Interaction of Apurinic/Apyrimidinic Endonucleases Nfo and ExoA with the DNA Integrity Scanning Protein DisA in the Processing of Oxidative DNA Damage during Bacillus subtilis Spore Outgrowth

Abstract

Oxidative stress-induced damage, including 8-oxo-guanine and apurinic/apyrimidinic (AP) DNA lesions, were detected in dormant and outgrowing Bacillus subtilis spores lacking the AP endonucleases Nfo and ExoA. Spores of the Δnfo exoA strain exhibited slightly slowed germination and greatly slowed outgrowth that drastically slowed the spores' return to vegetative growth. A null mutation in the disA gene, encoding a DNA integrity scanning protein (DisA), suppressed this phenotype, as spores lacking Nfo, ExoA, and DisA exhibited germination and outgrowth kinetics very similar to those of wild-type spores. Overexpression of DisA also restored the slow germination and outgrowth phenotype to nfo exoA disA spores. A disA-lacZ fusion was expressed during sporulation but not in the forespore compartment. However, disA-lacZ was expressed during spore germination/outgrowth, as was a DisA-green fluorescent protein (GFP) fusion protein. Fluorescence microscopy revealed that, as previously shown in sporulating cells, DisA-GFP formed discrete globular foci that colocalized with the nucleoid of germinating and outgrowing spores and remained located primarily in a single cell during early vegetative growth. Finally, the slow-outgrowth phenotype of nfo exoA spores was accompanied by a delay in DNA synthesis to repair AP and 8-oxo-guanine lesions, and these effects were suppressed following disA disruption. We postulate that a DisA-dependent checkpoint arrests DNA replication during B. subtilis spore outgrowth until the germinating spore's genome is free of damage.

Structural and Biochemical Analysis of the Essential Diadenylate Cyclase CdaA from Listeria monocytogenes

“…It was also observed that the DAC activity of DisA is strongly inhibited by the presence of non-standard DNA secondary structures like Holliday junctions (9). A recent study emphasized a function of DisA in controlling genome replication during outgrowth of B. subtilis spores to prevent propagation of damaged DNA to the germinating cells (12). Meanwhile, homologs of DisA have been identified in several other bacteria like Mycobacterium tuberculosis (13)(14)(15).…”

An Essential Poison: Synthesis and Degradation of Cyclic Di-AMP in Bacillus subtilis

“…1A). DisA is involved in the control of DNA integrity, and the activity of this enzyme is controlled by a protein-protein interaction with the DNA methyltransferase RadA, as first observed for Mycobacterium smegmatis (13)(14)(15). CdaA is the most widespread diadenylate cyclase, and it is the only such enzyme in pathogenic firmicutes.…”

Structural Studies of Potassium Transport Protein KtrA Regulator of Conductance of K+ (RCK) C Domain in Complex with Cyclic Diadenosine Monophosphate (c-di-AMP)

“…This interaction leads to a functional change of downstream effector proteins and enables control of specific cellular pathways. Although it was only recently identified as a second messenger, c-di-AMP was found to have central importance in numerous bacterial functions including stress responses, antibiotic resistance, cellular morphology, bacterial growth, and virulence (5)(6)(7)(8)(9)(10)(11)(12). Also, c-di-AMP secretion by Listeria monocytogenes, Mycobacterium tuberculosis, and Chlamydia trachomatis during infection induces an IFN-␤-mediated host immune response (13)(14)(15).…”