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Microbial Physiology > Lecture 2 - Exam 1 > Flashcards

Lecture 2 - Exam 1 Flashcards

1
Q

What are Pili?

A

Protein filaments on cell surface. Some pili are anchored in the cell membrane, but most are not and where they originate and how they are attached to the cell surface is unknown.

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2
Q

Where are Pili found (what kind of cells)?

A

Found almost exclusively in G- bacteria.
Gm + recently discovered in Corynebacterium, Actinobacteria, and Streptococcus species.

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3
Q

What are the characteristics of Pili?

A

Very diverse in length and thickness. Shorter, straighter, and more fragile than a flagellum. Usually present in large numbers on the surface.

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4
Q

What is another name for Pili?

A

Fimbriae

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5
Q

What is the production of pili like? Where are pili often found?

A

It is tightly regulated. Pili are often found in bacteria isolated directly from the environment. Pili are often lost when grown under laboratory conditions.

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6
Q

Pili will not grow in liquid culture. What does that tell us?

A

Prokaryotes need the pili in order to compete in their natural environment, but not in the culture. Hence, it is increases their fitness in their natural environment.

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7
Q

What are the various functions that Pili mediate?

A

Adhesion, twitching motility and social motility (type IV pili), phage receptor, conjugation (sex pili)

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8
Q

What type of Pili are responsible for adhesion?

A

Type 1 Pili

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9
Q

Adhesion (or attachment) is mediated by what on pili?

A

Adhesion is mediated by proteins located at the tip of the pili called adhesins. Adhesins recognize and bind to specific receptors on the surface of cells or other substrates.

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10
Q

Adhesins recognize and bind to specific receptors on the surface of cells or other substrates. However, some bacterial pathogens’ bind to what?

A

Some bacterial pathogens’ adhesins bind to glycolipids or oligosaccharides in cell surface receptors. These oligosaccharides can act as natural receptors for pathogens, whose pili bind to those receptors.

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11
Q

Type 1 Pili, or mannose-sensitive pili, attach to what?

A

Many strains of E.coli, Salmonella, and Shigella have pili that attach specifically to the mannose glycoside residue on cell membranes. This attachment can be prevented by the addition of mannose, because the mannose competes with the pili for the receptor. Mannose inhibits E. coli Type-1 pili-mediated attachment via FimH.

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12
Q

Different Pili types mediate attachment to different substrates. What can this do?

A

Can be responsible for host and tissue specificity.

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13
Q

What are the functions that Type IV pili are responsible for?

A

Social motility, twitching motility, serve as a receptor for a phage, and required for biofilm formation for P. aeruginosa.

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14
Q

What can phages do to a recipient cell? An example?

A

Introduce new genetic information. An example would be Lysogenic conversion.

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15
Q

What is Lysogenic Conversion? What is the best known example?

A

The introduction of phage DNA into the host genome, which can confer enhanced virulence. Best example: The binding of the CTX phage to the toxin-coregulated pili of Vibrio cholerae.

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16
Q

Virulent strains of Vibrio cholerae have a type IV pili called?

A

Toxin Coregulated Pili (TCP)

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17
Q

What are the two primary functions of Toxin Coregulated Pili (TCP)?

A
  1. Colonization of intestinal mucosa by attachment to specific cell receptors.
  2. Acts as the receptor for the cholera toxin phage (CTX). The CTX phage carries the cholera toxin genes, which converts the recipient cell into a toxin-producing strain. The cholera toxin phage recognized the TCP on the cell surface, binds to it, and translocates into the cell cytoplasm.
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18
Q

What is the Toxin Coregulated Pili (TCP) required for?

A

Virulence, without it, there would be no colonization and no cholera toxin.

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19
Q

What is twitching motility that involve Type IV pili? What is most well studied in?

A

Twitching motility is a surface motility mechanism mediated by Type IV pili in some Gm- bacteria…. Most well studied in: Pseudomonas aeruginosa and Neisseria gonorrhoeae.

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20
Q

When does twitching motility occur?

A

When type IV pili (often several bundled pili) are extended and adhere to a surface. The pili are then retracted, pulling the cell(s) forward. Twitching occurs on moist surfaces of moderate viscosity, equivalent to that of 1% agar.

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21
Q

What is social motility that involves type IV pili?

A

Another type of gliding motility used by Myxobacteria.

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22
Q

What are the two types of motility used by Myxobacteria?

A

A motility: Adventurous motility -> cells move individually with the aid of slime secreted by the cell, does not use pili.
S motility: Social motility -> cells are propelled forward by extension and retraction of Type Iv pili, but are also pushed forward by secretion of slime at the opposite pole of the cell.

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23
Q

What do type IV pili mediate in Myxobacteria?

A

Type Iv pili also mediate attachment to adjacent cells and coordinate movement as a wolfpack, or swarm, of Myxobacteria

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24
Q

Describe the conjugation function of pili.

A

Pili are important for horizontal transfer of genetic information via conjugation.

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25
Q

What do sex pili do during conjugation?

A

Sex pili are coded for by the F plasmid, and mediate conjugal transfer of plasmids.
Sex pilus grows from the “donor” cell and mediates cell-cell contact between donor and recipient bacterial cells.
Requires cell-to-cell contact.

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26
Q

What is a plasmid?

A

A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently.

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27
Q

Who will retain a plasmid after conjugation?

A

The donor and recipient will both retain a plasmid after conjugation since ssDNA is transferred.

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28
Q

How many plasmid-encoded functions can be transferred? Sex pili can be important for what?

A

Multiple plasmid-encoded functions can be transferred, and sex pili can be important for virulence.

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29
Q

What is the Glycocalyx?

A

Any material found external to the cell wall (CW).
Are predominately polysaccharides and/or proteins.
Protect bacteria from phagocytosis by host immune cells (acts as a protective coating and prevents opsonization).
Has nonspecific attachment (not recognizing/requiring receptor) to biotic and abiotic surfaces, have a role in biofilm formation (extracellular matric).
Protect bacteria from desiccation.
Includes S layers, capsules & slime or exopolysaccharides

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30
Q

What is the S-layer?

A

Array of proteins or glycoproteins external to the cell wall.

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31
Q

Where is the S-layer present?

A

Gm +, Gm -, and Archaea.
In some archaea, the S layer tightly covers the cell membrane and acts as the cell wall -> in these organisms, the S layer is not considered a glycocalyx.

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32
Q

What is a capsule?

A

It is a thick, structured layer of repeating polysaccharide units or glycoproteins at the cell surface. Capsule generally ensheath the cell.

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33
Q

What is a capsular polysaccharide?

A

Covalently attached to cell.

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34
Q

What may capsules act as? Example?

A

Capsules may act as virulence factors.
Example: E. coli strains have more than 80 different capsular polysaccharides, called K antigens!

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35
Q

What does the presence of K antigens in E. coli do?

A

It reduces opsonization which inhibits phagocytosis by immune cells.

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36
Q

What is an exopolysaccharide (EPS)?

A

Loosely adhered polysaccharide.

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37
Q

What does slime refer to?

A

Slime refers to other loosely adhered material that isn’t polysaccharides.

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38
Q

What does the cell wall protect bacteria from?

A

Turgor (most bacteria can not live without CW)

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39
Q

What does turgor result from?

A

Solute concentration difference between inside and outside of the cell.

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40
Q

Bacteria generally live in environments _____ dilute than the cytoplasm, resulting in ___________.

A

More.
Resulting in: a net influx of water and a large hydrostatic pressure (turgor) of several atmospheres (!) directed against the cell membrane.

41
Q

Describe Gm - cell wall.

A

Comprised of an outer membrane and a thin layer of peptidoglycan.

42
Q

Describe Gm + cell wall.

A

Comprised entirely of a thick layer of peptidoglycan.

43
Q

The cell wall determines what of the bacterial cell?
What is the cell wall made up of?

A

The cell wall determines the shape of the bacterial cell.
The cell wall is made up of many molecules that are found only in bacteria (ex: Peptidoglycan, LPS, etc.)

44
Q

What can disturb the cell wall?

A

Antibiotics (synthetic and natural). Antibiotics designed against many cell wall components do not affect the host cell because of the unique compounds present only in bacterial cell wall.

45
Q

What does Lysozyme do? What’s an example of where a lysozyme can be in?

A

Lysozyme hydrolyzes NAG-NAM glycosidic linkage B-lactams (Penicillin) and prevent transpeptidation (cross-linking). An example: in tears and saliva

46
Q

Do all prokaryotes have a cell wall?

A

No! Mollicutes are bacteria that lack a cell wall (Mycoplasma, Spiroplasma etc.)

47
Q

Describe Mollicutes.

A

Has the smallest genome of any free-living prokaryotes.
Their absence of a cell wall results in hypersensitivity to fluctuation in salt concentrations.
Instead of a cell wall, they have a strong membrane made of sterols.

48
Q

What are sterols (present in mollicutes)?

A

Are hydrophobic ringed structures that are normally found in eukaryotes and are rigid. Mycoplasma may have acquired sterols from a eukaryotic host.

49
Q

What is peptidoglycan (PG)?

A

Heteropolymer of glycan chains cross-linked by peptides that surrounds the whole cell to confer strength and rigidity to the cell wall.
It provides the strength and rigidity of the cell wall.
Forms a three-dimensional network surrounding the cell membrane, covalently bonded throughout by glycosidic and peptide linkages.

50
Q

What is peptidoglycan made out of?

A

It is composed of alternating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) attached each other via B-1,4 linkage

51
Q

What do antibiotics do to cells during peptidoglycan synthesis?

A

Antibiotics, such as penicillin, vancomycin, or bacitracin, that interfere with peptidoglycan synthesis cause the cells to swell and burst (from turgor pressure).

52
Q

What gives peptidoglycan its strength?

A

Its covalent bonds throughout.

53
Q

During cell growth, what must be broken within peptidoglycan?

A

Its stabilizing bonds within peptidoglycan.
Precise and tight regulation of bond hydrolysis during growth is vital importance to prevent cell destabilization and death.

54
Q

Discuss the glycan chains in Peptidoglycan.

A

The glycan chains are cross-linked from one NAM unit to another by a tetrapeptide.
In G-, this crosslinking between L-R3 and D-alanine is usually direct.
In G+, there is often an additional peptide bridge connecting the L-R3 and D-alanine.

55
Q

What forms are the amino acids in the tetrapeptide of Peptidoglycan?
Why are these forms important?

A

D-forms.
It matters if it is L or D amino acid because almost all L-amino acids are targeted by enzymes. The D-form is hardly targeted by degrative enzymes, so it is beneficial for the bacteria to adopt this form, as it allows them to be resistant to these enzymes.

56
Q

What are bacterial racemases?

A

They catalyze the conversion of the L-amino acid to the D-amino acid form required for peptidoglycan biosynthesis. These racemases are very important protective adaptation, protecting the bacteria from degrative enzymes.

57
Q

Discuss peptidoglycan in gram negative bacteria.

A

In G- Peptidoglycan is attached noncovalently to the outer envelope via lipoprotein.
G- PG can be isolated as a sac of pure PG “murein sacculus”

58
Q

Gram Negative:
Where do you find diaminopimelic acid (DAP)?
Cross-linking happens where?

A

Commonly found at R3 (other things are found at R3 in the molecule, too).
Cross-linking normally direct between D-Ala and DAP
Light cross-linking (~50% in E. coli)

59
Q

What would happen if there was a DAP mutation?

A

E. coli would not be able to grow. DAP would be need to be added into the media for it to grow.

60
Q

Discuss peptidoglycan in gram positive bacteria.

A

GM+ PG cannot be isolated as a sac.
PG is covalently bound to various polysaccharides and teichoic acids in the cell wall.

61
Q

Gram Positive:
Where do you find diaminopimelic acid (DAP)?
Cross-linking happens where?

A

Commonly found at R3 (other things are found at R3 in the molecule, too)
Cross-linking indirect between D-Ala and R3 may involve a peptide bridge.
Heavy cross-linking.
In some G+, PG = ~ 50% total cell weight and almost 90% of the cell wall weight.

62
Q

The degree of PG cross-linking determines what?

A

The degree of rigidity of the cell wall.
G+ = indirect cross-linking, heavy cross-linking, so more rigid
G- = direct cross-linking, lighter cross-linking, so less rigid

63
Q

What is the function of Peptidoglycan?

A

Mostly structural, lends rigidity and strength to cell wall

64
Q

What peptidoglycan degradative molecule exists?

A

Bacterial PG hydrolases

65
Q

NAM residues have an attached tetrapeptide with alternating L- and D- isomers of amino acids. What is the tetrapeptide conformation?

A

NAM – L-alanine – D-glutamate – L-R3 – D-Alanine

66
Q

Where are Peptidoglycan precursors made?

A

Uridine diphosphate (UDP) derivatives are made in the cytoplasm (UDP-NAG and UDP-NAM).
Here, a pentapeptide is added to UDP-NAM… the extra peptide added is another D-Alanine to the end, which requires a specific enzymes and ATP.
The Result is a UDP-NAM pentapeptide, referred to as Park’s Nucleotide.

67
Q

Each step of biosynthesis of peptidoglycan is catalyzed by what?

A

Each step is catalyzed by a different enzyme and requires ATP.

68
Q

Discuss every step of Peptidoglycan biosynthesis.

A

1) Synthesis of UDP-NAG and UDP-NAM pentapeptide (the precursors) in the cytosol.
2) UDP - NAM - Pentapeptide, often called Park’s Nucleotide.
3) A membrane associated lipid carrier (bactoprenol, aka Lipid P and undecaprenyl phosphate) attacks the phosphate in UDP-NAM-pentapeptide and displaces one of the phosphates. The result is the NAM-pentapeptide (NAMPP) is transferred to the lipid carrier, resulting in a
NAMPP - Lipid P + Pi complex (also called Lipid I)
4) A molecule of UDP-NAG is added to the NAMPP - Lipid PP complex. The resulting complex is NAG - NAMPP - Lipid PP (also called Lipid II) .
5) NAG - NAM - pentapeptide - Lipid PP monomers units synthesized in the cytoplasm are translocated across the cytoplasmic membrane to the periplasm by a flippase present in the cell membrane.
6) The NAG-NAM pentapeptide monomers are the cross-linked to pre-existing peptidoglycans by transglycosylation. Once the NAG-NAM pentapeptide monomers are translocated into the periplasm, the transglycoslase portion of the PBP releases the Lipid-PP and transfers the NAG-NAM subunits to the growing glycan chain.
NOTE: NO ATP IN PERIPLASM… The Lipid-PP is hydrolyzed to Lipid-P (bactoprenol) and phosphate. This hydrolysis results in a substantial release of energy that is used to complete the transglycosylation reaction. This hydrolysis also recycles the Lipid-P and phosphate needed for more glycan subunit transfers.
7) The PBP catalyzes transpeptidation, the peptide bond formation between NAM-monomer subunits.
8) Peptidoglycan relies on PBPs for covalent bond formation outside of the cytoplasm. However, as the name states, many antibiotics bind to PBPs and inhibit peptidoglycan synthesis.

69
Q

Peptidoglycan synthesis requires two activities. What are they?
These activities are carried out by what?

A

Transglycosylation to polymerize the glycan chains & Transpeptidation to catalyze peptide cross-linking between two adjacent glycan chains.
These activities are carried out by high molecular weight, bifunctional enzymes called penicillin-binding proteins (PBPs), anchored in the cell membrane. The PBPs have a transglycosylase domain and a transpeptidase domain to catalyze cross-linking.
In summary:
PBPs catalyze the transglycosylation and transpeptidation and they interact covalently with B-lactams.

70
Q

During peptidoglycan synthesis, there is a step where PBP catalyzes transpeptidation. Discuss the details of the transpeptidation reaction.

A

The transpeptidation reaction is a nucleophilic displacement reaction, where the peptide bond between the D-alanine - D-alanine in one pentapeptide is exchanged for a peptide bond between the penultimate D-alanine and the amino acid in L-R3 position, displacing the terminal D-alanine (yielding tetrapeptide). There is no energy required here because the D-alanine - D-alanine bond was made at the expense of ATP in the cytoplasm, and this is just the exchange of one bond for another.

71
Q

How many PBPs does E. coli have?

A

7

72
Q

Some PBPs (high molecular weight PBPs) are ________, while some low molecular weight PBPs are not required for ________.

A

Indispensable ; PG synthesis

73
Q

What are two of the major other polymers that may be present in G+ cell walls, that is covalently linked to the glycan chain of peptidoglycan?

A

Teichoic acid and lipoteichoic acid

74
Q

Describe Teichoic acid.

A

Found in almost all G+ bacteria and is covalently bound to peptidoglycan, meaning there is no removing teichoic acid from peptidoglycan without some serious work.
They are negatively charged polymers of glycerol-phosphate or ribitol-phosphate.

75
Q

Describe Lipoteichoic acid.

A

These are teichoic acids bound to membrane lipids and NOT covalently bound to PG.
They are amphipathic, with the lipid portion bound hydrophobically to the cell membrane and the phosphate extending into the cell wall.

76
Q

Describe the roles of “Wall teichoic acids.”

A
  • Regulation of cell morphology and division
  • Regulation of PG biosynthesis machinery
  • Protecting cells from host defenses and antibiotics
  • Contributing to virulence through biofilm formation and host cell adhesion and colonization
77
Q

Many G+ bacteria do not have lipoteichoic acid. What is the major example of this, and instead of lipoteichoic acid what is used?

A

Mycobacterium is the major example.
Instead of lipoteichoic acid, they have other amphiphilic glycolipids that may perform similar functions.

78
Q

What does the mycobacteria cell wall consist of?

A

Peptidoglycan, arabinogalactan (AG), and mycolic acid (MA), all covalently linked.

79
Q

What are Mycolic acids?

A

Waxy lipids found in Mycobacteria genus that provides protection from desiccation, hydrophobic antimicrobials, acids and bases. Mycolic acids form a hydrophobic layer on the external face of the cell wall.
Also, allows acid fast stains.

80
Q

Bacteria with mycolic acids in the cell wall are called?

A

Acid-fast bacteria.

81
Q

What is the major polysaccharide of the mycobacterial cell wall?

A

Arabinogalactan and it also anchors MA to PG.

82
Q

Mycolic acid may also be bound to __________.

A

Trehalose.

83
Q

What is trehalose?

A

It is a disaccharide of D-glucose, forming a compound called cord factor when MA is bound to trehalose.

84
Q

What does the presence of a cord factor do?

A

Gives Mycobacterium tuberculosis its characteristic appearance of long chains of cells forming serpentine, ropelike “cords.”

85
Q

Gram negative bacterial cells have a structurally and chemically complex cell wall composed of:

A

Outer membrane and underlying peptidoglycan layer. The PG layer lies within the periplasm.

86
Q

The gram negative bacterial cell wall does what?

A

Acts as a selective barrier (pores for transport of small hydrophilic solutes), contains phage receptors, has pathogenic properties, and acts as a barrier for periplasmic components (keeps things in).

87
Q

Describe lipopolysaccharides (LPS).

A

The outermost portion of the gram negative cell wall, attached to the extracellular portion of the outer membrane.
LPS: Lipid A + Core + O-antigen.
LPS are negatively charged with hydrophilic surface. Is a barrier to hydrophobic components like bile salts and cationic antibiotics.
The LPS is rigid because of saturated fatty acid in Lipid A.

88
Q

The Lipid A portion of LPS can do what?

A

It can have toxic effect when released from bacterial cells (like toxic shock).

89
Q

Describe Lipid A.

A

Lipid A portion of LPS is highly conserved.
It is hydrophobic, buried in the outer membrane, and pyrogenic (endotoxin & induces fever).

90
Q

Lipid A is an endotoxin. What does that mean?

A

It is not secreted by bacteria into the extracellular environment, but is released from dying bacteria.

91
Q

Toxicity from Lipid A is due to…?

A

Activation of complement and release of cytokines that results in localized high concentration of complement and cytokines.

92
Q

Describe biosynthesis of Lipid A.

A

The first four reactions in biosynthesis of peptidoglycan and Lipid A are the same:
Lipid A is synthesized in the cytoplasmic membrane.
The early steps of synthesis are catalyzed by three cytoplasmic enzymes ; two acyltransferases and a deacetylase.
Remember these are the four steps of peptidoglycan biosynthesis:
1) Synthesis of UDP-NAG and UDP-NAM pentapeptide (precursors).
2) UDP-NAM pentapeptides are transferred to the lipid carrier (bactoprenol).
3) Polymerization of UDP-NAG and NAM PP-lipid.
4) Translocation of NAG:NAM PP-lipid across the cell membrane (flippase).

93
Q

Describe the core region of LPS.

A

Comprised of the inner and outer core, attaching Lipid A and O antigen.

94
Q

Describe the O-Antigen.

A

O Antigen: repeat oligosaccharide of 4-6 sugar residues.
It is instrumental for host colonization and environmental niche adaptation.
It is highly variable (between and among bacterial species). One of the most diverse bacterial cell constituents.
It is highly immunogenic and targeted by adaptive immune system and phage.
Used for serotyping.
Many bacterial adaptations to alter O-antigen structure (O-antigen switching) to evade immune response,
It is synthesized as a separate polymer on a lipid carrier (bactoprenol), then transferred as a unit to the core-lipid A complex (instead of adding molecules one at a time, like the core).

95
Q

Describe LPS transport.

A

It is not know how LPS moves from the outer surface of the inner membrane into the outer membrane.
Two hypotheses:
1) A chaperone carrier transports LPS through the periplasm.
2) Adhesion sites between the inner and outer membrane may provide a channel through the periplasm.
BOTH METHODS WOULD REQUIRE ATP & REMEMBER: there periplasm does not contain ATP.

96
Q

What are some of the other components of gram negative cell envelope?

A

Phospholipids : lipids with covalently attached phosphate
Lipoproteins: small proteins with lipid tail at the N-terminus, lipid portion interacts with lipids in the OM, and protein end of some lipoproteins are covalently bound to peptidoglycan to anchor outer membrane to PG.
Outer Membrane Proteins:
-porins: form small non-specific hydrophilic pores for transport of small neutral and charged solutes. Porin synthesis is highly regulated.
-specific solute transport proteins: uptake of larger solutes.

97
Q

Summary of Bacterial Cell Wall for GM+ and GM-

A

Thickness of cell wall-
GM+: 20-80 nm GM-: 10 nm
PG content-
GM+: >50% GM-: 10-20%
Teichoic acid-
GM+: Present GM-: Not present
Lipid and lipoprotein content-
GM+: 0-3% GM-: >50%
Protein content-
GM+: <1% GM-: ~10%
LPS-
GM+: Not present GM-: 13%
Penicillin-
GM+: More sensitive GM-: Could or could not be sensitive
Lysozyme-
GM+: More sensitive GM-: Could or could not be sensitive

98
Q

Describe the Archaeal Cell wall.

A

It is complex. Has much different cell walls and their cell walls differ based on environment.
They have a S-layer (proteinaceous layer that is part of the cell wall).
NOTE: In bacteria, S-layer is in addition to the cell wall.

Microbial Physiology (22 decks)

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