Glutamate 83 and arginine 85 of helix H3 bend are key residues for FtsZ polymerization, GTPase activity and cellular viability of Escherichia coli: lateral mutations affect FtsZ polymerization and E. coli viability

BACKGROUND: FtsZ is an essential cell division protein, which localizes at the middle of the bacterial cell to mediate cytokinesis. In vitro, FtsZ polymerizes and induces GTPase activity through longitudinal interactions to form the protofilaments, whilst lateral interactions result within formation of bundles. The interactions that participate in the protofilaments are similar to its eukaryotic homologue tubulin and are well characterized; however, lateral interactions between the inter protofilaments are less defined. FtsZ forms double protofilaments in vitro, though the key elements on the interface of the inter-protofilaments remain unclear as well as the structures involved in the lateral interactions in vivo and in vitro. In this study, we demonstrate that the highly conserved negative charge of glutamate 83 and the positive charge of arginine 85 located in the helix H3 bend of FtsZ are required for in vitro FtsZ lateral and longitudinal interactions, respectively and for in vivo cell division. RESULTS: The effect of mutation on the widely conserved glutamate-83 and arginine-85 residues located in the helix H3 (present in most of the tubulin family) was evaluated by in vitro and in situ experiments. The morphology of the cells expressing Escherichia coli FtsZ (E83Q) mutant at 42°C formed filamented cells while those expressing FtsZ(R85Q) formed shorter filamented cells. In situ immunofluorescence experiments showed that the FtsZ(E83Q) mutant formed rings within the filamented cells whereas those formed by the FtsZ(R85Q) mutant were less defined. The expression of the mutant proteins diminished cell viability as follows: wild type > E83Q > R85Q. In vitro, both, R85Q and E83Q reduced the rate of FtsZ polymerization (WT > E83Q >> R85Q) and GTPase activity (WT > E83Q >> R85Q). R85Q protein polymerized into shorter filaments compared to WT and E83Q, with a GTPase lag period that was inversely proportional to the protein concentration. In the presence of ZipA, R85Q GTPase activity increased two fold, but no bundles were formed suggesting that lateral interactions were affected. CONCLUSIONS: We found that glutamate 83 and arginine 85 located in the bend of helix H3 at the lateral face are required for the protofilament lateral interaction and also affects the inter-protofilament lateral interactions that ultimately play a role in the functional localization of the FtsZ ring at the cell division site

Mutations in the Lipopolysaccharide Biosynthesis Pathway Interfere with Crescentin-Mediated Cell Curvature in Caulobacter crescentus

Bacterial cell morphogenesis requires coordination among multiple cellular systems, including the bacterial cytoskeleton and the cell wall. In the vibrioid bacterium Caulobacter crescentus, the intermediate filament-like protein crescentin forms a cell envelope-associated cytoskeletal structure that controls cell wall growth to generate cell curvature. We undertook a genetic screen to find other cellular components important for cell curvature. Here we report that deletion of a gene (wbqL) involved in the lipopolysaccharide (LPS) biosynthesis pathway abolishes cell curvature. Loss of WbqL function leads to the accumulation of an aberrant Opolysaccharide species and to the release of the S layer in the culture medium. Epistasis and microscopy experiments show that neither S-layer nor O-polysaccharide production is required for curved cell morphology per se but that production of the altered O-polysaccharide species abolishes cell curvature by apparently interfering with the ability of the crescentin structure to associate with the cell envelope. Our data suggest that perturbations in a cellular pathway that is itself fully dispensable for cell curvature can cause a disruption of cell morphogenesis, highlighting the delicate harmony among unrelated cellular systems. Using the wbqL mutant, we also show that the normal assembly and growth properties of the crescentin structure are independent of its association with the cell envelope. However, this envelope association is important for facilitating the local disruption of the stable crescentin structure at the division site during cytokinesis

Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin-binding proteins (PBPs) are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here, we developed a novel Förster resonance energy transfer-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and applied this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay will allow unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high-throughput screening for new antimicrobials

Insights into pneumococcal fratricide from the crystal structures of the modular killing factor LytC

7 pags, 3 figs, 1 tabThe first structure of a pneumococcal autolysin, that of the LytC lysozyme, has been solved in ternary complex with choline and a pneumococcal peptidoglycan (PG) fragment. The active site of the hydrolase module is not fully exposed but is oriented toward the choline-binding module, which accounts for its unique in vivo features in PG hydrolysis, its activation and its regulatory mechanisms. Because of the unusual hook-shaped conformation of the multimodular protein, it is only able to hydrolyze non-cross-linked PG chains, an assertion validated by additional experiments. These results explain the activation of LytC by choline-binding protein D (CbpD) in fratricide, a competence-programmed mechanism of predation of noncompetent sister cells. The results provide the first structural insights to our knowledge into the critical and central function that LytC plays in pneumococcal virulence and explain a long-standing puzzle of how murein hydrolases can be controlled to avoid self-lysis during bacterial growth and division. © 2010 Nature America, Inc. All rights reserved.This work was supported by grants from the Spanish Ministry of Science and Technology (BFU2008-01711, SAF2006-00390), EU-CP223111 (CAREPNEUMO, European Union), the COMBACT program (S-BIO-0260/2006) and CIBER de Enfermedades Respiratorias (CIBERES). CIBERES is an initiative of Instituto de Salud Carlos III. The work in the US was supported by the US National Institutes of Health. W.V. was supported by the European Commission (EUR-INTAFAR project). I.P.-D. was a fellow of the Consejo Superior de Investigaciones Científicas

The lytic transglycosylase MltB connects membrane homeostasis and in vivo fitness of Acinetobacter baumannii

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146339/1/mmi14000-sup-0001-Supinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146339/2/mmi14000.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146339/3/mmi14000_am.pd

Діагностична значимість полярізаційно-оптичних властивостей рогівки ока при патології внутрішньоочного тиску

Незважаючи на прогрес в методах лікування і діагностики, глаукома останніми роками стала головною причиною невиліковної сліпоти в розвинених країнах світу. За даними ВООЗ більше 67 млн. чоловік в світі хворіють на глаукому і до 2030 року ця цифра повинна подвоїтися. Підвищення внутрішньоочного тиску (ВОТ) є одною з основних клінічних ознак глаукомного процесу, реєстрація якого лежить в основі діагностики і вибору методу адекватного лікування. Практично всі існуючи в теперішній час методи виміру ВОТ засновані на різних впливах на око (вантажами, плунжерами або струменем повітря). Як було встановлено, результати таких вимірів ВОТ суттєво залежать від біомеханічних параметрів рогівки або ока

Slippery Liquid-Like Solid Surfaces with Promising Antibiofilm Performance in both Static and Flow Conditions

[Image: see text] Biofilms are central to some of the most urgent global challenges across diverse fields of application, from medicine to industries to the environment, and exert considerable economic and social impact. A fundamental assumption in anti-biofilms has been that the coating on a substrate surface is solid. The invention of slippery liquid-infused porous surfaces—a continuously wet lubricating coating retained on a solid surface by capillary forces—has led to this being challenged. However, in situations where flow occurs, shear stress may deplete the lubricant and affect the anti-biofilm performance. Here, we report on the use of slippery omniphobic covalently attached liquid (SOCAL) surfaces, which provide a surface coating with short (ca. 4 nm) non-cross-linked polydimethylsiloxane (PDMS) chains retaining liquid–surface properties, as an antibiofilm strategy stable under shear stress from flow. This surface reduced biofilm formation of the key biofilm-forming pathogens Staphylococcus epidermidis and Pseudomonas aeruginosa by three–four orders of magnitude compared to the widely used medical implant material PDMS after 7 days under static and dynamic culture conditions. Throughout the entire dynamic culture period of P. aeruginosa, SOCAL significantly outperformed a typical antibiofilm slippery surface [i.e., swollen PDMS in silicone oil (S-PDMS)]. We have revealed that significant oil loss occurred after 2–7 day flow for S-PDMS, which correlated to increased contact angle hysteresis (CAH), indicating a degradation of the slippery surface properties, and biofilm formation, while SOCAL has stable CAH and sustainable antibiofilm performance after 7 day flow. The significance of this correlation is to provide a useful easy-to-measure physical parameter as an indicator for long-term antibiofilm performance. This biofilm-resistant liquid-like solid surface offers a new antibiofilm strategy for applications in medical devices and other areas where biofilm development is problematic

Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ

Chlamydiae are important pathogens and symbionts with unique cell biological features. They lack the cell-division protein FtsZ, and the existence of peptidoglycan (PG) in their cell wall has been highly controversial. FtsZ and PG together function in orchestrating cell division and maintaining cell shape in almost all other bacteria. Using electron cryotomography, mass spectrometry and fluorescent labelling dyes, here we show that some environmental chlamydiae have cell wall sacculi consisting of a novel PG type. Treatment with fosfomycin (a PG synthesis inhibitor) leads to lower infection rates and aberrant cell shapes, suggesting that PG synthesis is crucial for the chlamydial life cycle. Our findings demonstrate for the first time the presence of PG in a member of the Chlamydiae. They also present a unique example of a bacterium with a PG sacculus but without FtsZ, challenging the current hypothesis that it is the absence of a cell wall that renders FtsZ non-essential

A Widespread Bacterial Type VI Secretion Effector Superfamily Identified Using a Heuristic Approach

SummarySophisticated mechanisms are employed to facilitate information exchange between interfacing bacteria. A type VI secretion system (T6SS) of Pseudomonas aeruginosa was shown to deliver cell wall-targeting effectors to neighboring cells. However, the generality of bacteriolytic effectors and, moreover, of antibacterial T6S remained unknown. Using parameters derived from experimentally validated bacterial T6SS effectors we identified a phylogenetically disperse superfamily of T6SS-associated peptidoglycan-degrading effectors. The effectors separate into four families composed of peptidoglycan amidase enzymes of differing specificities. Effectors strictly co-occur with cognate immunity proteins, indicating that self-intoxication is a general property of antibacterial T6SSs and effector delivery by the system exerts a strong selective pressure in nature. The presence of antibacterial effectors in a plethora of organisms, including many that inhabit or infect polymicrobial niches in the human body, suggests that the system could mediate interbacterial interactions of both environmental and clinical significance

The Pseudomonas aeruginosa T6SS Delivers a Periplasmic Toxin that Disrupts Bacterial Cell Morphology.

The type VI secretion system (T6SS) is crucial in interbacterial competition and is a virulence determinant of many Gram-negative bacteria. Several T6SS effectors are covalently fused to secreted T6SS structural components such as the VgrG spike for delivery into target cells. In Pseudomonas aeruginosa, the VgrG2b effector was previously proposed to mediate bacterial internalization into eukaryotic cells. In this work, we find that the VgrG2b C-terminal domain (VgrG2bC-ter) elicits toxicity in the bacterial periplasm, counteracted by a cognate immunity protein. We resolve the structure of VgrG2bC-ter and confirm it is a member of the zinc-metallopeptidase family of enzymes. We show that this effector causes membrane blebbing at midcell, which suggests a distinct type of T6SS-mediated growth inhibition through interference with cell division, mimicking the impact of β-lactam antibiotics. Our study introduces a further effector family to the T6SS arsenal and demonstrates that VgrG2b can target both prokaryotic and eukaryotic cells