Cell Septum Research Articles

First person – Shengnan Zheng

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Shengnan Zheng is first author on ‘ The Cdc42 GTPase-activating protein Rga6 promotes the cortical localization of septin’, published in JCS. Shengnan is a PhD student in the lab of Chuanhai Fu at University of Science and Technology of China, Hefei, Anhui, China, investigating how septin and microtubule cytoskeletons regulate cell septation and cell polarity.

FtsZ-Ring Regulation and Cell Division Are Mediated by Essential EzrA and Accessory Proteins ZapA and ZapJ in Streptococcus pneumoniae.

The bacterial FtsZ-ring initiates division by recruiting a large repertoire of proteins (the divisome; Z-ring) needed for septation and separation of cells. Although FtsZ is essential and its role as the main orchestrator of cell division is conserved in most eubacteria, the regulators of Z-ring presence and positioning are not universal. This study characterizes factors that regulate divisome presence and placement in the ovoid-shaped pathogen, Streptococcus pneumoniae (Spn), focusing on FtsZ, EzrA, SepF, ZapA, and ZapJ, which is reported here as a partner of ZapA. Epi-fluorescence microscopy (EFm) and high-resolution microscopy experiments showed that FtsZ and EzrA co-localize during the entire Spn cell cycle, whereas ZapA and ZapJ are late-arriving divisome proteins. Depletion and conditional mutants demonstrate that EzrA is essential in Spn and required for normal cell growth, size, shape homeostasis, and chromosome segregation. Moreover, EzrA(Spn) is required for midcell placement of FtsZ-rings and PG synthesis. Notably, overexpression of EzrA leads to the appearance of extra Z-rings in Spn. Together, these observations support a role for EzrA as a positive regulator of FtsZ-ring formation in Spn. Conversely, FtsZ is required for EzrA recruitment to equatorial rings and for the organization of PG synthesis. In contrast to EzrA depletion, which causes a bacteriostatic phenotype in Spn, depletion of FtsZ results in enlarged spherical cells that are subject to LytA-dependent autolysis. Co-immunoprecipitation and bacterial two-hybrid assays show that EzrA(Spn) is in complexes with FtsZ, Z-ring regulators (FtsA, SepF, ZapA, MapZ), division proteins (FtsK, StkP), and proteins that mediate peptidoglycan synthesis (GpsB, aPBP1a), consistent with a role for EzrA at the interface of cell division and PG synthesis. In contrast to the essentiality of FtsZ and EzrA, ZapA and SepF have accessory roles in regulating pneumococcal physiology. We further show that ZapA interacts with a non-ZapB homolog, named here as ZapJ, which is conserved in Streptococcus species. The absence of the accessory proteins, ZapA, ZapJ, and SepF, exacerbates growth defects when EzrA is depleted or MapZ is deleted. Taken together, these results provide new information about the spatially and temporally distinct proteins that regulate FtsZ-ring organization and cell division in Spn.

Analysis of conidiogenesis and lifelong conidial production from single conidiophores of Podosphaera aphanis on strawberry leaves using digital microscopic and electrostatic techniques

In this study, strawberry plants (Fragaria × ananassa Duchesne ex Rozier) grown via elevated cultivation in a greenhouse were heavily infested with powdery mildew. We isolated the powdery mildew fungus from strawberry leaves and identified the isolate as Podosphaera aphanis (Wallroth) U. Braun & S. Takamatsu var. aphanis KSP-7 N, based on its morphological characteristics and sequencing of the ribosomal DNA internal transcribed spacer region. The host range of KSP-7 N was assessed by inoculating 17 plant families comprising 49 species (a total of 110 cultivars) with conidia. The fungus caused severe powdery mildew on the tested strawberry plants (commercial, wild, and false strawberries), produced scattered conidia on conidiophores, and formed completely catenated conidia within approximately 27 h from conidiophore erection to the first release of mature conidia. Six conidia were produced on each conidiophore; only those at the apex reached maturity. The cycles of conidial release were repeated 17 to 21 times at intervals of approximately 6 h, during gradual upward elongation of the conidiophores. At the final stage, conidia were released without growth or septation of the generative cells. Conidiophores produced an average of 38 conidia during a 96-h period. These results will help to clarify the modes of conidiogenesis, the lifetime of conidiophores, and the production of conidia on a conidiophore among powdery mildew pathogens isolated from strawberry leaves.

The Ribbon-Helix-Helix Domain Protein CdrS Regulates the Tubulin Homolog ftsZ2 To Control Cell Division in Archaea.

Precise control of the cell cycle is central to the physiology of all cells. In prior work we demonstrated that archaeal cells maintain a constant size; however, the regulatory mechanisms underlying the cell cycle remain unexplored in this domain of life. Here, we use genetics, functional genomics, and quantitative imaging to identify and characterize the novel CdrSL gene regulatory network in a model species of archaea. We demonstrate the central role of these ribbon-helix-helix family transcription factors in the regulation of cell division through specific transcriptional control of the gene encoding FtsZ2, a putative tubulin homolog. Using time-lapse fluorescence microscopy in live cells cultivated in microfluidics devices, we further demonstrate that FtsZ2 is required for cell division but not elongation. The cdrS-ftsZ2 locus is highly conserved throughout the archaeal domain, and the central function of CdrS in regulating cell division is conserved across hypersaline adapted archaea. We propose that the CdrSL-FtsZ2 transcriptional network coordinates cell division timing with cell growth in archaea.IMPORTANCE Healthy cell growth and division are critical for individual organism survival and species long-term viability. However, it remains unknown how cells of the domain Archaea maintain a healthy cell cycle. Understanding the archaeal cell cycle is of paramount evolutionary importance given that an archaeal cell was the host of the endosymbiotic event that gave rise to eukaryotes. Here, we identify and characterize novel molecular players needed for regulating cell division in archaea. These molecules dictate the timing of cell septation but are dispensable for growth between divisions. Timing is accomplished through transcriptional control of the cell division ring. Our results shed light on mechanisms underlying the archaeal cell cycle, which has thus far remained elusive.

Regulation of filamentation by bacteria and its impact on the productivity of compounds in biotechnological processes.

The bacteria wall fulfills important physiological functions at the cell, depending on its composition and organization. Many researches focused their studies in understanding the change of its properties not only in strength and permeability, but also in morphological plasticity due to both chemical and physical stresses. In particular, filamentation morphology is a cryptic phenomenon, with involve for great variety of bacteria, which allow them to acquire adaptive benefits. This phenotypic alteration consists of an alteration or lack of cell septation during the cell growth, as consequence of DNA damage or development of stress, such as nutritional factors, antibiotic resistance, low temperature, non-availability of oxygen, high osmolarity, and antimicrobial agents. These cells result in modification of elongation 10-50 times, thickness, chemical composition, and extent of cross-linking of the cell wall polymers than normal-shaped cells. Moreover, the advancement in the morphology engineering permitted the manipulation of the genes encoding the proteins belonging to the plasma membrane or cytoplasm, to have the control over the bacterial shapes and of the its cytoplasmatic environment. In biotechnology application, the intracellular space is primary used for a greater accumulation of secondary products, such as polyhydroxyalkanoates (PHAs). This review provides an insight into environmental induction of filamentation morphology and its use in biotechnological process. KEY POINTS: • Environmental stresses inducing filamentation morphology • Morphology engineering in biotechnological processes • Increase of polyhydroxyalkanoates (PHAs) accumulation.

The MmpL3 interactome reveals a complex crosstalk between cell envelope biosynthesis and cell elongation and division in mycobacteria

Integral membrane transporters of the Mycobacterial Membrane Protein Large (MmpL) family and their interactome play important roles in the synthesis and export of mycobacterial outer membrane lipids. Despite the current interest in the mycolic acid transporter, MmpL3, from the perspective of drug discovery, the nature and biological significance of its interactome remain largely unknown. We here report on a genome-wide screening by two-hybrid system for MmpL3 binding partners. While a surprisingly low number of proteins involved in mycolic acid biosynthesis was found to interact with MmpL3, numerous enzymes and transporters participating in the biogenesis of peptidoglycan, arabinogalactan and lipoglycans, and the cell division regulatory protein, CrgA, were identified among the hits. Surface plasmon resonance and co-immunoprecipitation independently confirmed physical interactions for three proteins in vitro and/or in vivo. Results are in line with the focal localization of MmpL3 at the poles and septum of actively-growing bacilli where the synthesis of all major constituents of the cell wall core are known to occur, and are further suggestive of a role for MmpL3 in the coordination of new cell wall deposition during cell septation and elongation. This novel aspect of the physiology of MmpL3 may contribute to the extreme vulnerability and high therapeutic potential of this transporter.

Septins regulate the equatorial dynamics of the separation initiation network kinase Sid2p and glucan synthases to ensure proper cytokinesis.

Septins generally function as scaffolds and as cortical barriers to restrict the diffusion of membrane proteins. In the fission yeast Schizosaccharomyces pombe, septins form a ring structure at the septum after spindle breakdown during the constriction of the contractile actomyosin ring (CAR) and serve as a scaffold to recruit glucanases to mediate ultimate daughter cell separation. Despite this, it remains unclear if septins play any significant roles before the cell separation during cytokinesis. Employing live cell microscopy, we carefully examined SIN (Septation Initiation Network) signaling and glucan synthases, two key factors ensuring proper function of the CAR. In the absence of the core septin component Spn1p, the formation of a compact CAR is advanced and the CAR constriction rate is slightly but significantly decreased. Moreover, the SIN kinase Sid2p and the glucan synthases Bgs1p and Ags1p form an equatorial ring quite prematurely, but their maintenance at the equatorial region is diminished spn1Δ cells. These findings suggest that septins act as key players in an accurate establishment and the maintenance of CAR by orchestrating the equatorial dynamics of Sid2p and glucan synthases. Hence, this work demonstrates that, in addition to their function during ultimate cell septation, septins have important roles in regulating earlier cytokinetic events, including CAR assembly and constriction, SIN signaling, and the cortical dynamics of the glucan synthases.

Exposure to Sub-lethal 2,4-Dichlorophenoxyacetic Acid Arrests Cell Division and Alters Cell Surface Properties in Escherichia coli.

Escherichia coli is a robust, easily adaptable and culturable bacterium in vitro, and a model bacterium for studying the impact of xenobiotics in the environment. We have used correlative atomic force – laser scanning confocal microscopy (AFM-LSCM) to characterize the mechanisms of cellular response to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). One of the most extensively used herbicides world-wide, 2,4-D is known to cause hazardous effects in diverse non-target organisms. Sub-lethal concentrations of 2,4-D caused DNA damage in E. coli WM1074 during short exposure periods which increased significantly over time. In response to 2,4-D, FtsZ and FtsA relocalized within seconds, coinciding with the complete inhibition of cell septation and cell elongation. Exposure to 2,4-D also resulted in increased activation of the SOS response. Changes to cell division were accompanied by concomitant changes to surface roughness, elasticity and adhesion in a time-dependent manner. This is the first study describing the mechanistic details of 2,4-D at sub-lethal levels in bacteria. Our study suggests that 2,4-D arrests E. coli cell division within seconds after exposure by disrupting the divisome complex, facilitated by dissipation of membrane potential. Over longer exposures, 2,4-D causes filamentation as a result of an SOS response to oxidative stress induced DNA damage.

Pestalotiopsis-Like Species Causing Gray Blight Disease onCamellia sinensisin China

Gray blight of tea, caused by several Pestalotiopsis-like species, is one of the most destructive foliar diseases in tea cultivation yet the characteristics of these pathogens have not been confirmed until now. With morphological and multigene phylogenetic analyses, we have identified the gray blight fungi as Pseudopestalotiopsis camelliae-sinensis, Neopestalotiopsis clavispora, and Pestalotiopsis camelliae. Phylogenetic analyses derived from the combined internal transcribed spacer, β-tubulin, and translation elongation factor 1-α gene regions successfully resolved most of the Pestalotiopsis-like species used in this study with high bootstrap supports and revealed three major clusters representing these three species. Differences in colony appearance and conidia morphology (shape, size, septation, color and length of median cells, and length and number of apical and basal appendages) were consistent with the phylogenetic grouping. Pathogenicity tests validated that all three species isolated from tea leaves were causal agents of gray blight disease on tea plant (Camellia sinensis). This is the first description of the characteristics of the three species Pseudopestalotiopsis camelliae-sinensis, N. clavispora, and Pestalotiopsis camelliae as causal agents of tea gray blight disease in China.

The exocyst subunit Sec3 is regulated by a protein quality control pathway

Exocytosis involves fusion of secretory vesicles with the plasma membrane, thereby delivering membrane proteins to the cell surface and releasing material into the extracellular space. The tethering of the secretory vesicles before membrane fusion is mediated by the exocyst, an essential phylogenetically conserved octameric protein complex. Exocyst biogenesis is regulated by several processes, but the mechanisms by which the exocyst is degraded are unknown. Here, to unravel the components of the exocyst degradation pathway, we screened for extragenic suppressors of a temperature-sensitive fission yeast strain mutated in the exocyst subunit Sec3 (sec3-913). One of the suppressing DNAs encoded a truncated dominant-negative variant of the 26S proteasome subunit, Rpt2, indicating that exocyst degradation is controlled by the ubiquitin-proteasome system. The temperature-dependent growth defect of the sec3-913 strain was gene dosage-dependent and suppressed by blocking the proteasome, Hsp70-type molecular chaperones, the Pib1 E3 ubiquitin-protein ligase, and the deubiquitylating enzyme Ubp3. Moreover, defects in cell septation, exocytosis, and endocytosis in sec3 mutant strains were similarly alleviated by mutation of components in this pathway. We also found that, particularly under stress conditions, wild-type Sec3 degradation is regulated by Pib1 and the 26S proteasome. In conclusion, our results suggest that a cytosolic protein quality control pathway monitors folding and proteasome-dependent turnover of an exocyst subunit and, thereby, controls exocytosis in fission yeast.

Schizosaccharomyces japonicus: A Distinct Dimorphic Yeast among the Fission Yeasts.

Genomic sequencing data and morphological properties demonstrate evolutionary relationships among groups of the fission yeast, Schizosaccharomyces Phylogenetically, S. japonicus is the furthest removed from other species of fission yeast. The basic characteristics of cell proliferation are shared among all fission yeast, including the process of binary fission during vegetative growth, conjugation and karyogamy with horsetail movement, mating-type switching, and sporulation. However, S. japonicus also exhibits characteristics that are unique to filamentous fungi. S. japonicus is a nonpathogenic yeast that exhibits dimorphism. Depending on the environmental conditions, S. japonicus transforms from yeast cells into filamentous cells (hyphae), and blue light triggers synchronous septation of hyphal cells. A rough version of the whole-genome sequence is now available, facilitating genetic manipulation of S. japonicus. Furthermore, the extensive genetic knowledge available for S. pombe is aiding the development of genetic tools for analyzing S. japonicus. S. japonicus will help shed light on the evolutionary relationships among the fission yeast.

WhmD promotes the assembly of Mycobacterium smegmatis FtsZ: A possible role of WhmD in bacterial cell division

WhmD is considered to have a role in the septation and division of Mycobacterium smegmatis cells. Since FtsZ is the central protein of the septum, we determined the effect of WhmD on the assembly of Mycobacterium smegmatis FtsZ (MsFtsZ) in vitro. WhmD increased both the rate and extent of the assembly of MsFtsZ in vitro. WhmD also increased the amount of polymerized MsFtsZ as evident from a sedimentation assay. Further, the assembly promoting activity of WhmD occurred in the presence of GTP. MsFtsZ polymerized to form thin filaments in the absence of WhmD while MsFtsZ formed thick filaments in the presence of WhmD suggesting that WhmD enhanced the bundling of MsFtsZ filaments. Interestingly, WhmD neither suppressed the dilution-induced disassembly of FtsZ filaments nor significantly altered the GTPase activity of FtsZ. Using size exclusion chromatography, circular dichroism and fluorescence spectroscopy, WhmD was found to bind to MsFtsZ in vitro. The results showed that WhmD can promote the assembly of FtsZ and indicated that WhmD may play a role in the division of M. smegmatis cells by assisting the polymerization of FtsZ.

Biomineralization Patterns of Intracellular Carbonatogenesis in Cyanobacteria: Molecular Hypotheses

The recent discovery of intracellular carbonatogenesis in several cyanobacteria species has challenged the traditional view that this process was extracellular and not controlled. However, a detailed analysis of the size distribution, chemical composition and 3-D-arrangement of carbonates in these cyanobacteria is lacking. Here, we characterized these features in Candidatus Gloeomargarita lithophora C7 and Candidatus Synechococcus calcipolaris G9 by conventional transmission electron microscopy, tomography, ultramicrotomy, and scanning transmission X-ray microscopy (STXM). Both Ca. G. lithophora C7 and Ca. S. calcipolaris G9 formed numerous polyphosphate granules adjacent or engulfing Ca-carbonate inclusions when grown in phosphate-rich solutions. Ca-carbonates were scattered within Ca. G. lithophora C7 cells under these conditions, but sometimes arranged in one or several chains. In contrast, Ca-carbonates formed at cell septa in Ca. S. calcipolaris G9 and were segregated equally between daughter cells after cell division, arranging as distorted disks at cell poles. The size distribution of carbonates evolved from a positively to a negatively skewed distribution as particles grew. Conventional ultramicrotomy did not preserve Ca-carbonates explaining partly why intracellular calcification has been overlooked in the past. All these new observations allow discussing with unprecedented insight some nucleation and growth processes occurring in intracellularly calcifying cyanobacteria with a particular emphasis on the possible involvement of intracellular compartments and cytoskeleton.

In Fission Yeast the Constriction Rate is not Set by the Cytokinetic Ring, but by the Septum Growth Machinery

Constriction of the actomyosin contractile ring is often thought to drive division of the cell into two and to set the constriction rate. We measured a tension in the cytokinetic ring of fission yeast, where ring constriction is accompanied by septation, the growth of new cell wall in the wake of the constricting ring. Using a cell wall modulus ∼ 30 MPa, we conclude ring tension produces negligible septum strains ∼ 0.01% for a ring of radius and width ∼0.1 μm. Thus the inward septum growth rate determines the constriction rate, not the ring tension.Instead, ring tension maintains the circular shape of the septum, ensuring proper closure, using a mechanism that exploits the mechanosensitivity of septum growth. Hypothesizing mechanonsensitivity of the septum growth machinery, a mathematical model showed that ring tension suppresses septum edge roughness. Measurements of septum edge roughness in live cells agreed with model predictions, implying a mechanosensitivity per beta glucan synthase (Bgs) complex, the relative synthesis rate increase per force. Estimating the density of active Bgs complexes, a ring at constriction onset () therefore exerts centripetal force per Bgs, giving a small rate of synthesis increase. A rigorous calculation, from our mathematical model, shows little effect of tension on the constriction rate throughout constriction: ring radius versus time is almost linear with a small downward curvature.Thus, ring tension has little effect on the constriction rate, which is set by the intrinsic rate of synthesis by the cell's septum synthesis machinery.

CT-Based Morphological Analysis of Isolated Inferior and Medial Blow-out Orbital Fractures in Korean Adults

Purpose: To evaluate morphologic differences in isolated inferior medial orbital wall fractures (OWF) based on computed tomography scans.Methods: This was a retrospective observational case study of 22 patients with an isolated inferior OWF and 32 patients with an isolated medial fracture between January 2008 and August 2010. We analyzed patient demographics and bony radiologic characteristics on CT scans, including the length and height of the lamina papyracea, the number of ethmoid air cell septa, the length of the anterior and posterior border of the orbital floor, the thickness of the orbital floor maxillary bone, and the axial length of the eyeball.Results: There were no significant differences in sex, laterality, or concomitant intraocular injury between the two groups. The anteroposterior length (p = 0.391), the number of ethmoid septa (p = 0.869), and the thickness of the orbital floor (p = 0.419) did not differ significantly. The anterior (p < 0.001) or posterior (p = 0.014) height of the lamina papyracea, the lamina papyracea area (p < 0.001), and the lamina papyracea area/ethmoid air cell septa (p = 0.024) were significantly higher in the medial OWF group, while the anterior (p = 0.026) or posterior (p < 0.001) border length of the orbital floor and the axial length (p = 0.047) and volume (p = 0.034) of the eyeball were longer and smaller, respectively, in the inferior OWF group.Conclusions: Patients with a longer anterior or posterior border of the orbital floor, a shorter axial length, and a smaller eyeball volume are more likely to incur an isolated inferior OWF than an isolated medial OWF.

The functional dlt operon of Clostridium butyricum controls the d-alanylation of cell wall components and influences cell septation and vancomycin-induced lysis

Clostridium butyricum is a Gram-positive bacterium involved in the development of necrotizing enterocolitis (NEC) in preterm infants. To colonize the digestive tract, components of the cell wall of C. butyricum must interact with the intestinal mucosa. The d-alanylation of cell wall components such as teichoic acids results in a net positive charge on the cell wall, which is important for many functions of Gram-positive bacteria. Notably, d-alanylation mediates resistance to antimicrobial peptides and antibiotics. Here, we show that the dlt operon of C. butyricum encodes the enzymes responsible for the d-alanylation of cell wall components and influences the surface properties of the cell wall. We show that the d-alanylation of cell wall components controls the septation of C. butyricum, which is an essential mechanism during vegetative growth. Furthermore, we find that d-alanylation is involved in the resistance of C. butyricum to some cationic antimicrobial peptides (CAMPs) and lysozyme. Finally, we show that the d-alanylation of cell wall components influences vancomycin-induced lysis.

Essential protein SepF of mycobacteria interacts with FtsZ and MurG to regulate cell growth and division.

Coordinated bacterial cell septation and cell wall biosynthesis require formation of protein complexes at the sites of division and elongation, in a temporally controlled manner. The protein players in these complexes remain incompletely understood in mycobacteria. Using in vitro and in vivo assays, we showed that Rv2147c (or SepF) of Mycobacterium tuberculosis interacts with the principal driver of cytokinesis, FtsZ. SepF also interacts with itself both in vitro and in vivo. Amino acid residues 189A, 190K and 215F are required for FtsZ-SepF interaction, and are conserved across Gram-positive bacteria. Using Mycobacterium smegmatis as a surrogate system, we confirmed that sepFMSMEG is essential. Knockdown of SepF led to cell elongation, defective growth and failure of FtsZ to localize to the site of division, suggesting that SepF assists FtsZ localization at the site of division. Furthermore, SepF interacted with MurG, a peptidoglycan-synthesizing enzyme, both in vitro and in vivo, suggesting that SepF could serve as a link between cell division and peptidoglycan synthesis. SepF emerges as a newly identified essential component of the cell division complex in mycobacteria.

Digital microscopic analysis of conidiogenesis of powdery mildew pathogens isolated from melon leaves

Melons (Cucumis melo L.) grown hydroponically in a greenhouse were heavily infested with powdery mildew. We isolated powdery mildew pathogens from the melon leaves and identified the isolate as Podosphaera xanthii KMP-6N, based on morphological characteristics and sequences of ribosomal DNA internal transcribed spacer (rDNA-ITS) regions. Host ranges of KMP-6N were determined by estimating the infectivity or pathogenicity after inoculating the conidia onto multiple plant species. The fungi caused severe powdery mildew symptoms on Cucurbitaceae plants, producing scattered conidia on conidiophores. The goal of this study was to observe KMP-6N conidiogenesis on melon leaves. The pathogen formed completely catenated conidiophores approximately 24 h from conidiophore erection to release of mature conidia. Six conidia were produced on the conidiophores and only the conidia at the apex reached maturity. The cycles of conidial release were repeated on melon leaves 14 to 18 times, at approximately 6-h intervals. In the final stage, conidia were released without causing growth and septation of generative cells. Conidiophores produced an average of 36 conidia during a 90-h period. In our study, the modes of conidiogenesis, lifetime of conidiophores and productivity of conidia on a conidiophore were described for powdery mildew fungi.

Modulating the level of the Rpb7 subunit of RNA polymerase II affects cell separation in Schizosaccharomyces pombe

The rpb7+ gene encodes the seventh largest subunit of RNA polymerase II and is essential for survival of yeast cells. To gain insight into its functions, we expressed rpb7+ under the control of the nmt1 promoter and investigated its role in regulating multiple phenotypes in Schizosaccharomyces pombe. We observed that low rpb7+ levels resulted in slow growth of cells under optimum growth conditions. However, no growth defect was observed under different stress conditions tested in this study. Our results also showed that the most prominent phenotype of cells expressing reduced rpb7+ is a defect in cell separation. Quantitative real-time PCR analysis further revealed that the transcription of specific cell septation genes was significantly reduced in these cells. Collectively, results presented in this study highlight the distinct role of Rpb7p in regulating cell separation in S. pombe.

Biogenesis of YidC Cytoplasmic Membrane Substrates Is Required for Positioning of Autotransporter IcsA at Future Poles

Localization of proteins to specific sites within bacterial cells is often critical to their function. In rod-shaped bacteria, proteins involved in diverse and important cell processes localize to the cell poles. The molecular mechanisms by which these proteins are targeted to the pole, however, are poorly understood. The Shigella autotransporter protein IcsA, which is localized to the pole on the surface of the bacterium, is targeted to the pole in the cytoplasm by a mechanism that is conserved across multiple Gram-negative bacterial species and has thus served as an important and informative model for studying polar localization. We present evidence that in Escherichia coli, the establishment of polar positional information recognized by IcsA requires the activity of the cytoplasmic membrane protein insertase YidC. We show that the role of YidC in IcsA localization is independent of the cell septation and cytokinesis proteins FtsQ and FtsEX. FtsQ is required for polar localization of IcsA and, based on cross-linking studies, is inserted in the vicinity of YidC, but, we find, is not dependent on YidC for membrane insertion. FtsEX is a YidC substrate, but we find that it is not required for polar localization of IcsA. These findings indicate that polar positional information recognized by IcsA depends on one or more membrane proteins that require YidC for proper membrane insertion.