Functional Comparison of Human and Zebra Fish FKBP52 Confirms the Importance of the Proline-Rich Loop for Regulation of Steroid Hormone Receptor Activity.

Previous studies demonstrated that the 52-kDa FK506-binding protein (FKBP52) proline-rich loop is functionally relevant in the regulation of steroid hormone receptor activity. While zebra fish (Danio rerio; Dr) FKBP52 contains all of the analogous domains and residues previously identified as critical for FKBP52 potentiation of receptor activity, it fails to potentiate activity. Thus, we used a cross-species comparative approach to assess the residues that are functionally critical for FKBP52 function. Random selection of gain-of-function DrFKBP52 mutants in Saccharomyces cerevisiae identified two critical residues, alanine 111 (A111) and threonine 157 (T157), for activation of receptor potentiation by DrFKBP52. In silico homology modeling suggests that alanine to valine substitution at position 111 in DrFKBP52 induces an open conformation of the proline-rich loop surface similar to that observed on human FKBP52, which may allow for sufficient surface area and increased hydrophobicity for interactions within the receptor-chaperone complex. A second mutation in the FKBP12-like domain 2 (FK2), threonine 157 to arginine (T157R), also enhanced potentiation, and the DrFKBP52-A111V/T157R double mutant potentiated receptor activity similar to human FKBP52. Collectively, these results confirm the functional importance of the FKBP52 proline-rich loop, suggest that an open conformation on the proline-rich loop surface is a predictor of activity, and highlight the importance of an additional residue within the FK2 domain.

Gain-of-function variants of FtsA form diverse oligomeric structures on lipids and enhance FtsZ protofilament bundling.

Escherichia coli requires FtsZ, FtsA and ZipA proteins for early stages of cell division, the latter two tethering FtsZ polymers to the cytoplasmic membrane. Hypermorphic mutants of FtsA such as FtsA* (R286W) map to the FtsA self-interaction interface and can bypass the need for ZipA. Purified FtsA forms closed minirings on lipid monolayers that antagonize bundling of FtsZ protofilaments, whereas FtsA* forms smaller oligomeric arcs that enable bundling. Here, we examined three additional FtsA*-like mutant proteins for their ability to form oligomers on lipid monolayers and bundle FtsZ. Surprisingly, all three formed distinct structures ranging from mostly arcs (T249M), a mixture of minirings, arcs and straight filaments (Y139D) or short straight double filaments (G50E). All three could form filament sheets at higher concentrations with added ATP. Despite forming these diverse structures, all three mutant proteins acted like FtsA* to enable FtsZ protofilament bundling on lipid monolayers. Synthesis of the FtsA*-like proteins in vivo suppressed the toxic effects of a bundling-defective FtsZ, exacerbated effects of a hyper-bundled FtsZ, and rescued some thermosensitive cell division alleles. Together, the data suggest that conversion of FtsA minirings into any type of non-miniring oligomer can promote progression of cytokinesis through FtsZ bundling and other mechanisms.

Escherichia coli FtsA forms lipid-bound minirings that antagonize lateral interactions between FtsZ protofilaments.

Most bacteria divide using a protein machine called the divisome that spans the cytoplasmic membrane. Key divisome proteins on the membrane's cytoplasmic side include tubulin-like FtsZ, which forms GTP-dependent protofilaments, and actin-like FtsA, which tethers FtsZ to the membrane. Here we present genetic evidence that in Escherichia coli, FtsA antagonizes FtsZ protofilament bundling in vivo. We then show that purified FtsA does not form straight polymers on lipid monolayers as expected, but instead assembles into dodecameric minirings, often in hexameric arrays. When coassembled with FtsZ on lipid monolayers, these FtsA minirings appear to guide FtsZ to form long, often parallel, but unbundled protofilaments, whereas a mutant of FtsZ (FtsZ*) with stronger lateral interactions remains bundled. In contrast, a hypermorphic mutant of FtsA (FtsA*) forms mainly arcs instead of minirings and enhances lateral interactions between FtsZ protofilaments. Based on these results, we propose that FtsA antagonizes lateral interactions between FtsZ protofilaments, and that the oligomeric state of FtsA may influence FtsZ higher-order structure and divisome function.

Impact of Membrane Phospholipid Alterations in Escherichia coli on Cellular Function and Bacterial Stress Adaptation.

Bacteria have evolved multiple strategies to sense and rapidly adapt to challenging and ever-changing environmental conditions. The ability to alter membrane lipid composition, a key component of the cellular envelope, is crucial for bacterial survival and adaptation in response to environmental stress. However, the precise roles played by membrane phospholipids in bacterial physiology and stress adaptation are not fully elucidated. The goal of this study was to define the role of membrane phospholipids in adaptation to stress and maintenance of bacterial cell fitness. By using genetically modified strains in which the membrane phospholipid composition can be systematically manipulated, we show that alterations in major Escherichia coli phospholipids transform these cells globally. We found that alterations in phospholipids impair the cellular envelope structure and function, the ability to form biofilms, and bacterial fitness and cause phospholipid-dependent susceptibility to environmental stresses. This study provides an unprecedented view of the structural, signaling, and metabolic pathways in which bacterial phospholipids participate, allowing the design of new approaches in the investigation of lipid-dependent processes involved in bacterial physiology and adaptation.IMPORTANCE In order to cope with and adapt to a wide range of environmental conditions, bacteria have to sense and quickly respond to fluctuating conditions. In this study, we investigated the effects of systematic and controlled alterations in bacterial phospholipids on cell shape, physiology, and stress adaptation. We provide new evidence that alterations of specific phospholipids in Escherichia coli have detrimental effects on cellular shape, envelope integrity, and cell physiology that impair biofilm formation, cellular envelope remodeling, and adaptability to environmental stresses. These findings hold promise for future antibacterial therapies that target bacterial lipid biosynthesis.

Proximity Interactions among Basal Body Components in Trypanosoma brucei Identify Novel Regulators of Basal Body Biogenesis and Inheritance.

The basal body shares similar architecture with centrioles in animals and is involved in nucleating flagellar axonemal microtubules in flagellated eukaryotes. The early-branching Trypanosoma brucei possesses a motile flagellum nucleated from the basal body that consists of a mature basal body and an adjacent pro-basal body. Little is known about the basal body proteome and its roles in basal body biogenesis and flagellar axoneme assembly in T. brucei Here, we report the identification of 14 conserved centriole/basal body protein homologs and 25 trypanosome-specific basal body proteins. These proteins localize to distinct subdomains of the basal body, and several of them form a ring-like structure surrounding the basal body barrel. Functional characterization of representative basal body proteins revealed distinct roles in basal body duplication/separation and flagellar axoneme assembly. Overall, this work identified novel proteins required for basal body duplication and separation and uncovered new functions of conserved basal body proteins in basal body duplication and separation, highlighting an unusual mechanism of basal body biogenesis and inheritance in this early divergent eukaryote. IMPORTANCE: The basal body in the early-branching protozoan Trypanosoma brucei nucleates flagellum assembly and also regulates organelle segregation, cell morphogenesis, and cell division. However, the molecular composition and the assembly process of the basal body remain poorly understood. Here, we identify 14 conserved basal body proteins and 25 trypanosome-specific basal body proteins via bioinformatics, localization-based screening, and proximity-dependent biotin identification. We further localized these proteins to distinct subdomains of the basal body by using fluorescence microscopy and superresolution microscopy, discovered novel regulators of basal body duplication and separation, and uncovered new functions of conserved basal body proteins in basal body duplication and separation. This work lays the foundation for dissecting the mechanisms underlying basal body biogenesis and inheritance in T. brucei.

Semi-automated microplate monitoring of protein polymerization and aggregation.

Static light scattering (SLS) is a commonly used technique for monitoring dynamics of high molecular weight protein complexes such as protein oligomers or aggregates. However, traditional methods are limited to testing a single condition and typically require large amounts of protein and specialized equipment. We show that a standard microplate reader can be used to characterize the molecular dynamics of different types of protein complexes, with the multiple advantages of microscale experimental volumes, semi-automated protocols and highly parallel processing.

The bacterial divisome: ready for its close-up.

Bacterial cells divide by targeting a transmembrane protein machine to the division site and regulating its assembly and disassembly so that cytokinesis occurs at the correct time in the cell cycle. The structure and dynamics of this machine (divisome) in bacterial model systems are coming more clearly into focus, thanks to incisive cell biology methods in combination with biochemical and genetic approaches. The main conserved structural element of the machine is the tubulin homologue FtsZ, which assembles into a circumferential ring at the division site that is stabilized and anchored to the inner surface of the cytoplasmic membrane by FtsZ-binding proteins. Once this ring is in place, it recruits a series of transmembrane proteins that ultimately trigger cytokinesis. This review will survey the methods used to characterize the structure of the bacterial divisome, focusing mainly on the Escherichia coli model system, as well as the challenges that remain. These methods include recent super-resolution microscopy, cryo-electron tomography and synthetic reconstitution.

The Min system and other nucleoid-independent regulators of Z ring positioning.

Rod-shaped bacteria such as E. coli have mechanisms to position their cell division plane at the precise center of the cell, to ensure that the daughter cells are equal in size. The two main mechanisms are the Min system and nucleoid occlusion (NO), both of which work by inhibiting assembly of FtsZ, the tubulin-like scaffold that forms the cytokinetic Z ring. Whereas NO prevents Z rings from constricting over unsegregated nucleoids, the Min system is nucleoid-independent and even functions in cells lacking nucleoids and thus NO. The Min proteins of E. coli and B. subtilis form bipolar gradients that inhibit Z ring formation most at the cell poles and least at the nascent division plane. This article will outline the molecular mechanisms behind Min function in E. coli and B. subtilis, and discuss distinct Z ring positioning systems in other bacterial species.

A mutation in Escherichia coli ftsZ bypasses the requirement for the essential division gene zipA and confers resistance to FtsZ assembly inhibitors by stabilizing protofilament bundling.

The earliest step in Escherichia coli cell division consists of the assembly of FtsZ protein into a proto-ring structure, tethered to the cytoplasmic membrane by FtsA and ZipA. The proto-ring then recruits additional cell division proteins to form the divisome. Previously we described an ftsZ allele, ftsZL169R , which maps to the side of the FtsZ subunit and confers resistance to FtsZ assembly inhibitory factors including Kil of bacteriophage λ. Here we further characterize this allele and its mechanism of resistance. We found that FtsZL169R permits the bypass of the normally essential ZipA, a property previously observed for FtsA gain-of-function mutants such as FtsA* or increased levels of the FtsA-interacting protein FtsN. Similar to FtsA*, FtsZL169R also can partially suppress thermosensitive mutants of ftsQ or ftsK, which encode additional divisome proteins, and confers strong resistance to excess levels of FtsA, which normally inhibit FtsZ ring function. Additional genetic and biochemical assays provide further evidence that FtsZL169R enhances FtsZ protofilament bundling, thereby conferring resistance to assembly inhibitors and bypassing the normal requirement for ZipA. This work highlights the importance of FtsZ protofilament bundling during cell division and its likely role in regulating additional divisome activities.