Inositol Acylation of Phosphatidylinositol Mannosides: A Rapid Mass Response to Membrane Fluidization in Mycobacteria

Mycobacteria share an unusually complex, multilayered cell envelope, which contributes to adaptation to changing environments. The plasma membrane is the deepest layer of the cell envelope and acts as the final permeability barrier against outside molecules. There is an obvious need to maintain the plasma membrane integrity, but the adaptive responses of the plasma membrane to stress exposure remain poorly understood. Using chemical treatment and heat stress to fluidize the membrane, we show here that phosphatidylinositol (PI)-anchored plasma membrane glycolipids known as PI mannosides (PIMs) are rapidly remodeled upon membrane fluidization in Mycobacterium smegmatis. Without membrane stress, PIMs are predominantly in a triacylated form: two acyl chains of the PI moiety plus one acyl chain modified at one of the mannose residues. Upon membrane fluidization, we determined the fourth fatty acid is added to the inositol moiety of PIMs, making them tetra-acylated variants. Additionally, we show that PIM inositol acylation is a rapid response independent of de novo protein synthesis, representing one of the fastest mass conversions of lipid molecules found in nature. Strikingly, we found that M. smegmatis is more resistant to the bactericidal effect of a cationic detergent after benzyl alcohol pre-exposure. We further demonstrate that fluidization-induced PIM inositol acylation is conserved in pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. Our results demonstrate that mycobacteria possess a mechanism to sense plasma membrane fluidity change. We suggest that inositol acylation of PIMs is a novel membrane stress response that enables mycobacterial cells to resist membrane fluidization

Spatially distinct and metabolically active membrane domain in mycobacteria

Protected from host immune attack and antibiotic penetration by their unique cell envelope, mycobacterial pathogens cause devastating human diseases such as tuberculosis. Seamless coordination of cell growth with cell envelope elongation at the pole maintains this barrier. Unraveling this spatiotemporal regulation is a potential strategy for controlling mycobacterial infections. Our biochemical analysis previously revealed two functionally distinct membrane fractions in Mycobacterium smegmatis cell lysates: plasma membrane tightly associated with the cell wall (PM-CW) and a distinct fraction of pure membrane free of cell wall components (PMf). To provide further insight into the functions of these membrane fractions, we took the approach of comparative proteomics and identified more than 300 proteins specifically associated with the PMf, including essential enzymes involved in cell envelope synthesis such as a mannosyltransferase, Ppm1, and a galactosyltransferase, GlfT2. Furthermore, comparative lipidomics revealed the distinct lipid composition of the PMf, with specific association of key cell envelope biosynthetic precursors. Live-imaging fluorescence microscopy visualized the PMf as patches of membrane spatially distinct from the PM-CW and notably enriched in the pole of the growing cells. Taken together, our study provides the basis for assigning the PMf as a spatiotemporally distinct and metabolically active membrane domain involved in cell envelope biogenesis

Stress-Induced Reorganization of the Mycobacterial Membrane Domain

Cell 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. IMPORTANCE Mycobacteria include medically important species, such as the human tuberculosis pathogen Mycobacterium tuberculosis. The highly impermeable cell envelope is a hallmark of these microbes, and its biosynthesis is a proven chemotherapeutic target. Despite the accumulating knowledge regarding the biosynthesis of individual envelope components, the regulatory mechanisms behind the coordinated synthesis of the complex cell envelope remain elusive. We previously reported the presence of a metabolically active membrane domain enriched in the elongating poles of actively growing mycobacteria. However, the spatiotemporal dynamics of the membrane domain in response to stress have not been examined. Here, we show that the membrane domain is spatially reorganized when growth is inhibited in the stationary growth phase, under nutrient starvation, or in response to perturbation of peptidoglycan biosynthesis. Our results suggest that mycobacteria have a mechanism to spatiotemporally coordinate the membrane domain in response to metabolic needs under different growth conditions

Cell wall damage reveals spatial flexibility in peptidoglycan synthesis and a non-redundant role for RodA in mycobacteria [preprint]

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 for in vitro growth. However, RodA contributes to survival of Mycobacterium tuberculosis in 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 fortifies M. smegmatis against 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. Importance Peptidoglycan 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 challenged M. smegmatis with cell wall-damaging enzymes to characterize the roles of cell wall-building enzymes when the bacterium is under attack

Installation and Test of the ATLAS Muon Endcap Trigger Chamber Electronics

For the detector commissioning planned in 2007, a sector assembly of the ATLAS muon endcap trigger chambers is being progressed in CERN. Final technical test of the electronics mounted on a sector must be made at this stage. For systematic test of the electronics (sector test), we have developed a DAQ system on top of the ATLAS online software framework. The system is not dedicated only for this test, but can be used also for the front-end detector part of the overall ATLAS DAQ system. In the presentation, the procedure, meaning and results of the sector test are discussed after brief introduction of the TGC electronics and the sector structure as a construction unit. We introduce plans of further detailed and elaborated tests for the whole system using cosmic ray and single halo muons when all the TGC sub-detector part is completed as concluding remark

A study of the etapipi channel produced in central pp interactions at 450 GeV/c

The reaction pp -> pf (eta pi pi) ps has been studied at 450 GeV/c. There is clear evidence for an a2(1320)pi decay mode of the eta2(1645) and eta2(1870). In addition, there is evidence for an a0(980)pi$ decay mode of both resonances and an f2(1270)eta decay mode of the eta2(1870). No evidence is found for a JPC = 2++ a2(1320)pi wave.Comment: 15 pages, Latex, 4 Figures Branching ratio a2pi /f2 eta correcte

A search for charmonium states produced in central pp interactions at 450 GeV/c

A search for centrally produced charmonium states has been presented. There is no significant evidence for any charmonium production. An upper limit of 2 nb is found for the cross section of chic production using the decay chic(1P)-> J/psi gamma.Comment: 10 pages, Latex, 4 Figure