BIO425 Chapter 5. Control of Microbial Growth-1 PDF

Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 1 MICROBIOLOGY LECTURE Multiple Fission Chapter 5: Control of Microbial Growth Cyanobacteria Sources:...

Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 1 MICROBIOLOGY LECTURE Multiple Fission Chapter 5: Control of Microbial Growth Cyanobacteria Sources: PPT, Lecture, Book Progeny cells, baeocytes (baeo = small; cyte = cell), are held within the cell wall of parent cell EUKARYOTIC REPRODUCTION until they mature Alternate between diploid and haploid stages ○ Starts as small spherical cell, 1-2 um in in life cycle diameter, grows into a vegetative cell, Types: 13 um in diameter ○ As it is grows DNA is replicated over Sexual Asexual and over, cell produces a thick cellular matrix Meiosis Mitosis Haploid (n) Diploid (2n) - single set of - 2 complete sets chromosomes of chromosomes End product: organism Daughter cells are exact with traits coming from replicas both parents Gamete formation MICROBIAL REPRODUCTION Haploid only Asexual ○ Binary fission ○ Budding ○ Filamentous Commonality in all processes: ○ Genome of the cell must be replicated and segregated to form distinct nucleoids ○ Each nucleoid becomes enclosed within its own plasma membrane Budding Binary Fission Hyphomicrobium Cell elongates as new cell envelope material is ○ Prostheca (i.e. hypha filament) grows synthesized out on one end of the cell and the bud Subcellular structures like ribosomes and grows at the tip inclusions are abundant enough to be evenly ○ Separated by a long distance from the distributed in the cytoplasm mother cell Nucleoid ○ Present as a single entity ○ Replication and partitioning into half of elongated cell is a critical event ○ Contains all or most of genetic material, since they have no nucleus ○ Nucleus-like Septum ○ New cell wall formed between two daughter cells as a result of cell Multinucleoid Filaments division Streptomyces ○ Formed at mid cell ○ Specialized aerial branches that forms ○ Dividing the parent cell into 2 progeny spores cells ○ Spores are dispersed ○ Multinucleoid filaments → uninucleoid spores Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 2 Cell at early phase of the cycle Parent cell prepares for division by enlarging its cell wall, plasma membrane, and overall volume. DNA replication then starts BACTERIAL CELL CYCLE (BINARY FISSION) *Copying of DNA by Cell Cycle replication enzymes ○ Complete sequence of events beginning at the origin of replication, until extending form formation of a new cell entire chromosome is through the next division copied ○ Simple cycle Grown 2x starting size Septum begins to grow (increasing its volume) inward as the Split into two chromosomes move toward opposite ends of Synthesis of peptidoglycan during the cell the cell cycle is the target of numerous antibiotics used to treat bacterial infections Remain viable and competitive the bacteria should divide at the right time and place, must Other cytoplasmic provide each offspring with a complete copy of components are its essential genetic material distributed to the two developing cells Major phases 1. Period of growth after the cell is born Membrane pinches Similar to G1 phase of inward eukaryotic cycle Preparation for division Septum is synthesized 2. Chromosome replication and completely through the cell center, creating two partitioning separate cell chambers S and mitosis events of the M phase of eukaryotic cell cycle The daughter cells are Chromosome replication and divided. partitioning occur concurrently Some species separate, 3. Cytokinesis (cytoplasmic division) while others remain Septum and daughter cells are attached, forming formed chains, doublets, or Initial events of cytokinesis other cellular actually occur before arrangements chromosome replication and partitioning are complete Most bacteria have a single circular Initiate new rounds of chromosome found in the nucleoid replication before the first Cell cycle takes 60 mins to complete, but more round of replication and rapid rate of 20 mins can occur despite that cytokinesis is finished DNA replication requires at least 40 mins ○ Bacteria begins second round of DNA replication (sometimes 3rd and 4th) Image Description before the first round of replication is complete Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 3 ○ Progeny cells receive a chromosomes cells with two or more replication forks, and replication is continuous since cells are always copying their DNA Chromosome Partitioning ○ Chromosomes must be replicated and Chromosome Replication and Partitioning separated from mother cell into Origin of replication/Origin daughter cells ○ Single site at which replication starts Eukaryotic: mitotic spindle Replication is completed at the terminus, Prokaryotes: cytoskeletal located directly opposite the origin elements Stages: No mitotic spindle in 1. Chromosome is compacted and bacteria, hence they do oriented so that the origin and not undergo terminus are in opposite halves of the karyokinesis (i.e. cell division of the nucleus) 2. Origin and terminus move to midcell, Bacterial cytoskeleton and proteins needed for chromosome is actively involved in replication assemble at the origin ○ Chromosome Replisome segregation DNA synthesizing ○ Maintenance of machinery cell shape Group of proteins ○ Replisome pushes, or condensation of needed for DNA daughter chromosomes to opposite synthesis ends 3. DNA replication proceeds in both ○ MreB (murein cluster B) directions from the origin Widely conserved actin 4. As progeny chromosome is homolog, plays role in synthesized, the two origins move determination of cell shape as toward opposite ends of the cell, and spiral inside cell periphery, and the rest of each chromosome follows in chromosomes segregation an orderly fashion Important in elongation and maintenance of rod like shape (associated with bacilli (i.e. rod shaped bacteria)) If MreB is mutated, chromosomes do not segregate Governs cell elongation Cytokinesis Septation ○ Process of forming a cross wall between 2 daughter cells ○ Process 1. Selection site where the septum will be formed 2. Assembly of Z-ring (i.e. 1. Replication of circular chromosome at origin composed of the cytoskeletal of replication 2. Replication continues at both directions at protein—FtsZ) once Most critical 3. Cell begins to elongate Without the Z-ring 4. Duplicated chromosome separate and other steps would not continue to move away from each other to take place the opposite ends of the cell 3. Linkage of Z ring to plasma 5. Formation of septum membrane (cell wall) 6. Divides the cell 7. Cell pinches into two, forming 2 daughter 4. Assembly of the cell wall-synthesizing machinery Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 4 Synthesis of ○ Membrane protein complex with peptidoglycan and proteins at both sides of cytoplasmic other cell wall membrane constituents Gram negative: inner 5. Constriction of the cell and membrane septum formation ○ Ubiquitous in bacteria but varies among species Cytokinesis ○ Responsible for: ○ Producing 2 daughter cells Cell division Constriction of inner and outer Z-ring membranes during division ○ Needed at the proper place and time for Synthesis of peptidoglycan at correct septation the division site ○ Forms the scaffold for the complete cell division machinery At midcell attracts other cell division proteins to form divisome ○ Dynamic with portion being exchanged constantly with newly formed, shorted FtsZ polymers from cytosol ○ FtsA and ZipA proteins promote its attachment to the plasma membrane ○ Lack of MinCDE system limits the ring E.coli divisome formation to the center of the cell MinC, MinD, MinE oscillate from one side of cell to other Min C is highly Divisome Protein Function concentrated at the poles, where it prevents FtsA, ZipA Anchor Z ring to plasma formation of the Z ring membrane Z ring can only occur at FtsZ Forms Z ring mid cell since it lacks the MinCDE FtsK Chromosome segregation and Link Z ring to cell membrane separation of chromosome Z ring constricts and cell wall dimers synthesis of cell wall FtsQLB May provide a scaffold for assembly of proteins involved FtsZ Protein in peptidoglycan synthesis ○ Homologue of eukaryotic tubulin found in most bacteria and archaea Ftsl (also known as Peptidoglycan synthesis Highly conserved penicillin binding ○ Polymerizes to form filaments that protein 3), FtsW create the meshwork that constitutes FtsN Thought to trigger constriction the Z-ring initiation ○ Governs cell division Divisome CELLULAR GROWTH AND DETERMINATION OF CELL ○ Forms after Z ring forms SHAPE ○ Late division proteins Determined by peptidoglycan synthesis in ○ Essential protein assembly required for bacteria septum synthesis and cell separation ○ Strength of existing peptidoglycan ○ Complex machinery of bacteria must be maintained as new composed of 2 dozen proteins, peptidoglycan subunits are added ○ Peptidoglycan Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 5 Rigid envelope surrounding the 3. The divisome protein MurJ (not shown) “flips” cytoplasmic membrane lipid II across the plasma membrane so that Protect them from the NAM-NAG units are available for insertion environmental stress into the sacculus. Preserve cell morphology 4. Autolysins (blue balls labeled “A”) located at Synthesis is important in the divisome degrade bonds in the existing bacterial cell division peptidoglycan sacculus. This permits the ○ Involves many proteins such as PBPs insertion of new NAM-NAG units into the sacculus. Penicillin Binding Proteins (PBPs) ○ Capacity to bind penicillin ○ Group of enzymes that: Link the peptidoglycan strands Catalyze controlled degradation for new growth Hydrolysis of existing strands so new units can be inserted during cell growth ○ Autolysins PBP enzymes that hydrolyze bonds of peptidoglycans Process Disclaimer: Flow Chart based on the book’s discussion and on the team’s understanding of the process Cell Shape and its Process Cocci ○ New peptidoglycan forms only at the central septum ○ When daughter cells separate each has one new and one old hemisphere ○ Proper placement of septum depends 1. Peptidoglycan synthesis starts in the on the FtsZ localization cytoplasm with the attachment of uridine FtsZ determines site of cell wall diphosphate (UDP) to the sugar growth N-acetylglucosamine (NAG). Some of the FtsZ may recruit PBPs (and UDP-NAG molecules are converted to UDP-NAM other enzymes needed for the (N-acetylmuramic acid). Amino acid addition peptidoglycan synthesis to the to NAM is not shown for simplicity. divisome) for synthesis of 2. NAM is transferred from UDP to bactoprenol, a septum carrier embedded in the plasma membrane. NAG is then attached to bactoprenol-NAM generating bactoprenol-NAM-NAG (called lipid II) carries the NAM-NAG unit across the plasma membrane, delivering it to the periplasm. Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 6 confined to the midcell III. Division Daughter cells are formed with one new pole and one old pole Green: Old hemisphere Vibrio Red: New hemisphere ○ Comma-shaped bacteria ○ FtsZ - forms Z ring Rods ○ MreB - helical polymerization ○ Synthesized by 2 molecular throughout the cell machineries share many proteins but ○ Crescentin - intermediate filament differ in terms of placement and time homologue of function Localizes to short, curved side Elongasome of cell Responsible for Localizes to one side of the cell, elongation of the cell where it slows the insertion of that occurs prior to new peptidoglycan units into septum formation the peptidoglycan sacculus → Divisome Asymmetric cell wall synthesis Synthesizes → Forms inner curvature/curve peptidoglycan during cytokinesis ○ Similar to cocci but elongate prior to septation ○ MreB determines cell diameter and elongation as the Z ring forms in the center ENVIRONMENTAL FACTORS AFFECTING MICROBIAL GROWTH Most organisms grow fairly moderate environmental conditions Extremophiles ○ Microorganisms that grow in harsh conditions ○ Harsh conditions are normal Extreme temperature I. Side wall New cell wall is made Extreme pressure Elongation along the side of the cell All microbes have a characteristic range at but not at the poles. which growth occurs defined by high and low values beyond which the microbe cannot Determined by the survive position of MreB Optimal value homologues (i.e. ○ Value at which it grows best associated with cell elongation and and maintenance of cell Overview shape in rod shaped bacteria) II. Preseptal FtsZ polymerization Elongation forms a Z ring and new cell wall growth is Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 7 ○ Mechanosensitive (MS) channels Walled (additional protection) and wall-lacking microbes utilize this Lower solute concentration of plasma membrane Membrane stretches due to an increase in hydrostatic pressure and cellular swelling → MS opens and allow solutes to leave Acts as escape valves that prevent the cell from bursting ○ Contractile Vacuoles Protist Expel excess water Protective responses to hypertonic solution ○ Increase internal ions in order to remain hypertonic to their environment Osmosis and Water Activity Obtain solutes compatible with Changes in osmotic concentrations in the metabolism and growth environment may affect microbial cells Osmophiles Solutes in an aq sol’n (i.e. water solution) alter ○ Require high concentrations of sugars the behavior of water during osmosis Halophiles Osmosis ○ Require the presence of NaCl at a ○ Movement of fluid across the concentration above 0.2 M membrane in response to different ○ Categorized into 3 concentrations of solutes on the two Slight : 0.3-0.8 M salt content sides of the membrane Moderate: 0.8-3.4 salt content ○ Two solution are separated by a Extreme halophiles: semipermeable membrane that Discussion: 3.4 M to 5.1 allows the movement of water but not salt content of solutes concentrations of salt ○ Diffusion of water molecules (NaCl), more than 10% Movement along a of solution concentration gradient PPT: 2M - 6.2 M Water movement Prescott: 3M - 6.2M ○ Lower solute concentrations → Higher Cell walls, proteins, and solute concentration plasma membrane Osmotic concentration/Osmolarity require high salt ○ Solute concentration content to maintain stability and activity Hypotonic Sol’n Hypertonic Sol’n Lower solute Higher solute Effects of NaCl on Microbial Growth concentration concentration Water goes in the cell → Water goes out of the burst cell, shrink/plasmolysis Protective responses to hypotonic solution ○ Cell wall prevents overexpansion of the plasma membrane Not all microbes have cell walls Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 8 *only illustrative Halotolerant ○ Don’t require the presence of salt but can grow under saline conditions Water Activity (aw) ○ Degree of water availability pH Type Examples ○ Microorganisms take up water by moving it across the cell membrane 0 - 5.5 Acidophiles Most Fungi Depends on water activity (between pH: 4-6) gradient Photosynthetic Higher aw (outside the cell) → protist (slight Lower aw (within the cell) acidity) ○ Reduced by interaction with solute molecules (osmotic effect) Archaea ○ Higher [solute] → lower aw ○ When water activity outside is low it Sulfolobus acidocaldarius (a) causes osmotic stress → cell cannot take up water → becomes dormant Ferroplasma ○ Reduced by adsorption to surfaces acidarmanus (a) ○ aw sol’n = 1/100 relative humidity of sol’n = ratio of sol’n vapor pressure Picrophilus (Psoln) to pure water (Pwater) oshimae (a) 5.5 - 7.0 Neutrophiles Most known pH (Prescott: bacteria and Measure of relative acidity of a solution 8.0) protist pH = -log[H+] = log(1/[H+]) Each pH unit represents a 10-fold change in 8.5 - 11.5 Alkaliphiles Bacillus (b) (Prescott: (Alkalophiles) hydrogen ion concentration 8.0-11.5) Micrococcus (b) Pseudomonas (b) Streptomyces (b) Marine microorganisms (a) = archaea (b) bacteria Temperature Microbes are susceptible to their external temperature as they cannot regulate their internal temperature Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 9 Enzyme-catalyzed reactions are sensitive to temp ○ Each enzyme has temperature at which the can function optimally ○ Below optimum = cease to be catalytic/function ○ Beyond optimum = denaturation Cell membranes ○ Low temperatures = solidify ○ High temperatures = melts and disintegrates Cardinal temperatures ○ Characteristic temperature dependence Psychrotolerants Minimum: 0 ○ Growth range: -20 to over 120OoC (Psychrotrophs) ○ Three portions: Maximum: 35 Minimum Optimal Psychrophiles Minimum: 0 Generation time (g) is (Cryophiles) Optimal: 15 shortest Always closer to the Maximum: 20 maximum than the minimum Mesophiles Minimum: 15-20 Range: 0-75OC Optimal: 20-45 Maximum Depends on environmental factors such as pH Maximum: 45 and availability of nutrients ○ Crithidia fasciculata Thermophiles Minimum: 45 Simple medium: 22-27OC Optimal: 55-65 Medium w/ extra metals, amino acids, vitamins, and Maximum: 85 lipids: 33-34OC Hyperthermophiles Minimum: 55 Organism Temperature Optimal: 85-113 Protist > 50oC Psychrophiles Fungi 55 - 60oC ○ Isolated from Artic and Antartic habitats Bacteria & Archaea HIgher than eukaryotes ○ Main habitat: oceans 90% of ocean water is 5oC or Eukaryotes > 60oC below ○ Genera Terminology Vibrio Alcaligenes Type Temperature (OC) Bacillus Photobacterium Shewanella ○ Survival mechanisms Membranes have high levels of unsaturated fatty acids and remain semifluid when cold (Begin to leak cellular constituents at temperatures higher than 20oC) Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 10 Accumulate come solutes to Oxygen Concentration decrease the freezing point of Oxygen correlates with microbes energy cytosol conserving metabolic processes and the Use antifreeze proteins electron transport chain (ETC) and nature of Mesophiles terminal electron acceptor ○ Almost all human pathogens Energy-conserving metabolic processes involve Normal body temperature: 37oC, the moving of electrons through a series of thrive in the body membrane-associated electron carriers, Thermophiles electron transport chain (ETC) ○ Vast majority are from Bacteria or ○ Terminal acceptor is oxygen Archaea Hyperthermophiles Type Description ○ Pyrococcus abyssi ○ Pyrodictium occultum Survival mechanisms of thermophiles and hyperthermophiles in high temperatures ○ Heat stabilizing proteins Highly organized hydrophobic interiors More Hydrogen and noncovalent bonds Stabilize structure Proline (i.e. type of amino acid) make polypeptide chains less Aerobe - grows in presence of atmospheric oxygen flexible and more heat stable (O2) which is 20% O2 Chaperones Obligate Aerobes Oxygen serves as a Aid in folding and terminal electron stabilization of acceptor for ETC proteins Nucleoid-associated proteins / Aerobic respiration Histone-like proteins Stabilize the DNA Need oxygen to grow Reverse DNA gyrase Microaerobic O2 levels in range of Enzyme that changes 2-10% for growth the topology of their DNA & enhances their Damaged by stability atmospheric level of O2 (20%) ○ Membranes stabilized in variety of means Anaerobe - grows in absence of O2 Membranes of lipids are more saturated, branched, and Facultative Anaerobes Do not require O2 for higher molecular weight growth Increases melting point Grow better in the of membranes presence of O2 Ether linkages in archaeal membranes Aerotolerant Anaerobes Grow equally well Resistant to hydrolysis whether O2 is present or at high temperatures not Diglycerol tetraethers Can tolerate O2 but do observed in the not make use of it membranes of some archaeal thermophiles Obligate Anaerobes O2 is toxic; killed by span the membrane to prolonged exposure to form a rigid, stable oxygen monolayer May be recovered in Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 11 Land and water surface organisms can live habitats that appear to be oxic, associated with with pressure of 1 atm facultative anaerobes, Some bacteria and archaea live in deep sea that use the available O2 water (≥ 1000m) with very high hydrostatic pressure O2 response are determined by growing the Hydrostatic Pressure microbe in the medium thioglycollate broth, ○ Deep ocean contains a reducing agent to lower O2 levels ○ 600-1100 atm; temp: 2-3oC Fungi = aerobic ○ Affect membrane fluidity and Yeasts = facultative anaerobes membrane associated function Photosynthetic protists = obligate aerobes Barotolerant Reactive Oxygen Species ROS ○ Microbes found at great ocean depths ○ Toxic O2 derivatives ○ Adversely affected by pressure ○ Causes inactivation of proteins, when ○ Not as severely as non-tolerant sulfhydryls are oxidized organisms ○ Unpaired electrons in the outer shell of Barophilic (Piezophilic) Organisms oxygen, makes it inherently unstable ○ Organisms that have maximal growth ○ Formed when cellular proteins such as rate at pressures greater than 1 atm flavoproteins transfer electron to O3 ○ Require or grow more rapidly in the ○ Damages protein, lipids, and nucleic presence of increased pressure acid ○ Change membrane fatty acids to adapt ○ Ex to high pressures Superoxide radical Hydrogen peroxide RADIATION DAMAGE Hydroxyl radical (most Earth - bombarded with electromagnetic dangerous) radiation of various types ○ Utilized by neutrophils and Electromagnetic Spectrum - range of macrophages to destroy invading frequencies of electromagnetic radiation and pathogens respective wavelengths and photon energies Protection against ROS Radiation behaves as waves like those ○ Obligate aerobes and facultative traveling on the surface of water anaerobes ○ Sunlight ○ Enzymes = Destroyed ROS Major source of radiation on Superoxide Dismutase (SOD) = Earth superoxide radicals Includes: Catalase = hydrogen peroxide Visible light Hydrogen peroxide Ultraviolet (UV) decomposes to water radiation and oxygen Infrared rays Peroxidase = hydrogen peroxide Radio waves Strict Anaerobes ○ Lack or have low quantities of superoxide dismutase and catalase → cannot tolerate O2 ○ To grow microbes w/o O2 Candle jar method (time ni Maam Medina) Jar with a candle inside to eliminate oxygen, then it is closed Workstation with incubator Gaspak Anaerobic system Pressure Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 12 ○ Can harm microorganisms Many forms of electromagnetic radiation are Induce the breakdown of the very harmful to microorganisms amino acid tryptophan to toxic photoproducts Ionizing Radiation Toxic photoproducts + One of the most damaging forms of near-UV radiation = electromagnetic radiation breaks in DNA strands Radiation of very short wavelength and high energy Visible Light ○ Causes atoms to lose electrons When present in sufficient intensity, can Two forms: damage or kill microbial cells ○ X-rays - artificially produced Photosynthesizers (pigment) and 𝑂2 are ○ Gamma rays - emitted during natural involved radioisotope decay ○ Photosynthesizers: Effects: Can absorb light and become ○ Low levels: Mutations → indirectly excited/activated result in death (sterilization) Chlorophyll ○ High levels: directly lethal Bacteriochlorophyll Causes a variety of changes in cells Cytochromes ○ Breaks hydrogen bonds Flavins ○ Oxidizes double bonds 1 Generates singlet oxygen ( 𝑂2) ○ Destroys ring structures ○ Polymerizes some molecules at high intensities ○ Disrupts chemical structure of many Powerful oxidizing molecules (e.g. DNA) agent Damage may be repaired by Carotenoid pigments DNA repair mechanisms if Protect many small dose light-exposed Microorganisms - more resistant to ionizing microorganisms from radiation than larger organisms photooxidation ○ Can still be destroyed by a sufficiently large dose MICROBIAL GROWTH IN NATURAL ENVIRONMENTS Can be used to sterilize items Complex Bacterial endospores and bacteria like Constantly changing Deinococcus radiodurans Often contain low nutrient environment ○ Extremely resistant to DNA damage (Oligotrophic Environment) May expose microorganisms to overlapping Ultraviolet (UV) Radiation gradients of nutrients and environmental Another very damaging form of radiation factors that include Can kill microorganisms ○ Environmental parameters Short wavelength, high energy e.g. pH, temperature, etc. ○ (Approximately from 10 - 400 nm) ○ Inhibitory substances that limit Most lethal UV radiation microbial growth ○ Wavelength most effectively absorbed by DNA is 260 nm BIOFILMS Mutations → death Most microbes grow attached to surfaces Causes formation of thymine dimers in DNA (sessile) rather than free-flowing (planktonic) Requires direct exposure on microbial surface Biofilms DNA damage can be repaired by several repair ○ Complex, slime-enclosed communities mechanisms of microbes ○ Excessive exposure of microorganisms ○ Ubiquitous in nature in water to UV light outstrips the organism’s Often seen as layers of slime on ability to repair the damage and death rocks or other objects in water results or at water-air interfaces Longer wavelengths of UV light ○ near-UV radiation; 325 - 400 nm Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 13 ○ Clusters of bacteria that are attached to a surface or to each other and embedded in a self-produced matrix Biofilm matrix - substances like proteins, polysaccharides ○ Can be formed on any conditioned surface Interactions occur among the attached organisms ○ Exchanges take place metabolically, DNA uptake and communication Initially microbes attach to the conditioned surface but can readily detach Biofilm Microorganisms Microbes reversibly attach to conditioned EPS - Extracellular Polymeric Substances surface ○ Collective term for biofilm polymers ○ Release polysaccharides, proteins, and ○ Allows microbes to stick more stably to DNA to form the extracellular polymeric the surface substance (EPS) As biofilm thickens and matures, the microbes Additional polymers are produced as microbes reproduce and secrete additional polymers reproduce and biofilm matures Conditions at locations in the biofilm can become detrimental to the cells ○ Becomes beneficial for cells to detach and escape the biofilm Of considerable importance for medical device-associated biofilms (e.g. implants) Escaping cells - seed sites of infection elsewhere in the body and often lead to illness The EPS and change in attached organisms’ Mature Biofilm - a complex, dynamic physiology protects microbes from harmful community of microorganisms agents Heterogeneity - differences in metabolic ○ UV light activity and locations of microbes ○ Antibiotics ○ Some are persister cells ○ Antimicrobials “Persist” Sloughing off of organisms Bacterial cells that are ○ Can result in contamination of water identified by their ability to phase above the biofilm (e.g. in a survive exposure (e.g. drinking water system) antibiotic) Not growing cells that are CELL TO CELL COMMUNICATION WITHIN THE MICROBIAL thought to be dormant POPULATIONS Quorum Sensing ○ Communication in a density-dependent manner of bacterial cells in biofilms Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 14 ○ Cell communication that is widely used by bacterial pathogens to coordinate expression of several collective plates Production of multiple virulence factors Biofilm formation Swarming motility once the population threshold is reached ○ Bacteria monitor the density of the population (no. of bacteria in an area) Based on chemical signals When the signal reach a threshold level, all bacteria in the population will change their behavior or gene expression at the same time Produce small proteins that increase in concentration as microbes replicate and convert a microbe to a competent state ○ DNA uptake occurs, bacteriocins are Process regulated by quorum sensing involve released host-microbe interactions ○ Symbiosis Quorum Sensing Vibrio fischeri and Quorum sensing bacteria produce and release bioluminescence in hawaiian chemical-signaling molecules called bobtail squid autoinducers The marine ○ Increase in concentration as a function luminescence of Vibrio of cell density fischeri lives within the Acylhomoserine lactone (AHL) light organ of the squid ○ Autoinducer molecule produced by and in some fishes many gram-negative organisms Vibrio fischeri regulates Vary in length and substitution its luminescence by of the acyl side chain producing a small, Diffuses across plasma diffusible molecule membrane (autoinducer) Once inside the cell, induces ○ Other processes regulated by quorum expression of target genes sensing also has something to do with: regulating a variety of Pathogenicity and increased functions depending on the virulence factor production microbe DNA uptake for antibiotic resistance genes Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 15 Typical when one transfers the inoculum in a GROWTH particular medium Growth - one of the attributes in life ○ Still have to incubate the sample ○ No growth = no life before growth of microorganisms can ○ e.g. increase in height, increase in size be observed on the preferred culture Increase in cellular constituents that may medium result in: Varies in length ○ Increase in cell number ○ In some cases, can be very short or ○ Increase in cell size even absent Growth - population growth rather than growth of individual cells Exponential Phase Also called Log Phase (Logarithmic Phase) THE GROWTH CURVE Rate of growth and division - constant and Microorganisms cultivated in broth maximal ○ Observed when microorganisms are ○ Cells are actively dividing cultivated in batch culture No. of cell division > no. of cell death Incubated in a closed culture ○ Rapid increase in population vessel with a single batch of ○ More cells undergo cell division medium Population - most uniform in terms of Fresh medium - not provided chemical and physical properties during incubation Nutrient concentration Stationary Phase decline, waste Closed system population growth eventually concentration increase ceases Usually plotted as logarithm of cell number Total number of viable cells remains constant vs. time ○ Active cells stop reproducing Has four distinct phases ○ Reproductive rate = Death rate Additional phase in other sources Period of equilibrium ○ Long-Term Stationary Phase (Stationary ○ No. of cell division = no. of cell death Phase) ○ Cells are still dividing but there are also cells that start to die Possible Reasons: ○ Same reasons why cells are dying during the Death Phase Nutrient Limitation Essential nutrient is severely depleted, population growth will slow and eventually stop Limited Oxygen Availability Insoluble oxygen and may deplete quickly that only the surface of a culture will have an Lag Phase 𝑂2 concentration Cell synthesizing new components adequate for growth ○ To replenish spent materials Toxic Waste Accumulation ○ To adapt to new medium or other Limits the growth of conditions many cultures growing Phase where the cells are still adjusting to their in the absence of 𝑂2 new environment Period of adjustment Critical Population Density During this time, there is active synthesis reached taking place and most cells are not yet Growth may cease reproducing when a critical Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 16 population level is Protects DNA from reached various oxidative Stationary Phase and Starvation Response damage in ○ Entry into stationary phase due to stressed/starved cells starvation and other stressful Chaperone proteins prevent conditions activates survival strategy protein damage Morphological changes ○ Persister Cells Endospore formation Subpopulation of transiently ○ Bacillus and antibiotic-tolerant bacterial Clostridium - cells that are often growing two genera very slowly or have arrested capable of growth forming Able to resume endospores growth/reproduction Their after a little stress coping Long-term survival mecha Increased virulence nism due to Senescence and Death Phase nutrien No. of cell division < no. of cell death t Period of decline depleti ○ In the Log Phase, all the nutrients on needed by the microbes are present. In Decrease in size addition to these nutrients, the Protoplast shrinkage required environmental factors have Nucleoid condensation been met to make sure of the RpoS protein microbes’ growth. While RNA polymerase microorganisms are dividing, they sigma S consume the nutrients in the medium Also called katF while at the same time, releasing Encodes the sigma wastes. There is an accumulation of factor (sigma 38) toxic materials, and an increase in Regulate transcription density and overcrowding. in bacteria Two alternative hypotheses Can be activated in ○ Cells are viable but not culturable response to different (VBNC) environmental Some microbiologists think conditions that some cells are only Transcribed in late temporarily unable to grow at exponential phase least under laboratory Primary regulator of the conditions used stationary phase genes Once the appropriate Assists RNA conditions are polymerase in available, the VBNC transcribing genes for microbes resume starvation proteins growth Starvation Responses Cells are alive but dormant ○ Production of Starvation Proteins Capable of new growth when Increase cross-linking in cell conditions are right wall ○ Programmed cell death DPS protein (DNA binding Fraction of the population protein) genetically programmed to die Conserved protein (commit suicide) after growth found in most bacterial ceases species Dying cells sacrifice themselves for the Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 17 benefit of the larger population THE MATHEMATICS OF GROWTH During the exponential phase, each microorganism is dividing in constant intervals. Generation (doubling) time - specific length of time where the population doubles ○ Time required for the population to double in size ○ Varies depending on species of microorganism and environmental conditions ○ Range: 10 minutes for some bacteria Several days for some eukaryotic microorganisms MEASUREMENT OF MICROBIAL GROWTH There are many ways to measure microbial growth to determine growth rate constants and Suppose that a culture tube is inoculated with generation times one cell that divides every 20 minutes. The ○ Can measure changes in number of population will be 2 cells after 20 minutes, 4 cells in a population cells after 40 minutes, and so forth. Because ○ Can measure changes in mass of the population is doubling every generation, population the increase in population is always 2n where n Growth leads to increases in is the number of generations. The resulting both population increase is exponential (logarithmic). DIRECT MEASUREMENT Direct cell counts Exponential Population Growth ○ Counting Chambers Population is doubling every generation Petroff-Hausser Counting As one incubates the sample, the number of Chamber cells increases as well. Most commonly used in laboratory Most obvious way to determine microbial numbers Easy, inexpensive, and quick Useful for counting both eukaryotes and prokaryotes Cannot distinguish living from dead cells Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 18 assess cell morphology Cells filtered through special membrane that provides dark background for observing cells Cells are stained with fluorescent dyes Useful for counting bacteria With certain dyes, can distinguish living from dead cells VIABLE COUNTING Microbiologists have traditionally defined microbes as being dead when they could not be cultured. However, microbiologists have come to realize that cells may be “inactive” or “damaged” and therefore unable to reproduce temporarily. With time and appropriate conditions, the cells may recover and begin to reproduce. Whether or not a cell is alive or dead isn’t always clear in microbiology ○ Cells can exist in a variety of states between ‘fully viable’ and ‘actually dead’ ○ Electronic Counters Flow cytometry Creates a stream of cells so narrow that one cell at a time passes through a beam of laser light Scattered light is detected independently No. of light-scattering events = no. of cells in sample ○ On Membrane Filters In this technique, the sample is first filtered through a back polycarbonate membrane filter Bacteria are then stained with nucleic acid fluorescent stains Acridine orange DAPI Viable Counting Methods (4’,6-diamidino-2-phen Several plating methods can be used to ylindole) determine the number of viable microbes in a ○ Used to sample determine the ○ Viable Counting Methods number of ○ Standard Plate Counts nuclei and to Can both count only those cells able to reproduce when cultured Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 19 Spread and Pour Plate Techniques ○ Incubated until colonies grow on ○ Diluted sample of bacteria is spread membrane over solid agar surface or ○ Colony count ○ Mixed with agar and poured into petri Number of bacteria in sample plate Selective media - used to select for specific Spread Plate Pour Plate microorganisms Medium (solidified Sample first before agar) first before medium (not yet organism solidified) Utilizes glass spreader Does not utilize glass - Bent glass rod spreader, only pipette to - Slingshot-like transfer sample Introduce the The sample microorganism by (microorganism) is ‘spreading’ to the plate mixed along with the medium. Then, solidify. ○ After incubation (number of time needed for microorganisms to grow), the number of organisms are determined by Number of colonies · dilution factor GROWTH (FORMATION OF COLONIES) Sometimes, plate counts cannot be used to measure population size Spread Plate Pour Plate ○ If microbe cannot be cultured on plate media On the surface of the On the surface of the medium (agar). medium (agar) and Turbidity determined to yield the most within (below) the agar probable number (MPN) Dilutions - made and added to suitable media After incubation, each tube is examined to Disadvantage of Pour Plate: ○ determine if growth occurred If using differential media, one If no growth is observed, the tube is assumed will not be able to differentiate not to have received any cells the colonies formed because there are some colonies found MEASUREMENT OF CELL MASS underneath the agar. Techniques for measuring changes in cell ○ It is not possible to be certain that mass can also be used to determine each colony arose from an individual population size cell ○ Microbial Dry Weight Results expressed as colony Cells growing in liquid medium forming units (CFU) rather are: than the no. of microorganisms Collected by Membrane Filter Technique centrifugation ○ Another way of determining microbial Washed, growth Dried in an oven, and ○ Especially useful when one wants to Weighed analyze water purity Especially useful technique for ○ First, bacteria from aquatic samples measuring the growth of are trapped on membranes filamentous fungi ○ Membrane soaked in culture media or Time consuming placed on agar medium Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 20 Bacteria weigh so little, The second tube (more turbid) has more it may be necessary to microorganisms. centrifuge several ○ The number of cells increases, the hundred milliliters of population increases, the medium culture to collect a becomes more turbid so therefore more sufficient quantity light is scattered and the absorbance Not very sensitive reading is increased ○ Quantity of a Particular Cell Constituent MICROBIAL CONTROL Protein, DNA, ATP, or chlorophyll Different methods to control the growth of Useful if amount of substance microorganisms in each cell is constant Autoclaving - one way of patrolling growth of ○ Turbidimetric Measures (Light microorganisms Scattering) Different Microbial Control Methods: Spectrophotometry ○ Physical Agents more rapid and Heat sensitive method Dry Heat Depends on the fact Moist Heat that microbial cells Radiation scatter light that Ionizing strikes them Nonionizing Because microbial cells ○ Chemical Agents in a population are of Gasses roughly constant size, Liquids (e.g. alcohol, bleach) the amount of ○ Mechanical Removal Methods scattering is directly Filtration proportional to the ○ Biological Agents biomass of cells Predator present and indirectly Virus related to cell number Toxin Quick, easy, and sensitive Frequently Used Terms Sterilization - “destruction” ○ Sterilize by means of autoclaving ○ To “remove”, to “kill”, to “deactivate” all forms of life ○ Destruction or removal of all viable organisms Fungi Determination of microbial mass by Bacteria measurement of light absorption Spores As the population and turbidity increase, more Unicellular eukaryotic light is scattered and the absorbance reading organisms (Plasmodium) given by the spectrophotometer increases Arca, Bantilan, Embay, Ervas, Mendoza R. BIO425 | Finals Period | 21 Biological agents (e.g. preens) thereby limiting or preventing the present in a specific surface, harmful results of infection object, or fluid (e.g. food or ○ Prevention of infection of living tissue biological culture media) by microorganisms ○ Can be achieved through various ○ Antiseptics means Chemical agents that kill or Heat inhibit growth of Chemicals microorganisms when applied Irradiation to tissue High pressure Reduce total microbial Filtration population ○ Disinfection, sanitization, and pasteurization - “reduction” instead COMPARISON of elimination of all forms of life and other biological agents (Sterilization) Similarities Differences ○ After sterilization, an object is referred Disinfection - Both are used Disinfectant - to as being sterile or aseptic to destroy or more toxic to Disinfection inhibit humans ○ Application of a chemical agent microorganism ○ Killing, inhibition, or removal of s. Used on disease-causing (pathogenic) inanimate organisms - Alcohol can be objects both Destroy or inhibit the growth of disinfectant Example: microorganisms on the surface and antiseptic. bleach, lysol of an instrument or any device ○ Disinfectants Antisepsis Antiseptics - Agents, usually chemical, used less toxic to for disinfection humans Used on inanimate objects