Photorhabdus lux- operon heat shock-like regulation

Abstract

For decades, transcription of Photorhabdus luminescens lux-operon was considered being constitutive. Therefore, this lux-operon has been used for measurements in non-specific bacterial luminescent biosensors. Here, the expression of Photorhabdus lux-operon under high temperature was studied. The expression was researched in the natural strain Photorhabdus temperata and in the heterologous system of Escherichia coli. P. temperata FV2201 bacterium was isolated from soil in the Moscow region (growth optimum 28 °C). We showed that its luminescence significantly increases when the temperature rises to 34 °C. The increase in luminescence is associated with an increase in the transcription of luxCDABE genes, which was confirmed by RT-PCR. The promoter of the lux-operon of the related bacterium P. luminescens ZM1 from the forests of Moldova, being cloned in the heterologous system of E. coli, is activated when the temperature rises from room temperature to 42 °C. When heat shock is caused by ethanol addition, transcription of lux-operon increases only in the natural strain of P. temperata, but not in the heterologous system of E. coli cells. In addition, the activation of the lux-operon of P. luminescens persists in E. coli strains deficient in both the rpoH and rpoE genes. These results indicate the presence of sigma 32 and sigma 24 independent heat-shock-like mechanism of regulation of the lux-operon of P. luminescens in the heterologous E. coli system.

Keywords: Heat shock; Lux-operon; P. temperata; Promoter.

Fig. 1

G. mellonella infected with P. temperata. Shooting with a camera in the dark.

Fig. 2

Coefficients of induction of luminescence of E. coli MG1655 cells with pXen7, pXenP, and pDlac plasmids after 14–16 h at 28, 34, 37, and 42 °C without shaking. Data points and error bars correspond to means and standard deviations determined in three independent experiments.

Fig. 3

The pictures of plates with cells of E. coli MG1655 pXen7 and E. coli MG1655 pGrpE-lux after incubation overnight at 23, 28, 34, 37, and 42 °C. The A) and B) pictures are taken in the light and in the dark, respectively. Segments with cultures carrying pXen7 are outlined in green; pGrpE-lux ― red. Temperature of overnight incubation of every plate is indicated in the picture.

Fig. 4

Luminescence change kinetics of P. temperata FV2201 and E. coli MG1655 pXen7 cells after split and incubation at 23, 28, and 34 °C. Unit luminescence was calculated according to (1). The initial value corresponds to the moment of separation into equal samples when OD₆₀₀ ∼ 0.1 is reached.

Fig. 5

Induction coefficients of luminescence of E. coli MG1655 cells transformed with pGrpE-lux, pDlac, pXen7, or pXenP and P. temperata FV2201 cells after 2 h at room temperature, when ethanol was added to the medium at final concentrations of 1, 3, and 6%. Means and standard deviations were determined in three independent experiments.

Fig. 6

Induction coefficients of E. coli MG1655, K165 and MG1655 ΔrpoE with pXen7 and pGrpE-lux plasmids after 3 h at 28, 34, 37, and 42 °C. Means and standard deviations were determined in three independent experiments.