About rings and crossbands - Characterization of proteins involved in cell division and compartmentalization in Caulobacter crescentus

In most prokaryotes, cell division is accomplished by a multiprotein complex, called the divisome. The scaffold for assembling the divisome is provided by the Z-ring, which is a ring-like structure that forms during polymerization of FtsZ molecules at midcell. Coordination of Z-ring formation as well as divisome assembly and stability are crucial for successful proliferation. Several factors that stabilize the division apparatus have been characterized. However, these factors appear to be phylogenetically unrelated and to fulfill a rather species-specific function during cell division. This raises the question of what additional factors are required to ensure efficient cell division in C. crescentus. Here, I report the identification of the novel cell division protein CedX (cell division protein X). CedX is a proline-rich inner membrane protein that localizes in an FtsZ- and FtsN- dependent manner to the cell division plane. Interestingly, it was found that overproduction of CedX blocks cell division and causes the formation of several non-contractile Z-rings. Functional analysis of CedX mutant derivatives demonstrated that CedX requires its membrane anchor and proline-rich region for proper localization and protein-protein interaction. In addition, coimmunoprecipation and bacterial two-hybrid analyses suggest that CedX not only interacts with FtsZ and FtsN but also with several other late cell division proteins. Colocalization experiments with fluorescently tagged derivatives of FtsZ, FtsA, FtsN and CedX further support the notion that CedX is a late recruit to the cell division apparatus. However, it remains to be elucidated under which conditions CedX becomes essential for proper cell division. Collectively, these findings suggest that CedX is an accessory divisome component that presumably supports the assembly process of late divisome components by means of its unstructured proline-rich C-terminal tail. Apart from cell division, the formation of a prostheca, also known as stalk, is another characteristic change in cell morphology, which is a widespread phenomenon among bacteria and also an obligatory developmental checkpoint in the C. crescentus life cycle. In C. crescentus, the stalk represents a thin extension of the cell envelope that is free of DNA, ribosomes and most cytoplasmic proteins. It is segmented at irregular intervals by so-called crossbands, disk-like structures that traverse the entire width of the stalk perpendicular to the long-axis of the cell. Crossbands are generally thought to have an architectural, stabilizing function. Despite the fact that researchers have been trying to reveal the mechanisms underlying stalk formation, including the synthesis of the enigmatic crossbands, the biogenesis and function of these structures is still poorly understood. In an attempt to identify factors involved in stalk biogenesis and morphogenesis, four novel stalk proteins, StpABCD, were identified in C. crescentus. Synthesis of StpABCD is initiated at the onset of stalk outgrowth. It was found that StpABCD are specifically targeted to the inner membrane and the periplasmic space of the stalk, with StpA acting as a recruitment factor for StpBCD. Additionally, coimmunoprecipitation analysis supports the idea that StpABCD interact in vivo to form a multiprotein complex. The four proteins colocalize in the stalk in distinct foci that display the same subcellular distribution as crossbands. Noteably, electron cryo-tomography revealed that cells deficient in StpAB consistently lack crossbands. To test for a potential role of crossbands in cellular compartmentalization, the mobility of fluorescently labeled proteins was examined in wild-type or StpAB-deficient cells using fluorescence-loss-in-photobleaching (FLIP) and pulse-labeling experiments. Interestingly, these analyses demonstrated that crossbands act as diffusion barriers for periplasmic, inner and outer membrane proteins. Based on these findings, it can be hypothesized that StpABCD constitute the crossband structures, which act as a protein diffusion barrier to compartmentalize the periplasmic space of the stalk, thereby physically separating it from the cell body.