Fundamentals II:Bacterial Physiology and

Taxonomy

Janet Yother, Ph.D.Department of Microbiology

jyother@uab.edu4-9531

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

superoxide radical

O2 2H2O2 2H2O + O2

flavoproteins catalase

2O2 2O2- O2 + H2O2

Ferrous ion + 2H+

TOXIC

hydrogen peroxide

superoxide dismutase 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

• Log phase - exponential growth

• Stationary phase - slowed metabolic activity and growth; limiting nutrients or toxic products

• Death phase - exponential loss of viability; natural or induced by detergents, antibiotics, heat, radiation, chemicals

Growth rate dependent on bacterium, conditionsMaximum attainable cell density ~1010/ml (species-dependent)

lag

exponential (log)

stationary

death

time, hr

log C

FU/m

l

log

OD

OR

b-lactamseffective here

not here

Lysozyme – effective all

Bacterial Culture Systems• 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

lag

exponential (log)

stationary

death

time, hrlog

CFU

/ml

log

OD

OR

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 mediateda. facilitated diffusion b. active transport - group translocationc. 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 Substrate Example

Facilitated Diffusion no Trapped by P; equilibrated

Gly Gly-P

Active Transport(Group Translocation)

PEP, ATP Altered (P);Concentrated

Glc Glc-6-P(phosphotransferase system, PTS)

Active Transport(Substrate Translocation)

ATP, PMF

Unchanged;Concentrated

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 Eubacteria• Division Gracilicutes• Class Scotobacteria• Subclass• Order Spirochaetales• Family Spirochaetaceae• Tribe• Genus Borrelia • Species Borrelia burgdorferi

– Subspecies

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 Electrophoresis1 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

DNACut with restriction

enzyme

1 2 3 4

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 Hybridizationds DNA ss DNATotal DNA or specific sequence

+ labeled DNA (ss; 3H, fl) of known

heat

http://members.cox.net/amgough/Fanconi-genetics-PGD.htm

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 rRNArRNA - associated with ribosome; critical for protein

synthesis (DNA ------------> mRNA -------------> protein)

• 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

transcription translation

Translation Initiation

3’ 5’ A N U N

UCCUCCA5’-NNNNNNAGGAGGU-N5-10-AUG-NNNn-3’

3’ end of16S rRNA

mRNA

Shine-Delgarnosequence

InitiationCodon

Ribosome

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)