Exam 1: Lectures 1-7 Flashcards
Regulators for stationary phase gene expression
- sigma s
- cAMP-CRP complex (increase expression of genes, like ones that code for enzymes that can supply energy independent of glucose)
- Guanosine tetraphosphate, or alarmone (inhibits rRNA, ribosomal protein, and tRNA synthesis during amino acid starvation (stringent response), this also leads to decrease in other protein production
Role of D-alanine substitutions in innate immunity
D-ala substitutions add positive charges to LTA, increasing positive charge of cell wall
- Positive cell wall repels cationic antimicrobial peptides (CAMPs), such as polymyxin, beta-defensins
D-ala substitutions play an important role in immune recognition and cytokine production
- Toll-like receptor 2 is involved
Limitations of measuring growth via viable cell counts
(serial dilutions and determining colony-forming unit by plating)
will give an underestimation if cells are clumped or in chains because each clump or chain will give rise to a single colony
Function of peptidoglycan in mycobacterial cell wall
Provides rigidity
MIC, MBC
minimum inhibitory concentration
minimum bacteriocidal concentration
(relating to antibiotics)
LPS biosynthesis
Three parts created individually
Core oligosaccharide added to lipid A, then O-antigen added to lipid A-core oligosaccharide molecule by a ligase, then completed LPS molecule is translocated across periplasm and outer membrane by LPS transport proteins (Lpt A, B, C, etc.)
Function of surface proteins in mycobacterial cell wall
Variety of activities, e.g. act as enzymes and adhesins
Events before septum formation in bacterial cell division
- DNA synthesis initated when cell reaches critical mass
- Two copies of chromosomes are formed
- Chromosome partition occurs (sister chromosomes move to opposite cell poles)
- Replication origin anchored to cell poles, remaining chromosomes continue to migrate to daughter cells
BCG vaccine
live, attenuated bovine strain of tubercule bacillus (M. bovis)
Uncertain efficacy, not routinely used in US (low incidence, prevents use of tuberculin test to determine spread)
Phospholipid
Two fatty acid chains attached to 2 carbon atoms of glycerol by ester bonds, a phosphate attached to third carbon atom of glycerol
- small organic groups linked to phosphate group give variety
Amphipathic - fatty acid chains hydrophobic, head hydrophilic - form micelles when placed in aqueous environment
PBP3
Penicillin binding protein specifically important for peptidoglycan synthesis in the septum
Type 4 pili assembly
Major pilin (PilA) and minor pilin are proccessed by the inner membrane peptidase PilD
Processed pilins accumulate in inner membrane, where they are polymerized into a helical fiber by hexameric ATPase PilB (extension)
Depolymerization of pilins by the ATPases PilT and PilU results in pilus retraction
Polymerizeed fiber extends to the exterior via the secretion pore formed in the outer membrane by PilQ, and (hypothesized) with the help of actin-like PilM and inner membrane proteins PilN, PilO, and FimV, which form a complex with PilP, PilF, and PilQ
Pseudopeptidoglycan
Glycans:
- a beta-1,3 glycosidic linked NAG and N-acetyl-talosaminouronic acid (NAT)
- lysozyme cannot digest beta-1,3 glycosidic bonds
Peptides:
- only L-amino acids
- attached to NAT
- cross-linked directly between the lysine and glutamate residues
- penicillin is ineffective in inhibiting the cross-linking
Septum formation in bacterial cell division
- MinE forms a membrane-associated ring at cell centre, relieving inhibition of FtsZ ring formation by MinCD
- FtsZ recruited to cell centre and begins forming FtsZ ring (probably with help from ZipA)
- Other proteins (FtsA, FtsW) recruited to the ring to form the septal ring
- Further recruitment of other proteins (FtsK, FtsI, FtsN) and the septal ring constricts and MinE ring disassembles
- Constriction is completed, MinE forms a new ring at the centers of the daughter cells
Peptidoglycan biosynthesis (steps)
- UDP derivatives of amino sugars are made in the cytosol
- Amino sugars are transferred to a lipid carrier in the membrane, which carries them across the membrane
- The peptidoglycan is polymerized on the outer surface of the membrane
- A transpeptidation reaction cross-links the peptidoglycan with other peptidoglycan molecules
Phospholipid biosynthesis
ACP-fatty acid chains added on to glycerol-3-phosphate, forming phosphatidic acid
Phosphatidic acid is then coupled to CTP, releasing PPi and forming CDP-diglyceride
- Cytidine diphosphate (CDP) is used as a carrier
Head groups are added to CDP-diglyceride
- serine added, forming phosphatidylethanolamine (PE)
- G3P added, forming phosphatidylglycerol (PG)
Major porins in E. coli
OmpC, OmpF, PhoE
OmpC = smaller and more abundant than OmpF in high osmolarity media and in the intestine
PhoE is produced only under phosphate-limiting conditions to facilitate phosphate uptake
Twitching
Form of cell motility expressed as a jerky translocation over a solid surface
Results from constant extension and retraction of type 4 pili
Catalase
Enzyme produced by aerobes and facultative anaerobes to eliminate hydrogen peroxide by converting it into water and oxygen
Periplasmic space
The space between the outer and cytoplasmic membranes, has an oxidizing environment that allows disulfide bond formation (bacterial cytoplasm is a reducing environment)
Cytoplasmic steps of peptidoglycan synthesis
- NAG-1-phosphate attacks UTP, displacing pyrophosphate and forming UDP-NAG
- Some of the UDP-NAM is converted to UDP-NAM
i) PEP adds enolpyruvate to UDP-NAM, creating Pi and UDP-NAG-enolpyruvate
ii) NADPH reduces the enol derivative to the lactyl moiety (UDP-NAM)
3) sequential addition of L-ala, D-glutamate, L-R3, D-ala-D-ala, and 4ATP to create UDP-NAM-pentapeptide (aka Park’s nucleotide)
4) Parks nucleotide added to membrane lipid carrier
Autolysins
Cell wall hydrolases produced by the bacteria for breakdown of cell wall during cell division/separation, wall morphogenesis and turnover, biofilm formation, and DNA release
N-acetylglucosaminidase and N-acetylmuramidase cut the glycan backbone (which is made of NAG and NAM)
Endopeptidases cut the peptide
Bacitracin
Antibiotic that acts on the bacterial cell wall
Blocks release of Pi from lipid carrier (undecaprenyl-PP —> undecaprenyl-P + Pi), blocking the lipid carrier recycling
Toxic, so only used topically (eg polysporin ointment)
S. aureus
Gram positive bacterium
S layers
Regularly structured (RS) layer composed of proteins or glycoproteins arranged in hexamers, tetramers, or dimers Common in archeobacteria and many eubacteria Functions: protective barrier, maintain cell shape (in some archaea), potential virulence factor for Aeromonas salmonicida
Function of porins in mycobacterial cell wall
low number and long length (compared to gram negative) results in low permeability
Penicillins
AKA beta lactams b/c they all contain a lactam ring
Antibiotic class that acts on bacterial cell wall
Inhibit penicillin-binding proteins, preventing transpeptidation
Used clinically
Flagellar rings
Cytoplasmic membrane: - C ring (most near the base) - S-M ring Periplasm: - P ring (G- only) Outer membrane: - L ring (G- only)
Type 4 pili
Subtype of somatic pili expresesed by N. gonnorrhoeae and Ps. aeruginosa, mediate twitching motility on semisolid surface
Structure:
-Minor pilin subunit (PilE) on the tip - mediates adhesion to host
Retraction of pili mediates motility
CheA
Sensor histidine kinase, autophosphorylates upon sensing a signal, phosphorylates CheY
Surface proteins
Proteins part of cell wall
Can be associated with the cell wall by non-covalent interactions or by covalent bonds (e.g. LPXTG-linked cell wall protein)
Homopolysaccharide EPS types
Polysialic acid, dextrans (branched alpha-1,6 pollyglucose), levans (polyfructose), bacterial cellulose (beta-1,4 polyglucose)
Components of flagellum
Filament (tail), hook (hinge), basal body (anchor)
Categories of flagella arrangement
Monotrichous: monopolar, single
Lophotrichous: monopolar, clustered
Amphitrichous: dipolar, single or clustered
Peritrichous: Lateral (nonpolar - encircling)
Stator
Stationary portion of flagellar motor
The stator consists of multiple copies of the MotA and MotB proteins, forming a Mot complex that surrounds the S-M ring
Mot complex allows passage of protons that drive the attached rotor through rotational steps and generate torque
Beer-Lambert’s Law
I/I0 = 10^-x1
log(I0/I) = x1
-> I/I0 = fraction of light transmitted, LogI0/I = turbitity or optical density (OD)
-> OD is directly proportional to cell density if the mass per cell remains constant - can be measured by a spectrophotometer
I = transmitted light, I0 = incident light
x = cell density
Chlorhexidine
Antimicrobial that targets cell membrane
Cationic at physiological pH
Binds, inserts, disrupts membrane
Topical use only as antiseptic, or as an oral rinse to reduce oral bacterial load
Sigma factors
Bacterial transcription initiation factors that enable specific binding of RNA polymerase to gene promoters
Function of mycolic acids and other glycolipids in mycobacterial cell wall
impede entry of nutrients (causing slow growth) and harmful chemicals (antibiotics, lysosomal components of phagocytes)
Mycobacterial cell wall components
- waxy, hydrophobic, high lipid content
- up to 60% of dry weight can be mycolic acids (long chain, branched fatty acids)
- (high MW) mycolic acids are indirectly linked to peptidoglycan via arabinogalactan
- arabinogalactan/mycolic acid layer is overlaid with proteins and lipids
- Porins are present above the arabinogalactan layer to allow small hydrophilic molecules passage through the outer cell wall layer
- Other glycolipids present: lipoarabinomannan and phosphatidyinositol mannosides (PIM)
CheZ
Phosphatase that removes phosphate from CheY-P
Phosphomycin
Antibiotic that acts on bacterial cell wall
Inhibits MurA, preventing formation of UDP-NAM
Chemostat
Device used to achieve continuous growth
- reservoir feeds fresh medium continuously into growth chamber at a certain flow rate
- concentration of nutrient kept low soo it is the limiting nutrient for growth
- when a drop of medium enters, growth limiting nutrient is used up immediately and the cells cannot continue to grow until next drop of medium enters
- growth rate is thus controlled by flow rate, and growth yield is controlled by concentration of the growth-limiting nutrient
Polymyxin
Cationic antimicrobial peptide
(cyclic peptide with long fatty acid tail)
binds, inserts, disrupts bacterial membranes
topical use (polysporin ointment)
Braun’s lipoprotein
small but abundant molecules that attaches peptidoglycan to the outer membrane
C-terminal lysine is covalently bonded to meso-diaminopimelic acid (mesoDAP) on the peptidoglycan tetrapeptide
N-terminal cysteine is bonded to two fatty acid chains that are embedded in the inner leaflet oof the outer membrane
- N-term cysteine is part of lipoprotein motif “LXXC” - during protein secretion, LXX is cleaved and fatty acid chains are added to the cysteine
Components of Gram positive cell wall
Peptidoglycan, teichoic acid and lipoteichoic acid, surface proteins
Flagella
Long, threadlike proteinaceous structures
AKA H-antigen
Responsible for motility
Chlamydia
Gram negative obligate intracellular bacteria
Exists in two forms:
- elementary body - survives outside host cells, infectious form
- reticulate body - pleomorphic form inside host cells
Human pathogens:
- C. tracomatis: trachoma (eye infection), genital infections
- C. pneumonia: pneumonia, associated with atherosclerosis
Basal body
Base anchor portion of flagellum
Anchors filament and hook to cell envelope by a rod and either 2 (G+) or 4 (G-) rings of integral proteins
Motor present in the base of the basal body
Major porins in E. coli
OmpC, OmpF, PhoE
OmpC = smaller and more abundant than OmpF in high osmolarity media and in the intestine
PhoE is produced only under phosphate-limiting conditions to facilitate phosphate uptake
Hook
Hinge portion of flagellum
Made of copies of a single protein, with hook-associated proteins present to bridge the junction between hook and filament
Porins: structure
Usually homotrimeric proteins
Proteins are beta sheets linked together by beta turns
Beta sheets lie in an anti-parallel fashion and form a cylindrical tube, called a beta barrel
Porin beta sheets contain alternating polar and nonpolar amino acids - nonpolar residues face outwards to interact with membrane; polar face inward to the center of the barrel to form a hydrophilic channel
Porin channel is partially blocked by a loop, called the eyelet, which projects into the barrel - the eyelet defines the size of solute that can traverse the channel
Archaeal cell wall
Made of pseudopeptidoglycan (pseudomurein)
Mycobacterium
Aerobic and nonmotile rods
Considered to be Gram positive despite not being able to be gram stained (stained instead by acid-fast stain)
Grow slowly (weeks) and can survive within macrophages
Classification of prokaryotes based on how they obtain carbon and energy
Photoautotrophs
Chemoautotrophs
Photoheterotrophs
Chemoheterotrophs
Auto = intake carbon dioxide Hetero = intake organic compounds
Rod
Component of flagellar motor, acts as drive shaft that transmits rotation of the SM and C rings across the periplasm and out of the cell
Runs and tumbles
Runs = movements in a single direction for some time due to CCW (bundling) flagellar rotation
Tumbles = abrupt, random changes in direction due to CW (unbundling) flagellar rotation
- when bacteria is moving along desired gradient direction, runs are longer and tumbles delayed (and vice versa)
Pilus structure/composition
- typical pilus = ca. 1000 major pilin subunits arranged in a helical manner with an axial hole
- tip of pilus usually made of a minor pilin subunit, which confers adherence properties
Growth yield constant
When a single nutrient is the sole source of carbon and energy, and its quantity limits bacterial growth, it is possible to define a growth yield constant, Y
Y = amount of dry weight of cells produced per weight of nutrient
e.g. Yglucose for an aerobe = 0.5, which means 50% of glucose is converted into cellular materials and 50% is oxidized to CO2
Fatty acid biosynthesis
Malonyl-ACP and Acetyl-ACP are condensed together to form acetoacetyl-CoA (3-ketoacyl-SACP), releasing 1ACP and 1CO2
reduction (by NADPH), then dehydration, then another reduction to produce Acyl-ACP
Malonyl-ACP molecules repeatedly add 2 carbons at a time to the growing fatty acid chain, each time expelling 1CO2
Mycobacterial cell wall function
Responsible for resistance to antibiotics (except rifampin and streptomycin)
Responsible for ‘acid-fast’ nature –> positive stain fives a presumptive identification in samples
Has adjuvant properties, may be responsible for the development of delayed-type hypersensitivity (DTH)
Steady state growth
State at which all components of a cell culture double at each division - achieved either by maintaining culture in exponential growth by subculturing (not allowing to enter stationary phase) or during continuous growth achieved using a chemostat
EPS functions
Pathogenic determinant
- inhibit phagocytosis (masking cell surface antigen)
- adhesion and biofilm formation
- diffusion barrier (blocks antimicrobials)
Cell-cell recognition and symbiosis
Protection against dessication (drying out)
Carbon and energy storage
Metal (Mg2+) binding
Bacteriophage receptor
Industrial uses
- gelling agent, food thickener (xanthan gum), adhesive (dextran gum)
Archaeal cell membrane composition
Lipids consisting of either C20 isopranoid alcohols ether linked to a glycerol to form monoglycerol diethers, or C40 isopranoid alcohols either-linked to 2 glycerols to form diglycerol tetraethers
Variable head groups attached to the diether/tetraether
mono-di –> form lipid bilayer
di-tetra –> form monolayer
Core oligosacccharide
middle portion of LPS that extends into medium
~10 residues of 4-5 different sugars
- 2 unusual sugars generally present: C8 keto-deoxy-octonic acid (KDO) and C7 heptose
- KDO is commonly used as a marker for outer membrane in cellular fractionation experiment
Composition and arrangement generally the same in most Gram negative bacteria
Ethambutol
Drug used to treat TB
Interferes with the incorporation of arabinogalactan into cell wall
Pilins
Structural proteins that make up pili/fimbriae
Bacterial growth: stationary phase physiological changes
- morphological changes (smaller, from rod to coccoid)
- changes in surface properties (become hydrophobic, more adhesive)
- changes in membrane phospholipids (unsaturated FAs converted to cyclopropyl derivatives by methylation of double bonds)
- Increase in rate of turnover of protein and RNA
- Targeted new protein synthesis (e.g. PhoE porin synthesized under phosphate starvation)
- increase in resistance to environmental stress (some due to synthesis and activity of starvation sigma factor, sigmaS (KatF) encoded by rpoS (katF) gene;
Determination of cell membrane fluidity
Fatty acids more unsaturated = more fluid membrane
Fatty acids more saturated = more rigid membrane
Bacteria can modify the level of unsaturated fatty acids according to growth conditions (e.eg. low temp –> make more unsaturated or branched chain fatty acids)
Acid-fast staining of Mycobacterium: steps
- Heat-fix a smear of sputum sample
- Cover the smear with blotting paper and flood with carbol fuchsin (in phenol)
- Steam for 5 minutes by passing the slide through the flame of a gas burner
- Wash the slide with water
- Add the acid-alcohol drop-wise slowly until the dye no longer runs off from the smear
- Rinse with water
- Counter-stain with methylene blue
Bacteria look pink-ish
Mycolic acids
Long chain, branched fatty acids present in mycobacterial cell wall; used to distinguish between different mycobacteria
Core oligosaccharide biosynthesis
Not well understood
Thought to involve membrane-bound glycosyltransferases, which add one sugar at a time to growing glycan chain
Bacterial cytoplasmic membrane
Phospholipid bilayer composed of phospholipids and proteins
- integral proteins span the membrane, peripheral proteins are partially embedded
Extensive lateral mobility of bulk proteins and phospholipids
Functions as a selective barrier
- water, gases, small molecules can cross
- most polar compounds (amino acids, organic acids, inorganic salts) cannot cross and must be transported by proteins
Bacterial growth curve in a batch culture
Lag phase - no net growth - taking time to adapt to new environment, recovery from toxic products of metabolism, synthesis of new enzymes/coenzymes Log phase -exponential growth Stationary phase - maintenance metabolism - no net increase in cell numbers - nutrient depletion, oxygen limitation, accumulation of toxic metabolic products (stop growing) - state of most bacteria in nature Death phase - depletion of cellular energy, cell lysis due to autolytic enzymes
O-antigen biosynthesis
1&2: glycosyltransferase enzymes sequentially add sugar residues to the C55 lipid carrier to form the polymer
3&4: newly synthesized O antigen is exported across the inner membrane by the ABC transporter (Wzm, Wzt); ATP hydrolysis provides the energy
5: O antigen is ligated to lipid A-core oligosaccharide by a ligase
Heteropolysaccharide EPS types
Anionic (containing non-sugar components such as phosphate, acetate, pyruvate)
Simple (one chain of different sugars)
Complex (branched chains of different sugars)
Catalase test
Add hydrogen peroxide to bacterial sample: bubbles indicate production of oxygen by catalase (and thus presence of catalase in the sample)
Useful in differentiating Streptococcus (catalase negative) from staphylococcus (catalase positive)
Bacteriorhodopsin
A proton pump produced by some archaea to capture light energy for pumping out protons (to generate a proton motive force)
Cell wall steps of peptidoglycan biosynthesis
- Transpeptidation reaction where :NH2 group from diamino acid in 3rd position attacks the carbonyl carbon in the peptide bond holding the two D-alanine residues together in the pentapeptide, displacing the terminal D-ala and creating a peptide bond (inhibited by penicillin)
Taxis
Bacterial movement in response to a stimulus
- phototaxis (stimulus is light)
- chemotaxis (stimulus is chemicals)
- aerotaxis (stimulus is oxygen)
- magnetotaxis (stimulus is a magnetic field orientation)
Capsule
Extracellular polysaccharides (EPS) that are covalently attached to the cell surface (e.g. K antigen)
E. coli
Gram negative bacterium
Porins: location and function
Major proteins in G- outer membrane
Form small hydrophilic channels, allowing passage of low MW neutral and charged solutes (ions, sugars, amino acids, most antibiotics)
Sec system components
- leader peptide (on the membrane protein): important for initial stages of translocation through the membrane
- chaperone proteins (e.g. SecB): soluble protein that transports nascent preprotein from cytosol to the membrane, and prevents preprotein from assuming a tightly folded configuration or aggregating into a complex that can’t be translocated
- translocase: membrane-bound portion, SecYEG, and SecA, a peripheral ATPase which is attached to SecYEG (SecYEG forms a hydrophilic channel, SecA provides energy to push protein through)
Peptidoglycan structure
Two alternating sugars form the backbone (N-acetyl-D-glucosamine or NAG, N-acetyl-D-muramic acid or NAM), and a tetra-peptide (L-ala-D-glu-R-D-ala) is linked to NAM
Gram negative: R is usually mesoDap (D,L-diaminopimelic acid)
Gram positive: R is usually L-lys
Outer membrane functions
Selectively permeable barrier
Confers negative charge on bacteria
Contents of the periplasmic space
Hydrolytic enzymes involved in processing nutrients (e.g. amylases, alkaline phosphatases)
Nutrient binding proteins (e.g. maltose-binding protein) that bind and deliver nutrients to specific cell membrane transporters
Biosynthetic enzymes required for peptidoglycan synthesis
Proteins involved in fitness and survival to stress (e.g. DegP)
Cytochrome Cs that oxidize carbon compounds or inorganic compounds and deliver the electrons to the electron transport chain
Detoxifying agents (e.g. beta-lactamase that degrades penicillins)
Flagellar motor: components
Rotor - part that rotates
Stator - stationary component against which rotor rotates
Lipid A
Part of LPS that is embedded in membrane
Endotoxin, can be released with toxic effect
Hydrophilic head composed of 2 NAG-phosphate molecules joined by beta-1,6 glycosidic bond; 6 fatty acids linked to NAGs
4 of 6 fatty acids are C14 hydroxymyristic acids (not present in cytoplasmic membrane phospholipids)
NAM
N-acetyl-D-muramic acid
One of two basic units of the glycan component of peptidoglycan
CheR
Methyltransferase that methylates MCPs
Synthesis of Guanine tetraphosphate
Synthesized by Re1A (ppGpp synthase I) and SpoT (ppGpp synthase II)
Bayer’s junctions
zones of adhesion between inner and outer membranes of gram-negative bacteria
Culturing of E. Coli vs Streptococccus
E. coli can synthesize all 20 amino acids from glucose but Streptococcus can’t - therefore exogenous amino acids/peptides must be provided to culture streptococcus
Classification of microbes by pH requirement
Acidophiles
Neutrophiles (most microbes)
Alkaliphiles
PBPs
Penicillin binding proteins
(PBP1-6)
PBP1-3 = high MW, catalyze transglycosylation and transpeptidation in peptidoglycan synthesis
PBP4-6 = low MW, appear to be dispensable
Targets of penicillin antibiotics
NAG
N-acetyl-D-glucosamine
One of two basic units of the glycan component of peptidoglycan
Classification of microbes by temperature requirement
Psychrophiles (low temp)
Mesophiles (med temp)
Thermophiles (high temp)
Hyperthermophiles (extremely high temp)
Effect of attractants/repellants on chemotaxis
Attractants reduce rate of CheA autophosphorylation, preventing CW rotation and tumble which would change the bacteria’s course
Repellants increase rate of CheA autophosphorylation, causing CW rotation and tumble to change bacteria’s course
EPS synthesis outside the cell membrane
e. g. dextrans and levans produced by S. mutans
1. sucrose is cleaved into glucose and fructose
2. glucose is synthesized into dextran or mutant by glucosyltransferases (GTFs)
3. Fructose is made into levan by fructosyltransferases (FTFs)
Porins
Major proteins in G- outer membrane
Form small hydrophilic channels, allowing passage of low MW neutral and charged solutes (ions, sugars, amino acids, most antibiotics)
Most are heterotrimeric proteins
Outer membrane composition
Phospholipids (form inner leaflet)
Lipopolysaccharide (found only in outer leaflet)
Outer membrane proteins (OMP) - porins
Lipoprotein (Braun’s, also associated with the peptidoglycan)
Slime
Extracellular polysaccharides (EPS) that are free or loosely attached to the cell surface
ABC transporter-dependent EPS synthesis at the cell membrane: steps
- Sugar residues are polymerized at the inner leaflet of the membrane, possibly involving the lipid carrier
- Completed polysaccharide chain is exported across the cytoplasmic membrane via an ABC transporter
- In gram negatives, the polysaccharide is translocated to the cell surface possibly via Bayer’s junctions
Determination of generation time of a continuous culture
k (growth rate constant) = D (dilution rate)
D = F/V (F = flow rate, mL/h; V = culture volume, mL)
Membrane steps of peptidoglycan biosynthesis
Arrival at membrane: nucleotide sugars diffuse to the membrane, where undecaprenyl phosphate (lipid-P) attacks the UDP-NAM-pentapeptide, displacing UMP
- NAG is transferred from UDP-NAG to the NAM on the lipid carrier (C4 hydroxyl in NAM attacks C1 carbon in UDP-NAG, displacing UDP and forming beta 1-4 bond)
- Disaccharide-lipid molecule flips to other side of membrane
- Transglycosylation where C4 hydroxyl of incoming NAG attacks the C1 of the NAM in the glycan, transferring the NAM-pentapeptide-NAG off the lipid carrier and onto the growing peptidoglycan chain (catalyze by transglycosylase, inhibited by vancomycin)
- Lipid-PP released from glycan chain is hydrolyzed by a membrane bound phosphatase to lipid-P (recycled) and Pi (inhibited by bacitracin)
Barophiles
Category of microbes defined by hydrostatic pressure, like to grow in around 10atm pressure environment
Biological functions of TA and LTA
- Facilitate adhesion (role in colonization)
- Capture divalent cations such as Mg2+, Ca2+, which are important for metabolism and growth (provide negative charge)
- Binding site for some enzymes that cleave peptidoglycan
- Activate complement (proteins in blood, immune response to bacterial invader)
D-ala specific functions:
- Confer resistance to cationic antimicrobial peptides (e.g. polymyxin)
- Activate innate immune response (e.g. induce cytokine production)
Sec system
System used for bacterial translocation of membrane-bound proteins
Important Mycobacterium pathogens
M. tuberculosis - tuberculosis
M. bovis - tuberculosis
- (BCG vaccine)
M. leprae - leprosy
LPS biosynthesis
Three parts created individually
Core oligosaccharidee added to lipid A, then O-antigen added to lipid A-core oligosaccharide molecule by a ligase
Halophiles
Category of microbes defined by osmotic pressure requirement, grow in as high as 30% salt environment
LPS composition
Embedded in membrane:
Lipid A
Extend into medium:
Core poly/oligosaccharide
O-antigen (O-specific polysaccharide)
2 key differences between archaeal and bacterial membrane lipids
Bacteria: ester bond, fatty acids
Archaea: ether bond, isoprene (isopranoid alcohols)
Determining the site of septum formation
Septum formation takes place only at the center of the cell: determined by two systems: Min system and nucleoid occlusion system
Min system:
- MinCD prevent FtsZ ring formation; MinCD oscillate from one side of the MinE ring to the other
- In the MinE-negative mutant, MinCD distribute evenly over the membrane, cells cannot form FtsZ ring (thus septum) and become fillamentous
Nucleoid occlusion system:
- nucleoid mass interferes with FtsZ formation (mechanism unclear)
- mutants defective in DNA segregation form FtsZ rings on either side of the central unsegregated nucleoid
2 main mechanisms of beta lactam resistance
- Expression of beta lactamase
2. Mutations in penicillin binding proteins
Limitations of measuring growth with spectrophotometer
when cell density is too high, measurements become inaccurate, because some of the light is re-scattered and directed towards the detector, lowering turbidity reading
To correlate OD readings to cell numbers, a standard curve must be constructed by measuring the OD of several cell suspensions and counting the cell number independently (must be done for each organism to create standard values)
Lipoteichoic acids
Polyanionic teichoic acids found in most gram-positive cell walls
Linear polymer of phosphodiester-linked glycerol phosphate covalently bound to lipid
C2 position of the glycerol phosphate is usually glycosylated and/or D-alanylated
Amphipathic (neg. charged glycerol phosphate backbone, hydrophobic lipid)
- lipid portion bound hydrophobically to cell membrane, poly-glycerol phosphate portion extends into cell wall
Not covalently bound to peptidoglycan, in some bacteria LTA is secreted and thought to be used as an adhesin at the cell surface (becomes part of glycocalyx)
Flagellar switch
Located directly underneath basal body
Responsible for changing direction of flagellar rotation
Consists of at least 3 proteins: FliG, FliM, and FliN
- FliM and FliN form C ring, FliG binds S-M ring and C ring
-CheY-P binds to FliM to trigger rotational change during chemotaxis
Undecaprenyl phosphate
C55 isoprenoid alcohol phosphate
Synthesized from acetyl-CoA
Common lipid carrier for peptidoglycan, LPS, EPS, and teichoic acid synthesis - helps to solubilize hydrophilic sugars in the hydrophobic membrane
Turbidity
AKA optical density
Measurement whereby growth is determined by the amount of light scattered by a bacterial cell suspension (more dense culture = less light transmitted)
Turbidity = Log(I0/I), where I0 incident light, I = transmitted light
CheB
Response regulator protein, methyltransferase that removes methyl groups from methylated MCPs
Filament
Tail portion of flagellum
Hollow structure made of flagellin proteins arranged in a helix
Vancomycin
Antibiotic that acts on bacterial cell wall
Inhibits transglycosylation by complexing with the D-alanyl-D-alanyl portion of the pentapeptide
Mathematics of bacterial growth
k = (logNt - logN0)/0.301(t)
where: k = growth rate constant, Nt = number of cells at time t, N0 is number of cells at beginning, n = number of generations in time t
g = 1/k
where: g = generation time or doubling time (time for Nt to equal 2N0)
Mycoplasma cell membrane composition
Phospholipids and sterols, which are believed to hold cell shape in lieu of a cell wall
- sterols acquired from environment
- no cell wall means beta-lactam antibiotics are ineffective against Mycoplasma
Lipopolysaccharide alternate names
Endotoxin, Pyogen
Protein folding and stability
Once protein is at the membrane, it will fold into a confirmation that is most thermodynamically stable
- most amino acid side chains of transmembrane segments must be non-polar
- polar part of protein backbone of transmembrane segments must participate in hydrogen bonds. This is most easily accomplished with alpha-helices, for which all peptide bonds are H-bonded internally. It can also be accomplished with beta sheets provided that the beta strands form closed structures such as the beta barrel
Limitations of measuring growth via total cell counts
Does not distinguish live or dead cells (unless live/dead stains [e.g. BacLight] are used)
Cannot be performed on low cell density cell suspensions (not enough cells to be accurately counted)
Bacterial adaptation to chemotaxis signal
When a bacterium adapts to an attractant, it resumes tumbling and will stay in the area
It will run again if/when it encounters a higher concentration of attractant
In this way it remains always in the area with the highest attractant concentration
Mantoux test
Tuberculosis skin test
- small amount of tuberculin (mixture of cell wall components - proteins, mycolic acid, glycolipids - from M. tuberculosis)/PPD (purified protein derivative) is injected intradermally
- swelling is measured after 48-72 hours
Positive test: enlarged, reddened, and raised injection site >10mm (<4mm = negative, 5-10mm = retest needed)
Positive test indicates that the individual has cell-mediated immunity against antigens of M. tuberculosis (as a result of vaccination, infection, or exposure to mycobacteria)
Sigma s
Starvation sigma factor required for synthesis of at least 30 proteins (e.g. catalase) induced by carbon, phosphate, and nitrogen starvation
Peptidoglycan growth at the septum
- peptidoglycan triplet is formed by PBP1A and PBP1B
- the triplet is covalently attached to replace the docking strand of the preexisting peptidoglycan by PBP2 and PBP3 (PBP3 is FtsI)
- the docking strand is removed by PBP4 and PBP7
CheW
Membrane protein that controls rate of autophosphorylation of CheA
Isoniazid
Drug used to treat TB
Blocks the incorporation of mycolic acid into cell wall
Type 1 pili
Subtype of somatic pili expressed by most strains of E. coli, mediate adherence to D-mannose-containing surface receptors on host cells, which leads to bacterial persistence and infection of urinary bladder
Structure:
- Major pilin subunit FimA
- Minor pilin subunit FimH
- Mannose binding site
Freund’s complete adjuvant
Potent solution that contains killed mycobacterial cells, used for immunization
Torque generation by flagellar motor
Overview: protons follow a path within the stator, driving cyclical conformational changes in the stator that apply force to the rotor, causing rotation
- Proper alignment of the stator with the rotor allows entry of a proton which binds to Asp32 on Mot proteins
- Proton binding Asp32 causes a conformational change, causing rotor to drive rotation
- Following conformational change, proton dissociates and enters cytoplasm, reversing the conformational change and causing further rotation
Rotor
Motile portion of flagellar motor
Consists of FliG protein, C ring, and S-M ring
MCPs
Methyl-accepting chemotaxis proteins
Receptors that span inner membrane as dimers and transmit chemotactic signals to the cytoplasm
MCP glutamate residues (4-5 spots per MCP) are reversibly methylated (by CheR) following attractant/repellant binding so the cell can detect changes in their concentration and adjust its swimming style accordingly
Porins: structure
Usually homotrimeric proteins
Proteins are beta sheets linked together by beta turns
Beta sheets lie in an anti-parallel fashion and form a cylindrical tube, called a beta barrel
Porin beta sheets contain alternating polar and nonpolar amino acids - nonpolar residues face outwards to interact with membrane; polar face inward to the center of the barrel to form a hydrophilic channel
Porin channel is partially blocked by a loop, called the eyelet, which projects into the barrel - the eyelet defines the size of solute that can traverse the channel
Pilus types
- Sex pili
- involved in conjugation, facilitate transfer of genetic material - Somatic/common pili
- many subtypes, most mediate adherence to host tissue for colonization and biofilm formation
Chlamydia cell envelope composition
Outer membrane containing LPS and porins
- major outer membrane proteins (MOMPs) in porins are cross-linked by disulfide bonds
Lacks peptidoglycan
- instead has a P layer, consisting of cross-linked high MW cysteine-rich proteins (CRPs) that may be functionally equivalent to peptidoglycan
LPXTG-linked cell wall protein
Type of surface protein
Subtypes: collagen adhesion protein, iron-binding proteins, pilins (in pili), protein A
Have a C-terminal cell sorting signal within the protein
- (~35 aa stretch encompassing a conserved LPXTG motif (X = any amino acid)
- proteins are amide-bonded to the peptide cross-bridge of peptidoglycan by an enzyme called sortase (transpeptidase)
Teichoic acids
Polyanionic polymers of either ribitol phosphate or glycerol phosphate, joined by anionic phosphodiester bonds
Varying molecules may be bonded to glycerol/ribitol phosphate in the backbone, such as D-ala, glucose, NAM
Attached to peptidoglycan via covalent bonds between phosphate of glycerol/ribitol phosphate and the C6 hydroxyl of NAM
Lipid-carrier dependent EPS synthesis at the cell membrane: steps
- Sugar nucleotide precursors interact with undecaprenyl phosphate lipid carrier at the inner leaflet of membrane
- Sugar residues are taken up by the lipid carrier, nucleotide recycled
- Sugar residues are carried to the outer leaflet and added to the growing polysaccharide chain
- Lipid carrier returns to the inner leaflet to transport another molecule of sugar residue
- Completed polysaccharide chain is translocated to the cell surface
Cycloserine
Antibiotic that acts on the bacterial cell wall
Competitive inhibitor of the D-alanine racemase and D-ala-D-ala synthetase
Blocks synthesis of D-ala-D-ala
Functions of bacterial membrane proteins
- solute transport (e.g. ABC transporter for nutrient uptake)
- electron transport (e.g. cytochromes)
- ATP synthesis (e.g. ATPase)
- Protein secretion (e.g. Sec translocase)
- Motility (e.g. flagella rotor)
- sensing environmental signals (e.g. sensor histidine kinase)
- biosynthesis of cell wall polymers and lipids
CheY
Response regulator protein
After phosphorylation by CheA, CheY-P binds to flagellar motor switch to initiate the reverse (CW) rotation
Methods of measuring growth
- Spectrophotometer and measuring turbidity
- Total cell count
- Viable cell count with serial dilutions and determining colony-forming unit by plating
- Dry weight (samples must be free of media components and dried to a constant weight)
- Total proteins (Coomassie blue G250 dye binding assay with a protein standard; or total N content (Kjeldahl method))
2 most common phospholipids in bacterial membrane
Phosphatidylethanolamine, phosphatidylglycerol
Predominant fatty acids in bacteria
Saturated: palmitic acid (C16)
Unsaturated: oleic acids (C18 monounsaturated)
- polyunsaturated fatty acids are not found in bacteria; however they can be branched, hydroxylated, methylated, or contain cyclopropane rings
Classification of microbes based on oxygen requirement
Aerobes
- oxygen is essential; serves as final electron acceptor of electron transport chain
Anaerobes
- oxygen is a deadly poison (unable to eliminate toxic forms of oxygen (superoxide, hydroxyl radicals, hydrogen peroxide)
Facultative anaerobes
- grow in presence or absence of oxygen - don’t need it but it is not toxic to it either (produce superoxide dismutase and catalase to eliminate superoxide radicals and hydrogen peroxide, like aerobes do)
Superoxide dismutase
Enzyme produced by aerobes and facultative anaerobes to eliminate superoxide radicals, converting them into hydrogen peroxide + oxygen
Rhizobium-legume symbiosis
Rhizobium adheres to plant cells via interaction between its EPS and the plant protein called lectin
Adherence leads to the formation of root nodules
In the nodules, Rhizobium fixes N2 from the atmosphere, providing nitrogen for the plant in return for the plant providing it with carbon
Important role in agriculture (happens in legume plants)
OM-mediated antimicrobial resistance
Downregulation or mutation of porins, mutate amino acid residues in the beta-barrel –> slow down rate at which antibiotics. can enter the cell
