2 The bacteria Flashcards
Describe structure of bacteria
Prokaryotes
Double stranded DNA - no introns. DNA composed of continous coding sequence of genes. No nuclear membrane. Tightly coiled in region known as necleoid
Plasid - small ciruclar self replicating molecule
Ribosomes 70S in prokaryotes (80S in eukaryotes). S refers to how unit behaves when examined under centrifugal force. Aminoglycosides can target 70S ribosome
Metabolic functions carried out by cell membrane (instead of mitochondria)
Cell wall (except mycoplasma)
Cell capsule
Construction of gram positive bacteria cell wall
Which antibioitcs target this
Peptidoglycan forms thick 20-80nm wall
Peptidoglycan is combination of:
- mucopeptide or murein
- hexose sugars
- amino acids
Polysaccharides and charged amino acids make it highly polar, providing bacterium with thick hydrophilic surface Allows gram positive bacteria to resist activity of bile in intestine. Conversely, layer is digested by lysozyme, and susceptible to bactericidal properties.
Synthesis of peptidoglycan is disrupted by: beta-lactam Cephalosporins Carbapenems Glycopeptides
Construction of gram negative bacteria cell wall
Cell wall affects shape of bacteria, and generally classed as cooci/ bacilli/ spirilla
Peptidoglycan forms thin 5-10nm wall
Peptidoglycan is combination of:
- mucopeptide or murein
- hexose sugars
- amino acids
Overlaid by outer layer of lipopolysaccharides and lipoprotein
Outer membrane is hydrophilic, but lipids also give hydrophobic components. Entry of hydrophilic molecules (sugars/ amino acids) necessary for nutrition and achieved through special channels called porins.
Lipopolysaccharide membrane confers antigenic properties (O antigen from carbohydrate chain) and toxic properties (endotoxin from lipid A component)
Why doesn;t gram stain affect mycobacterium`
Mycobacteria also have outer membrane which contains variety of lipids (mycolic acids). This creates waxy layer, which alters staining (mycolic acids is why called acid-fast bacteria)
Function of capsule and composition
Composed of polysaccharides or amino acids
Prevents phagocytosis
Some bacteria have flagella made from protein (flagellins). They are strongly antigenic - these H antigens are targets for antibody response
What is function of this
Where can they be located
What biochemical process generates movement
Help bacteria move
Singular - monotrichous
Double at both ends - amphitrichous
Multiple at one end - lophotrichous
General surface - peritichous
Prokaryotes - driven by movement of hydrogen ions
Eukaryotes - ATP dependent
Function of pili (also called fimbriae)
Pili are made from proteins called adhesins
Some organisms such as gonococci can re-organise there genes coding for constant and variiable region of pili molecule
More rigid than flagella and function in attachment either to other bacteria (sex pili) or host cells (common pili)
Bacteria obtain nutrients mainly by taking up small molecules across cell wall - amino acids, small peptides, oligosaccharides. Gram negative can take up larger molecules and preilimnary digest in periplasmic space
Oxidative metabolism takes place at membrane-cytoplasm junction
How much ATP is produced from molecule of glucose in aerobic metabolism?
How is anaerobic metabolism different?
Aerobic - Glucose to pyruvate, then undergoes aerobic metabolism in mitochondria to create 38 molecules ATP
Anaerobic - glucose to pyruvate, then converted into lactate/ ethanol. Produces 2 molecules of ATP
Anaerobic is beneficial as can be done in absence of oxygen, and usually substrate available in host body. . Oxygen requirement of bacteria can be “obligate” or “facultative”
Describe metabolism of glucose
Glucose to pyruvate
Pyruvate can then be fermented and produce 2 ATP and alcohol
Pyruvate can also be turned in to acetyl-CoA, and move into electron transport system
If oxygen present - produce 38ATP, CO2, H2O and heat
If other inorganic compound used - 34ATP generated and other inorganic moleucle
Classification of bacteria based on response to environmental oxygen
Obligate aerobe
Microaerophile
Obligate anaerobe
Facultative (anaerobe/ aerobe)
Organisms which utilise oxygen have oxygen detoxyfying enzymes such as superoxide dismutase, catalase, peroxidase to prevent free-radical damage
Obligate aerobe - grows in oxygen, cannot grow without
Microaerophile - grows in low oxygen, cannot grow without
Obligate anaerobe - no growth in oxygen, growth without oxygen
Facultative (anaerobe/ aerobe) - can grow with or without oxygen
Growth/ division of bacteria depends on nutritional status of environment. E. Coli may divide in 20-30 mins if conditions correct, can take 2 hours if not ideal. Conversely mycobacterium only divide every 24 hours
What are bacterial growth curve steps
Lag phase - initial adjustment
Logarithmic phase - cell division rapidly occurs, with population doubling at constant rate (generation time)
Stationary phase - nutrients deplete, and cell growth slows to stop
Death phase - bacteria start to die
Bacterial circular DNA starts at origin of replication (termed OriC). Multienzyme replication complex binds to origin, and initiates unwinding and separation of DNA strands
Which enzymes are used for above step?
Separated DNA strands serve as template for DNA polymerase, which incorporates deoxyribonucleotides to correctly base pair with template DNA. DNA polymerase is capable of proofreading, and removing incorrect bases. Reduces error rate, and also accurate replication
Helicases Topoisomerases (e.g DNA gyrase)
What does process of cell division involve
Segregation of replicated genomes
Formation of septum in middle of cell
Division of cell to produce daughter cells
What is septum made from in cell division
Invagination of cytoplasmic membrane, and ingrowth of peptidoglycan cell wall. This is not very accurate, but usually daughter cells end up with one copy of DNA
Bacterial growth and division are important targets for antimicrobial agents - which antibiotics target this
Quinolones - ciprofloxacin/ levofloxacin - inhibit unwinding of DNA by DNA gyrase during DNA replication
Beta lactams (penicillin), cephalosporins, carbapenems, glycopeptide (vancomycin) all inhibit peptidoglycan cell wall synthesis
What is transcription
Performed by DNA-dependent RNA polymerase to produce RNA transcript. Polymerisation involves incorporation ribonucleiotides which correctly base pair with template DNA
Gene expression is decoding genetic information within gene, to produce function protein, or RNA.
Most genes transcribed into mRNA, which is then translated into proteins
Some genes transcribed into ribosomal RNA species 5S/ 16S/ 23S which provide scaffold for assembling ribosomal subunits
Some genes transcribed into tRNA which together with ribosomes participate in decoding mRNA into functional proteins
Where does transcription start
What can influence frequency of transcription
Initiated at promoters
Promoters are nucleotide sequences that can bind RNA polymerase
Exact DNA sequence of promoter site
Overall topology (supercoiling) of DNA
Presence of regulatory proteins - sigma factor is protein which enables binding of RNA polymerase to promoters. This protein allows bacteria to switch genes on/off by altering level of sigma factor for specific genes - e.g spore formation in gram positive bacteria
What terminates transcription
Uracil incorporation in mRNA termintates RNA polymerase activity
If certain protein rho formed, this will also terminate it
Bacterial arrangement for single genes is described as monocistronic.- produces one mRNA molecule. However, single promoter/ terminator may be flanked by multiple other genes. This is a polycystronic arrangement, also known as operons.
What is the benefit of having operons?
Multiple proteins/ protein sub units synthesised simultaneously for one process, are made together.
e.g proteins required for uptake and metabolism of lactose are encoded on lac operon
Cholera toxin from vibrio cholerae is an operon
Fimbriae of uropathogenic E. Coli cauae colonoisation
What is translation
Transcription and translation are important targets for antimicrobial agents
Inhibitors of RNA poloymerase - rifampicin
Protein synthesis inhibitors - macrolides, aminoglycosides, teracyclines, chloramphenicol, lincosamides, streptogamins, oxazolidinones
Each set of three bases (triplets) in mRNA corresponds to codon for specific amino acid
64 codons encode all 20 amino acids, as well as start and stop signal codons.
mRNA translated to tRNA, and then ribosome uses this to build protein.
Ribosome binds to specific mRNA sequence (Shine-Dalgarno sequence, and begin translation at start codon (AUG) which binds to tRNA. Ribosome moves along mRNA, and tRNA molcules (Carrying different amino acids) recognise codon triplets. Ribosome carry out condensation reaction which couples incoming amino acid with growing polypeptide chain
How does gene regulation help bacteria to adapt to environment
Changing temperature e.g from environment to 37deg, or increase in oxygen, can increase binding of RNA polymerase, thereby activating gene transcriptin
If new source of carbon or nitrogen, can activate new metabolic pathways
When compounds misisng from environment e.g amino acids, then can switch on enzymes that enable it to metabolise other molecules
Transcriptional regulation with three types of protein - what are they, and how do they work?
- activators increase rate transcription - e.g can encourage RNA polymerase to bind to promoter, and initiate mRNA production
- repressors inhibit transcription - can block RNA polymerase
- regulator protein - can control multiple genes simultaenously. Can be activators/ or repressors
What is quorum sensing?
Quroum sensing bacteria produce autoinducer signalling compounds, which bind to activate specific gene transcription in neighbouring bacteria. When bacteria in high enough concentration, then autoinducers can activate a specific effect, which may not be active at low concentration
For example, when pseudomonas numbers reach a certain point, they start to activate genes to produce biofilms
Other bacteria can produce toxins etc
When are endospores formed
Clostridium (tetanus) and bacillus (anthrax) species form spores readily, and convert to normal bacteria once in wounds
Endospores are highly resistant to environment, and allows survive adverse conditions. Formed when cells unable to grow e.g nutrients exhausted, environment changed
Complex multilayered coat surrounds cell - containing dipicolinic acid and high calcium content
Bacteria have most genes on chromosomes. Many bacteria have extrachromosomal nucleic acid molecules termed plasmids and bacteriophages
Plasmids are circular units of DNA, which can have genes confer antibiotic resistance, toxin production. Can be transferred horizontally between cells.
What are R plasmids
Give two common examples of virulence genes on plasmids
Carry resistance genes on self-transferrable plasmids.
e.g R100 carries genes for resistance to sulphonamides, aminoglycosides, chloramphenicol, tetracycline
E. Coli enterotoxin
Staph aureus enterotoxin - haemolysin, fibrinolysin
How are plasmids utilised in genetic research?
How is a gene inserted into plasmid?
Can use to transfer genes across species barriers so that defined gene products can be studied or synthesised in large quantities
Plasmid cleaved open by endonuclease.
DNA cleaved by endonuclease, isolate specifc portion of DNA.
DNA ligated into plasmid
Bacteriophages consits of protein coat or capsid, which surrounds either RNA or DNA.
What happens if bacteriophage takes virulent or lysogenic pathway?
Virulent pathway is when bacteriophages commandeer cellular processes and produce new viruses, which causes cell to lyse, and release virions into environment. Cycle then repeats.
Lysogenic pathway is when viral DNA can be inserted in bacterial chromosome. Viral DNA known as prophage. New characteristics of cell can occur - known as prophage conversion. For example, may increase bacteria virulence. Diptheria toxin resides on a prophage. Eventually this process will kill the bacteria, albeit slower than virulent pathway. Prophage DNA will be copied when bacteria replicates
What is genetic transposition
Segments of DNA that can jump from one site in one
DNA molecule to another in a cell.
Small areas of DNA are called insertion sequences. Larger areas coding multiple geners are called transposons.
- from host DNA to transposon on plasmid
- from one plasmid to another
- from plasmid to genomic DNA
Rapid transposition can disseminate antibiotic resistance among bacteria
Bacteria are haploid and chromosome contains one copy of each gene. Daughter cells have exact copy of chromosome.
What processes can give rise to change in genome
Mutation
Recombination
Mutation is change in nucleotide sequence which can occur spontaneously of under influence of external agents.
Can be spontaneous
Or driven by chemicals (mutagens) which can change base pairs, or interact with DNA helix.
What are examples of different types of mutation
Point mutation - change in single nucleotide.
- Can be silent mutation, if new codon codes for same amino acid.
- Missense mutation - amino acid substitution in translated protein which may later its stability or function
- Formation of stop codon - causes premature termination of protein
Comprehensive changes are likely to harm organism, but may cause radical beneficial changes Deletion Replacement Insertion Inversion
How do bacteria repair damaged DNA
Direct repair - reverse or remove damage. For example, UV exposure can cause abnormal pyrimidine base pairing, and light-dependent enzyme can repair this
Excision repair - damaged DNA strand is recognised by housekeeping enzymes. Repair polymerisation to fill gap using complementary DNA as template. This tries to remove errors prior to replication
Second line repair - when DNA damage has reached point where cannot be corrected easily, “cut and paste” can be used to insert correct sequence, and minimise total error. This tries to prolong cell survival
New genetic material can arrives when genetic material transferred from one bacterium to another. New genes expressed once genetic material inserted into genome, or as plasmid within cell.
What ways can DNA be transferred to donor cell 3 (horizontal gene transfer)
Transformation - pick up exogenous bacterial DNA
Transduction - bacteriophage viruses infect bacteria and replicate. New virions may incorporate bacterial genes form chromosme/ plasmid, and incoroporate in other bacteria
Conjugation - outgrowth of cell wall connects two bacteria. Plasmids pass from one bacterium to another
Transformation
Certain bacteria e/g s pneumoniae, bacilus, subtilis, haemophilus influenzae, neisseria gonorrhoeae can pick up DNA fragments through their cell wall. Fragments come from lysed bacteria.
If DNA related to bacteria, it can recombine into host DNA. If unrelated, can be uptaken as plasmid
Why is this least important method of gene transfer?
Once cells lysed, DNA is extracellular, and at risk of degradation by environment. So low chance of actually being successful in a patient with infection
Transduction involves transfer of genetic material by infection with bacteriophage.
During process of virulent bacteriophage replication other DNA in cell (genomic or plasmid) can be erroneously incorporated in virus head, creating a transducing particle, which can attach to and transfer DNA to recipient cell. This is called generalised transduction
What is specialised transduction?
Transduction has higher chance of succesful transfer and potential clinical releavance than transformation. However, bacteriophages are extremley host specific parasites and therefore unable to move DNA between species.
Temperate bacteriophages integrate into bacterial genome. Prophages produced prepare to enter lytic cycle, they occassionally incorrectly excise from the site of attachment.
Infection of recipient cell results in high frequency of successful recombination of prophage with receipient genome.
This always uses host genome DNA, no plasmid.
Conjugation is type of bacterial mating, in which DNA is transferred from one bacterium to another.
Describe how plasmid transferred
Describe how chromosomal transfer occurs
Conjugation is most rapid and highly efficient movement of genetic material through bacterial population
Tra genes on in conjugative plasmids encode instructions for bacteria to produce sex pilus, a tube-like appendage which allows cell to cell contact to ensure protected transfer of a plasmid DNA from donor to recipient
Conjugative plasmids can occasionaly be integrated into bacterial genome e.g fertility plasmid of E. Coli. Integrated plasmids are called episomes. When episomes attempt conjugative transfer, they can transfer episome and adjacted DNA. This allows larger portions of DNA to be transferred
Why is bacterial genome analysis important?
Can use PCR and nucleic acid probes for analysis
Identification and classification - genes encoding ribosomal RNA (16S, 23S, 5S) usually found together in operon where transcription coordinated. Genes are same for all of species, but intbetween these genes there is a variable region, which can change between bacterial isolates
Resistance to anmicrobial agents - some genes are recognised to confer resistance
Molecular epidemiology - can see evolution of bacteria
How to microarrays work in genome analysis
They can only identify previously known genomic regions, and this is a limitation, as unrecognised sequences are not detected or recognised.
Specific point mutations involve only one nucleotide change, and referred to as nucelotide polymorphisms. e.g quinolone resistance is single gene mutation
Provide global targetted genome analysis. General arrangement of samples put into matrix of known nucleotide sequence.
Different fluorescently labelled probes can identify if any binding has occurred.
This allows multiple genomes and whole genomes to be analysed, as opposed to other techniques where individual genes are analysed
What are benefits of whole genome sequencing
Allow old genomes to be compared to database of known organisms, and identifies any new mutations that are occuring
Allow new organisms to be classified/ identified
If bacteria cannot be identified on culture, what techniques can be used?
MALDI-TOF
16S gene sequencing - PCR of conserved ribosome sequence, and compare results to database
