A ProQ/FinO family protein involved in plasmid copy number control favours fitness of bacteria carrying mcr-1-bearing IncI2 plasmids

Abstract

The plasmid-encoded colistin resistance gene mcr-1 challenges the use of polymyxins and poses a threat to public health. Although IncI2-type plasmids are the most common vector for spreading the mcr-1 gene, the mechanisms by which these plasmids adapt to host bacteria and maintain resistance genes remain unclear. Herein, we investigated the regulatory mechanism for controlling the fitness cost of an IncI2 plasmid carrying mcr-1. A putative ProQ/FinO family protein encoded by the IncI2 plasmid, designated as PcnR (plasmid copy number repressor), balances the mcr-1 expression and bacteria fitness by repressing the plasmid copy number. It binds to the first stem-loop structure of the repR mRNA to repress RepA expression, which differs from any other previously reported plasmid replication control mechanism. Plasmid invasion experiments revealed that pcnR is essential for the persistence of the mcr-1-bearing IncI2 plasmid in the bacterial populations. Additionally, single-copy mcr-1 gene still exerted a fitness cost to host bacteria, and negatively affected the persistence of the IncI2 plasmid in competitive co-cultures. These findings demonstrate that maintaining mcr-1 plasmid at a single copy is essential for its persistence, and explain the significantly reduced prevalence of mcr-1 following the ban of colistin as a growth promoter in China.

Figure 1.

Effects of pHNSHP45-encoded ProQ/FinO family protein PcnR on bacterial growth and fitness cost. (A) Effects of pHNSHP45 encoded putative regulators on bacterial growth. These data represent the mean of three independent experiments. (B) Relative fitness of E. coli BW25113 harbouring pHNSHP45Δorf00050::kan, pHNSHP45Δorf00039::kan, pHNSHP45ΔpcnR::kan, and its complementation strain pHNSHP45ΔpcnR::kan-pHSG575-pcnR in vitro competition. The reference strain is BW25113/pHNSHP45. Complementation assays were performed by expressing pcnR from its native promoter on pHSG575-pcnR. All competitions assays were carried out with three biological replicates and the relative fitness of each strain was detected at 24, 48 and 72 h. (C) The colonial morphology of E. coli BW25113/pHNSHP45 and BW25113/pHNSHP45ΔpcnR on LB agar plate.

Figure 2.

The fitness cost of pHNSHP45ΔpcnR is associated with mcr-1. (A) Comparison of the nucleotide sequence between pHNSHP45ΔpcnR and plasmids in transposon mutants. From inside to outside, coding sequences of pHNSHP45 appear on the innermost circle in grey. The second circle in orange represents the nucleotide sequence of pHNSHP45ΔpcnR. The following circles in red, blue, purple, cyan, and green represent plasmids pTns1, pTns2, pTns3, pTns4 and pTns7, respectively. The 15–19 kb regions containing mcr-1 between nikB and pilP genes are missing from pTns1, pTns2, pTns3 and pTns7 plasmids. A point mutation (shown by black triangle) was located in the -10 region of mcr-1 promoter on pTns4 plasmid. The circular BLAST Atlas was computed by the GView server (https://server.gview.ca/), and each plasmid was mapped against pHNSHP45 (GenBank accession number: KP347127). (B) Effect of point mutation (T-C) in the -10 region of promoter on the expression of mcr-1. Error bars represent the SD and P-values were calculated by One-way ANOVA test. (C) Deletion of mcr-1 restores the growth of BW25113/pHNSHP45ΔpcnR. Values represent the mean of three independent experiments. (D) Deletion of mcr-1 restore the relative fitness of BW25113/pHNSHP45ΔpcnR. E. coli BW25113 harbouring pHNSHP45ΔpcnR::kan, pHNSHP45ΔpcnRΔmcr-1::kan and pHNSHP45ΔpcnRΔmcr-1::kan-pHSG575-mcr-1 were competed with the reference strain BW25113/pHNSHP45 in vitro separately. All competitions assays were carried out with three biological replicates and the relative fitness of each strain was detected at 24, 48 and 72h.

Figure 3.

The effect of antibiotic resistant genes (bla_CTX-M-55 or mcr-1) on the fitness of E. coli BW25113/pHN1122-1(A) and BW25113/pHNSHP45 (B). E. coli BW25113/pHNSHP45 and BW25113/pHN1122-1 were competed with E. coli BW25113/pHNSHP45Δmcr-1 and BW25113/pHN1122–1Δbla_CTX-M-55, respectively. All competitions assays were carried out with three biological replicates and last for 10 days. The relative fitness of each strain was detected every day (from D1 to D10). P-values were calculated by one-way ANOVA test.

Figure 4.

Regulation of RepA. (A) Schematic representation of the 5′untranslated region (UTR) of repA. The upstream nucleotide sequence (−497 to +45) of repA was fused with lacZ to monitor the expression of repA, and the resulting fusion was named P_repA-lacZ. The transcription of repA gene starts from the position -253, and the resulting polycistronic mRNA that contains ORFs encoding RepR and RepA is indicated by a purple arrow. The reading frame of repR overlaps the repA start codon (GUG). The transcription of antisense RNA (AS RNA) starts from the position -68, and a predicted rho-independent transcription terminator was located at the end of antisense RNA. The antisense RNA is indicated by red arrow. The -35 region of the promoter of antisense RNA (AS RNA) was mutated (CA-TT) to construct ΔAS RNA mutation. The rho-independent transcription terminator was predicted by ARNold: finding terminators web server (http://rssf.i2bc.paris-saclay.fr/toolbox/arnold/index.php). The upstream nucleotide sequence (−497 to +45) of repA is shown in Supplementary Figure S4. (B) Effect of pcnR on the expression of repA. Activity of P_repA was monitored from lacZ fusion (P_repA-lacZ) and pcnR is under control of its native promoter on pHSG575-pcnR plasmid. (C) The translation of RepA is coupled with RepR. Constructing T-A mutation at +23 position of repR to introduce a stop codon in repR open reading frame, and the resulting mutation was named repR (T23A). (D) Effect of antisense RNA (AS RNA) on the expression of RepA. Complementation assays were performed by expressing the AS RNA from its native promoter on pUC19-AS RNA. Error bars represent the SD and P-values were calculated by two-tailed t-tests.

Figure 5.

PcnR regulates the expression of RepA by binding to the repR mRNA. (A) Predicted RNA secondary structure of the repR mRNA. This structure was predicted by RNAfold web server. The Shine-Dalgarno (SD) sequence of repR is shown in green and the stem-loop structure containing the start codon of repR is highlighted in blue. The start codon of repR is shown in red, and A is numbered ‘+ 1’. The stop codon of repR is highlighted in purple. The mutational changes introduced for the present study are shown by arrows. The predicted secondary structure of the leader region of repA mRNA was shown in Supplementary Figure S8B. (B) The inhibition of PcnR on RepA expression is independent of antisense RNA. Activity of P_repA with ΔAS RNA mutation was monitored from the lacZ fusions, and expression of pcnR is controlled by P_ara with (+) or without (−) arabinose. Error bars represent the SD and P-values were calculated by two-tailed t-tests. (C) The inhibition of PcnR on RepA expression is dependent on the RNA secondary structure of repR mRNA. The stem structure of repR mRNA is predicted to be eliminated by GAAA-CTCT mutation and is denoted by stem mutation (GAAA-CTCT). The GA-TT mutation was located in the loop structure of repR mRNA and is denoted by loop mutation (GA-TT). The repR₉ fusion and repR₂₄ fusion represent the lacZ were fused with repR at + 9 and + 24 positions, respectively. Activity of P_repA with these mutations was monitored from the lacZ fusions. Expression of pcnR is controlled by P_ara with (+) or without (−) arabinose. Error bars represent the SD and P-values were calculated by two-tailed t-tests. (D) The first stem-loop structure of repR mRNA interacts with PcnR protein in vitro. 6 His-tagged PcnR was expressed in E. coli BL21(DE3) from pET28b-pcnR and purified by Ni-NTA affinity chromatography. Purified His-tag-PcnR was cut by thrombin to remove the His-tag and then purified PcnR was used for RNA-EMSA.

Figure 6.

Bacterial population dynamics in co-cultures with plasmid-free and plasmid-containing E. coli BW25113. Plasmid-containing strains including E. coli BW25113/pHNSHP45, BW25113/pHNSHP45ΔpcnR::kan, BW25113/pHNSHP45Δmcr-1::kan,BW25113/pHNSHP45ΔpcnRΔmcr-1::kan were mixed with a 1000-fold excess of plasmid-free E. coli BW25113 at the beginning of the invasion assay. E. coli BW25113 is indicated by black dot, BW25113/pHNSHP45 is indicated by green dot, BW25113/pHNSHP45Δmcr-1::kan is indicated by blue dot, BW25113/pHNSHP45ΔpcnR::kan is indicated by orange dot, and BW25113/pHNSHP45ΔpcnRΔmcr-1::kan is indicated by red dot. (A) Co-cultures with E. coli BW25113 and BW25113/pHNSHP45. (B) Co-cultures with E. coli BW25113 and BW25113/pHNSHP45ΔpcnR::kan. (C) Co-cultures with E. coli BW25113 and BW25113/pHNSHP45Δmcr-1::kan. (D) Co-cultures with E. coli BW25113 and BW25113/pHNSHP45ΔpcnRΔmcr-1::kan. (E) Co-cultures with E. coli BW25113, BW25113/pHNSHP45, and BW25113/pHNSHP45ΔpcnR::kan. (F) Co-cultures with E. coli BW25113, BW25113/pHNSHP45 and BW25113/pHNSHP45Δmcr-1::kan. (G) Co-cultures with E. coli BW25113, BW25113/pHNSHP45, and BW25113/pHNSHP45ΔpcnRΔmcr-1::kan. Bars represent SD of three biological replicates.

Figure 7.

The pHNSHP45 plasmid-encoded PcnR protein favours the fitness of bacteria carrying mcr-1-bearing IncI2 plasmids. (A) PcnR enhances the fitness of E. coli BW25113 harbouring pHNSHP45 by repressing the plasmid copy number. pHNSHP45 plasmid is the first discovered mcr-1⁺-IncI2 plasmid. Deletion of pcnR increases plasmid copy number by 10-fold and largely impairs the growth of the host. Deletion of mcr-1 completely rescues the growth of pcnR^– strain, although with high plasmid copy number. Complementation of mcr-1 in E. coli BW25113 harbouring pHNSHP45ΔpcnR confirmed that host bacteria are sensitive to multicopy mcr-1. Complementation of pcnR represses the copy number of pHNSHP45ΔpcnR. Summary: PcnR maintains a balance between the mcr-1 expression level and bacterial fitness by limiting the copy number of the IncI2 plasmid pHNSHP45 to ensure an appropriate expression level of mcr-1. (B) PcnR maintains the plasmid copy number at a single copy by inhibiting the translation of repA. Deletion of pcnR derepresses the expression of repA and leads to the plasmid copy number increase to ∼10 copies.