Study of involvement of ImuB and DnaE2 in stationary-phase mutagenesis in Pseudomonas putida

Koorits, Lauri; Tegova, Radi; Tark, Mariliis; Tarassova, Kairi; Tover, Andres; Kivisaar, Maia

doi:10.1016/j.dnarep.2007.01.010

Cited by 35 publications

(42 citation statements)

References 36 publications

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“…S2) and DnaE2 (Fig. S3) proteins with conflicting reports of biological function: specifically, the observation that ImuB and DnaE2 fulfill antagonistic roles in Pseudomonas putida (22), whereas the homologous Pseudomonas aeruginosa proteins are dispensable for damage tolerance (38) but not for induced mutagenesis (39). The results of Sanders et al (39) are more consistent with the data presented here in that they indicate essential, and interdependent, roles for DnaE2 and ImuB proteins in damage tolerance and induced mutagenesis.…”

Section: Discussionsupporting

confidence: 82%

“…The absence of the complete triad of active-site acidic residues is a feature of all ImuB homologs analyzed ( Fig. S2) and strongly implies that these proteins cannot function as DNA polymerases (22). In turn, this observation suggests a model for translesion synthesis in which DnaE2 itself catalyzes lesion bypass (6).…”

Section: Imua′ and Imub Are Individually Essential For Dnae2 Function Inmentioning

confidence: 95%

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Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

Warner

Ndwandwe

Abrahams

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

120

205

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In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2-type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA′) and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA′ and DnaE2, as well as with the β-clamp. Moreover, disruption of the ImuB-β clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA′, imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the α-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA′-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance. (1), a Cfamily DNA polymerase implicated in error-prone bypass of DNA lesions. Loss of DnaE2 activity renders Mtb hypersensitive to DNA damage and eliminates induced mutagenesis. Moreover, dnaE2 deletion attenuates virulence and reduces the frequency of drug resistance in vivo. Mtb contains two DnaE-type polymerases; the other, DnaE1, provides essential, high-fidelity replicative polymerase function (1). However, the basis for the functional specialization of the DnaE subunits remains unclear (2, 3). Although structural determinants such as active-site architecture contribute significantly to inherent fidelity, it is possible that differential interactions with other DNA metabolic proteins modulate polymerase function.Bacterial genomes containing a DnaE2-type DNA polymerase almost invariably encode a homolog of ImuB (4-6), a putative Yfamily polymerase that is usually present in a LexA-regulated imuA-imuB-dnaE2 gene cassette (5). In Caulobacter crescentus, both ImuB and ImuA are required for induced mutagenesis and damage tolerance (6) whereas plasmid-encoded DnaE2 and ImuB mediate UV-induced mutagenesis in Deinococcus deserti (7). Although distributed widely across the bacterial domain, the imuA-imuB-dnaE2 cassette is not found in organisms possessing umuDC homologs (5). This suggests that the encoded proteins perform an analogous function to DNA polymerase V (8), the Y-family member required for damage-induced mutagenesis in Escherichia coli (9).Mtb contains a putative SOS-inducible operon, Rv3395c-Rv3394c (1, 10), locat...

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Section: Discussionsupporting

confidence: 82%

Section: Imua′ and Imub Are Individually Essential For Dnae2 Function Inmentioning

confidence: 95%

Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

Warner

Ndwandwe

Abrahams

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

120

205

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“…Interestingly, the results of these studies also indicate that the lexA2 gene is always cotranscribed with three other DNA damage-inducible genes (PP_3117, PP_3118, and PP_3119, designated here, following the recently proposed nomenclature, imuA, imuB, and dnaE2) (13,17,20), which make up a cassette that has been shown to be widespread in the domain Bacteria and to be involved in DNA damagemediated mutagenesis (1,13,17). During its evolutionary history, this multiple-gene cassette has undergone a number of reorganizations, with three-gene, two-gene, and completely split cassettes reported in different phyla but always showing apparent regulation by LexA (13).…”

mentioning

confidence: 79%

“…Even though the SOS response has been thoroughly characterized in several microorganisms, such as E. coli (10), Pseudomonas aeruginosa (9), and the gram-positive organisms Bacillus subtilis (2) and Staphylococcus aureus (8), both of which have an SOS box (GAACN 4 GTTC) (8,40) markedly different from that of E. coli (38), recent work has offered a glimpse of the composition of the LexA regulon in several additional bacterial species (20). In contrast to the examples cited above, the genetic composition of the LexA network has been found to vary widely among phyla and even among species belonging to the same class (14,22).…”

mentioning

confidence: 99%

Cohabitation of Two DifferentlexARegulons inPseudomonas putida

et al. 2007

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In contrast to the vast majority of the members of the domain Bacteria, several Pseudomonas and Xanthomonas species have two lexA genes, whose products have been shown to recognize different LexA binding motifs, making them an interesting target for studying the interplay between cohabiting LexA regulons in a single species. Here we report an analysis of the genetic composition of the two LexA regulons of Pseudomonas putida KT2440 performed with a genomic microarray. The data obtained indicate that one of the two LexA proteins (LexA1) seems to be in control of the conventional Escherichia coli-like SOS response, while the other LexA protein (LexA2) regulates only its own transcriptional unit, which includes the imuA, imuB, and dnaE2 genes, and a gene (PP_3901) from a resident P. putida prophage. Furthermore, PP_3901 is also regulated by LexA1 and is required for DNA damage-mediated induction of several P. putida resident prophage genes. In silico searches suggested that this marked asymmetry in regulon contents also occurs in other Pseudomonas species with two lexA genes, and the implications of this asymmetry in the evolution of the SOS network are discussed.

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“…The requirement for an error-prone DNA polymerase and the requirement for stress responses are two features of mutagenesis in the Lac assay which are also found in many other stress-inducible mutagenesis mechanisms in E. coli and other species. DinB DNA polymerase is required for ciprofloxacin-induced resistance mutations in E. coli (10) and stationary-phase mutagenesis in starved cells of both Pseudomonas putida (38,74) and Bacillus subtilis (70). The SOS response is required for ciprofloxacin-induced resistance mutations (10), as well as mutagenesis in aging colonies (72), in E. coli.…”

mentioning

confidence: 99%

Stress-Induced β-Lactam Antibiotic Resistance Mutation and Sequences of Stationary-Phase Mutations in the Escherichia coli Chromosome

Petrosino

Galhardo²,

Morales³

et al. 2009

J Bacteriol

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In some enterobacterial pathogens, but not in Escherichia coli, loss-of-function mutations are a common route to clinically relevant ␤-lactam antibiotic resistance. We previously constructed an assay system for studying enterobacterial ␤-lactam resistance mutations using the well-developed genetics of E. coli by integrating enterobacterial ampRC genes into the E. coli chromosome. Like the cells of other enterobacteria, E. coli cells acquire ␤-lactam resistance by ampD mutation. Here we show that starvation and stress responses provoke ampD ␤-lactam resistance mutagenesis. When starved on lactose medium, Lac ؊ strains used in mutagenesis studies accumulate ampD ␤-lactam resistance mutations independent of Lac reversion. DNA double-strand break repair (DSBR) proteins and the SOS and RpoS stress responses are required for this mutagenesis, in agreement with the results obtained for lac reversion in these cells. Surprisingly, the stressinduced ampD mutations require DinB (DNA polymerase IV) and partially require error-prone DNA polymerase V, unlike lac mutagenesis, which requires only DinB. This assay demonstrates that real-world stressors, such as starvation, can induce clinically relevant resistance mutations. Finally, we used the ampD system to observe the true forward-mutation sequence spectrum of DSBR-associated stress-induced mutagenesis, for which previously only frameshift reversions were studied. We found that base substitutions outnumber frameshift mutations, as seen in other experimental systems showing stress-induced mutagenesis. The important evolutionary implication is that not only loss-of-function mutations but also change-of-function mutations can be generated by this mechanism.

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Study of involvement of ImuB and DnaE2 in stationary-phase mutagenesis in Pseudomonas putida

Cited by 35 publications

References 36 publications

Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

Cohabitation of Two DifferentlexARegulons inPseudomonas putida

Stress-Induced β-Lactam Antibiotic Resistance Mutation and Sequences of Stationary-Phase Mutations in the Escherichia coli Chromosome

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