Energy, Matter, and Enzymes

Energy, Matter, and Enzymes

• Metabolism involves both breaking down complex molecules (catabolism) and building complex molecules (anabolism).
• Autotrophs convert inorganic carbon dioxide into organic carbon, while heterotrophs use fixed organic carbon compounds.
• Phototrophs acquire energy from light, while chemotrophs obtain energy from chemical compounds; organotrophs use organic molecules, and lithotrophs use inorganic chemicals.
• Cellular electron carriers like FAD/FADH2, NAD+/NADH, and NADP+/NADPH accept high-energy electrons and serve as electron donors in redox reactions.
• Adenosine triphosphate (ATP) stores chemical energy in its phosphate bonds for driving energy-requiring processes.
• Enzymes are biological catalysts that accelerate chemical reactions by lowering activation energy.
• Exergonic reactions release energy and proceed without additional energy beyond activation energy.
• Endergonic reactions require energy beyond activation energy, and in cells, they are coupled to exergonic reactions for energetic favorability.
• Substrates bind to the enzyme's active site, altering the structures of both the active site and substrate, favoring transition-state formation, a process called induced fit.
• Cofactors are inorganic ions that stabilize enzyme conformation, while coenzymes are organic molecules (often from vitamins) needed for enzyme function.
• An enzyme without a cofactor or coenzyme is called an apoenzyme; with a bound cofactor or coenzyme, it's called a holoenzyme.
• Competitive inhibitors regulate enzymes by binding to the active site, preventing substrate binding.
• Noncompetitive (allosteric) inhibitors bind to allosteric sites, inducing a conformational change that prevents enzyme function.
• Feedback inhibition occurs when a metabolic pathway's product noncompetitively binds to an enzyme early in the pathway, preventing further synthesis of the product.

Catabolism of Carbohydrates

• Glycolysis is the initial step in glucose breakdown, producing ATP via substrate-level phosphorylation, NADH, and two pyruvate molecules; it doesn't use oxygen.
• After glycolysis, pyruvate is decarboxylated to a two-carbon acetyl group, coupled with NADH formation, and then attached to coenzyme A.
• Coenzyme A transports the acetyl group to the Krebs cycle, where it is oxidized, producing three NADH molecules, one FADH2, one ATP, and two CO2 molecules per cycle turn.
• The Krebs cycle intermediates are utilized to synthesize amino acids, chlorophylls, fatty acids, and nucleotides.

Cellular Respiration

• Most ATP during glucose cellular respiration is generated through oxidative phosphorylation.
• The electron transport system (ETS) consists of membrane-associated protein complexes and mobile electron carriers embedded in the cytoplasmic membrane of prokaryotes and the inner mitochondrial membrane of eukaryotes.
• Each ETS complex has a different redox potential, allowing electrons to move from carriers with more negative to positive redox potentials.
• Aerobic respiration requires oxygen as the final electron acceptor along with a complete Krebs cycle, cytochrome oxidase, and oxygen detoxification enzymes.
• Anaerobic respiration uses alternative electron transport system carriers for final electron transfer to non-oxygen electron acceptors.
• Variations in electron transport system composition among microbes can be used for diagnostic purposes to identify pathogens.
• Electrons passing from NADH and FADH2 through the ETS causes a loss in energy.
• This energy is used to pump H+ across the membrane, creating a proton motive force.
• The proton motive force drives hydrogen ions back through the membrane via chemiosmosis using ATP synthase, resulting in ATP production from ADP and Pi through oxidative phosphorylation.
• Aerobic respiration produces more ATP (up to 34 molecules) via oxidative phosphorylation compared to anaerobic respiration (1-32 ATP molecules).

Fermentation

• Fermentation uses an organic molecule as the final electron acceptor to regenerate NAD+ from NADH, allowing glycolysis to continue.
• Fermentation doesn't involve an electron transport system, and it makes little ATP (only two ATP molecules per glucose molecule) directly.
• Microbial fermentation is used in food and pharmaceutical production, as well as microbe identification.
• Lactic acid fermentation involves pyruvate accepting electrons from NADH and reduces lactic acid.
• Homolactic fermentation produces only lactic acid, while heterolactic fermentation produces a mixture of lactic acid, ethanol and/or acetic acid, and CO2.
• Lactic acid produced by normal microbiota prevents pathogen growth in certain body regions and contributes to gastrointestinal tract health.
• Ethanol fermentation involves decarboxylation of pyruvate (releasing CO2) to acetaldehyde, then reduction of acetaldehyde to ethanol by accepting electrons from NADH.
• Ethanol fermentation is used for production of alcoholic beverages, bread, and biofuel.
• Fermentation pathways provide distinctive flavors to food products and are used to produce chemical solvents and pharmaceuticals.
• Specific microbes can be distinguished by their fermentation pathways and products, as well as the substrates they can ferment.

Catabolism of Lipids and Proteins

• Microbes can degrade lipids and proteins; with catabolic pathways eventually connecting to glycolysis and the Krebs cycle.
• Triglycerides are degraded by extracellular lipases into fatty acids and glycerol, while phospholipids are degraded by phospholipases into fatty acids and a phosphorylated head group.
• Lipases and phospholipases act as virulence factors for pathogenic microbes.
• Fatty acids are degraded inside the cell through β-oxidation, removing two-carbon acetyl groups sequentially.
• Protein degradation involves extracellular proteases that degrade large proteins into peptides.
• Gelatinase and caseinase detection can differentiate clinically relevant bacteria.

How Microbes Grow

• Bacterial cells divide by binary fission, with generation time defined as the population's doubling time.
• Cells in a closed system follow a growth pattern with four phases: lag, logarithmic (exponential), stationary, and death.
• Cells can be counted by direct viable cell count using pour plate and spread plate methods to plate serial dilutions, or by using membrane filtration for dilute solutions.
• The most probable cell number (MPN) method estimates cell numbers without solid media.
• Indirect methods estimate culture density by measuring turbidity or metabolic activity.
• Other cell division patterns include multiple nucleoid formation, asymmetric division (budding), and hyphae and terminal spore formation.
• Biofilms are communities of microorganisms enmeshed in an extracellular polymeric substance matrix.
• Biofilm formation occurs when planktonic cells attach to a substrate and become sessile, coordinating activity through quorum sensing.
• Biofilms are common on surfaces in nature and in the human body, and pathogens associated with biofilms are often more resistant to antibiotics and disinfectants.

Oxygen Requirements for Microbial Growth

• Aerobic and anaerobic environments exist in diverse niches in nature, including within and on the human body.
• Microorganisms have varying requirements for molecular oxygen.
• Obligate aerobes depend on aerobic respiration and use oxygen as the terminal electron acceptor, and cannot grow without oxygen.
• Obligate anaerobes cannot grow in the presence of oxygen and depend on fermentation and anaerobic respiration with a non-oxygen electron acceptor.
• Facultative anaerobes show better growth with oxygen but can grow without it.
• Aerotolerant anaerobes grow in the presence of oxygen but do not perform aerobic respiration, and most test negative for catalase.
• Microaerophiles need oxygen to grow, at a lower concentration than 21% oxygen in air.
• Optimal oxygen concentration promotes the fastest growth rate.
• Minimum/maximum permissive oxygen concentrations are the lowest/highest oxygen levels the organism can tolerate.
• Peroxidase, superoxide dismutase, and catalase are key enzymes in detoxifying reactive oxygen species, and superoxide dismutase is present in cells that tolerate oxygen.
• All three enzymes are detectable in cells performing aerobic respiration to help produce more ROS.
• A capnophile requires a higher than atmospheric concentration of CO2 to grow.

The Effects of pH on Microbial Growth

• Bacteria are generally neutrophiles and grow best close to pH 7.0
• Acidophiles grow optimally close to pH 3.0
• Alkaliphiles grow optimally between pH 8 and 10.5
• Extreme acidophiles and alkaliphiles grow slowly or not at all near neutral pH
• Microorganisms grow best at their optimum growth pH

Temperature and Microbial Growth

• Microorganisms exist in different temperatures; Both extreme cold and hot temperatures require evolutionary adjustments to macromolecules and biological processes.
• Psychrophiles grow best between 0 and 15 °C whereas psychrotrophs thrive between 4 and 25 °C.
• Mesophiles grow best at moderate temperatures (20 °C to about 45 °C); pathogens are usually mesophiles.
• Thermophiles and hyperthermophiles are adapted to temperatures above 50 °C.
• Adaptations to cold and hot temperatures require changes in the composition of membrane lipids and proteins.

Other Environmental Conditions that Affect Growth

• Halophiles require high salt concentration, whereas halotolerant organisms can grow and multiply in the presence of high salt without requiring it for growth.
• Halotolerant pathogens are an important source of foodborne illnesses.
• Photosynthetic bacteria depend on visible light for energy.
• Most bacteria require high moisture to grow.

Media Used for Bacterial Growth

• Chemically defined media contain only chemically known components, and selective media favor the growth of some microorganisms while inhibiting others.
• Enriched media contain added essential nutrients a specific organism needs to grow.
• Differential media help distinguish bacteria by the color of the colonies or the change in the medium.

Using Microbiology to Discover the Secrets of Life

• DNA was discovered before its role in heredity was understood, and microbiologists played significant roles in demonstrating its hereditary information.
• Gregor Mendel demonstrated the heritability of specific observable traits using true-breeding garden peas in the 1850s and 1860s.
• Friedrich Miescher isolated and purified a phosphorus-rich compound from the nuclei of white blood cells, naming it nuclein, later renamed nucleic acid by his student Richard Altmann, and Albrecht Kossell characterized the nucleotide bases.
• Walter Sutton and Theodor Boveri proposed the Chromosomal Theory of Inheritance in 1902 and it was not scientifically demonstrated until the 1915 publication.
• Joachim Hämmerling demonstrated that the nucleus was the location of hereditary information using Acetabularia in the 1930s and 1940s.
• George Beadle and Edward Tatum showed that each protein's production was under the control of a single gene, demonstrating the "one gene-one enzyme" hypothesis, in the 1940s using Neurospora crassa mold.
• Frederick Griffith showed that dead encapsulated bacteria could pass genetic information to live nonencapsulated bacteria and transform them into harmful strains in 1928
• Oswald Avery, Colin McLeod, and Maclyn McCarty identified DNA as the compound in 1944.
• Alfred Hershey and Martha Chase demonstrated that DNA stores genetic information when labeled DNA from bacterial viruses entered and infected bacterial cells and the labeled protein coats did not participate in the transmission.

Structure and Function of DNA

• Nucleic acids are composed of nucleotides, each containing a pentose sugar, a phosphate group, and a nitrogenous base.
• Deoxyribonucleotides within DNA contain deoxyribose as the pentose sugar.
• DNA contains the pyrimidines cytosine and thymine, and the purines adenine and guanine.
• Nucleotides are linked by phosphodiester bonds between the 5' phosphate group of one nucleotide and the 3' hydroxyl group of another.
• A nucleic acid strand has a free phosphate group at the 5' end and a free hydroxyl group at the 3' end.
• Chargaff found that the amount of adenine matches thymine, and the amount of guanine is equal to cytosine in DNA and determined complementary base pairing.
• Watson and Crick proposed the double helix model and base pairing for DNA structure, building on work by Chargaff, Franklin, Gosling, and Wilkins.
• DNA is composed of two complementary strands oriented antiparallel to each other with the phosphodiester backbones on the exterior of the molecule.
• The nitrogenous bases of each strand face each other and complementary bases hydrogen bond stabilizing the structures.
• Heat or chemicals can break these bonds, denaturing DNA and cooling or removing chemicals can lead to renaturation or reannealing.

Structure and Function of RNA

• Ribonucleic acid (RNA) is single stranded and contains ribose as its pentose sugar and the pyrimidine uracil instead of thymine.
• An RNA strand can undergo significant intramolecular base pairing to takes on a three-dimensional structure.
• There are three main types of RNA: mRNA, rRNA, and tRNA.
• Messenger RNA (mRNA) serves as the intermediary between DNA and the synthesis of protein products during translation.
• Ribosomal RNA (rRNA) is a stable RNA that is a major constituent of ribosomes, ensuring the proper alignment of the mRNA and ribosomes during protein synthesis and catalyzing the formation of the peptide bonds between two aligned amino acids during protein synthesis.
• Transfer RNA (tRNA) is a small type of stable RNA that carries an amino acid to the corresponding site of protein synthesis in the ribosome, using base pairing between the tRNA and mRNA that allows for the correct amino acid to be inserted in the polypeptide chain.

Structure and Function of Cellular Genomes

• The genome is the entire genetic content of a cell.
• Genes code for proteins, or stable RNA molecules, each with a specific function.
• Genotype, the genetic makeup of a cell, remains constant, gene expression depends on environmental conditions.
• Phenotype refers to the observable characteristics of a cell and results from the genes being used.
• The majority of genetic material is organized into chromosomes with DNA that controls cellular activities.
• Prokaryotes are typically haploid, with a single circular chromosome found in the nucleoid.
• Eukaryotes are diploid, with multiple linear chromosomes found in the nucleus.
• Supercoiling and DNA packaging using DNA binding proteins allows lengthy molecules to fit inside a cell.
• Eukaryotes and archaea use histone proteins, and bacteria use different proteins with similar function.
• Prokaryotic and eukaryotic genomes contain noncoding DNA, which has some function in the formation of small noncoding RNA molecules that influence gene expression or contribute to chromosome structure.
• Extrachromosomal DNA in eukaryotes includes what is found within organelles of prokaryotic origin (mitochondria and chloroplasts); some viruses also maintain themselves extrachromosomally.
• Extrachromosomal DNA in prokaryotes consists of plasmids that encode nonessential genes that may be helpful under specific conditions and spread through horizontal gene transfer.

The Functions of Genetic Material

• The two important cellular functions of DNA are it is the genetic material passed from parent to offspring and it directs protein construction.
• The central dogma indicates that DNA genes specifies the sequences of messenger RNA (mRNA), which, in turn, specifies the amino acid sequence of proteins
• The genotype is the full collection of genes a cell contains and the phenotype is a cell's observable characteristics.

DNA Replication

• DNA replication results in two DNA molecules, each having one parental strands and one newly synthesized strand.
• In bacteria, DNA gyrase unwinds supercoiled DNA at the origin of replication, made single-stranded by helicase, and bound by single-stranded binding protein to maintain its single-stranded state.
• Primase synthesizes a short RNA primer, providing a free 3'-OH group to which DNA polymerase III can add DNA nucleotides.
• Leading strand is synthesized continuously from a single primer, but the lagging strand is synthesized discontinuously in Okazaki fragments, each requiring a primer.
• RNA primers are removed and replaced with DNA nucleotides by bacterial DNA polymerase I, and DNA ligase seals the gaps.
• Replication termination involves resolution of circular DNA concatemers by topoisomerase IV.
• Eukaryotes have multiple linear chromosomes, each with multiple origins of replication, with telomeres protecting genes near chromosome ends.
• Telomerase extends telomeres, preventing their degradation.
• Rolling circle replication is a type of rapid unidirectional DNA synthesis of a circular DNA molecule.

RNA Transcription

• RNA transcription involves the information encoded in DNA, making RNA.
• RNA polymerase synthesizes RNA, using the antisense strand of DNA as template by adding complementary RNA nucleotides to the 3' end of the growing strand.
• RNA polymerase binds to DNA at a promoter during the initiation of transcription.
• Genes encoding proteins of related functions are often transcribed with a single promoter in prokaryotes, resulting in the formation of a polycistronic mRNA molecule that encodes multiple polypeptides.
• Unlike DNA polymerase, RNA polymerase does not require a 3'-OH group to add nucleotides, so there is no primer.
• Termination of transcription in bacteria occurs when the RNA polymerase encounters specific DNA sequences that leads to polymerase stalling and transcript freeing.
• Eukaryotes have three different RNA polymerases and monocistronic mRNA, with each encoding a single polypeptide.
• Eukaryotic primary transcripts are processed: addition of a 5' cap and a 3'-poly-A tail, and splicing to generate a mature mRNA molecule that can be transported and protected by degradation.

Protein Synthesis (Translation)

• Polypeptides are synthesized using mRNA sequences and cellular machinery, including tRNAs that match mRNA codons to amino acids and ribosomes composed of RNA and proteins that catalyze the reaction.
• The genetic code is degenerate, mRNA codons code for the same amino acids
• Translation is universal among living organisms.
• Prokaryotic (70S) and cytoplasmic eukaryotic (80S) ribosomes are each composed of a large and small subunit.
• Organelle ribosomes in eukaryotic cells resemble prokaryotic ribosomes.
• Bacteria consist of some 60 to 90 species of tRNA exist in bacteria, each has a anticodon and a site that binds to with a cognate amino acid and all tRNAs with a specific anticodon will carry the same amino acid.
• Initiation of translation occurs when the small ribosomal subunit binds with initiation factors and an initiator tRNA at the start codon of an mRNA, then, large ribosomal subunit binds to the initiation complex.
• In prokaryotic cells, codes for N-formyl-methionine and the start codon codes for methionine carried by a special initiator tRNA in eukaryotic cells.
• Elongation: a charged tRNA binds to mRNA in the A site and occurs when a peptide bond is catalyzed between the two adjacent amino acids and the ribosome moves one codon along the mRNA.
• Termination: occurs when the ribosome encounters a stop codon, which does not code for a tRNA.
• Release factors cause the polypeptide to be released, and the ribosomal complex dissociates.
• In prokaryotes, transcription and translation may be coupled, meaning that translation of an mRNA molecule begins as soon as transcription allows translation prior to transcription termination.
• Polypeptides require one or more post-translational modifications to become biologically active.

Mutations

• A mutation is a heritable change in DNA and may lead to a change in the amino-acid and protein function.
• A point mutation, affecting a single base pair, may cause a silent mutation, a missense mutation or a nonsense mutation.
• Missense mutations may retain function
• Nonsense mutations produce truncated proteins.
• A frameshift mutation results from an insertion or deletion (not a multiple of three).
• Spontaneous mutations occur through DNA replication errors, whereas induced mutations occur through exposure to a mutagen.
• Mutagenic agents are frequently carcinogenic chemical mutagens include base analogs and chemicals that modify existing bases.
• Ionizing radiation leads to backbone breakage; Nonionizing radiation introduces pyrimidine dimers.
• DNA polymerase has proofreading activity and mismatch repair is a process to repair after completed replication.
• Enzymes detect distortions and replace the damaged strand by using non-compromised DNA as a template during nucleotide excision repair.
• Organisms may use direct repair, in which the photolyase enzyme, and break pyrimidines when there is visible light .

How Asexual Prokaryotes Achieve Genetic Diversity

• Specific loss-of-function mutants called auxotrophs can be identified through comparison of growth on the complete plate and lack of growth on media lacking specific nutrients
• The Ames test is a method that is used auxotrophic bacteria to measure mutagenicity of a chemical compound (cancer).
• Horizontal gene transfer is an way for reproducing organisms like prokaryotes to acquire new traits and occurs when bacteria uses transformation,transduction, and conjugation.
• Transformation occurs when cells take up naked DNA, released from other cells in the cytoplasm, where it may recombine with the host genome.
• In generalized transduction, any piece of chromosomal DNA can be accidentally packaged and degraded. Only chromosomal DNA close to the integration site of with phage may be transferred due to inaccurate excision of theprophage, This is known as specialized transduction.
• Conjugation is mediated by the F plasmid that conjoins two plasmid cells together within the vicinity.
• The integration of the F plasmid into the bacterial chromosome allows for transfer of chromosomal DNA from the donor to the recipient, if F plasmid not aligned, F’ may be transferred to a recipient by conjugation.
• R plasmids transfer promotes antibiotic resistance.
• Transposons are molecules of DNA with inverted repeats at their ends that encode the enzyme transposase, allowing movement from one location in DNA to another and transfers virulence factors, including resistance genes.

Gene Regulation

• Gene expression is regulated in the cell
• Gene expression in prokaryotes regulated is at the point of transcription and when in eukaryotes is regulated after transcription.
• Prokaryotic structural genes are organized into operons, controlled by transcription that binds and manipulates a promoter.
• Repressor binds to operator,blocking transcription of these operons and activator binds helping polymerase by enahncing transcriptions and is influenced by an inducer.
• The trp operon represses tryptophan and the lac operon is by the presence of Lactose, allolatose which allows transcription.
• When depleted, some cellular ATP is converted into cAMP, which binds to CAP activating transcription.
• Small intracellular molecules called alarmones when control environmental stressors.
• Transcription occurs rapidly.
• Prokaryotes have regulatory,stem loops which allow for transcription and translation.
• Eukaryotypes regulate by chemical modification

Controlling Microbial Growth

• Formites are inanimate items that may harbor microbes and aid in transmission.
• The CDC and NIH have established standards to ensure laboratory personal and community and are designed by infectivity of agent and potential severity.
• Disinfection is pathogenic removal, antisepsis are antimicrobial chemical safe enough for tissues but unable to retain bacteria and cleanliness sterilizes and helps sterilize critical and non critical items.
• Medical standards should be carried away with sterile field and sterilization for medical application.
• Methods to control the growth that results if microbe are death are the suffixs- cide or cidal wheras those that inhibit stat or static and d-value is microbial reduction.
• Factors for protocol include length,micro, intensity,interferrance,or effectivness.

Using Physical Methods to Control Microorganisms

• Heat eliminates microbial spread and moist sterilization is more effective.
• Pathos are killed during pasteurization such which are for HTS, and ultra high, refrigeration slows microbial or halts growth.
• High pressure is used to kill microbes and hyperbraic used to saturation.

Using Chemicals to Control Microorganisms

• Heavy Metals are mercury,silver,silver long used but have harmful effects and chlorine, fluorine, and iodine are used (halogens) along with iodine in forms antiseptic.
• Alcohols,including ethyl alcohol and isopropyl alcohol, are commonly used antiseptics that aid by denaturing proteins and disrupting membranes Phenolics are stable, long-acting disinfectants that denature proteins and disrupt membranes are found in antiseptics along also with triclosan that halt fatty synthesis.
• Surfactants and detergents lower tensions, soaps/ fatty vs synthetics
• Quaternary Ammonium disrupts membrane
• Bisbiguanides disrupt membranes and commonly washing scurbs
• Alkylating agents effectivly sterilize
• Preozygens which is peroxide and ozone

Testing the Effectiveness of Antiseptics and Disinfectants

• Chemical disinfectants are categorized by: Germacides, level germs. Effectiveness influenced disinfectant, exposure, temps,etc
• Phenols were compared with aureus along with typhil and disinfect effectiveness and dilusions.
• Disk dif is used to test chemicle effect vs microbes.
• Disenfectants are compared to phenol with the test to find aureus.
• Disk dif is used to view microbes under a particular test.
• Dilsution find effectiveness