Transcriptional coupling (Mfd) and DNA damage scanning (DisA) coordinate excision repair events for efficient Bacillus subtilis spore outgrowth

Abstract

The absence of base excision repair (BER) proteins involved in processing ROS‐promoted genetic insults activates a DNA damage scanning (DisA)‐dependent checkpoint event in outgrowing Bacillus subtilis spores. Here, we report that genetic disabling of transcription‐coupled repair (TCR) or nucleotide excision repair (NER) pathways severely affected outgrowth of ΔdisA spores, and much more so than the effects of these mutations on log phase growth. This defect delayed the first division of spore′s nucleoid suggesting that unrepaired lesions affected transcription and/or replication during outgrowth. Accordingly, return to life of spores deficient in DisA/Mfd or DisA/UvrA was severely affected by a ROS‐inducer or a replication blocking agent, hydrogen peroxide and 4‐nitroquinoline‐oxide, respectively. Mutation frequencies to rifampin resistance (Rifr) revealed that DisA allowed faithful NER‐dependent DNA repair but activated error‐prone repair in TCR‐deficient outgrowing spores. Sequencing analysis of rpoB from spontaneous Rifr colonies revealed that mutations resulting from base deamination predominated in outgrowing wild‐type spores. Interestingly, a wide range of base substitutions promoted by oxidized DNA bases were detected in ΔdisA and Δmfd outgrown spores. Overall, our results suggest that Mfd and DisA coordinate excision repair events in spore outgrowth to eliminate DNA lesions that interfere with replication and transcription during this developmental period.

c-di-AMP assists osmoadaptation by regulating the Listeria monocytogenes potassium transporters KimA and KtrCD

“…Bars, means (n Ͼ 3). p Ͻ 0.01 (**), p Ͻ 0.001 (***), and p Ͻ 0.0001 (****) compared with empty plasmid alone (F(8,20) ϭ 275.9, ANOVA with Dunnett's post hoc test).…”

Transcriptional coupling and repair of 8-OxoG activate a RecA-dependent checkpoint that controls the onset of sporulation in Bacillus subtilis

“…B. subtilis cells were grown and induced to sporulate in DSM. Cellular samples of the wild-type strain, ΔGO Δ mfd , ΔGO and Δ mfd mutants were collected at the appropriate times, washed twice with cold phosphate-buffered saline [PBS; 0.7% Na2HPO4, 0.3% KH2PO4, 0.4% NaCl (pH 7.5)] and were fixed as described previously 20 . Phase contrast microscopy was performed with a Zeiss LSM700 scanning laser confocal microscope with a LD A-Plan 40x/0.55 Ph2 objective.…”

“…However, two additional sporulation roles have been attributed to RecA, firstly as a factor that regulates the levels of phosphorylated Spo0A during the onset of sporulation, and secondly blocking replication and vegetative growth in further stages of this developmental pathway [15][16][17] . As noted above, Mfd and the NER system counteract the cytotoxic and genotoxic effects promoted by physical and chemical factors that promote bulky DNA lesions 13,[18][19][20] . However, in the absence of external DNA damaging factors, the sole absence of Mfd affected sporulation in B. subtilis 13 , suggesting that spontaneous DNA lesions in actively transcribed genes require Mfd for a proper sporogenesis.…”

Making and Breaking of an Essential Poison: the Cyclases and Phosphodiesterases That Produce and Degrade the Essential Second Messenger Cyclic di-AMP in Bacteria

“…Moreover, the branch migration transferase RadA, which is encoded in a conserved operon with DisA, interacts with DisA and inhibits its enzymatic activity in both B. subtilis and Mycobacterium tuberculosis (52-55) The DisA-produced c-di-AMP was proposed to control the initiation of sporulation in B. subtilis, as DNA damage results in reduced DisA activity, and the reduced c-di-AMP levels will then result in a sporulation delay (51). In addition, a role in DNA repair in outgrowing spores was proposed for DisA to ensure that DNA lesions are eliminated during outgrowth (33,56). Finally, DisA is required for DNA damage response also in vegetative cells of B. subtilis (57).…”