Evolved plasmid-host interactions reduce plasmid interference cost

Abstract

Antibiotic selection drives adaptation of antibiotic resistance plasmids to new bacterial hosts, but the molecular mechanisms are still poorly understood. We previously showed that a broad-host-range plasmid was poorly maintained in Shewanella oneidensis, but rapidly adapted through mutations in the replication initiation gene trfA1. Here we examined if these mutations reduced the fitness cost of TrfA1, and whether this was due to changes in interaction with the host's DNA helicase DnaB. The strains expressing evolved TrfA1 variants showed a higher growth rate than those expressing ancestral TrfA1. The evolved TrfA1 variants showed a lower affinity to the helicase than ancestral TrfA1 and were no longer able to activate the helicase at the oriV without host DnaA. Moreover, persistence of the ancestral plasmid was increased upon overexpression of DnaB. Finally, the evolved TrfA1 variants generated higher plasmid copy numbers than ancestral TrfA1. The findings suggest that ancestral plasmid instability can at least partly be explained by titration of DnaB by TrfA1. Thus under antibiotic selection resistance plasmids can adapt to a novel bacterial host through partial loss of function mutations that simultaneously increase plasmid copy number and decrease unfavorably high affinity to one of the hosts' essential proteins.

Fig 1. Improvement of plasmid persistence after plasmid adaptation to S. oneidensis

(A) Representative plasmid persistence curves in the absence of selection for the plasmid. Curves were redrawn based on data from our previous study (Sota et al., 2010). (B) Genes and sites around the trfA locus in wild-type pBP136 and its derived mini-replicon pMS0506, used in this study. Triangles indicate iterons and pentagons indicate DnaA-boxes. (C) Locations of TrfA1 mutations and conserved motif in Rep proteins. DBD1 and DBD2 both indicate iteron-binding motifs, M98 and M124 are start methionines for TrfA-33 and TrfA2, respectively. QLSLF is a motif associated with binding to sliding clamp (Kongsuwan et al., 2006).

Fig 2. Effect of TrfA1 mutations on maximum growth rates

Relative maximum growth rates of bacterial cells carrying a cloned trfA gene are shown. A: S. oneidensis; B. E. coli; C, P. putida; D, C. pinatubonensis. Open circles represent means and error bars standard deviations. The M124L mutation was introduced into trfA1 and all its variants. For S. oneidensis and P. putida, the strain carrying wild-type trfA1 had a significantly lower growth rate than the same strain with trfA1 variants (P < 0.001, in multiple comparisons in Dunnett’s test), but this was not the case for the other two hosts.

Fig 3. DnaB-loading and activation activity of TrfA1 and its variants

FI indicates the position of supercoiled substrate, pMS0506, containing oriV. FI* indicates the position of unwound substrate, and is indicative of DnaB-loading and activation activity. The 1st lane (left) indicates a control reaction without TrfA protein. To make constitutively monomeric TrfA, Q279D/S292L mutations were introduced into all TrfA proteins. A M124L mutation was also introduced into all TrfA1 variants to avoid production of TrfA2.

Fig 4. Analysis of DnaB helicases interactions with TrfA proteins

Interactions of DnaB helicase from S. oneidensis, E. coli or P. putida with monomeric forms of TrfA1, R31P, A25T, Δ43 and TrfA2 proteins were analyzed with Surface Plasmon Resonance. TrfA proteins were immobilized on the surface of CM5 sensorchip. Increasing amounts of DnaB proteins (7, 15, 30, 60, 125, 250, and 500 nM) were flown over the sensor chip. TrfA proteins contain the M124L/Q279D/S292L mutations.

Fig.5. Two models for the possible cause of plasmid interference cost associated with the replication initiation protein

Left: Titration model. TrfA1, which shows high affinity to DnaB DNA helicase, reduces the amount of free DnaB available for chromosomal replication and repair. Right: Inhibition model. The TrfA1-DnaB complex occurring at the replication fork arrests replication or repair, leading to exposure of single strand DNA followed by strand break. This may occur on both the plasmid and chromosome.

Fig 6. Effect of DnaB overproduction on plasmid persistence in S. oneidensis

The data points indicate means and standard deviations from triplicate assays. (A) Persistence of pMS0506. In this data set, the dnaB⁺ strain with 10 µM IPTG, and 100 µM IPTG showed improved persistence compared to the dnaB⁻ strain (See ΔBIC test in Table S3, and quasi-extinction time in Table S4). In a second set of experiments for 0 µM IPTG condition, a significant difference was observed between dnaB⁺ and dnaB⁻ strains (Fig. S5). (B) Persistence of pEvo-Sh1. (C) Relative dnaB mRNA levels determined by qPCR; the mean value for the dnaB⁻ strain, 0 µM IPTG, was represented as 1 in this plot. Error bars indicate SD. The host used were S. oneidensis HY1014 (chr::mini-Tn7-tac_p-dnaB; dnaB⁺), and S. oneidensis HY0759 (chr::mini-Tn7; dnaB⁻). For the dnaB⁻ strain, only IPTC 100 µM conditions were shown for simplicity.

Fig 7. Effect of TrfA1 mutations on plasmid copy number

(A) Relative plasmid copy number (PCN) in S. oneidensis. The copy number of pHY872 (Fig.1) for the TrfA1 condition is represented as 1. The data are shown on a logarithmic scale. (B) Relative PCN in E. coli. TrfA proteins were expressed from the chromosome (see materials and methods for details) ‘TrfA1+TrfA2’ indicates expression of two proteins from the wild-type trfA1 gene. The M124L mutation was introduced into TrfA1 and TrfA1 variants. In (B), the data for TrfA1+TrfA2, TrfA1, and TrfA2 were previously published (Yano et al., 2012). The trfA-tagged strains used were HY0321, HY0323, HY0325, HY0329, HY0333, HY0337, HY0341, HY0345 for panel (A), and HY0414, HY0390, HY0391, HY0392, HY0395, HY0408, HY0409, HY0475 for panel (B). Comparisons were made using the post-hoc Tukey test for the data before normalization to relative PCN (**, P <0.01; ***, P <0.001; n.s., not significant). Data are obtained from at least four independently grown cultures for each clone.