On the role of nucleotides and lipids in the polymerization of the actin homolog MreB from a Gram-positive bacterium.

In vivo, bacterial actin MreB assembles into dynamic membrane-associated filamentous structures that exhibit circumferential motion around the cell. Current knowledge of MreB biochemical and polymerization properties in vitro remains limited and is mostly based on MreB proteins from Gram-negative species. In this study, we report the first observation of organized protofilaments by electron microscopy and the first 3D-structure of MreB from a Gram-positive bacterium. We show that Geobacillus stearothermophilus MreB forms straight pairs of protofilaments on lipid surfaces in the presence of ATP or GTP, but not in the presence of ADP, GDP or non-hydrolysable ATP analogs. We demonstrate that membrane anchoring is mediated by two spatially close short hydrophobic sequences while electrostatic interactions also contribute to lipid binding, and show that the population of membrane-bound protofilament doublets is in steady-state. In solution, protofilament doublets were not detected in any condition tested. Instead, MreB formed large sheets regardless of the bound nucleotide, albeit at a higher critical concentration. Altogether, our results indicate that both lipids and ATP are facilitators of MreB polymerization, and are consistent with a dual effect of ATP hydrolysis, in promoting both membrane binding and filaments assembly/disassembly.

A High-Content Microscopy Screening Identifies New Genes Involved in Cell Width Control in Bacillus subtilis.

How cells control their shape and size is a fundamental question of biology. In most bacteria, cell shape is imposed by the peptidoglycan (PG) polymeric meshwork that surrounds the cell. Thus, bacterial cell morphogenesis results from the coordinated action of the proteins assembling and degrading the PG shell. Remarkably, during steady-state growth, most bacteria maintain a defined shape along generations, suggesting that error-proof mechanisms tightly control the process. In the rod-shaped model for the Gram-positive bacterium Bacillus subtilis, the average cell length varies as a function of the growth rate, but the cell diameter remains constant throughout the cell cycle and across growth conditions. Here, in an attempt to shed light on the cellular circuits controlling bacterial cell width, we developed a screen to identify genetic determinants of cell width in B. subtilis. Using high-content screening (HCS) fluorescence microscopy and semiautomated measurement of single-cell dimensions, we screened a library of ∼4,000 single knockout mutants. We identified 13 mutations significantly altering cell diameter, in genes that belong to several functional groups. In particular, our results indicate that metabolism plays a major role in cell width control in B. subtilis. IMPORTANCE Bacterial shape is primarily dictated by the external cell wall, a vital structure that, as such, is the target of countless antibiotics. Our understanding of how bacteria synthesize and maintain this structure is therefore a cardinal question for both basic and applied research. Bacteria usually multiply from generation to generation while maintaining their progenies with rigorously identical shapes. This implies that the bacterial cells constantly monitor and maintain a set of parameters to ensure this perpetuation. Here, our study uses a large-scale microscopy approach to identify at the whole-genome level, in a model bacterium, the genes involved in the control of one of the most tightly controlled cellular parameters, the cell width.

Methods for Studying Membrane-Associated Bacterial Cytoskeleton Proteins In Vivo by TIRF Microscopy.

MreB proteins are actin homologs present in nonspherical bacteria. They assemble into membrane-associated discrete filamentous structures that exhibit different dynamic behaviors along the bacterial sidewalls. Total internal reflection fluorescence (TIRF) microscopy, a sensitive method for studying molecular events at cell surfaces with high contrast and temporal resolution, is a method of choice to characterize the localization and dynamics of cortical MreB assemblies in vivo. This chapter describes the methods for visualizing fluorescently tagged MreB proteins in live Bacillus subtilis cells. We detail how to (1) grow B. subtilis strains for reproducible TIRF observations, (2) immobilize cells on agarose pads and (3) in CellASIC® microfluidic plates, and (4) acquire TIRF images and time lapses.

MreB Forms Subdiffraction Nanofilaments during Active Growth in Bacillus subtilis.

The actin-like MreB protein is a key player of the machinery controlling the elongation and maintenance of the cell shape of most rod-shaped bacteria. This protein is known to be highly dynamic, moving along the short axis of cells, presumably reflecting the movement of cell wall synthetic machineries during the enzymatic assembly of the peptidoglycan mesh. The ability of MreB proteins to form polymers is not debated, but their structure, length, and conditions of establishment have remained unclear and the subject of conflicting reports. Here we analyze various strains of Bacillussubtilis, the model for Gram-positive bacteria, and we show that MreB forms subdiffraction-limited, less than 200 nm-long nanofilaments on average during active growth, while micron-long filaments are a consequence of artificial overaccumulation of the protein. Our results also show the absence of impact of the size of the filaments on their speed, orientation, and other dynamic properties conferring a large tolerance to B. subtilis toward the levels and consequently the lengths of MreB polymers. Our data indicate that the density of mobile filaments remains constant in various strains regardless of their MreB levels, suggesting that another factor determines this constant.IMPORTANCE The construction of the bacterial cell envelope is a fundamental topic, as it confers its integrity to bacteria and is consequently the target of numerous antibiotics. MreB is an essential protein suspected to regulate the cell wall synthetic machineries. Despite two decades of study, its localization remains the subject of controversies, its description ranging from helical filaments spanning the entire cell to small discrete entities. The true structure of these filaments is important because it impacts the model describing how the machineries building the cell wall are associated, how they are coordinated at the scale of the entire cell, and how MreB mediates this regulation. Our results shed light on this debate, revealing the size of native filaments in B. subtilis during growth. They argue against models where MreB filament size directly affects the speed of synthesis of the cell wall and where MreB would coordinate distant machineries along the side wall.

Antibiotic sensitivity reveals that wall teichoic acids mediate DNA binding during competence in Bacillus subtilis.

Despite decades of investigation of genetic transformation in the model Gram-positive bacterium Bacillus subtilis, the factors responsible for exogenous DNA binding at the surface of competent cells remain to be identified. Here, we report that wall teichoic acids (WTAs), cell wall-anchored anionic glycopolymers associated to numerous critical functions in Gram-positive bacteria, are involved in this initial step of transformation. Using a combination of cell wall-targeting antibiotics and fluorescence microscopy, we show that competence-specific WTAs are produced and specifically localized in the competent cells to mediate DNA binding at the proximity of the transformation apparatus. Furthermore, we propose that TuaH, a putative glycosyl transferase induced during competence, modifies competence-induced WTAs in order to promote (directly or indirectly) DNA binding. On the basis of our results and previous knowledge in the field, we propose a model for DNA binding and transport during genetic transformation in B. subtilis.

PamR, a new MarR-like regulator affecting prophages and metabolic genes expression in Bacillus subtilis.

B. subtilis adapts to changing environments by reprogramming its genetic expression through a variety of transcriptional regulators from the global transition state regulators that allow a complete resetting of the cell genetic expression, to stress specific regulators controlling only a limited number of key genes required for optimal adaptation. Among them, MarR-type transcriptional regulators are known to respond to a variety of stresses including antibiotics or oxidative stress, and to control catabolic or virulence gene expression. Here we report the characterization of the ydcFGH operon of B. subtilis, containing a putative MarR-type transcriptional regulator. Using a combination of molecular genetics and high-throughput approaches, we show that this regulator, renamed PamR, controls directly its own expression and influence the expression of large sets of prophage-related and metabolic genes. The extent of the regulon impacted by PamR suggests that this regulator reprograms the metabolic landscape of B. subtilis in response to a yet unknown signal.

Contrasting mechanisms of growth in two model rod-shaped bacteria.

How cells control their shape and size is a long-standing question in cell biology. Many rod-shaped bacteria elongate their sidewalls by the action of cell wall synthesizing machineries that are associated to actin-like MreB cortical patches. However, little is known about how elongation is regulated to enable varied growth rates and sizes. Here we use total internal reflection fluorescence microscopy and single-particle tracking to visualize MreB isoforms, as a proxy for cell wall synthesis, in Bacillus subtilis and Escherichia coli cells growing in different media and during nutrient upshift. We find that these two model organisms appear to use orthogonal strategies to adapt to growth regime variations: B. subtilis regulates MreB patch speed, while E. coli may mainly regulate the production capacity of MreB-associated cell wall machineries. We present numerical models that link MreB-mediated sidewall synthesis and cell elongation, and argue that the distinct regulatory mechanism employed might reflect the different cell wall integrity constraints in Gram-positive and Gram-negative bacteria.

Structural rearrangements in the phage head-to-tail interface during assembly and infection.

Many icosahedral viruses use a specialized portal vertex to control genome encapsidation and release from the viral capsid. In tailed bacteriophages, the portal system is connected to a tail structure that provides the pipeline for genome delivery to the host cell. We report the first, to our knowledge, subnanometer structures of the complete portal-phage tail interface that mimic the states before and after DNA release during phage infection. They uncover structural rearrangements associated with intimate protein-DNA interactions. The portal protein gp6 of bacteriophage SPP1 undergoes a concerted reorganization of the structural elements of its central channel during interaction with DNA. A network of protein-protein interactions primes consecutive binding of proteins gp15 and gp16 to extend and close the channel. This critical step that prevents genome leakage from the capsid is achieved by a previously unidentified allosteric mechanism: gp16 binding to two different regions of gp15 drives correct positioning and folding of an inner gp16 loop to interact with equivalent loops of the other gp16 subunits. Together, these loops build a plug that closes the channel. Gp16 then fastens the tail to yield the infectious virion. The gatekeeper system opens for viral genome exit at the beginning of infection but recloses afterward, suggesting a molecular diaphragm-like mechanism to control DNA efflux. The mechanisms described here, controlling the essential steps of phage genome movements during virus assembly and infection, are likely to be conserved among long-tailed phages, the largest group of viruses in the Biosphere.

The nuclease domain of the SPP1 packaging motor coordinates DNA cleavage and encapsidation.

The large terminase subunit is a central component of the genome packaging motor from tailed bacteriophages and herpes viruses. This two-domain enzyme has an N-terminal ATPase activity that fuels DNA translocation during packaging and a C-terminal nuclease activity required for initiation and termination of the packaging cycle. Here, we report that bacteriophage SPP1 large terminase (gp2) is a metal-dependent nuclease whose stability and activity are strongly and preferentially enhanced by Mn(2+) ions. Mutation of conserved residues that coordinate Mn(2+) ions in the nuclease catalytic site affect the metal-induced gp2 stabilization and impair both gp2-specific cleavage at the packaging initiation site pac and unspecific nuclease activity. Several of these mutations block also DNA encapsidation without affecting ATP hydrolysis or gp2 C-terminus binding to the procapsid portal vertex. The data are consistent with a mechanism in which the nuclease domain bound to the portal switches between nuclease activity and a coordinated action with the ATPase domain for DNA translocation. This switch of activities of the nuclease domain is critical to achieve the viral chromosome packaging cycle.