Photorhabdus lux-operon heat shock-like regulation

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 Photorhabduslux-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.


Introduction
Enterobacteria of the genus Photorhabdus (formerly Xenorhabdus) mostly are terrestrial bioluminescent bacteria living in the intestines of entomopathogenic nematodes Heterorhabditis [1]. Previously Photorhabdus isolates were divided into 3 new species: P. luminescens, P. temperata and Photorhabdus asymbiotica [1]. P. luminescens (containing the type strain) and P. temperata are symbiotic representatives with max growth temperatures 35-39 • C and 33-35 • C respectively [1]. P. asymbiotica isolates have been collected from a human host [1,2]. P. luminescens cells can switch between two forms: the pathogenic to insects P-form and the mutualistic M-form, which appears in the intestines of nematodes. It was shown in Refs. [3,4] that the transition from one form to another is controlled by the orientation of the mad-operon promoter. In the P-form, these bacteria have a more pronounced luminescence compared to the M-form, release toxins to kill insects, antibiotics to fight other microorganisms, as well as biologically active substances for the successful reproduction of itself and the host nematodes [3,5]. Various substances that can act as antibiotics against both Gram-positive and Gram-negative bacteria are described in the review [6]. For example, novel antibiotic versus Gram-negative bacteria named Darobactin have been found from screening of Photorhabdus isolates [7]. Also, the possibility of application of Photorhabdus toxins against Colorado potato beetle and mosquitoes has been discussed [8,9].
Many bioluminescent bacteria are characterized by induction of lux-operon expression depending on cell density (quorum sensing) and environmental conditions [10][11][12]. Also, commonly the glow of bacteria depends on various factors: cAMP concentration, iron level, oxygen concentration, osmolarity, etc [13]. However, for some representatives of the luminescent microflora, the mechanisms of luminosity regulation have not been identified. In particular, it is believed that luminescence is constitutive among representatives of the genera Photorhabdus and Photobacterium [14][15][16][17][18][19][20].
Under heat shock conditions, the synthesis of many chaperones and proteases is activated. In E. coli, the main inducer of transcriptional synthesis of heat shock proteins is the σ 32 factor (RpoH) of RNA-polymerase [26]. The tenfold increase in the heat shock proteins synthesis is observed when temperature rises from 30 to 42 • C [27]. This effect is achieved due to the complex regulation of activity and synthesis of σ 32 [26]. In addition to σ 32 , E. coli cells synthesize the σ E factor, the activation of which is controlled by improperly folded periplasmic proteins (heat shock from 30 to 46 • C) [28]. Cells carrying P. luminescens lux-operon under control of its own promoter are often applied as non-specific biosensor for integral toxicity assessment [19,20,29,30]. Ability of native lux-operon to be induced under some conditions can interfere with such investigations. Here, we determined the influence of temperature conditions on the expression of lux-genes under native promoter in E. coli cells, carrying lux-operon from P. luminescens ZM1, and in the natural strain P. temperata. The expression of the lux-operon in cells was compared by unit luminescence at various temperatures ranging from room temperature to 42 • C, and at different concentrations of ethanol. To prove lux-operon promoter activation, transcription intensity of Photorhabdus lux-operons was analyzed by RT-PCR technique.

Bacterial strains and plasmids
Bacteria and plasmids are listed in Table 1 and Table 2, respectively. Construction of vectors is described in the supplementary materials. Primers used in the study are given in Table S1 (see Supplementary file).

Isolation of a natural strain of P. temperata
Soil samples weighing 2-3 kg were collected in sandy soil under young pine plantings near the territory of the Serebryany Bor Park (55.7807, 37.4281). Soil samples were delivered to the laboratory, where it was distributed in 500 mL plastic containers, filling it to the brim so that the lid pressing the soil did not leave empty spaces inside. 5 caterpillars of wax moth (large wax fireworm G. mellonella) were added to each container. After 3-4 days, the condition of the insects was checked, and if dead ones were found, evenly colored in red, dark gray or crimson, then such dead insects were placed singly on filter paper in a Petri dish with a diameter of 40 mm, which was wrapped with Parafilm (Parafilm ®) to prevent drying. Daily observations of the condition of the deceased insect were carried out, and at the first signs of the appearance of numerous invasive larvae on its surface (length about 700 μm), the lower part of a small Petri dish was transferred to a large cup filled with 1-2 mm of standing tap water. Upon the release of the formed larvae into the water, the suspension was transferred to plastic flattened flasks with screw caps (Falcon or similar). In this form, they were stored at 4-6 • C. The resulting larvae infected with bacteria had the ability to bioluminesce (Fig. 1).
The internal contents of the infected larva were dissolved in a liquid LB medium for liquid seeding on an agarized medium. As a result, separate luminous bacterial colonies were obtained, which were later used in the work. Sequencing of the 16S ribosomal gene using primers 16S_Ph_unidir and 16S_Ph_univer (Table S1) and comparison of the obtained sequence in the NCBI database by the BLAST method showed kinship to P. temperata.

Cultivation of bacterial cells
E. coli and P. temperata grew in LB medium (1% trypton, 0.5% yeast extract, 1% NaCl) with constant aeration at 200 rpm at the temperature specified in the experiment. The solid medium for Petri dishes was prepared with the addition of 1.5% agar.
The optical density was measured with a KFK-2 photometer (ZOMP, Russia).

DNA manipulation
Plasmid DNA was isolated with the use of GeneJET Plasmid Miniprep Kit (Thermo Scientific, Waltham, MA, USA). Cell transformation with hybrid plasmids, agarose gel electrophoresis, and isolation of DNA fragments from agarose gel were performed according to the Sambrook manual [33]. Restriction and ligation reactions were carried out using enzymes from Promega (Madison, WI, USA).

Luminescence measurement
The measurement of the luminescence level was conducted as described before [12]. In brief, a cell suspension was transferred into 200 μL tubes, then the tube was placed in a Biotox-7BM (BioPhysTech, Russia) luminometer to measure the total luminescence level (in relative units) at room temperature (23 • C). Measurements were conducted after 5 min of incubation at 23 • C to cool samples to standard conditions. Highly luminous samples were 10-or 100-fold diluted to match the dynamic range of the luminometer.

Design of experiments to evaluate luminescence dependence on temperature
Luminous bacterial cells were grown in liquid LB at 23 • C (28 • C in the measurements with ethanol in the medium) and 200 rpm to an optical density of OD 600 ~ 0.1. Then each investigated cell suspension was divided into equal portions, which were further incubated at different temperatures. At the end of incubation or at specific moments, the luminescence and optical density of cell cultures were measured.
To take into account the difference in growth rate of E. coli and P. temperata cells at different temperatures the unit luminescence was calculated for each point as follows: where UL -unit luminescence of sample, Lum -total luminescence produced by the sample, OD -optical density of cell culture in the To unify all experimental replicates coefficient of induction for each temperature was calculated as follows: Where K(T) -coefficient of induction of luminescence, T 0 -the lowest temperature in the experiment (23 or 28 • C). When ethanol was used to stimulate heat shock, the final concentration of ethanol in the medium was varied instead of temperature, and the luminescence of the sample without spirit was used in equation (2) in denominator.

RNA extraction and RT-PCR
Bacterial cells of E. coli MG1655 pXen7 and P. temperata grew at 28 • C and 200 rpm to an optical density of OD 600 ~ 0.1. Then the cell suspension was divided into samples at 28 and 34 • C and incubated for 3 h. After the time elapsed, the luminescence and optical density were measured. Next, the equivalent of 3 OD•ml of bacterial cells (volume depends on optical density) was harvested and total RNA was extracted according to BioRad Aurum Total RNA Kit manual.
Reverse transcription polymerase chain reaction (RT-PCR) was performed to estimate relative transcriptional activity of lux-operons of P. temperata and E. coli MG1655 pXen7 cells when they had been grown at higher temperature conditions. It was done for both samples by evaluating contents of target gene of lux-operons -luxC normalized to housekeeping 16S ribosomal gene from extracted total RNA. To show changes in transcription, ΔΔC T [34] was calculated: Where C T is a threshold cycle for exact probe, it was calculated using software Rotor-Gene 6000 (Corbett Research, Australia). RT-PCR was done using the OneTube RT-PCR SYBR kit (Eurogen, Moscow, Russia) according to the procedure specified by the manufacturer in Rotor-Gene Q amplifier. 0.5 ng of extracted total RNA was added to the finished mixture, the concentration of which was measured with a NanoPhotometer ® P 330 spectrophotometer. Primers Ph_DluxC and Ph_RluxC (Table S1) flanked a region of ~400 nucleotides of the target luxC gene. The fragment of ~400 nucleotides of conservatively expressed 16S ribosomal gene was amplified by universal primers 16SuniD and 16SuniRmidi (Table S1) Efficiency of amplification in the experiment wasn't measured however should be almost the same for primers with close annealing temperatures and similar length of PCR products. Therefore, the exact relevant amount of luxC at temperature 34 • C with respect to 28 • C is not calculated.

Statistics
All experiments with luminescence measurements with E. coli and P. temperata cell cultures were performed in triplicate. RNA extraction and RT-PCR analysis were done in duplicate. The error of induction coefficient and ΔΔC T were calculated by the formula of standard deviation on the basis of three or two replicates, respectively. Its value on the graphs is represented by error bars.

Activation of the lux-operon in the heterologous E. coli system
To investigate the temperature-dependent activation of expression of luminescence-encoding genes under the control of the promoter of lux-operon from Photorhabdus bacteria, E. coli MG1655 cells transformed with pXen7, pXenP, and pDlac plasmids grew for 14-16 h (overnight) at 23, 28, 34, 37, and 42 • C without shaking. Then the luminescence intensity was measured, as well as the optical density. On the histogram (Fig. 2) the coefficients of induction (3) are given.
As shown on the histogram (Fig. 2), E. coli cells with pXen7 and pXenP plasmids containing the lux-operon promoter P. luminescens ZM1 demonstrate an increase in the level of luminescence with increasing of temperature, in contrast to cells with pDlac plasmid, in which luxCDABE genes are under the control of non-activated heat shock, constitutive, under these conditions, the P lac promoter.
Induction of lux-genes expression under heat shock conditions can be observed clearly in Fig. 3, where E. coli MG1655 pXen7 and specific biosensor for heat shock E. coli MG1655 pGrpE-lux were grown overnight on plates at 23, 28, 34, 37, and 42 • C.

Induction of luminescence of P. temperata strain
For experiments with lux-operon in native host, we conducted a search for luminescent bacteria in the Moscow region and succeeded to isolate P. temperata FV2201 strain. It turned out that overnight incubation at 34 • C is very poorly for growth of P. temperata cells. Moreover, when incubated for 3 h at 37 • C, the unit luminosity is lower than at 34 • C, and at 40 • C they stop to luminesce at all. It is understandable, since the max growth temperature for P. temperata is 33-35 • C.
P. temperata FV2201 and E. coli MG1655 pXen7 cells suspensions after incubation were divided into equal samples, which continued growth at 23, 28, and 34 • C. Fig. 4 shows characteristic dependence of the specific luminescence (1) on the incubation time at the indicated temperatures. Additional experimental data could be found in the Supplementary materials (Table S2).
It can be observed that at 34 • C both E. coli MG1655 pXen7 and P. temperata cell suspension have more intensive expression of luxgenes with regard to lower temperatures 23 and 28 • C.

Induction of heat shock by ethanol
To test the protein-mediated response of lux-operons in the presence of ethanol, the following measures were conducted. E. coli MG1655 cells carrying plasmids pGrpE-lux, pDlac, pXen7 or pXenP, and P. temperata cell suspensions were divided into 200 μL portions and supplemented with ethanol at final concentrations of 0, 1, 3, and 6%. Resulting samples were incubated at room temperature for 2 h. The data obtained is shown in Fig. 5.
On the graph, the activation of luciferase synthesis in the presence of ethanol in the E. coli MG1655 pDlac cells does not occur. The specific biosensor for heat shock of E. coli MG1655 pGrpE-lux is reliably activated by all the ethanol concentrations studied. For cells with pXen7 and pXenP, the activation of luminescence at 1% ethanol is low and unreliable. Higher concentrations reduce luminescence due to toxicity. On the contrary, the synthesis of lux-genes of the P. temperata strain significantly increases at ethanol concentrations of 1 and 3%. A higher concentration of 6% reduces luminescence due to general toxicity.

Analysis of transcriptional activity
To assess changes in the transcription of lux-operon promoter, E. coli MG1655 pXen7 and P. temperata cells were incubated at 28 and 34 • C for 3 h, followed by total RNA extraction and RT-PCR. At these temperatures, there is still a small difference in the rate of cell growth, but a significant increase in luminescence is observed. The luminescence level and optical density of the samples were also  The results of the calculation of ΔΔC T (3) are as follows: ΔΔC T are − 3.54 ± 1.47 for P. temperata FV2201 and -1.53 ± 0.74 for E. coli MG1655 pXen7 cells. Comparison of unit luminescence (1) for these samples gave the following coefficients of induction: 12.5 ± 7.3 and 6.4 ± 2.4, respectively.
According to the data, both luxC gene transcription and unit luminescence increase with an increase in the incubation temperature 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 OD 600 ~ 0.1 is reached.  ranging from 28 to 34 • C. The values obtained are almost the same for P. temperata cells and the lux-operon of P. luminescens in a heterologous E. coli cell system. The bacterial luminescence is expected to be proportional to the square of the activity of luxAB transcription [35], thus taking into account that PCR effectiveness was lower, then doubling per cycle, the activation coefficients correspond to the values of ΔΔC T . This allows us to conclude that the luminescence enhancement described in this paper with an increase in temperature is due to the regulation of the lux-operon promoter. Moreover, this regulation persists even when the lux-operon is transferred to a heterologous system of an unrelated bacterial species.

Influence of σ 32 and σ 24 on activation of the lux-operon promoter
To investigate the effect of σ 32 and σ 24 subunits on the activation of luminescence gene synthesis at high temperatures in E. coli cells, strains K165 (σ 32 mutant), MG1655 ΔrpoE (σ 24 mutant) and wild-type strain MG1655 were transformed by pXen7 and pGrpE-lux plasmids. E. coli MG1655, K165 and MG1655 ΔrpoE cells with the pXen7 and pGrpE-lux plasmids were divided into equal samples and then were incubated at 23, 28, 34, 37, and 42 • C for 3 h. The coefficients of induction (2) are shown in Fig. 6. As can be seen on the histogram (Fig. 6), the activation of lux-operon in E. coli cells with pGrpE-lux plasmid does not occur in the K165 strain compared to MG1655, which is understandable, since the promoter of the GrpE gene is σ 32 dependent. At 42 • C, a drop is observed due to the sensitivity of the K165 strain to high temperatures. In E. coli MG1655, K165 and MG1655 ΔrpoE cells cloned with the pXen7 plasmid, the unit luminescence at 23 and 28 • C remains almost at the same level, while an increase is observed at 34 • C, and with temperature rising, continues to grow to 42 • C. The drop in the luminescence level at 42 • C for K165, is explained by the inability of bacteria to live at high temperatures. The key result of this experiment is the preservation of the induction of the P. luminescens lux-operon promoter in E. coli cells with the deleted genes of σ 32 and σ 24 RNA polymerase subunits.

Discussion
In this paper, it is shown that the lux-operon of P. luminescens ZM1 in the heterologous system of E. coli and lux-operon of the closely related natural strain P. temperata are activated in a temperature-dependent manner. Activation of the synthesis of luminescence genes occurs when the temperature rises from 23 to 34 • C for the P. temperata strain and from 23 to 42 • C in the E. coli MG1655 pXen7 strain (Figs. 4 and 6), and this activation is due to the lux-operon promoter (Fig. 2). Reliable activation of lux-genes expression with the addition of ethanol is observed only in P. temperata cells (Fig. 5). This effect can be explained by the fact that the heat shock response regulation systems of E. coli cell do not recognize the promoter of the lux-operon of P. luminescens, and the response to an increase in temperature is associated with another mechanism, for example, possible regulation at the translation level with the formation of RNAthermometers [26,[36][37][38][39]. To test this hypothesis, experiments were conducted in E. coli strains mutated by the genes of σ-heat shock factors. As it turned out, the temperature-dependent activation of the lux-operon of P. luminescens persists in strains deficient in both the rpoH and rpoE genes (Fig. 6). Thus, we can assume the presence of a secondary structure of the lux-operon mRNA, which melts regardless of the presence of σ-factors of heat shock in both E. coli and Photorhabdus cells, which in turn can regulate the translation of lux-genes depending on temperature. However, the presence of activation of the expression of the lux-operon of P. temperata with the addition of ethanol indicates in favor of protein-mediated regulation, since ethanol does not induce melting of RNA pins. Probably, in natural strains of Photorhabdus genus, there is a combined regulation involving both mechanisms.
Further investigations of Photorhabdus lux-operon regulation mechanisms could form the basis for the construction of the novel biosensor for detection of specific stress conditions [40]. Also, determination of molecular mechanism of this regulation can bring a new instrument for biotechnological genetic engineering, since many microbiological processes in biotechnology are conducted at 20-40 • C.

Conclusion
Induction of transcription of lux-operon from bacteria of Photorhabdus genus in response to heat shock is shown. Expression of both lux-operon of the P. luminescens ZM1 strain in the heterologous system of E. coli and lux-operon of the newly isolated closely related strain P. temperata FV2201 is activated with rising temperature from 23 to 42 • C and 23-34 • C respectively. Only P. temperata luxoperon expression is induced by ethanol. Transcriptional heat shock lux-operon activation is demonstrated by RT-PCR from 28 to 34 • C. Heat-shock activation promoter Photorhabdus lux-operon is shown, and it is not depend on E. coli transcriptional factors σ 32 and σ E .

Funding statement
The investigation of mechanisms of luminescence regulation was supported by Ministry of Science and Higher Education of the RF, project FSMF-2022-0007 "Development of technology for rational and highly productive use of agro-and bioresources, their efficient processing and obtaining safe and highquality sources of food and non-food products".
The search and characterization of luminescent bacteria in Moscow was supported by Russian Science Foundation [21-64-00018].
In this study the equipment of Applied Genetics Resource Facility of MIPT was used [075-15-2021-684].

Data availability statement
All experimental data is presented in the article and Supplementary meterials. The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.

Additional information
No additional information is available for this paper.

Declaration of interest's statement
The authors declare no conflict of interest.