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

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

Note