Microbial Growth & Binary Fission

Microbial Growth Applications

• Understanding microbial growth is crucial for genetic engineering and food preservation

Binary Fission

• Bacteria reproduce asexually through binary fission instead of mitosis
• Binary fission involves a bacterial cell growing, copying its DNA, and then dividing into two identical cells
• Each resulting daughter cell contains the parent cell's genome
• Cytokinesis, or the splitting of the cytoplasm, occurs during binary fission
• The protein FtsZ aids division by forming a Z ring

Bacterial Growth Curve Phases

• The growth curve models the number of cells over time and consists of phases:
  • Lag Phase: Bacteria prepare for growth by synthesizing proteins and adapting to their environment; cells are metabolically active but not dividing
  • Log Phase: Bacteria exhibit rapid and synchronized division, representing optimal growth
  • Stationary Phase: Limited resources cause growth to slow or stop; while some bacteria die, others remain actively dying; spore/antibiotic production ceases
  • Death Phase: The rate of bacterial death increases

Sustaining Microbial Growth

• A chemostat maintains bacteria in the log phase by continuously supplying nutrients and removing waste, useful for research and industrial applications
• Oxygen levels are controlled for aerobic bacteria in chemostats

Measuring Bacterial Growth

• Bacteria counts help assess the severity of infections
• Direct methods involve physically counting bacterial cells
• Indirect methods measure bacterial presence without counting individual cells, like fluorescent staining

Fluorescent Staining

• Fluorescent staining assesses bacterial viability
• Primary staining uses green fluorescence to stain both live and dead cells
• Secondary staining uses red fluorescence to identify dead bacteria by staining cells with damaged cell membranes

Indirect Cell Counts

• Transmittance occurs by increasing particle suspension amount then decreasing light reaching detector
• Absorbance relates to the amount of light absorbed by bacteria; higher bacterial concentration results in higher absorbance
• The Most Probable Number (MPN) technique estimates viable microorganisms by detecting growth through color change in water/food samples

Alternative Bacterial Growth Patterns

• Fragmentation involves parent filaments breaking apart into new cells
• Budding involves a small bud extending from the parent cell and eventually detaching
• Spore formation produces spores in aerial filaments, aiding survival and dispersal
• Biofilms are complex ecosystems on surfaces in liquid environments, like dental plaque
  • Biofilms offer protection, facilitate resource sharing, and enhance microbe survival
  • Biofilms consist of 50-90% protective extracellular polymeric substance (EPS) dry mass, shielding from threats and stress

Reactive Oxygen Species (ROS) and Microbes

• ROS are unstable molecules formed from partially reduced oxygen, damaging cell components
• Different microbes have varying oxygen requirements:
  • Obligate aerobes: Require high amount of oxygen
  • Facultative anaerobes: Grow with or without oxygen, like E. coli
  • Aerotolerant anaerobes: Do not use oxygen but are not harmed by its presence
  • Microaerophiles: Need low oxygen levels
  • Obligate anaerobes: Oxygen is lethal and only grow in its absence

Anaerobic Conditions and Obligate Anaerobes

• Anaerobic conditions exist in poorly vascularized tissues, ideal for obligate anaerobes like Clostridium spp. that form endospores
• C. difficile causes healthcare-related infections, often triggered by antibiotics
• C. tetani and C. perfringens cause severe infections in necrotic tissues
• Obligate anaerobes are studied in labs using anaerobic jars with chemical packets to absorb oxygen and release carbon dioxide, or in anaerobic chambers

Enzymes and ROS

• Enzymes reduce the toxicity of reactive oxygen species (ROS)
• Superoxide dismutase (SOD) breaks down superoxide anions
• Peroxidase reduces hydrogen peroxide to water
• Catalase converts hydrogen peroxide into water and oxygen, protecting cells
• Catalase tests differentiate bacteria based on their ability to break down hydrogen peroxide

Microbial Growth and pH

• Neutrophiles grow optimally around pH 7, which is where most bacteria are classified
• Acidophiles grow best around pH 3
• Alkaliphiles prefer pH levels above 9
• Extreme pH bacteria thrive in extremely acidic environments

Other Environmental Conditions Affecting Growth

• Halophiles thrive in high salt concentrations
• Halotolerant organisms survive and reproduce in elevated salt levels but don't require high salt
• Barophiles require high pressure for growth
• Phototrophs need light for growth, such as cyanobacteria, green sulfur bacteria, and purple nonsulfur bacteria
  • Photoautotrophs use light to drive carbon fixation
  • Photoheterotrophs use light for energy but also require organic carbon sources

Microbial Growth and Temperature

• Low temperatures slow microbial growth
• High temperatures increase the reaction rate
• Extreme temperatures will denature proteins
• Psychrophiles thrive in cold temperatures
• Mesophiles prefer moderate temperatures
• Thermophiles thrive in heat
• Hyperthermophiles survive in extreme heat

Taq Polymerase

• Taq Polymerase is a heat-resistant enzyme used in PCR

Nutritional Requirements

• Enriched media supports fastidious organisms
• Complex media contains digests/extracts with an imprecise composition
• Selective media suppress unwanted microbes while promoting target growth
• Chemically defined media has a precise composition
• Differential media distinguishes microbes by color changes

Eukaryotes

• Have Mitochondrion and Chloroplasts
• Microscopic forms of life
• Malaria caused by eukaryotic organism Plasmodium transmitted by mosquitoes
• Malaria leads to morbidity (illness) and mortality (death)

Unicellular Eukaryotic Parasites

• Microeukaryotes are diverse with complex lifecycles, unique morphology, and specific nutritional needs
• Protists are a broad eukaryotic group of single-celled organisms lacking specialized tissues
• Protist characteristics include multicellularity and feeding on small particulate food sources like bacteria
• Protists also:
  • Produce cysts with a protective wall; reproduce asexually via budding, binary fission, or schizogony where the nucleus splits multiple times before the cell splits
  • ingest food through phagocytosis using a cytostome and discard the waste via cytoproct
  • Moves using flagella, cilia, or pseudopodia
• Protozoans are nonphotosynthetic, motile unicellular protists described as "nothing"
  • Protozoans do not perform photosynthesis and are always unicellular
  • Holozoic protozoans ingest food through phagocytosis
  • Saprozoic protozoans ingest small, soluble food molecules
  • Protozoa's unique organelles include contractile vacuoles for osmoregulation and kinetoplastids or hydrogenosomes
• Protophytes are plant-like organisms, mostly simple algae that conduct photosynthesis and can be unicellular or multicellular
• Parasitism is when one organism benefits to the detriment of another
• Plankton are microorganisms that drift in water

Fungi

• Most fungi are opportunistic,but opportunistic, most are non-pathogenic
• Fungi act as decomposers in the environment
  • Cellulases degrade cellulose
  • Xylanases degrade xylan
  • Chitinases degrade chitin
• They are a source of antibiotics like penicillin
• Fungi can be dimorphic or polymorphic, appearing differently based on the environment
  • H.capsulatum causes lung disease histoplasmosis
  • C.albicans is related to vaginal yeast
  • Fungi contain chitin in their cell wall and ergosterol in their membrane, the later makes them good targets for anti-fungal medication
• Fungi are either haploid or diploid
• Asexual reproduction occurs through mitosis, budding, or fragmentation
• Fungi classification:
  • Zygomycota
  • Ascomycota includes edible mushroom fungi that cause food spoilage
    • Coccidoides immitis cause the lung disease "Valley Fever"
  • Basidiomycota has clubbed structures, a large group and important decomposers

Vertical and Horizontal Inheritance

• Vertical inheritance is parent to offspring
• Horizontal inheritance is between organisms

Discoveries of the Secrets of Life

• Acetabularia is a single-celled photosynthetic organism
• Hammerling found genetic information is needed for regeneration
• Griffith experiment showed a heat-killed S strain combined with live R strain killed mice
  • Injecting the S strain killed mice
  • A combined heat & live R strain kills the mouse
• Created the one gene – one enzyme hypothesis
  • Beadle and Tatum experiment – each gene is responsible for making a specific enzyme needed for life

DNA (deoxyribonucleic acid)

• double stranded molecule of nucleotides
• DNA has deoxyribose, which lacks -OH at the 2′Carbon
  • Purines are two ringed (Adenine and Guanine)
  • Pyrimidines are one ringed (Cytosine and Thymine) -A and T use 2 Hydrogen bonds -G and C use 3 Hydrogen bonds Read from 5’ end (phosphate group) to the 3’ end (hydroxyl group) Is a double helix structure Sugar phosphate backbones on the outside Nitrogenous bases form the rungs of the ladder RNA has a ribose (has OH at 2’) -RNA has uracil instead of thymine Nucleoside equals nitrogenous base + sugar Nucleotide equals nucleoside + phosphate DNA equals the material Chromosome defines an essential DNA molecule for replication Plasmid defines an accessory DNA that can replicate independently Gene defines a functional DNA region that codes for RNA/protein transcript Transcript (RNA): RNA made from DNA during transcription Protein: product of translation of RNA DNA gets transcribed RNA gets translated

DNA and RNA Bonds

• H-bonds between ds-DNA (denature) and ss-DNA (renaturation) -Double helix can be denatured to single stranded DNA through exposure of heat or chemicals, and then renatured cooling mRNA is translated into protein rRNA and tRNA are transcribed but not translated rRNA and tRNA are essential components of the protein synthesis machinery

Genetic Material

• Phenotype is defined as physical traits observed from gene expression
• Complete set of genes
• Gene regulation allows the production of different phenotypes based on environmental conditions. DNA (transcription) RNA (translation) Protein DNA Replication: dNTPs are the building blocks of the DNA

DNA Building Blocks

• 3’ hydroxyl group introduces a new nucleotide Four types of dNTPs
  • dATP, dTTP, dCTP, dGTP dNTPs work in replication by releasing pyrophosphate, creating a strong sugar phosphate bond
  • Replication starts at A/T pairs because they are easier to separate
  • Topoisomerase II uncoils, Helicase separates the strands, and Single stranded binding proteins keep strands separated Primase adds an RNA primer; DNA Polymerase III adds dNTPS to the 3’OH end, making a new 5’ to 3’ strand
    • DNA Pol 3 keeps moving along continuously to the leading strand
    • Okazaki fragments on lagging strand means it nees multiple new RNA primer

The Enzymes

Okazaki fragments where it needs new RNA primer, the DNA Polymerase I replaces DNA polymerase 3 when it reaches an RNA primer and removes it and fills the gap with DNA on the lagging strand Essentially DNA Ligase connects the Okazaki fragments by creating phosphodiester binds between them to complete the lagging strand -Topoisomerase untangles -Helicase unwinds strand -SSB Proteins keep DNA open -Primase lays down primer -DNA Pol III main builder -DNA Pol I cleaner -DNA Ligase glue everything together

RNA Transcription

• A gene codes for RNA RNA Polymerase enzyme reads DNA is Transcription 10 and 35 Boxes: Conserved sequences in bacterial promoters
• Messenger RNA carries genetic instructions from DNA tRNA:brings amino acids to ribosomes for protein synthesis ribosomes form peptide bonds to create protein

Translation

coding sequence: part of RNA that codes for a protein

• Ribosome attaches to the ribosome binding site to start Ribosome then searches downstream for the first AUG codon where it starts making protein Ribosome reads the RNA 3 letters at a time; codon Each codon codes for one amino acid or tells the ribosome to stop Ribosome reads them one after another, in succession

Coding (DNA) Sequence

• Code for protein Translation ends when ribosome hits the stop codon
• Release factors help complete the process ribosome separate mRNA released protein complete
• Translation:cytoplasm for both prokaryotes and eukaryotes

Mutations

Mutants can exist every where point mutation and change occur

• Silent mutation is when a change in nucleotide doesn’t do anything missense mutation occurs when results in the change of aminio acitids nonsense mutation: causes a stop codon. insertion/deletion: shifting the reading frame Spontaneous mutation is when DNA polymerase messes up. induced is when chemicals and radiation cause it Mutations cause disease, cancer, death mutagens will make typos

Effects of Mutations

Most of the mutation is neutral mutations become problematic beneficial mutants become stronger detrimental mutants get eliminated/weaker neutral mutants remain Auxotrphic mutants cannot recycle because need essentail nutrients Primary mutations cause an orginal problem revertions fix the problem Ames test tests what kind of mutations

Prokaryotes + Genetic Diversity

Vertical inhereitance is to the offspring by binary fission Horizontal Gene Transfer DNA from one bacteria cells transfer to each other Transformation by Bacteria picking up DNA from the environment Transtransduction viruses enter and pick up each others genes Conjugation 2 bacteria connect and pass over with pili

Genetics

Vertical:parent cells divide by transferring them over Horizontal is fast transfer Naked DMA will transfrom transduction is exchange in the virus with a Plasmid high frequency fragmented DNA recipent cells

Operon theory

Promoter controls gene to start being copied transcription starts with RNA Polymerase Structural Genes are set to transcirbe and code/ make prottein Contstructive cells always need protein. Genes will only use them when required opern will breaks down Lactose lacZ, lacy,lac are genes that help digest lactose

• With lactose present= lactose binds to Laci and prevents it from binding to DNA
• RNA polymersase will can and digest Lactose Cell knows if glucose is low and AT p is high and CAMP is low

Operon

Without the needed substrate= repressor protein binds to operator and doesnt help transcription happen with needed subsatnce/ inducer- bonds to repressror from operator.

• why= quick to adapt, saves energy, and adapt to changing conditions.

Acelluar Pathogens

virus and cells are replicated virus =genetic prion =infecrtios protiens that cause problems

Virus

can be seen with light micro cope, identifed with tvm all life forms cell alters direct contact indirect animals and ticks capsude =protection capsomere indeiflaide protein for protections, that contrtacs

Viral structure

sheath for bacteriapahej to inject nakes vsis = core/rna spike, glycopetine attahc envelope with lipids bacteriopaehs infext bacteria

Classaficitation of Virus

shapes and modes of inefcton and diesese mechanism Baltmarioes class of virus genera mRNA from HIV that retotr

Viral lifye cycles

lytic (viulent attach hots penetration rna and dna. the machinery ias maturation assembles virus lysis releases lysiogenic

the viral enetege into a prophgae enviormenntal causes = inducing lityccycles transfdutons

life cycle in eurotkic= endocytosis virus enter and integrates with rna tissue= spefici issues Flu hemagluttin key entry. neuraidines H5N = poss risks and mutation

hiv like tempetures and reverse with in unlike bacter a ecliple, butser and plaltue