
Unit 3
Unit 3
monomer/polymer chemistry 1
many biomolecules are large and complex, with thousands or millions of atoms
many important biomolecules are made up of smaller building block molecules
large biomolecules made using building block molecules called polumers
the building block biomolecules are called monomers
proteins - polymers made of amino acids
amino acids are monomers
nucleic acids - polymers of nucleotides
nucleotides are monomers
polysaccharides - polymers of sugar
sugar molecule (often glucose) are monomers
monomer structure
monomers are made of a carbon skeleton with functional group(s) attatched
functional froups are the sites wehre monomers attach to one another
functional groups have only a few atoms
monomers are bonded together at functional groups by a reaction called dehydration synthesis
linked monomers can be broken apart between functional groups by reaction called hydrolysis
monomers are building blocks
polysaccharides, nucleic acids, and proteins are large biomolecules (polymers) made of smaller building block molecules (monomers)
polysaccharides means "many sugars" and starch, a polysaccharide, is made up of long chains of thousands of glucose molecules
DNA and RNA are the nucleic acids, and they are made of long chains of building block molecules called nucleotides
proteins are also large biological molecules; proteins are polymers that are long chains of building block molecules; the building block molecules of proteins are the amino acids
like all monomers, each amino acid has its own important functional groups; in fact, they are called amino acids because their key functional groups are an amino group and a carboxylic acid group
genetic information in action
lactase is an enzyme (proteins)
the lactase gene is a DNA sequence with the recipe for making the lactase enzyme
lactase mRNA molecules are copies of the lactase gene, help cell make lactase
lactase molecules digest lactose molecules so they can be metabolized
enzymes are proteins that do a job
lactase: shaping up right
lactase molecules are proteins
lactase molecules have the right shape to fit together with lactose molecules
when a lactase molecule binds a lactose molecule, the lactase will digest the lactose molecule into its building blocks, the simple sugars glucose and galactose
cells can use the simple sugars for energy
protein conformation (shape)
proteins are molecules that gets things done
a protein molecule's ability to do its job depends on the protein's overall shape
for a protein to work, it has to attach to, or interact with other things; these interactions depend on the proper shape of the protein
conformation is overall shape of proteins; i.e. tertiary or quaternary structure
correct conformation is required for protein molecules to function properly
proper conformation required for:
enzymes to bind substrate
antibodies to bind antigens
bacterial exotoxins to poison cells
virus coat proteins to form the virus shell
a protein molecules ability to do its job depends on the protein's overall shape; the overall shape of a protein is its conformation
for a protein to work, it binds to or interacts with other substances; these interactions are totally dependent on proper protein shape
changes in protein conformation will destroy the ability of protein to function
protein denaturation
a denatured protein no longer has its characteristic three dimensional shape
denatured proteins are non-functional; they cannot work because lost conformation
people sometimes denature proteins on purpose; denatureation can be caused by cooking, autoclaving and many disinfectants and detergents
denatutring proteins kills germs
proteins and amino acids
all proteins are made of building block molecules called amino acids
because proteins are made of many smaller units (amino acids), they are called polymers
because amino acids are the building blocks fo proteins, they are called monomers
the terms building blocks and monomers have the same meaning
protein facts
all proteins made using universal set of twenty different amino acids
average protein: about 300 amino acids
there are very few proteins with < 50 amino acids
an E. coli cell can make about 5,000 different kinds of protein molecules
a typical bacterial cell contains hundreds of millions of protein molecules
amino acids
amino acids are so named because:
they all have an amino group (-NH2)
and a carboxylic acid group (-COOH)
these amino & carboxylic acid groups are the functional groups where amino acids are joined (dehydration synthesis) and where they are cleaved apart (hydrolysis)
some amino acids also have functional groups located in the variable region
amino acid structure
central carbon atom: (a=alpha)
a-carbon is bonded to four things
lone hydrogen atom (-H)
carboxylic acid functional group (-COOH)
amino functional group (-NH2)
variable group (-R)
variable group (R group) is different in the different amino acids; it gives the amino acid its identity; page 44, R groups in blue
peptide bond
the chemical bond between amino acids
the dehydration synthesis reaction forms peptide bonds between the amino acids
the carbon atom in the carboxyl group of one amino acid bonds to the nitrogen atom in the amino group of another amino acid
its dehydration synthesis reaction: the amino group loses -H; carboxyl group loses -OH
the (-H) and the (-OH) combine to form H2O
hydrolysis reactions
water molecules ionize in aqueous solutions; the equation is written: H2o -> H+ + OH-
so, in a volume of water, there is always a small but significant number of H+ and OH-
in a hydrolysis reaction, H+ will bond with an atom on one functional group and OH- will bond to an atom of the other functional group and break the bond between the monomers
biochemical pathways
many important metabolic processes have several steps; the steps are sequential
each step in the process is a single chemical reaction catalyzed by a specific enzyme
all the steps, from start to finish, are known as a biochemical pathway
glycolysis, the kreb's cycle and the calvin cycle are examples of biochemical pathways
each step is catalyzed by a different enzyme
product of enzyme A is substrate for enzyme B
product of enzyme B is substrate for enzyme C
example: making amino acids from sugar molecules, ammonia, and other chemicals; making fatty acids from sugar molecules
the enzymes involved are often held in place in order by being placed in cell membranes
chemical energy
there is an exchange of energy whenever chemical reactions occur called chemical energy
endergonic reaction: requires energy; products have more chemical energy than reactants
exergonic reactions: release energy; products have less chemical energy than reactants
in general, synthesis reactions require energy (endergonic) and decomposition reactions release energy (exergonic)
anabolism and catabolism
synthesis reactions- when atoms, ions, or molecules combine to make larger, more complex molecules; often referred to as an anabolic reaction in biology (anabolism)
decomposition reactions- when larger, more complex molecules are broken into smaller atoms, ions, or molecules; often called catabolic reactions (catabolism)
catabolism
catabolic reactions release energy
catabolism usually refers to the breakdown of nutrient molecules like simple sugars, amino acids and fatty acids to generate energy
digestion of a biological molecule like starch into its glucose building blocks isnt usually referred to as catabolism, even though it is a breakdown reaction; no energy generated
metabolism
metabolism is the sum of all anabolic and catabolic reactions that occur in an organism
anabolic reactions require energy and are used to building larger molecules
catabolic reactions release energy by breaking down smaller energy molecules
catabolism plus anabolism is metabolism
energy from catabolism used to make ATP
energy for anabolism comes from ATP
ATP and energy coupling
adenosine triphospate (ATP) is the main player in the transfer of energy in cells
the chemical energy released by burning nutrient molecules like glucose is used to generate ATP molecules
the cell uses ATP molecules to do work
the energy released burning nutrients has to be trapped in ATP before it can be used
ATP is an energy transfer intermediate
ATP transfers energy from where it is released by burning nutrients, to where it is needed and used to do work within the cell
the energy released from nutrients is temporarily trapped in ATP molecules
ATP molecules can deliver and release the trapped energy wehre it is needed to do work
this is called energy coupling
ATP and cellular work
ATP contains energy in a form the cell can use for work. What work does a cell do?
chemical work: sythesis of biomolecules like making proteins or copying nucleic acids
transport work: moving things across plasma membrane, like food in and wastes out
mechanical work: moving chromosomes, spinning flagella, contracting muscles
ATP provides useable energy for cell
the ATP energy molecule is like a rechargeable battery
an ATP molecule has a packet of energy
it's charged
this energy is released when bond between the 2nd and 3rd phosphates is hydrolyzed (broken)
when the 3rd phosphate is removed, then we don't have ATP anymore, but ADP (+P)
ADP no longer has the packet of energy
it's discharged
cell uses energy from breakdown of nutrients to recharge ADP (make it into ATP again)
ATP isn't exactly like a battery
ATP goes directly from fully charged to fully discharged in a single reaction; nanoseconds
ATP isn't used to store energy; ATP is made and used almost immediately; there is less than one minute between ATP formation and use
ADP is recharged in a single reaction; this takes virtually no time at all
ATP and energy
ATP provides energy that the cell can use
ATP -> ADP + P is an exergonic reaction
the energy released can do work for the cell
ADP has less energy than ATP
ADP + P -> ATP is an endergonic reaction
the energy needed to power this comes from oxidizing (burning) nutrients like glucose
ATP has more energy than ADP
ATP and metabolism
energy is released in catabolic reactions
energy is trapped briefly in ATP molecules
ATP provides energy for anabolic reactions that make large molecules and for things like muscle contractions, flagella movement etc
energy released from catabolic reactions is used to make ATP, then ATP is used to drive anabolic reactions and other forms of work
carbohydrate catabolism
carbohydrate catabolism is a key source of energy for ATP production in living things
three types of carbohydrate catabolism
aerobic respiration:
glucose + O2 -> CO2 + H2O (38 ATP)
anearobic respiration:
glucose + not O2 -> CO2 + not H2O (<38 ATP)
fermentation:
glucose -> organic by-product (2 ATP)
other energy nutrients
amino acids can be broken down to provide energy (make ATP) for the cell
fatty acids can be catabolized to provide energy to make ATP (from ADP and P)
our focus: the catabolism of glucose
polysaccharides like starch and glycogen are made of glucose; digested to form free glucose and glucose catabolized
NADH: a high energy molecule
NADH is a key player in ATP production
NADH: nicotinamide adenine dinucleotide, high energy (known as the reduced form)
like ATP, NADH has a low energy form: NAD+ (known as the oxidized form)
during some catabolic reactions, the energy released is used by the cell to make NADH from NAD+ and H (hydrogen)
NADH has high energy electrons that will be used later to help make ATP
aerobic cellular respiration
often called aerobic respiration
aka oxidative catabolism
requires molecular oxygen (O2)
purpose: generate useable energy (ATP)
virtually all eukaryotes, some prokaryotes
occurs in 3 stages
glycolysis / kreb's cycle / electron transport
each stage is a biochemical pathway
gylcolysis
first stage of aerobic respiration
glycolysis is conversion of glucose (6-C) to two pyruvic acid (3-C) molecules
biochemical pathway; 10 enzymatic steps
glucose starting material; initial reactant
pyruvic acid (pyruvate) is final product
kreb's cycle
second stage of aerobic respiration
pyruvate initial reactant; CO2 and H products
biochemical pathway; 8 enzymatic steps
remember 2 pyruvates per glucose enter
pyruvic acid converted to acetyl-CoA and enters Kreb’s cycle called prep phase
at the end of Kreb’s cycle all three carbons of pyruvate have been converted to CO2
cell gets one ATP and 5 NADH per pyruvate
electron transport chain
third stage in aerobic respiration
biochemical pathway with proteins called electron carriers, as well as enzymes
the ETC is where molecular oxygen is used; oxygen is the final electron acceptor
the energy that was temporarily held in NADH is finally used
cell gets 34 ATPs per glucose
NADH from glycolysis and Kreb's cycle donates their high energy electrons to ETC
electrons pass down the chain of electron carriers releasing energy
released energy used to make ATP from ADP and P via ATP synthase (an enzyme)
in last steps, electrons from ETC hook up with H+ and O2, forming H2O
chemiosmosis: the parts
the chemiosmotic theory explains how the electron transport chain generates ATP
the electron carriers (7 total) are embedded in the membrane in sequential order
some of the electron carriers are proton pumps that transport H+ across membrane
the symbol H+ represents the hydrogen ion
ATP synthase is an enzyme that joins ADP and phosphate to make ATP molecules
chemiosmosis: the process
NADH gives its electrons to first electron carrier
as electrons pass down the chain, they pass through carriers called proton pumps
the proton pumps actively transport H+ to the other side of the membrane; the energy to do this comes from the flow of electrons down the ETC
the H+ concentration on the other side of the membrane builds up to high levels; the uneven distribution of H+ is potential energy and is called the proton gradient (an H+ is a proton)
the protons can flow back across the membrane only through special channels that contain the enzyme ATP synthase
when H+ flows back across the membrane through the channels, ATP synthase uses the energy from the flow of protons to make ATP from ADP and phosphate
anaerobic cellular respiration
stages the same as in aerobic respiration
gylcolysis, Kreb's cycle, electron transport chain
ATP yield is variable, depends on bacteria and growth conditions; often approaches 38 ATP
key difference is that an inorganic molecule other than O2 is final electron acceptor
final electron acceptors: nitrate (NO3), sulfate (SO4), carbonate (CO3) and others
some anaerobic bacteria
different anaerobic bacteria use different chemicalsas final electron acceptors
Desulfovibrio: sulfate (SO4) is final electron acceptor, produce hydrogen sulfide (H2S)
Methanobacterium: carbonate (CO3) is final electron acceptor, produce methane (CH4)
anaerobic bacteria do not need O2 to live and grow because they can generate ATP without using O2 (molecular oxygen)
fermentation
fermentation occurs in two stages
glycolysis: the conversion of glucose to pyruvate
fermentation: conversion of pyruvate to the fermentation end product
many types of fermentation, named after the fermentation end product
lactic acid fermentation: lactic acid is fermentation end product
acetic acid fermentation: acetic acid is fermentation end product
alcoholic fermentation: ethanol is fermentation end product
does not require oxygen
can occur in the presence of oxygen
yeilds two ATPs from each glucose
not all bacteria are capable of fermentation
regenerating NAD+ is purpose of converting pyruvate to fermentation end product
NAD+ is converted to NADH during glycolysis and the cell has only so much NAD+
fermentation example
brewers' yeast ferments glucose to ethanol
stage 1 is glycolysis
glucose -> 2 pyruvate and 2 NAD+ +2 H -> 2 NADH
stage 2 is fermentation
2 pyruvate -> 2 ethanol + 2 CO2 and 2 NADH -> 2 NAD+ + 2 H
purpose of stage 2 is to regenerate NAD+
purpose of stage 1 is to make the two ATPs
catabolic energy production and O2
some key facts about molecular oxygen
some bacteria do not need O2: ones that are capable of anaerobic respiration; ones that are fermentative
oxygen is toxic to some bacteria: they lack enzymes to neutralize toxic forms; have only anaerobic pathways
some bacteria require oxygen: ones that have only the pathways of aerobic respiration; obligate aerobes
some bacteria, the facultative anaerobes and the aerotolerant anaerobes, can live with or without oxygen
the air is 20% oxygen
five categories based on O2
obligate aerobes
facultative anaerobes
obligate anaerobes
aerotolerant anerobes
microaerophiles
obligate aerobes
require oxygen to live
can only perform aerobic respiration
Moraxella species (spp.) are aerobic; they cause conjuctivitis; the conjunctiva is the membrane covering the eyes and eyelids
Neisseria spp. are aerobic bacteria; one species causes gonorrhea; a different species causes meningitis; meninges are membranes around brain and spinal cord
facultative anaerobes
all can perform aerobic respiration
can switch to fermentation if oxygen is absent; so they can live without molecular oxygen
growth is usually rapid in presence of oxygen and limited in its absence
Escherichia coli and many other enteric bacteria, like Klebsiella pneumoniae
Saccharomyces cerevisiae bewer's yeast
a few facultative anaerobes switch from aerobic respiration to anaerobic respiration
sometimes growth rate is not much affected by the absence of molecular oxygen
usually the term facultative anaerobe refers to bacteria that can switch from aerobic respiration to fermentation
obligate anaerobes
oxygen is toxic to them; air kills them
performs anaerobic respiration onlu
some can form endospores to escape oxygen
Closstridium spp. can cause tetanus, botulism, food poisoning, gangrene; form endospores
Bacteroides spp. non-endospore forming obligate anaerobes that inhabit digestive tract of humans and other animals
aerotolerant anaerobes
do not use molecular oxygen
these microbes perform only fermentation
grown equally well in presence or absence of molecular oxygen; but they grow slowly
Streptococcus spp. are important human pathogens; strep throat and typical pneumonia
Lactobacillus spp. used in dairy products, ferment carbohydrates to lactic acid
microaerophiles
perform only aerobic respiration; use oxygen
normal oxygen levels (20%) are harmful to these cells; usually grow best between 2%-5% oxygen
often live in locations, or niches, within the host where oxygen levels are low
Campylobacter is a leading cause of acute gastroenteritis; often contaminates poultry
some reasons for growing bacteria
vaccine production
grow pathogenic bacteria in culture, then kill them and use killed bacteria as a vaccine; old pertussis vaccine
grow bacteria in culture, then purify a part of the cell, like capsule, and use as a vaccine, pneumococcal pneumonia
diagnosis of infection
traditional methods of identifying bacteria first require isolating and culturing bacteria in pure culture
antibiotic production
nearly half of antibiotics are extracted from cultures of antibiotic-producing bacteria; tetracycline, erythromycin
genetic engineering (biotechnology)
used to manufacture useful proteins
bacterial cells are given the gene (DNA) for useful protein and bacteria make the protein; human insulin
useful protein purified from bacterial culture
use in food and industry
to make many types of food, especially dairy foods
medical research
to learn more about microbes and how they cause disease; how they live; how they can be controlled
pure bacterial cultures
culture containing only one bacterial species
pure cultures are essential for work involving bacteria, including traditional identification
clinical samples may contain dozens of different bacteria; one must isolate pathogen and grow it in a pure culture for successful ID
streak plate technique often used here
culturing bacteria successfully
proper physical conditions for growth
temperature
pH
osmotic pressure
isotonic, hypertonic, hypotonic
proper chemical requirements
macronutrients
growth factors
trace elements
laboratory conditions optimized for maximum growth
temperature - for most clinical bacteria, it's near body temperature (98F or 37C)
osmotic pressure- near isotonic; the solute concentrations inside and outside cell are nearly equal; growth media is near isotonic
pH- the pH of culture media is near neutral; media generally has pH between 6 & 8; pH 7 is neutral
bacterial growth
bacterial growth really means multiplication
bacteria reproduce by binary fission
one cell becomes two, two becomes four, etc
bacteria can reproduce in a short time; time between cycles is called generation time
generation time for most bacteria is between twenty and sixty minutes
m x 2^n = p; m = # of starting cells, n = # of generations, p = # of cells in population
chemical growth requirements
CHONPS - carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur; called macronutrients; about 98% of the cell
organic growth factors - pre-made amino acids, nucleotides, and vitamins; required for growth of some microbes
trace elements - minerals and metals; calcium, zinc, magnesium
macronutrients
carbon: carbon atoms in carbon skeleton
hydrogen: fills in needed bonds
oxygen: two kinds are important
o2 from air: reactant in cellular respiration
o as in h2o; c6h12o6
nitrogen: amino acids, nucleotides, and ATP
phosphorus: nucleotides and phospholipids
sulfur: in some amino acids (proteins)
biomolecule breakdown
proteins: contain COHNS
starch and most carbohydrates: contain CHO
nucleic acids: contain CHONP
phospholipids: contain CHONP
lipids: contain CHO
peptidogylcan: contains CHON
culture media for bacteria
complex media - aka general purpose media; made from natural products like plants or animals
anaerobic media - for growing anaerobes; has chemicals that trap molecular oxygen
selective media - selects for different types of bacteria; grows one type better than others
differential media - differentiates between bacteria; the media (or the growth) will appear different if the bacteria growing in the media have different biochemical characteristics
complex media
made from natural products like animal, plant, or yeast extracts; peptones often added
lots of CHONPS in the media, these elements are part of different biomolecules, like proteins, amino acids, and carbohydrates found in the extracts
examples: nutrient broth, nutrient agar, tryptic soy broth, tryptic soy agar, brain heart infusion
complex media are for routine culturing of bacteria; aka general purpose media
anaerobic media
in addition to nutrients like shown above, anaerobic media contains chemicals that remove or soak up molecular oxygen (o2)
the chemicals that remove molecular oxygen are called reducing agents
it's oxygen free due to the reducing agent
used to culture obligate anaerobes, which are killed by molecular oxygen; Clostridium
selective media
used to grow desired bacteria by inhibiting the growth of undesired bacteria
used to separate (or select) desired type of bacteria from mixtures of bacteria
most selective media will grow only gram positive or only gram negative bacteria
selective media select for a particular kind of bacteria; media x selects for gram+, which means that g+ bacteria grow and g- bacteria dont
examples of selective media
brilliant green agar- contains a dye that prevents peptidoglycan layering; this slows or prevents the growth of gram - positives; BGA selects for gram negative bacteria
phenyl ethyl alcohol agar - contains PEA, which dissolves the outer membrane of the gram negative cell wall; prevents the growth of gram negatives; gram positives do grow; we say it selects for gram positive bacteria
differential media
will grow most, if not all, types of bacteria
used to differentiate between species of bacteria based on bacterial biochemistry
usually contain chemical indicators that change color in the presence of specific substances made during bacterial growth
widely used in bacterial identification; used to determine specific biochemical characteristics of unknown bacteria
example of differential media
phenol red fermentation tubes
media contains a known carbohydrate, broth with non-carbohydrate nutrients, phenol red
phenol red is a pH indicator
red at pH 7 and turns yellow at acidic pH (below pH 5)
if bacterium growing in this broth ferments the carbohydrate to acid, the media will turn yellow
if bacterium can't ferment carbohydrate, it grows on the non-carbohydrate nutrients (peptones) present in the broth and the media stays red
blood agar
this is a TSA based media that contains sheep red blood cells; media is reddish brown in color
some bacteria can lyse blood cells and degrade the hemoglobin (hg); hg makes blood red
these hemolytic bacteria will be surrounded by a tan colored halo on blood agar
non hemolytic bacteria will not have the halo
blood agar differentiates between the two types
combined selective and differential
some media are selective and differential
mannitol salt agar (MSA)
salt (NaCl) in the media selects for salt tolerant bacteria; meaning that only bacteria that can tolerate high salt levels will grow
the media also has mannitol (a carbohydrate) and phenol red; if the bacteria ferments the mannitol, the pH drops and media will turn from pink to yellow; no fermentation, stays pink
Unit 3
monomer/polymer chemistry 1
many biomolecules are large and complex, with thousands or millions of atoms
many important biomolecules are made up of smaller building block molecules
large biomolecules made using building block molecules called polumers
the building block biomolecules are called monomers
proteins - polymers made of amino acids
amino acids are monomers
nucleic acids - polymers of nucleotides
nucleotides are monomers
polysaccharides - polymers of sugar
sugar molecule (often glucose) are monomers
monomer structure
monomers are made of a carbon skeleton with functional group(s) attatched
functional froups are the sites wehre monomers attach to one another
functional groups have only a few atoms
monomers are bonded together at functional groups by a reaction called dehydration synthesis
linked monomers can be broken apart between functional groups by reaction called hydrolysis
monomers are building blocks
polysaccharides, nucleic acids, and proteins are large biomolecules (polymers) made of smaller building block molecules (monomers)
polysaccharides means "many sugars" and starch, a polysaccharide, is made up of long chains of thousands of glucose molecules
DNA and RNA are the nucleic acids, and they are made of long chains of building block molecules called nucleotides
proteins are also large biological molecules; proteins are polymers that are long chains of building block molecules; the building block molecules of proteins are the amino acids
like all monomers, each amino acid has its own important functional groups; in fact, they are called amino acids because their key functional groups are an amino group and a carboxylic acid group
genetic information in action
lactase is an enzyme (proteins)
the lactase gene is a DNA sequence with the recipe for making the lactase enzyme
lactase mRNA molecules are copies of the lactase gene, help cell make lactase
lactase molecules digest lactose molecules so they can be metabolized
enzymes are proteins that do a job
lactase: shaping up right
lactase molecules are proteins
lactase molecules have the right shape to fit together with lactose molecules
when a lactase molecule binds a lactose molecule, the lactase will digest the lactose molecule into its building blocks, the simple sugars glucose and galactose
cells can use the simple sugars for energy
protein conformation (shape)
proteins are molecules that gets things done
a protein molecule's ability to do its job depends on the protein's overall shape
for a protein to work, it has to attach to, or interact with other things; these interactions depend on the proper shape of the protein
conformation is overall shape of proteins; i.e. tertiary or quaternary structure
correct conformation is required for protein molecules to function properly
proper conformation required for:
enzymes to bind substrate
antibodies to bind antigens
bacterial exotoxins to poison cells
virus coat proteins to form the virus shell
a protein molecules ability to do its job depends on the protein's overall shape; the overall shape of a protein is its conformation
for a protein to work, it binds to or interacts with other substances; these interactions are totally dependent on proper protein shape
changes in protein conformation will destroy the ability of protein to function
protein denaturation
a denatured protein no longer has its characteristic three dimensional shape
denatured proteins are non-functional; they cannot work because lost conformation
people sometimes denature proteins on purpose; denatureation can be caused by cooking, autoclaving and many disinfectants and detergents
denatutring proteins kills germs
proteins and amino acids
all proteins are made of building block molecules called amino acids
because proteins are made of many smaller units (amino acids), they are called polymers
because amino acids are the building blocks fo proteins, they are called monomers
the terms building blocks and monomers have the same meaning
protein facts
all proteins made using universal set of twenty different amino acids
average protein: about 300 amino acids
there are very few proteins with < 50 amino acids
an E. coli cell can make about 5,000 different kinds of protein molecules
a typical bacterial cell contains hundreds of millions of protein molecules
amino acids
amino acids are so named because:
they all have an amino group (-NH2)
and a carboxylic acid group (-COOH)
these amino & carboxylic acid groups are the functional groups where amino acids are joined (dehydration synthesis) and where they are cleaved apart (hydrolysis)
some amino acids also have functional groups located in the variable region
amino acid structure
central carbon atom: (a=alpha)
a-carbon is bonded to four things
lone hydrogen atom (-H)
carboxylic acid functional group (-COOH)
amino functional group (-NH2)
variable group (-R)
variable group (R group) is different in the different amino acids; it gives the amino acid its identity; page 44, R groups in blue
peptide bond
the chemical bond between amino acids
the dehydration synthesis reaction forms peptide bonds between the amino acids
the carbon atom in the carboxyl group of one amino acid bonds to the nitrogen atom in the amino group of another amino acid
its dehydration synthesis reaction: the amino group loses -H; carboxyl group loses -OH
the (-H) and the (-OH) combine to form H2O
hydrolysis reactions
water molecules ionize in aqueous solutions; the equation is written: H2o -> H+ + OH-
so, in a volume of water, there is always a small but significant number of H+ and OH-
in a hydrolysis reaction, H+ will bond with an atom on one functional group and OH- will bond to an atom of the other functional group and break the bond between the monomers
biochemical pathways
many important metabolic processes have several steps; the steps are sequential
each step in the process is a single chemical reaction catalyzed by a specific enzyme
all the steps, from start to finish, are known as a biochemical pathway
glycolysis, the kreb's cycle and the calvin cycle are examples of biochemical pathways
each step is catalyzed by a different enzyme
product of enzyme A is substrate for enzyme B
product of enzyme B is substrate for enzyme C
example: making amino acids from sugar molecules, ammonia, and other chemicals; making fatty acids from sugar molecules
the enzymes involved are often held in place in order by being placed in cell membranes
chemical energy
there is an exchange of energy whenever chemical reactions occur called chemical energy
endergonic reaction: requires energy; products have more chemical energy than reactants
exergonic reactions: release energy; products have less chemical energy than reactants
in general, synthesis reactions require energy (endergonic) and decomposition reactions release energy (exergonic)
anabolism and catabolism
synthesis reactions- when atoms, ions, or molecules combine to make larger, more complex molecules; often referred to as an anabolic reaction in biology (anabolism)
decomposition reactions- when larger, more complex molecules are broken into smaller atoms, ions, or molecules; often called catabolic reactions (catabolism)
catabolism
catabolic reactions release energy
catabolism usually refers to the breakdown of nutrient molecules like simple sugars, amino acids and fatty acids to generate energy
digestion of a biological molecule like starch into its glucose building blocks isnt usually referred to as catabolism, even though it is a breakdown reaction; no energy generated
metabolism
metabolism is the sum of all anabolic and catabolic reactions that occur in an organism
anabolic reactions require energy and are used to building larger molecules
catabolic reactions release energy by breaking down smaller energy molecules
catabolism plus anabolism is metabolism
energy from catabolism used to make ATP
energy for anabolism comes from ATP
ATP and energy coupling
adenosine triphospate (ATP) is the main player in the transfer of energy in cells
the chemical energy released by burning nutrient molecules like glucose is used to generate ATP molecules
the cell uses ATP molecules to do work
the energy released burning nutrients has to be trapped in ATP before it can be used
ATP is an energy transfer intermediate
ATP transfers energy from where it is released by burning nutrients, to where it is needed and used to do work within the cell
the energy released from nutrients is temporarily trapped in ATP molecules
ATP molecules can deliver and release the trapped energy wehre it is needed to do work
this is called energy coupling
ATP and cellular work
ATP contains energy in a form the cell can use for work. What work does a cell do?
chemical work: sythesis of biomolecules like making proteins or copying nucleic acids
transport work: moving things across plasma membrane, like food in and wastes out
mechanical work: moving chromosomes, spinning flagella, contracting muscles
ATP provides useable energy for cell
the ATP energy molecule is like a rechargeable battery
an ATP molecule has a packet of energy
it's charged
this energy is released when bond between the 2nd and 3rd phosphates is hydrolyzed (broken)
when the 3rd phosphate is removed, then we don't have ATP anymore, but ADP (+P)
ADP no longer has the packet of energy
it's discharged
cell uses energy from breakdown of nutrients to recharge ADP (make it into ATP again)
ATP isn't exactly like a battery
ATP goes directly from fully charged to fully discharged in a single reaction; nanoseconds
ATP isn't used to store energy; ATP is made and used almost immediately; there is less than one minute between ATP formation and use
ADP is recharged in a single reaction; this takes virtually no time at all
ATP and energy
ATP provides energy that the cell can use
ATP -> ADP + P is an exergonic reaction
the energy released can do work for the cell
ADP has less energy than ATP
ADP + P -> ATP is an endergonic reaction
the energy needed to power this comes from oxidizing (burning) nutrients like glucose
ATP has more energy than ADP
ATP and metabolism
energy is released in catabolic reactions
energy is trapped briefly in ATP molecules
ATP provides energy for anabolic reactions that make large molecules and for things like muscle contractions, flagella movement etc
energy released from catabolic reactions is used to make ATP, then ATP is used to drive anabolic reactions and other forms of work
carbohydrate catabolism
carbohydrate catabolism is a key source of energy for ATP production in living things
three types of carbohydrate catabolism
aerobic respiration:
glucose + O2 -> CO2 + H2O (38 ATP)
anearobic respiration:
glucose + not O2 -> CO2 + not H2O (<38 ATP)
fermentation:
glucose -> organic by-product (2 ATP)
other energy nutrients
amino acids can be broken down to provide energy (make ATP) for the cell
fatty acids can be catabolized to provide energy to make ATP (from ADP and P)
our focus: the catabolism of glucose
polysaccharides like starch and glycogen are made of glucose; digested to form free glucose and glucose catabolized
NADH: a high energy molecule
NADH is a key player in ATP production
NADH: nicotinamide adenine dinucleotide, high energy (known as the reduced form)
like ATP, NADH has a low energy form: NAD+ (known as the oxidized form)
during some catabolic reactions, the energy released is used by the cell to make NADH from NAD+ and H (hydrogen)
NADH has high energy electrons that will be used later to help make ATP
aerobic cellular respiration
often called aerobic respiration
aka oxidative catabolism
requires molecular oxygen (O2)
purpose: generate useable energy (ATP)
virtually all eukaryotes, some prokaryotes
occurs in 3 stages
glycolysis / kreb's cycle / electron transport
each stage is a biochemical pathway
gylcolysis
first stage of aerobic respiration
glycolysis is conversion of glucose (6-C) to two pyruvic acid (3-C) molecules
biochemical pathway; 10 enzymatic steps
glucose starting material; initial reactant
pyruvic acid (pyruvate) is final product
kreb's cycle
second stage of aerobic respiration
pyruvate initial reactant; CO2 and H products
biochemical pathway; 8 enzymatic steps
remember 2 pyruvates per glucose enter
pyruvic acid converted to acetyl-CoA and enters Kreb’s cycle called prep phase
at the end of Kreb’s cycle all three carbons of pyruvate have been converted to CO2
cell gets one ATP and 5 NADH per pyruvate
electron transport chain
third stage in aerobic respiration
biochemical pathway with proteins called electron carriers, as well as enzymes
the ETC is where molecular oxygen is used; oxygen is the final electron acceptor
the energy that was temporarily held in NADH is finally used
cell gets 34 ATPs per glucose
NADH from glycolysis and Kreb's cycle donates their high energy electrons to ETC
electrons pass down the chain of electron carriers releasing energy
released energy used to make ATP from ADP and P via ATP synthase (an enzyme)
in last steps, electrons from ETC hook up with H+ and O2, forming H2O
chemiosmosis: the parts
the chemiosmotic theory explains how the electron transport chain generates ATP
the electron carriers (7 total) are embedded in the membrane in sequential order
some of the electron carriers are proton pumps that transport H+ across membrane
the symbol H+ represents the hydrogen ion
ATP synthase is an enzyme that joins ADP and phosphate to make ATP molecules
chemiosmosis: the process
NADH gives its electrons to first electron carrier
as electrons pass down the chain, they pass through carriers called proton pumps
the proton pumps actively transport H+ to the other side of the membrane; the energy to do this comes from the flow of electrons down the ETC
the H+ concentration on the other side of the membrane builds up to high levels; the uneven distribution of H+ is potential energy and is called the proton gradient (an H+ is a proton)
the protons can flow back across the membrane only through special channels that contain the enzyme ATP synthase
when H+ flows back across the membrane through the channels, ATP synthase uses the energy from the flow of protons to make ATP from ADP and phosphate
anaerobic cellular respiration
stages the same as in aerobic respiration
gylcolysis, Kreb's cycle, electron transport chain
ATP yield is variable, depends on bacteria and growth conditions; often approaches 38 ATP
key difference is that an inorganic molecule other than O2 is final electron acceptor
final electron acceptors: nitrate (NO3), sulfate (SO4), carbonate (CO3) and others
some anaerobic bacteria
different anaerobic bacteria use different chemicalsas final electron acceptors
Desulfovibrio: sulfate (SO4) is final electron acceptor, produce hydrogen sulfide (H2S)
Methanobacterium: carbonate (CO3) is final electron acceptor, produce methane (CH4)
anaerobic bacteria do not need O2 to live and grow because they can generate ATP without using O2 (molecular oxygen)
fermentation
fermentation occurs in two stages
glycolysis: the conversion of glucose to pyruvate
fermentation: conversion of pyruvate to the fermentation end product
many types of fermentation, named after the fermentation end product
lactic acid fermentation: lactic acid is fermentation end product
acetic acid fermentation: acetic acid is fermentation end product
alcoholic fermentation: ethanol is fermentation end product
does not require oxygen
can occur in the presence of oxygen
yeilds two ATPs from each glucose
not all bacteria are capable of fermentation
regenerating NAD+ is purpose of converting pyruvate to fermentation end product
NAD+ is converted to NADH during glycolysis and the cell has only so much NAD+
fermentation example
brewers' yeast ferments glucose to ethanol
stage 1 is glycolysis
glucose -> 2 pyruvate and 2 NAD+ +2 H -> 2 NADH
stage 2 is fermentation
2 pyruvate -> 2 ethanol + 2 CO2 and 2 NADH -> 2 NAD+ + 2 H
purpose of stage 2 is to regenerate NAD+
purpose of stage 1 is to make the two ATPs
catabolic energy production and O2
some key facts about molecular oxygen
some bacteria do not need O2: ones that are capable of anaerobic respiration; ones that are fermentative
oxygen is toxic to some bacteria: they lack enzymes to neutralize toxic forms; have only anaerobic pathways
some bacteria require oxygen: ones that have only the pathways of aerobic respiration; obligate aerobes
some bacteria, the facultative anaerobes and the aerotolerant anaerobes, can live with or without oxygen
the air is 20% oxygen
five categories based on O2
obligate aerobes
facultative anaerobes
obligate anaerobes
aerotolerant anerobes
microaerophiles
obligate aerobes
require oxygen to live
can only perform aerobic respiration
Moraxella species (spp.) are aerobic; they cause conjuctivitis; the conjunctiva is the membrane covering the eyes and eyelids
Neisseria spp. are aerobic bacteria; one species causes gonorrhea; a different species causes meningitis; meninges are membranes around brain and spinal cord
facultative anaerobes
all can perform aerobic respiration
can switch to fermentation if oxygen is absent; so they can live without molecular oxygen
growth is usually rapid in presence of oxygen and limited in its absence
Escherichia coli and many other enteric bacteria, like Klebsiella pneumoniae
Saccharomyces cerevisiae bewer's yeast
a few facultative anaerobes switch from aerobic respiration to anaerobic respiration
sometimes growth rate is not much affected by the absence of molecular oxygen
usually the term facultative anaerobe refers to bacteria that can switch from aerobic respiration to fermentation
obligate anaerobes
oxygen is toxic to them; air kills them
performs anaerobic respiration onlu
some can form endospores to escape oxygen
Closstridium spp. can cause tetanus, botulism, food poisoning, gangrene; form endospores
Bacteroides spp. non-endospore forming obligate anaerobes that inhabit digestive tract of humans and other animals
aerotolerant anaerobes
do not use molecular oxygen
these microbes perform only fermentation
grown equally well in presence or absence of molecular oxygen; but they grow slowly
Streptococcus spp. are important human pathogens; strep throat and typical pneumonia
Lactobacillus spp. used in dairy products, ferment carbohydrates to lactic acid
microaerophiles
perform only aerobic respiration; use oxygen
normal oxygen levels (20%) are harmful to these cells; usually grow best between 2%-5% oxygen
often live in locations, or niches, within the host where oxygen levels are low
Campylobacter is a leading cause of acute gastroenteritis; often contaminates poultry
some reasons for growing bacteria
vaccine production
grow pathogenic bacteria in culture, then kill them and use killed bacteria as a vaccine; old pertussis vaccine
grow bacteria in culture, then purify a part of the cell, like capsule, and use as a vaccine, pneumococcal pneumonia
diagnosis of infection
traditional methods of identifying bacteria first require isolating and culturing bacteria in pure culture
antibiotic production
nearly half of antibiotics are extracted from cultures of antibiotic-producing bacteria; tetracycline, erythromycin
genetic engineering (biotechnology)
used to manufacture useful proteins
bacterial cells are given the gene (DNA) for useful protein and bacteria make the protein; human insulin
useful protein purified from bacterial culture
use in food and industry
to make many types of food, especially dairy foods
medical research
to learn more about microbes and how they cause disease; how they live; how they can be controlled
pure bacterial cultures
culture containing only one bacterial species
pure cultures are essential for work involving bacteria, including traditional identification
clinical samples may contain dozens of different bacteria; one must isolate pathogen and grow it in a pure culture for successful ID
streak plate technique often used here
culturing bacteria successfully
proper physical conditions for growth
temperature
pH
osmotic pressure
isotonic, hypertonic, hypotonic
proper chemical requirements
macronutrients
growth factors
trace elements
laboratory conditions optimized for maximum growth
temperature - for most clinical bacteria, it's near body temperature (98F or 37C)
osmotic pressure- near isotonic; the solute concentrations inside and outside cell are nearly equal; growth media is near isotonic
pH- the pH of culture media is near neutral; media generally has pH between 6 & 8; pH 7 is neutral
bacterial growth
bacterial growth really means multiplication
bacteria reproduce by binary fission
one cell becomes two, two becomes four, etc
bacteria can reproduce in a short time; time between cycles is called generation time
generation time for most bacteria is between twenty and sixty minutes
m x 2^n = p; m = # of starting cells, n = # of generations, p = # of cells in population
chemical growth requirements
CHONPS - carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur; called macronutrients; about 98% of the cell
organic growth factors - pre-made amino acids, nucleotides, and vitamins; required for growth of some microbes
trace elements - minerals and metals; calcium, zinc, magnesium
macronutrients
carbon: carbon atoms in carbon skeleton
hydrogen: fills in needed bonds
oxygen: two kinds are important
o2 from air: reactant in cellular respiration
o as in h2o; c6h12o6
nitrogen: amino acids, nucleotides, and ATP
phosphorus: nucleotides and phospholipids
sulfur: in some amino acids (proteins)
biomolecule breakdown
proteins: contain COHNS
starch and most carbohydrates: contain CHO
nucleic acids: contain CHONP
phospholipids: contain CHONP
lipids: contain CHO
peptidogylcan: contains CHON
culture media for bacteria
complex media - aka general purpose media; made from natural products like plants or animals
anaerobic media - for growing anaerobes; has chemicals that trap molecular oxygen
selective media - selects for different types of bacteria; grows one type better than others
differential media - differentiates between bacteria; the media (or the growth) will appear different if the bacteria growing in the media have different biochemical characteristics
complex media
made from natural products like animal, plant, or yeast extracts; peptones often added
lots of CHONPS in the media, these elements are part of different biomolecules, like proteins, amino acids, and carbohydrates found in the extracts
examples: nutrient broth, nutrient agar, tryptic soy broth, tryptic soy agar, brain heart infusion
complex media are for routine culturing of bacteria; aka general purpose media
anaerobic media
in addition to nutrients like shown above, anaerobic media contains chemicals that remove or soak up molecular oxygen (o2)
the chemicals that remove molecular oxygen are called reducing agents
it's oxygen free due to the reducing agent
used to culture obligate anaerobes, which are killed by molecular oxygen; Clostridium
selective media
used to grow desired bacteria by inhibiting the growth of undesired bacteria
used to separate (or select) desired type of bacteria from mixtures of bacteria
most selective media will grow only gram positive or only gram negative bacteria
selective media select for a particular kind of bacteria; media x selects for gram+, which means that g+ bacteria grow and g- bacteria dont
examples of selective media
brilliant green agar- contains a dye that prevents peptidoglycan layering; this slows or prevents the growth of gram - positives; BGA selects for gram negative bacteria
phenyl ethyl alcohol agar - contains PEA, which dissolves the outer membrane of the gram negative cell wall; prevents the growth of gram negatives; gram positives do grow; we say it selects for gram positive bacteria
differential media
will grow most, if not all, types of bacteria
used to differentiate between species of bacteria based on bacterial biochemistry
usually contain chemical indicators that change color in the presence of specific substances made during bacterial growth
widely used in bacterial identification; used to determine specific biochemical characteristics of unknown bacteria
example of differential media
phenol red fermentation tubes
media contains a known carbohydrate, broth with non-carbohydrate nutrients, phenol red
phenol red is a pH indicator
red at pH 7 and turns yellow at acidic pH (below pH 5)
if bacterium growing in this broth ferments the carbohydrate to acid, the media will turn yellow
if bacterium can't ferment carbohydrate, it grows on the non-carbohydrate nutrients (peptones) present in the broth and the media stays red
blood agar
this is a TSA based media that contains sheep red blood cells; media is reddish brown in color
some bacteria can lyse blood cells and degrade the hemoglobin (hg); hg makes blood red
these hemolytic bacteria will be surrounded by a tan colored halo on blood agar
non hemolytic bacteria will not have the halo
blood agar differentiates between the two types
combined selective and differential
some media are selective and differential
mannitol salt agar (MSA)
salt (NaCl) in the media selects for salt tolerant bacteria; meaning that only bacteria that can tolerate high salt levels will grow
the media also has mannitol (a carbohydrate) and phenol red; if the bacteria ferments the mannitol, the pH drops and media will turn from pink to yellow; no fermentation, stays pink