Functional Characterization of the Role of the Chromosome I Partitioning System in Genome Segregation in Deinococcus radiodurans

Abstract

Deinococcus radiodurans, a radiation-resistant bacterium, harbors a multipartite genome. Chromosome I contains three putative centromeres (segS1, segS2, and segS3), and ParA (ParA1) and ParB (ParB1) homologues. The ParB1 interaction with segS was sequence specific, and ParA1 was shown to be a DNA binding ATPase. The ATPase activity of ParA1 was stimulated when segS elements were coincubated with ParB1, but the greatest increase was observed with segS3. ParA1 incubated with the segS-ParB1 complex showed increased light scattering in the absence of ATP. In the presence of ATP, this increase was continued with segS1-ParA1B1 and segS2-ParA1B1 complexes, while it decreased rapidly after an initial increase for 30 min in the case of segS3. D. radiodurans cells expressing green fluorescent protein (GFP)-ParB1 produced foci on nucleoids, and the ⌬parB1 mutant showed growth retardation and ϳ13%-higher anucleation than the wild type. Unstable mini-F plasmids carrying segS1 and segS2 showed inheritance in Escherichia coli without ParA1B1, while segS3-mediated plasmid stability required the in trans expression of ParA1B1. Unlike untransformed E. coli cells, cells harboring pDAGS3, a plasmid carrying segS3 and also expressing ParB1-GFP, produced discrete GFP foci on nucleoids. These findings suggested that both segS elements and the ParA1B1 proteins of D. radiodurans are functionally active and have a role in genome segregation.

Divisome and segrosome components of Deinococcus radiodurans interact through cell division regulatory proteins

“…Depletion of ParB and/ or increase in ParA could inhibit Z-ring formation and cell division in C. crescentus (Mohl et al, 2001) and Mycobacterium smegmatis (Ginda et al, 2013). Similar observation has also been reported in D. radiodurans where the role of ParA of chromosome II (ParA2) in the regulation of cell division, in the context of its stoichiometric balance with ParB2, was observed (Charaka & Misra, 2012). DivIVA a tropomyocinlike coiled-coil protein appears as a bifunctional protein with distinct roles in division-site selection as well as during chromosome segregation at least in B. subtilis (Thomaides et al, 2001).…”

“…The mechanisms underlying maintenance and faithful inheritance of its multipartite genome and the regulation of cell division are not known in this bacterium and would be worth investigating. Characterization of chromosome I partitioning proteins and their functional interaction with newly identified centromeric sequences in chromosome I of D. radiodurans have been reported recently (Charaka & Misra, 2012). Further, deinococcal FtsZ (DrFtsZ) and FtsA (DrFtsA) have been characterized both in vitro and in vivo .…”

Phosphorylation of Deinococcus radiodurans RecA Regulates Its Activity and May Contribute to Radioresistance

“…Wild type and their respective derivatives were grown aerobically in TGY (0.5% Bacto-tryptone, 0.3% Bacto-yeast extract, and 0.1% glucose) broth or on an agar plate at 32°C in presence of antibiotics as required. Shuttle expression vectors pRADgro (40) and pVHS559 (41) and their derivatives were maintained in E. coli strain DH5␣ as described earlier. Molecular biology grade chemicals and enzymes were procured from Sigma, Roche Applied Science, New England Biolabs, Cell Signaling, and Merck.…”

“…For functional complementation studies, the coding sequences of wild type recA and its Y77F, Y77E, T318A, T318E, and Y77F/ T318A mutant versions were PCR-amplified from respective pET derivatives using T7-F and T7-R primers (Table 2) and cloned at NdeI and XhoI sites in deinococcal shuttle expression vector pVHS559 (41), resulting in pVHSRecA, pVHSY77F, pVHSY77E, pVHST318A, pVHST318E, and pVHSY77T318. These recombinant plasmids were transformed to the recA30 mutant of D. radiodurans, the recombinant clones were scored on TGY agar plates supplemented with spectinomycin (75 g/ml), and cloning was confirmed by restriction analysis.…”

DR1769, a Protein with N-Terminal Beta Propeller Repeats and a Low-Complexity Hydrophilic Tail, Plays a Role in Desiccation Tolerance of Deinococcus radiodurans

“…Fluorescence microscopy of D. radiodurans cells was carried out on a Zeiss AxioImager (model LSMS10 META; Carl Zeiss) microscope equipped with a 254 Zeiss AxioCam HRm camera as described earlier (31). The D. radiodurans cells desiccated for 14 days at ϳ5% humidity were allowed to grow for 24 h, and cells were collected by centrifugation and washed twice with 10 mM MgCl 2 .…”