Microbial and Bacterial Cell Growth

Microbial Growth

• Microbial growth marks a cell's life process, essential for species survival through the multiplication of cells.
• An understanding of microbial growth is key to controlling it, influencing disease treatment and sterilization techniques.
• Studying microbial growth in bacteria provides insights applicable to cell division in higher organisms.
• Growth relies on energy transformation, synthesis of building block molecules, and enzymatic reactions.
• Polymerization is a key reaction where macromolecules are made from monomers.
• Newly synthesized macromolecules form structures like cell walls, membranes, flagella, and ribosomes.

Bacterial Cell Division Process

• Growing rod-shaped cells elongate and divide into two via binary fission.
• Division involves septum formation, where the cell membrane and wall grow inward, eventually pinching off two daughter cells.
• Variations exist like septum formation without cell wall constriction in Bacillus subtilis, or constriction without septum in Caulobacter.
• The period for a cell to separate into two is known as generation time.
• Balanced growth occurs when all cellular components increase proportionally during a generation.
• Each daughter cell inherits a chromosome, ribosomes, and essential molecules.
• DNA partitioning involves chromosomal attachment to the cytoplasmic membrane.
• Constriction leads to chromosome separation.
• Generation time varies among species based on nutritional, genetic, and temperature factors.
• E. coli has a generation time of ≈20min under optimal conditions.
• Natural microbial growth is slower than maximum rates due to varied conditions and resource availability.

Fts proteins

• Fts proteins are vital for cell division across all Bacteria.
• Fts stands for Filamentous Temperature Sensitive, describing a mutant phenotype of long cells that cannot divide.
• FtsZ is an essential Fts protein well-studied in E. coli.
• FtsZ is found in all prokaryotes and in eukaryotic mitochondria and chloroplasts.
• FtsZ is related to tubulin in eukaryotes.

Fts Proteins and Cell Division within Bacteria,

• Fts proteins construct a cell-division apparatus termed the divisome.
• In rod-shaped cells, FtsZ molecules gather at the cell's center, marking the division site.
• In E. coli, ≈10,000 FtsZ molecules form a ring that recruits FtsA and ZipA.
• ZipA acts as an anchor to connect and stabilize the FtsZ ring with the cytoplasmic membrane.
• FtsA, related to actin, links the FtsZ ring to the membrane and recruits other proteins.
• The divisome forms after cell elongation and DNA replication.
• The peptidoglycan synthesis protein FtsI is present in the divisome.
• Penicillin-binding proteins, including FtsI, are inhibited by penicillin.
• The divisome coordinates the synthesis of new cytoplasmic membrane and cell wall for the division septum.

Factors Influencing FtsZ Ring Assembly

• Min proteins (MinC, MinD, MinE) aid in the division, especially MinD forms a spiral on the inner membrane and oscillates.
• MinD aids localization of MinC, which inhibits FtsZ ring formation.
• MinE sweeps MinC and D aside as it oscillates, resulting in lower MinCD concentration at the cell center.
• Consequently, the cell center becomes the favorable site for FtsZ ring assembly and division plane definition.
• FtsK and other proteins help separate chromosomes during cell constriction.

FtsZ Ring Dynamics and Clinical Relevance

• The FtsZ ring disassembles during cell constriction, which induce cell wall material growth to form the septum.
• GTP hydrolysis by FtsZ provides energy for polymerization and depolymerization.
• Detailed bacterial division knowledge may lead to new antibacterial drugs.

Cell Shape Determination

• Proteins dictate cell shape in prokaryotes, sharing similarities with eukaryotic cytoskeletal proteins.
• Prokaryotes possess a dynamic and multifaceted cytoskeleton.
• The MreB protein is an actin analog that determines cell shape in prokaryotes.
• MreB forms spiral bands inside the cell, contacting the cytoplasmic membrane.
• The MreB cytoskeleton recruits proteins for specific cell wall growth patterns.
• Bacteria missing the gene for MreB become coccoid.
• MreB aids chromosome segregation and Par proteins, mimicking eukaryotic mitotic apparatus, separate chromosomes.

MreB Mechanism and Crescentin Function

• MreB defines cell shape by localizing peptidoglycan synthesis to specific points.
• New cell wall material forms at MreB helix contact points.
• Cell wall synthesis occurs at multiple points along the long axis of a rod-shaped cell, guided by MreB.
• Caulobacter crescentus uses crescentin, along with MreB, to produce its vibrio shape.
• Crescentin organizes into filaments on the concave side of the curved cell which contributes to their morphology.

Parallels in cell division and cell shape between prokaryotes and eukaryotes

• Parallels exist in division and morphology between prokaryotes and eukaryotes.
• MreB relates to eukaryotic actin (microfilaments) and FtsZ relates to tubulin (microtubules).
• Caulobacter crescentin relates to eukaryotic keratin.
• Most eukaryotic cell division and shape proteins originate from prokaryotes.

Peptidoglycan Synthesis in Cell Division

• Cell division needs the production of new cell wall material.
• Cell walls grow outward from the FtsZ ring in cocci and along the cell length in rods.
• Preexisting peptidoglycan must be cut before new peptidoglycan insertion.
• Autolysins create small wall gaps at the FtsZ ring, by breaking glycosidic bonds.
• New wall material adds across gaps.
• The junction generates a wall band in gram-positive bacteria.

Disrupting Peptidoglycan Synthesis

• Failing to coordinate peptidoglycan splicing can cause a breach in cell wall integrity, leading to autolysis and cell lysis.
• Synthesis involves precursors called N-acetylmuramic acid/N-acetylglucosamine/tetrapeptide units.
• Peptidoglycan is a stress-bearing layer.
• New peptidoglycan needs controlled cutting of old peptidoglycan via autolysins alongside the insertion of precursors
• Bactoprenol, a lipid carrier molecule, is vital to this process.
• The total cell wall cell synthesis is transpeptidation.
• Transpeptidation: peptide cross-links between muramic acid residues.
• Gram-negative bacteria like E. coli form cross-links between diaminopimelic acid (DAP) on one peptide and D-alanine on the other.
• The transpeptidation is necessary, supplying energy for reaction outside the cytoplasm.

Transpeptidation medically relevant

• Transpeptidation inhibited by penicillin binding to penicillin-binding proteins.
• Penicillin's success is its nontoxicity to humans and ability to target peptidoglycan in bacteria.
• Overuse of penicillin causes resistant mutants with catalytically active penicillin-binding proteins that no longer bind penicillin.

Population Growth Basics

• Microbial growth involves increasing cell numbers over a lifespan.
• Exponential growth doubles cell numbers in a given time, called the generation time.
• Semilogarithmic plots are used to plot cells growing exponentially, it reflects a population doubling
• Semilogarithmic plots are also usable to estimate the generation time of a microbial culture.
• Generation times differ widely among organisms and are affected by medium and conditions.

Exponential Growth Explained

• Exponential growth is the doubling of cells at a constant time interval.
• Arithmetic plots of such populations yield curves with increasing slopes.
• Logarithmic plots yield straight lines.
• The equation to define that is N = No2n
• N is the final cell number and No is the initial one
• From both, you can derive n, the number of generations.
• The generation time (g=t/n,) of growth can be calculated
• Growth starts rather slow and ends up growing really rapidly.

Practical Implications of Exponential Growth

• Milk spoilage illustration: Bacteria contaminating fresh milk exponentially grows at different temperature, hence the difference in spoilage
• Microbe-count differences in milk two weeks apart will lead to taste differences.
• Semilogarithmic plots are used to work out generation rate etc.

Mathematics of Exponential Growth

• Expressing N = No2n logarithmically, we can better derive relationships of growth, as expressed by: log N - log No = n log 2
• Another such equation is n = 3.3 (log N - log No)
• When graphing, picking one doubling results in a generation time between X intercepts.
• Growth increases at a faster rate during exponential growth's later stages.

Additional Growth Metrics

• Specific growth rate: k = 0.301/g
• Division rate: v = 1/g
• The values can help when trying to control growth for a specific organism

Microbial Growth Cycle

• Batch cultures do not sustain exponential growth over extended periods.
• Instead, an entire growth cycle occurs encompassing exponential growth with other phases.
• Overall, the growth curve covers lag, exponential, stationary, and death phases.

Growth Phase Breakdown

• Inoculation into a fresh medium yields a period of adaptation called the lag phase.
• Lag time adapts to medium vs no lag if conditions are identical.
• Nutrient depletion or toxic buildup ends the rate, reaching a stationary state in what is called the stationary phase.
• Zero growth results no net increase or decrease, but continuing cell functions.
• Cryptic growth occurs when some cells grow, and others die, balancing each other.
• Constant incubation yields a death phase.
• Death is exponential but is slower than exponential growth
• Microbes need a good medium and condition to grow correctly.

Continuous Culture: The Chemostat

• Batch cultures are closed off so the composition and conditions change rapidly to create the environment the experimenter desires.
• Continuous culture maintains constant conditions.
• Its volume remains balanced via consistent media infusion/removal.
• Chemostats control rates depending on resource in vessel, creating a steady state

Chemostat Operation

• Two variables govern the rate, (1) dilution rate the spent medium removed, and, (2) the specific limiting nutrient concentration
• Affecting either the flow rate or nutrient has impacts as shown
• Too high of a rate causes a lack of growth or washout. Too low a rate causes starvation as its not fast enough to maintain cell rate

Chemostat Uses

• Allows you to maintain exponential growth, or to re-do experiments in the same conditions
• Can mimic ecology, so people use them combined with things like gene tracking to observe communities
• You can start with a real world thing then mess with the dilution rate until something emerges, using the chemostat for isolation purposes

Counting Measures

• There are two measure, either by change of cell number or by physical component (nucleic acids, protein etc)
• Or you can do cell counts, or turbidity, the mass of cells.

Optical measurement

• Optical density (OD) at a wave helps enumerate cells in sample
• 12 cells over so and so (see slide) means that the number is what you measure by area etc and convert it.