Temperature Restriction in Entomopathogenic Bacteria

Abstract

Temperature plays an important role in bacteria-host interactions and can be a determining factor for host switching. In this study we sought to investigate the reasons behind growth temperature restriction in the entomopathogenic enterobacterium Photorhabdus. Photorhabdus has a complex dual symbiotic and pathogenic life cycle. The genus consists of 19 species but only one subgroup, previously all classed together as Photorhabdus asymbiotica, have been shown to cause human disease. These clinical isolates necessarily need to be able to grow at 37°C, whilst the remaining species are largely restricted to growth temperatures below 34°C and are therefore unable to infect mammalian hosts. Here, we have isolated spontaneous mutant lines of Photorhabdus laumondii DJC that were able to grow up to 36-37°C. Following whole genome sequencing of 29 of these mutants we identified a single gene, encoding a protein with a RecG-like helicase domain that for the majority of isolates contained single nucleotide polymorphisms. Importantly, provision of the wild-type allele of this gene in trans restored the temperature restriction, confirming the mutations are recessive, and the dominant effect of the protein product of this gene. The gene appears to be part of a short three cistron operon, which we have termed the Temperature Restricting Locus (TRL). Transcription reporter strains revealed that this operon is induced upon the switch from 30 to 36°C, leading to replication arrest of the bacteria. TRL is absent from all of the human pathogenic species so far examined, although its presence is not uniform in different strains of the Photorhabdus luminescens subgroup. In a wider context, the presence of this gene is not limited to Photorhabdus, being found in phylogenetically diverse proteobacteria. We therefore suggest that this system may play a more fundamental role in temperature restriction in diverse species, relating to as yet cryptic aspects of their ecological niches and life cycle requirements.

Keywords: Photorhabdus; evolution; mutants; pathogenicity; temperature restriction.

FIGURE 1

(A) Genetic organization of the temperature restriction locus. (B) Domain architecture of TrlG and the presence of substitution mutations in the temperature tolerant clones. Vertical lines indicate the positions of the mutations; red depicts stop mutations. The colored boxes show the domains identified by Pfam; Magenta: Alba_2 (Pfam domain PF04326, at amino acid positions 14–128) and light blue: HATPase_c_4 (Pfam domain PF13749, at amino acid positions 298–365). (C) (Left) I-TASSER simulation of TrlG with the distribution of mutations mapped. Orange indicates SNP positions, whilst red depicts stop mutations. (Right) The same model as in (Left) with homology domains mapped in color. Images prepared with UCSF Chimera.

FIGURE 2

(A) Growth of the 30 sequenced tolerant clones and P. laumondii DJC (WT) on LB agar at 28 and 36°C. (B) Growth of the additional two clones isolated independently (JA and JB) compared to P. laumondii DJC (WT) as well as of the Δtrl deletion strain compared to the isogenic rifampicin resistant P. laumondii DJC (DJCRif) at 28 and 36°C.

FIGURE 3

Growth of the Δtrl deletion mutant complemented with either the empty pBCSK′ vector or pBCSK′ carrying the TRL operon on (A) LB agar and (B) liquid LB media at 28 and 36°C. The asterisk denotes significant difference at p_adj < 0.05. Results show the averages and standard error from four biological replicates.

FIGURE 4

Virulence of the trl mutants at room temperature. The results show the number of surviving Galleria mellonella larvae infected with either the WT strain, the S1 SNP mutant, the RifR WT strain or the Δtrl deletion mutant at different time points. G. mellonella larvae injected with PBS were used as control. Twenty larvae were used for each strain and the experiment was repeated with similar results.

FIGURE 5

Up-regulation of the promoter of the TRL operon upon shift to 36°C. (A) Schematic diagram showing the different regions used in the promoter reporter constructs. The expanded panel shows the region between trlF (PluDJC_RS01880) and polA with the predicted promoters based on BPROM indicated as arrows on top of the sequence. At the bottom are the regions amplified and introduced separately into pGAG1 upstream of the gfpmut3 gene to create promoter constructs P_AGFP-P_DGFP. The diagrams are color coded to reflect the colors of the data plotted below. (B) Left: Fluorescence of the P. laumondii DJC reporter strains following a shift in temperature from 28 to 36°C after 5 h of growth as indicated by the arrow. Right: Fluorescence of the P. laumondii DJC reporter strains grown continuously at 28°C. Results on the secondary axis are arbitrary fluorescence units following a subtraction of the fluorescence of the P. laumondii DJC carrying pGAG1 in the absence of any promoter to account for any autofluorescence and they represent the mean of three biological replicates ± standard error. (C)The growth and rate of change in fluorescence in P. laumondii DJC carrying pGAG1(P_CGFP), calculated by subtracting the fluorescence at a given time point by the fluorescence of the previous time point; left: following a shift in temperature from 28 to 36°C after 5 h of growth as indicated by the arrow; right: at 28°C. Results represent the mean of three biological replicates ± standard error.

FIGURE 6

Maximum likelihood tree constructed using the Photorhabdus recA nucleotide sequences from RefSeq genomes. Black circles indicate branch support over 80%. The scale bar represents the number of substitutions per nucleotide position. Highlighted are the strains that were found to contain a copy of the trlG gene.

FIGURE 7

Maximum likelihood tree constructed using protein sequences with over 60% sequence identity to the Photorhabdus laumondii TrlG protein (with a query cover >90%). Nodes with support values higher than 80% are indicated with black circles. The external colored ring represents different genera. The scale bar represents the branch length, indicating the number of substitutions per amino acid position. Protein identifiers for all the entries can be found in Supplementary Table 1.

FIGURE 8

Conservation and genetic organization of the TRL operon in representative species as detected by MultiGeneBlast. In red is trlG, in green is trlF and in blue is trlR; genes colored identically in other organisms denote the identified homologs of trlG, trlF, and trlR, respectively.