Bio 301 - Ch. 3 Book Notes

Bacteria & Archaea

• both classified as prokaryotes

• bacteria have phospholipid bilayers similar to the ones eukaryotes have

  • thick, complex outer envelope that protects the cell from environmental stress

  • contains a compact genome which maximizes the production of cells from limited resources

  • tightly coordinated functions to form a highly coordinated mechanism

• archaea have unique membrane and envelope structures

  • an example is ether membranes

  • archaea live in moderate environments

Eukaryotic cells

• possess extensive membranous organelles

  • endoplasmic reticulum & Golgi complex

• mitochondria and chloroplasts evolved by endosymbiosis with engulfed bacteria

Model of a bacterial cell

• a cell is composed of a cell membrane and cytoplasm which form the physical qualities of a cell which are reinforced by inner and outer membranes

  • the inner membrane is made up of phospholipids, transporter proteins, and other molecules; functions to prevent cytoplasmic proteins from escaping and maintains a gradient of ions & nutrients

• the cell wall lies between the inner and outer membrane of the cell which is formed by sugar chains linked covalently by peptides

  • limits the expansion of the cytoplasm and keeps the cell membrane intact when water flows in (turgor pressure)

  • a gram-positive species would have the cell wall outside its one plasma membrane

  • a gram-negative species has a cell wall that lies within the periplasm and has phospholipids & lipopolysaccharides (LPS) outside the cell wall

• the bacterial envelope includes cell-surface proteins that enable the bacterium to interact with specific host organisms

• motile bacteria has an array of chemoreceptors the bind molecules from outside the cell or the periplasm and converts this binding intel into signals within the cytoplasm

  • the signaling molecules direct the rotation of flagella to propel the corresponding movement

the membrane is a 2D fluid of lipids and proteins

• a phospholipid bilayer that has lipid-soluble proteins

  • the bilayer behaves as a 2D fluid which lipids and proteins can diffuse across

• proteins embedded in the cell membrane often function as a complex

  • the subunits of a complex are usually adjacent and fit together like a puzzle (eg. ATP synthase)

• the cell membrane and envelope provide an attachment point for one or more chromosomes

  • organized as a system of looped coils, nucleoids, which are not enclosed by a membrane

  • instead, the loops of DNA extend throughout the cytoplasm & can be transcribed by RNA polymerase to form mRNA, rRNA, and tRNA

  • recall during the transcription process, chaperones are present to aid in the proper DNA folding

biochemical composition of bacteria

• all cells share common chemical components:

  

  • water - the fundamental solvent of life

  • essential ions - these include K+, Mg2+, and Cl-

  • small organic molecules - these include lipids & sugars which are incorporated in numerous cell structures and provide nutrition by catabolism

  • macromolecules - these include nucleic acids and proteins which contain information, catalyze reactions and mediate transport

  • small molecules & ions - these include phospholipids, enzyme cofactors, and charged organic molecules

• the cell’s genomic DNA directs expression of its proteins

  • a cell uses different genes to make different proteins while factors such as temperature, nutrient levels, and entry to a host organism are accounted for

  • the proteins expressed by a cell under given conditions are known as a proteome

• another important component of cells is the bacterial cell wall that consists of peptidoglycan

  • this component limits the volume of the enclosed cell meaning water rushing in will create high turgor pressure

cell fractionation

• a procedure to separate cell components that often includes ultracentrifugation

• this process also provides purified proteins that act as antigens for candidate vaccines

• the process of ultracentrifugation was refined

  • the gravitational force separates molecules by weight and density

• cell fractionation requires techniques that lyse the cell

  • there must be enough force to separate the membrane lipids but not enough to disintegrate complexes of protein and RNA

  • for gram-negative cells, the method requires more specificity to separate the compartments because it has inner & outer membranes, the cytoplasm and the periplasm

• the membrane vesicles proteins are analyzed on gel electrophoresis

  • these proteins can be identified by bands on the gel by enzyme digestion and mass spectrometry

limitations to cell fractionation

• provides little information about processes that require an intact cell (eg cell divison)

• an alternation approach to studying a portion of the cell without breaking it open would be genetic analysis

  • this process includes mutating a strain so it alters a gene and then select the mutant strains for loss of a given function

  • the phenotype of the mutant can provide insight about the function of the altered part

steps for cell wall lysis and spheroplast formation

• 1: permeabilize the bacterial outer membrane by removing the Mg2+ & Ca2+

  • this allows sucrose to cross and fill the periplasm which maintains an osmotically stable solution

• 2: lysozyme cleaves peptidoglycan and breaks down the cell wall

  • without the cell wall, the cell forms into a spheroplast

  • these spheroplasts can be seen by TEM

• 3: to isolate the periplasmic contents, the spheroplasts are transferred to distilled water

  • water rushes in through the EDTA-weakened outer membrane

  • this causes osmotic shock of the periplasmic compartment, but the inner membrane remains intact

• 4: after osmotic shock, the spheroplasts undergo ultracentrifugation to separate the periplasmic contents from the other three type of cell compartments

• 5: the membranes are then broken open by a French press device

• 6: a second step of ultracentrifugation now pellets the inner and outer membrane vesicles while removing the cytoplasm in the supernatant

• 7: the inner and outer membranes are separated by density gradient ultracentrifugation

  • the gradient is created by a solute concentration

  • the lower-density fractions contain inner membrane vesicles

  • the higher-density fractions contain outer membrane vesicles

membrane lipids

• the phospholipid bilayer creates membrane fluidity and gives the cell consistent thickness

• a phospholipid consists of glycerol with ester links to each of two fatty acids and a phosphoryl polar head group (phosphatide)

  • the negatively charged head group of a phosphatide can contain various organic groups or have a side chain with a positive charge (typically on amine group)

  • lipid biosynthesis is a key process that can make some cells vulnerable to antibiotics

factors that can effect the formation of membrane lipids to maintain structural integrity and function & uniform thickness

• environmental stress

  • starvation stress increases bacterial production of lipids with an unnatural type of phosphoryl head group

  • cardiolipin, a double phospholipid linked by a glycerol, concentration will increase in bacteria grown to starvation

  • cardiolipin helps define the polar structure of a bacterial cell and diffuses in concentration patches called “domains” near the cell poles

  • at the cell pole cardiolipin binds certain environmental stress proteins and a phospholipid can have specific functions associated with specific membrane proteins

• the fatty acid components of phospholipids varies between being saturated and unsaturated (most are cis - forms a kink)

  • the enhanced fluidity of a kinked phospholipid improves the function of the membrane at low temperatures

• cyclization of the part of the chain to form a stiff planar ring with decreased fluidity

  • the double bond of unsaturated fatty acids can generate a cyclopropane fatty acid

• stiff planar molecules can reinforce the membrane and reduce the membrane fluidity

  • for eukaryotes reinforcing agents are sterols like cholesterol

  • in some bacteria, reinforcing agents are hopanoids (five ring hydrocarbons)

• archaea have unique membrane lipids

  • these phospholipids replace the ester link between the glycerol and fatty acid with an ether link

  • ethers are more stable than esters which hydrolyze easily in water

  • archaeal phospholipids have hydrocarbon chains are branched terpenoids which limits the movement of the membrane

membrane proteins

• structural support

  • these proteins can anchor together different layers of the cell envelope

  • these proteins can attach the membrane to the cytoskeleton or form the base of structures extending out from the cell

• detection of environmental signals

• secretion of virulence factors and communication factors

  • membrane protein complexes export toxins and cell signals across the envelope

• ion transports and energy storage

  • transport of ions across a membrane generates a transmembrane gradient that stores energy

• there is a requirement of a portion of hydrophobic amino-acid side chains that are soluble

molecules cross the cell membrane

• because cell membranes act as a barrier to contain the cell contents and exclude extracellular material, selective transport is essential for cell survival

  • the ability to acquire nutrients, transport waste, and transmit signals to neighbor cells

• passive diffusion

  • small, uncharged molecules like diatomic oxygen and carbon dioxide can easily permeate the membrane

  • large, strongly polar molecules like sugar and charged molecules like amino acids cannot penetrate the membrane and require transportation

  • water molecules can permeate the membrane, but their passage is increased by protein channels called aquaporins

• osmosis

  • the internal concentration of water is lower than the concentration outside the cell

  • the solute concentration is higher inside the cell than outside

  • because of this, water tends to diffuse across the membrane into the cell which causes the cell volume to expand

  • osmotic pressure will cause a cell to lyse in the absence of a countering pressure such as that provided by the cell wall

• membrane-permeant weak acids and bases

  • these particles can only cross the membrane in their uncharged form which is HA for weak acids and B for weak bases

  • membrane-permeant acids conduct H+ ions across the membrane which can cause acidic stress (higher H+ concentration outside the cell drives weak acids into the cell)

  • membrane-permeant bases conduct OH- ions across the membrane which can cause alkali stress

• transmembrane ion gradients

  • molecules that carry a fixed charge cannot cross the membrane like H+ and Na+

  • an ion gradient across the cell membrane can store energy for nutrition or to drive the transport of other molecules

  • inorganic and charged organic ions require transport proteins (passive | active)

  • a transport protein obtains energy for active transport by cotransport of another substance down its gradient from higher to lower concentration or by coupling transport to a chemical reaction

protective layers of the cell envelope

• cell wall & some common structural support is an S-layer (outer membrane)

• the bacterial cell wall, the sacculus, consists of a single interlinked molecule that envelopes the cell

  • typically encloses maximal volume with minimal surface area

• the sacculus is a single molecule cage-like structure, highly porous to ions and organic molecules

  • the form is not rigid; it is a flexible mesh bag with unbreakable joints

  • turgor pressure within the enclosed cytoplasm fills the cell’s shape

• peptidoglycan / murein structure

  • this component is unique to bacteria; the molecule consists of parallel polymers of disaccharides called glycan chains cross-linked with peptides of four to six amino acids

  • the peptide extension can form cross-bridges connecting parallel strands of glycan

peptidoglycan synthesis as a target for antibiotics

• the enzymes required to bind the antibiotic penicillin, termed penicillin-binding proteins, hold the function of building the peptide bonds and sealing the cross-bridges

  • peptidoglycan is a trait unique to bacteria which makes it a target for new antibiotics (despite the resistance that some strains of bacteria have formed against commonly prescribed antibiotics

• the overall extension of the cell wall by the peptidoglycan layer is organized by a protein complex that includes MreB

  • this component polymerizes a helical direction along ana arc beneath the plasma membrane

cell envelope of bacteria

• most bacteria have additional envelope layers that provide structural support and protection from predators and host defenses

  • additional molecules are attached to the cell wall and cell membrane and some thread through the layers

• Gram-positive bacteria have a thick cell wall with 3-20 layers of peptidoglycan which are interpenetrated by teichoic acids

• Gram-negative bacteria have a thin cell wall with 1-3 layers of peptidoglycan which are enclosed by an outer membrane

  • lipoproteins link the outer membrane to the peptidoglycan layer

  • they are bound to the periplasm before the inner membrane

firmicute cell envelope — Gram-positive

• the multiple layer of peptidoglycan are reinforced by teichoic acids threaded through its multiple layers

  • the qualities of teichoic acids that help retain the Gram stain are the negatively charged cross-threads and the overall thickness of the Gram-positive cell wall

• the cell wall attaches to extracellular structures through an enzyme, sortase, which forms a peptide bond from a cell wall cross-bridge to a protein extending from the cell

  • proteins attached by the sortases can help the cell acquire nutrients or help the cell adhere to a substrate

• S-layer

  • this layer is composed of protein subunits that fit together like tiles which provides defense against phages or predators

  • this layer is rigid but it flexes and allows the passage of substances in either direction

• capsule

  • a slippery outer layer composed of polysaccharides that surrounds the cell envelope of some bacteria

proteobacterial cell envelope — Gram-negative

• the cell envelope of these bacteria includes 1-3 layers of peptidoglycan covered by an outer membrane

  • the outer membrane confers defensive abilities and toxigenic properties on many pathogens

• lipoprotein and lipopolysaccharide (LPS)

  • in Gram-negative bacteria, the inward facing leaflet of the outer membrane has a phospholipid composition similar to one of the inner membranes

  • the outer membrane’s inward-facing leaflet includes lipoproteins that connect the outer membrane to the peptide bridges of the cell wall

  • murein lipoprotein consists of a protein with an N-terminal cysteine attached to three fatty acid side chains

  • LPS’s act as endotoxins which are cell compartments that are harmless if the pathogen remains intact but when lysed, the endotoxins induce a potentially lethal shock to the host

• outer membrane proteins

  • Gram-negative bacterial cells have porins that permit the entry of nutrients such as nutrients like sugars and peptides

  • outer membrane porins have limited specificity, allowing passive uptake of various molecules including antibiotics

  • to prevent the entry of dangerous molecules, cells express different outer membrane porins under different environmental conditions

  • in dilute environments, cells express porins of large pore size to maximize the uptake of nutrients

  • in rich environments, cells down-regulate the expression of large porins & express porins of smaller pore size to select only smaller nutrients as to avoid uptake of toxins

• periplasm

  • this portion of the cell contains specific enzymes and nutrient transporters not found within the cytoplasm

  • these proteins in the periplasm are subjected to pH and salt concentration fluctuations because the outer membrane is porous to ions

• capsule

  • some Gram-negative bacteria have capsules made of loose glycolipids

mycobacterial cell envelope

• have effective defenses against host defenses & the gram stain is not applicable to use

• the mycobacterial envelope includes features of both Gram-positive and Gram-negative cells

  • the peptidoglycan layer is linked to chains of galactose called galactans

  • the galactans are attached to arabinans

• mycolic acids provide the basis for acid-fast staining due to the ester links that the arabinans form with mycolic acid

  • this function retains the dye carbolfuchsin

bacterial cytoskeleton

• to determine the shape of bacteria, aside from turgor pressure, they possess protein cytoskeletal components

• the functions of cytoskeletal proteins are probed by fluorescent protein fusions

  • in both spherical bacteria and rod-shaped bacilli requires cell division with the FtsZ protein

  • this encodes for a Z-ring to form and determine the cell diameter & manages the growth of the dividing partition - the septum

  • for rod-shaped bacteria, there is a requirement of elongation to polymerization of MreB where MreB travels in a helical arc beneath the cell membrane

  • if the rod-shaped bacteria is curved and forms a crescent shape, crescentin, polymerizes along the inner curve of the crescent

bacterial cell division by septation

• in prokaryotes, the cell divides by a process called septation which forms a partition that divides the envelope

  • septation requires rapid biosynthesis of all envelope components including membranes and the cell wall

  • envelope expansion must coordinate the extension of all layers—and regulate the placement and timing of the septum

• the overall process of septation is managed by a protein complex: divisome

  • this component manages assembly of the septum with its two envelopes back-to-back

  • FtsZ, a critical part to the divisome, polymerizes to form the Z-ring

  • FtsN helps regulate the timing of constriction of the septum

• cocci shaped bacteria can split on any plane (diagonal, horizontal, vertical) but rod-shaped bacteria only split vertically

DNA is organized in the nucleoid by domains (loops)

• the midpoint on the DNA is the origin of replication which is attached to the cell envelope at a point on the cell’s equator

  • the DNA may be looped back to the center of the cell, near the origin of replication

  • within the domains, the DNA is compacted by supercoils which causes portions of DNA to double back and twist upon themselves to result in compaction of the chromosome

  • DNA is also compacted by DNA-binding proteins

• to initiate DNA replication, the DNA double helix at the origin is opened by binding proteins, and then DNA polymerase synthesizes new strands in both directions

• in rapidly growing bacteria, the DNA is transcribed and the messenger RNA is translated to proteins while the DNA itself is being replicated

  • this phenomenon explains why bacterial cells can divide in as little as 10 mins

  • some of the newly translated proteins are made to function within the membrane and are synthesized in association with the membrane; they are directed there by signal recognition particles

DNA replication regulates cell division

• completion of replication triggers Z-ring formation

• bacterial cell size

  • cell size depends on genetic regulators and environmental constraints

  • more resources will lead to cell elongation occurring quicker and reaching larger sizes before septation and division

  • with less resources, cell growth slows & early division produces smaller cells

bacterial cell differentiation

growth asymmetry and polar aging

• cell division generates two daughter cell with chemically different poles

  • under environmental stress, at each cell division, some members of a population die of polar old age

  • the cause of this death is by the preferential accumulation of protein aggregates

  • a protein is more likely to aggregate under stress conditions such as low pH or antibiotic presence

• one consequence of polar aging is cells of different polar ages may differ in their resistance to antibiotics

• an extreme form of asymmetrical growth is endospore formation

  • under starvation, desiccation, or other stress conditions, a bacterium can undergo an asymmetrical cell division to develop an endospore at one end

  • this requires an extreme form of cellular altruism where the mother cell sacrifices itself for the spore-forming cell to generate an endospore capable of remaining dormant but viable for years

membrane vesicles

• functions that vesicle production has to outweigh the loss of resources

  • attraction of partner heterotrophs — because heterotrophs are attracted by the released carbon sources & consume excess oxygen and reactive oxygen species, for some bacteria, it is a requirement to have a partner for growth

  • phage decoys — the bacterial membrane vesicles have envelope receptors for phages which can trap the phages and prevent them from infecting cells

  • DNA transfer — the DNA released in cytoplasmic vesicles may provide useful information to encode for genetic traits for other members of the population as a form of horizontal gene transfer

membrane extensions and nanotubes

• bacteria may possess cell extensions such as filaments and “pearling” chains of vesicles

• nanotubes enable bacteria to directly share proteins and mRNA that encodes products useful under hostile conditions

• another remarkable feat that derives from the presence of nanotubes is the fact that bacteria of different species can share beneficial components of cytoplasm

  • the nanotubes facilitate exchange of different amino acids between the two species

  • the nanotubes only form when the two types of cells each produce an amino acid lacking in the other which prompts metabolic cross-feeding

• archaea show various kinds of intracellular nanotubes that are essential parts of the cell

thylakoids, carboxysomes, and storage granules

• cyanobacteria need to maximize the amount of light that is necessary to drive photosynthesis & they do this with the presence of thylakoids

  • thylakoids consist of layers of folded sheets (lamellae) or tubes of membranes packed with chlorophylls and electron carriers

  • thylakoids conduct only the light reactions of photon absorption and energy storage

  • the energy obtained is spent fixing CO2

• to stay at the top of water columns, some bacteria and archaea form gas vesicles to increase buoyancy

  • the gases are hydrogen or carbon dioxide produced by the cell’s metabolism

• when light is scarce, cyanobacteria might digest their thylakoids for energy and as a source of nitrogen

  • they might also digest energy-rich materials from storage granules: PHB & PHA

  • these are polymers that of interest in biodegradable plastics since bacteria have been engineered to produce them industrially

• another type of storage device is sulfur

  • granules of elemental sulfur produced by purple & green phototrophs through photolysis of hydrogen sulfide

  • the granules may be used as an oxidant when reduced substrates are available

  • the presence of potentially toxic sulfur granules may help cells avoid predation

pili and stalks

• pili are constructed of straight filaments of protein monomers called pilin

  • short attachment of pili are called fimbriae

• pili can provide a form of motility called “twitching” in which the pili act as limbs to “walk” the bacterium across a substrate

• in Gram-negative enteric bacteria, pili of a different kind (sex pili) attach a donor cell to a recipient cell for transfer of DNA

  • the transfer of DNA is conjugation

• stalks are another type of attachment organelle which is an extension of the envelope and cytoplasm

  • the tip of the stalk secretes adhesion factors that form a “holdfast” to firmly attach the bacterium in a favorable environment

  • a stalk and holdfast enable some types of bacteria to form large biofilms in streams

rotary flagella

• many bacteria and archaea can swim by means of rotary flagella which can benefit the microorganism to disperse the population and decrease competition

  • movement can also prompt cells to swim towards a favorable habitat

• flagellar movement

  • different bacterial species have different numbers and arrangements of flagella

• how rotary flagellum work

  • each flagellum has a spiral filament of protein monomers called flagellin (protein FliC)

  • the filament rotates by means of a motor driven by the cell’s transmembrane proton current

  • the flagellar motor is embedded in the layers of the cell envelope & the motor possesses an axle and rotary parts

  • another protein, FliG, forms part of the device that generates torque (rotary force)

• how to cells decide where to swim

  • most flagellated cells have an elaborate sensory system for taxis, the ability to swim towards favorable environments

  • taxis to specific chemicals is called chemotaxis which requires receptors that tell the bacterium when it is swimming toward a source of attractant or repellent

  • these molecules are detected by arrays of chemoreceptors that are located near a cell pole

• another function of flagellum is the adherence of cells to a substrate to begin forming a biofilm