A natural system of chromosome transfer in Yersinia pseudotuberculosis

Abstract

The High Pathogenicity Island of Yersinia pseudotuberculosis IP32637 was previously shown to be horizontally transferable as part of a large chromosomal segment. We demonstrate here that at low temperature other chromosomal loci, as well as a non-mobilizable plasmid (pUC4K), are also transferable. This transfer, designated GDT4 (Generalized DNA Transfer at 4°C), required the presence of an IP32637 endogenous plasmid (pGDT4) that carries several mobile genetic elements and a conjugation machinery. We established that cure of this plasmid or inactivation of its sex pilus fully abrogates this process. Analysis of the mobilized pUC4K recovered from transconjugants revealed the insertion of one of the pGDT4-borne ISs, designated ISYps1, at different sites on the transferred plasmid molecules. This IS belongs to the IS6 family, which moves by replicative transposition, and thus could drive the formation of cointegrates between pGDT4 and the host chromosome and could mediate the transfer of chromosomal regions in an Hfr-like manner. In support of this model, we show that a suicide plasmid carrying ISYps1 is able to integrate itself, flanked by ISYps1 copies, at multiple locations into the Escherichia coli chromosome. Furthermore, we demonstrate the formation of RecA-independent cointegrates between the ISYps1-harboring plasmid and an ISYps1-free replicon, leading to the passive transfer of the non-conjugative plasmid. We thus demonstrate here a natural mechanism of horizontal gene exchange, which is less constrained and more powerful than the classical Hfr mechanism, as it only requires the presence of an IS6-type element on a conjugative replicon to drive the horizontal transfer of any large block of plasmid or chromosomal DNA. This natural mechanism of chromosome transfer, which occurs under conditions mimicking those found in the environment, may thus play a significant role in bacterial evolution, pathogenesis, and adaptation to new ecological niches.

Figure 1. Transfer frequencies at various temperatures of three distantly located chromosomal loci.

The donor 637-irp2^K-ureB^S-5076^T and recipient 637ΔHPI-Nal^R strains were co-incubated in LB-αα' with agitation. Transfer frequency was calculated as the number of Nal^R (or Kan^R, Spe^R, Tmp^R) and Rif^S transconjugants per Rif^R donor cells. Shown are mean values of transfer frequencies (vertical bars) and standard error of the mean (sem, vertical lines) of two independent experiments at each temperature. Mean transfer frequencies (±sem) at 4°C: 1.3(±0.7)×10⁻⁸ for irp2 ^K, 0.7(±0.3)×10⁻⁸ for ureB ^S and 0.4(±0.4)×10⁻⁸ for or5075 ^T, and at 12°C: 0.03(±0.02)×10⁻⁸ for the three loci. Transfer frequencies at temperatures ≥20°C were systematically below the detection limit (10⁻¹⁰).

Figure 2. Conjugative properties of pGDT4.

(A) Comparison of the efficiency of transfer of pGDT4 and the irp2^K chromosomal locus. The donor 637-irp2^K-ureB^S(pGDT4^T) and the recipient 637-Nal^R were co-incubated at 4°C in LB-αα' and independent experiments were performed 4 to 5 times. (B) Efficiency of transfer of pGDT4 to various recipient strains. The donor 637-irp2^K-ureB^S(pGDT4^T) was co-incubated at 4°C with either 637-Nal^R, 953-Nal^R or 637c-Nal^R. The results of two independent experiments were combined. (C) Efficiency of transfer of pGDT4 at various temperatures in liquid and on solid media. The experiments were performed twice independently. Shown are mean values of transfer frequencies (vertical bars) and standard error of the mean (sem, vertical lines). The detection limit was 10⁻¹⁰.

Figure 3. Genetic map of pGDT4.

Figure 4. Electron microscopy of IP32637 grown at 4°C in LB with agitation.

White arrows point at bridge-like structures.

Figure 5. Schematic representation of seven recombinant pUC4K molecules recovered from transconjugants.

Ten transconjugants resulting from the co incubation of 637(pUC4K) and 637-Nal^R were analyzed. The donor plasmid pGDT4 harbors two copies of ISYps1 in direct orientation and one copy in opposite direction, two copies of ISYps3 transposon including ISYps3 and ISYps2 transposases, one ISL3 and one ISNCY. ISYps1° is a truncated copy of ISYps1. Stars indicate XhoI restriction sites. Genes used to search by PCR for the presence of inter-ISYps1 regions are boxed.

Figure 6. Model proposed for pUC4K mobilization based on the transposition mechanism of the IS6 family.

Transposase-mediated replicon fusion of the two plasmid molecules generates a cointegrate carrying an additional copy of ISYps1 in the same orientation. Although only one type of cointegrate is represented here, different types of cointegrates mediated by each ISYps1 copy can be generated (indicated by dashed arrows). RecA-dependent homologous recombination between any two copies of ISYps1 present on the cointegrate will either regenerate the donor plasmid, leaving a single IS copy in the target pUC4K or create a rpUC4K containing a portion of pGDT4. Figure adapted from Mahillon J. and Chandler M. .