Bacterial Structure and Classification

Question 1

What are the two major shapes of bacteria?

Answer 1

rods (bacilli) and spheres (cocci).

Question 2

Bacilli can be found in chains, but chains of cocci are more often noted and are called _______.

Answer 2

streptococci

Question 3

How can cocci present?

Answer 3

Cocci can also be found in pairs (diplococci) in addition to chains, e.g., the classic “kissing kidney beans” appearance of Neisseria gonorrhoeae, or in “grape-like clusters” characteristic of staphylococci

Question 4

What are some other bacterial shapes?

Answer 4

comma-shaped vibrio species, e.g., Vibrio cholerae, the causative agent of a watery diarrhea, and corkscrew-shaped spirilli and spirochetes, for which the etiologic agent of syphilis, Treponema pallidum, is an example.

Question 5

What are the major differences between eukaryotic and prokaryotic cells (i.e. bacteria)?

Answer 5

1. Bacteria are much smaller (yet are significantly larger than viruses)
2. Bacteria lack a formal nucleus and other membrane-bound organelles associated with eukaryotes. The bacterial genome usually comprises a single, circular DNA chromosome that lacks introns and is not bound by histone proteins.
3. While animal cells lack cell walls, bacteria synthesize cell walls composed of peptidoglycan, which distinguishes them from the cell walls of plants (composed of cellulose) and fungi (chitin)

Question 6

What structure of bacteria is a major target for therapeutic intervention? Why?

Answer 6

because peptidoglycan is unique to bacteria, the cell wall and its biosynthetic machinery serve as major targets for antimicrobial therapy

NOTE: The ribosome, smaller in bacteria than animal cells, RNA polymerase, and topoisomerases are also good targets

Question 7

What cell wall characteristics distinguish gram- positive bacteria from gram-negative bacteria?

Answer 7

it is thick and heavily cross-linked in Gram positive bacteria and thin and lightly cross-linked in Gram negative bacteria

Question 8

Significant points about the cell membranes of bacteria (2 things)

Answer 8

1. Bacteria cell membranes lack sterols in their membranes.
2. Gram negative bacteria possess a second, outer membrane

Question 9

What is the major structural difference between gram-positive and gram-negative bacteria?

Answer 9

the major difference is the additional outer membrane of Gram negative bacteria

Question 10

What are the major roles of pili in bacteria?

NOTE: they are found in both gram-positive and negative bacteria, but are much more common in gram-negative bacteria

Answer 10

The “sex pilus” is used to transmit genetic material from one bacterium to another during conjugation.

The major role of pili in pathogenesis, however, is to attach bacteria to host cell surfaces. Without firm attachment to some tissues, colonization is difficult.

Question 11

What kind of antigen so flagella possess? Do all bacteria have flagella?

Answer 11

flagella bear the “H” antigen used in serotyping. Not all bacteria are motile nor do all possess pili for attachment.

Question 12

What are capsules and what do they do?

Answer 12

Capsules are external structures composed of either polysaccharide or polypeptides, and serve to protect encapsulated bacteria from phagocytosis.

Question 13

Do all bacteria contain capsules?

Answer 13

Not all bacteria are encapsulated, but virtually every bacterium that causes meningitis contains a capsule.

Question 14

What are spores?

Answer 14

Spores (or endospores) are dehydrated, dormant forms that allow potential pathogens to survive (in some instances, for centuries) during harsh conditions. The Bacillus species that causes anthrax is a prime example.

Question 15

What kinds of bacteria can form spores?

Answer 15

gram-positive only

Question 16

What is the composition of gram-positive cell walls?

Answer 16

Up to 50% of the Gram positive cell wall is composed of teichoic acid or lipoteichoic acid, polymers of glycerol phosphate or ribitol phosphate

lesser species such as the carbohydrate Lancefield antigens of streptococci (important in serotyping) and M proteins of group A streptococci.

Question 17

Where is the peptidoglycan layer of gram-negative bacteria found?

Answer 17

generally only two layers deep, lightly cross-linked, and lies between the outer and cytoplasmic membranes, a region called the periplasmic space

Question 18

What is the purpose of the outer membrane in gram negative bacteria?

Answer 18

is negatively charged, which helps these bacteria evade phagocytosis, hinder antibiotic uptake, and avoid the action of complement.

Question 19

How do nutrients enter gram negative bacteria?

Answer 19

To allow the import of nutrients, the outer membrane contains porins, molecular sieves that allow substrates such as sugars to be transported across the membrane. The sugars are then bound by periplasmic binding proteins and delivered to the cytoplasmic membrane, where they are again transported into the cytoplasm.

Question 20

What is a major component of the OUTER membrane of gram negative bacteria?

Answer 20

lipopolysaccharide (LPS), found in the outer leaflet of the membrane.

Question 21

What is the composition of LPS?

Answer 21

LPS contains lipid A, a toxic phospholipid also known as endotoxin, a core polysaccharide, and O antigen polysaccharide, the major surface antigen of Gram negative bacteria and an important component for serotyping some species

Question 22

How does the body react to Lipid A?

Answer 22

Lipid A is recognized by the innate immune response to elicit a “cytokine storm” which leads to sepsis or septic shock.

NOTE: Gram positive bacteria are now the major cause of sepsis despite lacking lipid A; the eliciting antigen is not clearly understood.

Question 23

T or F. The successful pathogenesis by some bacteria depends on their ability to secrete proteins or other substances from the cell.

Answer 23

T

Question 24

How do gram negative bacteria secrete proteins from the cell?

Answer 24

To date, at least five such systems have been described, but three are most significant to viability and pathogenesis: Types I, II, and III

Question 25

How do Type I secretion systems work in gram-negative bacteria?

Answer 25

Type I secretion systems are members of the ABC (ATP binding cassette) family of transporters. All Gram negative bacteria appear to encode a Type I system, through which proteins are directly secreted into the extracellular environment from the cytoplasm (i.e. don’t stop in the periplasm).

Question 26

What is the significance of Type I secretion systems to bacterial pathogenesis?

Answer 26

Antimicrobial drugs can be expelled from the cell using these systems, thus promoting resistance to the antibiotic.

Question 27

Are Types I and II secretion systems found in ALL gram negative bacteria?

Answer 27

Yes, ALL. No gram positive bacteria

Question 28

What are Type II secretory systems used for?

Answer 28

Type II systems serve as the general secretory pathway, used to deliver proteins to the periplasm and extracellular spaces. In the Type II system, proteins are first secreted to the periplasm before being secreted across the outer membrane

Question 29

Where are Type III systems found?

Answer 29

Type III protein secretion systems are found only in pathogens

Question 30

Where are Type III systems referred to as “molecular syringes”?

Answer 30

because their structure allows the direct injection of toxins and other virulence factors into the cytosol of a targeted animal cell.

Question 31

How are gram stains performed?

Answer 31

In the Gram method, a suspension of bacterial cells is dried and heat-fixed to a glass slide. A purple stain, crystal violet, is applied to the
cells, and then “fixed” to the cells by the addition of a mordant, typically iodine. The cells are then destained by the application of alcohol to the cells. The extensive cell wall of Gram positive bacteria retains the crystal violet while the thin layer of peptidoglycan found in Gram negative bacteria does not. As a result, Gram positive bacteria stain purple/blue in the Gram protocol. Gram negative bacteria are counterstained with safranin, a red/pink dye, so that they can be seen under a microscope.

Question 32

What happens when the cell wall of enzymes is broken down?

Answer 32

When the cell wall is disrupted under physiologic conditions the bacterial cell lyses.

peptidoglycan is a prominent target for antimicrobial therapy

Question 33

What is peptidoglycan composed of?

Answer 33

Peptidoglycan is composed of a backbone of repeating disaccharides comprising N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG)

Question 34

The disaccharides of peptidoglycan are linked to form long chains by an enzyme termed a _____.

Answer 34

transglycosylase

Question 35

How are peptidoglycan molecules attached to one another?

Answer 35

A pentapeptide composed of L- and D-isomers of amino acids is attached to the NAM residue of each disaccharide, and these pentapeptides cross-link to one another to bridge the sugar backbones.

Question 36

What enzymes facilitate the cross-linking of peptidoglycan molecules?

Answer 36

transpeptidases and carboxypeptidases, the former often referred to as penicillin-binding proteins because this is how they were first discovered: they are the targets of the antibiotic penicillin.

Question 37

T or F. The cross-linking differs between Gram positive and Gram negative bacteria

Answer 37

T. it is a simple bond in Gram negative bacteria, but in Gram positive bacteria, a pentaglycine interpeptide links the polypeptides

Question 38

NAM-pentapeptides contain what at their terminal end?

Answer 38

a terminal D-alanine pair. These amino acids are critical to the cross-linking process and the ultimate D-alanine is removed by the carboxypeptidases. The pair is also a target of antimicrobials.

Question 39

What does Cycloserine do?

Answer 39

inhibits the incorporation of the a terminal D-alanine pair into the pentapeptide side chain

Question 40

What do glycoproteins such as Vancomycin do?

Answer 40

bind the terminal D-alanine pair, masking their availability for cross-linking.

Question 41

How does cell wall assembly occur?

Answer 41

Cell wall assembly begins in the cytoplasm, where individual NAM-NAG disaccharides linked to a pentapeptide side chain are synthesized and attached via a diphosphate linkage to a lipid carrier. The lipid carrier is localized in the cytoplasmic membrane and is used to transport individual disaccharide-pentapeptide subunits across the membrane to the external face of the cytoplasmic membrane. There the subunit is incorporated into the growing cell wall by the action of glycosidases and transpeptidases.

Question 42

What happens once the lipid carrier drops off its dissacharide to the growing cell wall?

Answer 42

The lipid carrier then recycles to the interior leaflet of the cytoplasmic membrane to recharge with new cargo, but only after dephosphorylation of the carrier has occurred.

Question 43

What drug targets/inhibits the dephosphorylation of the lipid carrier during cell wall assembly?

Answer 43

Bacitracin

Question 44

What is Lysozyme?

Answer 44

Lysozyme is a natural defense that targets the peptidoglycan cell wall. Lysozyme is a glycosidase that hydrolyzes the bond between NAM and NAG, thus disrupting the sugar backbone instead of the pentapeptide cross-links

Question 45

Where is lysozyme present?

Answer 45

Lysozyme is present in tears, saliva, and in the lysosomes of phagocytic cells, and is particularly effective against Gram positive bacteria.

Question 46

What are B-lactams? How do they work?

Answer 46

Antibiotics tat are centered on the β-lactam ring, which structurally resembles the D-alanine D-alanine terminal pair of the pentapeptide side chains. Because of this resemblance, the β-lactams are bound by the transpeptidases (i.e., penicillin-binding proteins) of the bacteria and prevent cross-linking from occurring

Thus, these are competitive inhibitors

Question 47

What are some examples of B-lactams?

Answer 47

penicillin G, isolated from Penicillium chrysogenum, cephalosporins, carbapenems, and monobactams

Question 48

How can bacteria be resistant to B-lactams?

Answer 48

The β-lactam ring is cleaved by a number of β-lactamases produced in resistant bacteria OR other resistant bacteria produce mutated transpeptidases that no longer bind β-lactams.

Question 49

How does Vancomycin resistance occur?

Answer 49

occurs when D-alanine D-lactone is synthesized by bacteria instead of D-alanine D-alanine. The new pair is still recognized by transpeptidases, but not vancomycin.

Question 50

What do B-lactams require for activity?

Answer 50

Under normal growth conditions, bacterial autolysins act much like lysozyme to cleave cell walls at sites for new subunit insertion. When β-lactams then inhibit the cross-linking of new subunits, cells lyse due to a loss of structural integrity. In non-dividing cells, the autolysins do not act to cleave cell walls, so inhibition by β-lactams does not have deleterious effects.

Question 51

What types of bacteria do not stain using a gram stain?

Answer 51

Mycoplasma species, the smallest free-living organisms, lack a cell wall, and Chlamydia species lack peptidoglycan even though they contain the double membrane structure of Gram negative bacteria. Neither of these genera stains by the Gram method.

Members of the genus Mycobacterium contain a cell wall that is covered with a waxy coat containing mycolic acid; this structure interferes with proper Gram staining

Question 52

How are mycobacteria identified?

Answer 52

using a method that allows a red stain, carbol fuchsin, to penetrate the cells.

Once stained, the waxy surface of mycobacteria prevents the cells from being destained with acid-alcohol, a reagent that efficiently destains Gram organisms. Hence, mycobacteria are said to be acid-fast. The red stained cells are generally observed in contrast to a blue- or green-stained background. Prominent Mycobacterium species cause tuberculosis or leprosy.

Question 53

The growth property most associated with classification is what?

Answer 53

the bacterium’s response to oxygen.

Aerobic bacteria (aerobes) require oxygen and use respiration for growth. Anaerobic bacteria (anaerobes) are inhibited or killed in the presence of oxygen and use fermentation exclusively for metabolism. Facultative bacteria (facultative anaerobes) represent the majority of pathogens and use respiration in the presence of oxygen and use fermentation in its absence.

Question 54

What are some factors used for classification of bacteria?

Answer 54

• Morphology
• Arrangement
• Staining properties
• Growth properties
• Fermentation properties
• Other enzymatic and virulence properties
• Antigenicity
• Genotype

Question 55

What are serotyping and what is it used for?

Answer 55

Serotyping is in some cases the major way in which members of a single genus are classified. The cell wall carbohydrate Lancefield antigen of Streptococcus species is used to distinguish the major pathogenic members of the group

For example, Streptococcus pyogenes (“strep throat”) expresses the Group A Lancefield antigen and is commonly referred to as GAS for “Group A Streptococcus” while S. agalactiae (a cause of newborn meningitis) expresses the Group B Lancefield antigen and is hence known as GBS. Still other Streptococcus species lack the Lancefield antigen and are classified accordingly.

Serotyping is also used to distinguish strains within a genus. This can be extremely important, especially during outbreaks, to determine the source and possible severity of the infections. For example, Escherichia coli O157:H7 causes severe diarrheal outbreaks, the result of contaminated food or water. Escherichia is of course the genus name, coli is the species name, “O157” refers to the specific O antigen found in the lipopolysaccharide of this strain, and “H7” refers to the specific H antigen found on the flagella of this strain. Serotyping is necessary for these outbreaks because the O157:H7 serotype confers much more serious disease to those infected than other serotypes of similar E. coli.