2 The bacteria

Question 1

Describe structure of bacteria

Answer 1

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

Question 2

Construction of gram positive bacteria cell wall

Which antibioitcs target this

Answer 2

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

Question 3

Construction of gram negative bacteria cell wall

Cell wall affects shape of bacteria, and generally classed as cooci/ bacilli/ spirilla

Answer 3

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)

Question 4

Why doesn;t gram stain affect mycobacterium`

Answer 4

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)

Question 5

Function of capsule and composition

Answer 5

Composed of polysaccharides or amino acids

Prevents phagocytosis

Question 6

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

Answer 6

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

Question 7

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

Answer 7

More rigid than flagella and function in attachment either to other bacteria (sex pili) or host cells (common pili)

Question 8

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?

Answer 8

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”

Question 9

Describe metabolism of glucose

Answer 9

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

Question 10

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

Answer 10

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

Question 11

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

Answer 11

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

Question 12

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

Answer 12

Helicases
Topoisomerases (e.g DNA gyrase)

Question 13

What does process of cell division involve

Answer 13

Segregation of replicated genomes
Formation of septum in middle of cell
Division of cell to produce daughter cells

Question 14

What is septum made from in cell division

Answer 14

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

Question 15

Bacterial growth and division are important targets for antimicrobial agents - which antibiotics target this

Answer 15

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

Question 16

What is transcription

Performed by DNA-dependent RNA polymerase to produce RNA transcript. Polymerisation involves incorporation ribonucleiotides which correctly base pair with template DNA

Answer 16

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

Question 17

Where does transcription start

What can influence frequency of transcription

Answer 17

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

Question 18

What terminates transcription

Answer 18

Uracil incorporation in mRNA termintates RNA polymerase activity

If certain protein rho formed, this will also terminate it

Question 19

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?

Answer 19

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

Question 20

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

Answer 20

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

Question 21

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

Answer 21

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

Question 22

Transcriptional regulation with three types of protein - what are they, and how do they work?

Answer 22

• 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

Question 23

What is quorum sensing?

Answer 23

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

Question 24

When are endospores formed

Clostridium (tetanus) and bacillus (anthrax) species form spores readily, and convert to normal bacteria once in wounds

Answer 24

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

Question 25

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

Answer 25

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

Question 26

How are plasmids utilised in genetic research?

How is a gene inserted into plasmid?

Answer 26

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

Question 27

Bacteriophages consits of protein coat or capsid, which surrounds either RNA or DNA.

What happens if bacteriophage takes virulent or lysogenic pathway?

Answer 27

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

Question 28

What is genetic transposition

Answer 28

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

Question 29

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

Answer 29

Mutation

Recombination

Question 30

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

Answer 30

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

Question 31

How do bacteria repair damaged DNA

Answer 31

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

Question 32

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)

Answer 32

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

Question 33

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?

Answer 33

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

Question 34

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.

Answer 34

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.

Question 35

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

Answer 35

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

Question 36

Why is bacterial genome analysis important?

Can use PCR and nucleic acid probes for analysis

Answer 36

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

Question 37

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

Answer 37

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

Question 38

What are benefits of whole genome sequencing

Answer 38

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

Question 39

If bacteria cannot be identified on culture, what techniques can be used?

Answer 39

MALDI-TOF

16S gene sequencing - PCR of conserved ribosome sequence, and compare results to database