Bacterial Cell Growth, Metab, Genetics

Front 1

How bacterial cells grow, divide and die.

Back 1

Binary fission: 1) cell elongates and DNA is replicated; 2) cell wall and plasma membrane begin to divide; 3) cross-wall forms completely around divided DNA; 4) cells separate

Front 2

Each phase of the bacterial growth curve and what happens during each of them.

Back 2

1) lag phase- turning on metabolic and replication enzymes; no or little cell division
2) log phase- rapid exponential cell growth; generation time/doubling time (time req for a cell to divide)
3) stationary phase- no more nutrient; accumulation of toxic metabolites; the rate of cell division balances the rate of cell death
4) death phase- nutrients depleted and cell number declines exponentially

Front 3

The nutritional requirements of bacteria as well as the different mechanisms involved in nutrient uptake.

Back 3

1. water
2. carbon source- (glucose yields energy production + synthesis of cellular organic macromolecules
3. nitrogen source- aa’s; some enteric bacteria can use NH4+
4. Sulfur – in the form of methionine or cysteine for synthesis of sulfur-containing macromolecules
5. Iron – siderophores (bacteria make and excrete them, and get them back) = small molecules that bind iron; siderophores + bound iron are internalized via receptors by the bacterial cell
6. Other cations: Ca, P, Mn, Mg
7. Growth factors – vitamins…

Front 4

The differences between obligate aerobes, obligate anaerobes and facultative anaerobes.
Why is it important to know about metabolic requirements?
KNOW TABLE ON SLIDE #19

Back 4

1. heterotrophic - All pathogen bacteria are heterotrophic
 i.e. obtain energy by oxidizing organic compounds
 usually metabolize sugars, fats and proteins
2. Parasites (Most pathogens) – i.e. can live on dead and living tissue
3. Obligate parasites – i.e. can live only on living tissue
 to better understand pathogenesis
 for classification & diagnosis purpose
4. Obligate aerobes
 require O2 to grow
 catalase(+) & superoxide dismutase(+)
 aerobic respiration
 no fermentation pathway
5. Obligate anaerobes
 do NOT grow in presence of O2
 catalase(-) & superoxide dismutase(-) Æ killed by O2
 anaerobic respiration or fermentation
6. Facultative anaerobes
 grow best when O2 present, but can grow without O2 as well
 aerobic respiration, anaerobic respiration or fermentation depending on conditions
 catalase(+) & superoxide dismutase(+)
7. Others (DON’T NEED TO KNOW SO MUCH)
 Aerotolerant anaerobes
 Microaerophiles
• To better understand pathogenesis; for classification and diagnosis purpose

Front 5

Describe nutrient uptake

Back 5

PASSIVE TRANSPORT:
• No energy required
• Movement of solute from higher to lower concentration
o Diffusion- Transport dependent on the permeability of the cell membrane
o Facilitated diffusion- Protein channel or carrier protein embedded within the cell membrane
ACTIVE TRANSPORT;
• transport against concentration gradient; carrier protein + energy are required
o proton-gradient active transport
o ATP-dependant active transport
o breakdown of other high-energy compound e.g. phosphoenolpyruvate
GROUP TRANSPORTATION:
o chemical conversion of transported molecule
o e.g. phosphotransferase systems

Front 6

Describe catabolism in bacteria

Back 6

• Catabolism of proteins, polysaccharides, and lipids
o glucose, pyruvate, or intermediates of the tricarboxylic acid (TCA) cycle
o energy in the form of adenosine triphosphate (ATP) or the reduced form of nicotinamide-adenine dinucleotide (NADH)
• Formation of basic subunits used for synthesis of major cellular constituents

Front 7

The difference between respiration (aerobic or anaerobic) and fermentation in bacteria.

Back 7

Aerobic Respiration: occurs when oxygen is available; produces 34 ATP;
Anaerobic respiration: occurs when oxygen is not available; Respiration in which an inorganic molecule other than O2 is the final electron acceptor
• Like aerobic respiration, it involves: glycolysis, TCA cycle, and electron transport chain
• Theoretical maximum energy yield is 36 ATP/glucose or less
• Examples: bacteria called
• sulfate reducers can transfer electrons to sulfate (SO42-) reducing it to H2S
• nitrate reducers can transfer electrons to NO3- reducing it to NO2-
Fermentation:
• In the cytosol
• In absence of O2
• Organic molecules are used as electron acceptors to recycle NADH
• Energy yield is 2 ATP/glucose
• Fermentation of pyruvate by different microorganisms results in different end products
• End products can be used for identification of many bacteria

Front 8

Describe the different responses to oxygen

Back 8

O2 may be a poison for many bacteria
o because O2 generates toxic products - hydrogen peroxide & superoxide anion
• To survive oxygen, bacteria must have
o superoxide dismutase
o peroxidase [e.g. catalase]
O2- + 2H+ SOD H2O2 catalase (peroxidase) H2O + ½ O2

Front 9

Describe Replication, transcription and translation in bacteria

Back 9

1. Replication
• Replication complex binds to origin of replication
o helicases & topoisomerases unwind and separate the 2 DNA strands
o DNA polymerase – high proof-reading capability
• Bi-directional Æ2 replication forks in opposite directions
• One parental DNA gives rise to two identical copies of daughter DNAs
• Semi-conservative – one new stand & one parental strand
2. Transcription
• synthesis of mRNA (majority), tRNA, rRNA
• RNA polymerase – recognizes and binds to specific DNA sites (promoter) to start transcription
3. Translation
• protein synthesis
• ribosome moves along mRNA
• tRNA reads genetic code in mRNA and moves in a specific amino acid
*In prokaryotes mRNA transcription and translation occur simultaneously

Front 10

Describe Gene expression in bacteria

Back 10

• Operons - simultaneous expression of genes; polycistronic mRNA
o of a biochemical pathway
o of protein subunits that make up particular enzyme complexes
• Sigma factors
o bind to RNA polymerase core enzyme & recognize promoter
o different σ subunits Æ different promoters Æ different genes expressed
• Some sigma factors of E. coli
o σ70 - primary sigma factor
o σS - stationary phase sigma factor
o σ24 - extracytoplasmic stress sigma factor
• Many operons contain an operator region where a regulator protein binds
• Regulator protein can be
o activator: binding of activator Æbinding of RNA polymerase to promoter Æ initiates transcription
o repressor: binding of repressor to operator Æ inhibition of binding of RNA polymerase Æ blocks transcription; transcription only on absence of repressor
• Regulon = set of genes needed for a particular response that are in different operons but under the control of one common regulator protein

Front 11

The mechanisms involved in bacterial gene transfer:

Back 11

1. Transduction
2. Conjugation
3. Transformation

Front 12

Prophage

Back 12

• Definition: Repressed temperate phage DNA inserted into the bacterial chromosome
• Prophage DNA encodes for a repressor protein Æ lysogenic pathway
• If repressor protein not expressed Æ lytic (pathogenic) pathway
• Prophage DNA may also for other proteins that make the bacteria more virulent = lysogenic conversion
Examples:
Corynebacterium diphtheriae ’ diphtheria toxin
Clostridium botulinum ’ botulinum toxin
Streptococcus pyogenes ’ erythrogenic toxins

Front 13

Conjugation and Hfr bacteria

Back 13

• Exchange of genetic information directly from one cell (the donor) to another (the recipient)
• One way transfer through the sex pilus (F pilus)
• Conjugation genes are on the conjugation plasmid (F plasmid)
Hfr conjugation:
• Occasionally, the F factor integrates into a the bacterial chromosome Æ the bacterial cell is then called Hfr (High-frequency recombinant) instead of F+
• The Hfr cell is still able to initiate conjugation with an F- cell
• The first DNA to be transferred is chromosomal DNA
• The transfert is almost never complete Æ F factor itself is almost never transferred
• Integration of the DNA fragmant via homologous recombination

Front 14

Recombination and homologous recombination

Back 14

• Rearrangement of DNA within the bacteria
• 2 types of recombination: Homologous and Site-Specific
Homologous Recombination
o Gene exchange process
o Can follow transformation, conjugation or transduction
o Requires homology or near homology between the DNA strands
o Requires recombination enzymes (rec genes: A, B, C, D)
Site-Specific Recombination
o Integration of a DNA molecule into another – mechanism used to combine circular pieces of DNA: e.g. plasmids, temperate phage, transposons
o No homology required except for the attachment site & no DNA is lost
o Requires restriction endonucleases and attachement sites on both DNA

Front 15

Transposition

Back 15

• Changes in the nucleotide sequence of chromosomal DNA
o spontaneous – errors in DNA replication
o under influence of external agents (mutagens) – direct changes in DNA
• Point mutation: change of a single nucleotide
o missense mutation – different amino acid encoded
o nonsense mutation – formation of a stop codon
o silent mutation – mutated codon codes for the same amino acid
o Frameshift mutation: change in reading frame (deletion, insertion, inversion of several bases)
o DNA repair machinery – protection against mutation

Front 16

Mechanisms involved in the acquisition of resistance to antibiotics.

Back 16

1. Inhibition of cell wall synthesis: penicillings, cephalosporins, bacitracin, vancomycin
2. Inhibition of nucleic acid replication and transcription: quinolones, rifampin
3. Inhibition of protein synthesis: chloramphenicol, erythromycin, tetracyclines, streptomycin
4. Inhibition of synthesis of essential metabolites: sulfanilamide, trimethoprim
5. Injury to plasma membrane: polymyxin B
 These mechanisms can either:
 chemically modify the antibiotic
 render it inactive through physical removal from the cell
 modify target site so that it is not recognized by the antibiotic.
Inherent resistance:
Acquired resistance:
 Vertical gene transfer: spontaneous mutation
 Horizontal gene transfer: transduction, transformation or conjugation

Front 17

transduction

Back 17

Transduction – transfer of genetic information from one bacterium to another by a bacteriophage; phage-mediated gene transfer; bacteriaphage is an obligate intracellular parasite that multiplies inside bacteria by making use of some or all of the host biosynthetic machinery; general and specific transduction
Lifecycle of bacteriophage: lysogenic vs lytic/virulent pathway; depends on the type of phage
General transduction: host DNA is packaged into virus head: “transducing particle”
transducing particles can attach to a new recipient cell and transfer the DNA
DNA incorporation by homologous recombination; plasmid DNA can also be transduced
Specialized transduction: prophage integration at specific sites of bacterial chromosome = attachment site; occasionally bacterial genes adjacent to attachment site are transferred with viral DNA

Front 18

transformation

Back 18

Transformation – transfer of naked DNA from environment (ex. DNA released from lysed cells, plasmids, transposons); artificial transformation in laboratory; (1) donor cell (2) cell lysis and release of DNA fragments (3) DNA enters recipient cell and integrates into DNA
-competence: ability to pick up env’t DNA; encoded by chromosomal genes (ex. Steptococcus pneumonia, Haemophilus influenza);

Front 19

conjugation

Back 19

Conjugation – gene transfer through the sex pilus
• Genetic materiel transferred
• Bacteriophages – viruses that infect bacteria and can infect by lysogenic or lytic/virulent pathway
• Plasmids
• Transposons – small segments of DNA that can move from one region of a DNA to another
• Recombination = incorporation of extrachromosomal DNA into the chromosome