Bacterial Sporulation. Steps and Molecular Mechanism

Abstract.

Sporulation is a type of bacterial reaction towards stressful environmental conditions in order to survive. When bacteria face a nutrient-deficient environment, they start to form an endospore, a type of dormant cell that can live under extremely harsh conditions. Due to their high resistance properties, these spores are of high interest as they not only cause food contamination but also cause various diseases in the human body when they start to grow. Thus, in order to develop a specific mechanism to get rid of these spores and to provide a hygienic environment, it is necessary to understand all steps and mechanisms through which a spore is formed.

Introduction:

Sporulation of Gram-positive bacteria is a tightly controlled process that have several stages. Sporulation of Bacillus subtilis includes regulatory elements such as SpoOA (transcriptional regulator of sporulation initiation), sigma factors, and some other auxiliary regulators. A well-documented gene expression of sporulation for B. subtilis and for some Clostridia like (Pepto)Clostridium difficile. As a result of this sporulation morphogenesis, it leads to the formation of bacterial spores that are encased in two protective layers (cortex and coat).

Speaking about the life-span time of a spore, a spore can survive from 1000s up to millions of years, depending upon various factors such as the extent of compaction of chromosomal DNA and dehydration of the spore. These dormant cells can re-initiate the growth by a process known as germination. This process occurs as a result of environmental signals.
This spore formation phenomenon is somewhat different from the normal growing cells. In sporulation, an asymmetric division occurs of the cell near one of its poles. This asymmetric division causes the formation of two unequal compartments, the larger one is known as the mother cell while the smaller one is called a forespore. The septum that is formed during sporulation is different from the normal cell division septum. The sporulation septum contains peptidoglycan and some other associated protein that are present only on one side of the septum. This means that there is not an equal distribution of molecules across each compartment. After this, the mother cell engulfs the smaller spore cell.
Among all these, the most important factor that regulates sporulation is the SpoOA factor. THE phosphorylation of SpoOA, with the help of phosphatases and kinases inhibitor proteins, causes the initiation of sporulation by binding on the promoter region and thus responsible for controlling gene expression.

Role of Kinases in sporulation:

Kinases are responsible for the phosphorylation of the SpoOA protein. The level of phosphorylated SpoOA will decide the type of event to occur. If SpoOA is less phosphorylated, then there will a biofilm formation occur. Similarly, the medium level of phosphorylated SpoOA will cause the initiation of cannibalism. High phosphorylated SpoOA will result in the formation of sporulation.

KinA is the major kinases that are responsible for the production of spores and they are over secretion results in the initiation of sporulation. A cell can lead to sporulation of the KinA is overproduced regardless of the nutrient availability.

Kin-A is further composed of three domains i.e., PAS-A, PAS-B, and PAS-C. These domains work synergistically and complement each other. Any of them become non-functional can retard the sporulation or delay the phenomenon. Loss of PAS-A domain does not have to severe effect as compared to other two domain if lost. The effect of KinC on sporulation is weaker than KinA and KinB. Recently KinD was observed to delay the sporulation process if a bacterium lacks this KinD molecule.

Kinase activity gets restricted by regulatory factors such as Sda and Kipl. Overexpression of Kipl causes the blockage of KinA autophosphorylation.

 

Activation of SpoOA and sporulation:

Activation of SpoOA is controlled by phosphorelay (consists of kinases and phosphatases). The SpoOA-p regulates the expression of 121 genes that in turn regulate the sporulation process. When there is a stressful environment, SpoOF gets phosphorylated by the help of the KinA molecule and gets converted into SpoOF-P.  SpoOF-P then phosphorylates the SpoOB. In the end, the SpoOA gets phosphorylated by the help of SpoOB. This SpoOA can also get phosphorylated with the help of KinC that bypasses all the previous steps and directly phosphorylate the SpoOA.

KinD is the only kinase that act against the SpoOA-p. This molecule reverses the reaction and convert SpoOA-p to SpoOA. Similarly other phosphatases e.g., RapA, B, E, and H and SpoOE work against SpoOA-P.

 

Axial filament and Septum Formation.

The first step during chromosome segregation happens when the replication origins get anchored at the poles of the cell.
Rac-A protein present at the replication origin helps in the attachment of these origins to the Div-IV-A. Div-IV-A protein is present at the poles of a bacterial cell that gets attached with RacA protein.
Two rings are formed near the pole of the cell out of which 1-Z ring gets activated and septum at the pole while other Z-ring dissemble.

As a result of this asymmetric division, Sigma-F gets activated by dephosphorylating SpoII-A.  Sigma is mostly bound with SpoII-AB that gets free after reacting with SpoII-A. The activation of Sigma-F gets activated only in the fore-spore. Only 1/3 of the chromosome gets received by forespore.

Sigma-F gets activated after it senses that there is a septum is formed. As Sigma-F gets activated, this activates the Sigma-E in the mother cell.

When the forespore cells get engulfed by the mother cell this results in the activation of Sigma-G in the forespore cell by Sigma-E. Sigma-G in turn activate the sigma-K molecule.