Download Sophomore Dental and Optometry Microbiology

Fundamentals II:
Bacterial Physiology and
Taxonomy
Janet Yother, Ph.D.
Department of Microbiology
[email protected]
4-9531
Learning Objectives
• Requirements for bacterial growth
• Culturing bacteria in the lab
• Bacterial mechanisms for transporting
substrates
• Methods for identifying, classifying bacteria
Bacterial Growth and
Metabolism
Growth Requirements
• Water - 70 to 80% of cell
• Carbon and energy source (may be same)
– Most bacteria, all pathogens = chemoheterotrophs (use
organic molecules for carbon and energy sources)
– monosaccharides - glucose, galactose, fructose, ribose
– disaccharides - sucrose (E. coli can't use), lactose (S.
typhimurium can't use)
– organic acids - succinate, lactate, acetate
– amino acids - glutamate, arginine
– alcohols - glycerol, ribitol
– fatty acids
Growth Requirements - Nitrogen
• Inorganic source
– Ammonia (NH4+)  glutamate, glutamine
– Nitrogen fixation N2  NH4+  Glu, Gln
– Nitrate (NO3-) or nitrite (NO2-)
• Nitrate reduction NO3  NO2  NH4+
• Denitrification NO3   N2 (use NO3 as electron
acceptor under anaerobic conditions, give off N2)
• Organic source
– amino acids, e.g. (Glu, Gln, Pro)
Growth Requirements - Oxygen
• Aerobe (strict) - requires O2
– Cannot ferment (i.e., transfer electrons and protons
directly to organic acceptor); always transfers to
oxygen (respires)
• Anaerobe (strict) - killed in O2
– lack enzymes necessary to degrade toxic O2
metabolites; always ferment
O2
2O2
flavoproteins
TOXIC
2H2O2
catalase
hydrogen peroxide
+ 2H+
Ferrous ion
2O2-
superoxide
radical
superoxide
dismutase
2H2O + O2
O2 + H2O2
hydrogen peroxide
Growth Requirements - Oxygen
• Aerobe (strict) - requires O2
– Cannot ferment (i.e., transfer electrons and protons
directly to organic acceptor); always transfers to
oxygen (respires)
• Anaerobe (strict) - killed by O2
– lack superoxide dismutase, catalase; always ferment
• Facultative - grows + or - O2 (respire or ferment)
• Aerotolerant anaerobe - grows + or - O2 (always
ferments)
• Microaerophilic - grows best with low O2; can
grow without
Growth Requirements
• Temperature
– Thermophiles - >50oC
– Psychrophiles - 4oC to 20oC
– Mesophiles - 20oC to 40oC
• pH - mostly 6 to 8; can vary with environment
• Other
– Sulfur, phosphorous, minerals (K, Mg, Ca, Fe), growth
factors (aa, vitamins)
Bacterial Growth in Culture
• Lag phase - actively
metabolizing; gearing up for
active growth
stationary
b-lactams
effective here
growth
• Stationary phase - slowed
metabolic activity and growth;
limiting nutrients or toxic
products
• Death phase - exponential
l og OD
OR
log CFU/ m l
• Log phase - exponential
death
exponential (log)
not here
lag
loss of viability; natural or
induced by detergents,
antibiotics, heat, radiation,
chemicals
Growth rate dependent on bacterium, conditions
Maximum attainable cell density ~1010/ml (species-dependent)
Lysozyme – effective all
time, hr
Bacterial Culture Systems
stationary
death
l og OD
OR
log CFU/ m l
• Closed system (batch
culture) - typical growth
curve
• Open system (continuous
culture) - chemostat.
Constant source of fresh
nutrients - growth rate
doesn’t change (linear).
• Synchronous growth - all
cells divide at same time
exponential (log)
lag
time, hr
Bacterial Growth on Solid (Agar)
Medium
Each colony arose
from a single bacterial
cell (or chain for
streptococci, cluster
for staphylococci)
Nutrient Uptake
1. Hydrolysis of nonpenetrating nutrients by
proteases, nucleases, lipases
2. Cytoplasmic membrane transport - protein
mediated
a. facilitated diffusion
b. active transport - group translocation
c. active transport - substrate translocation
Facilitated Diffusion
• Passive mediated transport
• No energy required
• Carrier protein equilibrates [substrate]
in/out of cell
• Phosphorylation traps substrate in cell
• Glycerol = example
Active Transport - Group
translocation
• Requires energy (PEP, ATP)
• Carrier protein concentrates substrates in
cell
• Substrate altered and trapped in cell
• Glucose = example
Active Transport - Substrate
Translocation
• Requires energy (proton gradient or ATP)
• Carrier protein concentrates substrate in cell
• Substrate unchanged. Transport system has
higher affinity for substrate outside cell.
Protein-Mediated Transport (Uptake)
Mechanisms
Energy
Facilitated Diffusion no
Active Transport
(Group
Translocation)
Active Transport
(Substrate
Translocation)
Substrate
Trapped by P; Gly  Gly-P
equilibrated
PEP, ATP Altered (P);
Concentrated
ATP,
PMF
Example
Unchanged;
Concentrated
Glc  Glc-6-P
(phosphotransferase
system, PTS)
Mal, aa, peptides
(ABC transporters)
Bacterial Taxomony
How bacteria are named, classified,
and identified
Bacterial Taxonomy
• Nomenclature - assignment of names by international
rules. Latinized, italicized (Escherichia coli, E. coli)
• Classification - arrangement into taxonomic groups based
on similarities.
• Identification - determining group to which new isolate
belongs
• Bergey’s Manual of Systematic Bacteriology standard reference
Bacterial Nomenclature
•
•
•
•
•
•
•
•
•
Kingdom
Division
Class
Subclass
Order
Family
Tribe
Genus
Species
– Subspecies
Eubacteria
Gracilicutes
Scotobacteria
Spirochaetales
Spirochaetaceae
Borrelia
Borrelia burgdorferi
Numerical Classification enumerates similarities and differences
• Morphology
– Microscopic - size, shape, motility, spores,
stains (gram, acid fast, capsule, flagella)
– Colony - shape, size, pigmentation
• Biochemical, physiological traits - growth
under different conditions (sugars, C, pH, temp,
aeration)
Serological Classifications
• Reactivity of specific antibodies with
homologous antigens of different bacteria
• Usually surface antigens - capsules, flagella,
LPS (O-Ag), proteins, polysaccharide, pili
• Important in epidemiology (E. coli O157:H7)
Genetic relatedness
• DNA base composition - %GC
– Very different - unrelated
– Very similar - may be related
• Multilocus enzyme electrophoresis
• Ability to exchange and recombine DNA
• DNA restriction profile
Genetic relatedness
• DNA base composition - %GC
– Very different - unrelated
– Very similar - may be related
• Multilocus enzyme electrophoresis
• Ability to exchange and recombine DNA
• DNA restriction profile
Multilocus Enzyme Electrophoresis
1
2
ref
Starch gel; enzyme assays to detect proteins;
shifts in mobility due to changes in protein (amino acid) sequence
Genetic relatedness
• DNA base composition - %GC
– Very different - unrelated
– Very similar - may be related
• Multilocus enzyme electrophoresis
• Ability to exchange and recombine DNA
• DNA restriction profile
Restriction Fragment Length
Polymorphism (RFLP) analysis
1
2
3
4
DNA
Cut with restriction
enzyme
Agarose gel stained with ethidium bromide
Genetic relatedness
• DNA sequence - genes, whole genomes; true %
identity
• DNA hybridization - total or specific sequences
• DNA-RNA homology - hybridization between
DNA and rRNA (highly conserved, small part of
genetic material)
• rRNA sequence - most useful
– Determine sequence of DNA encoding rRNA
DNA Hybridization
ds DNA
heat
ss DNA
Total DNA or
specific sequence
+ labeled
http://members.cox.net/amgough/
Fanconi-genetics-PGD.htm
DNA (ss; 3H, fl) of known
DNA Hybridization - PCR
http://www.246.ne.jp/~takeru/chalk-less/lifesci/images/pcr.gif
Genetic relatedness
• DNA sequence - genes, whole genomes; true %
identity
• DNA hybridization - total or specific sequences
• DNA-RNA homology - hybridization between
DNA and rRNA (highly conserved, small part of
genetic material)
• rRNA sequence - most useful
– Determine sequence of DNA encoding rRNA
Sensitivity of rRNA
rRNA - associated with ribosome; critical for protein
synthesis
transcription
translation
(DNA ------------> mRNA -------------> protein)
• binds initiation site (Ribosome binding site, ShineDelgarno sequence) in mRNA
• must have 2o structure (base pairs with self)
• Changes in critical areas likely detrimental
• DNA that encodes rRNA is highly conserved among
bacteria of common ancestry
Phylogenetic trees are based on rRNA sequences
Translation Initiation
Ribosome
3’ end of
16S rRNA
3’
A
5’
N
U
N
UCCUCCA
mRNA 5’-NNNNNNAGGAGGU-N5-10-AUG-NNNn-3’
ShineInitiation
Delgarno
Codon
sequence
Ribosome Binding Site
Sensitivity of rRNA
rRNA critical for protein synthesis
• binds initiation site (Ribosome binding site,
Shine-Delgarno sequence) in mRNA
• must have 2o structure (base pairs with self)
• Changes in critical areas likely detrimental
• DNA that encodes rRNA is highly
conserved among bacteria of common
ancestry
Phylogentic trees are based on rRNA sequences
http://asiago.stanford.edu/RelmanLab/supplements/Nikkari_EID_8/nikkari2002.html
Sensitivity of rRNA
rRNA critical for protein synthesis
• binds initiation site (Ribosome binding site,
Shine-Delgarno sequence) in mRNA
• must have 2o structure (base pairs with self)
• Changes in critical areas likely detrimental
• DNA that encodes rRNA is highly
conserved among bacteria of common
ancestry
Phylogenetic trees are based on rRNA sequences
Domains (Kingdoms)
Based on evolutionary relationships
• Eukaryote (Plants, Animals, Protists, Fungi)
• Eubacteria (Eubacteria)
• Archaea (Archaea)