Questions 7-13

Question 1

Genetic recombination in bacteria – principles and possibilities

Answer 1

There is a donor cell and a recipient cell, there is a parasexual transfer that happens, and a recombination within the recipient cell.

As for parasexual transfer, there is transduction, transformation and conjugation

Transduction
There is a transfer of DNA by bacteriophages (virus that infect bacteria)

• Virulent bacteriophages: lytic cycle (host DNA is digested and used for new phage DNA, translation into proteins → new phages, destroy cell
• Temperate bacteriophages: lysogenic cycle (phage DNA integrates into bacterial chromosome, becomes noninfective prophage → gives cell new properties)

Conjugation
F+ cell forms sexpilus, gives F- cell one strand of plasmid, both cell synthetise second strand, both F+

Transposon
Jumping genes, can transpose between nucleoid/plasmids/prophages, has specific insertion sequences, often resistance genes

Question 2

What are the principal mechanisms of antibiotics?

Answer 2

Mechanisms of action (interfere with)
- Cell wall synthesis
- Folic acid metabolism
- Cytoplasmic membrane structure
- DNA gyrase
- RNA elongation
- DNA-directed RNA polymerase
- Protein synthesis (50S inhibitors)
- Protein synthesis (30S inhibitors)
- Protein synthesis (tRNA)

Question 3

What are some resistance mechanisms of bacteria?

Answer 3

Enzymatic inactivation: β-lactamases, acetyltransferases

Decrease of intracellular concentration by control of influx and amplified efflux (influx, efflux)
! Antibiotics, heavy metals and desinfectants can induce efflus pumps → resistance can develop

Changing of target structures: rRNA methylases, alternative PBP (Penicillin Binding Proteins, f.e. MRSA = Methillicin Resistant Staphylococcus Aureus)

Question 4

What are the bacterial structures and their function?

Answer 4

Cytoplasm
All of the material within a cell, enclosed by the cell membrane, except for the cell nucleus (cytosol, organelles, cytoplasmic inclusions)

Genome
- Nucleoid: Circular or linear chromosome (double-stranded DNA), free in cytoplasm, only a few proteins (histones), housekeeping genes, mostly singly copy genes: no compensation after mutation

• Plasmid: circular, linear, extrachromosomal DNA (virulence/resistance genes, non-essential), independent replication, conjugative plasmids control own transfer

Capsule
Extracellular substances: anti-dehydration, permeability barrier, adhesion, protection against phagocytosis, biofilm

Cell wall
Shaping exoskeleton, permeability barrier, adhesion to host cells, virulence factors, antigens

Cytoplasmic membrane
Permeability barrier, transport processes,…

Ribosome
Protein synthesis

Pili
Tubular hair-like protein structures: 1-2 per cell; F-Pilus, Sexpilus for conjugation

Fimbriae
Long protein filaments for adhesion, F-antigens

Flagella
Motility of bacteria, taxis possible, virulence factor, antigens, powered by protons

Plasmid
Circular DNA molecules, linear plasmids, not essential, replication-independent from the nucleoid, virulence/ resistance genes

Question 5

Examples for spore-forming bacteria

Answer 5

Sporulation due to unfavourable life conditions

Bacillus + Clostridium

Vegetative cell → sporulation → free spore → germination → vegetative cell

You need 135°C, 3 bar, 30 min in autoclave to kill spores

Question 6

What are structures of viruses and their function?

Answer 6

Genome
- DNA or RNA: ss/ds, linear/segmented/circular, +/- sense.
- Contains genetic information

Capsid
- Cubic, helical or complex protein structure, can include enzymes

Structural protein
Protect, bind, penetrate cell membrane

Non-structural proteins
Replication, modify host cell metabolism

Envelope
Double lipid membrane encoded by the host cell, contains virus-specific (glyco)proteins

Nucleocapsid
Genome + capsid, envelope optional

Subunit construction
- Translation produces small proteins that can be assembled in various ways
- Necessity: at best a nucleic acid can only code for 15% of its weight as a protein, viruses are composed of 50 – 90 % protein by weight
- Advantages: self assembly, fidelity (smaller genes + proteins → less error), economy (wrong proteins for trash are smaller), complexity (larger number of subunits → more stable capsid

Question 7

How do we “diagnose” bacteria?

Answer 7

Direct detection of pathogens

Microscopy + good for viruses, unique shape
- Light or fluorescence microscopy (min. 0.2µm)
- Electron microscopy (min. 0.0001 µm): transmission (2D, thin layer, for viruses) or scanning (structure, bigger samples coated in gold/silver, laser scans surface)

Culture → differentiation + good for bacteria
- Liquid: motility?, gas production?, need oxygen?
- Energy recovery from CH/AA?, change of pH/color?
- Solid: to produce pure cultures on agar plates, size? Colour? Shape/edge/elevation?
- Serological: glass slide agglutination: antibodies bind to O-antigens of Lipopolysaccharid of Salmonella → flaky look
- Phage typing: Lysis or resistant to phages?

Molecular methods
- PCR: detection of species-specific genes/virulence or resistance genes
PCR + sequencing: variability in phylogenetic conserved genes? (16S rRNA …)
RFLP = restriction fragment length polymorphism
– PCR needs DNA-template, hard to tell whether MO is already dead

Indirect detection of pathogens
- Antibodies, cellular immunity

Specifically viruses
Antigen detection, PCR, virus isolation, serology (Adenovirus) ELISA, RT-PCR, real-time RT-PCR (genetic diversity can make detection difficult)