1_Bacteriology I Flashcards
bacteria:
define
- small, single cell organisms
- 0.1-10 micrometers in length
- seen w/ light microscope
how do bacteria differ from eukaryotic cells?
8 differences
- all are:
- smaller
- have cell wall
- different ribosome structure (70S in bacteria vs. 80S in ribosomes)
- lack
- nucleus
- other organelles (membrane bound vesicles)
- sterols in membranes
- some can
- produce flagella and pili/fimbriae
- produce capsules and extracellular matrix for biofilms
describe basic structure of bacterial cell wall
- composed of peptidoglycan
- pattern
- alternating residues of NAG and NAM
- NAG: N-acetylglucosamine
- NAM: N-acetylmuramic
- peptide chain of 3 variant amino acids w/ a terminal D-Ala, which can be crosslinked to 3rd amino acid of a neighboring chain
- alternating residues of NAG and NAM
purpose of cross-linking in the bacterial cell wall?
how can the cross-link vary?
- fxn: cross-linking adds strength to the peptidoglycan
- the cross-link b/w 4th D-Ala and the 3rd amino acid can be either:
- direct, or
- via multi-amino acid bridge
purpose of gram-staining
divides and identifies pathogenic bacteria into two different classes on the basis of cell wall and membrane structure.
steps of gram staining, and colors that result
- staining with crystal violet
- both purple: Gram positive and Gram negative cells appear purple under oil-immersion light microscopy
- treatment with iodine aggregates the crystal violet
- both purple: Gram positive and Gram negative cells appear purple
- washing with ethanol collapses peptidoglycan and extracts lipids from the outer membrane
- Purple/Colorless: Gram positive cells appear purple; Gram negative cells are colorless
- counterstaining with Safarnin
- Purple/Red: Gram positive cells appear purple; Gram negative cells appear red
after which steps of gram staining will both the gram positive and gram negative bacteria be purple?
Steps 1 and 2
- staining with crystal violet - both purple under oil-immersion light microscopy
- treatment with iodine aggregates the crystal violet - both purple
just list the 4 steps of gram staining, not the color that results
“Color Is Everything #Science”
- staining with crystal violet
- treatment with iodine aggregates the crystal violet
- washing with ethanol collapses peptidoglycan and extracts lipids from the outer membrane
- counterstaining with Safarnin
after which step of gram staining yields both purple and red staining?
After counterstaining w/ Safarnin
Yields purple gram-positive, and red gram-negative
compare cell wall of Gram-Positive and Gram-Negative (outer to inner layers)
and example of bacteria
Gram-positive:
- peptidoglycan cell wall
- inner plasma membrane
- E.g. S. aureus
Gram-negative:
- cell wall:
- outer membrane +
- periplasmic space containing peptidoglycan layer
- inner plasma membrane
- e.g. E. coli
which organisms do NOT stain well with Gram reagents?
why?
- Mycobacteria – high lipid content in wall
- Rickettsia, Chlamydia – obligate intracellular parasites, Gram-negative cell wall structure
- Mycoplasma, Ureaplasma – lack cell wall –> too small for standard light microscopy
- Spirochetes – most are too slender for standard LM
can growth state affect gram-staining?
yes!
e.g. old cultures – stationary phase and some species of bacteria when directly stained in clinical specimens
how to visualize:
mycobacteria
acid-fast stain
(cannot be gram-stained due to high lipid content in cell wall)
how to visualize:
rickettsia
Giminez stain or acridine orange
(can’t be gram-stained bc it is an obligate parasite; gram negative cell wall structure)
how to visualize:
chlamydia
DNA probes used for rapid ID;
intracellular includions may be visualized w/ fluorescent antibody; can also use giemsa, giminez, or other stains
(can’t be gram-stained bc it is an obligate parasite; gram negative cell wall structure)
how to visualize:
mycoplasma or ureaplasma
culture and patient serology
(can’t use gram staining bc lacks cell wall; too small for standard LM)
how to visualize:
spirochetes
darkfield microscopy used to be standard,
but now ELISA is used for treponema and borrelia
(can’t use gram-staining bc most are too slender for standard LM)
what factor confers cell shape and arrangement of bacteria?
cell wall (specifically peptidoglycan)
basic bacterial shapes
- cocci: roughly spherical
- bacillus: rod-shaped
- vibrio: curved-rod shape (comma shape)
- fusiform: tapered ends
- spirochete: flexible spirally twisted
- filamentous: long, branching structure
- pleomorphic: alter their shape or size in response to environmental condition
basci bacterial arrangements
- diplo: pair
- tetrad: four
- sarcinae: arranged cubes of eight
- strepto-: arranged in chains
- staphylo-: arranged in irregular, often grape-like clusters
how do these bacterial arrangements differ?
sarcinae, strepto-, staphylo-
- sarcinae: arranged cubes of eight
- strepto-: arranged in chains
- staphylo-: arranged in irregular, often grape-like clusters
under normal conditions, what causes a cell to lyse?
cells w/o a cell wall would lyse bc the membrane cannot withstand the internal turgor pressure
how can bacteria stay stable even w/o a cell wall?
some can undergo a programmed change in their membrane so the membranes are stable w/ a reduced (cell-wall defective form) or no cell wall (L-form)
what confers a negative charge to the cell wall?
what determines the amount of this polysaccharide?
- teichoic acids attached to the wall confer a negative charge
- amount of teichoic acid depends on:
- bacterial species and the
- amount of peptidoglycan
teichoic acid:
define
- acidic polysaccharide covalently linked to NAM (N-acetylmuramic) –> gives typical negative charge to more abundant Gram+ bacteria
- can act as barrier to penetration of negatively charged molecules
cell-wall associated proteins:
define
- proteins covalently linked to peptidoglycan in place of cross-linking
- (KC: the cell wall can have proteins covalently attached to the peptide side chain in place of a peptidoglycan crosslink (predominantly in Gram positive bacteria)
*steps of cell wall biosynthesis
- cytoplasmic steps (synthesis of peptidoglycan subunits)
- NAG –> makes NAM
- A pentapeptide chain is attached to NAM (AA1-AA2-AA3-D-Ala-D-Ala).D-Ala is derived from L-Ala by a racemase.
- cell membrane step
- an undecaprenyl carrier transfers the NAG-NAM-pentapeptide subunit to the outer leaflet of the cytoplasmic (inner membrane)
- cell wall steps
- existing cell wall is cleaved by autolysins and the addition of new subunits is carried out by penicillin binding proteins (PBPs).
- The new subunits are added by transglycosylation (sugar bonds) and transpeptidation (peptide bond) reactions carried out by penicillin binding proteins.
Abx that inhibit cell wall synthesis, and where they act
- Phosphomycin/Fosfomycin is a PEP analog that inhibits the synthesis of NAM from NAG.
- D-cycloserine is an analog of D-Ala and inhibits the racemase reaction and addition of D-Ala to the AA1-AA2-AA3 chain
- Bacitracin binds undecaprenyl-PP and inhibits recycling of the carrier.
- Vancomycin (glycopeptide antibiotic) binds D-Ala-D-Ala and sterically hinders PBPs from carrying out transpeptidation and transglycosylation reactions.
- b-lactams are structurally related to D-Ala and tightly bind PBPs inhibiting their transglycosylation and transpeptidation activity.
what conditions are needed for inhibitory antibiotics to work?
these require growing cells for activity
- Growing cells need to increase their cell wall, hence they produce autolysins to cleave the wall prior to inserting new peptidoglycan.
- In the absence of new subunits, the autolysins will eventually destroy the cell wall. These resulting cells are osmotically fragile.
how can bacteria become resistant to cell-wall-inhibitory antibiotics?
- enter a quiescent state (learn later)
- convert to L-forms – undergo programmed change in cell membrane so they don’t lyse
- Mycoplasma lacks cell wall
key components of bacterial membranes?
what do these membranes lack, as compared to eukaryotes?
- PHOSPHOLIPIDS, w/ hydrophilic head/ hydrophobic tail:
- made up primarily of phosphotidylethanolamine and phosphotidylglycerol
- LACK sterols
- *exception is Mycoplasma, which incorporate sterols from their environment into their membranes
components of gram-positive membrane
- lipoproteins
- lipoteichoic acid
- membrane proteins
components of gram-negative membrane
- both leaflets of the cytoplasmic (inner membrane) are made of a mix of phosphotidylglycerol and phosphotidylethanolamine
- inner leaflet of the outer membrane made of predominantly phosphotidylethanolamine
- outer leaflet of the outer membrane is composed of LPS (lipopolysaccharide)
which leaflets have:
- mix of phosphotidylglycerol and phosphotidylethanolamine
- predominantly phosphotidylethanolamine
- lipopolysaccharide
- mix of phosphotidylglycerol and phosphotidylethanolamine
- both inner and outer leaflets of inner membrane
- predominantly phosphotidylethanolamine
- inner leaflet of outer membrane
- lipopolysaccharide (LPS)
- outer leaflet of outer membrane
lipopolysaccharide is composed of:
- lipid A
- core glycolipid
- O-specific oligosaccharide subunit composed of repeating sugar subunits
which bacteria are capable of causing endotoxic shock?
why?
- Only Gram-negative bacteria are capable of causing endotoxic shock, a type of septic shock.
- Due to presence of Lipopolysaccharide in the outer leaflet of the outer membrane –> upon cell lysis and release of LPS
two types of porins, and where are they found?
- types:
- non-specific, (water-filled channels)
- specific
- found in outer membrane of Gram negative bacteria
where are transporters located in gram-positive and gram negative bacteria?
A number of transporters are located in the cytoplasmic membrane of Gram negative and Gram positive bacteria
how does the gram-negative outer membrane affect Abx?
acts as an additional barrier;
example of intrinsic antibiotic resistance
polymixin:
define and characteristics
- antibiotics, used predominantly as topical antibiotic (due to toxicity for human cells)
- composed of a fatty acid tail and cyclic peptide head group
- disrupts bacterial membrane causing release of cytoplasmic components
- will destroy quiescent or dormant (non-replicating) cells
polymixins have a high affinity for…
LPS and phosphatidylethanolamine
rank the effectiveness of polymixins from high to low:
human cells, gram positive, gram negative
High to Low
Gram negative (most effective) > Gram positive > Human cells
capsule:
define and functions
- discrete layer assoc. w/ individual cells
- functions:
- mediate adherence
- protect from engulfment by phagocytes
- protect from drying/ dessication
matrix:
define and functions
- (slime layer) secreted layer which embeds cells allowing for biofilm formation
- functions:
- carbohydrate reservoir
- matrix for biofilm formation
fimbriae/ pili:
define and functions
- protein structures
- functions:
- involved in attachment, adherence, conjugation (DNA exchange)
- motility (gliding/twitching motility)
flagella:
define and functions
- composed of flagellin
- function:
- uses the proton motive force to power bacterial motility
- 3 arrangements:
- Monotrichous (polar)
- Lophotrichous
- Peritrichous
describe the DNA organization in bacteria
- Bacterial cells have tightly coiled DNA that can be referred to as a nucleoid.
- nucleoid has no membrane, so it is not a nucleus
- Most bacterial chromosomes are circular (supercoiled and compacted; a number of structural maintenance proteins aid in this process)
which proteins allow for replication starting at the origin of replication (ORI), and are target of antibiotics?
- DNA gyrase and topoisomerase IV
DNA gyrase:
function
- binds to and relaxes DNA, and catalyzes strand cleavage and exchange to affect DNA supercoiling and compaction –> to fit DNA into bacterial cell;
- makes DNA accessible for transcription and replication
which drugs bind to DNA gyrase?
- quinolones and floroquinolines bind the DNA gyrase: ATP complex thus blocking transcription and DNA replication
- novobiocin inhibits DNA gyrase activity
principle behind PCR
- Many pathogens have genes unique to only the single species
- The ability to PCR amplify that gene can be used as a diagnostic indicator that the specific pathogen is present
