Identification of novel prophage regions in Xenorhabdus nematophila genome and gene expression analysis during phage-like particle induction

Prophage region analysis

Prophage regions present in the genomes of Xenorhabdus spp. were identified using PHASTER (PHAge Search Tool Enhanced Release) (Arndt et al., 2016). PHASTER classified and detected prophage region by completeness (incomplete, questionable and intact) thus we focused on regions with higher score detected as “intact” (Arndt et al., 2016). Annotation of the proteins localized in these regions were confirm by homology searches between the protein sequences using blastp (Camacho et al., 2009) and hidden Markov Models (HMM) profile using hmmscan in the HMMER web server (Potter et al., 2018).

Bacterial strain, culture conditions and mitomycin C-induced or UV-induced cultures

Cultures of Xenorhabdus nematophila strain AN6/1 were considered in the current study that are maintained in P. Stock’s laboratory (University of Arizona). The bacteria were grown in 10 mL Luria Bertani (LB) broth supplemented with 0.1% (w/v) sodium pyruvate (LBP) in a 50 ml centrifuge tube with agitation at 28 °C overnight for 12–16 h. Culture growth was monitored based on 600 nm optical density (OD600). Then, the bacteria were subculture (1:5 dilution) and grown at 28 °C to reach OD600 of 0.4–0.5. Two types of induction were tested: (a) with mitomycin C at a concentration of 5 μg ml⁻¹ and (b) 10 min exposure to UV 6 μJ/cm². Negative controls consisted of cultures not induced. Both induced and uninduced cultures were incubated at 28 °C in an incubator with moderate shaking (1 g). Two independent experiments were performed to assess: (1) late effect of induction with two studied time points: 2 and 10 h after induction, and (2) early effect of mitomycin C induction with two studied time points: 15 and 40 min after induction. For both experiments, OD600 was quantified at each time point and results were plotted using scatterplot in R environment (Fox & Weisberg, 2019).

RNA extraction and RT-PCR

At each time point, three replicates of 1.5 mL of cultures were collected, centrifugated at 3,080 g for 10 min. The supernatant was discarded and 750 μL Trizol was added to the pellet. Then, 150 μL chloroform was added to the bacteria mix and incubated on ice 15 min. After centrifugation at (20,821 g for 15 min at 4 °C) the aqueous phase was transferred to a new tube and 375 μL of isopropanol were added. After 10 min of incubation on ice, samples were centrifuge for 10 min at 20,821 g at 4 °C. The supernatant was discarded, and 1 mL of 75% ethanol was added to rinse the samples which were then vortexed, followed by centrifugation for 5 min at 7,885 g at 4 °C. The rinse step was repeated, and the RNA was air-dry and resuspend in water RNase-free. An additional incubation step for 10 min at 55 °C was considered to improve yield of the RNA elution. The RNA was quantified using a Nanodrop and 300 ng of RNA was used as the template for cDNA synthesis. cDNA was synthesized using the Bioline SensiFast cDNA Synthesis kit following manufacturer’s protocols.

qPCR of prophage region genes

Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the relative gene expression across induction conditions. Each primer set condition was optimized using Bioline SensiFast No ROX Sybr Master Mix and summarized in the Table S1. Primers were designed based on the genome reference of Xenorhabdus nematophila AN6/1 (NZ_LN681227) using AmplifX (v2.0.7) (Nicolas Jullien; https://inp.univ-amu.fr/en/). A total of 10 pairs primers were used in this study (Table S1). We normalized the gene expression using the DNA gyrase subunit A, gyrA as housekeeping gene. We used the Pfaffl model to calculate the relative gene expression using the formula: ${\displaystyle \frac{({E_{GOI)}}^{\mathrm{C}\mathrm{t}\mathrm{G}\mathrm{O}\mathrm{I}}}{({E_{Ref)}}^{CtRef}}}$ (Pfaffl, 2001). Primer efficiency was determined for each gene by running a standard curve and converted primers efficiency (E) was calculated as follows: (primer efficiency (%)/100) + 1. The calculated Ct from uninduced cultures was used as a calibrator when calculating the ∆Ct values for all the samples. The relative gene expression values were transformed by a logarithmic base 2 function to be plot as Boxplot in R environment. The distribution of the data was not normal, thus the variance of the relative expression with the non-parametric Kruskal-wallis rank sum associated with multiple pairwise comparisons was considered using the Dunn’s test in the R environment (dunn.test package) (Dunn, 1964).

Phage particles preparation and SDS-PAGE gels

Three time points were selected to purified induced phage particles: the two time points of the early induction experiment, 15 and 40 min after induction, and the last time point of the late induction experiment (10 h after induction). A total of 8 mL of induced and uninduced cultures were centrifuged at 3,080 g for 10 min to remove cellular debris. The supernatants were filtered using 0.22 μm pore size filter. Then, NaCl and polyethylene glycol 8000 (PEG 8000) solution were added to reach respectively 1 M and 10% (wt/vol) final concentration to precipitate the phage particles. The solution was incubated overnight at 4 °C. The particles were collected by centrifugation of 27,720 g 30 min at 4 °C and suspended in 200 μL in LBP. Manufacturer’s instructions were followed for the electrophoresis of phage particle samples using the Mini-PROTEAN TGXTM precast gels system (Bio-Rad, Hercules, CA, USA), followed by Coomassie blue staining to visualize precipitated proteins.

Four intact prophage regions are present in genomes of X. nematophila strain AN6/1

Of the 13 prophage regions identified by PHASTER in the studied genomes of X. nematophila strain AN6/1 (NZ_LN681227), only four were detected as potential “intact” prophage regions. Two of them are the prophage regions previously described: Xnp1 (localized at 1.05 M to 1.09 M), the P2-like remnant phage previously described as responsible of bacteriocin production; Xnp2 (localized at 3.03 M to 3.06 M), the P2-like complete phage previously described as not involved in bacteriocin production (Fig. 1). These two regions display similarities with P2-like bacteriophages. Our analysis also suggests strong similarities with the bacteriophage RE-2010 isolated from Salmonella enterica Serovar Enteritidis strain LK5 (NC_019488). As previously described, the prophage region xnp1 is characterized by the absence of genes coding head capsids proteins, as well as portal proteins or terminases. Another difference between the two P2-like prophages, is the region containing genes responsible to lysogeny control: Xnp2 region exhibited C immunity repressor and Cox protein while Xnp1 exhibited CI repressor (Fig. 1; Table S3).

The two newly identified prophage regions are “intact” regions and are designated as Xnp3 and Xnp4 to aligned with previously used nomenclature. Region Xnp3 (localized at 3.28 M to 3.35 M) presents strong similarities with Mu-like phage, while region Xnp4 (localized at 1.86 M to 1.90 M) is a remnant phage presenting similarities with Siphoviridae-like bacteriophages (Fig. 1). Compared to the other prophage regions, the Xnp4 region contained higher number of transposases and insertion sequence elements (IS), few phage-related proteins were clearly identified (only tail proteins) and no regulator or repressor protein was detected (Fig. 1; Table S3).

Our analysis also suggests strong similarities between Xnp3 and the SfMu bacteriophage isolated from Shigella flexneri (NC_027382) and the D108 bacteriophage isolated from Escherichia coli (NC_013594). PHASTER also detected another prophage region of 67.4 kb with two distinct prophage regions: (1) a 33.4 kb region (localized at 3.283 M to 3.316 M) which is like a complete Mu-like bacteriophage and (2) a 22.6 kb region (localized at 3.319 M to 3.341 M) which seems to be a remnant prophage (Fig. 1; Table S3). We focused on the first region (like Mu-like phage), which when compared to closely related bacteriophages, lacks the terminal part including one tail collar-fiber protein (S), as well as the DNA-binding transcriptional regulator Com and mom proteins (Fig. 2). This prophage region is not only present in X. nematophila but seems to be present (or has similar prophage regions) in the three other Xenorhabdus species including: Xenorhabdus cabanillasii strain JM26, Xenorhabdus miraniensis strain DSM17902 and Xenorhabdus thuongxuanensis strain 30TX1 (Fig. 2; Table S4).

Characterization of effect of mitomycin C and UV exposure

Our results showed that bacterial grow is not disrupted by mitomycin C induction at early stages of induction (15 and 45 min) (Fig. 3). Contrastingly, both mitomycin C and UV triggered growth difference in both uninduced cultures and induced cultures in late induction stages (Fig. 3). Similarly, after 2 h, bacterial cells density was observed (a slightly lower with cultures exposed to UV), but after 10 h a high mortality rate was observed for the cultures exposed to mitomycin whereas a strong growth reduction was observed for the cultures exposed to UV (Fig. 3).

SDS-page electrophoresis showed the presence of phage-particles after 10 h in lysates of both mitomycin C induced cultures and UV-induced cultures (Fig. 4). We also observed four subunits more abundant: (1) below 37 kDa, (2) above 37 kDa, (3) above 50 kDa and (4) below 75 kDa. We speculate that the subunit above 37 kDa might be the outer membrane porin (OpnP) proteins based on previous reported data (Kim et al., 2003; Leisman, Waukau & Forst, 1995; Morales-Soto & Forst, 2011). Three other subunits (between 75–125 Dk) were detected but less abundant. We also observed low signal of phage-particles in lysate of mitomycin-induced cultures after 15 and 40 min of induction.

Genetic mechanisms induced by mitomycin C and UV exposure

Regarding the level of gene expression after mitomycin C exposure, our preliminary analyses showed increase of the expression of recA and lexA at 45 min and 2 h after induction. We observed a down-regulation of lexA 10 h after both mitomycin C and UV induction, while a down-regulation of recA 10 h after induction was only observed with UV induction (Fig. 5).

We were able to study the expression of the three prophages regions, Xnp1, Xnp3 and Xnp4 but the low level of expression of the Xnp2 did not allow presentation of robust data. It is consistent with previous studies, genes encoding the tube and sheath protein in Xnp2 prophage region were expressed at barely detectable levels (Morales-Soto & Forst, 2011).

We successfully quantified the relative expression of two genes in Xnp1 prophage regions, the replication gene, GpA, and the baseplate assembly protein W, GpW, as well as the putative CI repressor gene (Table S5). We observed up-regulation of GpA and GpW at 2 and 10 h after mitomycin exposure. With respect to UV exposure, the up regulation of these genes was observed only at 2 h post exposure. No significant differences were observed with the controls (uninduced cultures) at early time points. Regarding the CI repressor contained in the Xnp1 region, no significant differences were observed, however a tendency for up-regulation was observed in mitomycin-induced cultures (at 2 and 10 h post exposure) (Fig. 5).

Regarding the Xnp3 prophage regions, we quantified the relative expression of the major head protein, as well as the putative CI repressor (Table S5). We observed an up-regulation of the gene encoding the major head protein at 2 and 10 h post mitomycin C exposure; and an up-regulation only after 2 h for the UV exposure (Fig. 5). With respect to the CI repressor, an up-regulation was observed after 2 h of UV exposure and thereafter, a down-regulation after 10 h was denoted for both mitomycin and UV exposure. However, no significant differential expression of the CI repressor was observed at early time post exposure (15 or 40 min).

With respect to Xnp4 prophage regions, we quantified the relative expression of the tail fiber protein (Table S5). We observed an up-regulation of the gene that encodes the tail fiber protein only 2 h post exposure to either mitomycin or UV. A down-regulation of the gene was observed at 10 h post-exposure for the UV-induced cultures only.