Lecture 8 - Bacterial Growth and Cell Division

Question 1

Growth of a bacterial population

Answer 1

LAG PHASE. Essentially a period of adjustment that follows the introduction of microbes into fresh culture medium. Length of lag phase depends on the history of the inoculum. For example, if the inoculum is from an exponentially-growing culture, there is no lag phase and exponential growth will occur immediately. However, if the inoculum is from a stationary phase culture those cells will be depleted of certain essential constituents, and time is needed for their biosynthesis before exponential growth can occur.

EXPONENTIAL PHASE. Typically when bacteria are in their ‘healthiest’ state. A rapid period of growth, the duration of which will depend on available nutrients. Rates of exponential growth vary greatly between different bacterial species – influenced by environmental conditions and genetics.

STATIONARY PHASE. Growth ultimately becomes limited as essential nutrients are used up and as microbial waste products accumulate in the environment. In stationary phase, there is no net increase or decrease in cell number (although many cellular functions do continue). Some cells may divide at stationary phase, but this is balanced by the fact that others die.

DEATH PHASE. Cells will eventually start dying if maintained in stationary phase. In some cases this is accompanied by actual cell lysis.

Question 2

Cell growth & binary fission

Answer 2

In a growing rod-shaped cell, elongation continues until cell division into two new cells (“binary fission”)

In the process of septation, a cross wall (the “septum”) is formed between the two daughter cells
Septation involves the inward growth of cytoplasmic membrane & cell wall from opposing directions

Under optimal conditions, the generation time of E. coli can be 20 minutes

Question 3

Coordinating DNA replication and cell division

Answer 3

Within the one cell cycle, approximately two-thirds of the time is required for DNA replication and partitioning of the chromosomes.

1. Initiation mass reached
2. Initiation of replication (replisome) (cell elongates)
3. Threshold cell length reached (chromosomes separate)
4. Initiation of septum formation (cells divide)

Question 4

Septation and the Z-ring

Answer 4

Septation can be divided into several steps:

1. Selection of the site where the septum will be formed
2. Assembly of the Z-ring, composed of the cytoskeletal protein FtsZ
3. Assembly of the cell wall synthesizing machinery
4. Constriction of the cell and septum formation

Assembly of the FtsZ-based Z-ring is critical for septation
FtsZ is found in the majority of prokaryotes, including Archaea
FtsZ is related to tubulin, the protein that polymerizes to form microtubules of the eukaryotic cytoskeleton
Like tubulin, FtsZ polymerizes to form the Z-ring

Question 5

Identifying the midpoint of the cell

Answer 5

In E. coli, the MinCDE system is responsible for localizing Z-ring formation
The MinC, MinD & MinE proteins form a complex that oscillates from one end of the cell to the other

The MinCDE complex inhibits formation of the Z-ring
As the MinCDE concentration is lowest at the midpoint of the cell, the midpoint is most permissive for Z-ring formation

Question 6

Visualizing assembly of the Z-ring

Answer 6

The Z-ring appears as the nucleoids start to segregate, with the full ring forming as the cell elongates

At cell division, the Z-ring depolymerizes, constricting the ring & triggering inward growth of wall material to form the septum

Question 7

The Z-ring and the divisome

Answer 7

Once the Z-ring forms, the rest of the division machinery is assembled
Referred to as the divisome

Fts, Filamentous Temperature Sensitive

Fts mutants fail to divide, instead forming long filamentous cells

Question 8

The divisome as an antimicrobial target

Answer 8

PC190723 – an FtsZ inhibitor

PC190723 exhibits bactericidal activity against staphylococci, including methicillin-resistant and multi-drug resistant strains
Confers protection in mice inoculated with lethal dose of S. aureus

Latest generation FtsZ inhibitors are moving towards clinical trials

Question 9

Determining cell morphology – role of MreB

Answer 9

MreB is an actin-like protein of bacteria, in which it forms a simple cytoskeleton that plays a role in determining shape

MreB forms spiral-shaped bands around the inside of the cell
Plays a role in recruitment of proteins required for determining cell shape & those involved in cell wall synthesis
Variations in MreB arrangement are believed to give rise to varying cellular morphologies of prokaryotic cells

Question 10

MreB protein, cell growth & morphology

Answer 10

Inactivation of MreB in rod-shaped bacteria causes the cell to become coccoid
Naturally coccoid bacteria lack MreB, indicating that the “default” shape for a bacterium is most likely coccoid

Question 11

Spatial organisation of peptidoglycan synthesis

Answer 11

The primary determinant of cell shape is the peptidoglycan exoskeleton found outside of the cytoplasmic membrane
Rod-shaped cells become spherical in the absence of this peptidoglycan layer
The shape of rod-shaped cells is dependent on enzymes responsible for longitudinal peptidoglycan synthesis

MreB organises peptidoglycan biosynthetic enzymes into a helical pattern that is oriented along the long axis of the cell

Question 12

Peptidoglycan synthesis & cell growth in cocci

Answer 12

In coccoid cells, the cell walls typically grow in opposite directions outward from the FtsZ ring

Irrespective of the differences between rod-shaped and coccoid bacteria, cell growth requires newly synthesized peptidoglycan precursors to be spliced into pre-existing peptidoglycan

The insertion of newly synthesized peptidoglycan precursors into pre-existing peptidoglycan must be carefully coordinated so as to avoid a breach in the integrity of the cell wall.

The “wall bands” shown above are ridges formed on the surface of Gram-positive bacteria at the junction between old and new peptidoglycan.

Question 13

Peptidoglycan synthesis & cell division

Answer 13

1. Autolysin activity
   Hydrolyzes the bonds that connect NAG and NAM in the peptidoglycan backbone
2. Transglycosylase activity
   Links old peptidoglycan with new
3. Transpeptidase activity
   The final step. Forms peptide cross-links between NAM residues in adjacent glycan chains.

FtsZ & MreB both play key roles in organising peptidoglycan biosynthetic enzymes

NAG = N-acetylglucosamine
NAM = N-acetylmuramic acid

Bactoprenol is a lipid carrier molecule that plays a critical role in the transport of peptidoglycan precursors across the cytoplasmic membrane so that they can be incorporated into the growing cell wall. The bactoprenol renders the precursors sufficiently hydrophobic to pass through the membrane interior. Once in the periplasm, the bactoprenol interacts with the transglycosylase enzymes that insert the cell wall precursors into the growing cell wall.

It is the transpeptidase reaction that is inhibited by penicillin antibiotics. The inhibition of the transpeptidase reaction whilst autolysin activity continues ultimately leads to catastrophic weakening of the bacterial cell wall and eventual lysis.

Question 14

Cell polarity and protein localization – how??

Principles of protein localization in bacteria

Answer 14

The localization of many proteins in bacteria is believed to occur by diffusion & capture

A protein is synthesized in one location and then diffuses (in cytoplasm or within membrane) until it recognizes and binds to a localized cue

The location of the cue dictates the ultimate location of the protein

Three distinct types of cue are recognised:
Geometrical cues
Lipids
Landmark proteins

Question 15

Geometrical cues for protein localization

Answer 15

Membrane curvature
The cytosolic side of the bacterial cytoplasmic membrane is concave, and has a negative curvature

However, in rod-shaped bacteria, curvature is not uniform
Curvature at cell poles and the division septum is approx. 2-fold higher than along the lateral sides

Both positive and negative curvature can dictate localization of proteins

Question 16

Membrane curvature & protein localization

Answer 16

Bacillus subtilis DivIVA protein
DivIVA intrinsically recognizes membranes of negative curvature
If we make Bacillus spherical, DivIVA becomes uniformly distributed
Recruits proteins involved in cell division (incl. the MinCDE complex), cell wall biosynthesis & chromosome segregation
DivIVA directly recognizes membranes of negative curvature and is highly conserved in Gram-positive bacteria. Membrane binding and curvature sensitivity appear to be intrinsic features of DivIVA, as it has been shown that DivIVA of Bacillus subtilis also localizes to curved membranes when expressed in other, nonrelated species, including yeast cells.

Bacillus subtilis SpoVM protein
In contrast, SpoVM protein recognizes positive curvature and is critical for spore formation in Bacillus
Discriminates between the positive curvature of the developing spore and the negative curvature of the cytoplasmic membrane of the mother cell
Sporulation involves an inner cell maturing into a spore and an outer cell nurturing the developing spore. SpoVM is produced in the outer cell, where it embeds in the membrane that surrounds the inner cell but not in the cytoplasmic membrane of the outer cell. SpoVM achieves this by discriminating between the positive curvature of the membrane surrounding the inner cell and the negative curvature of the cytoplasmic membrane.

Question 17

Lipids and protein localization

Answer 17

Lipids, that makeup lipid bilayers of membranes, comprise a hydrophilic head group attached to hydrophobic hydrocarbon chains (the tails)
Differing properties of lipid molecules (particularly the head-to-tail ratio) influence their localisation within curved membranes

Lipid head-to-tail ratio close to 1: Favours planar structures

Small lipid head-to-tail ratio (head smaller than the tail)
: Found in curved structures

Question 18

Lipids and protein localization

Answer 18

E. coli membranes consist of three main types of lipid:
~ 5% cardiolipin (CL)
20–25% phosphatidylglycerol (PG)
70–80% phosphatidylethanolamine (PE)

Cardiolipin has a small head-to-tail ratio and consequently localises to the curved poles, creating cardiolipins-rich domains

Certain proteins colocalize with cardiolipin, and thus localize to the poles

Question 19

Landmark proteins and cell polarity

Answer 19

Polarity is an intrinsic feature of bacterial cells

Cytokinesis in rod-shaped bacteria occurs in the middle, so daughter cells have an “old” pole and a “new” pole
The old poles are zones of inert peptidoglycan resulting from the absence of new synthesis

Similarly, the two hemispheres of coccoid cells have different ages

Proteins associated with the divisome complex will become localized at the “new” cell pole of the daughter cell
Referred to as landmark proteins (or “birth scars”)

Question 20

Cell polarity in Caulobacter crescentus

Answer 20

C. crescentus is dimorphic, existing as two cell types during its life cycle:
A sessile stalked cell that attaches to a substrate and does replicate
A motile non-replicating swarmer cell with a polar flagellum

C. crescentus exists in two cell types: a motile, DNA replication-quiescent ‘‘swarmer cell’’ and a sessile, DNA replication-competent ‘‘stalked cell’’; the
former is important for dispersion and the latter for reproduction.

During the cell cycle, the swarmer cell, which has a single flagellum and several pili at one cell pole, grows into a stalked cell. During this swarmer-to-stalked
cell transition, the flagellum and pili are lost, and a stalk, a thin extension of the cell body, elongates at the pole originally occupied by the flagellum.

The stalked cell then initiates chromosome replication and cell division.

During the predivisional stage, a new flagellum and a pilus secretion apparatus are assembled specifically at the opposite pole from the stalk (the new pole). This results in a morphologically asymmetric “predivisional cell” that has a flagellum at one pole and a stalk at the other. Subsequent cell division produces a swarmer cell with the polar flagella and a stalked cell.

With each division, the identity of the poles (new versus old) must be redefined to restore the polarity axis in the stalked progeny and to reverse it in the swarmer progeny.

Question 21

TipN acts as a landmark protein in Caulobacter

Answer 21

In wild-type cells, TipN at the new pole provides a positional cue to orient and maintain the correct polarity axis, which is important for polar morphogenesis (including the formation of the polar flagella) and for the correct placement of the division site.

The relocation of TipN to the division site in the late predivisional cell stage redefines the identity of the poles by marking the birth site of the future progeny’s new poles. Thus, TipN acts as a landmark from the previous division cycle to orient the polarity axis in the daughter cells. In the swarmer progeny, this results in a reversal of the polarity axis.

TipN mutants display morphological defects as they are unable to correctly assign the polarity axis.

It is predicted that correct localization of TipN depends on a protein complex called TolA-Pal. In turn, the correct localization of the TolA and Pal proteins is dependent on the FtsZ ring. Thus, it all comes back to the idea of ‘birth scars’ at the site of septation.

Question 22

Conclusions

Answer 22

Bacterial cell division is a tightly regulated process, dependent on the coordinated actions of numerous proteins that constitute the divisome
Components of the divisome are attractive antimicrobial targets

Polarity is an intrinsic feature of bacterial cells, enabling discrimination between ‘old’ and ‘new’ poles

The diffusion and capture process is central to the proper localization of proteins and protein complexes, and can be facilitated by geometrical, lipid and landmark protein cues