Completion of engulfment and cortex synthesis (Stages III-IV)
5. Completion of engulfment and cortex synthesis (Stages III-IV)
Completion of engulfment is marked by the release of the forespore in the cytoplasm of the mother cell by separation of the engulfing membranes from the mother cell membrane. The forespore is now a separate entity within the mother cell and is surrounded by its own double membrane. For a detailed description of membrane movement mechanisms mediating engulfment, we refer to the manuscript of Broder and Pogliano 91 and the recent review of Higgins and Dworkin 89.
Stage IV sporulation is characterized by the formation of a thick layer of peptidoglycan between the two forespore membranes, forming the spores' cortex, and the construction of a thick proteinaceous protective coat surrounding the forespore. A new set of sporulation-specific sigma factors is activated that is responsible for the expression of the final clusters of genes required for events during stage IV and later stages of sporulation. Firstly, under the influence of σE in the mother cell, σG is activated in the forespore – an event that precedes and is required for the subsequent activation of σK in the mother cell (reviewed in 33 and 84 ). Recently, several reports provided evidence for a SpoIIQ-SpoIIIAH channel (or so-called 'feeding tube') spanning both forespore membranes, which accommodates communication between the two cellular compartments supporting these activation events 130, 131, 132, 133. Premature accumulation of active σG and σK levels is lethal for the cell and is prevented by both genetic and posttranslational control systems to ensure proper timing of progression into further stages of sporulation 116, 121, 135, 136.
Sigma factor switch in RNA polymerase
After asymmetric cell division almost all gene transcription is compartmentalized and results in two different parallel transcriptional processes operating under the control of location-specific sigma factors and secondary regulators. The overlap between forespore and mother cell regulons is limited to eight genes only 101. The compartmentalized gene expression has been described as a hierarchical regulatory cascade, consisting of σF→ RsfA/σG→ SpoVT in the forespore and σE→ GerR/SpoIIID →σK→ GerE in the mother cell 101. Sequential activation of these regulators depends on signals derived from their predecessors and tight regulatory control systems are put in motion to prevent inappropriate activation and subsequent gene expression. As soon as σG and σK are activated, their predecessors σF and σE are inactivated. This leads to a switch in RNA polymerase specificity 97, 137. The exact mechanism of the inactivation of displaced sigma factors remains largely unclear. Theories on protein degradation or regulation at the genetic level as well as affinity competition for RNA polymerase have been suggested 116, although prevention of activity through binding by an inhibitory protein seems a more plausible mechanism 138, 139.
Fine-tuning of late stage sporulation gene expression
Shortly after activation, σG in the forespore and σK in the mother cell are responsible for the production of additional regulators SpoVT and GerE, respectively. Similar to RsfA, SpoIIID and GerR, these regulators are responsible for fine-tuning of sporulation-specific gene expression and the establishment of feed-forward loops. Additionally, a novel transcription modulating protein called YlyA was recently described 140. This RNA polymerase-binding protein stimulates σG-directed gene transcription, especially those involved in spore germination, and enhances fine-tuning of gene expression together with σG and SpoVT. It is at this stage that most structural proteins that make up the spore coat and cortex layers are produced 141, as well as proteins that play important roles in spore resistance and spore germination. Transcription of genes encoding proteins involved in metabolism is mainly restricted to the mother cell compartment 101.
Genes transcribed during this stage can be divided into several different groups: (i) those that rely solely on late sigma factors σG or σK, (ii) those that are activated by both early and late sigma factors, (iii) genes that are shortly activated by σG and σK and hereafter repressed by secondary regulators SpoVT or GerE and (iv) those that require both sigma factors and secondary regulators for their expression 33, 100, 101. In this way the timing of sporulation-specific gene expression is tightly controlled by a number of parallel regulatory mechanisms adding to the complexity of gene transcription during later stages of sporulation.