Application of nanotags and nanobodies for live cell single-molecule imaging of the Z-ring in Escherichia coli.

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.

A dynamic, ring-forming MucB / RseB-like protein influences spore shape in Bacillus subtilis.

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.

Structural insights into ring-building motif domains involved in bacterial sporulation.

Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for "Ring-Building Motifs". During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the ß-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions.

The Ethanologenic Bacterium Zymomonas mobilis Divides Asymmetrically and Exhibits Heterogeneity in DNA Content.

The alphaproteobacterium Zymomonas mobilis exhibits extreme ethanologenic physiology, making this species a promising biofuel producer. Numerous studies have investigated its biology relevant to industrial applications and mostly at the population level. However, the organization of single cells in this industrially important polyploid species has been largely uncharacterized. In the present study, we characterized basic cellular behavior of Z. mobilis strain Zm6 under anaerobic conditions at the single-cell level. We observed that growing Z. mobilis cells often divided at a nonmidcell position, which contributed to variant cell size at birth. However, the cell size variance was regulated by a modulation of cell cycle span, mediated by a correlation of bacterial tubulin homologue FtsZ ring accumulation with cell growth. The Z. mobilis culture also exhibited heterogeneous cellular DNA content among individual cells, which might have been caused by asynchronous replication of chromosome that was not coordinated with cell growth. Furthermore, slightly angled divisions might have resulted in temporary curvatures of attached Z. mobilis cells. Overall, the present study uncovers a novel bacterial cell organization in Z. mobilisIMPORTANCE With increasing environmental concerns about the use of fossil fuels, development of a sustainable biofuel production platform has been attracting significant public attention. Ethanologenic Z. mobilis species are endowed with an efficient ethanol fermentation capacity that surpasses, in several respects, that of baker's yeast (Saccharomyces cerevisiae), the most-used microorganism for ethanol production. For development of a Z. mobilis culture-based biorefinery, an investigation of its uncharacterized cell biology is important, because bacterial cellular organization and metabolism are closely associated with each other in a single cell compartment. In addition, the current work demonstrates that the polyploid bacterium Z. mobilis exhibits a distinctive mode of bacterial cell organization, likely reflecting its unique metabolism that does not prioritize incorporation of nutrients for cell growth. Thus, another significant result of this work is to advance our general understanding in the diversity of bacterial cell architecture.

Spatial separation of FtsZ and FtsN during cell division.

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.

Super-resolution images of peptidoglycan remodelling enzymes at the division site of Escherichia coli.

Bacterial cells need to divide. This process requires more than 30 different proteins, which gather at the division site. It is widely assumed that these proteins assemble into a macromolecular complex (the divisome), but capturing the molecular layout of this complex has proven elusive. Super-resolution microscopy can provide spatial information, down to a few tens of nanometers, about how the division proteins assemble into complexes and how their activities are co-ordinated. Herein we provide insight into recent work from our laboratories, where we used super-resolution gSTED nanoscopy to explore the molecular organization of FtsZ, FtsI and FtsN. The resulting images show that all three proteins form discrete densities organised in patchy pseudo-rings at the division site. Significantly, two-colour imaging highlighted a radial separation between FtsZ and FtsN, indicating that there is more than one type of macromolecular complex operating during division. These data provide a first glimpse into the spatial organisation of PG-synthesising enzymes during division in Gram-negative bacteria.

Ultrastructure of macromolecular assemblies contributing to bacterial spore resistance revealed by in situ cryo-electron tomography.

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.

FtsK and SpoIIIE, coordinators of chromosome segregation and envelope remodeling in bacteria.

The translocation of DNA during bacterial cytokinesis is mediated by the SpoIIIE/FtsK family of proteins. These proteins ensure efficient chromosome segregation into sister cells by ATP-driven translocation of DNA and they control chromosome dimer resolution. How FtsK/SpoIIIE mediate chromosome translocation during cytokinesis in Gram-positive and Gram-negative organisms has been the subject of debate. Studies on FtsK in Escherichia coli, and recent work on SpoIIIE in Bacillus subtilis, have identified interactions between each translocase and the division machinery, supporting the idea that SpoIIIE and FtsK coordinate the final steps of cytokinesis with completion of chromosome segregation. Here we summarize and discuss the view that SpoIIIE and FtsK play similar roles in coordinating cytokinesis with chromosome segregation, during growth and differentiation.

SmdA is a Novel Cell Morphology Determinant in Staphylococcus aureus.

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. IMPORTANCE 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 ß-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.

Genetic Screens Identify Additional Genes Implicated in Envelope Remodeling during the Engulfment Stage of Bacillus subtilis Sporulation.

During bacterial endospore formation, the developing spore is internalized into the mother cell through a phagocytic-like process called engulfment, which involves synthesis and hydrolysis of peptidoglycan. Engulfment peptidoglycan hydrolysis requires the widely conserved and well-characterized DMP complex, composed of SpoIID, SpoIIM, and SpoIIP. In contrast, although peptidoglycan synthesis has been implicated in engulfment, the protein players involved are less well defined. The widely conserved SpoIIIAH-SpoIIQ interaction is also required for engulfment efficiency, functioning like a ratchet to promote membrane migration around the forespore. Here, we screened for additional factors required for engulfment using transposon sequencing in Bacillus subtilis mutants with mild engulfment defects. We discovered that YrvJ, a peptidoglycan hydrolase, and the MurA paralog MurAB, involved in peptidoglycan precursor synthesis, are required for efficient engulfment. Cytological analyses suggest that both factors are important for engulfment when the DMP complex is compromised and that MurAB is additionally required when the SpoIIIAH-SpoIIQ ratchet is abolished. Interestingly, despite the importance of MurAB for sporulation in B. subtilis, phylogenetic analyses of MurA paralogs indicate that there is no correlation between sporulation and the number of MurA paralogs and further reveal the existence of a third MurA paralog, MurAC, within the Firmicutes. Collectively, our studies identify two new factors that are required for efficient envelop remodeling during sporulation and highlight the importance of peptidoglycan precursor synthesis for efficient engulfment in B. subtilis and likely other endospore-forming bacteria. IMPORTANCE In bacteria, cell envelope remodeling is critical for cell growth and division. This is also the case during the development of bacteria into highly resistant endospores (spores), known as sporulation. During sporulation, the developing spore becomes internalized inside the mother cell through a phagocytic-like process called engulfment, which is essential to form the cell envelope of the spore. Engulfment involves both the synthesis and hydrolysis of peptidoglycan and the stabilization of migrating membranes around the developing spore. Importantly, although peptidoglycan synthesis has been implicated during engulfment, the specific genes that contribute to this molecular element of engulfment have remained unclear. Our study identifies two new factors that are required for efficient envelope remodeling during engulfment and emphasizes the importance of peptidoglycan precursor synthesis for efficient engulfment in the model organism Bacillus subtilis and likely other endospore-forming bacteria. Finally, our work highlights the power of synthetic screens to reveal additional genes that contribute to essential processes during sporulation.

Molecular Analysis of pSK1 par: A Novel Plasmid Partitioning System Encoded by Staphylococcal Multiresistance Plasmids.

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.

Chromosome Segregation and Peptidoglycan Remodeling Are Coordinated at a Highly Stabilized Septal Pore to Maintain Bacterial Spore Development.

Asymmetric division, a hallmark of endospore development, generates two cells, a larger mother cell and a smaller forespore. Approximately 75% of the forespore chromosome must be translocated across the division septum into the forespore by the DNA translocase SpoIIIE. Asymmetric division also triggers cell-specific transcription, which initiates septal peptidoglycan remodeling involving synthetic and hydrolytic enzymes. How these processes are coordinated has remained a mystery. Using Bacillus subtilis, we identified factors that revealed the link between chromosome translocation and peptidoglycan remodeling. In cells lacking these factors, the asymmetric septum retracts, resulting in forespore cytoplasmic leakage and loss of DNA translocation. Importantly, these phenotypes depend on septal peptidoglycan hydrolysis. Our data support a model in which SpoIIIE is anchored at the edge of a septal pore, stabilized by newly synthesized peptidoglycan and protein-protein interactions across the septum. Together, these factors ensure coordination between chromosome translocation and septal peptidoglycan remodeling to maintain spore development.

Spo0J and SMC are required for normal chromosome segregation in Staphylococcus aureus.

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.

Cell shape-independent FtsZ dynamics in synthetically remodeled bacterial cells.

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.

First-generation hybrid MEMS gas chromatograph.

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.