ParA proteins of secondary genome elements cross-talk and regulate radioresistance through genome copy number reduction in Deinococcus radiodurans

Abstract

Deinococcus radiodurans, an extremely radioresistant bacterium has a multipartite genome system and ploidy. Mechanisms underlying such types of bacterial genome maintenance and its role in extraordinary radioresistance are not known in this bacterium. Chromosome I (Chr I), chromosome II (Chr II) and megaplasmid (Mp) encode its own set of genome partitioning proteins. Here, we have characterized P-loop ATPases of Chr II (ParA2) and Mp (ParA3) and their roles in the maintenance of genome copies and extraordinary radioresistance. Purified ParA2 and ParA3 showed nearly similar polymerization kinetics and interaction patterns with DNA. Electron microscopic examination of purified proteins incubated with DNA showed polymerization on nicked circular dsDNA. ParA2 and ParA3 showed both homotypic and heterotypic interactions to each other, but not with ParA1 (ParA of Chr I). Similarly, ParA2 and ParA3 interacted with ParB2 and ParB3 but not with ParB1 in vivo. ParB2 and ParB3 interaction with cis-elements located upstream to the corresponding parAB operon was found to be sequence-specific. Unlike single mutant of parA2 and parA3, their double mutant (ΔparA2ΔParA3) affected copy number of cognate genome elements and resistance to γ-radiation as well as hydrogen peroxide in this bacterium. These results suggested that ParA2 and ParA3 are DNA-binding ATPases producing higher order polymers on DNA and are functionally redundant in the maintenance of secondary genome elements in D. radiodurans. The findings also suggest the involvement of secondary genome elements such as Chr II and Mp in the extraordinary radioresistance of D. radiodurans.

Characterization of ori and parS-like functions in secondary genome replicons in Deinococcus radiodurans

“…The anchoring of the chromosome to membrane-proximal region through SMC proteins has been reported in other bacteria (Ben-Yehuda et al, 2005;Thanbichler & Shapiro, 2006;Yamaichi et al, 2012). It would be worth mentioning that ParAs encoded on ChrII and MP are found to be functionally redundant (Maurya et al, 2019a), while ParB2 and ParB3 are not (Maurya et al, 2019b), and now cis-elements are differentially regulating both replication and segregation of cognate replicons in this bacterium.…”

“…radiodurans, an extraordinary radioresistant bacterium is characterized for its peculiar cytogenetic features like the multipartite genome, ploidy, and tetrad cell morphology. Mechanisms underlying DNA replication and genome segregation have not been studied in detail in this bacterium, and very limited information is available in other MGH bacteria such as V. cholerae, R. sphaeroides, S. meliloti, and D. radiodurans (Fogel & Waldor, 2005;Charaka & Misra, 2012;Dubarry et al, 2019;Maurya et al, 2019a). Recently, the nucleoid dynamics and radial positioning of ori and ter in chrI of D. radiodurans have been demonstrated as a function of cell cycle (Floc'h et al, 2019).…”

“…The loss of γ radiation resistance in cis mutants that apparently had shown the reduction in copy number in nearly half of the population, and the other half were anucleated for cognate replicons, suggested the role of chrII and MP in radioresistance of this bacterium. Earlier, it was shown that the reduction in copy number of chrII and MP in ΔparA2ΔparA3 double mutants has affected γ radiation response in D. radiodurans (Maurya et al, 2019a). The mechanisms responsible for the underlying loss of radioresistance in cells that have either a reduced copy number or the complete loss of chrII and MP are not understood.…”

“…DNA binding activity of DnaA, ParB2, and ParB3 was monitored using electrophoretic mobility shift assay as described earlier (Charaka & Misra, 2012;Maurya et al, 2019a) with different versions of cis-element as DNA substrates. In brief, the full-length cisII and cisMP and their repeat derivatives (Fig S4A and B) were PCR-amplified using sequence-specific primers (Table S1) except the two repeats and one repeat that were chemically synthesized and annealed with the complementary strand.…”

Guanine Quadruplex DNA Regulates Gamma Radiation Response of Genome Functions in the Radioresistant Bacterium Deinococcus radiodurans

“…Here, we also observed that the arrest of G4 DNA structure dynamics significantly affected the copy number for small plasmid but less significantly for chromosomes or megaplasmid. Recently, it has been demonstrated that the primary chromosome contributes maximally to normal growth, while secondary genome elements such as chromosome II and megaplasmid are implicated in stress tolerance phenotypes in D. radiodurans (48). The molecular basis of the G4 DNA structure affecting the copy number of the small plasmid and a little less of other genome elements is not clear and needs independent investigations.…”