Microbial Growth

Binary Fission

Binary fission is the method by which prokaryotes produce new individuals that are genetically identical to the parent organism.

Learning Objectives

Describe the process of binary fission in prokaryotes

Key Takeaways

Key Points

  • In bacterial replication, the DNA is attached to the plasma membrane at about the midpoint of the cell.
  • The origin, or starting point of bacterial replication, is close to the binding site of the DNA to the plasma membrane.
  • Replication of the bacterial DNA is bidirectional, which means it moves away from the origin on both strands simultaneously.
  • The formation of the FtsZ ring, a ring composed of repeating units of protein, triggers the accumulation of other proteins that work together to acquire and bring new membrane and cell wall materials to the site.
  • When new cell walls are in place, due to the formation of a septum, the daughter cells separate to form individual cells.

Key Terms

  • mitotic spindle: the apparatus that orchestrates the movement of DNA during mitosis
  • karyokinesis: (mitosis) the first portion of mitotic phase where division of the cell nucleus takes place
  • binary fission: the process whereby a cell divides asexually to produce two daughter cells

Binary Fission

Prokaryotes, such as bacteria, propagate by binary fission. For unicellular organisms, cell division is the only method used to produce new individuals. In both prokaryotic and eukaryotic cells, the outcome of cell reproduction is a pair of daughter cells that are genetically identical to the parent cell. In unicellular organisms, daughter cells are individuals.

Due to the relative simplicity of the prokaryotes, the cell division process, or binary fission, is a less complicated and much more rapid process than cell division in eukaryotes. The single, circular DNA chromosome of bacteria is not enclosed in a nucleus, but instead occupies a specific location, the nucleoid, within the cell. Although the DNA of the nucleoid is associated with proteins that aid in packaging the molecule into a compact size, there are no histone proteins and thus, no nucleosomes in prokaryotes. The packing proteins of bacteria are, however, related to the cohesin and condensin proteins involved in the chromosome compaction of eukaryotes.

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Binary Fission: These images show the steps of binary fission in prokaryotes.

The bacterial chromosome is attached to the plasma membrane at about the midpoint of the cell. The starting point of replication, the origin, is close to the binding site of the chromosome at the plasma membrane. Replication of the DNA is bidirectional, moving away from the origin on both strands of the loop simultaneously. As the new double strands are formed, each origin point moves away from the cell wall attachment toward the opposite ends of the cell. As the cell elongates, the growing membrane aids in the transport of the chromosomes. After the chromosomes have cleared the midpoint of the elongated cell, cytoplasmic separation begins. The formation of a ring composed of repeating units of a protein, FtsZ, directs the partition between the nucleoids. Formation of the FtsZ ring triggers the accumulation of other proteins that work together to recruit new membrane and cell wall materials to the site. A septum is formed between the nucleoids, extending gradually from the periphery toward the center of the cell. When the new cell walls are in place, the daughter cells separate.

Mitotic Spindle Apparatus

The precise timing and formation of the mitotic spindle is critical to the success of eukaryotic cell division. Prokaryotic cells, on the other hand, do not undergo karyokinesis and, therefore, have no need for a mitotic spindle. However, the FtsZ protein that plays such a vital role in prokaryotic cytokinesis is structurally and functionally very similar to tubulin, the building block of the microtubules that make up the mitotic spindle fibers that are necessary for eukaryotes. FtsZ proteins can form filaments, rings, and other three-dimensional structures that resemble the way tubulin forms microtubules, centrioles, and various cytoskeletal components. In addition, both FtsZ and tubulin employ the same energy source, GTP (guanosine triphosphate), to rapidly assemble and disassemble complex structures.

FtsZ and tubulin are homologous structures derived from common evolutionary origins. In this example, FtsZ is the ancestor protein to tubulin (a modern protein). While both proteins are found in extant organisms, tubulin function has evolved and diversified tremendously since evolving from its FtsZ prokaryotic origin. A survey of mitotic assembly components found in present-day unicellular eukaryotes reveals crucial intermediary steps to the complex membrane-enclosed genomes of multicellular eukaryotes.

Fts Proteins and Cell Division

FtsZ is a protein encoded by the ftsZ gene that assembles into a ring at the future site of the septum of bacterial cell division.

Learning Objectives

Evaluate the role of Fts proteins in cell division

Key Takeaways

Key Points

  • FtsZ has been named after “Filamenting temperature-sensitive mutant Z”.
  • During cell division, FtsZ is the first protein to move to the division site, and is essential for recruiting other proteins that produce a new cell wall between the dividing cells.
  • FtsZ’s role in cell division is analogous to that of actin in eukaryotic cell division, but unlike the actin-myosin ring in eukaryotes, FtsZ has no known motor protein associated with it.

Key Terms

  • segrosomes: multiprotein complexes that partition chromosomes/plasmids in bacteria.
  • cell division: a process by which a cell divides into two cells.
  • septum: a partition that separates the cells of a (septated) fungus
  • cytoskeleton: A cellular structure like a skeleton, contained within the cytoplasm.

FtsZ is a protein encoded by the ftsZ gene that assembles into a ring at the future site of the septum of bacterial cell division. This is a prokaryotic homologue to the eukaryotic protein tubulin. FtsZ has been named after “Filamenting temperature-sensitive mutant Z”. The hypothesis was that cell division mutants of E. coli would grow as filaments due to the inability of the daughter cells to separate from one another.

FtsZ was the first protein of the prokaryotic cytoskeleton to be identified. During cell division, FtsZ is the first protein to move to the division site, and is essential for recruiting other proteins that produce a new cell wall between the dividing cells. FtsZ’s role in cell division is analogous to that of actin in eukaryotic cell division, but unlike the actin-myosin ring in eukaryotes, FtsZ has no known motor protein associated with it. The origin of the cytokinetic force thus remains unclear, but it is believed that the localized synthesis of new cell wall produces at least part of this force. It is interesting to note that L-form bacteria that lack a cell wall do not require FtsZ for division, which implies that bacteria may have retained components of an ancestral mode of cell division.

Much is known about the dynamic polymerization activities of tubulin and microtubules, but little is known about these activities in FtsZ. While it is known that single-stranded tubulin protofilaments form into 13 stranded microtubules, the multistranded structure of the FtsZ-containing Z-ring is not known. It is only speculated that the structure consists of overlapping protofilaments. Recently, proteins similar to tubulin and FtsZ have been discovered in large plasmids found in Bacillus species. They are believed to function as components of segrosomes, which are multiprotein complexes that partition chromosomes/plasmids in bacteria. The plasmid homologs of tubulin/FtsZ seem to have conserved the ability to polymerize into filaments.

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FtsZ Filaments: The Z-ring forms from smaller subunits of FtsZ filaments. These filaments may pull on each other and tighten to divide the cell.

FtsZ has the ability to bind to GTP, and also exhibits a GTPase domain that allows it to hydrolyze GTP to GDP and a phosphate group. In vivo, FtsZ forms filaments with a repeating arrangement of subunits, all arranged head-to-tail. These filaments form a ring around the longitudinal midpoint, or septum, of the cell. This ring is called the Z-ring. The GTP hydrolyzing activity of the protein is not essential to the formation of filaments or division. Mutants lacking the GTPase domain form twisted and disordered septa. These cells with irregular septa can still divide, although abnormally. It is unclear as to whether FtsZ actually provides the physical force that results in division or serves as a marker for other proteins to execute division.

The Z-ring forms from smaller subunits of FtsZ filaments. These filaments may pull on each other and tighten to divide the cell. If FtsZ does provide force that divides the cell, it may do so through the relative movement of subunits. In this model, FtsZ scission force comes from the relative lateral movement of subunits. Lines of FtsZ would line up together parallel and pull on each other creating a “cord” of many strings that tightens itself. In other models, FtsZ does not provide the contractile force but provides the cell a spatial scaffold for other proteins to execute the division of the cell. This is akin to the creating of a temporary structure by construction workers to access hard-to-reach places of a building. The temporary structure allows unfettered access and ensures that the workers can reach all places. If the temporary structure is not correctly built, the workers will not be able to reach certain places, and the building will be deficient.

This “scaffold theory” is supported by information that shows that the formation of the ring and localization to the membrane requires the concerted action of a number of accessory proteins. ZipA or the actin homologue FtsA permit initial FtsZ localization to the membrane. Following localization to the membrane, division proteins of the Fts family are recruited for ring assembly. Many of these proteins, such as FtsW, FtsK, and FtsQ are involved in stabilization of the Z ring and may also be active participants in the scission event. The formation of the Z-ring closely coincides with cellular processes associated with replication.

MreB and Determinants of Cell Morphology

MreB is a protein found in bacteria homologous to actin.

Learning Objectives

Explain the role of MreB in cell morphology determination

Key Takeaways

Key Points

  • MreB proteins polymerize to form filaments that are similar to actin microfilaments.
  • MreB controls the width of rod-shaped bacteria, such as Escherichia coli.
  • Bacteria that are naturally spherical do not have the gene encoding MreB.

Key Terms

  • peptidoglycan: A polymer of glycan and peptides found in bacterial cell walls.
  • cell wall: A thick, fairly rigid layer formed around individual cells of bacteria, Archaea, fungi, plants, and algae, the cell wall is external to the cell membrane and helps the cell maintain its shape and avoid damage.
  • cytoskeleton: A cellular structure like a skeleton, contained within the cytoplasm.
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Atomic structure of MreB, a prokaryotic structural protein: Procaryotic MreB in cartoon representation. The fold of the protein is similar to its eukaryotic counterpart

MreB is a protein found in bacteria that has been identified as a homologue of actin, as indicated by similarities in tertiary structure and conservation of active site peptide sequence. The conservation of protein structure suggests the common ancestry of the cytoskeletal elements formed by actin and MreB, found in prokaryotes. Indeed, recent studies have found that MreB proteins polymerize to form filaments that are similar to actin microfilaments.MreB controls the width of rod-shaped bacteria, such as Escherichia coli. A mutant E. coli that creates defective MreB proteins will be spherical instead of rod-like. Also, bacteria that are naturally spherical do not have the gene encoding MreB. Prokaryotes carrying the mreB gene can also be helical in shape. MreB has long been thought to form a helical filament underneath the cytoplasmic membrane. However, this model has been brought into question by three recent publications showing that filaments cannot be seen by electron cryotomography and that GFP-MreB can be seen as patches moving around the cell circumference. It has also been shown to interact with several proteins that are proven to be involved in length growth (for instance PBP2). Therefore, MreB probably directs the synthesis and insertion of new peptidoglycan building units into the existing peptidoglycan layer to allow length growth of the bacteria.

MreB is a cytoskeleton element that assembles into filamentous structures within the bacterial cytoplasm. MreB and its homologs have been shown to interact and co-localize with cytoplasmic protein( MurB-G), membrane-imbedded proteins ( MreD, MraY and RodA), as well as other molecules with large periplasmic domain in organism. Recent research shows that peptidoglycan precursors are inserted into cell wall following helical pattern which is dependent on MreB, and it’s reported that MreB also promote the GT activity of PBPs. This ability of MreB is because of RodZ, an inner membrane protein containing an 80-residue, N-terminal cytoplasmic region, and a 200-amino acid periplasmic C-terminal tail. RodZ co-localizes with MreB helices in a manner that is strictly dependent on its cytoplasmic region. MreB- RodZ complexes act as a major stabilizing factor in bacterial cell wall and ensure the insertion of new peptidoglycan in a spiral like fashion into the cell wall.

Peptidoglycan Synthesis and Cell Division

Peptidoglycan, also known as murein, is a polymer and consists of sugars and amino acids which form the cell walls of bacteria.

Learning Objectives

Examine Peptidoglycan synthesis during cell division

Key Takeaways

Key Points

  • The sugar component of peptidoglycan consists of alternating residues of β-(1,4) linked N-acetylglucosamine and N-acetylmuramic acid.
  • Peptidoglycan serves a structural role in the bacterial cell wall, giving structural strength but not shape, and counteracting the osmotic pressure of the cytoplasm.
  • Peptidoglycan is also involved in binary fission during bacterial cell reproduction.

Key Terms

  • peptidoglycan: A polymer of glycan and peptides found in bacterial cell walls.
  • MreB: MreB is a protein found in bacteria that has been identified as a homologue of actin, as indicated by similarities in tertiary structure and conservation of active site peptide sequence.

Peptidoglycan, also known as murein, is a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of bacteria (but not Archaea; []), forming the cell wall. The sugar component consists of alternating residues of β-(1,4) linked N-acetylglucosamine and N-acetylmuramic acid. Attached to the N-acetylmuramic acid is a peptide chain of three to five amino acids. The peptide chain can be cross-linked to the peptide chain of another strand forming the 3D mesh-like layer. Some Archaea have a similar layer of pseudopeptidoglycan or pseudomurein, where the sugar residues are β-(1,3) linked N-acetylglucosamine and N-acetyltalosaminuronic acid. That is why the cell wall of Archaea is insensitive to lysozymes, which are present in human sweat and tears as part of innate immunity.

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Simplified sc hematic of a cell wall in a Gram-positive bacteria: Cross-linking between amino acids in different linear amino sugar chains occurs with the help of the enzyme transpeptidase and results in a 3-dimensional structure that is strong and rigid.

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Peptidoglycan structure: The peptidoglycan layer in the bacterial cell wall is a crystal lattice structure formed from linear chains of two alternating amino sugars, namely N-acetylglucosamine (GlcNAc or NAG) and N-acetylmuramic acid (MurNAc or NAM).

Peptidoglycan serves a structural role in the bacterial cell wall giving it strength, as well as counteracting the osmotic pressure of the cytoplasm. A common misconception is that peptidoglycan gives the cell its shape. However, it is actually the MreB protein that facilitates cell shape. Peptidoglycan is also involved in binary fission during bacterial cell reproduction.

The peptidoglycan layer is substantially thicker in Gram-positive bacteria (20 to 80 nanometers) than in Gram-negative bacteria (7 to 8 nanometers), with the attachment of the S-layer. Peptidoglycan forms around 90% of the dry weight of Gram-positive bacteria but only 10% of Gram-negative strains. Thus, presence of high levels of peptidoglycan is the primary determinant of the characterisation of bacteria as gram-positive. In Gram-positive strains, it is important in attachment roles and stereotyping purposes. For both Gram-positive and Gram-negative bacteria, particles of approximately 2 nm can pass through the peptidoglycan. Gram-positive and Gram-negative bacteria are sensitive to different types of antiobiotics.

Generation Time

Bacterial growth occurs by the division of one bacterium into two daughter cells in a process called binary fission.

Learning Objectives

Examine microbial generation times

Key Takeaways

Key Points

  • The doubling time is the generation time of the bacteria.
  • The measurement of an exponential bacterial growth curve can be done by cell counting, colony counting, or determining the turbidity of bacterial cultures.
  • Bacterial growth in batch culture can be modeled with four different phases: lag phase, exponential or log phase, stationary phase, and death phase.

Key Terms

  • bacterium: A single celled organism with no nucleus.
  • bacterial growth: Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission.
  • doubling time: The doubling time is the period of time required for a quantity to double in size or value. It is applied to population growth, inflation, resource extraction, consumption of goods, compound interest, the volume of malignant tumours, and many other things which tend to grow over time.
  • lag phase: the period of bacterial growth in which bacteria adapt themselves to growth conditions; the individual bacteria are maturing and not yet able to divide

Bacterial growth is the division of one bacterium into two daughter cells in a process called binary fission. Providing no mutational event occurs the resulting daughter cells are genetically identical to the original cell. Therefore, “local doubling” of the bacterial population occurs. Both daughter cells from the division do not necessarily survive. The doubling time is the generation time of the bacteria. If the number surviving exceeds unity on average, the bacterial population undergoes exponential growth.

The measurement of an exponential bacterial growth curve in batch culture was traditionally a part of the training of all microbiologists. The basic means requires bacterial enumeration (cell counting) by direct and individual (microscopic, flow cytometry), direct and bulk (biomass), indirect and individual (colony counting), or indirect and bulk (most probable number, turbidity, nutrient uptake) methods. In autecological studies, bacterial growth in batch culture can be modeled with four different phases: lag phase, exponential or log phase, stationary phase, and death phase.

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Bacterial Growth Curve: This chart shows the logarithmic growth of bacteria. Note the Y-axis scale is logarithmic meaning that the number represents doubling. The phases of growth are labelled on top.

During lag phase, bacteria adapt themselves to growth conditions. It is the period where the individual bacteria are maturing and not yet able to divide. During this phase of the bacterial growth cycle, synthesis of RNA, enzymes, and other molecules occurs. The exponential phase (sometimes called the log phase or the logarithmic phase) is a period characterized by cell doubling. The number of new bacteria appearing per unit time is proportional to the present population. If growth is not limited, doubling will continue at a constant rate so both the number of cells and the rate of population increase doubles with each consecutive time period. For this type of exponential growth, plotting the natural logarithm of cell number against time produces a straight line. The slope of this line is the specific growth rate of the organism, which is a measure of the number of divisions per cell per unit time.

The actual rate of this growth (i.e. the slope of the line in the figure) depends upon the growth conditions, which affect the frequency of cell division events and the probability of both daughter cells surviving. However, exponential growth cannot continue indefinitely because the medium is soon depleted of nutrients and enriched with wastes. Finally, the stationary phase is due to a growth-limiting factor, such as depletion of a nutrient and/or the formation of inhibitory products such as organic acids. Death of cells as a function of time is rather unpredictable and very difficult to explain. At death phase, bacteria run out of nutrients and die. This basic batch culture growth model draws out and emphasizes aspects of bacterial growth which may differ from the growth of macrofauna. It emphasizes clonality, asexual binary division, the short development time relative to replication itself, the seemingly low death rate, the need to move from a dormant state to a reproductive state or to condition the media, and finally, the tendency of lab adapted strains to exhaust their nutrients. In reality, even in batch culture, the four phases are not well defined.