophila type IV secretion channel.

Type IV secretion systems (T4SSs) are complex machines used by bacteria to deliver protein and DNA complexes into target host cells 1–5. Conserved ATPases are essential for T4SS function, but how they coordinate their activities to promote substrate transfer remains poorly understood. Here, we show that the DotB ATPase associates with the Dot/Icm T4SS at the Legionella cell pole through interactions with the DotO ATPase. The structure of the Dot/Icm apparatus was solved in situ by cryo-electron tomography at 3.5 nanometer resolution and the cytoplasmic complex was solved at 3.0 nanometer resolution. These structures revealed a cell-envelope-spanning channel that connects to the cytoplasmic complex. Further analysis revealed a hexameric assembly of DotO dimers associated with the inner membrane complex, and a DotB hexamer associated with the base of this cytoplasmic complex. The assembly of a DotB/DotO energy complex creates a cytoplasmic channel and directs the translocation of substrates through the T4SS. These data define distinct stages in Dot/Icm machine biogenesis, advance our understanding of channel activation, and identify an envelope-spanning T4SS channel.

Asymmetric polar localization dynamics of the serine chemoreceptor protein Tsr in Escherichia coli.

The spatial location of proteins in living cells can be critical for their function. For example, the E. coli chemotaxis machinery is localized to the cell poles. Here we describe the polar localization of the serine chemoreceptor Tsr using a strain synthesizing a fluorescent Tsr-Venus fusion at a low level from a single-copy chromosomal construct. Using photobleaching and imaging during recovery by new synthesis, we observed distinct asymmetry between a bright (old) pole and a dim (new) pole. The old pole was shown to be a more stable cluster and to recover after photobleaching faster, which is consistent with the hypothesis that newly synthesized Tsr proteins are inserted directly at or near the old pole. The new pole was shown to be a less stable cluster and to exchange proteins freely with highly mobile Tsr-Venus proteins diffusing in the membrane. We propose that the new pole arises from molecules escaping from the old pole and diffusing to the new pole where a more stable cluster forms over time. Our localization imaging data support a model in which a nascent new pole forms prior to stable cluster formation.

Is Longitudinal Division in Rod-Shaped Bacteria a Matter of Swapping Axis?

The morphology of bacterial species shows a wealth of variation from star-shaped to spherical and rod- to spiral-shaped, to mention a few. Their mode of growth and division is also very diverse and flexible ranging from polar growth and lateral surface increase to midcell expansion and from perpendicular to longitudinal asymmetric division. Gammaproteobacterial rod-shaped species such as Escherchia coli divide perpendicularly and grow in length, whereas the genetically very similar rod-shaped symbiotic Thiosymbion divide longitudinally, and some species even divide asynchronously while growing in width. The ovococcal Streptococcus pneumoniae also lengthens and divides perpendicularly, yet it is genetically very different from E. coli. Are these differences as dramatic as is suggested by visual inspection, or can they all be achieved by subtle variation in the regulation of the same protein complexes that synthesize the cell envelope? Most bacteria rely on the cytoskeletal polymer FtsZ to organize cell division, but only a subset of species use the actin homolog MreB for length growth, although some of them are morphologically not that different. Poles are usually negative determinant for cell division. Curved cell poles can be inert or active with respect to peptidoglycan synthesis, can localize chemotaxis and other sensing proteins or other bacterial equipment, such as pili, depending on the species. But what is actually the definition of a pole? This review discusses the possible common denominators for growth and division of distinct and similar bacterial species.

Intracellular localization of sirtuin and cell length analysis of Lactobacillus paracasei suggest possible role of sirtuin in cell division and cell shape regulation.

Sirtuin has been associated in prolonging lifespan of different model organisms. It has been shown to have an enzymatic activity of NAD+-dependent protein deacetylation targeting acetylated proteins. To determine targets and possible roles of sirtuin (LpSirA) in the Lactobacillus paracasei BL23, deletion (ΔsirA), sirtuin overexpressor (highsirA) and GFP fusion (highsirA-Venus) strains were generated, and microscopic localization and cell length analysis were done. Microscopic analysis revealed localization of LpSirA at cell division plates, at cell poles and all throughout the cell length in a spiral manner. Cell length analysis revealed that 46.9% of the ΔsirA cells were observed to be shorter (<2μm), whereas 12.6% of the highsirA cells were observed to be longer (>4μm) in comparison with the wild-type with only 17.1% short cells and 5.3% long cells. Our results suggest that sirtuin may have an essential role in cell division and cell shape regulation.

Phosphatidylserine and GTPase activation control Cdc42 nanoclustering to counter dissipative diffusion.

The anisotropic organization of plasma membrane constituents is indicative of mechanisms that drive the membrane away from equilibrium. However, defining these mechanisms is challenging due to the short spatiotemporal scales at which diffusion operates. Here, we use high-density single protein tracking combined with photoactivation localization microscopy (sptPALM) to monitor Cdc42 in budding yeast, a system in which Cdc42 exhibits anisotropic organization. Cdc42 exhibited reduced mobility at the cell pole, where it was organized in nanoclusters. The Cdc42 nanoclusters were larger at the cell pole than those observed elsewhere in the cell. These features were exacerbated in cells expressing Cdc42-GTP, and were dependent on the scaffold Bem1, which contributed to the range of mobility and nanocluster size exhibited by Cdc42. The lipid environment, in particular phosphatidylserine levels, also played a role in regulating Cdc42 nanoclustering. These studies reveal how the mobility of a Rho GTPase is controlled to counter the depletive effects of diffusion, thus stabilizing Cdc42 on the plasma membrane and sustaining cell polarity.

Membrane Curvature and the Tol-Pal Complex Determine Polar Localization of the Chemoreceptor Tar in Escherichia coli.

Chemoreceptors are localized at the cell poles of Escherichia coli and other rod-shaped bacteria. Over the years, different mechanisms have been put forward to explain this polar localization, including stochastic clustering, membrane curvature-driven localization, interactions with the Tol-Pal complex, and nucleoid exclusion. To evaluate these mechanisms, we monitored the cellular localization of the aspartate chemoreceptor Tar in different deletion mutants. We did not find any indication for either stochastic cluster formation or nucleoid exclusion. However, the presence of a functional Tol-Pal complex appeared to be essential to retain Tar at the cell poles. Interestingly, Tar still accumulated at midcell in tol and in pal deletion mutants. In these mutants, the protein appears to gather at the base of division septa, a region characterized by strong membrane curvature. Chemoreceptors, like Tar, form trimers of dimers that bend the cell membrane due to a rigid tripod structure. The curvature approaches the curvature of the cell membrane generated during cell division, and localization of chemoreceptor tripods at curved membrane areas is therefore energetically favorable, as it lowers membrane tension. Indeed, when we introduced mutations in Tar that abolish the rigid tripod structure, the protein was no longer able to accumulate at midcell or the cell poles. These findings favor a model where chemoreceptor localization in E. coli is driven by strong membrane curvature and association with the Tol-Pal complex.IMPORTANCE Bacteria have exquisite mechanisms to sense and adapt to the environment they live in. One such mechanism involves the chemotaxis signal transduction pathway, in which chemoreceptors specifically bind certain attracting or repelling molecules and transduce the signals to the cell. In different rod-shaped bacteria, these chemoreceptors localize specifically to cell poles. Here, we examined the polar localization of the aspartate chemoreceptor Tar in E. coli and found that membrane curvature at cell division sites and the Tol-Pal protein complex localize Tar at cell division sites, the future cell poles. This study shows how membrane curvature can guide localization of proteins in a cell.

Absence of the Min System Does Not Cause Major Cell Division Defects in Agrobacterium tumefaciens

In A. tumefaciens, the essential FtsZ protein is located at the growth pole before shifting to the mid-cell right before division. Loss of FtsZ causes a halt in cell separation and lysis of cells. To understand how FtsZ polymerization is regulated to properly localize the FtsZ ring at the mid-cell, we have conducted a systematic characterization of the Min system in A. tumefaciens. Our findings indicate that the Min system is not required for cell survival. Yet, we find that the deletion of either minE or minCDE results in a broad cell size distribution, including an increase in the proportion of short and long cells. We observe that the site of constriction is misplaced in the minE or minCDE deletion strains allowing for short cells to arise from sites of constriction near the cell poles. Remarkably, the short cells are viable and contain DNA. In order to observe chromosome replication and segregation in these strains, YFP-ParB is used as a proxy to track the origin of replication as cells elongate and divide. In the absence of the Min proteins, duplication and segregation of the origin of replication is frequently delayed. Taken together, our data suggest that the Min system contributes to the proper regulation of FtsZ placement and subsequent cell division. Furthermore, the failure to precisely place FtsZ rings at mid-cell in the min mutants impacts other cell cycle features including chromosome segregation.

MinC and FtsZ mutant analysis provides insight into MinC/MinD-mediated Z ring disassembly

The Min system negatively regulates the position of the Z ring, which serves as a scaffold for the divisome that mediates bacterial cytokinesis. In Escherichia coli, this system consists of MinC, which antagonizes assembly of the tubulin homologue FtsZ. MinC is recruited to the membrane by MinD and induced by MinE to oscillate between the cell poles. MinC is a dimer with each monomer consisting of functionally distinct MinCN and MinCC domains, both of which contact FtsZ. According to one model, MinCC/MinD binding to the FtsZ tail positions MinCN at the junction of two GDP-containing subunits in the filament, leading to filament breakage. Others posit that MinC sequesters FtsZ-GDP monomers or that MinCN caps the minus end of FtsZ polymers and that MinCC interferes with lateral interactions between FtsZ filaments. Here, we isolated minC mutations that impair MinCN function and analyzed FtsZ mutants resistant to MinC/MinD. Surprisingly, we found mutations in both minC and ftsZ that differentiate inhibition by MinC from inhibition by MinC/MinD. Analysis of these mutations suggests that inhibition of the Z ring by MinC alone is due to sequestration, whereas inhibition by MinC/MinD is not. In conclusion, our genetic and biochemical data support the model that MinC/MinD fragments FtsZ filaments.

Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation

Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation.

Chemotaxis arrays in Vibrio species and their intracellular positioning by the ParC/ParP system.

Most motile bacteria are able to bias their movement towards more favorable environments or to escape from obnoxious substances by a process called chemotaxis. Chemotaxis depends on a chemosensory system that is able to sense specific environmental signals and generate a behavioral response. Typically, the signal is transmitted to the bacterial flagellum, ultimately regulating the swimming behavior of individual cells. Chemotaxis is mediated by proteins that assemble into large, highly ordered arrays. It is imperative for successful chemotactic behavior and cellular competitiveness that chemosensory arrays form and localize properly within the cell. Here we review how chemotaxis arrays form and localize in Vibrio cholerae and Vibrio parahaemolyticus We focus on how the ParC/ParP-system mediates cell cycle-dependent polar localization of chemotaxis arrays and thus ensures proper cell pole development and array inheritance upon cell division.

Size Laws and Division Ring Dynamics in Filamentous Escherichia coli cells

Our understanding of bacterial cell size control is based mainly on stress-free growth conditions in the laboratory [1-10]. In the real world, however, bacteria are routinely faced with stresses that produce long filamentous cell morphologies [11-28]. Escherichia coli is observed to filament in response to DNA damage [22-25], antibiotic treatment [11-14, 28], host immune systems [15, 16], temperature [17], starvation [20], and more [18, 19, 21], conditions which are relevant to clinical settings and food preservation[26]. This shape plasticity is considered a survival strategy [27]. Size control in this regime remains largely unexplored. Here we report that E.coli cells use a dynamic size ruler to determine division locations combined with an adder-like mechanism to trigger divisions. As filamentous cells increase in size due to growth, or decrease in size due to divisions, its multiple Fts division rings abruptly reorganize to remain one characteristic cell length away from the cell pole and two such length units away from each other. These rules can be explained by spatiotemporal oscillations of Min proteins. Upon removal of filamentation stress, the cells undergo asequence of division events, randomly at one of the possible division sites, on average after the time required to grow one characteristic cell size. These results indicate that E.coli cells continuously keep track of absolute length to control size, suggest a wider relevance for the adder principle beyond the control of normally sized cells, and provide a new perspective on the function of the Fts and Min systems.

Perspective: challenges and opportunities for the study of cardiolipin, a key player in bacterial cell structure and function.

Cardiolipin (CL) is a key player in bacterial cell biology. CL accumulates at the poles of rod-shaped cells; the polar localization and function of diverse bacterial proteins are CL-dependent. Cardiolipin (CL) is an unusual phospholipid comprised of a glycerol headgroup coupled with two phosphatidate moieties. CL-rich membrane domains are often visualized with the fluorescent indicator 10-N-nonyl-acridine orange (NAO). Recent data show that NAO can also indicate phosphatidylglycerol localization under different experimental conditions, in the absence of CL. The formation of CL-rich membrane domains at bacterial cell poles was predicted to occur spontaneously, by lipid microphase separation arising from the conical CL shape. New data reveal that membrane-anchored cardiolipin synthase A is targeted to the cytoplasmic membrane surface at bacterial cell poles. Thus, localized CL synthesis, interaction of CL with ClsA, and membrane curvature could all contribute to retention of CL at cell poles. These observations provide new insight regarding the mechanism for assembly of CL-rich membrane domains in prokaryotes and eukaryotes.

Probing Asymmetric Behavior of a Cell Cycle Regupatory Protein in Live Caulobacter using Single-Molecule Imaging

Asymmetric cell division is a fundamental event in developmental programs where two daughter cells are produced and exhibit distinct characteristics and fates. Remarkably, asymmetric cell division is also observed in the bacterium Caulobacter crescentus (∼3μm x 0.5μm). Caulobacter regulates cell cycle events through its master regulator CtrA, which is specifically degraded at one cell pole (stalked) during the swarmer-to-stalk (G1-to-S) transition and in late predivisional cell to enable replication onset and the transcription of over 90 genes. Surprisingly, the CtrA degradation adaptor protein, PopA, localizes symmetrically to both cell. PopA binds a secondary messenger molecule, c-di-GMP (cdG). cdG accumulates asymmetrically at only one of the two daughter cells (stalked). (Christen et al. 2010, Science) We hypothesized that the differential distribution of cdG could imply that PopA proteins at the two cell poles are intrinsically different. We report the first single-molecule tracking studies in live Caulobacter of wild type (WT) and mutant PopA that lacks cdG binding capability. By quantifying our single-molecule trajectories, we found that PopA that cannot bind cdG diffuses 1.28x faster than the WT. The difference in the diffusion coefficients is consistent with the possibility that WT, when cdG is bound, forms a complex twice the size of mutant that does not bind cdG. We also obtained evidence for preferential dimerization of PopA at the stalked pole, the site of cdG synthesis, using bimolecular fluorescence complementation. Furthermore, the different diffusion coefficients of PopA at the two poles, as well as the distinct impact on the diffusion coefficient when cdG cannot bind, suggest different localization mechanisms at the two poles. We propose that by forming different oligomeric states, PopA can perform multiple functions at the two poles and contribute to asymmetric cell division.

Morphodifferentiation of Gené’s organ in engorged Amblyomma sculptum Berlese, 1888 female ticks (Acari: Ixodidae)

The Gené’s organ (GO) secretes a waxy substance on eggs that reduces water loss and has antimicrobial properties. The current study evaluated morphological and histochemical aspects of GO in Amblyomma sculptum from the period of post-feeding – when ticks detach from the host – to the stage just before oviposition. In this species, GO is composed of a corpus and two pairs of glands, namely, cranial and caudal. Glandular cells are joined laterally by a system of interdigitating membranes with junctional complexes. Histochemistry showed that lipid droplets became more evident as GO developed, while glycogen gradually disappeared, and proteins were detected only near the onset of oviposition. The ultrastructural results revealed a marked distension of the cuticle filled with an amorphous material. Glandular cells showed poor endoplasmatic reticulum, many mitochondria mainly in the basal cell poles and a very developed basal labyrinth. We concluded that the development of GO in A. sculptum ticks was continuous and progressive, and it started after detachment from the host. Additionally, the ultrastructure study suggests that gland cells have an important absorption ability and a low synthetic activity, which indicates that the majority of wax precursors are derived from haemolymph.

Clostridial DivIVA and MinD interact in the absence of MinJ

One of the key regulators ensuring proper Z-ring placement in rod-shaped bacteria is the Min system. It does so by creating a concentration gradient of the MinC septation inhibitor along the cell axis. In Escherichia coli, this gradient is established by a MinE-mediated pole-to-pole oscillation of the MinCDE complex. In Bacillus subtilis, the creation of an inhibitory gradient relies on the MinJ and DivIVA pair of topological determinants, which target MinCD to the newly formed cell poles. Introducing the E. coli oscillating Min system into B. subtilis leads to a sporulation defect, suggesting that oscillation is incompatible with sporulation. However, Clostridia, close endospore-forming relatives of Bacilli, do encode oscillating Min homologues in various combinations together with homologues from the less dynamic B. subtilis Min system. Here we address the questions of how these two systems could exist side-by-side and how they influence one another by studying the Clostridium beijerinckii and Clostridium difficile Min systems. The toolbox of genetic manipulations and fluorescent protein fusions in Clostridia is limited, therefore B. subtilis and E. coli were chosen as heterologous systems for studying these proteins.In B. subtilis, MinD and DivIVA interact through MinJ; here, however, we discovered that the MinD and DivIVA proteins of both C. difficile, and C. beijerinckii, interact directly, which is surprising in the latter case, since that organism also encodes a MinJ homologue. We confirm this interaction using both in vitro and in vivo methods. We also show that C. beijerinckii MinJ is probably not in direct contact with DivIVACb and, unlike B. subtilis MinJ, does not mediate the MinDCb and DivIVACb interaction. Our results suggest that the Clostridia Min system uses a new mechanism of function.

Stress-Induced Reorganization of the Mycobacterial Membrane Domain.

ABSTRACTCell elongation occurs primarily at the mycobacterial cell poles, but the molecular mechanisms governing this spatial regulation remain elusive. We recently reported the presence of an intracellular membrane domain (IMD) that was spatially segregated from the conventional plasma membrane in Mycobacterium smegmatis. The IMD is enriched in the polar region of actively elongating cells and houses many essential enzymes involved in envelope biosynthesis, suggesting its role in spatially restricted elongation at the cell poles. Here, we examined reorganization of the IMD when the cells are no longer elongating. To monitor the IMD, we used a previously established reporter strain expressing fluorescent IMD markers and grew it to the stationary growth phase or exposed the cells to nutrient starvation. In both cases, the IMD was delocalized from the cell pole and distributed along the sidewall. Importantly, the IMD could still be isolated biochemically by density gradient fractionation, indicating its maintenance as a membrane domain. Chemical and genetic inhibition of peptidoglycan biosynthesis led to the delocalization of the IMD, suggesting the suppression of peptidoglycan biosynthesis as a trigger of spatial IMD rearrangement. Starved cells with a delocalized IMD can resume growth upon nutrient repletion, and polar enrichment of the IMD coincides with the initiation of cell elongation. These data reveal that the IMD is a membrane domain with the unprecedented capability of subcellular repositioning in response to the physiological conditions of the mycobacterial cell.

Phenotypic Heterogeneity in Sugar Utilization by E. coli Is Generated by Stochastic Dispersal of the General PTS Protein EI from Polar Clusters.

Although the list of proteins that localize to the bacterial cell poles is constantly growing, little is known about their temporal behavior. EI, a major protein of the phosphotransferase system (PTS) that regulates sugar uptake and metabolism in bacteria, was shown to form clusters at the Escherichia coli cell poles. We monitored the localization of EI clusters, as well as diffuse molecules, in space and time during the lifetime of E. coli cells. We show that EI distribution and cluster dynamics varies among cells in a population, and that the cluster speed inversely correlates with cluster size. In growing cells, EI is not assembled into clusters in almost 40% of the cells, and the clusters in most remaining cells dynamically relocate within the pole region or between the poles. In non-growing cells, the fraction of cells that contain EI clusters is significantly higher, and dispersal of these clusters is often observed shortly after exiting quiescence. Later, during growth, EI clusters stochastically re-form by assembly of pre-existing dispersed molecules at random time points. Using a fluorescent glucose analog, we found that EI function inversely correlates with clustering and with cluster size. Thus, activity is exerted by dispersed EI molecules, whereas the polar clusters serve as a reservoir of molecules ready to act when needed. Taken together our findings highlight the spatiotemporal distribution of EI as a novel layer of regulation that contributes to the population phenotypic heterogeneity with regard to sugar metabolism, seemingly conferring a survival benefit.

Ultrastructural Analysis of Cell Envelope and Accumulation of Lipid Inclusions in Clinical Mycobacterium tuberculosis Isolates from Sputum, Oxidative Stress, and Iron Deficiency.

Introduction: Mycobacteria have several unique cellular characteristics, such as multiple cell envelope layers, elongation at cell poles, asymmetric cell division, and accumulation of intracytoplasmic lipid inclusions, which contributes to their survival under stress conditions. However, the understanding of these characteristics in clinical Mycobacterium tuberculosis (M. tuberculosis) isolates and under host stress is limited. We previously reported the influence of host stress on the cell length distribution in a large set of clinical M. tuberculosis isolates (n = 158). Here, we investigate the influence of host stress on the cellular ultrastructure of few clinical M. tuberculosis isolates (n = 8) from that study. The purpose of this study is to further understand the influence of host stress on the cellular adaptations of clinical M. tuberculosis isolates.Methods: We selected few M. tuberculosis isolates (n = 8) for analyzing the cellular ultrastructure ex vivo in sputum and under in vitro stress conditions by transmission electron microscopy. The cellular adaptations of M. tuberculosis in sputum were correlated with the ultrastructure of antibiotic sensitive and resistant isolates in liquid culture, under oxidative stress, iron deficiency, and exposure to isoniazid.Results: In sputum, M. tuberculosis accumulated intracytoplasmic lipid inclusions. In liquid culture, clinical M. tuberculosis revealed isolate to isolate variation in the extent of intracytoplasmic lipid inclusions, which were absent in the laboratory strain H37Rv. Oxidative stress, iron deficiency, and exposure to isoniazid increased the accumulation of lipid inclusions and decreased the thickness of the cell envelope electron transparent layer in M. tuberculosis cells. Furthermore, intracytoplasmic compartments were observed in iron deficient cells.Conclusion: Our ultrastructural analysis has revealed significant influence of host stress on the cellular adaptations in clinical M. tuberculosis isolates. These adaptations may contribute to the survival of M. tuberculosis under host and antibiotic stress conditions. Variation in the cellular adaptations among clinical M. tuberculosis isolates may correlate with their ability to persist in tuberculosis patients during antibiotic treatment. These observations indicate the need for further analyzing these cellular adaptations in a large set of clinical M. tuberculosis isolates. This will help to determine the significance of these cellular adaptations in the tuberculosis treatment.