Roles of long and short replication initiation proteins in the fate of IncP-1 plasmids

Abstract

Broad-host-range IncP-1 plasmids generally encode two replication initiation proteins, TrfA1 and TrfA2. TrfA2 is produced from an internal translational start site within trfA1. While TrfA1 was previously shown to be essential for replication in Pseudomonas aeruginosa, its role in other bacteria within its broad host range has not been established. To address the role of TrfA1 and TrfA2 in other hosts, efficiency of transformation, plasmid copy number (PCN), and plasmid stability were first compared between a mini-IncP-1β plasmid and its trfA1 frameshift variant in four phylogenetically distant hosts: Escherichia coli, Pseudomonas putida, Sphingobium japonicum, and Cupriavidus necator. TrfA2 was sufficient for replication in these hosts, but the presence of TrfA1 enhanced transformation efficiency and PCN. However, TrfA1 did not contribute to, and even negatively affected, long-term plasmid persistence. When trfA genes were cloned under a constitutive promoter in the chromosomes of the four hosts, strains expressing either both TrfA1 and TrfA2 or TrfA1 alone, again, generally elicited a higher PCN of an IncP1-β replicon than strains expressing TrfA2 alone. When a single species of TrfA was produced at different concentrations in E. coli cells, TrfA1 maintained a 3- to 4-fold higher PCN than TrfA2 at the same TrfA concentrations, indicating that replication mediated by TrfA1 is more efficient than that by TrfA2. These results suggest that the broad-host-range properties of IncP-1 plasmids are essentially conferred by TrfA2 and the intact replication origin alone but that TrfA1 is nonetheless important to efficiently establish plasmid replication upon transfer into a broad range of hosts.

Fig 1

(A) Schematic representation of the diversity in TrfA structure. M98 and M124 indicate the start methionines of TrfA-33 in RK2 and TrfA2 in pBP136, respectively. QLSLF indicates the conserved amino acid sequence motif responsible for interaction with DNA polymerase III (33). DBD1 and DBD2 indicate DNA-binding (winged-helix) domains deduced from secondary structure comparisons (22, 49). (B) The trfA gene locus of pBP136 and its mini-pBP136 derivative pMS0506. A+T and G+C indicate the A/T- and G/C-rich regions, respectively. Triangles and pentagons indicate iterons (TrfA-binding motif) and DnaA-boxes (DnaA-binding motif), respectively. Flags indicate promoters. The region cloned in pHY872 is indicated. (C) Western blots showing TrfA production from pMS0506 in three hosts: E. coli, P. putida, and S. japonicum. The quantities of cell extracts were normalized to total protein content using a Bradford assay (11). Exp, exponential phase; Stat, stationary phase.

Fig 2

Persistence of pMS0506 and pEvo-Sh15 (the trfA1 frameshift variant) in four different bacterial hosts. Each data point represents the mean value obtained from four replicate assays for E. coli, S. japonicum, and C. necator, and three assays for P. putida.

Fig 3

Plasmid copy numbers in the presence of TrfA1 alone, TrfA2 alone, or both proteins. (A) Experimental design. TrfA proteins were expressed in the presence of 10 μM IPTG from the tac promoter inserted at the attTn7 site in the chromosomes. Plasmid copy number was determined as the ratio of the copy number of tetA in the introduced IncP-1β replicon pHY872 to the copy number of a gene in the chromosomal oriC region at stationary phase. (B) Plasmid copy numbers and TrfA levels in the four hosts. In the graphs, the value and the error bars indicate the mean and standard error of the mean obtained from at least three replicate experiments. Significance was determined by multiple comparisons (Tukey's test): *, P < 0.05; **, P < 0.01; ***, P < 0.001. The lower panels show the Western blots of the corresponding TrfA production under each condition. For each strain, the same amount of total protein was loaded in each lane of the SDS-PAGE gel: 6 μg for E. coli and P. putida, 3 μg for S. japonicum, and 25 μg for C. necator. Total TrfA levels in three hosts under this experimental condition were as follows (in fmol/μg of total protein, blots from left to right, respectively): 27, 12, and 68 for E. coli; 47, <10, and 30 for P. putida; 160, 80, and 290 for S. japonicum.

Fig 4

Plasmid copy numbers at different TrfA concentrations in E. coli. (A) Experimental design. Expression of TrfA1(M124L) and TrfA2 was induced from the pBAD24C constructs, pHY819 and pHY820, respectively, by addition of arabinose. Plasmid copy number was determined as the ratio of the copy number of tetA in the introduced IncP-1β replicon pHY872 to the copy number of a gene in the chromosomal oriC region at stationary phase. (B) A plot showing relationship between PCN and TrfA concentrations. Arabinose was added at the following concentrations (%, wt/vol): 0.2 × 10⁻⁵, 2 × 10⁻⁴, and 2 × 10⁻³ (data points from left to right). Each data point with error bars represents the mean and standard error of the mean obtained from three total DNA samples and more than three quantitative Western blot experiments. Note that the TrfA1(M124L) level without arabinose was equivalent to the TrfA1 level in E. coli harboring pMS0506.