PROKARYOTES

1
PROKARYOTES
NUTRITION
ECOLOGY AND GROWTH
2
BACTERIAL UBIQUITY

• Prokaryotes are found almost everywhere
• From deep oceans to volcanoes
• From polar regions to equator
• From the Great Salt Lake to freshwater streams
• Individual species differ, and grow in a limited
  set of environments
• May grow at temperatures near boiling, but not at
  moderate temperatures
• May grow in the Great Salt Lake but not in
  freshwater streams

3
BACTERIAL UBIQUITY

• Growth is dependent upon many factors
• Host organism
• Temperature
• pH
• Available nutrients
• Etc.

4
CULTURING BACTERIA

• Microorganisms grow in mixed populations in
  nature
• Joint contributions to numerous processes
• Microorganisms are grown in pure culture in the
  laboratory
• Requires isolation from a mixed culture
• Facilitates study of functions of a particular
  species

5
CULTURING BACTERIA

• Only 0.1 of prokaryotes can be grown in pure
  culture
• Most environmental microbes are difficult to
  study
• Growth requirements difficult to determine
• Most medically important bacteria can be grown in
  pure culture
• Why do you suppose this is the case?

6
CULTURING BACTERIA

• Various techniques of culturing microorganisms
• Equipment must be sterile
• Free of microorganisms
• Aseptic technique employed
• Minimize chance of inadvertent introduction of
  microorganisms
• Various types of media
• Contain necessary nutrients
• Solid or liquid

7
MEDIA

• Liquid media
• Nutrients required for growth
• Different requirements for different species
• Cannot be used to isolate pure cultures
• Solid media
• Liquid media plus solidifying agent agar
• Allows isolation of pure cultures
• Containers
• Petri dish (plates), tubes (deeps, slants)
• Not airtight (allows gas exchange)
• Excludes airborne microorganisms

8
AGAR

• Resistant to bacterial degradation
• Survives high-temperature treatment
• Can be sterilized in autoclave
• Liquefies at high temperatures
• Can be poured into convenient containers
• Remains liquid until below 45oC
• Once solidified, remains solid over a wide range
  of temperatures
• Melts at 95oC
• Translucent
• Colonies are visibly apparent

9
PURE CULTURE

• Bacteria are separated and placed on solid medium
• Various types of dilutions and plating techniques
  employed
• Individual bacteria divide to form visible
  colonies
• Visible at gt 1 million cells
• Genetically identical
• Clone
• Each clone is a pure culture

Streak-Plate Isolation
10
STOCK CULTURES

• After obtaining a pure culture, it is maintained
  as a stock culture
• Maintained as an inoculum for later study
• Various means of storage
• Agar slant
• Frozen in glycerol solution
• Glycerol prevents damage to cells from ice
  crystals
• Lyophilized (freeze-dried)

11
BACTERIAL GROWTH

• Defines as an increase in cell number
• Division by binary fission
• Simpler than mitotic division
• Cell size doubles
• Cell components doubled
• Cell divides
• Repeat
• Growth is exponential
• 1?2?4?8, etc.

12
BACTERIAL GROWTH

• Doubling time / generation time
• Time required for population to double in number
• Variable, dependent upon
• Species
• Growth conditions (nutrients, temp, etc.)
• Escherichia coli optimally 20 minutes
• Mycobacterium tuberculosis 12 24 hours
• Longer under suboptimal conditions

E. coli dividing
13
EXPONENTIAL GROWTH

• How to get rich quick
• On the first day of the month, put a penny in a
  jar
• On the second day, double this amount (2 cents)
• On the third day, double this amount (4 cents)
• Etc.

• Day 1 0.01
• Day 5 0.16
• Day 10 5.12
• Day 15 163.84
• Day 20 5,242.88
• Day 25 167,772.16
• Day 28 1,342,177.28
• Day 30 5,368,709.12

14
EXPONENTIAL GROWTH

• Bacteria have the ability to increase
  exponentially
• Exponential growth increases numbers incredibly
  quickly
• Exponential growth is not always possible
• Growth can be limited by various factors
• Environmental factors
• Nutrient availability

15
ENVIRONMENTAL FACTORS

• Various environmental factors affect prokaryotic
  growth
• Temperature requirements
• O2 requirements
• pH requirements
• Water availability
• Interactions with other organisms

16
TEMPERATURE REQUIREMENTS

• All species can grow within a particular range of
  temperatures
• Enzymes denatured above this range
• Optimal temperature within this range
• Divided into groups based on temperature
  optimum
• How does temperature affect the rate of chemical
  reactions?
• How would the enzyme DNA polymerase differ
  between mesophiles and thermophiles, etc.?

17
TEMPERATURE REQUIREMENTS

• Psychrophiles (-5ºC 15ºC)
• Psychrotrophs prefer gt 15ºC, but tolerate lower
• e.g., Listeria monocytogenes (food poisoning)
• Mesophiles (25ºC 45ºC)
• e.g., Escherichia coli
• Most other common bacteria, most human pathogens
• Thermophiles (45ºC 70ºC)
• Thermus aquaticus from thermal springs
• Hyperthermophiles (70ºC 110ºC)
• Members of Archaea
• Many members of Archaea are extremophiles
• Pyrolobus fumarii isolated from a hydrothermal
  vent has max growth temp of 113ºC

18
TEMPERATURE AND DISEASE

• Leprosy (Hansens disease)
• Caused by Mycobacterium leprae
• Typically affects body extremities
• Ears, hands, feet, fingers
• Syphilis
• Caused by Treponema pallidum
• Lesions generally appear on genitalia, lips,
  tongue, throat
• Early treatments involved induction of fever by
  deliberate infection with malaria parasite
• Antibiotics are a kinder, gentler treatment
• What can you determine regarding the temperature
  requirements of these bacteria?

19
OXYGEN REQUIREMENTS

• O2 can be useful
• e.g., aerobic cellular respiration
• O2 can be damaging
• Readily converted into toxic compounds
• esp. superoxide (O2-) and hydrogen peroxide
  (H2O2)
• Enzymes detoxify these toxic compounds
• Superoxide dismutase O2- ? H2O2
• Catalase H2O2 ? H2O O2
• Not all bacteria possess these enzymes
• Other enzymes exist that detoxify these compounds

20
OXYGEN REQUIREMENTS

• O2 levels vary widely in different environments
• Earths atmosphere is 20 O2
• O2 is absent from some environments
• e.g., swamps, beneath soil surface, human
  intestines, etc.
• Bacteria differ in their requirements for O2
• e.g., Some bacteria absolutely require O2 for
  cellular respiration
• Obligate aerobes
• Typically possess enzymes detoxifying O2- and
  H2O2
• Bacteria differ in their tolerance of O2
• e.g., Some bacteria cannot tolerate O2
• Obligate anaerobes
• Typically lack enzymes detoxifying O2- and H2O2

21
OXYGEN REQUIREMENTS

• O2 requirements determined using a shake tube
• (Could also use thioglycollate broth)
• Sterile tube of nutrient agar boiled
• Agar melted, O2 driven off
• Cooled to 50oC, bacteria added
• Agar hardens
• Incubate, note areas of growth
• What enzymes do you think each of these bacteria
  possess?
• Why do facultative anaerobes grow better when O2
  is present?

22
pH REQUIREMENTS

• All species can grow within a particular range of
  pH values
• Optimal pH within this range
• Divided into groups based on pH optimum
• Acidophiles (optimal pH lt 5.5)
• Neutrophiles (optimal pH near neutral)
• Alkalophiles (optimal pH gt 8.5)
• Internal pH constant and typically near neutral
• Often maintained by ion pumps

23
WATER AVAILABILITY

• Water is absolutely required for growth
• Constitutes 70 of cell
• May be present but osmotically unavailable
• High solute environment removes H2O from cell

Solar Evaporation Pond
(Red color due to pigments of halophilic
organisms)
24
WATER AVAILABILITY

• Microbes differ in their ability to live in high
  salt environments
• Two main ways to deal with high osmolarity in the
  environment
• Actively pump ions into cell
• e.g., K
• Produce small solute molecules to increase
  internal osmolarity
• e.g., proline

25
WATER AVAILABILITY

• Osmotolerant
• Organisms able to tolerate salt concentrations up
  to 10, but not requiring high salt
  concentrations
• a.k.a. facultative halophiles
• Halophiles
• a.k.a. obligate halophiles
• Organisms requiring high levels of NaCl to grow
• 3 9 minimum NaCl required
• Frequently spoilage organisms in high-salt or
  high-sugar preserved foods
• Examples of such foods?
• Many extreme halophiles belong to domain Archaea

26
MICROBIAL ASSOCIATIONS

• Microbes do not grow in pure culture in nature
• Live in shared habitats and interact with other
  organisms
• Symbiosis
• Close partnership between organisms
• Three main types
• Mutualism
• Commensalism
• Parasitism
• Non-symbiotic relationships
• Synergism
• Antagonism

27
MUTUALISM

• Association between organisms in which both
  benefit
• Lichen consist of a fungus and an alga (or
  cyanobacterium)
• What does the fungus gain?
• What does the photosynthetic partner gain?
• Protozoa in a termites gut hydrolyze cellulose
• What does the termite gain?
• What does the protozoan gain?

28
COMMENSALISM

• Association between two organisms in which one
  partner benefits and the other is unaffected
• e.g., Satellitism
• One bacterium produces a growth factor required
  by the second bacterial species
• Small colonies of the second species are able to
  grow near colonies of the first
• See figure 7.11, p. 206 Talaro Talaro

29
PARASITISM

• Association between two organisms in which one
  partner benefits and the other is harmed

Oral syphilis lesions
Bacteriophage infection
Tobacco mosaic virus
Ergot fungus on rye
Chlamydia attached to oviduct mucosa
30
NON-SYMBIOTIC ASSOCIATIONS

• Synergism
• Interrelationship between two or more free-living
  organisms of benefit to all
• Relationship not necessary for survival
• Similar to mutualism
• Antagonism
• Association between free-living species arising
  from competition
• One organism secretes a substance that inhibits
  or destroys other organisms
• e.g., antibiotics

31
MICROBIAL NUTRITION

• Process by which chemical substances (nutrients)
  are acquired from the environment and used in
  cellular activities
• Nutritional requirements
• Source of elements
• Source of energy
• Substances required by an organisms are termed
  essential nutrients

32
MICROBIAL NUTRITION

• Energy source
• Phototroph
• Derives energy from sunlight
• Chemotroph
• Derives energy from chemicals

• Carbon source
• Autotroph
• Obtains carbon as CO2
• Heterotroph
• Obtains carbon in organic forms

33
NUTRIENTS

• Macronutrients
• Required in relatively large amounts
• Principle roles in cell structure and metabolism
• e.g., energy source
• e.g., carbohydrates, proteins, etc.
• Micronutrients
• a.k.a. trace elements
• Enzyme cofactors, etc.
• e.g., Mg, Zn, Ni, etc.
• Requirements differ between species

• Organic nutrients
• Contain both C and H
• e.g., carbohydrates, lipids, nucleic acids,
  proteins, CH4, etc.
• Not required by all microorganisms
• Inorganic nutrients
• Lack either C or H
• e.g., metals, salts, gases, water
• Natural reservoirs are mineral deposits, water,
  air
• Required by all microorganisms

34
CELLULAR CHEMISTRY

• Cytoplasmic composition of Escherichia coli
• Aside from the 70 H2O
• 97 organic molecules (mainly proteins)
• What are the four major classes of biological
  macromolecules?
• 96 composed of six elements (CHNOPS)
• 5,000 different compounds
• Nutritional requirements very minimal
• Water, glucose, and a few salts
• No growth factors required (more on these later)

35
REQUIRED ELEMENTS

• Major elements
• Elements comprising cell constituents
• e.g., C, H, N, O, P, S, etc.
• Must be supplied in a usable form
• e.g., CO2 is usable by some (not all) organisms
• e.g., N2 is usable by very few organisms
• Trace elements
• Required in minute amounts
• e.g., Co, Zn, Cu, Mb, Mn, etc.
• Typically enzyme cofactors

36
GROWTH FACTORS

• Some bacteria cannot synthesize some of their
  cell constituents
• e.g., certain amino acids, vitamins, etc.
• A supply of these compounds is required for
  growth
• These required compounds are termed growth
  factors

• Not all bacteria require growth factors
• Growth factor requirements differ for different
  bacteria
• e.g., E. coli requires no growth factors
• e.g., species of Neisseria require 40 growth
  factors
• 7 vitamins, 20 amino acids, etc.
• Bacteria requiring many growth factors are termed
  fastidious

37
NUTRIENT SOURCES

• Ultimately derived from an inorganic reservoir
• Source of nutrients
• Replenished by organisms
• Nutrient cycling is a critically important
  ability of microorganisms

38
CARBON

• Heterotroph
• Requires carbon in an organic form
• e.g., proteins, carbohydrates, etc.
• Nutritionally dependent upon other life forms
• Not all organic carbon us usable by all organisms
• Did you ever eat a pine tree?
• Autotroph
• Uses CO2 as its carbon source
• Converts CO2 into organic carbon
• Not nutritionally dependent upon other life forms

39
NITROGEN

• Important component of proteins, DNA, RNA
• Primary nitrogen source for heterotrophs
• Main inorganic reservoir is atmospheric gas (N2)
• 79 of Earths atmosphere
• N2 can be used by a small number of
  microorganisms
• Nitrogen fixation
• N2 ? NH3

Rhizobium on clover root
40
NITROGEN

• Decomposers convert organic nitrogen into NH3
• Ammonification
• NH3 can be converted to other inorganic forms of
  nitrogen by various microorganisms
• NO3-, NO2-, NH3
• Many organisms can use one or more of these
  inorganic forms of nitrogen

41
OXYGEN

• Required for aerobic cellular respiration
• Major component of various macromolecules
• Even anaerobes require oxygen in some form
• Main inorganic reservoirs
• Free oxygen (O2) constitutes 20 of Earths
  atmosphere
• Oxygen is also present in many inorganic salts
• e.g., sulfates, phosphates, nitrates, etc.

42
HYDROGEN

• Major element in all organic molecules
• esp. carbohydrates, proteins, etc.
• Also important element in many inorganic
  compounds
• e.g. H2O, many salts
• Important element in certain gases
• H2S, CH4, H2
• These gases both used and produced by microbes

43
PHOSPHORUS

• Important component of certain molecules
• DNA, RNA, ATP, phospholipids, etc.
• Main inorganic source is phosphate (PO43-)
• Present in rocks ad other mineral deposits
• Generally very scarce in environment
• Scarcity of phosphorus is typically limiting
  factor for growth
• Overabundance in water leads to eutrophication

44
SULFUR

• Essential component of some vitamins and amino
  acids
• Mineral form widely distributed in environment
• Hydrogen sulfide gas (H2S)
• Elemental sulfur (S)
• Sulfides (e.g., FeS)
• Sulfate (SO42-)
• Various forms usable by various microorganisms

• Acid drainage from a mine
• Bacteria oxidize metal sulfides, producing
  sulfuric acid (low pH)

45
MEASURING GROWTH

• Direct cell counts
• Counts all cells alive or dead
• Viable cell counts
• Counts only cells able to multiply
• Measuring biomass
• Measures turbidity, total weight, or nitrogen
• Measuring cell products
• Byproducts of metabolism

46
DIRECT CELL COUNTS

• Direct microscopic count
• Bacteria placed in counting chamber
• Holds known volume of liquid
• Counted under microscope
• Requires gt107 cells/ml
• Cell-counting instruments
• Coulter counter
• Counts cells as they pass through a small hole
  single file
• Cells interrupt an electrical current
• Flow cytometer
• Similar to Coulter counter
• Measures passing cells by scattering of light
  from a laser

47
VIABLE CELL COUNTS

• Plate counts
• Prepare serial dilutions
• Known volume added to plate
• Pour plate or spread plate
• Incubate, count colonies
• Isolated cell ? colony
• Colony-forming unit
• Requires gt100 cells/ml
• Membrane filtration
• Used when cell concentration is low
• Known volume of liquid passed through filter
• Bacteria retained by filter, plated

48
VIABLE CELL COUNTS

• Most Probable Number (MPN)
• Prepare serial dilutions
• Incubate tubes
• Note growth
• Compare results to MPN table
• Gives statistical estimate of cell
  concentration
• Frequently used to quantify fecal coliforms

49
MEASURING BIOMASS

• Measuring turbidity
• Spectrophotometer measures light transmittance
  through specimen
• Requires gt107 cells/ml

• Measuring total weight
• Wet weight
• Dry weight
• Measuring N content
• Treat cells with H2SO4
• Nitrogen converted to NH3
• Assay NH3
• Compute biomass
• Cells are typically 14 NH3
• Cannot differentiate between living and dead
  cells

50
MEASURING CELL PRODUCTS

• Acid production
• Acids often produced as a result of the breakdown
  of sugars
• Detected by pH indicators
• Gas production
• Visually detected in Durham tubes
• Detected by pH indicators
• CO2 slightly reduces pH
• CO2 H2O ? H2CO3 ? H HCO3-
• ATP production
• Add firefly enzyme luciferase
• Fluorescence requires ATP hydrolysis
• Fluorescence quantified

51
BACTERIAL GROWTH

• Bacteria have the ability to increase
  exponentially
• Exponential growth increases numbers incredibly
  quickly
• Exponential growth is not always possible
• Growth can be limited by various factors
• Environmental factors
• Nutrient availability

52
GROWTH CURVES

• Populations typically a predictable pattern of
  growth over time
• Growth curve
• Four distinct phases
• Lag phase
• Log (exponential) phase
• Stationary phase
• Death phase

53
LAG PHASE

• No cell division
• Preparation for cell division
• Synthesis of macromolecules
• Length of phase depends on conditions
• e.g., age of cells, richness of medium, storage
  temperature, etc.

54
LOG PHASE

• Exponential growth
• Cell division exceeds cell death
• Shortest generation time measured here
• More susceptible to antibiotics and other
  chemicals

55
STATIONARY PHASE

• Resources depleted or toxins accumulate
• Cell division cell death
• Total number of viable cells remains relatively
  constant
• Cells more resistant to antibiotics and other
  chemicals

56
DEATH PHASE

• Cell death exceeds cell division
• Cells die at a fairly constant rate
• Slope generally less steep than growth phase

57
COLONY GROWTH

• Growth in colonies follows a growth curve
• Different parts of the colony are at different
  points in the growth curve
• Resources depleted sooner in center of colony
• Reach stationary phase sooner
• Exponential growth continues at edges

58
MIXED MICROBIAL COMMUNITIES

• In nature, species live in close association with
  other species
• e.g. mouth, intestines, soil, water
• Interactions between species can optimize
  environment for other species
• e.g. aerobes use up O2, reducing O2 levels for
  anaerobes
• e.g. metabolic wastes may provide nutrients for
  another species
• Difficult to reproduce in laboratory
• Generally hard to grow these species

59
BIOFILMS

• Bacteria attached to surfaces encased in
  polysaccharide
• Slippery rocks in streams
• Slime in kitchen drain
• Scum in toilet bowls
• Plaque on teeth
• Etc.

Dental plaque, a biofilm
60
BIOFILM FORMATION

• Bacteria adhere to surface
• Loose glycocalyx produced
• Other species may attach to glycocalyx and grow
• Cells form characteristic architecture with open
  channels for nutrients and waste
• Cell-cell communication important in structure

Latex bead moving through biofilm channel
(time-lapse sequence)