FtsZ is the main regulator of bacterial cell division. It has been implicated in acting as a scaffolding protein for other division proteins, a force generator during constriction, and more recently, ...as an active regulator of septal cell wall production. FtsZ assembles into a heterogeneous structure coined the Z-ring due to its resemblance to a ring confined by the midcell geometry. Here, to establish a framework for examining geometrical influences on proper Z-ring assembly and dynamics, we sculpted Escherichia coli cells into unnatural shapes using division- and cell wall-specific inhibitors in a micro-fabrication scheme. This approach allowed us to examine FtsZ behavior in engineered Z-squares and Z-hearts. We use stimulated emission depletion (STED) nanoscopy to show that FtsZ clusters in sculpted cells maintain the same dimensions as their wild-type counterparts. Based on our results, we propose that the underlying membrane geometry is not a deciding factor for FtsZ cluster maintenance and dynamics in vivo.
The fabrication, assembly, and initial testing of a hybrid microfabricated gas chromatograph (microGC) is described. The microGC incorporates capabilities for on-board calibration, sample ...preconcentration and focused thermal desorption, temperature-programmed separations, and "spectral" detection with an integrated array of microsensors, and is designed for rapid determinations of complex mixtures of environmental contaminants at trace concentrations. Ambient air is used as the carrier gas to avoid the need for on-board gas supplies. The microsystem is plumbed through an etched-Si/glass microfluidic interconnection substrate with fused silica capillaries and employs a miniature commercial pump and valve subsystem for directing sample flow. The latest performance data on each system component are presented followed by first analytical results from the working microsystem. Tradeoffs in system performance as a function of volumetric flow rate are explored. The determination of an 11-vapor mixture of typical indoor air contaminants in less than 90 s is demonstrated with projected detection limits in the low part-per-billion concentration range for a preconcentrated air-sample volume of 0.25 L.
Bacterial chromosome segregation is an essential cellular process that is particularly elusive in spherical bacteria such as the opportunistic human pathogen Staphylococcus aureus. In this study, we ...examined the functional significance of a ParB homologue, Spo0J, in staphylococcal chromosome segregation and investigated the role of the structural maintenance of chromosomes (SMC) bacterial condensin in this process. We show that neither spo0J nor smc is essential in S. aureus; however, their absence causes abnormal chromosome segregation. We demonstrate that formation of complexes containing Spo0J and SMC is required for efficient S. aureus chromosome segregation and that SMC localization is dependent on Spo0J. Furthermore, we found that cell division and cell cycle progression are unaffected by the absence of spo0J or smc. Our results verify the role of Spo0J and SMC in ensuring accurate staphylococcal chromosome segregation and also imply functional redundancy or the involvement of additional mechanisms that might contribute to faithful chromosome inheritance.
Chromosome segregation is an essential but poorly understood process in spherical bacteria such as Staphylococcus aureus. Here, we show that the staphylococcal ParB homologue, Spo0J, and the structural maintenance of chromosomes (SMC) protein act in concert to achieve efficient chromosome segregation in S. aureus. We demonstrate that Spo0J and SMC form a complex that is dependent on correct localization of Spo0J and propose that additional mechanisms are likely involved in accurate staphylococcal chromosome segregation.
Summary
The division of Escherichia coli is mediated by a collection of some 34 different proteins that are recruited to the division septum and are thought to assemble into a macromolecular complex ...known as ‘the divisome’. Herein, we have endeavored to better understand the structure of the divisome by imaging two of its core components; FtsZ and FtsN. Super resolution microscopy (SIM and gSTED) indicated that both proteins are localized in large assemblies, which are distributed around the division septum (i.e., forming a discontinuous ring). Although the rings had similar radii prior to constriction, the individual densities were often spatially separated circumferentially. As the cell envelope constricted, the discontinuous ring formed by FtsZ moved inside the discontinuous ring formed by FtsN. The radial and circumferential separation observed in our images indicates that the majority of FtsZ and FtsN molecules are organized in different macromolecular assemblies, rather than in a large super‐complex. This conclusion was supported by fluorescence recovery after photobleaching measurements, which indicated that the dynamic behavior of the two macromolecular assemblies was also fundamentally different. Taken together, the data indicates that constriction of the cell envelope is brought about by (at least) two spatially separated complexes.
In order to divide, bacterial cells assemble a macromolecular machine, termed ‘the divisome’. Herein we used super‐resolution microscopy investigate the organization of two key divisome proteins; FtsZ and FtsN. We found that these proteins were both circumferentially and radially separated into individual protein assemblies. Thus, our data indicates that division is brought about by a collection of independently operating protein assemblies, rather than a single macromolecular machine.
Understanding where proteins are localized in a bacterial cell is essential for understanding their function and regulation. This is particularly important for proteins that are involved in cell ...division, which localize at the division septum and assemble into highly regulated complexes. Current knowledge of these complexes has been greatly facilitated by super-resolution imaging using fluorescent protein fusions. Herein, we demonstrate with FtsZ that single-molecule PALM images can be obtained
in-vivo
using a genetically fused nanotag (ALFA), and a corresponding nanobody fused to mEos3.2. The methodology presented is applicable to other bacterial proteins.
Bacterial spores owe their incredible resistance capacities to molecular structures that protect the cell content from external aggressions. Among the determinants of resistance are the quaternary ...structure of the chromosome and an extracellular shell made of proteinaceous layers (the coat), the assembly of which remains poorly understood. Here, in situ cryo-electron tomography on lamellae generated by cryo-focused ion beam micromachining provides insights into the ultrastructural organization of Bacillus subtilis sporangia. The reconstructed tomograms reveal that early during sporulation, the chromosome in the forespore adopts a toroidal structure harboring 5.5-nm thick fibers. At the same stage, coat proteins at the surface of the forespore form a stack of amorphous or structured layers with distinct electron density, dimensions and organization. By analyzing mutant strains using cryo-electron tomography and transmission electron microscopy on resin sections, we distinguish seven nascent coat regions with different molecular properties, and propose a model for the contribution of coat morphogenetic proteins.
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•The prototypical multiresistance plasmid pSK1 encodes a single par protein, pSK1 Par.•Unlike typical par systems, requiring 2 proteins, pSK1 Par alone drives pSK1 partition.•pSK1 ...Par-DNA structure reveals distinct CBP fold and centromere binding mechanism.•Biochemical analyses show pSK1 Par forms extended coiled-coiled oligomer.•In vivo data show pSK1 Par enhances plasmid separation of plasmids from clusters.
The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system.
Cell division and cell wall synthesis in staphylococci need to be precisely coordinated and controlled to allow the cell to multiply while maintaining its nearly spherical shape. The mechanisms ...ensuring correct placement of the division plane and synthesis of new cell wall have been studied intensively. However, hitherto unknown factors and proteins are likely to play key roles in this complex interplay. Here, we identified and investigated a protein with a major influence on cell morphology in Staphylococcus aureus. The protein, named SmdA (for staphylococcal morphology determinant A), is a membrane protein with septum-enriched localization. By CRISPRi knockdown and overexpression combined with different microscopy techniques, we demonstrated that proper levels of SmdA were necessary for cell division, including septum formation and cell splitting. We also identified conserved residues in SmdA that were critical for its functionality. Pulldown and bacterial two-hybrid interaction experiments showed that SmdA interacted with several known cell division and cell wall synthesis proteins, including penicillin-binding proteins (PBPs) and EzrA. Notably, SmdA also affected susceptibility to cell wall targeting antibiotics, particularly in methicillin-resistant S. aureus (MRSA). Together, our results showed that S. aureus was dependent on balanced amounts of membrane attached SmdA to carry out proper cell division.
Staphylococcus aureus is an important human and animal pathogen. Antibiotic resistance is a major problem in the treatment of staphylococcal infections, and cell division and cell wall synthesis factors have previously been shown to modulate susceptibility to antibiotics in this species. Here, we investigated the function of a protein named SmdA, which was identified based on its septal localization and knockdown phenotype resulting in defective cellular morphologies. We demonstrated that this protein was critical for normal cell division in S. aureus. Depletion of SmdA sensitized resistant staphylococci to β-lactam antibiotics. This work revealed a new staphylococcal cell division factor and a potential future target for narrow-spectrum antimicrobials or compounds to resensitize antibiotic-resistant staphylococcal strains.
How organisms develop into specific shapes is a central question in biology. The maintenance of bacterial shape is connected to the assembly and remodelling of the cell envelope. In endospore-forming ...bacteria, the pre-spore compartment (the forespore) undergoes morphological changes that result in a spore of defined shape, with a complex, multi-layered cell envelope. However, the mechanisms that govern spore shape remain poorly understood. Here, using a combination of fluorescence microscopy, quantitative image analysis, molecular genetics and transmission electron microscopy, we show that SsdC (formerly YdcC), a poorly-characterized new member of the MucB / RseB family of proteins that bind lipopolysaccharide in diderm bacteria, influences spore shape in the monoderm Bacillus subtilis. Sporulating cells lacking SsdC fail to adopt the typical oblong shape of wild-type forespores and are instead rounder. 2D and 3D-fluorescence microscopy suggest that SsdC forms a discontinuous, dynamic ring-like structure in the peripheral membrane of the mother cell, near the mother cell proximal pole of the forespore. A synthetic sporulation screen identified genetic relationships between ssdC and genes involved in the assembly of the spore coat. Phenotypic characterization of these mutants revealed that spore shape, and SsdC localization, depend on the coat basement layer proteins SpoVM and SpoIVA, the encasement protein SpoVID and the inner coat protein SafA. Importantly, we found that the ΔssdC mutant produces spores with an abnormal-looking cortex, and abolishing cortex synthesis in the mutant largely suppresses its shape defects. Thus, SsdC appears to play a role in the proper assembly of the spore cortex, through connections to the spore coat. Collectively, our data suggest functional diversification of the MucB / RseB protein domain between diderm and monoderm bacteria and identify SsdC as an important factor in spore shape development.
The design, fabrication, and characterization of a multiple-stage Si microfabricated preconcentrator-focuser (/spl mu/PCF) for a micro gas chromatography (/spl mu/GC) system that can provide ...real-time quantification and identification of complex organic vapor mixtures are presented. The /spl mu/PCF consists of a Si microheater loaded with Carbopack B, Carbopack X, and Carboxen 1000 carbon adsorbent granules, and a Si micromachined cover plate. Deep reactive ion etching is utilized to produce mechanically robust fluidic interconnection adapters hermetically sealed to fused silica capillary tubing for connection to the other components in the /spl mu/GC. This three-stage device is designed to capture compounds spanning up to 4 orders of magnitude in volatility. The dead volume, thermal mass, heating efficiency, and pressure drop of the three-stage /spl mu/PCF are improved significantly over its single-stage /spl mu/PCF predecessor. We demonstrate the successful capture, desorption, and high-resolution chromatographic separation of a mixture of 30 common organic vapors using our three-stage /spl mu/PCF in a conventional GC system. The peak width at half height is <2.05 s for all compounds after elution from the GC column.
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