Role of Bacillus subtilis DNA Glycosylase MutM in Counteracting Oxidatively Induced DNA Damage and in Stationary-Phase-Associated Mutagenesis.

UNLABELLED: Reactive oxygen species (ROS) promote the synthesis of the DNA lesion 8-oxo-G, whose mutagenic effects are counteracted in distinct organisms by the DNA glycosylase MutM. We report here that in Bacillus subtilis, mutM is expressed during the exponential and stationary phases of growth. In agreement with this expression pattern, results of a Western blot analysis confirmed the presence of MutM in both stages of growth. In comparison with cells of a wild-type strain, cells of B. subtilis lacking MutM increased their spontaneous mutation frequency to Rif(r) and were more sensitive to the ROS promoter agents hydrogen peroxide and 1,1'-dimethyl-4,4'-bipyridinium dichloride (Paraquat). However, despite MutM's proven participation in preventing ROS-induced-DNA damage, the expression of mutM was not induced by hydrogen peroxide, mitomycin C, or NaCl, suggesting that transcription of this gene is not under the control of the RecA, PerR, or σ(B) regulons. Finally, the role of MutM in stationary-phase-associated mutagenesis (SPM) was investigated in the strain B. subtilis YB955 (hisC952 metB5 leuC427). Results revealed that under limiting growth conditions, a mutM knockout strain significantly increased the amount of stationary-phase-associated his, met, and leu revertants produced. In summary, our results support the notion that the absence of MutM promotes mutagenesis that allows nutritionally stressed B. subtilis cells to escape from growth-limiting conditions. IMPORTANCE: The present study describes the role played by a DNA repair protein (MutM) in protecting the soil bacterium Bacillus subtilis from the genotoxic effects induced by reactive oxygen species (ROS) promoter agents. Moreover, it reveals that the genetic inactivation of mutM allows nutritionally stressed bacteria to escape from growth-limiting conditions, putatively by a mechanism that involves the accumulation and error-prone processing of oxidized DNA bases.

Predicting Sjögren's syndrome in patients with recent-onset SLE.

OBJECTIVE: To determine the prevalence of SS in a cohort of recent-onset SLE patients and evaluate the clinical and immunological variables that may identify SLE patients prone to develop SS. METHODS: A total of 103 patients participating in a prospective cohort of recent-onset SLE were assessed for fulfilment of the American European Consensus Group criteria for SS using a three-phase approach: screening (European questionnaire, Schirmer-I test and wafer test), confirmation (fluorescein staining test, non-stimulated whole-salivary flow and anti-Ro/La antibodies) and lip biopsy. Anti-Ro/SSA and anti-La/SSB antibodies and RF were measured at entry into the cohort and at SS assessment. RESULTS: Ninety-three females and 10 males were included. Mean age at lupus diagnosis was 25.9 ± 8.9 years, and lupus duration at SS assessment was 30.9 ± 9.1 years. SS was diagnosed in 19 (18.5%) patients, all female, and the patients were older at SLE diagnosis than patients without SS (30.8 ± 9.3 vs 24 ± 8.8 years, P = 0.004). Anti-Ro/SSA antibody was more common in SLE-SS patients (84% vs 55%, P = 0.02, LR + 1.53, 95% CI 1.14, 2.04). In the multivariate analysis, age ≥25 years and anti-Ro/SSA antibodies at SLE diagnosis were identified as predictors of SLE-SS, while the absence of anti-Ro/SSA, anti-La/SSB and RF seems to be protective (LR- 0.14, 95% CI 0.02, 0.95). CONCLUSION: The overlap of SLE and SS occurs in almost one-fifth of SLE patients and presents early during its evolution. SLE onset at age ≥25 years plus the presence of anti-Ro/SSA antibody at diagnosis are useful predictors, while the absence of anti-Ro/SSA, anti-La/SSB and RF identifies patients at lowest risk.

Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways.

In replication-limited cells of Bacillus subtilis, Mfd is mutagenic at highly transcribed regions, even in the absence of bulky DNA lesions. However, the mechanism leading to increased mutagenesis through Mfd remains currently unknown. Here, we report that Mfd may promote mutagenesis in nutritionally stressed B. subtilis cells by coordinating error-prone repair events mediated by UvrA, MutY and PolI. Using a point-mutated gene conferring leucine auxotrophy as a genetic marker, it was found that the absence of UvrA reduced the Leu⁺ revertants and that a second mutation in mfd reduced mutagenesis further. Moreover, the mfd and polA mutants presented low but similar reversion frequencies compared to the parental strain. These results suggest that Mfd promotes mutagenic events that required the participation of NER pathway and PolI. Remarkably, this Mfd-dependent mutagenic pathway was found to be epistatic onto MutY; however, whereas the MutY-dependent Leu⁺ reversions required Mfd, a direct interaction between these proteins was not apparent. In summary, our results support the concept that Mfd promotes mutagenesis in starved B. subtilis cells by coordinating both known and previously unknown Mfd-associated repair pathways. These mutagenic processes bias the production of genetic diversity towards highly transcribed regions in the genome.