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.

An OregonGreen488-labelled d-amino acid for visualizing peptidoglycan by super-resolution STED nanoscopy.

Fluorescent d-amino acids (FDAAs) are molecular probes that are widely used for labelling the peptidoglycan layer of bacteria. When added to growing cells they are incorporated into the stem peptide by a transpeptidase reaction, allowing the timing and localization of peptidoglycan synthesis to be determined by fluorescence microscopy. Herein we describe the chemical synthesis of an OregonGreen488-labelled FDAA (OGDA). We also demonstrate that OGDA can be efficiently incorporated into the PG of Gram-positive and some Gram-negative bacteria, and imaged by super-resolution stimulated emission depletion (STED) nanoscopy at a resolution well below 100 nm.

Streptococcus mutans yidC1 and yidC2 Impact Cell Envelope Biogenesis, the Biofilm Matrix, and Biofilm Biophysical Properties.

Proper envelope biogenesis of Streptococcus mutans, a biofilm-forming and dental caries-causing oral pathogen, requires two paralogs (yidC1 and yidC2) of the universally conserved YidC/Oxa1/Alb3 family of membrane integral chaperones and insertases. The deletion of either paralog attenuates virulence in vivo, but the mechanisms of disruption remain unclear. Here, we determined whether the deletion of yidC affects cell surface properties, extracellular glucan production, and/or the structural organization of the exopolysaccharide (EPS) matrix and biophysical properties of S. mutans biofilm. Compared to the wild type, the ΔyidC2 mutant lacked staining with fluorescent vancomycin at the division septum, while the ΔyidC1 mutant resembled the wild type. Additionally, the deletion of either yidC1 or yidC2 resulted in less insoluble glucan synthesis but produced more soluble glucans, especially at early and mid-exponential-growth phases. Alteration of glucan synthesis by both mutants yielded biofilms with less dry weight and insoluble EPS. In particular, the deletion of yidC2 resulted in a significant reduction in biofilm biomass and pronounced defects in the spatial organization of the EPS matrix, thus modifying the three-dimensional (3D) biofilm architecture. The defective biofilm harbored smaller bacterial clusters with high cell density and less surrounding EPS than those of the wild type, which was stiffer in compression yet more susceptible to removal by shear. Together, our results indicate that the elimination of either yidC paralog results in changes to the cell envelope and glucan production that ultimately disrupts biofilm development and EPS matrix structure/composition, thereby altering the physical properties of the biofilms and facilitating their removal. YidC proteins, therefore, represent potential therapeutic targets for cariogenic biofilm control.IMPORTANCE YidC proteins are membrane-localized chaperone insertases that are universally conserved in all bacteria and are traditionally studied in the context of membrane protein insertion and assembly. Both YidC paralogs of the cariogenic pathogen Streptococcus mutans are required for proper envelope biogenesis and full virulence, indicating that these proteins may also contribute to optimal biofilm formation in streptococci. Here, we show that the deletion of either yidC results in changes to the structure and physical properties of the EPS matrix produced by S. mutans, ultimately impairing optimal biofilm development, diminishing its mechanical stability, and facilitating its removal. Importantly, the universal conservation of bacterial yidC orthologs, combined with our findings, provide a rationale for YidC as a possible drug target for antibiofilm therapies.

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.

Coordinated disassembly of the divisome complex in Escherichia coli.

The divisome is the macromolecular complex that carries out cell division in Escherichia coli. Every generation it must be assembled, and then disassembled so that the sequestered proteins can be recycled. Whilst the assembly process has been well studied, virtually nothing is known about the disassembly process. In this study, we have used super-resolution SIM imaging to monitor pairs of fluorescently tagged divisome proteins as they depart from the division septum. These simple binary comparisons indicated that disassembly occurs in a coordinated process that consists of at least five steps: [FtsZ, ZapA] ⇒ [ZipA, FtsA] ⇒ [FtsL, FtsQ] ⇒ [FtsI, FtsN] ⇒ [FtsN]. This sequence of events is remarkably similar to the assembly process, indicating that disassembly follows a first-in, first-out principle. A secondary observation from these binary comparisons was that FtsZ and FtsN formed division rings that were spatially separated throughout the division process. Thus the data indicate that the divisome structure can be visualized as two concentric rings; a proto-ring containing FtsZ and an FtsN-ring.

The bacterial divisome: more than a ring?

Bacterial cells are critically dependent on their ability to divide. The process of division is carried out by a large and highly dynamic molecular machine, known as the divisome. An understanding of the divisomes' architecture is highly sought after, as it is essential for understanding molecular mechanisms and potentially designing antibiotic molecules that curb bacterial growth. Our current view, which is mainly based on high-resolution imaging of Escherichia coli, is that it is a patchy ring or toroid structure. However, recent super-resolution imaging has shown that the toroid structure contains at least three concentric rings, each containing a different set of proteins. Thus, the emerging picture is that the divisome has different functional modules that are spatially separated in concentric rings.

Disassembly of the divisome in Escherichia coli: evidence that FtsZ dissociates before compartmentalization.

In most bacteria cell division is mediated by a protein super-complex called the divisome that co-ordinates the constriction and scission of the cell envelope. FtsZ is the first of the divisome proteins to accumulate at the division site and is widely thought to function as a force generator that constricts the cell envelope. In this study we have used a combination of confocal fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) to determine if divisome proteins are present at the septum at the time of cytoplasmic compartmentalization in Escherichia coli. Our data suggest that many are, but that FtsZ and ZapA disassemble before the cytoplasm is sealed by constriction of the inner membrane. This observation implies that FtsZ cannot be a force generator during the final stage(s) of envelope constriction in E. coli.

Autotransporter-based antigen display in bacterial ghosts.

Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage ϕX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered the Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.

Multispecies bacterial invasion of human host cells.

Urinary tract infection (UTI), one of the most common bacterial infections worldwide, is a typical example of an infection that is often polymicrobial in nature. While the overall infection course is known on a macroscale, bacterial behavior is not fully understood at the cellular level and bacterial pathophysiology during multispecies infection is not well characterized. Here, using clinically relevant bacteria, human epithelial bladder cells and human urine, we establish co-infection models combined with high resolution imaging to compare single- and multi-species bladder cell invasion events in three common uropathogens: uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae and Enterococcus faecalis. While all three species invaded the bladder cells, under flow conditions the Gram-positive E. faecalis was significantly less invasive compared to the Gram-negative UPEC and K. pneumoniae. When introduced simultaneously during an infection experiment, all three bacterial species sometimes invaded the same bladder cell, at differing frequencies suggesting complex interactions between bacterial species and bladder cells. Inside host cells, we observed encasement of E. faecalis colonies specifically by UPEC. During subsequent dispersal from the host cells, only the Gram-negative bacteria underwent infection-related filamentation (IRF). Taken together, our data suggest that bacterial multispecies invasions of single bladder cells are frequent and support earlier studies showing intraspecies cooperation on a biochemical level during UTI.

Patterns of host-parasite associations between marine meiofaunal flatworms (Platyhelminthes) and rhytidocystids (Apicomplexa).

Microturbellarians are abundant and ubiquitous members of marine meiofaunal communities around the world. Because of their small body size, these microscopic animals are rarely considered as hosts for parasitic organisms. Indeed, many protists, both free-living and parasitic ones, equal or surpass meiofaunal animals in size. Despite several anecdotal records of "gregarines", "sporozoans", and "apicomplexans" parasitizing microturbellarians in the literature-some of them dating back to the nineteenth century-these single-celled parasites have never been identified and characterized. More recently, the sequencing of eukaryotic microbiomes in microscopic invertebrates have revealed a hidden diversity of protist parasites infecting microturbellarians and other meiofaunal animals. Here we show that apicomplexans isolated from twelve taxonomically diverse rhabdocoel taxa and one species of proseriate collected in four geographically distinct areas around the Pacific Ocean (Okinawa, Hokkaido, and British Columbia) and the Caribbean Sea (Curaçao) all belong to the apicomplexan genus Rhytidocystis. Based on comprehensive molecular phylogenies of Rhabdocoela and Proseriata inferred from both 18S and 28S rDNA sequences, as well as a molecular phylogeny of Marosporida inferred from 18S rDNA sequences, we determine the phylogenetic positions of the microturbellarian hosts and their parasites. Multiple lines of evidence, including morphological and molecular data, show that at least nine new species of Rhytidocystis infect the microturbellarian hosts collected in this study, more than doubling the number of previously recognized species of Rhytidocystis, all of which infect polychaete hosts. A cophylogenetic analysis examining patterns of phylosymbiosis between hosts and parasites suggests a complex picture of overall incongruence between host and parasite phylogenies, and varying degrees of geographic signals and taxon specificity.

Sequential closure of the cytoplasm and then the periplasm during cell division in Escherichia coli.

To visualize the latter stages of cell division in live Escherichia coli, we have carried out fluorescence recovery after photobleaching (FRAP) on 121 cells expressing cytoplasmic green fluorescent protein and periplasmic mCherry. Our data show conclusively that the cytoplasm is sealed prior to the periplasm during the division event.

Assembly dynamics of FtsZ and DamX during infection-related filamentation and division in uropathogenic E. coli.

During infection of bladder epithelial cells, uropathogenic Escherichia coli (UPEC) can stop dividing and grow into highly filamentous forms. Here, we find that some filaments of E. coli UTI89 released from infected cells grow very rapidly and by more than 100 µm before initiating division, whereas others do not survive, suggesting that infection-related filamentation (IRF) is a stress response that promotes bacterial dispersal. IRF is accompanied by unstable, dynamic repositioning of FtsZ division rings. In contrast, DamX, which is associated with normal cell division and is also essential for IRF, is distributed uniformly around the cell envelope during filamentation. When filaments initiate division to regenerate rod cells, DamX condenses into stable rings prior to division. The DamX rings maintain consistent thickness during constriction and remain at the septum until after membrane fusion. Deletion of damX affects vegetative cell division in UTI89 (but not in the model E. coli K-12), and, during infection, blocks filamentation and reduces bacterial cell integrity. IRF therefore involves DamX distribution throughout the membrane and prevention of FtsZ ring stabilization, leading to cell division arrest. DamX then reassembles into stable division rings for filament division, promoting dispersal and survival during infection.

Signal amplification of araC pBAD using a standardized translation initiation region.

araC pBAD is a genetic fragment that regulates the expression of the araBAD operon in bacteria, which is required for the metabolism of L-arabinose. It is widely used in bioengineering applications because it can drive regulatable and titratable expression of genes and genetic pathways in microbial cell factories. A notable limitation of araC pBAD is that it generates a low signal when induced with high concentrations of L-arabinose (the maximum ON state). Herein we have amplified the maximum ON state of araC pBAD by coupling it to a synthetically evolved translation initiation region (TIREVOL ). The coupling maintains regulatable and titratable expression from araC pBAD and yet increases the maximal ON state by >5-fold. The general principle demonstrated in the study can be applied to amplify the signal from similar genetic modules. Graphical Abstract.

Rapid separation of bacteria from primary nasal samples using inertial microfluidics.

Microbial populations play a crucial role in human health and the development of many diseases. These diseases often arise from the explosive proliferation of opportunistic bacteria, such as those in the nasal cavity. Recently, there have been increases in the prevalence of these opportunistic pathogens displaying antibiotic resistance. Thus, the study of the nasal microbiota and its bacterial diversity is critical in understanding pathogenesis and developing microbial-based therapies for well-known and emerging diseases. However, the isolation and analysis of these populations for clinical study complicates the already challenging task of identifying and profiling potentially harmful bacteria. Existing methods are limited by low sample throughput, expensive labeling, and low recovery of bacteria with ineffective removal of cells and debris. In this study, we propose a novel microfluidic channel with a zigzag configuration for enhanced isolation and detection of bacteria from human clinical nasal swabs. This microfluidic zigzag channel separates the bacteria from epithelial cells and debris by size differential focusing. As such, pure bacterial cell fractions devoid of large contaminating debris or epithelial cells are obtained. DNA sequencing performed on the separated bacteria defines the diversity and species present. This novel method of bacterial separation is simple, robust, rapid, and cost-effective and has the potential to be used for the rapid identification of bacterial cell populations from clinical samples.

Treadmilling FtsZ polymers drive the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism.

The FtsZ protein is a central component of the bacterial cell division machinery. It polymerizes at mid-cell and recruits more than 30 proteins to assemble into a macromolecular complex to direct cell wall constriction. FtsZ polymers exhibit treadmilling dynamics, driving the processive movement of enzymes that synthesize septal peptidoglycan (sPG). Here, we combine theoretical modelling with single-molecule imaging of live bacterial cells to show that FtsZ's treadmilling drives the directional movement of sPG enzymes via a Brownian ratchet mechanism. The processivity of the directional movement depends on the binding potential between FtsZ and the sPG enzyme, and on a balance between the enzyme's diffusion and FtsZ's treadmilling speed. We propose that this interplay may provide a mechanism to control the spatiotemporal distribution of active sPG enzymes, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacteria.

Nanoscale dynamics of peptidoglycan assembly during the cell cycle of Streptococcus pneumoniae.

Dynamics of cell elongation and septation are key determinants of bacterial morphogenesis. These processes are intimately linked to peptidoglycan synthesis performed by macromolecular complexes called the elongasome and the divisome. In rod-shaped bacteria, cell elongation and septation, which are dissociated in time and space, have been well described. By contrast, in ovoid-shaped bacteria, the dynamics and relationships between these processes remain poorly understood because they are concomitant and confined to a nanometer-scale annular region at midcell. Here, we set up a metabolic peptidoglycan labeling approach using click chemistry to image peptidoglycan synthesis by single-molecule localization microscopy in the ovoid bacterium Streptococcus pneumoniae. Our nanoscale-resolution data reveal spatiotemporal features of peptidoglycan assembly and fate along the cell cycle and provide geometrical parameters that we used to construct a morphogenesis model of the ovoid cell. These analyses show that septal and peripheral peptidoglycan syntheses first occur within a single annular region that later separates in two concentric regions and that elongation persists after septation is completed. In addition, our data reveal that freshly synthesized peptidoglycan is remodeled all along the cell cycle. Altogether, our work provides evidence that septal peptidoglycan is synthesized from the beginning of the cell cycle and is constantly remodeled through cleavage and insertion of material at its periphery. The ovoid-cell morphogenesis would thus rely on the relative dynamics between peptidoglycan synthesis and cleavage rather than on the existence of two distinct successive phases of peripheral and septal synthesis.

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.

Detecting Gold Biomineralization by Delftia acidovorans Biofilms on a Quartz Crystal Microbalance.

The extensive use of gold in sensing, diagnostics, and electronics has led to major concerns in solid waste management since gold and other heavy metals are nonbiodegradable and can easily accumulate in the environment. Moreover, gold ions are extremely reactive and potentially harmful for humans. Thus, there is an urgent need to develop reliable methodologies to detect and possibly neutralize ionic gold in aqueous solutions and industrial wastes. In this work, by using complementary measurement techniques such as quartz crystal microbalance (QCM), atomic force microscopy, crystal violet staining, and optical microscopy, we investigate a promising biologically induced gold biomineralization process accomplished by biofilms of bacterium Delftia acidovorans. When stressed by Au3+ ions, D. acidovorans is able to neutralize toxic soluble gold by excreting a nonribosomal peptide, which forms extracellular gold nanonuggets via complexation with metal ions. Specifically, QCM, a surface-sensitive transducer, is employed to quantify the production of these gold complexes directly on the D. acidovorans biofilm in real time. Detailed kinetics obtained by QCM captures the condition for maximized biomineralization yield and offers new insights underlying the biomineralization process. To the best of our knowledge, this is the first study providing an extensive characterization of the gold biomineralization process by a model bacterial biofilm. We also demonstrate QCM as a cheap, user-friendly sensing platform and alternative to standard analytical techniques for studies requiring high-resolution quantitative details, which offers promising opportunities in heavy-metal sensing, gold recovery, and industrial waste treatment.