Use source transformation to find in the circuit of Fig. 4.100.
Lecture 22 • Environmental Effects of Microbial Growth • The environment and pH • pH scale 014, 7 is neutral, below 7 is acidic, above 7 is alkaline, is a log scale (10 fold change) • most environment has a pH between 4 and 9, most organisms like 68 • Ocean acidification, seawater has pH of 8.1 • Neutrophiles: organisms that grow optimally at pH values of 5.5 to 7.9 • Acidophiles: organisms that grow best below pH 5.5 • most are moderately acidic, but some can grow below pH 3 or 2 • most cannot grow at pH 7 or 2 pH units greater than their optimum • membrane stability is critical; some organisms require acidic environments • Alkaliphiles: organisms with growth optima of pH 8 or greater • found in soda lakes and high carbonate soils; can also be halophiles (salt loving) ex. Great Salt Lake in Utah • can produce enzymes of industrial use (laundry detergents); some use Na+ ions for their proton motive force (cant use OH or H+ cause its either too much or too little) • pH references external environment, all cells usually have neutral intracellular pH (DNA and RNA would degrade in acidic or alkaline environments, respectively) • some cells can exhibit changes, pH 5 and 9 are the limits, if you go past that DNA and RNA degrade • Buffers are used to prevent major shifts in pH. • Osmolarity and Microbial Growth • solutes bind water making water less available to organisms • Water activity (asubw) = expression of water availability in physical terms; the ratio of vapor pressure of the air in equilibrium with a substance or solution to the vapor pressure of pure water • Ranges from 01: will never be 0, 1 is pure water (0.7 is like dried fruit, hard candy, most things are above this) • water diffuses from higher concentration (low solute conc) to lower water concentration (high solute conc) • cytoplasm has a higher solute concentration than environment, so water diffuses into the cell: called positive water balance • In environments where solute concentration exceeds that of the cytoplasm, water diffuses out, causes dehydration problems. • Halophiles and related organisms: • Seawater contains ~3% NaCl and many organisms need this salt • Halophiles: require some NaCl for growth • Halotolerant: organisms can tolerate a reduction in water activity of their environment, but grow best without the solute • Extreme halophiles: requires 1530% NaCl for optimal growth • Osmophiles: organisms that live in environments high in sugar as a solute • Xerophiles: organisms able to grow in very dry environments • Compatible Solutes • If living in a medium with low aw, cells can only get water by increasing their internal solute conc. and thus moving water in via osmosis • 2 mechanisms to increase internal solute conc. • 1. Pump solutes into cell from outside • 2. Synthesizing a solute • Solutes in the cell must be non inhibitory to macromolecules in the cell —> called compatible solutes • Typically consist of water soluble molecules like sugars, alcohols, or aa derivatives • examples, sucrose, mannitol, glycine betaine, KCl, Glycerol Lecture 23: • Oxygen and Microbial Growth: • Oxygen is poorly soluble in water, and can often be exhausted in bodies of water • anoxic environments are common in nature (muds, sediments, bogs, marshes, intestinal tracts) • Oxygen Classes of Microorganisms: • Aerobes: require oxygen to live • Anaerobes: do not require oxygen and may be killed by oxygen exposure • Facultative organisms: can live with or without oxygen • Aerotolerant anaerobes: can tolerate oxygen and grow in its presence even though they cant use it. • Obligate anaerobes: inhibited or killed by oxygen • Microaerophiles: can use oxygen only when it is present at levels reduced from that in air. • Use a thioglycolate broth to tell you when oxygen is present, where organisms grow helps us see what it is • Culture techniques for aerobes and anaerobes • may need to use aeration to provide enough O2 for aerobes; can bubble gas through or use shaking as diffusion often isn't sufficient • For anaerobes, need to exclude O2 • can use candle jars, tubes filled to the top with medium, jars with a palladium catalyst, or glove bags • Indicator dyes like resazurin, a reducing agent, will interact with O2 and change color to show where O2 is present. • Why is Oxygen Toxic • Molecular oxygen (O2) is not toxic, but it is converted to toxic oxygen byproducts • Include superoxide anion (O2), hydrogen peroxide (H2O2), and hydroxide radical (OH•) • All form from the reduction of O2 to H2O in respiration, so all organisms will experience these toxic by products • Enzymes can destroy toxic oxygen compounds: • Catalase: breaks H2O2 into O2 and H2O • Peroxidase: requires a reductant (usually NADH) and produces H2O from H2O2 • Superoxide Dismutase: destroys superoxides by generating H2O2 and O2 from 2O2, works with catalase, essential for aerobes • Superoxide Reductase: reduces superoxide to H2O2 without evolving O2, so found in anaerobes • Control of Microbial Growth • Sterilization: killing or removal of all microorganisms • Decontamination: treatment of an object or surface to make it safe to handle • Disinfection: process that targets pathogens for killing, but not necessarily all microorganisms • Control by Heat: • all organisms have a maximum growth temp, makes heat useful to control • Moist heat is better at penetrating than dry heat: more effective • Killing spores is much more difficult (dehydrated), temps need to reach 121˚C for at least 15 minutes to kill spores • pH and concentration of sugars and fats are also important • Autoclave: sealed heating device that uses steam under pressure to kill microorganisms • Larger volumes need longer times • Pasteurization: precisely controlled heat to significantly reduce the number of microorganisms, but not all • focuses on killing pathogenic bacteria and slowing growth of spoilage organisms • Control by radiation • UV radiation, Xrays, and gamma rays can kill microbes • UV is absorbed by DNA, causes mutations that lead to death • Good for surfaces but poor penetration into objects • Ionizing radiation is electromagnetic radiation that produces ions and reactive species (OH·) that kills cells • Control by Filtration: • Method used for heat sensitive liquids and gases • Pore size of .2um is preferred but wont trap viruses • Antimicrobial agent: natural or synthetic chemical that kills or controls growth of microorganisms • agents that kill are termed cidal, agents that inhibit growth are termed static • effects can be seen visually in viable counts and changes in turbidity • bacteriostatic inhibitors bind weakly and can be removed • bacteriocidal bind tightly to their cellular targets and kill (but don't lyse) the cell • bacteriolytic agents kill cells by rupturing them • Minimum Inhibitory Concentration: smalles amount of material needed to inhibit growth of a test organism • can be done in liquid or agar plates, can use discs with known conc. of antimicrobial agents applied to surfaces • as agent diffuses from disc, creates a concentration gradient, results in a zone of inhibition Lecture 24: • Genome: organisms entire complement of genetic information, including genes, regulatory sequences, and noncoding DNA • Genomics: discipline of mapping, sequencing, analyzing and comparing genomes • first bacteriaial genome was sequenced in 1995 • Human genome has ~3 billion bp and ~25,000 proteincoding genes • E: Endosymbiont, P: Parasitic, FL: Freeliving • E has small genomes, require host. P prefer a host but can live on their own little bigger, FL has largest genomes, need to make everything to live on their own • Sequencing: determining the precise order of nucleotides in a DNA or RNA molecule • Sanger dideoxy method was first widely applied method and employed sequencing by DNA synthesis rather than degradation, used dideoxynucleotides to block chain extension and used labeled precursors to allow detection • Primers: short segments of DNA (or RNA) that initiate the synthesis of new strands of nucleic acids • Uses DNA pol III to add new nucleotides to growing chain • Now fluorescent labels, not radioactivity like the old days • Shotgun Sequencing: • Most common today; uses computer to search for overlapping sequences and assemble fragments in correct order; called multifold coverage. • SecondGeneration DNA sequencing: • Major changes in technology that increased speed and accuracy (lowered costs) by using massively parallel methods • Large number of samples sequenced side by side in same machine • Miniaturization and increased computing power sequence data 100x faster • 454 method: DNA broken into small fragments and attached to small beads, each bead is placed in a well on a fiberoptic plate before DNA is amplified using PCR. • each time a base is added, light is released which is measured by a sensor (enzyme is luciferase) • Third and Fourth Generation DNA Sequencing: • Third gen: sequence single molecules of DNA; fluorescent tags are monitored on a microscope and computer assembles fragments • Fourth generation doesn’t use optical detection, uses semiconductor to measure release of protons (ion torrent) • Moore’s Law: decreasing cost by half every two years, didn’t come close, in 2008 major developments • Genome Assembly: • Typically use overlapping regions to assemble; needs to be annotated to identify genes after assembly. • Usually there are still gaps in sequence, called a draft genome. If complete with no gaps it is called a closed genome. • Bioinformatics: use of computers to store and analyze the structures of nucleic acids and proteins for comparative purposes • Annotation: converting raw sequence data into a list of genes present within the genome; currently the ‘bottleneck’ in genomics • Prokaryotic genes encode proteins with little noncoding sequences • Open Reading Frames (ORFs): sequence of DNA or RNA that can be translated to yield a polypeptide • How to find an ORF: • Begin by looking for start and stop codons in the same reading frame • Next look for size —> most proteins are ~100 or more amino acids so longer than 300 nucleotides • Next, look for ribosome binding site (Shine Dalgarno sequence) in bacteria to determine if the ORF is functional • Calculate codon bias: differential codon use • 70% of ORF’s can be clearly identified, Unknown ORFs are said to encode hypothetical proteins that are likely nonessential (most essential have been identified). Thought that there is 10% error for genes in databases • Some genes encode RNA molecules that are not translated so lack start codons and may have multiple stop codons • Include things like tRNAs and rRNAs: highly conserved but know them. Were tough to identify • Genomes of prokaryotes show a correlation between genome size and ORF content • 1 Mbp encodes ~1000 ORFs, allows scientists to examine minimum number of genes necessary for a cell to exist free living (~1.3 Mbp and 1350 genes) Lecture 25: • Small Genomes: • Smallest belong to prokaryotes that are parasitic or endosymbiotic • Obligate parasitic have genomes from 490 kbp to 4400 kbp • 250300 genes are thought to be the minimum for a cell to be viable • Large Genomes: • Larges prokaryotic genomes are similar to eukaryotes and have more genes • Largest is Sorangium Cellulosum at 13.3 Mbp; has 14.5% noncoding DNA (high for bacteria) • Largest archaeal genome is ~5 Mbp, but far fewer have been sequenced • Gene Conent: • can use information about organisms lifestyle or environment to predict what genes are present • Core cellular process genes show minor variation in gene number; cells with big genomes have lots of regulatory genes to adapt to environmental changes • Many genes can be identified by homology to genes in other organisms through comparative analysis. • Help us predict metabolic pathways and transport systems present. • As # of ORFs increases, so does ability to diversify (transcription and signal transduction) • Genomes of Organelles: • Mitochondria and Chloroplasts arose from endosymbiotic bacteria and have their own genomes. • Each has the necessary machinery for protein synthesis, including ribosomes and tRNAs • Chloroplasts Genomes • closely related to bacterial genomes; circular, typically 120160 bp with 2 inverted repeats that encode copies of the 3 rRNA genes • Many genes encode proteins for photosynthesis and autotrophy (CO2 fixation) • selfsplicing introns are common • Mitochondrial Genomes: • smaller than chloroplast genomes and primarily encode genes for proteins in oxidative phosphorylation • animal mitochondrial genomes are much smaller than plant mitochondrial; some are linear and some contain plasmids • Many genes have been translocated to the nucleus • Variability in Genetic Code: • Some mitochondria and a few cells use slight variations on the “universal” code • stop codons are not used to stop translation but rather infer an amino acid, making base pairing between codon and anticodon more flexible • result of selective pressure for smaller genomes in nutrient rich environments • Symbionts and Organelles: • Genomes of symbionts range in size dramatically similar to freeliving bacteria down to ~140kbp • Some symbiont genomes contain fewer genes than organelles and viruses • Most small genomes have a very high AT content (80%) except for 2 smallest genomes • Also have lost genes previously regarded as essential for replication (FtsZ) • Symbionts are usually restricted to a few tissues within host, and little evidence of transfer of symbiont genes to host or vice versa • We don't know whats different between a symbiont and organelle, no easy answer • Eukaryotic Microbial Genomes: • Vary widely, some have significantly more genes than humans • Most parasitic have genomes of 1030 Mbp and 4,000 to 11,000 genes, often on multiple chromosomes • Smalles freeliving eukaryote is Ostreococcus tauri, high gene density • Largest eukaryotic genome is Trichomonas vaginalis • The Yeast Genome: • Saccharomyces cerevisiae is eukaryotic model organism • haploid yest genome has 16 chromosomes, 220 kbp to 2352 kbp • Encodes 6000 ORFs, 4000 of which has known functions • 900 ORFS are essential for growth and survival, lots more than prokaryotes • Contain lots of repetitive DNA • Functional Genomics: • To understand cellular functions, need to investigate gene expression (transcription) and the functions of gene products • Transcriptome: entire complement of RNA produced under a given set of conditions • Can use microarrays or RNASeq to investigate transcriptomes • Microarray: solid supports to which genes or segments of genes are fixed and arrayed spatially, also called gene chips • rely on RNADNA hybridization where ds DNA is separated into single strands • single strands form hybrid ds molecules with other single stranded DNA or RNA molecules by nearly complementary base pairing • Nucleic Acid probes: segments of singlestranded nucleic acid with known sequences, usually radioactive or fluorescent • By varying conditions, can change exactness of complementarity (lower or raise temp) Lecture 26: • Applications of Gene Chips: Gene Expression • Can monitor global gene expression (RNA) in a single experiment, providing insight to transciptome • limited only by the gene fragments attached to chip • can also be used to identify microorganisms so array contains characteristic DNA sequences from a variety of microbes • Often used in food and medical industries but some environmental • RNASeq analysis • Sequences all RNA molecules from a cell, showing which genes and how many copies of each were made (quantitative) • useful for measuring expression of mRNA and identifying small noncoding RNAs • Requires highthroughput sequencing • rRNA dominates within a cell, masks mRNA so methods developed to remove rRNA • Metagenomics: genomic analysis of pooled DNA or RNA from an environment • RNAseq useful to determine likely nutrients for previously uncultured bacteria • Proteomics: genome wide study of structure, function, and activity of a cells proteins • Proteome: broad reference to all proteins encoded by an organisms genome, or only those proteins present at a given time • 2D polyacrylamide gel electrophoresis provides separation, and measurement of proteins in a sample. • Proteins can also be excised from the gel and sequenced to get the primary structure, or identified using mass spectrometry or liquid chromatography • Comparative Genomics: • Proteins with >50% sequence identity in aa composition usually have similar function, while >70% almost always have similar fxn • Conservation of function based on conservation of protein domains • ex) zinc binding domain present in almost all enzymes (highly conserved) • Structural proteomics determines 3D structure of proteins, uses conservation of structure • The Interactome: complete set of interactions among the macromolecules within a cell. • Data are expressed in network diagrams, nodes represent proteins and lines represent interactions • Metabolome: complete set of metabolic intermediates and other small molecules in an organism • More difficult than other omics, extreme diversity of small metabolites • MALDITOF useful for study of secondary metabolites (not essential) • Systems Biology: the integration of different fields of research (proteomics, transcriptomics, metabolomics) to provide an overview of entire system • System can be cells, organisms, species, or ecosystems • Predicted properties from the models are called emergent properties and may provide broader understanding • Metagenomics: environmental genomics in which the genomes of multiple organisms are examined simultaneously • Metagenome: total gene content of the organisms inhabiting the environment • Metatranscriptome: the total genes expressed within entire sample • Easier to do with less diversity (3 species vs 30 species) • Much of environmental DNA is not in living cells • Functional Redundancy: different organisms carry out same functions in human microbiome • Homologs: homologous genes related in sequence due to shared ancestry • Gene Families: groups of gene homologs • larger genomes are going to have more member from certain gene families • Paralogs: genes within an organism whose similarity to one or more genes is the result of a gene duplication • Orthologs: genes found in one organism thats similar to those in another organism but differ due to speciation • Gene Duplication: • mechanism by which most new genes evolve • allows one copy to evolve while other maintains original fxn • Deletions can eliminate gene if no longer needed • Gene analysis across members of 3 domains of life suggest many genes present in all organisms have common evolutionary roots, also indicates instances where genes were transferred laterally Lecture 27: • Horizontal Gene Transfer: transfer of genetic information from one cell to another in prokaryotes, not from parents. 3 Methods: • Transformation: “slurping up” free genetic information in the environment. • Transduction: cell infected with a virus, and virus confers new genetic information. • Conjugation: Sex pillus transfers genetic information to a new cell. • Detected by presence of genes found in distant species, difference in GC content, code for functions not involved in survival, and involvement in virulence • Mobile DNA: segments of DNA that move from one location to another within host DNA molecules. • Consist mostly of transposable elements, include insertion sequences and viral genomes. Important in genome evolution • Transposons: mobile DNA that moves between different host DNA molecules, including chromosomes, plasmids, and viruses through the activity of a transposase enzyme. • Responsible for horizontal gene transfer and large scale chromosomal changes. • Core Genome: that shared by all strains of a given species • Pan genome: the core genome plus all of the optional extra genes present in one or more strains, but not all strains of that species. • Allows for major differences in total amount of DNA and capabilities of individual strains of a single species. • Can be extremely large. • Chromosomal Islands: blocks of genetic material that are part of the chromosome (rather than a plasmid or integrated virus) that contain clusters of genes for specialized functions not needed for simple survival. • Two strains of same species can have very different genome sizes. • Have been found with functions to increase pathogenicity, encode biodegradation of pollutants, provide genes for symbiosis with rhizobia, and encode formation of magnetosomes. • Have a “foreign origin” because: • Often flanked by repeated inverts, have different base composition and codon bias from core genome, found in some strains but not others. • Some carry a gene for integrase enzyme. • Gradually accumulate mutations and lose their ability to move after being inserted into a host. • Pathogenicity Islands: virulence genes clustered in chromosomal regions. A type of chromosomal island • Accounts for over 20% more DNA and genes in E. coli than in nonpathogenic strain. • Proteins needed in the cell at similar levels under all growth conditions (constant expression) are called constitutive. • Other proteins are needed only under certain conditions, so regulation of genes helps to conserve both energy and resources. • Cells can either: 1. Control the activity of an enzyme or protein 2. Control the amount of an enzyme or protein: makes more sense • Activity control can only be regulated after it has been synthesized —> posttranslation. • Can be done rapidly • Synthesis takes time for each mRNA and more time for a sufficient amount to be present. Also takes time to remove an enzyme from a cell. • Major modes of regulation: • structural genes encode the gene product and its expression by sequences in the upstream region • 2 major levels: • 1. Regulate activity of preexisting enzymes (post translationally: quick) • 2. Regulate amount of an enzyme (regulate transcription; degradation) • Transcriptional regulation is the major mode of regulation in prokaryotes • halflife of mRNA is short (a few minutes) so organisms can respond quickly to environment • For a gene to be transcribe, RNA pol must recognize a specific promoter on DNA. • DNA Binding Proteins: proteins that regulate transcription of DNA • require regulatory sequences, often adjacent to the promoter • Interactions between protein and DNA can be specific or nonspecific. • Histones: nonspecific binding protein • Cause RNA pol to not be able to bind DNA • Most DNA binding proteins are specific: • Major groove of DNA is main site for protein binding • Inverted repeats are frequently binding sites for reg. proteins • Helixturnhelix structure: 1st helix is recognition helix that interacts with DNA while 2nd helix is the stabilizing helix; helices are separated by a turn consisting of 3 amino acids. Lecture 28: • DNA Binding Proteins: • Helixturnhelix recognize sequences by noncovalent interactions (Hbonds) • Commonly found in repressor proteins like lac and trp in E.coli • Zinc Fingers: bind a zinc ion; part of the finger of amino acids forms an ahelix which interacts with major groove of DNA. 23 fingers • Leucinezipper: leucine residues spaced every 7 amino acids, resembles a zipper. • Doesn't interact with DNA, but holds 2 recognition helices in proper orientation. • DNA binding can: • Catalyze reactions, block transcription, or activate transcription. • Negative Control: regulation by repression and induction • Negative control prevents transcription. Will always have a repressor in both repression and induction. • Repression: preventing synthesis of an enzyme in response to a signal; usually affects anabolic enzymes which are not synthesized when they aren't needed. • Prevents wasting of energy • Repressor is not always bound, presence of corepressor (substance that stops enzyme synthesis) changes shape and causes binding of repressor to DNA. • Induction: conceptually opposite of repression; enzyme is produced in response to a signal; usually affects catabolic enzymes that are made only when needed. • Repressor is always bound until presence of inducer causes shape change and release of repressor from DNA. Allows RNA pol to transcribe. • Inducer: substance that promotes enzyme synthesis • Effectors: Substances that work indirectly on DNA, corepressors and inducers. • Effectors bind to specific repressor proteins and change conformation of protein, called allosteric binding. • Positive Control of Transcription: • Positive control uses a regulatory protein called an activator that enhances binding of RNA pol to DNA. • Requires the binding of an activator protein. • Cannot bind to DNA unless it is firs bound to the inducer. • Once bound to DNA allows transcription to begin • Binds to activatorbinding site (typically upstream of the promoter) and controls transcription. • Promoters of positively controlled operons are poor matches to consensus sequence and RNA pol binds weakly. • Activator protein helps RNA pol recognize promoter and bind (It “holds its hand”). • Does this by creating change in DNA structure, allowing RNA pol to bind more readily or by interacting with RNA pol directly • Doesn’t have to be directly upstream, could be hundreds of bp away, requires looping of DNA. • Many genes have both positive and negative control, very complex! • Operons vs. Regulons: • Operon: series of genes under control by same promoter. • Each operon has an activator binding site but uses same maltose activator protein • Controls transcription of more than 1 operon (by same activator protein) • Regulon: multiple operons controlled by same regulatory protein. • The genes for maltose utilization are encoded by the maltose regulon • Also exist for negatively controlled systems; DNA binding proteins binds only at operon it controls, regardless of being an activator or repressor, but does not affect other operons. Lecture 29: • Global Control and the lac Operon: • Need to regulate unrelated genes simultaneously • Global Control Systems: regulatory mechanisms that respond to environmental signals by regulating expression of many different genes. • Catabolite Repression: mechanism of global control that decides between utilizing different available carbon sources. • Best available Carbon and energy source is used first. (Prefers glucose, will deregulate enzymes for other sugars if glucose is present) • Flagellar genes are controlled by catabolite repression: don't want to swim away from environment rich in nutrients! • Results in two exponential growth phases: Diauxic growth: cells grow first on preferred C source, when it is depleted, go through a lag phase, then growth resumes on another C source. • Catabolite repression relies on an activator protein and is a form of positive control. • Activator is cyclic AMP receptor protein (CRP) • Genes encoding a cataboliterepressible enzyme are only expressed if CRP binds to DNA in promoter. CRP can only bind to DNA when its bound to cyclic adenosine monophosphate (cAMP). • Is a key molecule in metabolite control and is considered a regulatory nucleotide. • Synthesized from ATP by enzyme adenylate cyclase. • Glucose inhibits synthesis of cAMP and stimulates its transport out of cell, so when it is present, cAMP is lowered so is not available to bind to CRP which then cannot bind to DNA —> no transcription of gene • Is an indirect result of the presence of a better energy source (glucose) • Lac Operon is controlled by catabolite repression • For lac genes to be transcribed, 2 things are necessary: • 1. cAMP must be available so that CRP protein can bind to the promoter site for the lac genes • 2. Lactose or allolactose that can act as an inducer must be present to prevent lactose repressor from blocking transcription by binding to operator • (Have to remove repressor AND activator protein to bring in RNA pol as well) • Archaea use DNAbinding proteins to control transcription much like bacteria and not like Eukarya. • Few arterial repressor or activator proteins have been characterized • Repressor proteins either block binding of RNA pol OR block binding of TATA binding protein and transcription factor B (protein that interacts w/ DNA) • Activators recruit TFB to the promoter, facilitate transcription • NrpR represses genes involved in nitrogen metabolism • Form of negative induction • Sensing and Signal Transduction: • Prokaryotes regulate cellular metabolism in response to environmental fluctuations (temp, pH, O2, and nutrient availability) • Some signals are small molecules that enter the cell and function as effectors • Some signals are external and not directly transmitted to regulatory protein, but are detected by a sensor that transmits the signal to the regulatory machinery —> Signal Transduction. • Most signal transduction systems have two parts and called twocomponent regulatory systems: • Include: • 1. Specific sensor kinase protein (usually in cytoplasmic membrane) • 2. Response regulator protein (usually in cytoplasm) • Kinase: enzyme that phosphorylates compounds using PO4 from ATP • Sensor kinases detect signals from environmen and phosphorylate themselves, use a specific histidine residue so called histidine kinases. • Sensor kinase then transfers PO4 to regulator protein • Regulator protein is typically a DNA binding protein that regulates transcription • Are also one component systems where 1 protein detects signal and carries out response, work by binding signal and then binding DNA • Removal of PO4 from response regulator resets system • Two component systems common in bacteria but rare in archaea and bacteria that are parasites: don't usually have changing environments. • Usually has to do with detecting phosphate limitation, nitrogen limitation, and osmotic pressure • Regulation of Chemotaxis: • Chemotaxis: movement towards or away from a chemical (attract or repel) • Organisms sense change in concentration of a chemical over time rather than absolute concentration of stimulus, employ a modified 2component system to regulate flagellation • Occurs in three steps: • 1. Response to signal: sensory transmembrane proteins called methyl accepting chemotaxis proteins (MCPs) in the cytoplasmic membrane ‘sense’ the presence of chemical and bind it, triggers interactions • MCPs contact cytoplasmic proteins CheA and CheW • CheA is sensor kinase and becomes phosphorylated; attractants decrease rate of phosphorylation, repellents increase it • PO4 passed to CheY, which is response regulator that controls flagellation. • 2. Controlling flagellar rotation: CheY controls direction of rotation; counter clockwise creates a run, clockwise makes a tumble. • CheYP induces clockwise (tumble), but CheY unphosphorylated cannot bind motor and causes counterclockwise (run) • CheZ dephosphorylates CheY allowing cell to run • Repellants increase CheYP, creating more tumbles. Attractants lower CheYP, leading to runs • 3. Adaptation: Once organism has responded, it must stop responding and reset the system, employs a feedback loop. • uses CheB, which removes methyl groups from MCPs that were added by CheR (functions at a constant rate). • CheB works faster when phosphorylated • MCPs respond differently when they are methylated (no longer respond to attractants but more sensitive to repellants) so varying the level allows adaptation to sensory signals. • High attractant at constant levels causes tumbles, only swims if higher levels of attractant are detected. • Quorum sensing: mechanism to evaluate density of cells of the same kind; assess population density. • Used to assess cell numbers prior to starting activities that require certain cell density. • Widespread in both gram + and bacteria • Autoinducer: specific signal molecule synthesized by participating organism that freely diffuses across cell envelope in both directions • Only reaches high concentration within cell when many other cells are nearby • Autoinducer binds to activator protein and triggers transcription of quorom dependent genes • First discovered for regulating light emission in bioluminescent bacteria • Only occurs at high density Lecture 30 • Biofilm Formation • Numerous signals, including cellcell communication, allow suspended cells to grow on a semisolid matrix or biofilm. • Genes necessary for biofilm formation are often triggered by quorum sensing • Cyclicdiguanosine monophosphate: important regulatory nucleotide that provides physiological changes and expression of virulence genes. • Formation of biofilms in many bacteria is triggered by accumulation of cyclic diGMP • Global Control Networks • Global networks can include activators, repressors, signal molecules, two component regulatory systems, regulatory RNA, and alternative sigma factors. • The heat shock response is widespread in all 3 domains of life and is controlled by alternative sigma factors. • Heat Shock Proteins: proteins made by heat stressed cells to help counteract the damage caused by heat. • Also induced by other stressors: exposure to chemicals and high doses of UV radiation. • In E.coli, heat shock response is controlled by alternative sigma factors that are not readily degraded under high temps. • Allows some genes to be more highly expressed under heat shock conditions. • Regulation of Development in Model Bacteria: • Differentiation and development largely found in multicellular Eukarya. • Differentiation: one cell gives rise to two genetically identical descendants that perform different roles and must therefore express different genes. • Two main examples: formation of endospores in gram+ bacillus and formation of two cell types in gram — caulobacter cells. • Sporulation in Bacillus: • Carried out in response to adverse growth conditions; when beneficial conditions restored, organism resumes normal life. • Endospore: spores formed inside a mother cell; requires that cell divides asymmetrically and the smaller cell develops into endospore. • Bacillus cells use 5 sensor kinases to monitor their environment, work via a phosphotransfer relay system more complex than 2component systems. • Multiple adverse conditions result in phosphorylation of sporulation factor proteins. • When Spo0A is highly phosphorylated, sporulation starts b/c it controls the expression of genes including SpoIIE, which removes phosphate from SpoIIAA. • SpoIIAA then removes the antisigma factor from SpoIIAB, and liberates the sigma factor F. • σF works to start a cascade of 3 additional sigma factors, some in forming endospore and some in mother cell. • Form of cannibalism in that cells with active Spo0A secrete a protein that lyses nearby cells of same species that are not yet activated; only cells expressing Spo0A are protected and used killed cells as a source of nutrients for their endospores. • Caulobacter Differentiation • Caulobacter is a member of proteobacteria and is commonly found in nutrient poor aquatic environments • 2 Life stages: • Swarmer: free swimming cells that move via flagella; function in dispersal as these cells cannot divide or replicate DNA. • Stalked (attached) cells: lack flagella and are attached to a surface via a stalk with a holdfast; reproductive stage. • Life cycle controlled by 23 major regulatory proteins expressed at different levels at different times. • GcrA, CtrA (swarmer cells), and DnaA all are transcriptional regulators that are active at specific stages. • Nitrogen Fixation, Nitrogenase, and Heterocyst Formation: • Nitrogen fixation is catalyzed by nitrogenase in a very energetically expensive process that is highly regulated. • Nitrogenase is inactivated by oxygen so to allow nitrogen fixation to occur simultaneously with photosynthesis, dedicated cells for nitrogen fixation are formed. • Heterocysts: cells where N2 fixation occurs, arise via monitoring of external conditions and cellcell signaling. • Genes for N2 fixation are in a regulon spanning 24 kb and containing 20 genes in operons. • Nitrogenase is repressed by O2 and by availability of certain N forms. • NifA serves as a positive regulator for expression of if genes, while NifL is a negative regulator. • If O2 is present, NifL is oxidized and the protein represses transcription of other nif genes. • The presence of ammonia also regulates N2 fixation through availability of NtrC when there is little ammonia, NtrC is active and promotes transcription of NifA. • Heterocyst Formation: • Heterocyst formation is highly regulated and must be tightly controlled to prevent nearby vegetative cells from also becoming heterocysts. • Initiated by nitrogen limitation which is ‘sensed’ by the cell as elevated α ketoglutarate levels. • These elevated levels activate the transcriptional regulator NtcA which activates transcription of hetR gene (controls heterocyst formation and genes to eliminate O2 and express nitrogenase). • PatS peptide produced in heterocyst diffuses into adjacent cells and is thought to prevent additional heterocyst formation. Lecture 31: • RNABased Regulation: • All of what we have discussed to date utilizes regulatory proteins to interact with DNA • RNA is also capable of regulating gene expression, both at transcription and translation • Noncoding RNA (ncRNA): RNA molecules that are not translated into proteins • rRNA and tRNA are ncRNAs • ncRNA also includes signal recognition particles used in protein secretion, RNA used for splicing of mRNA in eukaryotes, and small RNA (sRNA) molecules of 40400 nucleotides in length, and used to regulate gene expression • Most common way for regulatory RNAs to work is by base pairing with other RNA molecules • Need regions of complementary sequence but can prevent translation of mRNAs as double traded RNA that cant be translated • Antisense RNA: small RNAs with complementary sequences to the the coding sense of their mRNA • Small RNAs can 1) bind to block the RBS, 2) open up the RBS, 3) increase or decrease degradation of the transcript • Transcription of antisense RNA is enhanced when gene needs to be turned off • TranssRNAs are encoded in intergenic regions so are spatially separated from their mRNA targets; usually limits complementarity to their targets so use a small protein (Hfq) to facilitate interaction • Hfq and similar proteins are called RNA chaperones as they help sRNA maintain correct structure. • Riboswitch: RNA molecule that resembles a repressor or activator; binds metabolites like a. acids or vitamins and regulates gene expression. Located at 5’ end of mRNA • Some mRNAs contain regions upstream of the coding sequences that can fold into 3D structures and bind small molecules; exist as 2 structures 1 with small molecule bound and 1 without small molecule • Presence of small molecule determines whether or not mRNA is translated • Riboswitch control is analogous to negative control and often controlled by availability of small metabolite • Although part of mRNA, some can control transcription by causing premature termination of mRNA synthesis • Currently only found in some bacteria, plants and fungi. Possibly remnants of the RNA world • Attenuation: form of transcriptional control by early termination of mRNA synthesis. Initiated after start but before completion of transcription. • Results in fewer completed transcripts than number of attempts • First portion of mRNA made is leader region, which can fold into 2 secondary structures, one allows further transcription while second causes premature termination. • Attenuation is found in bacteria and archaea but phys. separation of transcription and translation in eukarya prevents it • Tryptophan operon contains structural genes for 5 proteins and promoter and regulatory sequences at beginning of operon • Leader sequence: sequence in the operon that encodes a short leader peptide, contains tandem tryptophan codons near its terminus which act as an attenuator. • If tryptophan is plentiful, enough charged trp and leader peptide is synthesized. Results in termination of transcription for rest of operon • If trp is scarce, leader peptide not synthesized and rest is transcribed • Attenuation is possible b/c transcription and translation are not separate —> as mRNA is released from DNA, ribosome binds to it and immediately starts translation • Transcription is attenuated b/c a portion of mRNA folds into a unique stem loop structure that inhibits RNA pol • Folding is based on complementary base pairing and doesnt always cause termination • Stalled ribosomes (due to lack of charged tRNAs) can result in stem loop formation that prevents formation of terminal stemloop • Regulation of Enzymes and Other Proteins: • Focus on controlling activity of enzymes already present in cell • 2 Major processes: • 1. Feedback Inhibition • Temporarily shuts off rxns in an entire biosynthetic pathway due to excess end product inhibiting early enzyme in pw • Reversible as once end product becomes limiting, pathway starts to work again • Works by allosteric enzymes: two binding sites: the active site for substrate and allosteric site for end product. • Binding to allosteric site changes conformation so substrate can no longer bind at active site • Some feedback inhibition pathways use isoenzymes: different enzymes that catalyze same reaction but are controlled differently • Affect branched pathways • Often diminish enzyme activity incrementally, in order to get zero activity, must shut off all possible pathways • 2. Posttranslational regulation • Addition or removal of a small molecule to the enzyme that affects its activity • Binding of a molecule changes conformation of enzyme, inhibiting catalytic activity • Removal of molecule returns enzyme to active state • Common additions include AMP, ADP, PO42, and CH3 grps • ex) Glutamine synthase: AMP grps added and removed by other enzymes controlled by NH3 levels • The more AMP you add, the less active it becomes (0 12; where 0 is fully active and 12 is inactive)