CroSR391 , an ortholog of the λ Cro repressor, plays a major role in suppressing polVR391 -dependent mutagenesis

Abstract

When subcloned into low-copy-number expression vectors, rumAB, encoding polV_R391 (RumA'₂ B), is best characterized as a potent mutator giving rise to high levels of spontaneous mutagenesis in vivo. This is in dramatic contrast to the poorly mutable phenotype when polV_R391 is expressed from the native 88.5 kb R391, suggesting that R391 expresses cis-acting factors that suppress the expression and/or the activity of polV_R391 . Indeed, we recently discovered that SetR_R391 , an ortholog of λ cI repressor, is a transcriptional repressor of rumAB. Here, we report that CroS_R391 , an ortholog of λ Cro, also serves as a potent transcriptional repressor of rumAB. Levels of RumA are dependent upon an interplay between SetR_R391 and CroS_R391 , with the greatest reduction of RumA protein levels observed in the absence of SetR_R391 and the presence of CroS_R391 . Under these conditions, CroS_R391 completely abolishes the high levels of mutagenesis promoted by polV_R391 expressed from low-copy-number plasmids. Furthermore, deletion of croS_R391 on the native R391 results in a dramatic increase in mutagenesis, indicating that CroS_R391 plays a major role in suppressing polV_R391 mutagenesis in vivo. Inactivating mutations in CroS_R391 therefore have the distinct possibility of increasing cellular mutagenesis that could lead to the evolution of antibiotic resistance of pathogenic bacteria harboring R391.

Keywords: DNA polymerase V; R391; SOS response; integrating conjugative element; mutagenesis.

FIGURE 1

Cartoon of the rumAB promoter region. A single SetR/CroS binding site is shown in blue color. This site partially overlaps with the −35 promoter element, shown in gold. A LexA binding site is shown in green, which partially overlaps with the −10 promoter element (also shown in gold). The ribosome binding site (RBS) is shown in purple, and the first two codons of RumA are shown in red

FIGURE 2

Spontaneous mutagenesis promoted by R391, or pRW290, in different genetic backgrounds. Cells were plated on minimal low histidine agar plates as described in Section 4.3: Qualitative analysis of spontaneous reversion of the hisG4(Oc) allele. His⁺ revertants appear as creamy white colonies against the dark background behind the agar plate. As observed, the 88.5‐kb R391 promotes low levels of spontaneous mutagenesis in all genetic backgrounds. This is in contrast to pRW290, which only expresses the rumAB _R391 operon from a low‐copy‐number vector. Although the rumAB operon is subject to transcriptional regulation by the LexA repressor, there is little difference in mutagenesis between lexA ⁺ (RW120) and lexA51(Def) (RW546) strains. Mutagenesis increases significantly when RecA is partially activated for co‐protease functions (recA718; MVG114) or fully activated for co‐protease functions (recA730; RW578)

FIGURE 3

RumAB_R391‐dependent mutagenesis is regulated by croS _R391. The histidine reversion assay was performed on the E. coli strain MVG114 or MVG114 strains transformed with pRLH421 or various croS _R391 or setR _R391 wild‐type or deletion combinations. (wt/ΔC) represents MVG114 transformed with the plasmid pRLH421 that contains ~21.5 kb of R391 and harbors a C‐terminal deletion of the croS _R391 gene. MVG114 was transformed with plasmids pJM1355, pJM1356, pJM1359 and pJM1360, and the croS _R391 or setR _R391 genotypes are indicated. Error bars indicate standard error of the mean (SEM)

FIGURE 4

CroS_R391 trans‐regulation of RumAB_R391‐dependent mutagenesis. (a) Spontaneous histidine reversion mutagenesis assays utilizing MVG114 strains harboring pJM1378 alone (−/−), a pCC1 derivative (copy‐control plasmid) carrying the rumAB _R391 operon, were transformed with low‐copy pGB2 derivatives (pJM1365, pJM1366, pJM1367, and pJM1368) carrying various iterations of the croS _R391/setR _R391 operon (Table 1). The genotypes of croS _R391 or setR _R391, either wild‐type or deleted, are indicated. (b) Western blot analysis using an anti‐CroS antibody indicating that only strains harboring a plasmid with a wild‐type croS _R391 gene express any CroS protein. (c) Western blot using an anti‐RumA antibody indicating that strains that express the CroS protein have significantly reduced levels of the RumA protein. However, strains that express only SetR show no reduction in the level of RumA protein. Numbers reported for the levels of RumA and RumA′ are relative to RumA in track 1

FIGURE 5

CroS_R391 regulation of RumAB_R391‐dependent mutagenesis is dependent on the rumAB _R391 promoter region. (a) Spontaneous histidine reversion mutagenesis assays utilizing MVG114 strains harboring pJM1467 (−/−), a pCC1 derivative carrying the rumAB _R391 operon under the control of the recA promoter, were transformed with low‐copy pGB2 derivatives (pJM1365, pJM1366, pJM1367, and pJM1368) carrying various iterations of the croS _R391‐setR _R391 operon (Table 1). The genotypes of croS _R391 or setR _R391, either wild‐type or deleted, are indicated. The histogram illustrates the mean colony count for each indicated strain (n = 5). Error bars represent the standard error of the mean (SEM). An unpaired two‐tailed t test was used to compare the mean colony counts for the ΔsetR ΔcroS and the ΔsetR croS ⁺ strains. * = p < .05. (b) Western blot using an anti‐RumA antibody indicating that the level of RumA expressed from the E. coli recA promoter does not change appreciably in the presence, or absence, of SetR or CroS. Numbers reported for the expression levels of RumA and RumA′ are relative to RumA in the left‐hand lane

FIGURE 6

Expression of RecA, RumA, and RumA′ in wild‐type recA ⁺ lexA ⁺ cells after exposure to the SOS‐inducing antibiotic, Ciprofloxacin. Western blot analysis was performed on whole‐cell protein extracts from RW520 (recA ⁺ lexA ⁺) harboring pRLH421 derivatives (pJM1355, pJM1356, pJM1359, or pJM1360), with various croS _R391 or setR _R391 wild type, or deletion combinations as indicated. To induce the SOS response, cells were treated with 30 ng ml^–1 Ciprofloxacin for various times, as indicated in the figure. Levels of RecA and RumA/ RumA′ were detected using affinity purified polyclonal rabbit antibodies to RecA and RumA proteins. The number reported for the level of RumA or RumA’ is relative to a cross‐reacting band in the same track

FIGURE 7

The galK2(Oc) reversion papillation assay of R391 or R391ΔcroS strains. R391 or R391ΔcroS were moved into MVG114 by conjugal transfer. Cells were plated onto MacConkey‐galactose agar media and grown for 8 days at 37℃. Pictures of representative colonies from each strain were taken showing the appearance of Gal+ papillae indicating the level of mutagenesis occurring within the colonies. The MVG114/R391ΔcroS colonies contain approximately 10−50 times the number of revertant papillae, when compared to the MVG114/R391 colonies

FIGURE 8

Rifampicin mutagenesis in R391 and R391ΔcroS strains. MVG114, MVG114/R391, and MVG114/R391ΔcroS cultures were started from with approximately 1,000 cells, or less, and grown overnight to stationary phase. Cells were plated onto LB agar plates containing rifampicin to select for rifampicin‐resistant mutants. Appropriate dilutions were plated to LB agar plates to determine viable counts, and the frequency of mutagenesis to rifampicin resistance was calculated. Error bars represent the standard error of the mean (SEM). The MVG114/R391ΔcroS strain exhibits an ~10‐fold higher frequency of mutagenesis to rifampicin resistance than the MVG114/R391 strain