Lectures 1-3 Flashcards
1
Q
How do bacteria differ from eukaryotic cells ?
A
Smaller, Lack nucleus, lack organelles, contain cell wall, lack sterols in membranes, some produce flagella, some produce capsules/ECM for biofilms, different ribosomes structure (70s vs 80s)
2
Q
What is the bacterial cell wall made up of?
A
Peptidoglycan, alternating residues of N-Acetylmuramic acid (NAM) peptide chain- 3 variant amino acids with a terminal D-ala that can be cross-linked to the 3rd AA of a neighboring chain.
3
Q
What ways can a crosslink occur between the 4th D-Ala and the 3rd AA ?
A
direct or via a multi AA bridge. Crosslinking adds strength to the peptiodglycan.
4
Q
Gram neg bacteria usually have what type of crosslinking?
A
intra peptide bridge
5
Q
Gram positive bacteria differ where? What form of cross linking do they have?
A
Differ in the AA of the side chain and often use peptide bridges
6
Q
What two classes does gram staining divide into? What factors contribute to classification?
A
Gram positive and negative, based on cell wall and membrane structure
7
Q
What are differences between gram + and gram - bacteria?
A
Gram + bacteria have a thick peptidoglycan layer and an inner cytoplasmic membrane. Gram - bacteria have an outer membrane, thin peptidoglycan layer, and an inner cytoplasmic membrane.
8
Q
What are the steps of the gram stain for each type of bacteria?
A
Staining with crystal violet, both gram + and - cells appear purple under oil-immersion light microscopy. Treatment with IODINE aggregates the crystal violet both types appear PURPLE. Washing with ETHANOL collapses peptidoglycan and extracts lipids from the outer membrane . Gram + cells appear PURPLE, gram - cells appear COLORLESS. ***
9
Q
Counterstaining with safarnin changes the staining to what?
A
gram + cells appear purple, gram - appear red.
10
Q
What are some exceptions to gram staining bacteria?
A
Growth state can affect the gram stain. Some exceptions include:
mycobacteria- acid-fast stain, high lipid content
rickettsia- acridine orange stain, intracellular parasites
chlamydia- dna probes, may be visualized with fluorescent antibodies, giminez stain, obligate intrace. parasites. Mycoplasma/ureaplasma- culture used for ID, lack cell wall, too small for standard light microscopy. Spirochetes-darkfield microscopy , too slender for standard microscopy
11
Q
What confers cell shape and arrangement?
A
bacterial wall (peptidoglycan)
12
Q
What are the basic bacterial shapes?
A
cocci, bacillus, vibrio, fusiform, spirochete, filamentous, pleomorphic.
13
Q
What are basic bacterial arrangements?
A
diplo, tetrad, sarcinae, strepto, staphylo
14
Q
Can bacteria form stable wall-defective cells?
A
Yes, some bacteria can. Some bacteria can undergo a programmed change in their membrane so the membranes are stable with a reduced (cell wall defective) form or no cell wall ( L -form)** enterococcus faecalis, norcardia, steptococcus pyogenes, proteus mirabilis, bacillus sui, staphylococcus epidermidis
15
Q
What charge do teichoic acids confer?
A
negative charge when attached to the wall
16
Q
What does the amount of teichoic acid depend on ?
A
The bacterial species, and the amount of peptidoglycan. The cell wall can have proteins covalently attached to the peptide side chain in place of a peptidoglycan crosslink ( predom. in gram + bacteria)
17
Q
What are cell wall associated proteins?
A
Proteins covalently linked to peptidoglycan in place of crosslinking. The cell wall can have proteins cov. attached to the peptide side chain in place of a peptido. crosslink. ( predom in gram + bacteria)
18
Q
What is the first step of cell wall biosynthesis?
A
- cytoplasmic steps( synthesis of peptidoglycan subunits) NAM is synthesized from NAG
A pentapeptide chain is attached to NAM. D-ALA is derived from L-ALa by racemase.
19
Q
What is the second step of cell wall biosynthesis?
A
Cell membrane step, an undecaprenyl carrier transfers the NAG-NAM pentapeptide subunit to the outer leaflet of the cytoplasmic (inner membrane)
20
Q
What is the third step of cell wall biosynthesis?
A
Cell wall steps. Existing cell wall is cleaved by autolysins and the addition of new subunits is carried out by penicillin binding proteins. (PBPs)
21
Q
What is the fourth step of cell wall biosynthesis?
A
The new subunits are added by transglycosylation (sugar bonds) and transpeptidation (peptide bond) reactions carried out by PBPs.
22
Q
What antibiotics inhibit cell wall synthesis?
A
Phosphomycin , D-Cycloserine, Bacitracin, Vancomycin, B-lactams
23
Q
What does Phosphomycin/ fosfomycin do to the cell?
A
It is a PEP analog that inhibits the synthesis of NAM from NAG
24
Q
What does D-cycloserine do?
A
analog of D-alanine that inhibits the racemase reaction and addition of D-Ala to the AA1-AA2-AA3 chain.
25
What does Bacitracin do?
binds undecaprenyl -PP and inhibits recycling of the carrier.
26
What does Vancomycin do?
It is a glycopeptide antibiotic that binds D Ala D Ala and sterically hinders PBPs from carrying out transpeptidation and transglycosylation reactions.
27
What do B-Lactams do?
B-lactams are structurally related to D-Ala and tightly bind PBPs inhibiting their transglycosylation and transpeptidation activity.
28
How do bacterias become resistant to cell wall inhibitory antibiotics?
Enter a quiescent state, convert to L forms( undergo programmed change in cell membrane so they dont lyse) , Mycoplasma lack cell walls
29
What do cell wall synthesis inhibitory antibiotics require ?
They require growing cells for activity. Growing cells need to increase their cell wall, hence they produce autolysins that 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.
30
What are bacterial membranes made up of?
Phospholipids
31
What substances do bacterial membranes contain?
phophotidylethanolamine, phosphotidylglycerol
32
What do membranes NOT contain?
They do not contain sterols with the exception of Mycoplasma that incorporate sterols from their environment into their membranes.
33
What is the structure of gram positive bacteria?
Gram + membrane structure contains: peptidoglycan, lipoproteins, lipoteichoic acid, and membrane proteins
34
What does Gram - structure contain?
Both leaflets of the cytoplasmic (inner mem) are made of a mix of phosphotidylglycerol and phosphotidylethanolamine. The inner leaflet of the outer membrane is made pred. of phosphotidylethanolamine. The outer leaflet of the outer membrane is composed of LPS. Lipoproteins are anchored in the membrane by a lipid tail.
35
Endotoxin LPS is composed of what?
Lipid A , core glycolipid, O-specific oligiosaccharide subunit composed of repeating sugar subunits. ** Only gram - bacteria are capable of causing endotoxic shock, a type of septic shock.
36
What does the outer membrane of gram - bacteria contain for transport?
Porins non specific(water filled channels) or specific A number of transporters are located in the cytoplasmic membrane of gram - and gram + bacteria.
37
What can cause intrinsic resistance to antibiotics?
The gram negative outer membrane is an additional barrier for antibiotics. This is an example of intrinsic antibiotic resistance.
38
What are polymyxin membranes?
Fatty acid tails attached to a large cyclic head group. Disrupts the bacterial membrane causing release of cytoplasmic components. Have the ability to destroy quiescent or dormant (non replicating) cells. target non-growing cells, bind best to LPS
39
What is the effectiveness of polymyxin ?
Gram negative outer membrane LPS phosphatidylethanolamine > gram positive > human mamalian cells. It is used predominantly as a topical antibiotic because of toxicity for human cells.
40
What is a capsule and what is its function?
Discrete layer associated with individual cells. Fxns to mediate adherence, protect from phagocytosis, protect from desiccation/drying.
41
What is the matrix layer?
Slime layer which is a secreted layer that embeds the cells allowing for biofilm formation. Biofilm matrix: secreted layer embeds cells FXNS: reserve of carb for subsequent metabolism, matrix for formation of biofilms
42
What are Pili/Fimbriae? FXNS?
protein structures involved in attachment , adherence, conjugation, motility, gliding/twitching Fxns: attachment, conjugation (DNA exchange), motility (gliding and twitching)
43
What are flagella? FXNS?
Composed of flagellin , uses the proton motive force to power baterial motility. 3 arrangements: 1. Monotrichous (polar) 2. lophotichous 3. peritrichous
FXN: motility, attachment
44
Do bacteria have organelles?
NO , no nucleus/organelles, they have tightly coiled DNA that can be referred to as a nucleoid. The nucleoid has no membrane so it is not a nucleus.
45
How is DNA arranged in the bacterial cell?
DNA is compacted to fit in the bacterial cell. Most bact. chromosomes are circular. DNA is supercoiled and compacted. Structural maintenance proteins aid in this process.
46
What are the functions of DNA gyrase and topoisomerase?
They allow for replication starting at the origin of replication. These are targets for antibiotics.
47
What do DNA gyrases specifically do?
DNA gyrases bind to DNA and catalyze strand cleavage and exchange to affect DNA supercoiling. **IT IS NEEDED TO make DNA accessible for transcription and replication.
48
What binds to DNA gyrase:ATP complex?
quinolones and floroquinolines bind to the DNA gyrase :ATP complex thus blocking transcription and DNA replication.
49
What inhibits DNA gyrase activity?
Novobiocin
50
What occurs when Novobiocin binds ?
Gyrase is inhibited and causes the cell to become bacteriocidal which leads to cell death due to collisions of replication machinery with gyrases and blockage of further DNA replication.
51
What type of test can be used to identify a specific gene in a pathogen of interest?
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.
52
What are the three phases of bacterial growth?
1. Lag- before cells start to grow ( can mimic adaptation to a new body site) 2. log- exponential growth in the host but results in increased bacterial numbers. 3. stationary- can be survival = balanced slow growth in a subpopulation( no net gain in numbers) death= culture slowly dies out or cells in stationary phase can be hypermutable and accumulate mutations
53
What does exponential growth reflect
Binary fission of bacteria generally into two daughter cells
54
What is the doubling time?
The doubling time (generation time) of the culture is the time it takes for the number of cells to increase by a factor of 2 . ( in graph - 30 mins)
55
What are some factors that slow bacterial growth in the host?
nutrient availability, bacterial cell density= competition, build up of toxic byproducts, immune system
56
What pathway is used to convert glucose to pyruvate?
the Embden-Meyerhof- Parnas pathway converts glucose to pyruvate.
57
What is consumed in the EMP pathway?
2 high energy phosphates, ATP , high energy phosphate PTS system- group translocation.
58
What is produced in the EMP pathway?
4 ATP are produced. NADH+ H+ is produced and must be oxidized by either reduction of pyruvate (fermentation/oxidation) or by being used in an electron transport system ( respiration)
59
What alternative sugars can be used by bacteria?
hexoses, complex sugars, citrate
60
What are the various end products of fermentation?
acids , alcohols, gas. ( lactic acid, ethanol, butyric acid)
61
What can some bacteria use for respiration?
NADH+ H+
62
How does bacteria breakdown pyruvate via the TCA cycle?
Bacteria that respire transform pyruvate into acetyl co-A and further break it down to Co2. This provides bacteria with NADPH for biosynthesis, additional molecules of NADH+ H+ for use in generating a proton motive force, and FADH+H+ that can also be used for the generation of a proton motive force.
63
How is a proton motive force generated?
A proton motive force is produced by passing the electrons from NADH+ H+ to a series of electron carriers that use the XS energy to pump protons(H+) outside the cell.
64
What are some carriers used to use the XS energy to pump protons out of the cell?
Carriers include cytochromes, quinones, flavoproteins (Fe-S proteins)
65
Fermentation yields more or less energy than respiration?
LESS energy
66
What are some anaerobic respiration final electron acceptors?
thiosulfate, NO3, SO4, C02
67
Can both aerobic and anaerobic bacteria respire?
YES
68
What does the proton motive force do in bacteria?
It is used to produce ATP through F0/F1 Atpase synthase that transport ions and flagellar rotation (motility) in bacteria.
69
How does ATP synthase work in reverse?
It works in reverse to charge the membrane in bacteria that obtain energy only from fermentation.
70
The re-internalization of the protons through ATPase is used to do what?
This creates a membrane potential and a pH gradient . The re-internalization of the protons through the ATPase is used to produce ATP, transport small molecules/ions, and to power flagella for motility.
71
What benefit does running a pump backward have?
Bacteria that use fermentation only can run the pump backwards, using ATP to pump protons out of the cell and charge the membrane.
72
What are some physical parameters for bacterial growth?
pH- most pathogens are neutrophiles or acidophiles , salt concentration- most pathogens are non-halophillic or halotolerant, temp- most pathogens are mesophils although cold-tolerant bacteria and psychophiles are important in food spoilage. ( grow best at 37 celsius)
73
Where do anaerobes grow?
ONLY in absence of oxygen
74
What are strict anaerobes?
killed by O2
75
What are aerotolerant anaerobes?
They can withstand O2
76
What are microaerophillic anaerobes?
prefers reduced O2 tension for growth
77
What are aerobes?
Grow ONLY in the presence of OXYGEN
78
What are obligate aerobes?
only use O2 as terminal electron acceptor, must have oxygen
79
What are faculatative aerobes?
can grow with or without O2
80
What are the different types of growth media classifications?
complex- numerous non chemically defined nutrient sources ( yeast), defined- all components are present in known amounts, enriched- has components to help bacterial growth (blood) differential- allows differentiation of bacteria selective- allows growth of only certain bacteria
81
What are differences between selective and differential MacConkey's agar?
selective- bile salts, crystal violet( accumulates in cell to inhibit most gram + bacteria), lactose - blocks growth. Differential - lactose as a C+E source and neutral red to detect acid production. Will show if fermentation is or isnt used , enables one to tell the difference.
82
Can a single medium have multiple characteristics
yes, differential and selective
83
What is unique about fastidious bacteria?
They have special growth requirements. Very specific
84
What are blood agar plates used for?
to determine if bacteria produce hemolysins.
85
What are the three types of hemolysis reactions?
gamma hemolysis (non hemolytic) no detectable lysis, alpha hemolysis-partial lysis of RBCs surrounding the bacterial colony indicated by a green zone around the colony , beta hemolysis- complete lysis of RBCs surrounding the bacterial colony.
86
What are bacterial siderophores used for?
To provide Fe2+ , siderophores allow bacteria to scavenge iron from human proteins and from other bacteria.
87
What is the 70s ribosome made up of ?
A 30S and 50S ribosomal subunit composed of multiple proteins.
88
What rRNAs are associated with the 50s and 30 s subunit?
23S rRna is associated with the 50S subunit and a 16S rRNA is associated with the 30S ribosomal subunit.
89
What are the 4 basic steps of bacterial translation?
1. formation of the initiation complex 50S and 30S subunits 2. transfer of a transfer RNA bound to an AA into the acceptor A site 3. formation of the peptide bond-peptidyl transfer 4. translocation of the growing peptide to the P site to restart the cycle.
90
What are Aminoglycosides?
bind 16S rRNA and A site of 30S ribosomal subunit blocking loading of charged tRNA
91
Oxazolidinones- zyvox ( linezolid)
Interacts with 50S ribosomal subunit block formation of the initiation complex active against MRSA , VRE and multiresistant S pneum.
92
Tetracycline
interacts with 16S rRNA and 30S ribosomal subunit weakens codon-anti codon interaction
93
Chloramphenicol
binds 30S rRNA and 50S subunit and blocks catalytic center for peptidyl transfer reaction (peptide bond formation)
94
Lincosamides
binds 30S rRNA 50S subunit and inhibit peptide bond formation
95
Macrolides (Erythromicin)
binds 50S subunit causing release of the peptide chain
96
Streptogramins
bind 50S subunit and block translocation of the peptide chain to the E (P) site
97
What do type 1 transporters do?
export small molecules , can act as nonspecific pumps that can also pump antibiotics out the cell conferring resistance. ONLY in gram negative bacteria. Can be drug efflux pumps.
98
What is type II secretion?
general secretory proteins Sec and Tat secrete most proteins in gram + bacteria. They are coupled to Type II secretion in gram negative bacteria.
99
What is type III secretion?
delivers toxins to eukaryotic cells . Proteins are synthesized in the cell, but they are not secreted until the bacterial cell comes in contact with a eukaryotic cell. Secreted proteins often include proteins that form a channel in the euk. membrane for the "injection" of toxins directly into the eukaryotic cell.
100
What is type IV /V secretion?
Allows for the secretion of self-assembling cellular structures( pilin), DNA , Toxins, also depend on Sec and Tat in gram + and gram - bacteria.
101
Why are RNA polymerases a target for antibiotics?
They have the same basic function as eukaryotic cells, but proteins are different , making it a target for antibiotics.
102
What directs the RNA polymerase to the promoter?
the sigma factor, assembly in bacteria does not happen without the sigma factor
103
What are the steps to bacterial transcription?
the RNA polymerase assembles at the promoter forming the closed complex. DNA is melted forming an open complex. The core transcribes mRNA and the sigma factor disassociates. In prokaryotes, there is no nuclear membrane so transcription and translation is coupled.
104
What is Rifampicin?
Rifampicin binds the closed complex of RNA polymerase and sigma prevents melting. This inhibits bacterial transcription. RNA polymerase still binds but transcription is not initiated.
105
How are transcription and translation related?
they are coupled , when translation stops, RNA can form terminator. Because bacteria do not have a nucleus, transcrip and trans. can be coupled with the ribosome binding the mRNA before transcription is complete.
106
What are polycistronic messages?
bacteria can have these polycistronic messages (operons) that encode for more than one protein. The long mRNA is produced and ribosomes are loaded at individual start initiation sites. (Shine Delgarno and ATG start codon) ends at individual stop codons for each protein on the long piece of mRNA. Overlap between stop and start codons can lead to translation of an upstream gene regulating translation of a downstream gene.
107
What are planktonic cells?
Planktonic cells refer to growth not in biofilms, usually in liquid culture or in the blood during septicemia.
108
What are adherent cells?
Refer to growth attached to a surface but in the absence of matrix and biofilm formation.
109
What are biofilms?
cells organized in a structure with a matrix material.
110
What is quorum sensing?
A type of cell-cell signaling. The ability to determine the number of cells present of the same or different species. Small diffusible signals are produced at a constant rate. The concentration increases in proportion to the total number of bacteria. When the concentration surpasses a critical threshold a signal is transduced. Signals can be homoserine lactones in gram negatives, peptides in gram positives, and SAM derivatives of both.
111
What does biofilm formation require?
1. attachment/adhesion, 2. growth to form microcolonies, 3. Mature 3D biofilm, secretion of matrix, maturation of the biofilm, 4. cellular detachment/dispersal
112
What is important in biofilm formation? What changes in many physiologic properties are present?
Quorum sensing is important. Cells in biofilms are heterogenic. phys properties include: UV light ( survival in the environment) increased genetic exchange (virulence genes and antibiotic resistance genes) Reduced sensitivity to antibiotics NOT due to resistance genes.
113
What can quorum sensing be used to detect?
can detect gram negatives (homoserine lactone derivatives) and gram positives (modified peptides) within the same species. It can also be used to sense many species using molecules like AI-2 , a common metabolic byproduct of SAM biosyn.
114
What are the different types of signals?
oligopeptides: gram positive bacteria, Nacyl homoserine lactone- gram negative bacteria, and Autoinducer-2 - interspecies cross talk SAM derivative metabolic by product between species
115
How do biofilms protect bacteria from the immune response?
antibodies do not bind efficiently and phagocytic cells cannot engulf the biofilm structures. Protects from antibody deposition and phagocytosis.
116
Where do biofilms form?
They can form on natural (biotic) surfaces and implants (abiotic) surfaces. Natural surfaces: normal flora, chronic infections, CF, endocarditis, otitis media, rhinosinusitis ABIOTIC- indwelling devices
117
What commonly grows in biofilms?
Microbiota ( normal flora) including GI tract microbiota, dental plaque. Greater than 700 species , vary between individuals. Often, these are mixed species biofilms with hundreds of different species. Composition of dental flora can differ between people.
118
What do implants colonized with biofilms cause ?
device failure, detachment of organisms cause classical planktonic infection, and release of endotoxin( if gram - bacteria are present)
119
Why are some biofilms very resistant to antibiotics?
inhibition of antibiotic penetration, changes in metabolism of bacteria, changes in membrane permeability, increased expression of efflux pumps.
120
How does a cycle of septicemia occur?
1. biofilm on a device 2. biofilm bacteria shed= septicemia 3. treat septicemia but biofilm is resistant, 4. biofilm regrows- sheds, etc
121
The conditions in biofilm are heterogenic in respect to what?
oxygen, quorum signals, nutrient availability, and secreted factors such as metabolites.
122
Biofilm resistance to antibiotics can lead to what?
infection cycles : resistance: 1. inhibition of antibiotics , 2. metabolic changes, 3. changes in membrane permeability, 4. increase in efflux pumps
123
Differences in secreted factors, bacterial density, and nutrient availability, can lead to different phenotypes. How are these phenotypes regulated?
Transcriptionally
124
Cells in stationary phase can become...
Can become hypermutable and accumulate mutations
125
What does stochastic gene regulation ( biphasic /bimodal) expression do?
Genes turn on and off in a small percent of the population. These become persisters.
126
What are persisters?
Dormant or slow growing cells that are present due to stochastic gene expression where bistable switches natural fluctuations in gene expression allow a small subpopulation of bacteria to switch from one stable phenotype to another. Persisters can be present in any population in any growth phase.
127
How does resistance occur?
Biofilms can be highly resistant to antibiotics. Resistance can come from physical protection due to interaction of the antibiotic and the matrix material.
128
What causes resistance?
Resistance is often due to changes in the physiologic state of the bacteria including: increases in efflux pump expression, changes in membrane permeability, changes in physiologic activity due to changes in oxygen, bacterial numbers, and nutrient availability and formation of persisters.
129
What is sporulation?
Some bacteria can undergo a programmed cell development cycle. Under conditions of high cell density ( sensed by quorum sensing) and nutrient starvation, the cells develop into spores. Vegetative growth= high growth
130
What are some characteristics of spores?
They are dormant and resistant. They are dehydrated with multiple layers protecting them. They are difficult to kill and require autoclaving and bleach (hypochorite) to eliminate them
131
What does RNA polymerase do?
Carries out bacterial transcription
132
What is the RNA polymerase core made up of?
2 alpha, a beta and a beta prime subunit. The holoenzyme consists of sigma factor associated with the core. The structure of bacterial RNA polymerase is distinct and is a target of antibiotics.
133
What does the sigma factor do?
The sigma factor targets the RNA polymerase to the promoter gene.
134
Where are bacterial promoters located?
They are located around -10 and -35 upstream of the transcription site.
135
What does sporulation use?
A cascade of sigma factors . Bacteria can contain specialized sigma factors, which can be activated during different growth phases and under various stress conditions
136
Where does the sigma factor bind?
IT binds to consensus -10 and -35 sequences and activate numerous genes ( promoter regions)
