Successful Host Adaptation of IncK2 Plasmids

Abstract

The IncK plasmid group can be divided into two separate lineages named IncK1 and IncK2. IncK2 is found predominantly in poultry while IncK1 was reported in various mammals, including animals and humans. The physiological basis of this distinction is not known. In this manuscript we examined fitness cost of IncK1 and IncK2 plasmids at 37 and 42°C, which resembles mammalian and chicken body temperatures, respectively. We analyzed conjugation frequency, plasmid copy number and plasmid fitness cost in direct competition. Additionally, we measured levels of σ-32 in Escherichia coli carrying either wild type or conjugation-deficient IncK plasmids. The results show that IncK2 plasmids have a higher conjugation frequency and lower copy number at 42°C compared to IncK1. While the overall fitness cost to the host bacterium of IncK2 plasmids was higher than that of IncK1, it was not affected by the temperature while the fitness cost of IncK1 was shown to increase at 42°C compared to 37°C. These differences correlate with an increased expression of σ-32, a regulator of heat-shock protein expression, in E. coli with IncK2 compared to cells containing IncK1. This effect was not seen in cells containing conjugation deficient plasmids. Therefore, it is hypothesized that the assembly of the functional T4S may lead to these increased levels of σ-32. Increased activation of CpxR at 42°C may explain why IncK2 plasmids, and not IncK1, are predominantly found in chicken isolates.

Keywords: IncK2; chicken; conjugation; plasmid; sigma-32.

FIGURE 1

Triplicate measures of conjugation rates of IncK plasmids at 37 and 42°C. Bars depict the mean and standard deviation. ^∗∗ depict statistical significance (p = 0.0039).

FIGURE 2

IncK1 and IncK2 plasmid copy numbers at 37 and 42°C. All the measurements were performed in triplicate. Bars depict the mean and standard deviation. ^∗∗ depict statistical significance (p = 0.0039).

FIGURE 3

Fitness cost of IncK1 and IncK2 plasmids measured at 37 and 42°C. All the measurements were performed in triplicate. Bars depict the mean and standard deviation. (A) Comparison of plasmid fitness cost by temperature. ^∗ depict statistical significance (p = 0.0201). (B) Comparison of plasmid fitness cost by plasmid type. ^∗ depict statistical significance (p = 0.0495).

FIGURE 4

(A) σ-32 fold increase between 37 and 42°C in Escherichia coli carrying wt plasmids. ^∗ depict statistical significance (p = 0.037). (B) σ-32 fold increase between 37 and 42°C in E. coli carrying plasmids with mutated TraY; Bars depict the mean and standard deviation.

FIGURE 5

Schematic presentation of the environmental context to the plasmid induced bacterial stress model adapted from Zahrl et al. (2006), which explains the high prevalence of IncK2 (and not IncK1) in poultry isolates. E. coli carrying an IncK2 plasmid that enters the chicken gut expresses the tra proteins. Assembly of a T4S induces extracytoplasmic stress sensed by CpxR. CpxR, which was associated with increased bacterial colonization, induces the σ-32 response encoded by rpoH gene, resulting in a further upregulation of cytoplasmic stress proteins. ^∗Arrow pointing up means less decrease in conjugation rate of IncK2 plasmid compared to IncK1 at 42°C.