3_Bacteriology III

Question 1

describe the subunits of bacterial ribosomes

Answer 1

• The bacteria ribosome significantly differs from the human ribosome.
• The 70S ribosome is made up of a 30S and 50S ribosomal subunit composed of multiple proteins
• There is a 23S rRNA associated with the 50S subunit and a 16S rRNA associated with the 30S ribosomal subunit

(*16S rRNA is used for identification of bacteria in the microbiome)

Question 2

four basic steps of bacterial translation

Answer 2

1. formation of initiation complex
2. transfer of a transfer RNA bound to an amino acid into the acceptor (A) site
3. formation of the peptide bond- peptidyl transfer
4. translation of the growing peptide to the P-site to restart the cycle

Question 3

aminoglycosides:

how does this ribosome/translation inhibitor work?

Answer 3

bind 16S rRNA and A site of 30S ribosomal subunit (*Sherris Fig incorrect ) blocking loading of charged tRNA

Question 4

Oxazolidinones - Zyvox (linezolid)

how does this ribosome/translation inhibitor work?

Answer 4

Interacts with 50S ribosomal subunit block formation of the initiation complex

active against MRSA, VRE and multiresistant S. pneumoniae

Question 5

tetracycline:

how does this ribosome/translation inhibitor work?

Answer 5

interacts with 16S rRNA and 30S ribosomal subunit weakens codon-anticodon interaction

Question 6

chloramphenicol:

how does this ribosome/translation inhibitor work?

Answer 6

binds 30S rRNA and 50S subunit and blocks catalytic center for peptidyl transfer reaction (peptide bond formation)

Question 7

lincosamides:

how does this ribosome/translation inhibitor work?

Answer 7

binds 30S rRNA 50S subunit and inhibit peptide bond formation

Question 8

macrolides:

how does this ribosome/translation inhibitor work?

Answer 8

bind 50S subunit causing release of the peptide chain

Question 9

streptogramins:

how does this ribosome/translation inhibitor work?

Answer 9

bind 50S subunit and block translocation of the peptide chain to the E (P) site

Question 10

type I transporters:

function

Answer 10

• export small molecules.
  • act as non-specific pumps that can also pump Abx out the cell –> conferring resistance.
  • *Only Gram-negative bacteria
  • Can be drug efflux pumps
• Coupled to Type II secretion in Gram-negative bacteria.

Question 11

which general secretory proteins secrete most proteins in gram-positive bacteria?

Answer 11

• General secretory proteins (Sec and Tat) secrete most proteins in Gram-positive bacteria.
• These are also coupled to Type II secretion in gram-negative bacteria

Question 12

Type III secretion:

function

Answer 12

• important in the delivery of toxins to eukaryotic cells.
  • The proteins are synthesized in the cell, but are not secreted until the bacterial cell comes in contact with a eukaryotic cell.
  • The secreted proteins often include proteins that form a channel in the eukaryotic membrane for the “injection” of toxins directly into the eukaryotic cell.

Question 13

Type IV/V secretion:

function

Answer 13

allows for the secretion of:

• self-assembling cellular structures (e.g. pilin)
• DNA
• Toxins
• also depend on Sec and Tat in Gram-positive and Gram-negative bacteria

Question 14

how does RNA polymerase in bacteria differ from eukaryotes?

what is clinical significance of this?

Answer 14

RNA polymerase has the same basic function as in eukaryotic cells,

but the proteins are different, making it a target for antibiotics

Question 15

describe RNA polymerase characteristics of bacteria

Answer 15

• alpha 2 beta beta ‘ - core
• alpha 2 beta beta ‘ sigma - enzyme

Question 16

what are the steps in bacterial transcription?

Answer 16

1. the sigma factor directs the RNA polymerase to the promoter – assembly in bacteria does not happen without the sigma factor
2. the RNA polymerase assembles at the promoter forming the closed complex
3. DNA is melted forming an open complex
4. the core transcribes mRNA and the sigma factor disassociates
5. in prokaryotes there is no nuclear membrane so transcription and translation is coupled

Question 17

how does Rifampicin/Rifampin work?

Answer 17

• binds the closed complex of RNA polymerase and sigma and prevents melting
• inhibits bacterial transcription.

Question 18

polycistronic messages of bacteria:

define

Answer 18

encode for more than one protein (operons)

Question 19

process of using polycistronic messages (operons)

Answer 19

• 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 (cistron) 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.

Question 20

why can transcription and translation be coupled in bacteria?

Answer 20

1. because bacteria do not contain nucleus;
2. transcription & translation can be coupled w/ the ribosome binding the mRNA before transcription is complete;
3. when translation stops, RNA can form terminator

Question 21

planktonic cells:

define

Answer 21

• refer to growth not in biofilms usually in a liquid culture or in the blood during septicemia
• e.g. in blood during septicemia

Question 22

adherent cells:

define

Answer 22

refer to growth attached to a surface but in the absence of matrix and biofilm formation

Question 23

biofilms:

define

Answer 23

are cells organized in a structure with a matrix material.

Question 24

quorum sensing:

define

Answer 24

• a type of cell-cell signaling.
• It is the ability to determine the number of cells present of the same or different species.

Question 25

how does **quorum sensing** work?

Answer 25

* Process 1. Small diffusible signals are produced at a constant rate. 2. The concentration increases in proportion to the total number of bacteria. 3. When the **concentration surpasses a critical threshold a signal is transduced.** * Important in biofilm formation * Signals can be homoserine lactones in Gram-negatives, peptides in Gram-positives, and SAM-derivatives in both

Question 26

what does biofilm formation require?

Answer 26

* attachment * growth to form microcolonies * secretion of matrix * maturation of the biofilm * cellular detachment/dispersal (in some bacterial species)

Question 27

cells in biofilms are heterogenic. how does being in a biofilm change the physiologic properties of bacteria?

Answer 27

* **UV light** – survival in the environment * **Increased genetic exchange** – virulence genes and antibiotic resistance genes * **Reduced sensitivity to antibiotics** NOT due to resistance genes

Question 28

key point about bacteria living in the microbiota

Answer 28

these bacteria live in a multispecies economies

Question 29

quorum sensing can be used for the following:

Answer 29

* can be used to detect both within the same species: * Gram-negatives will use homoserine lactone derivatives * Gram-positives will use modified peptides * can also be used to sense many species using molecules like AI-2 (common metabolic byproduct of S-adenosyl-methionine biosynthesis)

Question 30

how does the biofilm help protect bacteria from the immune response?

Answer 30

* antibodies do not bind efficiently * phagocytic cells cannot engulf the biofilm structures

Question 31

where can biofilms form?

Answer 31

* natural (biotic) surfaces: * normal flora * chronic infections (e.g. cystic fibrosis, endocarditis, otitis media, rhinosinusitis, caries, periodontal disease) * implants (abiotic) surfaces: * indwelling devices

Question 32

microbiota (normal flora) often grows in biofilms, describe the characteristics of this

Answer 32

* Often these are mixed species biofilms with hundreds of different species. * For example, dental plaque has over 700 species of bacteria, many of which have not been cultured in the laboratory. The composition of dental normal flora can differ between people and between individual teeth in a single person.

Question 33

microbiota: characteristics

Answer 33

* \>700 species * vary between individuals * many species cannot be cultured

Question 34

what are the symptoms resulting from biofilms colonizing on implants

Answer 34

* device failure * detachment of organisms leading to a planktonic infection * release of endotoxin (if Gram-negative bacteria are present)

Question 35

what makes biofilms resistant to antibiotics?

Answer 35

* inhibition of antibiotic penetration * changes in metabolism of the bacteria * changes in membrane permeability * increased expression of efflux pumps

Question 36

describe the cycle of septicemia

Answer 36

1. biofilm on a device 2. biofilm bacteria shed = septicemia 3. treat septicemia but biofilm is resistant 4. biofilm regrows – sheds – etc.

Question 37

what conditions in a biofilm are heterogenic?

Answer 37

* oxygen needs/concentration * quorum signals - *bacterial density* * nutrient availability - *deep in biofilm vs. channels* * secreted factors such as metabolites; from other bacteria

Question 38

what are the different phenotypic states in biofilms? what is the clinical application of this?

Answer 38

* phenotypic states: * transcriptional regulation * mutation and selection * stochastic gene switching (biphasic switch) * differences can lead to different phenotypes in diff't parts of the biofilms * bacteria on the outside have more nutrients and may still be growing whereas deep in biofilm cells may be in stationary phase * can be transcriptionally regulated

Question 39

what can happen to cells in the **stationary phase?**

Answer 39

* can beocme hypermutable * accumulate mutations

Question 40

what is **stochastic** gene regulation?

Answer 40

* Cells can undergo stochastic gene regulation (biphasic/bistability/bimodal gene expression) * genes turn on and off in a small percent of the population. * these become persisters.

Question 41

persisters: define

Answer 41

* **dormant or slow growing cells** that are present due stochastic gene expression where bistable switches where 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.

Question 42

how resistant can biofilms be to antibiotics?

Answer 42

up to 1000-fold increases in the dose needed which is often above a dose that is safe for treatment

Question 43

what allows for resistance of biofilms?

Answer 43

* can come from physical protection due to interaction of the antibiotic and the matrix material. * can be due to changes in the physiologic state of the bacteria * increases in efflux pump expression * changes in membrane permeability * changes in physiologic activity due to changes in oxygen, bacterial numbers (cell density/quorum), and nutrient availability and formation of persisters

Question 44

sporulation: define

Answer 44

* a programmed cell development cycle. * Under conditions of high cell density (sensed by quorum sensing) and nutrient starvation, **the cells develop into spores**

Question 45

describe the characteristics of **spores**

Answer 45

* resistant and dormant * dehydrated w/ multiple layers protecting them * may survive in boiling water * difficult to kill and require autoclaving and bleach (hypochlorite) to eliminate them

Question 46

describe transcription by RNA polymerase

Answer 46

* RNA polymerase: * core is made up of two alpha, a beta and a beta prime subunit * holoenzyme consists of sigma factor associated with the core * structure of bacterial RNA polymerase is distinct and is a target of antibiotics. * The sigma factor targets the RNA polymerase to the promoter of the genes.

Question 47

sigma factor binds promoter consensus sequence where?

Answer 47

Bacterial promoters a located around -10 and -35 upstream of the transcription start site.

Question 48

bacteria contain specialized sigma factors; function

Answer 48

* these sigma factors can control large numbers of genes during processes like stationary phase and spore formation * sporulation uses a cascade of sigma factors