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

Abstract

During sporulation Bacillus subtilis Mfd couples transcription to nucleotide excision repair (NER) to eliminate DNA distorting lesions. Here, we report a significant decline in sporulation following Mfd disruption, which was manifested in the absence of external DNA-damage suggesting that spontaneous lesions activate the function of Mfd for an efficient sporogenesis. Accordingly, a dramatic decline in sporulation efficiency took place in a B. subtilis strain lacking Mfd and the repair/prevention guanine oxidized (GO) system (hereafter, the ∆GO system), composed by YtkD, MutM and MutY. Furthermore, the simultaneous absence of Mfd and the GO system, (i) sensitized sporulating cells to H2O2, and (ii) elicited spontaneous and oxygen radical-induced rifampin-resistance (Rifr) mutagenesis. Epifluorescence (EF), confocal and transmission electron (TEM) microscopy analyses, showed a decreased ability of ∆GO ∆mfd strain to sporulate and to develop the typical morphologies of sporulating cells. Remarkably, disruption of sda, sirA and disA partially, restored the sporulation efficiency of the strain deficient for Mfd and the ∆GO system; complete restoration occurred in the RecA− background. Overall, our results unveil a novel Mfd mechanism of transcription-coupled-repair (TCR) elicited by 8-OxoG which converges in the activation of a RecA-dependent checkpoint event that control the onset of sporulation in B. subtilis.

8-OxoG-Dependent Regulation of Global Protein Responses Leads to Mutagenesis and Stress Survival in Bacillus subtilis

“…As shown in this work, these processes can be subjected to regulation with Mfd ( Figure 2 ), a transcription factor commonly associated with the repair of bulky DNA lesions in bacteria [ 81 ]. However, recent evidence has revealed that Mfd functions as a global regulator of gene transcription and cell differentiation in B. subtilis [ 34 , 82 ]. Therefore, elucidating the Mfd-dependent mechanisms that cells employ to directly or indirectly regulate the expression of the factors associated with the HPM and HPHR phenotypes will contribute to understanding the novel aspects of bacterial physiology in stressed bacterial cells.…”

“…The strain B. subtilis PS832 (Wild-type trp + revertant of strain 168 [ 35 ]), and its derivative, PERM603 ( ytkD :: neo mutM :: tet mutY :: sp . Neo R Tc R Sp R ∆GO; [ 34 ]), were employed in this study. The routinely used growth medium was the Luria–Bertani (LB) medium.…”

“…Spontaneous or H 2 O 2 -induced mutation frequencies were determined, as previously described [ 34 ]. Cells were propagated in an LB medium at 37 °C to an OD 600 of 1.0, and they were split into two subcultures.…”

“…Several lines of evidence associate the GO system with distinct cellular processes of B. subtilis , including stress-associated mutagenesis (SAM), transcriptional mutagenesis, and sporulation [ 31 , 32 , 33 , 34 ]. The inactivation of this system increases by 5–6 log orders, with regard to the production of His + and Met + (but not Leu + ) prototrophs in nutritionally stressed cells of B. subtilis YB955 ( hisC952 , metB5 , leuC427 ) [ 32 ].…”

“…In addition to its mutagenic effects, 8-OxoG was shown to affect developmental pathways in B. subtilis . Accordingly, a recent report showed that the processing of 8-OxoG through Mfd activated a RecA-dependent checkpoint that regulates the sporulation onset in this microorganism [ 34 ].…”