RpoS, the Stress Response Sigma Factor, Plays a Dual Role in the Regulation of Escherichia coli 's Error-Prone DNA Polymerase IV

Abstract

RpoS, Escherichia coli's general stress response sigma factor, regulates error-prone DNA polymerase IV (Pol IV) (encoded by the dinB gene). Pol IV is induced in stationary-phase cells, and thereafter, levels of the protein remain elevated for several days of continuous incubation. This induction and persistence in stationary-phase cells are dependent on RpoS. Data presented here show that this regulation is direct via the RpoS-directed transcription of the dinB gene. However, a loss of RpoS also results in a decrease in Pol IV-dependent mutation when Pol IV is overexpressed from an RpoS-independent promoter in exponentially growing cells. The loss of RpoS also increases cell sensitivity to 4-nitroquinoline-1-oxide, indicating that RpoS affects the ability of Pol IV to bypass DNA lesions. Thus, in addition to directly driving the transcription of the dinB gene in stationaryphase cells, RpoS regulates the activity of Pol IV in exponentially growing cells via a second, indirect pathway.In their natural environments bacteria are constantly assaulted by a variety of stresses that include nutritional deprivation, temperature fluctuation, desiccation, and exposure to DNA-damaging agents such as UV light and reactive oxygen species. To survive such challenges, bacteria have evolved coordinated cellular defense mechanisms, two of which are the RpoS-regulated response to nutritional deprivation and the SOS response to DNA damage.Sigma factors are the subunits of RNA polymerase that recognize the promoter regions of genes and direct the holoenzyme to them. Escherichia coli has seven sigma factors, each of which is responsible for gene regulation under specific conditions. The vegetative sigma factor RpoD (also called D and 70 ) directs the RNA polymerase holoenzyme (E D ) to transcribe housekeeping genes and the genes specifically expressed during the exponential phase. A second major sigma factor, RpoS (also called S and 38 ), regulates the cell's response to nutritional deprivation. RpoS is activated as cells enter stationary phase and is responsible, either directly or indirectly, for the regulation of as many as 500 genes (33,40,41). Because of their similar recognition sequences, RpoS and RpoD often direct the RNA polymerase holoenzyme to bind to the same promoters (17). However, promoter binding by the RpoS-containing RNA polymerase holoenzyme (E S ) is enhanced in stationary phase by the accumulation of accessory factors, such as ppGpp, and/or by changes in DNA topology (7,17,23,24).DNA damage in E. coli and its relatives results in the upregulation of more than 40 genes (11) that include the genes encoding DNA polymerase II (Pol II), Pol IV, and Pol V (8,19). Pol IV, encoded by dinB, and Pol V, encoded by umuDC, are members of the Y family of specialized DNA polymerases (for a review, see reference 30). These polymerases are found in organisms from all three domains of life and can replicate damaged DNA, a process called translesion synthesis. Because of its ability to bypass many types of DNA lesions, Pol V has a ...

The SMC-Like Protein Complex SbcCD Enhances DNA Polymerase IV-Dependent Spontaneous Mutation inEscherichia coli

“…For example, the presence of a copy of the dinB gene on the FЈ episome in addition to the copy on the chromosome results in 4-fold more Pol IV and a 2-to 3-fold increase in mutation frequencies (22,36). The presence of the dinB gene on a multicopy plasmid results in 10-to 20-fold more Pol IV (36,73) and, depending on the mutational target, 5-to 200-fold increases in mutation frequencies (37,39,63,65,70,73,75). These observations strongly suggest that the mutagenic activity of Pol IV normally is tightly regulated in growing cells but that even a modest increase in abundance allows Pol IV to, at least partially, escape this regulation.…”

“…In addition, RpoS has been found by transcription microarray analysis to regulate directly or indirectly almost 300 genes in exponential-phase cells (14a). Recently, we found that RpoS drives the transcription of dinB in stationary-phase cells but not in exponential-phase cells; and yet, RpoS still affects Pol IV activity in exponential phase (63). In exponentially growing cells, overexpression of Pol IV from an RpoS-independent promoter increases the growth-dependent mutation rate 10-fold, but in cells lacking RpoS, this increase is only 4-fold, even though the amount of Pol IV is unchanged (63).…”

“…Because of this strong phenotype, adaptive mutation in FC40 is often used as an assay for Pol IV's mutagenic activity (e.g., see reference 23). Several factors existent in stationary-phase cells contribute to this high rate of Pol IV-dependent adaptive mutation: (i) transcription of the dinB gene is induced approximately 3-fold under the control of the stationary-phase sigma factor RpoS (42,63), (ii) the Pol IV protein is stabilized by the chaperone GroEL (43), (iii) Pol IV activity is enhanced by cellular polyphosphate (65), and (iv) proposed inhibitors of Pol IV activity, such as UmuD, may be less active or abundant in stationary-phase cells (23). This growth phase regulation suggests that Pol IV's mutagenic activity may serve an important function during nutrient-limited conditions.…”

Impact of a stress-inducible switch to mutagenic repair of DNA breaks on mutation in Escherichia coli

“…Both confer up-regulation of the DinB error-prone DNA polymerase, which then makes errors that become mutations in acts of DSB repair via homologous recombination (HR) (7). Whereas up-regulation of DinB is the sole role of SOS in DSB-dependent SIM (8), RpoS additionally licenses the use of DinB (7,9,10) and other low-fidelity DNA polymerases (10) in DSB repair by a mechanism not yet elucidated. This process causes a switch from highfidelity to mutagenic DSB repair under RpoS-inducing stress (7).…”

Stress-Induced Mutation Rates Show a Sigmoidal and Saturable Increase Due to the RpoS Sigma Factor in Escherichia coli

“…An approach to circumventing the problem of selection and varying stress level is to use identical growth conditions, but with bacteria in which the level of SIM is under genetic control. A way of doing this is to control a major input into SIM regulation; RpoS has been implicated as a controller of SIM in several laboratories (Galhardo et al 2007;Storvik and Foster 2010;Gutierrez et al 2013). RpoS (also known as s S ) is an alternative RNA polymerase sigma factor and the master stress regulator in E. coli, which is highly variable and dependent on both environmental signals and growth phase (Battesti et al 2011;Hengge 2011).…”