Role of Ribonucleotide Reductase in Bacillus subtilis Stress-Associated Mutagenesis

Abstract

The Gram-positive microorganism Bacillus subtilis relies on a single class Ib ribonucleotide reductase (RNR) to generate 2=-deoxyribonucleotides (dNDPs) for DNA replication and repair. In this work, we investigated the influence of RNR levels on B. subtilis stationary-phase-associated mutagenesis (SPM). Since RNR is essential in this bacterium, we engineered a conditional mutant of strain B. subtilis YB955 (hisC952 metB5 leu427) in which expression of the nrdEF operon was modulated by isopropyl-␤-D-thiogalactopyranoside (IPTG). Moreover, genetic inactivation of ytcG, predicted to encode a repressor (NrdR) of nrdEF in this strain, dramatically increased the expression levels of a transcriptional nrdE-lacZ fusion. The frequencies of mutations conferring amino acid prototrophy in three genes were measured in cultures under conditions that repressed or induced RNR-encoding genes. The results revealed that RNR was necessary for SPM and overexpression of nrdEF promoted growth-dependent mutagenesis and SPM. We also found that nrdEF expression was induced by H 2 O 2 and such induction was dependent on the master regulator PerR. These observations strongly suggest that the metabolic conditions operating in starved B. subtilis cells increase the levels of RNR, which have a direct impact on SPM.IMPORTANCE Results presented in this study support the concept that the adverse metabolic conditions prevailing in nutritionally stressed bacteria activate an oxidative stress response that disturbs ribonucleotide reductase (RNR) levels. Such an alteration of RNR levels promotes mutagenic events that allow Bacillus subtilis to escape from growth-limited conditions. KEYWORDS Bacillus subtilis, stress-associated mutagenesis, ribonucleotide reductase R ibonucleotide reductases (RNRs) are essential enzymes present in all life forms and are responsible for the conversion of ribonucleotides to 2=-deoxyribonucleotides, the precursors for the replication and repair of DNA. Three different RNR classes (classes I, II, and III) differing in the metal cofactor required for their catalytic activity, allosteric regulation, and quaternary structure have been described (1, 2). Class I RNRs are composed of two homodimeric proteins, ␣ 2 and ␤ 2 ; the larger subunit, ␣, contains the catalytic and the allosteric site that controls the specificity of reduction, while the smaller subunit, ␤, contains a stable tyrosyl radical and a dinuclear metal center (3, 4). In Bacillus subtilis, the nrdE and nrdF genes encode the ␣ and ␤ subunits of the class Ib RNR (3,4). Although the activity of the RNRs is controlled mainly by allosteric regulation in Escherichia coli and other bacteria, the intracellular levels of this enzyme can also be regulated by different transcriptional factors, including DnaA, Fur, and NrdR. Such transcription factors respond to changes in metabolic or environmental conditions and mediate the repression or induction of RNR-encoding genes (2, 3, 5-7). The transcrip-

Role of Mfd and GreA in Bacillus subtilis Base Excision Repair-Dependent Stationary-Phase Mutagenesis

“…Mfd and GreA prevent mutagenesis in growing B. subtilis cells with disabled base excision repair (BER) systems. The conditions that prevail in growing bacteria are conducive to high-fidelity repair of ROS-promoted genetic lesions (40,41). Congruently, our results revealed that in actively growing B. subtilis strain YB955, the simultaneous absence of Nfo, ExoA, and Nth significantly increased the reversion rates of the hisC952, metB5, and leuC427 alleles ( Fig.…”

“…We speculate that the metabolic conditions operating in growing cells of B. subtilis, including a properly balanced pool of precursors for RNA and DNA synthesis (41,52), may elicit GreA-and Mfd-dependent high-fidelity repair in actively transcribed genes (17,18,30). In contrast, in nutritionally stressed bacteria, biased dNTP and NTP pools (41,52) and/or the recruitment of low-fidelity DNA polymerases (32,49) promotes error-prone repair and genetic variability that increases the likelihood of escaping growth-limiting conditions. Additionally, the production of Mfd-dependent Leu ϩ revertants was found to require the PolA and YqjH DNA polymerases in B. subtilis (32).…”

“…Following a GreA-dependent backtracking event (48), the RNAP activates a 3=-5= exonucleolytic event to eliminate the misaligned mRNA (30). In growth-compromised bacteria, these events provide an opportunity for error-prone incorporation of nucleotides from a biased nucleoside triphosphate (NTP) pool (41) and bypass of the DNA lesion, which generates a mutant mRNA pool that promotes SAM through retromutagenesis. It is also possible that the stalled GreA-RNAP complex can signal the recruitment of Mfd to dissociate RNAP and engage components of the BER system to incise/excise the DNA lesion(s) and resynthesize a DNA patch in an error-prone manner.…”

“…Furthermore, SA his, met, and leu reversions in AP endonuclease-deficient B. subtilis cells required the low-fidelity polymerase PolX (42). Finally, a perturbed balance in the NTP levels associated with increased expression of ribonucleotide reductase (RNR) has promoted SAM in starved B. subtilis cells (41).…”

Irregularities in genetic variation and mutation rates with environmental stresses

“…D). A form of SIM called stress‐associated mutagenesis has been reported in Bacillus subtilis (Castro‐Cerritos et al ., ) but the generality or otherwise of the phenomena discussed here (spectrum and rate differences) is vital in assessing the evolutionary significance of the mutation–stress relationship. At this stage, it is not clear whether other bacteria besides E. coli respond in an environment‐specific manner in terms of mutation rates or mutational spectra.…”