An SOS-regulated operon involved in damage-inducible mutagenesis in Caulobacter crescentus

Abstract

DNA polymerases of the Y-family, such as Escherichia coli UmuC and DinB, are specialized enzymes induced by the SOS response, which bypass lesions allowing the continuation of DNA replication. umuDC orthologs are absent in Caulobacter crescentus and other bacteria, raising the question about the existence of SOS mutagenesis in these organisms. Here, we report that the C.crescentus dinB ortholog is not involved in damage-induced mutagenesis. However, an operon composed of two hypothetical genes and dnaE2, encoding a second copy of the catalytic subunit of Pol III, is damage inducible in a recA-dependent manner, and is responsible for most ultraviolet (UV) and mitomycin C-induced mutations in C.crescentus. The results demonstrate that the three genes are required for the error-prone processing of DNA lesions. The two hypothetical genes were named imuA and imuB, after inducible mutagenesis. ImuB is similar to proteins of the Y-family of polymerases, and possibly cooperates with DnaE2 in lesion bypass. The mutations arising as a consequence of the activity of the imuAB dnaE2 operon are rather unusual for UV irradiation, including G:C to C:G transversions.

Figure 1

Prototypical genomic organizations of dnaE2 and accompanying imuA and imuB genes. The same organization found in C.crescentus is also present in α-proteobacterial genomes, Pseudomonas aeruginosa, Vibrio vulnificus, Vibrio parahaemolyticus, Ralstonia solanacearum, Bordetella bronchiseptica and Bordetella parapertussis. A duplication of lexA preceding the operon, as found in P.putida, is also present in Methylococcus capsulatus, Pseudomonas syringae, Xanthomonas campestris and Xanthomonas axonopodis. The M.tuberculosis gene arrangement is conserved in all species of the genera Mycobacterium and Corynebacterium, and in Nocardia farcinica. Small boxes represent known SOS operators, and interrupted lines indicate that the genes are not contiguous in the genome. Loci numbers as provided by the genome annotation are indicated above, the arrows representing genes, and our annotation is given inside the arrows. Different original gene annotations are indicated in parentheses.

Figure 2

Phylogenetic relationships between DnaE and DnaE2. Phylogenetic analyses included DnaE and DnaE2 proteins of all fully sequenced genomes where such duplication could be found. Only M.tuberculosis and Mycobacterium bovis were included for simplification, although the duplication can be found in several other Mycobacterial genomes. The names of the ORFs and species name abbreviations shown are the same adopted in the genome annotation of each organism. Abbreviations of organism names are as follows: AGR, A.tumefaciens str. C58; b, E.coli K12; BB, B.bronchiseptica RB50; bll, Bradyrhizobium japonicum USDA 110; BMA, Burkholderia mallei ATCC 23344; BMEI, Brucella melitensis 16M; BPP, B.parapertussis 12822; BPSL, Burkholderia pseudomallei K96243; BR, Brucella suis 1330; CC, C.crescentus CB15; CE, Corynebacterium efficiens YS-314; DIP, Corynebacterium diphtheriae NCTC 13129; Mb, M.bovis AF2122/97; MCA, M.capsulatus str. Bath; mll and mlr, Mesorhizobium loti MAFF303099; NCgl, Corynebacterium glutamicum ATCC 13032; Nfa, N.farcinica IFM 10152; PA, Pseudomonas aeruginosa PAO1; PP, P.putida KT2440; PPA, P.acnes KPA171202; PSPTO, P.syringae pv. tomato str. DC3000; RB, R.baltica SH 1; RPA, Rhodopseudomonas palustris CGA009; Rsc and Rsp, R.solanacearum GMI1000; Rv, M.tuberculosis H37Rv; SAV, S.avermitilis MA-4680; Smc and Sma, S.meliloti 1021; VP, V.parahaemolyticus RIMD 2210633; VV, V.vulnificus YJ016; XAC, X.axonopodis pv. citri str. 306; and XCC, Xanthomonas campestris pv. campestris str. ATCC 33913. The numbers indicate the bootstrap values >50%.

Figure 3

Phylogenetic relationships between ImuB and DinB. Phylogenetic analyses included ImuB and DinB proteins of all fully sequenced genomes where the imuB gene could be found. Only M.tuberculosis and M.bovis were included for simplification, although imuB can be found in several other Mycobacterial genomes. The names of the ORFs and species name abbreviations shown are the same adopted in the genome annotation of each organism. Abbreviations of organism names are indicated in the legend of Figure 2. The numbers indicate the bootstrap values >50%.

Figure 4

Expression of the imuAB dnaE2 operon. (A) Schematic diagram for this operon indicating the position of the oligonucleotides (small arrows) used in the RT–PCR strategy to detect the co-expression of the three genes. Template RNA was extracted from exponentially growing cells, both from non-irradiated cultures and from cultures irradiated with 60 J/m² of UV and recovered after 45 min. The RT–PCR products were visualized after electrophoresis in 1% agarose gel stained with ethidium bromide. (B) Expression of uvrA and imuA promoters in transcriptional fusions with the lacZ gene. β-Galactosidase activity was measured 90 min after irradiation with UV light, both in the wild-type and recA strains.

Figure 5

Quantitative RT–PCR analysis of UV-induced gene expression. RNAs were extracted from exponentially growing recA and wild-type cells before and 45 min after irradiation with 60 J/m² UVC. Relative levels of expression shown represent the average of two independent experiments done in triplicate.

Figure 6

Effects of imuAB, dnaE2 and dinB mutations on UV resistance and mutagenesis in C.crescentus. (A) Survival curves. (B) Frequency of Rif^r mutations induced by UV in the rpoB gene. The results shown are the mean of at least five independent experiments done in triplicate. Error bars indicate the SD.

Figure 7

Effects of imuAB, dnaE2 and dinB mutations on mit C resistance and mutagenesis in C.crescentus. (A) Survival curves. (B) Frequency of Rif^r mutations induced by mit C in the rpoB gene. The results shown are the mean of at least four independent experiments done in triplicate. Error bars indicate the SD.

Figure 8

UV mutation spectra in the rpoB gene. Nucleotides 1557–1625, comprising the cluster II of rifampicin resistance, are shown in this figure. All mutants analyzed had mutations in this region of the rpoB gene. Mutations in shaded background correspond to repetitions of the same base substitution, and tandem mutations are underlined. The total number of independent mutations sequenced is indicated for each strain.