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, 42 (4), 2366-79

Identification of Genes Involved in Low Aminoglycoside-Induced SOS Response in Vibrio Cholerae: A Role for Transcription Stalling and Mfd Helicase

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Identification of Genes Involved in Low Aminoglycoside-Induced SOS Response in Vibrio Cholerae: A Role for Transcription Stalling and Mfd Helicase

Zeynep Baharoglu et al. Nucleic Acids Res.

Abstract

Sub-inhibitory concentrations (sub-MIC) of antibiotics play a very important role in selection and development of resistances. Unlike Escherichia coli, Vibrio cholerae induces its SOS response in presence of sub-MIC aminoglycosides. A role for oxidized guanine residues was observed, but the mechanisms of this induction remained unclear. To select for V. cholerae mutants that do not induce low aminoglycoside-mediated SOS induction, we developed a genetic screen that renders induction of SOS lethal. We identified genes involved in this pathway using two strategies, inactivation by transposition and gene overexpression. Interestingly, we obtained mutants inactivated for the expression of proteins known to destabilize the RNA polymerase complex. Reconstruction of the corresponding mutants confirmed their specific involvement in induction of SOS by low aminoglycoside concentrations. We propose that DNA lesions formed on aminoglycoside treatment are repaired through the formation of single-stranded DNA intermediates, inducing SOS. Inactivation of functions that dislodge RNA polymerase leads to prolonged stalling on these lesions, which hampers SOS induction and repair and reduces viability under antibiotic stress. The importance of these mechanisms is illustrated by a reduction of aminoglycoside sub-MIC. Our results point to a central role for transcription blocking at DNA lesions in SOS induction, so far underestimated.

Figures

Figure 1.
Figure 1.
Plasmid pTOX-SOS. Described in the text. Briefly, this plasmid codes for a CcdAB toxin–antitoxin system derived from the plasmid F toxin–antitoxin module. The CcdB toxin blocks the gyrase activity and is lethal, except when bound by the CcdA antitoxin. The ccdA antitoxin gene is placed here under the control of a lac promoter that can be repressed by LacI, and the LacI production is placed under the control of the SOS-inducible recN promoter. When SOS is induced, lacI transcription from the recN promoter is induced. LacI represses the lac promoter and CcdA expression. The absence of CcdA expression results in CcdB toxicity for the cell carrying this construct. In summary, when SOS is induced, the CcdB toxin kills the bacteria, and when SOS is repressed, the CcdB toxin is inactivated. Thus, only bacteria unable to induce SOS survive to form colonies on sub-MIC antibiotics.
Figure 2.
Figure 2.
V. cholerae mutants unable to induce SOS after sub-MIC tobramycin treatment. GFP fused to the SOS-induced intIA promoter (p4640 in Table 3) was used to compare the fluorescence of various mutants with the wild-type V. cholerae. Histogram bars represent the ratio of GFP fluorescence in the presence of antibiotic over fluorescence in LB and thus reflect the induction of SOS by TOB or MMC. Error bars represent standard deviation. Each strain was tested at least four times. TOB was used at 0.01 µg/ml. Mitomycin C was used at 0.1 µg/ml. (A) Deletion of identified 3R genes (Table 1) in wild-type V. cholerae. WT stands for wild-type V. cholerae. 3R stands for replication recombination repair mutants. (B) Over-expression of genes identified in Table 2 in wild-type V. cholerae. p_empty stands for empty circularized pTOPO plasmid. p_VCxxx stands for pTOPO expressing the indicated gene.
Figure 3.
Figure 3.
Model for SOS induction by sub-MIC aminoglycosides. Adapted from (10). In the presence of sub-MIC AGs, DNA damage is induced by 8-oxo-G incorporation or through impaired action of DNA replication and repair proteins. When present and stable, RpoS protects cells from this type of DNA damage (10). Possible implications of the 3R mutants are discussed in the text.
Figure 4.
Figure 4.
VC1636 YejHvc has no RNA degrading activity at RNA–DNA hybrids when over-expressed in E. coli. Cells were propagated at 30°C for 1 h 30 min and were shifted to 42°C (time 0). Appropriate dilutions were plated at the indicated times and incubated at 30°C to allow growth. The Y axis shows survival at 42°C. Error bars represent standard deviation. Each strain was tested three times.
Figure 5.
Figure 5.
VC1636 YejHvc over-expression can complement the UV sensitivity of an E. coli Δmfd strain. Serial dilutions of exponential cultures (OD ≈ 0.5) of different strains were plated. Plates were UV irradiated at 0/40/60 J/m2 and incubated for 24 h at 37°C. The ratios of the numbers of colonies on irradiated plates to those on non-irradiated plates were calculated. The Y axis shows survival, i.e. the ratio of colony-forming units (cfu) at the indicated UV dose over cfu of non-irradiated culture. Error bars represent standard deviation. Each strain was tested at least three times.
Figure 6.
Figure 6.
Impact of 3R gene inactivations in V. cholerae on growth in sub-MIC tobramycin. Histogram bars represent the ratio of doubling time of the mutant over isogenic wild-type strain in LB or TOB, used at 0.25 µg/ml, which corresponds to 25% of the MIC. ΔrpoS where SOS is induced by TOB was used as control. ∞ means that bacteria did not grow. Error bars represent standard deviation. Each strain was tested at least three times.
Figure 7.
Figure 7.
Role of the NER and MMR pathways on sub-MIC tobramycin mediates SOS induction. Histogram bars represent the percentage of GFP fluorescence (i.e. SOS induction) in indicated V. cholerae strains in LB or sub-MIC TOB. gfp fusion with the SOS-induced promoter of intIA is carried by plasmid p4640 as described in Table 3. TOB was used at 0.01 µg/ml. Error bars represent standard deviation. Each strain was tested at least three times.
Figure 8.
Figure 8.
Model proposed for the involvement of RNAP in SOS induction after sub-MIC AG treatment. Discussed in the text.

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