Defining Membrane Protein Localization by Isopycnic Density Gradients.

Bacterial membrane proteins account for around one-third of the proteome in many species and can represent much more than half of the mass of the membranes. Classic techniques in cell biology can be applied to characterize bacterial membranes and their membrane protein constituents, and here we describe a protocol for the purification of outer membranes and inner membranes from Escherichia coli. This allows for compositional analysis of the membranes as well as functional analyses.

Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined.

Three-Dimensional Micropatterning Deters Early Bacterial Adherence and Can Eliminate Colonization.

Developing strategies to prevent bacterial infections that do not rely on the use of drugs is regarded globally as an important means to stem the tide of antimicrobial resistance, as argued by the World Health Organization (WHO) (Mendelson, M.; Matsoso, M. P.

The multifunctional enzyme S-adenosylhomocysteine/methylthioadenosine nucleosidase is a key metabolic enzyme in the virulence of Salmonella enterica var Typhimurium.

Key physiological differences between bacterial and mammalian metabolism provide opportunities for the development of novel antimicrobials. We examined the role of the multifunctional enzyme S-adenosylhomocysteine/Methylthioadenosine (SAH/MTA) nucleosidase (Pfs) in the virulence of S. enterica var Typhimurium (S.

Filamentous phages: masters of a microbial sharing economy.

Bacteriophage ("bacteria eaters") or phage is the collective term for viruses that infect bacteria. While most phages are pathogens that kill their bacterial hosts, the filamentous phages of the sub-class Inoviridae live in cooperative relationships with their bacterial hosts, akin to the principal behaviours found in the modern-day sharing economy: peer-to-peer support, to offset any burden. Filamentous phages impose very little burden on bacteria and offset this by providing service to help build better biofilms, or provision of toxins and other factors that increase virulence, or modified behaviours that provide novel motile activity to their bacterial hosts.

FusC, a member of the M16 protease family acquired by bacteria for iron piracy against plants.

Iron is essential for life. Accessing iron from the environment can be a limiting factor that determines success in a given environmental niche. For bacteria, access of chelated iron from the environment is often mediated by TonB-dependent transporters (TBDTs), which are β-barrel proteins that form sophisticated channels in the outer membrane.

An investigation into the Omp85 protein BamK in hypervirulent Klebsiella pneumoniae, and its role in outer membrane biogenesis.

Members of the Omp85 protein superfamily have important roles in Gram-negative bacteria, with the archetypal protein BamA being ubiquitous given its essential function in the assembly of outer membrane proteins. In some bacterial lineages, additional members of the family exist and, in most of these cases, the function of the protein is unknown. We detected one of these Omp85 proteins in the pathogen Klebsiella pneumoniae B5055, and refer to the protein as BamK.

The WD40 Protein BamB Mediates Coupling of BAM Complexes into Assembly Precincts in the Bacterial Outer Membrane.

The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E.

Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections.

Structure and Membrane Topography of the Vibrio-Type Secretin Complex from the Type 2 Secretion System of Enteropathogenic Escherichia coli.

The β-barrel assembly machinery (BAM) complex is the core machinery for the assembly of β-barrel membrane proteins, and inhibition of BAM complex activity is lethal to bacteria. Discovery of integral membrane proteins that are key to pathogenesis and yet do not require assistance from the BAM complex raises the question of how these proteins assemble into bacterial outer membranes. Here, we address this question through a structural analysis of the type 2 secretion system (T2SS) secretin from enteropathogenic O127:H6 strain E2348/69.

Structural Basis of Type 2 Secretion System Engagement between the Inner and Outer Bacterial Membranes.

Sophisticated nanomachines are used by bacteria for protein secretion. In Gram-negative bacteria, the type 2 secretion system (T2SS) is composed of a pseudopilus assembly platform in the inner membrane and a secretin complex in the outer membrane. The engagement of these two megadalton-sized complexes is required in order to secrete toxins, effectors, and hydrolytic enzymes.

Defining Membrane Protein Localization by Isopycnic Density Gradients.

In many bacteria, membrane proteins account for around one-third of the proteome and can represent much more than half of the mass of a membrane. Classic techniques in cell biology can be applied to characterise bacterial membranes and their membrane protein constituents. Here we describe a protocol for the purification of outer and inner membranes from Escherichia coli.

Super-Resolution Imaging of Protein Secretion Systems and the Cell Surface of Gram-Negative Bacteria.

Gram-negative bacteria have a highly evolved cell wall with two membranes composed of complex arrays of integral and peripheral proteins, as well as phospholipids and glycolipids. In order to sense changes in, respond to, and exploit their environmental niches, bacteria rely on structures assembled into or onto the outer membrane. Protein secretion across the cell wall is a key process in virulence and other fundamental aspects of bacterial cell biology.

Effective assembly of fimbriae in Escherichia coli depends on the translocation assembly module nanomachine.

Outer membrane proteins are essential for Gram-negative bacteria to rapidly adapt to changes in their environment. Intricate remodelling of the outer membrane proteome is critical for bacterial pathogens to survive environmental changes, such as entry into host tissues(1-3). Fimbriae (also known as pili) are appendages that extend up to 2 μm beyond the cell surface to function in adhesion for bacterial pathogens, and are critical for virulence.

Antibiotic regimen based on population analysis of residing persister cells eradicates Staphylococcus epidermidis biofilms.

Biofilm formation is a major pathogenicity strategy of Staphylococcus epidermidis causing various medical-device infections. Persister cells have been implicated in treatment failure of such infections. We sought to profile bacterial subpopulations residing in S.

In vivo polyester immobilized sortase for tagless protein purification.

Background: Laboratory scale recombinant protein production and purification techniques are often complicated, involving multiple chromatography steps and specialized equipment and reagents. Here it was demonstrated that recombinant proteins can be expressed as covalently immobilized to the surface of polyester (polyhydroxyalkanoate, PHA) beads in vivo in Escherichia coli by genetically fusing them to a polyester synthase gene (phaC). The insertion of a self-cleaving module, a modified sortase A (SrtA) from Staphylococcus aureus and its five amino acid recognition sequence between the synthase and the target protein led to a simple protein production and purification method.

Searching for new strategies against polymicrobial biofilm infections: guanylated polymethacrylates kill mixed fungal/bacterial biofilms.

Objectives: Biofilm-related human infections have high mortality rates due to drug resistance. Cohabitation of diverse microbes in polymicrobial biofilms is common and these infections present additional challenges for treatment compared with monomicrobial biofilms. Here, we address this therapeutic gap by assessing the potential of a new class of antimicrobial agents, guanylated polymethacrylates, in the treatment of polymicrobial biofilms built by two prominent human pathogens, the fungus Candida albicans and the bacterium Staphylococcus aureus.

Assembly of the secretion pores GspD, Wza and CsgG into bacterial outer membranes does not require the Omp85 proteins BamA or TamA.

In Gram-negative bacteria, β-barrel proteins are integrated into the outer membrane by the β-barrel assembly machinery, with key components of the machinery being the Omp85 family members BamA and TamA. Recent crystal structures and cryo-electron microscopy show a diverse set of secretion pores in Gram-negative bacteria, with α-helix (Wza and GspD) or β-strand (CsgG) transmembrane segments in the outer membrane. We developed assays to measure the assembly of three distinct secretion pores that mediate protein (GspD), curli fibre (CsgG) and capsular polysaccharide (Wza) secretion by bacteria and show that depletion of BamA and TamA does not diminish the assembly of Wza, GspD or CsgG.

Membrane-anchored MucR mediates nitrate-dependent regulation of alginate production in Pseudomonas aeruginosa.

Alginates exhibit unique material properties suitable for medical and industrial applications. However, if produced by Pseudomonas aeruginosa, it is an important virulence factor in infection of cystic fibrosis patients. The alginate biosynthesis machinery is activated by c-di-GMP imparted by the inner membrane protein, MucR.

Bioengineering of bacteria to assemble custom-made polyester affinity resins.

Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique.

Genetics and regulation of bacterial alginate production.

A vast range of extracellular polysaccharides are produced by bacteria in order to adapt to and thrive in diverse environmental niches. Many of these polymers have attracted great attention due to their implication in biofilm formation, capsule formation, virulence, or for their potential medical and industrial uses. One important exopolysaccharide, alginate, is produced by various Pseudomonas spp.

Microbial alginate production, modification and its applications.

Alginate is an important polysaccharide used widely in the food, textile, printing and pharmaceutical industries for its viscosifying, and gelling properties. All commercially produced alginates are isolated from farmed brown seaweeds. These algal alginates suffer from heterogeneity in composition and material properties.

Insights into the assembly of the alginate biosynthesis machinery in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen of particular significance to cystic fibrosis patients. This bacterium produces the exopolysaccharide alginate, which is an indicator of poor prognosis for these patients. The proteins required for alginate polymerization and secretion are encoded by genes organized in a single operon; however, the existence of internal promoters has been reported.

Structural basis for alginate secretion across the bacterial outer membrane.

Pseudomonas aeruginosa is the predominant pathogen associated with chronic lung infection among cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by the overproduction of the exopolysaccharide alginate.

Identification of a periplasmic AlgK-AlgX-MucD multiprotein complex in Pseudomonas aeruginosa involved in biosynthesis and regulation of alginate.

The opportunistic human pathogen Pseudomonas aeruginosa produces an extracellular polysaccharide called alginate. This is especially relevant in pulmonary infection of cystic fibrosis patients where it protects the bacteria from the hosts' immune system and the diffusion of antibiotics. Here a connection between the stability of a proposed alginate polymerisation/secretion complex and the regulation of the operon encoding these proteins was assessed.