The LexA/RecA-type SOS response is the only characterized mechanism of DNA damage response in bacteria. It regulates cell cycle by attenuating the functions of cell division protein FtsZ and inducing the expression of DNA repair proteins. There are bacteria, including Deinococcus radiodurans, that do not show this classical SOS response. D. radiodurans is known for its extraordinary resistance to gamma radiation, and a DNA damage-responsive Ser/Thr protein kinase (RqkA) has been characterized for its role in radioresistance. RqkA phosphorylates a large number of proteins in solution. The phosphorylation of RecA and PprA by RqkA enhanced their activities. FtsZ phosphorylation is inducible by gamma radiation in wild-type D. radiodurans but not in an rqkA mutant. Phosphorylation affected the interaction of FtsZ and FtsA in this bacterium. This study, therefore, brought forth some findings that might lead to the discovery of a new mechanism regulating the bacterial cell cycle in response to DNA damage.

Guanine quadruplex (G4) DNA/RNA are secondary structures that regulate the various cellular processes in both eukaryotes and bacteria. Deinococcus radiodurans, a Gram-positive bacterium known for its extraordinary radioresistance, shows a genomewide occurrence of putative G4 DNA-forming motifs in its GC-rich genome. N-Methyl mesoporphyrin (NMM), a G4 DNA structure-stabilizing drug, did not affect bacterial growth under normal conditions but inhibited the postirradiation recovery of gamma-irradiated cells. Transcriptome sequencing analysis of cells treated with both radiation and NMM showed repression of gamma radiation-responsive gene expression, which was observed in the absence of NMM. Notably, this effect of NMM on the expression of housekeeping genes involved in other cellular processes was not observed. Stabilization of G4 DNA structures mapped at the upstream of recA and in the encoding region of DR_2199 had negatively affected promoter activity in vivo, DNA synthesis in vitro and protein translation in Escherichia coli host. These results suggested that G4 DNA plays an important role in DNA damage response and in the regulation of expression of the DNA repair proteins required for radioresistance in D. radiodurans. IMPORTANCE Deinococcus radiodurans can recover from extensive DNA damage caused by many genotoxic agents. It lacks LexA/RecA-mediated canonical SOS response. Therefore, the molecular mechanisms underlying the regulation of DNA damage response would be worth investigating in this bacterium. D. radiodurans genome is GC-rich and contains numerous islands of putative guanine quadruplex (G4) DNA structure-forming motifs. Here, we showed that in vivo stabilization of G4 DNA structures can impair DNA damage response processes in D. radiodurans. Essential cellular processes such as transcription, DNA synthesis, and protein translation, which are also an integral part of the double-strand DNA break repair pathway, are affected by the arrest of G4 DNA structure dynamics. Thus, the role of DNA secondary structures in DNA damage response and radioresistance is demonstrated.

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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.

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The polymerization/depolymerization dynamics of FtsZ play a pivotal role in cell division in the majority of the bacteria. Deinococcus radiodurans, a radiationresistant bacterium, shows an arrest of growth in response to DNA damage with no change in the level of FtsZ. This bacterium does not deploy LexA/RecA type of DNA damage response and cell cycle regulation, and its genome does not encode SulA homologues of Escherichia coli, which attenuate FtsZ functions in response to DNA damage in other bacteria. A radiation-responsive Ser/Thr quinoprotein kinase (RqkA), characterized for its role in radiation resistance in this bacterium, could phosphorylate several cognate proteins, including FtsZ (drFtsZ) at Serine 235 (S235) and Serine 335 (S335) residues. Here, we reported the detailed characterization of S235 and S335 phosphorylation effects in the regulation of drFtsZ functions and demonstrated that the phospho-mimetic replacements of these residues in drFtsZ had grossly affected its functions that could result in cell cycle arrest in response to DNA damage in D. radiodurans. Interestingly, the phospho-ablative replacements were found to be nearly similar to drFtsZ, whereas the phospho-mimetic mutant lost the wild-type protein's signature characteristics, including its dynamics under normal conditions. The kinetics of post-bleaching recovery for drFtsZ and phospho-mimetic mutant were nearly similar at 2 h post-irradiation recovery but were found to be different under normal conditions. These results highlighted the role of S/T phosphorylation in the regulation of drFtsZ functions and cell cycle arrest in response to DNA damage, which is demonstrated for the first time, in any bacteria.

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