DivIVA Regulates Its Expression and the Orientation of New Septum Growth in Deinococcus radiodurans

Abstract

In rod shaped Gram-negative bacteria, FtsZ localization at mid cell position is regulated by the gradient of MinCDE complex across the poles. In round shaped bacteria, which lack predefined poles, the next plane of cell division is perpendicular to previous plane and the determination of site for FtsZ assembly is still intriguing. Deinococcus radiodurans, a coccus bacterium, is characterized by its extraordinary resistance to DNA damage. DivIVA, a putative component of Min system in this bacterium, interacts with cognate cell division and genome segregation proteins. Here, we report that deletion of chromosomal copy of DivIVA was possible only when wild type copy of DivIVA was expressed in trans on the plasmid. However, the deletion of C-terminal domain of DivIVA (CTD mutant) was possible but produced distinguishable phenotypes like smaller cells, slower growth and tilted septum orientation in D. radiodurans. In trans expression of DivIVA in CTD mutant could restore these features of wild type. Interestingly, the overexpression of DivIVA led to delayed separation of tetrad from octet state in both trans-complemented divIVA mutant and wild type cells. The CTD mutant showed upregulation of yggS-divIVAN operon. Both wild type and CTD mutant formed FtsZ foci, however unlike wild type, the position of foci in the mutant cells was found to be away from conjectural mid-cell position in cocci. Notably, DivIVA-RFP localizes to septum during cell division at the new division site. These results suggested that DivIVA is an essential protein in D. radiodurans and its C-terminal domain plays an important role in the regulation of its expression and orientation of new septal growth in this bacterium. Importance: In rod-shaped Gram-negative bacteria, the mid-cell position for binary fission is relatively easy to model. In cocci that do not have predefined poles, the plane of next cell division is shown to be perpendicular to the previous plane. However, the molecular basis of perpendicularity is not known in cocci. The DivIVA protein of Deinococcus radiodurans, a coccus bacterium, physically interacts with septum and establishes macromolecular interactions with genome segregation proteins through its N-terminal domain and with MinC through C-terminal domain. Here, we have brought forth some evidence to suggest that DivIVA is essential for growth, plays an important role in cell-polarity determination and its C-terminal domain plays a crucial role in the growth of new septum in correct orientation as well as regulation of its expression.

FtsK, a DNA motor protein, coordinates the genome segregation and early cell division processes in Deinococcus radiodurans

“…To understand it better, we expressed FtsZ-GFP (pVHZGFP) in ΔftsK mutant and observed that a significant population (∼10%) in ftsK deletion mutant got affected in the temporal and spatial regulation of FtsZ functions in D. radiodurans as seen by FtsZ-GFP ring misplacement (Figure 5G). Earlier, it was shown that deletion of the C-terminal region of DivIVA protein in D. radiodurans generates the bent septum (Chaudhary et al, 2021). So, the presence of bent septum in ΔftsK mutant may be attributed to its probable role in the positioning of the pole determining protein and divisome components in this bacterium.…”

FtsZ phosphorylation brings about growth arrest upon DNA damage in Deinococcus radiodurans

“…These PCR products were cloned in pRadgro (Misra et al, 2006) at Apa I- Eco RV yielding pRGZGP and pRGZDDGFP. These recombinant plasmids are further transformed in D. radiodurans using the protocol as mentioned in Chaudhary et al, 2021.…”

“…Immunoblotting of total proteins was carried out as described earlier (Chaudhary et al, 2021). In brief, E. coli cells expressing pDSW208, pDsZ, pDsZAA and pDsZDD were grown at 37°C.…”

“…The upstream and downstream fragments of ftsZ were cloned at Apa I- EcoR I and Bam HI- Xba I sites in pNOKOUT (Kan R ) to give pNKZ. For the expression of FtsZ in trans , the recombinant plasmid pVHZGFP expressing FtsZ-GFP under “Spac” promoter was used and replacement of chromosomal copy of ftsZ with nptII using pNKZ was achieved as described earlier (Chaudhary et al, 2021). The clones having replacement of chromosal copy of ftsZ in the presence of episomal copy of wild type protein was named as conditional null mutant of ftsZ (Δ ftsZ ).…”

“…Immunoblotting of total proteins was carried out as described earlier (Chaudhary et al, 2021). In brief, E. coli cells expressing pDSW208, pDsZ, pDsZAA and pDsZDD were grown at 37 o C. The optical density of bacterial cultures were adjusted to 1.0 and cells were centrifuged.…”