3_Bacteriology III Flashcards
describe the subunits of bacterial ribosomes
- 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)
four basic steps of bacterial translation
- formation of initiation complex
- transfer of a transfer RNA bound to an amino acid into the acceptor (A) site
- formation of the peptide bond- peptidyl transfer
- translation of the growing peptide to the P-site to restart the cycle
aminoglycosides:
how does this ribosome/translation inhibitor work?
bind 16S rRNA and A site of 30S ribosomal subunit (*Sherris Fig incorrect ) blocking loading of charged tRNA
Oxazolidinones - Zyvox (linezolid)
how does this ribosome/translation inhibitor work?
Interacts with 50S ribosomal subunit block formation of the initiation complex
active against MRSA, VRE and multiresistant S. pneumoniae
tetracycline:
how does this ribosome/translation inhibitor work?
interacts with 16S rRNA and 30S ribosomal subunit weakens codon-anticodon interaction
chloramphenicol:
how does this ribosome/translation inhibitor work?
binds 30S rRNA and 50S subunit and blocks catalytic center for peptidyl transfer reaction (peptide bond formation)
lincosamides:
how does this ribosome/translation inhibitor work?
binds 30S rRNA 50S subunit and inhibit peptide bond formation
macrolides:
how does this ribosome/translation inhibitor work?
bind 50S subunit causing release of the peptide chain
streptogramins:
how does this ribosome/translation inhibitor work?
bind 50S subunit and block translocation of the peptide chain to the E (P) site
type I transporters:
function
- 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.
which general secretory proteins secrete most proteins in gram-positive bacteria?
- 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
Type III secretion:
function
- 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.
Type IV/V secretion:
function
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
how does RNA polymerase in bacteria differ from eukaryotes?
what is clinical significance of this?
RNA polymerase has the same basic function as in eukaryotic cells,
but the proteins are different, making it a target for antibiotics
describe RNA polymerase characteristics of bacteria
- alpha 2 beta beta ‘ - core
- alpha 2 beta beta ‘ sigma - enzyme
what are the steps in bacterial transcription?
- the sigma factor directs the RNA polymerase to the promoter – assembly in bacteria does not happen without the sigma factor
- 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
how does Rifampicin/Rifampin work?
- binds the closed complex of RNA polymerase and sigma and prevents melting
- inhibits bacterial transcription.
polycistronic messages of bacteria:
define
encode for more than one protein (operons)
process of using polycistronic messages (operons)
- 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.
why can transcription and translation be coupled in bacteria?
- because bacteria do not contain nucleus;
- transcription & translation can be coupled w/ the ribosome binding the mRNA before transcription is complete;
- when translation stops, RNA can form terminator
planktonic cells:
define
- refer to growth not in biofilms usually in a liquid culture or in the blood during septicemia
- e.g. in blood during septicemia
adherent cells:
define
refer to growth attached to a surface but in the absence of matrix and biofilm formation
biofilms:
define
are cells organized in a structure with a matrix material.
quorum sensing:
define
- a type of cell-cell signaling.
- It is the ability to determine the number of cells present of the same or different species.
how does quorum sensing work?
- Process
- 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.
- Important in biofilm formation
- Signals can be homoserine lactones in Gram-negatives, peptides in Gram-positives, and SAM-derivatives in both
what does biofilm formation require?
- attachment
- growth to form microcolonies
- secretion of matrix
- maturation of the biofilm
- cellular detachment/dispersal (in some bacterial species)
cells in biofilms are heterogenic.
how does being in a biofilm change the physiologic properties of bacteria?
- UV light – survival in the environment
- Increased genetic exchange – virulence genes and antibiotic resistance genes
- Reduced sensitivity to antibiotics NOT due to resistance genes
key point about bacteria living in the microbiota
these bacteria live in a multispecies economies
quorum sensing can be used for the following:
- 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)
how does the biofilm help protect bacteria from the immune response?
- antibodies do not bind efficiently
- phagocytic cells cannot engulf the biofilm structures
where can biofilms form?
- natural (biotic) surfaces:
- normal flora
- chronic infections (e.g. cystic fibrosis, endocarditis, otitis media, rhinosinusitis, caries, periodontal disease)
- implants (abiotic) surfaces:
- indwelling devices
microbiota (normal flora) often grows in biofilms,
describe the characteristics of this
- 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.
microbiota:
characteristics
- >700 species
- vary between individuals
- many species cannot be cultured
what are the symptoms resulting from biofilms colonizing on implants
- device failure
- detachment of organisms leading to a planktonic infection
- release of endotoxin (if Gram-negative bacteria are present)
what makes biofilms resistant to antibiotics?
- inhibition of antibiotic penetration
- changes in metabolism of the bacteria
- changes in membrane permeability
- increased expression of efflux pumps
describe the cycle of septicemia
- biofilm on a device
- biofilm bacteria shed = septicemia
- treat septicemia but biofilm is resistant
- biofilm regrows – sheds – etc.
what conditions in a biofilm are heterogenic?
- oxygen needs/concentration
- quorum signals - bacterial density
- nutrient availability - deep in biofilm vs. channels
- secreted factors such as metabolites; from other bacteria
what are the different phenotypic states in biofilms?
what is the clinical application of this?
- 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
what can happen to cells in the stationary phase?
- can beocme hypermutable
- accumulate mutations
what is stochastic gene regulation?
- 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.
persisters:
define
- 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.
how resistant can biofilms be to antibiotics?
up to 1000-fold increases in the dose needed which is often above a dose that is safe for treatment
what allows for resistance of biofilms?
- 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
sporulation:
define
- a programmed cell development cycle.
- Under conditions of high cell density (sensed by quorum sensing) and nutrient starvation, the cells develop into spores
describe the characteristics of spores
- 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
describe transcription by RNA polymerase
- 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.
sigma factor binds promoter consensus sequence where?
Bacterial promoters a located around -10 and -35 upstream of the transcription start site.
bacteria contain specialized sigma factors;
function
- these sigma factors can control large numbers of genes during processes like stationary phase and spore formation
- sporulation uses a cascade of sigma factors
