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.

Conjugation dynamics depend on both the plasmid acquisition cost and the fitness cost

“…This procedure is ideal for our purposes since it minimizes growth and adaptation during the conjugation window, ensuring that subsequent dynamic characterization of de novo T captures emergent phenotypic changes. In contrast, adapted T can be isolated by streaking conjugation mixtures onto dual antibiotic agar plates; individual clones can then be grown and stored for subsequent testing (Dahlberg & Chao, 2003; Rozwandowicz et al , 2019). These established transconjugants exhibit stably reproducible growth rates and are often used to quantify fitness costs and/or determine the timescale of compensatory mutations (Harrison et al , 2015; Hall et al , 2020).…”

Estimating plasmid conjugation rates: a new computational tool and a critical comparison of methods

“…dimensionless Liquid [17,25]; Filter [18,22,24] (Plasmid) transfer frequency [22]; Gene transfer frequency [24]; Conjugation frequency [17,18], Recombinant yield [25], Plasmid transfer efficiency [15] T N…”

“…dimensionless Plate [12] Exconjugant frequency [12] T R dimensionless Filter [23] Gene transfer frequency [23] T D dimensionless Liquid [16,24]; Filter [17,21,23] (Plasmid) transfer frequency [21]; Gene transfer frequency [23]; Conjugation frequency [16,17], Recombinant yield [24], Plasmid transfer efficiency [14] T N dimensionless Liquid [20] Conjugation frequency based on total bacterial count [20] T +D R dimensionless Liquid microcosms [25] Plasmid prevalence [25] T +D N dimensionless Soil microcosms [26] Frequency of plasmid carriage [26] T R+T dimensionless Liquid [8,25], Mouse gut [8,11] Proportion of transconjugants [11,25], Fraction of transconjugants (in recipient population) [8] log 10 T √ DR dimensionless Liquid [27] (Logarithm of) Conjugation rate [27] T DR ml CFU Filter [28] Transconjugant frequency [28] ψmax…”

“…36 In addition, this ratio is not only determined by a plasmid's conjugation rate, but also by its clonal 37 expansion [14]. As such, the resulting measurements are not a priori comparable across experimen- 38 tal conditions that could affect the growth rate, including differing nutrient conditions [20], recipient 39 species [8,12,21], temperatures [16], and (sublethal) antibiotic exposure [17]. This limits the predic-40 tive power of conjugation proficiency when expressed as a ratio of population densities [14].…”

“…Differences in the output of such conjugation assays are then further exacerbated when the measured population densities are related to the amount of conjugation that occurred. Indeed, there is no consensus on what to call this quantity: commonly used phrases include conjugation frequency [21, 22], plasmid transfer rate constant [13, 23], or transfer efficiency [19, 20]. More than 10 different methods to quantify conjugation are currently found in the literature (see Table 1).…”