VALDOSTA STATE UNIVERSITY

Biology 2900--Spring 2005

PART II. MICROBIAL PHYSIOLOGY AND GROWTH

1. Bacterial growth
1. most bacteria grow by binary fission; therefore, when all bacteria reproduce the population doubles
1. counting bacteria
1. direct counts (microscopic or using a cell-counter (such as a Coulter counter))
2. plate counts with serial dilution
3. most probable number (and serial dilution)
4. turbidity
5. total mass
6. DNA, peptidoglycan, etc.
2. in culture, growth follows characteristic growth curve
1. lag phase -- adjust to conditions
2. exponential phase (log phase) -- when doubling with constant doubling time as above; produce normal (primary) metabolites
3. stationary phase -- growth slows, some cells dying, some reproducing
4. death phase -- culture begins to decline
5. prolonged decline phase and the concept of viable but not culturable
   
3. key number in looking at growth bacteria is the doubling time during exponential growth (time taken for population to double)
1. if you know initial population, the doubling time, and the total time for growth, it is easy to determine the size of the final population:  N_final = N_initial x 2^{number of doublings}  where the number of doublings is found by dividing the total time for growth by the doubling time
2. possible to detemine the doubling time if know the size of the initial and final populations and the time the bacterial were let grow: doubling time =  {time x log(2)}/{log(N_final) - log(N_initial)}
1. simpler if the ratio of the final population to the initial population population is a power of 2; in this case you can compare the exponents directly
1. example:  suppose N_final / N_initial = 1024 = 2¹⁰; then 2¹⁰ = 2^{number of doublings} = 2^{total time/doubling time}; if we compare exponents, 10 = total time/doubling time; solving for the doubling time gives doubling time = total time/10
4. the doubling time depends on the type of bacteria and on the enivornmental conditions
2. key environmental conditions
1. growth range vs optimum conditions
2. oxygen
1. uses of oxygen in cells -- energy metabolism (fermentation vs respiration)
2. harmful effects -- forms hydrogen peroxide, superoxides, which can attack and destroy organic molecules; catalase and superoxide dismutase are regarded as defences against the harmful effects, more common in aerobic organisms than in anaerobic organisms
3. categories of bacteria
1. oligate anaerobes -- cannot grow in the presence of oxygen (Clostridium)
2. aerotolerant anaerobes -- do not use oxygen, but not harmed (Streptococcus)
3. facultative anaerobes -- can grow without oxygen, but does much better with; capable of switching to aerobic respiration if oxygen present, ferments otherwise (Escherichia)
4. microaerophiles -- require small amount of oxygen, but cannot handle high concentrations
5. obligate aerobes -- like us, require oxygen, cannot survive on just fermentation (Pseudomonas)
4. note: oxygen is not the only important atmospheric gas; some bacteria, called capnophiles, require increased carbon dioxide (Neisseria and Haemophilus)
3. CHNOPS and energy sources (later)
   
4. water availability
1. almost all microbes require liquid water at some concentration; dried foods or other materials do not support bacterial growth (lyophilization)
   
2. most bacteria require relatively pure water; adding sugar or salt can control bacterial growth
1. osmotolerant microbes can survive concentrations as high at 10% salt (concentrations?)
2. halophiles require 3% or higher salt
5. temperature conditions
1. temperature affects protein, cell membranes; high temperatures more likely to cause permanent changes in proteins and cell death, low temperatures often just slow processes down; optimum temperatures tend to be near high end of range
2. categories of bacteria
1. psychrophiles -- opt. temp. between -5 and 15
2. psychrotrophs -- opt. temp. between 20 and 30, temperature range goes much lower
3. mesophiles -- opt. temp. between 25 and 45; most common in human infections (although a few disease-causing organisms (syphilis for example) prefer lower temperatures
4. thermophile -- opt. temp. between 45 and 70
5. hyperthermophiles -- opt. temp between 70 and 110
2. Basic ideas concerning metabolism
1. driving rules -- laws of mass conservation, thermodynamics
1. we can group all organisms according to where they get their energy, their carbon atoms, their atoms of the rest of CHNOPS:  photoautotrophs (photolithotrophs); chemolithotrophs (chemoautotrophs); chemoorganotrophs (chemoheterotrophs), etc.
2. sources of major nutrients (NPS)
3. minor nutrients (growth factors, minerals) and fastidious organisms
   
2. metabolic processes are grouped into pathways: substrate to intermediates to product
1. energy of chemical reactions -- endergonic vs exergonic; free energy; redox reactions
   
2. each step of the process is governed by an enzyme; enzymes reduce the activation energy of reactions, some couple reactions together; many enzymes are regulated by chemicals in their environment (competitive and non-competitive inhibitors, activators), these have potential use as poisons
   
3. some steps require additional energy in the form of ATP, NADH, etc.
3. key catabolic metabolic pathways
1. glycolysis (Embden-Meyerhoff pathway) - break-up of glucose into smaller molecules, with the formation of ATP and NADH
1. glucose is converted to glucose-phosphate to fructose-phosphate to PGAL to PGA to PEP to pyruvate; all of these can take part in anabolic pathways
2. pentose phosphate pathway - an alternate pathway for the break-up of glucose; key products (besides NADPH) are ribose phosphate and erythrose phosphate; some products can feed into glycolysis
3. TCA cycle - completes the oxidation of glucose with the formation of ATP and NADH;
1. begins with acetyl-CoA (made from pyrvate) joining with oxaloacetate to make citrate; continues through apha-ketoglutarate, succinate, malate, and oxaloacetate again; intermediates can be used in anabolic pathways
4. fermentation, respiration and ATP production
1. fermentation regenerates NADH, using the energy from pyruvate; many versions; no ATP made
1. lactic acid fermentation -- cheese, yoghurt, pickles, sausage
   
2. ethanolic fermentation -- beer, bread
3. proprionic acid -- swiss cheese
   
4. 2-3-butanediol fermentation -- Voges-Proskauer test to differentiate enterbacteriaceae
5. mixed acid fermentation -- methyl red test
   
2. respiration uses NADH to build up the proton motive force (regenerating NADH in the process); uses the proton motive force and a special enzyme called ATP synthetase to join ADP and phosphate to make ATP
1. to build PMF, electrons from NADH are given to molecules in the cell membrane, which pass them to other molecules, which pass them to other molecules, etc., down to an electron acceptor; if the acceptor is oxygen, this is called aerobic respiration; if not oxygen, it is some sort of anaerobic respiration
1. imple chain in E. coli: NADH dehydrogenase to ubiquinone to ubiqinol oxidase
1. Pseudomonas has more complex chain with cytochrome c oxidase (basis of oxidase test)
   
2. in some cases (chemolithotrophs), other molecules can feed into the PMF besides NADH
3. Regulating metabolism -- basic concepts
   
1. the genetic material -- nucleic acids
   
1. nucleotides
2. DNA vs RNA
1. DNA is double stranded; strands anti-parallel; held together by strict base-pairing rules
2. RNA generally single stranded although may be double stranded completely or in parts; bases pair with either DNA or RNA nucleotides following the same rules
   
3. DNA replication
   
1. enzymes -- DNA polymerase, DNA gyrase, helicases, ligases, primases
2. process -- primers, leading strand, lagging strand, Okazaki fragments; build 5' to 3', read 3' to 5'
3. application -- PCR
   
2. protein synthesis
1. transcription
1. (-) and (+)  strands of DNA; (+) and (-) strands of RNA
2. promoter sequence
3. RNA polymerase and sigma factor
4. terminator sequence
2. translation
1. reading frames and codons; the genetic code
   
2. tRNA and anti-codons
3. ribosomes -- A, P and E sites
3. post-translational folding, chaperones, and signal sequences
3. regulating protein synthesis
1. constituitive genes
2. operons -- regulate related genes through repressor proteins that bind to DNA and block the attachment of RNA polymerase
   
1. lac operon -- genes for bringing lactose into the cell then using it for energy; regulated by glucose and lactose
1. if glucose present lots of ATP, little cyclic AMP; low cAMP means an activator protein can't bind (needs to be activated itself by cAMP) so RNA polymerase can't bind
2. if no glucose, lots of cAMP, activator binds to cAMP and DNA, so RNA polymerase can bind
1. if no lactose, repressor still sitting on the operator sequence so no transcription
2. if lactose (allolactose) present, it binds to repressor, repressor falls off DNA and transcription occurs
   
4.  
   

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