Genetic analysis of the septal peptidoglycan synthase FtsWI complex supports a conserved activation mechanism for SEDS-bPBP complexes.

SEDS family peptidoglycan (PG) glycosyltransferases, RodA and FtsW, require their cognate transpeptidases PBP2 and FtsI (class B penicillin binding proteins) to synthesize PG along the cell cylinder and at the septum, respectively. The activities of these SEDS-bPBPs complexes are tightly regulated to ensure proper cell elongation and division. In Escherichia coli FtsN switches FtsA and FtsQLB to the active forms that synergize to stimulate FtsWI, but the exact mechanism is not well understood. Previously, we isolated an activation mutation in ftsW (M269I) that allows cell division with reduced FtsN function. To try to understand the basis for activation we isolated additional substitutions at this position and found that only the original substitution produced an active mutant whereas drastic changes resulted in an inactive mutant. In another approach we isolated suppressors of an inactive FtsL mutant and obtained FtsWE289G and FtsIK211I and found they bypassed FtsN. Epistatic analysis of these mutations and others confirmed that the FtsN-triggered activation signal goes from FtsQLB to FtsI to FtsW. Mapping these mutations, as well as others affecting the activity of FtsWI, on the RodA-PBP2 structure revealed they are located at the interaction interface between the extracellular loop 4 (ECL4) of FtsW and the pedestal domain of FtsI (PBP3). This supports a model in which the interaction between the ECL4 of SEDS proteins and the pedestal domain of their cognate bPBPs plays a critical role in the activation mechanism.

The architecture of the Gram-positive bacterial cell wall.

The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics1,2. Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor1,3. In Gram-positive bacteria, peptidoglycan is tens of nanometres thick, generally portrayed as a homogeneous structure that provides mechanical strength4-6. Here we applied atomic force microscopy7-12 to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface13,14, providing information complementary to traditional structural biology approaches.

Higher order assembling of the mycobacterial polar growth factor DivIVA/Wag31.

DivIVA or Wag31, which is an essential pole organizing protein in mycobacteria, can self-assemble at the negatively curved side of the membrane at the growing pole to form a higher order structural scaffold for maintaining cellular morphology and localizing various target proteins for cell-wall biogenesis. The structural organization of polar scaffold formed by polymerization of coiled-coil rich Wag31, which is implicated in the anti-tubercular activities of amino-pyrimidine sulfonamides, remains to be determined. A single-site phosphorylation in Wag31 regulates peptidoglycan biosynthesis in mycobacteria. We report biophysical characterizations of filaments formed by mycobacterial Wag31 using circular dichroism, atomic force microscopy and small angle solution X-ray scattering. Atomic force microscopic images of the wild-type, a phospho-mimetic (T73E) and a phospho-ablative (T73A) form of Wag31 show mostly linear filament formation with occasional curving, kinking and apparent branching. Solution X-ray scattering data indicates that the phospho-mimetic forms of the Wag31 polymers are on average more compact than their phospho-ablative counterparts, which is likely due to the extent of bending/branching. Observed structural features in this first view of Wag31 filaments suggest a basis for higher order Wag31 scaffold formation at the pole.

Peptidoglycan layer and disruption processes in Bacillus subtilis cells visualized using quick-freeze, deep-etch electron microscopy.

Peptidoglycan, which is the main component of the bacterial cell wall, is a heterogeneous polymer of glycan strands cross-linked with short peptides and is synthesized in cooperation with the cell division cycle. Although it plays a critical role in bacterial survival, its architecture is not well understood. Herein, we visualized the architecture of the peptidoglycan surface in Bacillus subtilis at the nanometer resolution, using quick-freeze, deep-etch electron microscopy (EM). Filamentous structures were observed on the entire surface of the cell, where filaments about 11 nm wide formed concentric circles on cell poles, filaments about 13 nm wide formed a circumferential mesh-like structure on the cylindrical part and a 'piecrust' structure was observed at the boundary. When growing cells were treated with lysozyme, the entire cell mass migrated to one side and came out from the cell envelope. Fluorescence labeling showed that lysozyme preferentially bound to a cell pole and cell division site, where the peptidoglycan synthesis was not complete. Ruffling of surface structures was observed during EM. When cells were treated with penicillin, the cell mass came out from a cleft around the cell division site. Outward curvature of the protoplast at the cleft seen using EM suggested that turgor pressure was applied as the peptidoglycan was not damaged at other positions. When muropeptides were depleted, surface filaments were lost while the rod shape of the cell was maintained. These changes can be explained on the basis of the working points of the chemical structure of peptidoglycan.

Analysis of an improved Cyanophora paradoxa genome assembly.

Glaucophyta are members of the Archaeplastida, the founding group of photosynthetic eukaryotes that also includes red algae (Rhodophyta), green algae, and plants (Viridiplantae). Here we present a high-quality assembly, built using long-read sequences, of the ca. 100 Mb nuclear genome of the model glaucophyte Cyanophora paradoxa. We also conducted a quick-freeze deep-etch electron microscopy (QFDEEM) analysis of C. paradoxa cells to investigate glaucophyte morphology in comparison to other organisms. Using the genome data, we generated a resolved 115-taxon eukaryotic tree of life that includes a well-supported, monophyletic Archaeplastida. Analysis of muroplast peptidoglycan (PG) ultrastructure using QFDEEM shows that PG is most dense at the cleavage-furrow. Analysis of the chlamydial contribution to glaucophytes and other Archaeplastida shows that these foreign sequences likely played a key role in anaerobic glycolysis in primordial algae to alleviate ATP starvation under night-time hypoxia. The robust genome assembly of C. paradoxa significantly advances knowledge about this model species and provides a reference for exploring the panoply of traits associated with the anciently diverged glaucophyte lineage.

Illuminating vital surface molecules of symbionts in health and disease.

The immunomodulatory surface molecules of commensal and pathogenic bacteria are critical to microorganisms' survival and the host's response1,2. Recent studies have highlighted the unique and important responses elicited by commensal-derived surface macromolecules3-5. However, the technology available to track these molecules in host cells and tissues remains primitive. We report, here, an interdisciplinary approach that uses metabolic labelling combined with bioorthogonal click chemistry (that is, reactions performed in living organisms)6 to specifically tag up to three prominent surface immunomodulatory macromolecules-peptidoglycan, lipopolysaccharide and capsular polysaccharide-either simultaneously or individually in live anaerobic commensal bacteria. Importantly, the peptidoglycan labelling enables, for the first time, the specific labelling of live endogenous, anaerobic bacteria within the mammalian host. This approach has allowed us to image and track the path of labelled surface molecules from live, luminal bacteria into specific intestinal immune cells in the living murine host during health and disease. The chemical labelling of three specific macromolecules within a live organism offers the potential for in-depth visualization of host-pathogen interactions.

Nanoscale-length control of the flagellar driveshaft requires hitting the tethered outer membrane.

The bacterial flagellum exemplifies a system where even small deviations from the highly regulated flagellar assembly process can abolish motility and cause negative physiological outcomes. Consequently, bacteria have evolved elegant and robust regulatory mechanisms to ensure that flagellar morphogenesis follows a defined path, with each component self-assembling to predetermined dimensions. The flagellar rod acts as a driveshaft to transmit torque from the cytoplasmic rotor to the external filament. The rod self-assembles to a defined length of ~25 nanometers. Here, we provide evidence that rod length is limited by the width of the periplasmic space between the inner and outer membranes. The length of Braun's lipoprotein determines periplasmic width by tethering the outer membrane to the peptidoglycan layer.

Ultrastructural analysis of bacteriophage Φ29 during infection of Bacillus subtilis.

Recent advances in cryo-electron tomography (cryo-ET) have allowed direct visualization of the initial interactions between bacteriophages and their hosts. Previous studies focused on phage infection in Gram-negative bacteria but it is of particular interest how phages penetrate the thick, highly cross-linked Gram-positive cell wall. Here we detail structural intermediates of phage Φ29 during infection of Bacillus subtilis. Use of a minicell-producing strain facilitated in situ tomographic reconstructions of infecting phage particles. Φ29 initially contacts the cell wall at an angle through a subset of the twelve appendages, which are attached to the collar at the head proximal portion of the tail knob. The appendages are flexible and switch between extended and downward conformations during this stage of reversible adsorption; appendages enzymatically hydrolyze wall teichoic acids to bring the phage closer to the cell. A cell wall-degrading enzyme at the distal tip of the tail knob locally digests peptidoglycan, facilitating penetration of the tail further into the cell wall, and the phage particle reorients so that the tail becomes perpendicular to the cell surface. All twelve appendages attain the same "down" conformation during this stage of adsorption. Once the tail has become totally embedded in the cell wall, the tip can fuse with the cytoplasmic membrane. The membrane bulges out, presumably to facilitate genome ejection into the cytoplasm, and the deformation remains after complete ejection. This study provides the first visualization of the structural changes occurring in a phage particle during adsorption and genome transfer into a Gram-positive bacterium.

The bacterial tubulin FtsZ requires its intrinsically disordered linker to direct robust cell wall construction.

The bacterial GTPase FtsZ forms a cytokinetic ring at midcell, recruits the division machinery and orchestrates membrane and peptidoglycan cell wall invagination. However, the mechanism for FtsZ regulation of peptidoglycan metabolism is unknown. The FtsZ GTPase domain is separated from its membrane-anchoring C-terminal conserved (CTC) peptide by a disordered C-terminal linker (CTL). Here we investigate CTL function in Caulobacter crescentus. Strikingly, production of FtsZ lacking the CTL (ΔCTL) is lethal: cells become filamentous, form envelope bulges and lyse, resembling treatment with ß-lactam antibiotics. This phenotype is produced by FtsZ polymers bearing the CTC and a CTL shorter than 14 residues. Peptidoglycan synthesis still occurs downstream of ΔCTL; however, cells expressing ΔCTL exhibit reduced peptidoglycan crosslinking and longer glycan strands than wild type. Importantly, midcell proteins are still recruited to sites of ΔCTL assembly. We propose that FtsZ regulates peptidoglycan metabolism through a CTL-dependent mechanism that extends beyond simple protein recruitment.

Association between the Brazilian Breastfeeding Network implementation and breastfeeding indicators / Associação entre a implantação da Rede Amamenta Brasil e indicadores de aleitamento materno

OBJECTIVE: To estimate the association between the implementation of the Brazilian Breastfeeding Network and prevalence of breastfeeding in a medium-size city in southern Brazil. METHODS: This was a cross-sectional study involving 405 children under 1 year who participated in the second phase of the multivaccination campaign in 2012. Children's consumption of food on the day before the interview was obtained through interviews with mothers or guardians. The manager and one health professional from every health facility that joined the Network were interviewed in order to investigate the process of implementation of this initiative. The association between prevalence of breastfeeding and exclusive breastfeeding and adherence to the Network implementation process was tested using Poisson regression with robust variance. RESULTS: Multivariate analysis revealed that among the children assisted by health facilities who joined the Network and those attending services that did not adhere to this strategy, the prevalence of breastfeeding (74% and 70.4% among children under 1 year, respectively) and exclusive breastfeeding (43.3% and 38.1% among children under 6 months, respectively) did not differ significantly. Difficulties in implementing the Network, such as high turnover of professionals, not meeting the criteria for accreditation, and insufficient participation of tutors in the process were identified. CONCLUSION: Contrary to the hypothesis of this study, there was no significant association between the implementation of the Brazilian Breastfeeding Network and prevalence of breastfeeding and exclusive breastfeeding in the studied city. It is possible that the difficulties found in implementing the Network in this city have influenced this result. .

OBJETIVO: Estimar a associação entre a implementação da Rede Amamenta Brasil e as prevalências de aleitamento materno (AM) em um município de médio porte do sul do Brasil. MÉTODOS: Estudo transversal que envolveu 405 crianças menores de um ano que participaram da segunda fase da campanha de multivacinação de 2012. O consumo de alimentos pela criança no dia anterior à entrevista foi obtido mediante entrevistas com as mães ou os responsáveis. Para investigar o processo de implementação da rede foram entrevistados o gerente e um profissional de saúde de cada unidade que aderiu a esse processo. A associação entre as prevalências de AM e AM exclusivo e a adesão ao processo de implementação da rede foi testada com a regressão de Poisson com variância robusta. RESULTADOS: A análise multivariada revelou que entre as crianças assistidas por unidades que aderiram ao processo de implementação da rede e as que frequentam serviços que não aderiram a essa estratégia as prevalências de AM (74% e 70,4% em menores de um ano, respectivamente) e AME (43,3% e 38,1% em menores de seis meses, respectivamente) não diferiram significativamente. Foram identificadas dificuldades na implementação da rede, tais como alta rotatividade dos profissionais, não cumprimento dos critérios para certificação e acompanhamento insuficiente das unidades pelos tutores da rede. CONCLUSÃO: Contrariando a nossa hipótese, não houve associação significativa entre a implementação da Rede Amamenta Brasil e as prevalências de AM e AME no município estudado. É possível que as dificuldades encontradas na implementação da rede nesse município tenham influenciado esse resultado. .

Imaging bacterial peptidoglycan with near-infrared fluorogenic azide probes.

Fluorescent probes designed for activation by bioorthogonal chemistry have enabled the visualization of biomolecules in living systems. Such activatable probes with near-infrared (NIR) emission would be ideal for in vivo imaging but have proven difficult to engineer. We present the development of NIR fluorogenic azide probes based on the Si-rhodamine scaffold that undergo a fluorescence enhancement of up to 48-fold upon reaction with terminal or strained alkynes. We used the probes for mammalian cell surface imaging and, in conjunction with a new class of cyclooctyne D-amino acids, for visualization of bacterial peptidoglycan without the need to wash away unreacted probe.

Different walls for rods and balls: the diversity of peptidoglycan.

Peptidoglycan performs the essential role of resisting turgor in the cell walls of most bacteria. It determines cell shape, and its biosynthesis is the target for many important antibiotics. The fundamental chemical building blocks of peptidoglycan are conserved: repeating disaccharides cross-linked by peptides. However, these blocks come in many varieties and can be assembled in different ways. So beyond the fundamental similarity, prodigious chemical, organizational and architectural diversity is revealed. Here, we track the evolution of our current understanding of peptidoglycan and underpinning technical and methodological developments. The origin and function of chemical diversity is discussed with respect to some well-studied example species. We then explore how this chemistry is manifested in elegant and complex peptidoglycan organization and how this is interpreted in different and sometimes controversial architectural models. We contend that emerging technology brings about the possibility of achieving a complete understanding of peptidoglycan chemistry, through architecture, to the way in which diverse species and populations of cells meet the challenges of maintaining viability and growth within their environmental niches, by exploiting the bioengineering versatility of peptidoglycan.

Architecture and assembly of the Gram-positive cell wall.

The bacterial cell wall is a mesh polymer of peptidoglycan--linear glycan strands cross-linked by flexible peptides--that determines cell shape and provides physical protection. While the glycan strands in thin 'Gram-negative' peptidoglycan are known to run circumferentially around the cell, the architecture of the thicker 'Gram-positive' form remains unclear. Using electron cryotomography, here we show that Bacillus subtilis peptidoglycan is a uniformly dense layer with a textured surface. We further show it rips circumferentially, curls and thickens at free edges, and extends longitudinally when denatured. Molecular dynamics simulations show that only atomic models based on the circumferential topology recapitulate the observed curling and thickening, in support of an 'inside-to-outside' assembly process. We conclude that instead of being perpendicular to the cell surface or wrapped in coiled cables (two alternative models), the glycan strands in Gram-positive cell walls run circumferentially around the cell just as they do in Gram-negative cells. Together with providing insights into the architecture of the ultimate determinant of cell shape, this study is important because Gram-positive peptidoglycan is an antibiotic target crucial to the viability of several important rod-shaped pathogens including Bacillus anthracis, Listeria monocytogenes, and Clostridium difficile.

Visualizing the production and arrangement of peptidoglycan in Gram-positive cells.

Decades of study have revealed the fine chemical structure of the bacterial peptidoglycan cell wall, but the arrangement of the peptidoglycan strands within the wall has been challenging to define. The application of electron cryotomography (ECT) and new methods for fluorescent labelling of peptidoglycan are allowing new insights into wall structure and synthesis. Two articles in this issue examine peptidoglycan structures in the model Gram-positive species Bacillus subtilis. Beeby et al. combined visualization of peptidoglycan using ECT with molecular modelling of three proposed arrangements of peptidoglycan strands to identify the model most consistent with their data. They argue convincingly for a Gram-positive wall containing multiple layers of peptidoglycan strands arranged circumferentially around the long axis of the rod-shaped cell, an arrangement similar to the single layer of peptidoglycan in similarly shaped Gram-negative cells. Tocheva et al. examined sporulating cells using ECT and fluorescence microscopy to demonstrate the continuous production of a thin layer of peptidoglycan around the developing spore as it is engulfed by the membrane of the adjacent mother cell. The presence of this peptidoglycan in the intermembrane space allows the refinement of a model for engulfment, which has been known to include peptidoglycan synthetic and lytic functions.

Peptidoglycan transformations during Bacillus subtilis sporulation.

While vegetative Bacillus subtilis cells and mature spores are both surrounded by a thick layer of peptidoglycan (PG, a polymer of glycan strands cross-linked by peptide bridges), it has remained unclear whether PG surrounds prespores during engulfment. To clarify this issue, we generated a slender ΔponA mutant that enabled high-resolution electron cryotomographic imaging. Three-dimensional reconstructions of whole cells in near-native states revealed a thin PG-like layer extending from the lateral cell wall around the prespore throughout engulfment. Cryotomography of purified sacculi and fluorescent labelling of PG in live cells confirmed that PG surrounds the prespore. The presence of PG throughout engulfment suggests new roles for PG in sporulation, including a new model for how PG synthesis might drive engulfment, and obviates the need to synthesize a PG layer de novo during cortex formation. In addition, it reveals that B. subtilis can synthesize thin, Gram-negative-like PG layers as well as its thick, archetypal Gram-positive cell wall. The continuous transformations from thick to thin and back to thick during sporulation suggest that both forms of PG have the same basic architecture (circumferential). Endopeptidase activity may be the main switch that governs whether a thin or a thick PG layer is assembled.

Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria.

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms.

Outer membrane continuity and septosome formation between vegetative cells in the filaments of Anabaena sp. PCC 7120.

Anabaena sp. PCC 7120 is a prototype filamentous nitrogen-fixing cyanobacterium, in which nitrogen fixation and photosynthesis are spatially separated. Recent molecular and cellular studies have established the importance of molecular exchange between cells in the filament, but the routes involved are still under investigation. Two current models propose either a continuous periplasm or direct connections between adjacent cells whose integrity requires the protein SepJ. We used electron tomography to analyze the ultrastructure of the septum between vegetative cells in the Anabaena filament and were able to visualize intercellular connections that we term 'SEPTOSOMES'. We observed that, whereas the existence of the septosome does not depend on the presence of SepJ, the spacing between the two plasma membranes of the septum was significantly decreased in a ΔsepJ mutant. In addition, we observed that the peptidoglycan layer of each cell enters the septum but the outer membrane does not. Thus, each cell in the filament is individually surrounded by a plasma membrane and a peptidoglycan layer, and physical cell-cell contacts are mediated by the septosome.

The caulobacter Tol-Pal complex is essential for outer membrane integrity and the positioning of a polar localization factor.

Cell division in Caulobacter crescentus involves constriction and fission of the inner membrane (IM) followed about 20 min later by fission of the outer membrane (OM) and daughter cell separation. In contrast to Escherichia coli, the Caulobacter Tol-Pal complex is essential. Cryo-electron microscopy images of the Caulobacter cell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. In wild-type cells, components of the Tol-Pal complex localize to the division plane in early predivisional cells and remain predominantly at the new pole of swarmer and stalked progeny upon completion of division. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane-associated polar proteins tested. Coimmunoprecipitation experiments show that both TolA and Pal interact directly or indirectly with TipN. We propose that disruption of the trans-envelope Tol-Pal complex releases TipN from its subcellular position. The Caulobacter Tol-Pal complex is thus a key component of cell envelope structure and function, mediating OM constriction at the final step of cell division as well as the positioning of a protein localization factor.

Comparison of the envelope architecture of E. coli using two methods: CEMOVIS and cryo-electron tomography.

Cryo-electron microscopy of vitreous sections (CEMOVIS) and cryo-electron tomography (cryo-ET) of vitrified specimens are gradually gaining popularity. However, similar to the conventional methods, these techniques tend to produce different images of the same sample. In CEMOVIS, the mechanical stress caused by sectioning may cause inaccuracies smaller than those caused by crevasses. Therefore, we examined Escherichia coli cells by using CEMOVIS and cryo-ET to determine the differences in the computed sizes of the envelope layers, which are smaller than crevasses. We found that the width of the periplasmic space in vitreous sections and tomograms was 12 and 14 nm, respectively; furthermore, while the distance between the outer membrane (OM) and the peptidoglycan (PG) layer was almost equal (11 nm) in the two techniques, that between the plasma membrane (PM) and PG was clearly different. Thus, the observed size difference can be mainly attributed to the PM-PG distance. Since our data were obtained from images acquired using the same microscope in the same conditions, the size differences cannot be attributed to microscope-related factors. One possible factor is the angle of the cutting plane against the long axis of the cell body in CEMOVIS. However, the same PG-OM distance in both methods may exclude the variations caused by this factor. Furthermore, the mechanical stress caused by vitreous sectioning or high-pressure freezing may result in shrinkage. If this shrinkage is responsible for the nanometre-scale deformation in CEMOVIS, this factor will have to be considered in determining the molecular resolution obtained by CEMOVIS.

Curvature of synthetic and natural surfaces is an important target feature in classical pathway complement activation.

The binding of Abs to microbial surfaces followed by complement activation constitutes an important line of defense against infections. In this study, we have investigated the relationship between complement activation and the binding of human IgM Abs to surfaces with different curvatures. IgM Abs to dextran were shown to activate complement potently on dextran-coated particles having a diameter around 250 nm, whereas larger (600 nm) particles were less potent activators. This selectivity regarding particle dimension was also found for complement activation by colloidal substances of microbial origin. Peptidoglycan (PGN) is the major chemical component in the cell wall of Gram-positive bacteria. Fragments of purified PGN with sizes of approximately 100 nm promoted complement activation effectively through the classical pathway. By contrast, larger or smaller fragments of PGN did not activate complement strongly. A careful analysis of PGN fragments released during planctonic growth of Staphylococcus aureus showed that these include curvatures that would permit strong IgM-mediated complement activation, whereas the curvature of intact cells would be less effective for such activation. Consistently, we found that the suspended PGN fragments were strong activators of complement through the classical pathway. We suggest that these fragments act as decoy targets for complement activation, providing protection for S. aureus against the host immune response to infection.