Chapter 12. Measure of peptidoglycan hydrolase activity Running head: peptidoglycan remodelling enzymes

International audienceMost of the gene clusters encoding multiprotein complexes of the bacterial cell envelope, such as conjugation and secretion systems, Type IV pili and flagella, bear a gene encoding an enzyme with peptidoglycan hydrolase activity. These enzymes are usually glycoside hydrolases that cleave the glycan chains of the peptidoglycan. Their activities are spatially controlled to avoid cell lysis and to create localized rearrangement of the cell wall. This is assured by interaction with structural subunits of the apparatus. Here, we describe protocols to test the peptidoglycan hydrolase activity of these proteins in vitro and in solution

Cell width dictates Type VI secretion tail length

International audienceThe type VI secretion system (T6SS) is a multiprotein apparatus that injects protein effectors into target cells, hence playing a critical role in pathogenesis and in microbial communities [1, 2, 3, 4]. The T6SS belongs to the broad family of ontractile injection systems (CISs), such as Myoviridae bacteriophages and R-pyocins, that use a spring-like tail to propel a needle loaded with effectors [5, 6]. The T6SS tail comprises an assembly baseplate on which polymerizes a needle, made of stacked Hcp hexamers, tipped by the VgrG-PAAR spike complex and wrapped by the contractile sheath made of TssB and TssC [7, 8, 9, 10, 11, 12, 13]. The T6SS tail is anchored to the cell envelope by a membrane complex that also serves as channel for the passage of the needle upon sheath contraction [14, 15, 16]. In most CISs, the length of the tail sheath is invariable and is usually ensured by a dedicated protein called tape measure protein (TMP) [17, 18, 19, 20, 21, 22]. Here, we show that the length of the T6SS tail is constant in enteroaggregative Escherichia coli cells, suggesting that it is strictly controlled. By overproducing T6SS tail subunits, we demonstrate that component stoichiometry does not participate to the regulation of tail length. The observation of longer T6SS tails when the apparatus is relocalized at the cell pole further shows that tail length is not controlled by a TMP. Finally, we show that tail stops its elongation when in contact with the opposite membrane and thus that T6SS tail length is determined by the cell width

Domestication of a housekeeping transglycosylase for assembly of a Type VI secretion system

International audiencehe Type VI secretion system (T6SS) is an anti-bacterial weapon comprising a contractile tail anchored to the cell envelope by a membrane complex. The TssJ, TssL and TssM proteins assemble a 1.7-MDa channel complex that spans the cell envelope, including the peptidoglycan layer. The electron microscopy structure of the TssJLM complex revealed that it has a diameter of ∼ 18 nm in the periplasm, which is larger that the size of peptidoglycan pores (∼ 2 nm), hence questioning how the T6SS membrane complex crosses the peptidoglycan layer. Here, we report that the MltE housekeeping lytic transglycosylase (LTG) is required for T6SS assembly in enteroaggregative E. coli. Protein-protein interactions studies further demonstrated that MltE is recruited to the periplasmic domain of TssM. In addition, we show that TssM significantly stimulates MltE activity in vitro and that MltE is required for the late stages of T6SS membrane complex assembly. Collectively, our data provide the first example of domestication and activation of a LTG encoded within the core genome for the assembly of a secretion system

In vivo TssA proximity labeling reveals temporal interactions during Type VI secretion 1 biogenesis and TagA, a protein that stops and holds the sheath.

International audienceThe Type VI secretion system (T6SS) is a multiprotein weapon used by bacteria to destroy competitor cells. The T6SS contractile sheath wraps an effector-loaded syringe that is injected into the target cell. This tail structure assembles onto the baseplate that is docked to the membrane complex. In entero-aggregative Escherichia coli TssA plays a central role at each stage of the T6SS assembly pathway by stabilizing the baseplate and coordinating the polymerization of the tail. Here we adapted an assay based on APEX2-dependent biotinylation to identify the proximity partners of TssA in vivo. By using stage-blocking mutations, we define the temporal contacts of TssA during T6SS biogenesis. This proteomic mapping approach also revealed an additional partner of TssA, TagA. We show that TagA is a cytosolic protein tightly associated with the membrane. Analyses of sheath dynamics further demonstrate that TagA captures the distal end of the sheath to stop its polymerization and to maintain it under the extended conformation

Role and recruitment of the TagL peptidoglycan-binding protein during Type VI secretion system biogenesis

International audienceThe type VI secretion system (T6SS) is an injection apparatus that uses a springlike mechanism for effector delivery. The contractile tail is composed of a needle tipped by a sharpened spike and wrapped by the sheath that polymerizes in an extended conformation on the assembly platform, or baseplate. Contraction of the sheath propels the needle and effectors associated with it into target cells. The passage of the needle through the cell envelope of the attacker is ensured by a dedicated trans-envelope channel complex. This membrane complex (MC) comprises the TssJ lipoprotein and the TssL and TssM inner membrane proteins. MC assembly is a hierarchized mechanism in which the different subunits are recruited in a specific order: TssJ, TssM, and then TssL. Once assembled, the MC serves as a docking station for the baseplate. In enteroaggregative Escherichia coli, the MC is accessorized by TagL, a peptidoglycan-binding (PGB) inner membrane-anchored protein. Here, we show that the PGB domain is the only functional domain of TagL and that the N-terminal transmembrane region mediates contact with the TssL transmembrane helix. Finally, we conduct fluorescence microscopy experiments to position TagL in the T6SS biogenesis pathway, demonstrating that TagL is recruited to the membrane complex downstream of TssL and is not required for baseplate docking

Caractérisation moléculaire du mécanisme de contrôle de la taille du système de type VI

Les bactéries ont développé une variété de structures macromoléculaires spécialisées impliquées dans la motilité, l'adhésion cellulaire, la sécrétion de protéines / ADN ou la destruction bactérienne. Ces structures s'assemblent pour former des super-complexes, soit ancrés dans l'enveloppe bactérienne ou libérés dans l'environnement. Parmi eux, le système de sécrétion de type VI (T6SS) représente l'une des machines les plus puissantes pour tuer des compétiteurs. Ce système est composé d'un complexe membranaire inséré dans l'enveloppe bactérienne qui ancre une structure reliée aux bactériophages contractiles. Représenté comme une nano-arbalète, la contraction de cette structure permet la translocation d'effecteurs toxiques directement dans la cellule cible. Au cours de la biogenèse du T6SS, la queue du T6SS forme une longue structure traversant la cellule. Alors que la détermination de la longueur du T6SS n'était pas connue, mon projet de doctorat a été dédié à déterminer son mécanisme de contrôle . J'ai d'abord démontré que la largeur de la cellule dicte la longueur de la queue du T6SS. En utilisant un test de marquage de proximité in vivo , j'ai ensuite identifié un nouveau composant du T6SS, TagA.La caractérisation de TagA a révélé qu'il capture l'extrémité distale de la queue du T6SS une fois qu'il s'approche de la membrane opposée, empêchant l'incorporation d'autres sous-unités de la queue et maintenant la structure en mode «pré-tir». Au cours de mes trois années dans le laboratoire, j'ai fourni quelques indices sur le mécanisme précis de contrôle de la taille du T6SS, permettant de compléter le tableau des étapes tardives de la biogenèse T6SS.Bacteria have developed a variety of specialized macromolecular structures involved in motility, cell adhesion, protein/DNA secretion or bacterial killing. These structures assemble to form super complexes, either anchored into the bacterial envelope or released in the environment. Among them, the type VI secretion system (T6SS) represents one of the most powerful machineries to efficiently kill competitors. This system is composed of a membrane complex anchored into the bacterial envelope that docks a contractile bacteriophage-related tail structure. Depicted as a nano-crossbow, the contraction of the T6SS tail structure allows effector delivery directly into the target cell. During the T6SS biogenesis, the T6SS tail forms a long structure spanning the cell. Since the T6SS length determination was not known, my Ph.D. project has been dedicated to determining the size-control mechanism of the T6SS tail. I first demonstrated that the cell width dictates the T6SS tail length, as tail polymerization is arrested at the opposite membrane. By using a in vivo proximity-labeling assay, I then identified a novel T6SS player, TagA. Characterization of TagA revealed that it captures the distal end of the T6SS tail once it approaches to the opposite membrane, preventing further tail subunits incorporation and maintaining the structure under a “pre-firing” mode. I then conducted a structure-function analysis of the TagA domains to better understand their contribution during T6SS activity. Over my three years in the lab, I provided some clues about the precise T6SS size-control mechanism, allowing to complete the picture of the late stages of T6SS biogenesis

Modulation of prey size reveals adaptability and robustness in the cell cycle of an intracellular predator.

Despite a remarkable diversity of lifestyles, bacterial replication has only been investigated in a few model species. In bacteria that do not rely on canonical binary division for proliferation, the coordination of major cellular processes is still largely mysterious. Moreover, the dynamics of bacterial growth and division remain unexplored within spatially confined niches where nutrients are limited. This includes the life cycle of the model endobiotic predatory bacterium Bdellovibrio bacteriovorus, which grows by filamentation within its prey and produces a variable number of daughter cells. Here, we examined the impact of the micro-compartment in which predators replicate (i.e., the prey bacterium) on their cell-cycle progression at the single-cell level. Using Escherichia coli with genetically encoded size differences, we show that the duration of the predator cell cycle scales with prey size. Consequently, prey size determines predator offspring numbers. We found that individual predators elongate exponentially, with a growth rate determined by the nutritional quality of the prey, irrespective of prey size. However, the size of newborn predator cells is remarkably stable across prey nutritional content and size variations. Tuning the predatory cell cycle by modulating prey dimensions also allowed us to reveal invariable temporal connections between key cellular processes. Altogether, our data imply adaptability and robustness shaping the enclosed cell-cycle progression of B. bacteriovorus, which might contribute to optimal exploitation of the finite resources and space in their prey. This study extends the characterization of cell cycle control strategies and growth patterns beyond canonical models and lifestyles

Coevolution-guided mapping of the Type VI secretion membrane complexbaseplate interface

The type VI secretion system (T6SS) is a multiprotein weapon evolved by Gram-negative bacteria to deliver effectors into eukaryotic cells or bacterial rivals. The T6SS uses a contractile mechanism to propel an effector-loaded needle into its target. The contractile tail is built on an assembly platform, the baseplate, which is anchored to a membrane complex. Baseplate-membrane complex interactions are mainly mediated by contacts between the C-terminal domain of the TssK baseplate component and the cytoplasmic domain of the TssL inner membrane protein. Currently, the structural details of this interaction are unknown due to the marginal stability of the TssK-TssL complex. Here we conducted a mutagenesis study based on putative TssK-TssL contact pairs identified by co-evolution analyses. We then evaluated the impact of these mutations on T6SS activity, TssK-TssL interaction and sheath assembly and dynamics in enteroaggregative Escherichia coli. Finally, we probed the TssK-TssL interface by disulfide cross-linking, allowing to propose a model for the baseplate-membrane complex interface