microbio le2

Microbial Nutrition

Cell metabolism

  • All life requires:

    • Electoral flow to drive all life processes

    • Energy to move electrons

    • Nutrients to make the cell parts

    • Sum total of all chemical reax that occur in a cell

Catabolism

  • From a larger molecule to smaller

  • Energy-releasing metabolic reactions

  • Breaks down molecular structures into smaller units

  • Supplies precursors for metabolism

anabolic reactions

  • Synthesis of smaller molecules to form larger molecules 

  • Energy requiring metabolic reactions 

  • Builds larger molecules from smaller molecules with input of energy 

Requires electron flow 

  • Source of electrons 

    • Litotrophs - Inorganic molecule as electron donors 

    • Organotrophs -  organic  molecule as electron donors

  •  ultimate electron acceptor

    •  inorganic molecules -   respiration

    •  organic molecules -   fermentation

 source of energy

  •  Phototrophs

    •  light energy excites electrons

    •  excited molecules are electron donors

  •   Chemotrophs

    •  chemicals areelectron donors

    •  oxidation of chemicals

 nutrition and cell chemistry

  •  Nutrients

    •  supply of monomers (or precursor of) required by cells for growth

    •  supplied from environment

  •   Macronutrients

    •  Nutrients

    •  ions necessary for protein functions

  • Micronutrients

    •  nutrients required in trace amounts

  biochemical components of cell

  •  Water:  80% of wet weight

  •  dry weight

    •  Protein:  40 to 70%

    •  nucleic acid:  13 to 34%

    •  Lipid:  10 to 15%

    •  monomers intermediates and inorganic ions

 Macronutrients

  •  cells make proteins,  nucleic acid and lipids

  •  Macronutrients

    •  macromolecules, metabolism

    • C, H, O, N, S, P, K, Mg, Fe

  • Sources 

    • Organic compounds

    •  inorganic salts

 Micronutrients

  •  elements needed in trace quantities

    • Co, Cu, Mn, Zn, V

    •  Enzymes

    •  tap water

  •  Growth Factors

    •  organic compounds

    •  Vitamins

      •  most function as coenzymes 

Other important nutrients 

  • Sodium Na

    •  important for certain types of cell transports

  •  calcium ca

    •  stabilizer of cell wall and endospores of bacteria

  •  magnesium Mg 

    • Component of chlorophyll and a stabilizer of membranes and ribosomes 

  • Iron Fe 

    • Component of cytochrome proteins of cell respiration

  •  Zinc Zn 

    • Essential regulatory element of eukaryotic genetics

    • major component of “zinc fingers”, binding factors that help enzymes adhere to specific sites on DNA 

Define media

  • Prepared with precise amounts of chemicals

  •  known composition

 complex media

  •  exact  composition unknown

  •  digests of beef soybean yeast

 other culturing considerations

  •  Ph

  •  oxygen concentration

  •  Temperature

  •  light or carbon dioxide

 simple diffusion ( passive)

  •  Fundamental  property of atoms and molecules that exist in a state of random motion

  •  energy required

    •  None

    •  substances move on a gradient from higher concentration to lower concentration

 facilitated diffusion ( passive)

  •   molecule binds to a specific receptor and membrane and is carried to the other side

    •  molecule- specific. Goes both directions.  rate of transport is limited by the number of binding site on transport proteins

  •  energy required

    •  None

    •  substances move on a gradient from higher concentration to lower concentration

 carrier-mediated active transport

  •  Atom  or molecules are pumped into or out of the cell by specialized receptors

  •  energy required

    •  driven by atp or proton motive force

Group translocation 

  • Molecule is moved across membrane and simultaneously converted to a metabolically useful substance 

  • Energy required

    •  Atp

bulk transport 

  •  mass transport of large particles cells liquids by engulfment and vesicle formation.  processes generally called endocytosis. phagocytosis moves solids into cell pinocytosis moves liquids into cells. 

Temperature

 

 cardinal temperature

  •  range of temperature for the growth of given microbial species

  minimum temperature

  •  lowest temperature that permits a microbes continued growth and metabolism

 maximum temperature

  •  highest temperature at which growth and metabolism can

  •  above maximum proteins are  denatured

 optimum temperature

  •  Intermediate,  promotes fastest rate of growth and metabolism

 temperature labels for microorganisms

 Psychrophiles

  •  optimum temperature: 15C

  •  capable of growth at 0C

  •  obligate with respect to cold ( cannot grow above 20C)

  •  natural habitat:  lakes, rivers, snowfields, polar eyes and deep ocean

  •  rarely pathogenic

Psychrotrophs

  •  gross slowly in the cold but have an optimum temperature of 15C and  30C

  •  Staphylococcus aureus and listeria monocytogenes are able to grow at refrigerator temperatures and cause foodborne disease

 Mesophiles

  •  majority of medically significant microorganisms

  •  grow 20 at intermediate temperatures between 20C  and 40C

  •  inhabits animals and plants  as well as soil and water and temperate, subtropical and tropical regions

  •  human pathogens; have optimal temperatures between 30C and 40C

 thermoduric 

  • Can survive short exposure to high temperatures but are normally mesophils

  •  common contaminants of heated or pasteurized foods

  •  examples are heat resistant cysts like Giardia and spore formers such as bacillus and costridium 

 Thermophile

  •  grows optimally at temperature is greater than 45C

  •  lives in soil and water associated with volcanic activity, compost piles, and habitats directly exposed to the sun

  •  very in heat requirements with the range growth of 45C to 80C

  •  most eukaryotic forms cannot survive above 60C

extreme thermophile 

  •  Grows between 80C and 121C

 ranking from cold to hot

  • Psychrophiles

  • Psychrotrophs

  • Mesophile

  • Thermoduric

  • Thermophile

  • Extreme thermophile

Environmental factor 

Gases 

  • O2 and co2 

    • Influence microbial growth

    •  o2 has the greatest impact on microbial growth

    •  important respiratory gas and powerful oxidizing agent

  •  microbes fall into one of three categories

    •  use oxygen and detoxify it

    •  does not use oxygen and does not detoxify it

    •  use does not use oxygen and detoxifies

 how microbes process oxygen

  •  transformed into several toxic products

    •  singlet oxygen: extremely reactive molecule that can damage and destroy a cell by oxidation of membrane lipids

    •  superoxide ion ( o2): highly reactive

    •  hydrogen peroxide ( h2o2):  toxic to cells and used as a disinfectant

    •  hydroxyl radicals (OH): also highly reactive

oxygen usage patterns in microbes

 Aerobes

  •  use gaseous oxygen in their metabolism

  •  possesses enzyme needed to process toxic oxygen products

    Obligate aerobe

  •  cannot grow without oxygen

  •  example of this is fungi protozoa and many bacterias

 Microaerophils

  •  do not grow at normal atmospheric concentrations of oxygen but require a small amount of it in its metabolism

  •  Example

    •  those that live in soil or water or in mammalian hosts that are not directly exposed to the atmosphere

 facultative anaerobes

  • Do not require oxygen for metabolism, but uses it when it's present

  •  can also perform anaerobic metabolism

  •  in thioglycollate medium, growth is throughout but heavier in the aerobic portion

Anaerobes (obligate anaerobe)

  • lack the metabolic enzyme for using oxygen in respiration

  • also lack the enzyme for processing toxic oxygen and die in its presence

  • ex. many oral bacteria, intestinal bacteria

Aerotolerant anaerobes

  • do not use oxygen but can survive and grow to a limited extent in its presence

  • not harmed by oxygen

  • possesses alternate mechanisms for breaking down peroxides and super oxide

  • ex

    • certain lactobacilli and streptococci, clostridial species

carbon dioxide

capnophiles

  • grow best at higher CO2 levels than is normally present in the atmosphere

  • important in the initial isolation of the ff:

    • neisseria

    • brucella

    • streptococcus pneumonias

oxygen

effect of O2

  • protein-inactivation

  • effect of toxic oxygen-derivatives (toxic and powerful oxidizing agents):

    • flavoprotein: hydrogen peroxide

    • superoxide radicals (O2-)

    • hydroxy radical (OH-)

protection of bacteria

  • superoxide dismutase

  • catalase

  • peroxidases

environmental factor: pH

  • degree of acidity or alkalinity of a solution

  • expressed by the pH scale

  • number ranging from 0-14

  • most organisms live/grow in habitats between 6 & 8 because strong acids and bases can be damaging to enzymes and other cellular structures

Acidophiles

  • thrives in acidic environments

obligate acidophiles

  • Euglena mutabilis: acid pools of pH 0-1

  • Thermoplasma: coal piles pH ½

  • Picrophilus: thrives at pH7 but can live at pH 0

Alkalinophiles

  • thrive in alkaline conditions

  • natronomonas: hot pools and soils at pH 12

  • proteus: can create alkaline conditions to neutralize urine and colonize and infect the urinary system

Osmophiles

  • live in habitats with high solute concentrations

halophiles

  • prefer high concentrations of salt

obligate halophiles

  • halobacterium and halococcus grow optimally at solutions of 25% NaCl but require at least 9% NaCl

Facultative halophiles

  • highly resistant to salt, even though they do not normally reside in high salt environments

  • staphylococcus aureus: grow on NaCl media ranging from 0.1% to 20%

environmental factor: pressure

barophiles

  • exist under pressure that range from a few times to over 1000 times the pressure of the atmosphere

  • deep sea microbes

  • strictly adapted to high pressure

  • will rupture when exposed to normal atmospheric pressure

thermobarophiles

  • pyrococcus spp.

radiation

  • ionizing radiation 

  • effect

    • cause mutations and directly cause death

    • destroys ring-structure, breaks hydrogen bonds, oxidizes double bond & polymerizes certain molecules

  • ultraviolet radiation

    • form thymine dimers in DNA causing DNA damage

Bacterial growth

  • increase in the number of cells (population increase)

  • growth

    • enlarge

    • cytoplasm

    • replicate DNA

binary fission

  • dna replication

  • cell elongation

  • septum formation

  • completion of septum with formation of distinct walls

  • cell separation

cell multiply

  • initiation maass reached

  • initiation of replication

  • origins separate 

  • cell elongates as replication continues

  • threshold cell reached

  • chromosomes separate

  • initiation of septum formation

  • cells divide

Filamentous temperature sensitive proteins and cell division

  • ”fts”

  • archaea and bacteria

  • interact to form division apparatus: divisome

  • has many different types

    • FtsZ: structurally similar to eukaryotic tubulin

      • also observed in mitochondria and chloroplasts

      • defines division plane in the cell

      • FtsZ ring forms after DNA replication

      • responsible for initiating the formation of the septal ring

    • FtsA: possesses an amino acid sequence domain found in ATPases

    • ZipA (FTsZ interacting protein A): cell membrane protein in E.coli that binds via C-terminal end to the FtsZ ring

    • Ftsl and FtsW: located at the site of septum formation

      • ftsl gene (phpB): codes for the Ftsl protein (PBP3) which is an enzyme required for the formation of peptide cross-links during peptidoglycan

    • FtsB, FtsK, FtsL, FtsN, and FtsQ

      • protein products are all membrane associated at the division site

      • has a cytoplasmic domain at the N-terminal end, a single transmembrane domain and a C-terminal domain in the periplasm

Min proteins

  • MinD

    • forms spiral structure on the inner surface of the cytoplasmic membrane and localize MinC

    • back and forth

  • MinC

    • binds to MinD

    • serves as FtsZ inhibitor, preventing it from polymerization

  • MinE

    • oscillates from pole to pole

    • sweep MinC and MinD aside

Aside from Min proteins, what other elements determines the site of septum formation?

  • nucleoid occlusion: dense nucleoid mass interferes with the polymerization of FtsZ ring on the inner surface of the cell membrane

Cell morphology

  • MreB

    • major shape-determining proteins in rods, spirillum, spirochetes

    • absent in cocci

    • forms actin-like cytoskeleton

Population Growth

  • generation time or doubling time

    • time taken by the bacteria to double in number

  • mathematics of exponential growth

    • exponential growth or logarithmic

    • population doubles every generation

    • 100% increase in population every generation

  • bacterial growth curve

Measurements of microbial growth: direct

  • total counts

    • microscopic counts

      • counting chamber

      • DAPI - 4’, 6-diaminodphenylindole - DNA

      • Acridine orange

    • flow cytometer

  • viable cell count

    • viability staining

      • based on presence of intact cytoplasmic membrane (live or dead baclighttm viability stain)

      • red dye with propidium iodide: penetrates those without intact cytoplasmic membrane dead

      • green dye: penetrates all cells

    • plate count

      • serial dilution

microbial growth: the chemostat

  • continuous culture

  • maintains culture in exponential phase for long periods - days, weeks

  • in a chemostat, the rate at which the culture is diluted governs the growth rate and growth yield