Lecture 4. Bacteriology IV - Microbiology PDF

BIOL2038/2044 Microbiology Bacteriology IV: Cell membrane/wall & cellular locomotion Learning outcomes: Understand the general structure of the cell membrane and wall. Describe the functions of both structures. Discuss the different types of bacterial motility and locomotion. Understand...

BIOL2038/2044 Microbiology Bacteriology IV: Cell membrane/wall & cellular locomotion Learning outcomes: Understand the general structure of the cell membrane and wall. Describe the functions of both structures. Discuss the different types of bacterial motility and locomotion. Understand different arrays of flagella and the basics of flagella structure. Consider chemotaxis behaviour. Consider the role of fimbriae and pili. Understand the differences between gliding, twitching and swarming motility. CYTOPLASMIC MEMBRANE – several roles, main being as the ‘gatekeeper’ for diffusion into and out of cell. CELL WALL – provides structural strength, resist osmotic pressure. 4 Cytoplasmic membrane Structurally weak but important to provide selective permeability. Phospholipid bilayer with embedded proteins – hydrophobic and hydrophilic components. Adapted from Brock Book of Microorganisms 5  Peripheral proteins Adapted from Brock Book of Microorganisms 6 3 main functions: Selective permeability – barrier to diffusion of polar & charged molecules in particular. Transport – use of transport proteins to accumulate solutes against concentration gradient. Allow for sufficient nutrients to perform biochemical reactions. Requires energy. Major site of energy conservation & consumption – proton motive force analogous to potential energy in a charged battery. 7 From Brock Book of Microorganisms 8 Cell wall - peptidoglycan Rigid polysaccharide = structural strength. Strands of peptidoglycan form a sheet around a cell, connected by cross links forming a polymer. Can be 90% of Gram-positive cell wall. Adapted from Brock Book of Microorganisms 9 Other components: In Gram-negative bacteria, outer membrane that can also contain polysaccharide. – Lipopolysaccharide layer or LPS. – Little structural role but is an effective barrier. Understanding these general differences is important when targeting bacteria – e.g. many antibiotics that are effective against Gram-positive bacteria show little activity against Gram-negative species. 10 Microbial locomotion Most bacteria are motile. Often due to the presence of specific structures; flagella. Can be due to the presence of gas vesicles – allow regulation of position in water column in some aquatic species. Some have gliding or twitching or swarming motility. 11 Flagella Long, thin appendages – organelles defined by function rather than structure (flagella differ between bacteria/archea/eukaryotes). Bacterial flagella are around 20 nm thick, contain the protein flagellin. Have a helical shape, with the base having a different structure that links to the ‘motor’. The array of proteins differs between Gram-negative and Gram-positive cells. 12 Structure of the flagellum in gram-negative bacteria. L-ring is embedded in the LPS, and the P-ring in the peptidoglycan. The M-S-ring is embedded in the cytoplasmic membrane. There is a narrow channel in the filament which flagellin molecules diffuse to reach the site of flagella synthesis. The Mot proteins function as the flagellar motor, while the Fli proteins function as the motor switch. The flagellar motor rotates the filament to propel the cell. Flagella ASM blog 13 Jianhua Xing et al. PNAS 2006;103:5:1260-1265 & Brock’s Biology of Microorganisms Arrangement of flagella Monotrichous – single flagellum. Lophotrichous – multiple flagella from the same location, forming a ‘tuft’. Amphitrichous – a single flagellum at each end. Peritrichous – numerous flagella around the cell structure. All are important in motility but are also used for classification. Remember, can also have atrichous – no flagellum! 14 Tortora's Microbiology 15 16 Chemotaxis: Chemotaxis of E. coli. (a) When no attractant is present E. coli switches from direct swimming to tumbling randomly. (b) In the presence of an attractant E. coli moves through the gradient in the direction of the attractant. (Attractant gradient is shown in green.) http://2012.igem.org/Team:Goettingen/Project 17 Other forms of locomotion: Gliding motility – independent of structures such as flagella, pili and fimbriae. Twitching motility – involves Type IV pili, important in pathogenicity and biofilm formation (e.g. Pseudomonas aeruginosa). Swarming motility – rapid, coordinated movement. Multicellular behavior. 18 Fimbriae and pili Fimbriae & pili are similar in structure to flagella. Fimbriae are sometimes referred to as the ‘attachment pili’. Important in adherence mechanisms. Pili – two main types; conjugation (sex) pili and type IV pili. 19 Schematic drawing of bacterial conjugation. 1- Donor cell produces pilus. 2- Pilus attaches to recipient cell, brings the two cells together. 3- The mobile plasmid is nicked and a single strand of DNA is then transferred to the recipient cell. 4- Both cells recircularize their plasmids, synthesize second strands, 20 and reproduce pili; both cells are now viable donors. Intestinal adherence associated with type IV pili of enterohemorrhagic Escherichia coli O157:H7 J Clin Invest DOI: 10.1172/JCI30727 21 Twitching motility The tug-of-war model of twitching motility. Cells (such as the shown N. gonorrhoeae diplococcus) extend pili (green) that attach themselves to locations in the surrounding environment (blue circles). Pili experience tension due to activation of the retraction machinery. Upon detachment or rupture of a pilus (red), the cell quickly jerks into a new position based on the resulting balance of forces acting through the remaining pili. 22 Swarming is multicellular surface movement powered by rotating helical flagella. Swimming is individual movement in liquid powered by rotating flagella. Twitching is surface movement powered by the extension and retraction of pili. Gliding is active surface movement that does not require flagella or pili and involves focal adhesion complexes. Sliding is passive surface translocation powered by growth and facilitated by a surfactant. The direction of cell movement is indicated by a grey arrow and the motors that power the movement are indicated by coloured circles. Kearns 2010 23 By Fortinda - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=53871041 24 Bacteriology IV Biofilms Biofilms Learning objectives: Understand what biofilms and how to define them. Discuss how biofilms impact on the environment, industrial processes and are important clinically. Consider the stages in the biofilm ‘life-cycle’. Briefly describe the events involved in attachment of bacteria to a surface. Biofilms – structured populations of microorganisms E. P. T. Tolker- Greenberg Nielsen A. Spormann M. Giskov Definition of a biofilm: A biofilm is a microbial, sessile community characterised by cells that are: Irreversibly attached to a substratum, interface or to each other. Are embedded in a self-produced matrix of extracellular polymeric substances (EPS), and In comparison to planktonic cells, they exhibit an altered phenotype with respect to: – Growth rate Donlan and Costerton (2002): Biofilms: Survival – Gene transcription Mechanisms of Clinically Relevant Microorganisms. Clin Microbiol Rev, 15, 167-193 Biofilms: the social life of microbes Biofilms refer to many different microbial aggregates which all share the following characteristics: – Organisms are embedded in a hydrogel formed by EPS. – Long retention time of cells next to each other = microconsortia. – Heterogeneity in space and time. – High biodiversity: strong gradients, high habitat variability, subpopulations. – Retention of exoenzymes. – Retention of nucleic acids – large gene pool. – Increased resistance to biocides, desiccation and other stress. – Access to degradation of particulate matter. – Sorption of dissolved and particulate nutrients from environment. – Physiological differences between planktonic and biofilm cells. Living in biofilms is an important microbial survival strategy Biofilms affect all aspects of life Dental plaque So many different kinds of biofilms…. Chronic infections, e.g. cystic fibrosis, chronic wounds, otitis media (glue ear). Flocs and granules in wastewater treatment processes. Anaerobic digester granules. Food-associated systems. Marine snow – loose associations of microbes with organic detritus. Pellicles: predominantly 2D structures forming on surfaces of liquids. Sludge and sediments, thick slime matrices. Wimpenny, 2000 The biofilm ‘life-cycle’ Steps in biofilm formation 1. & 2. Adhesion, reversible & irreversible (Marshall et al 1972). 3. Maturation 1: formation of microcolonies, surrounded by EPS (Sauer et al 2002). 4. Maturation 2: development of a continuous biofilm (Sauer et al 2002). 5. Dispersion and sloughing off; including due to programmed cell death and lytic phage expression or nitric oxide signalling (Webb et al 2003, 2006). 6. Transport of biofilm particles (flocs); dispersed organisms phenotypically similar to planktonic cells (Webb et al 2003). The glue: extracellular polymeric substances (EPS) – the ‘house’ of biofilm cells: Biopolymers of microbial origin – Polysaccharides – Proteins – Glycolipids, phospholipids, LPS – Nucleic acids Biofilm cells are embedded in EPS, which fundamentally influences their micro-environment. Centre for Biofilm Engineering, Montana State University. Oxygen gradient profiles Antimicrobial tolerance in biofilms How do microorganisms accumulate and bind at surfaces? Initial, reversible adhesion Adhesion at a distance of 5 – 20 nm. A result of forces that operate at long distances, i.e. van der Waals forces. Little energy needed to remove bacteria, e.g. the kinetic energy produced by turning the flagella leads to desorption. The theory for these forces describes the behaviour for colloidal particles – DLVO theory. Irreversible adhesion Binding is mediated by polymer bridging, achieved by reduced radius of body. Irreversible binding. Specific receptor/adhesion based processes. Can involved specific structural components. Achieved by bacteria but not colloids, i.e. the DLVO theory no longer helpful. Polymers involved in specific irreversible adhesion Bacteria possess a range of surface structures that allow for polymer bridging. – Exopolymers (exopolysaccharides, fibrillar proteins) – Fimbriae – Flagella – Stalks – Lipoteichoic acids (LTA) – Lipopolysaccharides (LPS) – Surface localised proteins – Surface localised pigments – A-layers – S-layers