Bacterial Physiology Test 1

Prokaryotic Organism

Prokaryotic Organism

• unicellular

• no nuclear membrane, mitochondria, Golgi bodies, or ER

• reproduce by asexual division

Lag phase

initial period of adjustment

Log phase

• cell division occurs rapidly

• doubling of population

Stationary phase

cell growth slows to a stop

Death

decline in population

Aerobic metabolism

is complete utilization of an energy source such as glucose

• produces 38 molecules of ATP

  • final electron acceptor is oxygen

• requirement of oxygen during aerobic respiration may be obligate or facultative

Anaerobic metabolism

• is utilizing an inorganic molecule other than oxygen as the final hydrogen acceptor

  • incomplete

• can be used in the absence of oxygen when the appropriate substrates are available

• produces fewer ATP molecules than aerobic respiration

Fermentation

• is an anerobic metabolism that utilizes an organic final hydrogen acceptor is much less efficient

• produces only 2 molecules of ATP

• uses pyruvate in secondary fermentation to generate additional energy

Peptidoglycan

the main structural component of the cell wall

• mixed polymer of hexose sugars and amino acids

• N-acetylglucosamine (NAG)

• N-acetylmuramic acid (NAM)

can be degraded by lysozyme

• in tears and mucus

Gram-positive bacteria

• thick, multilayered cell wall consisting mainly of peptidoglycan

  • surrounds the cytoplasmic membrane

• cell wall can also contain teichoic and lipotechoic acid

• complex polysaccharides may be present

Gram-negative bacteria

• thin peptidoglycan cell wall

  • surrounded by an outer membrane

• produce lipopolysaccharide (LPS)

  • aka endotoxin

  • found in the outer leaflet of the outer membrane

• transport/secretion systems

  • type I-V

Mycobacterium species

have a peptidoglycan layer that is intertwines with and covalently attached to an arabinogalactan polymer

• surrounded by mycolic acid layer

Mycoplasma

have no peptidoglycan

Spheres

coccus, cocci, diplococcus

Rods

bacilli, bacillus

Spiral

spirochete

Virulence

the ability to cause damage to the host and to what degree

Virulence factors

structures, molecules, regulatory systems, etc. that allow the microbe to establish infection

Capsule

• usually made of high molecular weight polysaccharide

  • can be made of amino acids, hyaluronic acid, etc.

• gives a slimy surface

• provides protection against phagocytosis by host cells

  • important to determine virulence

• few capsulated organisms can cause a fatal disease while unencapsulated mutants are avirulent

  • Streptococcus pneumoniae

Flagella

• long, helical filaments extending out from the surface

  • allow for movement in the environment— motility

• can be restricted to the poles of the cell

  • polar— single flagellum

  • lophotrichous— tufts

  • can be distributed all over the surface— peritrichous

Motility

• is driven by movement of hydrogens across the cell membrane

• allows positive and negative responses to environmental stimuli

  • response to chemicals=chemotaxis

Pili

• more rigid than flagella

  • function in attachment

• adherence to host cells involve specific interaction

  • adhesins and the host cell membrane

• antigens can be changed

  • allow bacteria to avoid immune recognition

  • antigenic variation

Lipopolysaccharide

• LPS (aka endotoxin)

  • made by Gram negatives

  • composed of lipid A, core polysaccharide, and an O antigen

  • Lipid A is the toxic component

• powerful stimulator of innate and adaptive immune responses

  • activates B cells

  • induces macrophages and dendritic cells

  • causes the release of IL-1, IL-6, TNF-α

• can induce fever and shock

Lipoologiosaccharide

• LOS

• truncated version of LPS

  • produced by the Neisseria species

• results in fever and very severe symptoms

Biofilm

• some bacteria produce a polysaccharide layer

• protects bacteria from antibiotics and host immune defenses

• need sufficient number of bacteria (quorum sensing)

  • small molecules are released to innate production

• tooth plaque

Spores

• produced by some Gram-positive bacteria

• under harsh environmental conditions these bacterial cells convert from vegetative to dormant states

• dormant spore is protected by a keratin-like protein coat

• can remain viable for years

Quorum Sensing

mechanism by which specific gene transcription is activated in response to bacterial concentration

Bacterial Physiology

the biochemistry of processes and mechanisms for cellular adjustments to an everchanging environment

Most bacteria have a single circular DNA molecule

exceptions:

1. actively replicating cells

2. Borrelia burgdorferi: 22 DNA molecules— 13 linear, 9 circular

3. Brucella species: 1 large circular chromosome, 1 small circular chromosome

Elemental Requirements of Prokaryotes

C, H, O, N, S, P, K, Mg, Fe, Na, Cl

Mn, Co, Ni, Cu, Zn, Se, Mo, W

Periplasm

a gel-like substance that maintains turgor pressor

• 10-30% of cell weight of the enterics

• classified separately in Gram-positive bacteria

Gram Stain

• invented in 1884 by Danish physician Christian Gram

  • amount of peptidoglycan determines stain retention

• “fixed” bacteria are stained with crystal violet

• iodine is added

• cells are “washed” with alcohol

• counterstain with safranin

Teichuronic acids

• phosphate-free, uronic acid-containing polysaccharide

• polyanoinic acidic polysaccharides

• some have NAG or D-glucuronic acid

Neutral Polysaccharides

• particularly important for classification of streptococci and lactobacilli

• used to divide genus into serological groups

Mycoplasma

• species do not have a cell wall

  • membrane contains similar amounts of lipids and proteins as other bacteria

• most have 25-30% cholesterol— similar to eukaryotes

  • acquired from medium— not synthesized

• most bacteria do not have cholesterol or sterols

Mycobacterium

• species have a cell wall consisting of waxy lipids

  • 40% of the dry weight

• Mycolic acids are the main waxy lipids

  • branched chain hydroxy fatty acids

  • form a hydrophobic layer on the external face of the cell wall

  • hydrophobic layer can be esterified to arabinogalactan or trehalose

• does retain gram stain well unless the wall lipids are removed with alkaline ethanol

Acid-Fast Stain

• carbol fuchsin is lipid-soluble and contains phenol

  • helps the stain penetrate the cell wall

• ability to resist decolorization with acid-alcohol indicates “acid-fastness”

• Mycobacterium species are difficult to stain due to waxy cell wall

Function of Peptidoglycan

• helps maintain the shape of the cell

  • also responsible for cellular morphogenesis

• can be destroyed by lysozyme

  • found in tears, saliva, breast milk, mucus

  • hydrolyzes the glycosidic linkages

• target of many antibiotics

  • penicillin, vancomycin, bacitracin interfere with synthesis

  • inability of the cell wall to restrain turgor pressure

Oligosaccharides

• thought to be involved in osmotic regulation

• cells grown in high osmolarity have decreased oligosaccharide

Solute-binding proteins

• assist in solute transport

• bind to and deliver solutes to specific transporters in the cell membrane

Cytochrome C

• oxidize carbon compounds or inorganic compounds— periplasmic oxidations

• deliver electrons to the ETC in the cell membrane

Hydrolytic enzymes

degrade nutrients to smaller molecules that can be transported across the cell membrane by specific transporters

Detoxifying agents

periplasmic enzyme β-lactamase degrades penicillins/β-lactams

Neutral lipids

free fatty acids, sterol esters, etc.

Polar lipids

phospholipids, glycolipids, etc.

Lipoconjugates

contain lipid and polysaccharide or protein; amphiphilic

Amphipathic/Amphiphilic Lipids

• ability to spontaneously aggregate

• nonpolar fatty acids interacting by hydrophobic bonds

• polar phosphorylated regions face the aqueous phase

Phospholipids

• predominate form of lipid found in bacterial membranes

• glycerol serves as backbone

  • support attachment of fatty acids, alcohols or phosphates

• fatty acids are bound at the C-1 or C-2 of glycerol

  • long chains of hydrocarbons that end with carboxyl group

  • 10-20 carbons— most are 16-20

• fatty acids in positions R1 and R2 can differ

Phosphate

is bound to the C-3 in the glycerol backbone

• makes this part of the molecule very polar

• negative charge on ionized phosphate group

Phospholipid facts

• 65-75% of total phospholipids are found in the cell membrane

  • remaining phospholipids are in outer membrane and cytoplasm

• influence charge density on the membrane

• important in signal transduction processes

  • chemotaxis

• plays a role in translocation of proteins across membrane by systems that involve molecular chaperones

• may have a role in DNA replication

Bacterial plasma membrane structure

• consist primarily of phospholipids and proteins

  • in a fluid mosaic structure— phospholipids form a bilayer

• usually referred to as lipoprotein bilayer

Integral proteins

• embedded in the membrane

• bound to fatty acids of phospholipids by hydrophobic bonding

• removal by detergents

Peripheral proteins

• attached at membrane surfaces to phospholipids by ionic interactions

• removal by washing with salt solutions

Aquaporins

• water channels

• enhance rapid equilibrium of water across cell membranes

Mechanosensitive channels

open under conditions of hypo-osmotic stress

Osmosensory transporters

• senses increasing osmolarity

• facilitates the uptake or organic osmolytes (proline and betaine)

Osmolytes

are important for increasing the cytoplasmic osmolarity

• prevents water from rushing out during hyperosmotic stress

Intracytoplasmic Membranes

• often connected to the cell membrane

  • believed to be derived from invagination of chemically modified areas of the cell membrane

• connections to the cell membrane are not always seen

• can be synthesized independently of cell membrane

  • thylakoids of cyanobacteria

• many prokaryotes have intracytoplasmic membranes that have specialized physiological functions

Methanotrophs

diverse group of Gram negative bacteria

• bacteria that grow on methane as their sole source of carbon

Azobacter vinelandii (N-fixing bacterium)

• intracytoplasmic membranes increase with aeration of growing cultures

• respiratory activity is localized in the membranes

• increase cellular respiratory activity to provide more ATP for N-fixation

  • also removes O2 from nitrogenase

Phototrophs

bacteria that use light as a source of energy for growth

Cytoplasm

• everything enclosed by the cell membrane

• viscous material contain a heavy concentration of protein, salts, and metabolites

• large aggregates of protein complexes designed for specific metabolic functions, various inclusions, and highly condensed DNA

• soluble portion is called cytosol

  • liquid portion

Inclusion bodies

specialized compartments in the cytoplasm

• not surrounded by a lipid-bilayer

• may have a membrane or coat

numerous large aggregate and multienzyme complexes

Gas Vesicles

• hollow, spindle-shaped structures filled with gas in equilibrium with the gases dissolved in the cytoplasm

• allow bacteria to float in lakes and ponds at depths that support growth

• collapsed vesicles do not recover

Carboxysomes

• large polyhedral protein-walled microcompartments

• most oceanic microorganisms that fix CO2 utilize carboxysomes

• stores ribulose-1,5-biphosphate carboxylase (RuBP carboxylase)

• may serve to concentrate CO2 inside the structure

Ellipsoid Inclusion

• lies immediately underneath the cytoplasmic membrane

• surrounded by a nonunit membrane of galactolipid

Chlorosomes

• ellipsoid inclusion

• stores the major light-harvesting photopigments

  • during photosynthesis light is absorbed by pigments in the chlorosomes

  • energy is transmitted to photosynthetic reaction centers in the cell membrane

• found in green photosynthetic bacteria and green sulfur photosynthetic bacteria

Magnetosomes

• chains of membrane-bound organelles

• strings of crystals of iron oxide, magnetite (Fe3O4) or sulfide, greigite (Fe3S4)

• influence the direction of swimming with respect to the earth’s magnetic field (magnetotaxis)

Magnetosomes

• found in certain marine and freshwater bacteria

  • magnetotactic bacteria

  • microaerophilic or anaerobic

• magnetotaxis results in “diving”

  • lower levels are beneficial=less oxygen at greater depths

Bacterial Ribosomes

• site of protein synthesis

• bacterial ribosomes are called 70S ribosomes

• differences in bacterial and eukaryotic ribosome can be exploited

  • aminoglycosides, macrolides

Nucleoid

• site of DNA and RNA synthesis

• an amorphous mass of DNA

  • approximately in the center of the cell— not membrane bound

• “fast” growing bacteria may contain more than one

  • each nucleoid has one chromosome

  • all chromosomes are identical

• DNA is tightly coiled

Multienzyme Complexes

• enzymes in the cytoplasm are not a random mixture of proteins

• enzymes in the same pathway can form stable multienzyme complexes

  • strong intermolecular bonding

• enzyme complexes catalyze consecutive series of biochemical reactions

  • facilitates the channeling of metabolites

  • increases the efficiency of catalysis

Glycocalyx

• often describes all extracellular material that is external to the cell wall

  • all bacteria are probably surrounded by glycocalyces when growing in nature

  • lose this layer when cultivated in the laboratory

• predominant polymers are polysaccharides and/or proteins

• extracellular polymers may be in the form of S layers, capsules, slime, or a loose network of fibrils

Role of Glcocalyx

• adhesion

• protection from phagocytosis

• dehydration

General Structure of Flagella

• basal body

• hook

• filament

• motor

• switch

• export apparatus

• capping proteins

• junction proteins

Basal Body

• found at the base of the flagellum

  • embedded in the membrane

• in gram-negatives— consists of 3 stacked rings and central rod

  • C, M, and S rings

• M and S rings are joined together as a single ring (MS ring)

  • made of different domains of the FliF protein

  • MS indicates position— membrane and supramembranous

Motor

consists of two parts

1. stator

2. rotor

Motor— Stator

• MotA and MotB exists as particles of multiple complexes

  • (MotA)4 (MotB)2

  • spans the cell membrane and surround the MS ring

• large periplasmic domain of MotB is attached noncovalently to the peptidoglycan

  • does not rotate when the motor turns

• MotA/MotB conduct protons from outside the cell to inside

  • across the membrane

  • use proton movement to provide the torque to rotate the rotor

Motor—Rotor

• MS and C rings together are often considered the rotor

• FliG is an essential rotor component

  • interacts with the Mot proteins

  • transmits torque generated by the Mot complex to rotate the rotor

• FliG proteins are part of the C ring

  • attach the C ring to the MS ring

• C ring functions as a switch that reverses the direction of rotation of the rotor

The Switch

• the motor can spontaneously change its direction rotation periodically

• FliG, FliM, and FliN— complex of switch proteins

• FliG is bound to the MS ring itself

• FliM and FliN form the C ring

• a regulator protein (CheY) binds to FliM

CheY

is important for regulating flagellar switching during chemotaxis

The Hook

• the central flagellar rod is attached to an external curved flexible hook

• is made of multiple copies of FlgE

• HAPs are necessary to form the junction between the hook and filament

• FlgE proteins fill the C ring and are transferred into the growing hook through the export apparatus

Filament and Capping Proteins

• a rigid, hollow, helical filament that is attached to the hook

• acts as a propeller when it rotates and pushes the cell forward

• comprised of flagellin

• the C- and N- terminals do share some homology

Spirochete flagella

• flagella are in the periplasm

  • do not protrude from the cell

  • flagella are wrapped around the length of the protoplasmic cylinder

• most spirochetes are helically coiled and have 2 or more flagella

• some have a flat meandering waveform

• the number of them inserted at opposite poles is the same

• are usually more than half the length of the cell

• is often covered by a proteinaceous sheath

Borrelia burgdorferi

• axial filaments are not surrounded by proteinaceous sheaths

• has planar waveform shape rather then corkscrew type

• rotation of the rigid periplasmic flagella between the outer membrane sheath and the cell cylinder propagates a helical wave

  • propels the cell forward

  • allows the cells to corkscrew through viscous media

Flagella Insertion

• site and number of flagella vary with bacterial species

• subpolar flagella

• medial flagella

Subpolar flagella

inserted near the poles

Medial flagella

seen in curved bacteria

Role of Flagella

• tactic response

• swimming response due to environmental signals

• virulence

Tactic response

• swim towards favorable environments

  • higher nutrients, appropriate light, electron acceptors

• swim away from toxic or less favorable environments

Chemotactic Process

• CheA is bound to the receptor

• Phosphorylated CheA then transfers phosphate to aspartate residue in CheY

• phosphorylated CheY interacts with FliM

Regulation of Chemotaxis

• phosphorylated CheA activates CheB (methyltransferase)

• CheR (methylesterase) acts to add CH3 to the MCPs

• MCPs that are methylated cause a CW rotation

Swarming

• “social swimming”

• allows bacterial populations to rapidly spread as biofilm, agar plates, etc.

• cells are morphologically different than swimming cells seen in liquid

• facilitated by the production of surfactant

• surfactant is a component of the extracellular slime

• isolated swarmer cells rarely move

Fimbriae

• protein fibrils extending from the surface

• often observed on the surface of most Gram-negative bacteria

• size can vary

• most seem to not originate in the cell membrane

• not always present

• often seen in freshly isolated samples— natural state

• lost during laboratory growth

Antigenic Variation

• diversification of pilin

• number of genes and recombination

• recombination results in chimeric pilin types

Sex pili

• conjugation or bacterial mating

• grows on the “male” strain— donor

• encoded by a conjugative transmissible plasmid— F plasmid

Bacterial cytoskeletal components

• provide scaffolding for peptidoglycan-synthesis machinery

• placement of the peptidoglycan machinery along the scaffolding determines the shape of the cell

FtsZ

• bacterial cytoskeletal component related to tubulin

• at the site of cell division— assembles as a ring (z ring)

  • recruits other proteins to form a contractile septal ring that constructs the cell during division

• FtsI and FtsW are recruited

  • necessary for peptidoglycan synthesis in the septum

  • necessary for cell division

Z ring

• important for cell separation

  • recruits peptidoglycan hydrolases

  • splits the septal peptidoglycan into 2— allows daughter cells to separate

• required for relocation of enzymes necessary for lateral peptidoglycan expansion

  • PBP1B— transglucosylase

• RodA and PBP2 are also required for peptidoglycan synthesis

  • important for cells to elongate as rods

Mre

• required for cells to elongate

  • sequence pattern similar to actin

• found in many rod-shaped, filamentous, and helical bacteria

• thought to organize the peptidoglycan biosythetic machinery so the cells grow as rods

• found in E. coli and Caulobacter crescentus

  • B. subtilis has three paralogues