A single promoter inversion switches Photorhabdus between pathogenic and mutualistic states

Abstract

Microbial populations stochastically generate variants with strikingly different properties, such as virulence or avirulence and antibiotic tolerance or sensitivity. Photorhabdus luminescens bacteria have a variable life history in which they alternate between pathogens to a wide variety of insects and mutualists to their specific host nematodes. Here, we show that the P. luminescens pathogenic variant (P form) switches to a smaller-cell variant (M form) to initiate mutualism in host nematode intestines. A stochastic promoter inversion causes the switch between the two distinct forms. M-form cells are much smaller (one-seventh the volume), slower growing, and less bioluminescent than P-form cells; they are also avirulent and produce fewer secondary metabolites. Observations of form switching by individual cells in nematodes revealed that the M form persisted in maternal nematode intestines, were the first cells to colonize infective juvenile (IJ) offspring, and then switched to P form in the IJ intestine, which armed these nematodes for the next cycle of insect infection.

Fig. 1

Photorhabdus cells that initiate mutualism in the nematode intestine are small-cell variant M-form cells. (A) Photorhabdus cells that initiate mutualism (green) on the ninth intestinal ring cells, left and right, INT9L and INTR, posterior intestinal cells and transients (red) are present throughout the lumen of the nematode. (B)M-form cells develop small colonies, and transient P-formcells develop large colonies. (C) Small colonies consist of primarily small cells, and large colonies consist of primarily larger cells.

Fig. 2

Expression of the mad fimbrial locus and madO are required for M formation. (A) Physical map of the mad locus required for maternal adhesion, which is an initial step of nematode mutualism. madA to K are co-transcribed and expressed by inversion of the madswitch promoter located between madR and madA. madR is predicted to encode a FimB-type site-specific recombinase (i.e., invertase). (B) Expression of mad is essential for M formation. The majority of TT01 wild-type P-form cells switch to the M form 4 days after insect infection. Marked mutations (e.g., ΔmadA::Gm^R) of madA, H, J, and an orphan FimB-type recombinase madO failed to switch to the M form at detectable levels. ゔmadR::Gm^R switched to the M form. In-frame deletions in madA and madH, but not madJ, led to a switch to the M form. Error bars represent standard deviation. (C to H) (Left) Colonies 4 days after P-form infection of insects, except the locked strains (D and E); (right) selection of the M form during maternal adhesion in nematodes. (C) TT01 wild type switched to the M form in insects and in nematodes. (D) No M-form colonies (N = 883) and nomaternal adhesion were detected in the locked L/P form. (E) Locked L/M form grew as M-form colonies (N = 1656) lacking sectors of the P form and adhering to the posterior nematode intestine. (F) Deleting the MadR invertase did not result in loss of switching to the M form in insects or nematodes. (G) Deleting the MadO invertase prevented switching to the M form in insects and nematodes. (H) Deleting MadJ resulted in no detectable M form in insects and 54%of maternal nematodes lacking adherent bacteria. i, intestine; r, rectum.

Fig. 3

Change in phenotypes, pathogenicity, secondary metabolite production, and persistence between P and M forms. (A) to (E) Quadrants on Petri dishes containing the P form (left), M form (right), unlocked forms (top), and locked forms (bottom). (A) Bioluminescence produced by the P form, which is absent in the M form (note that bioluminescence from P-form revertants is visible in the M form). (B) The P form is yellow and opaque, and the M form is unpigmented and transparent. (C) Antimicrobial activity produced at 48 hours of growth on LBP by the P form and not the M form against a softagar overlay containing Micrococcus luteus indicator bacteria. (D) Production of siderophore iron-chelating activity by the P form and not the M form indicated as a zone of clearing as iron is removed from chrome azural S chelator. (E) Hemolytic activity on sheep blood agar produced by the P form but not the M form. (F) Virulence of the L/P form and not the L/M form after injection into Galleria mellonella. (G) Metabolite analysis detected the production of rhabduscin (1), two anthraquinone pigment molecules (2 and 3) and two hydroxystilbene molecules (4 and 5), which were mostly absent in the L/M form. (H) There are 50 times more persister cells tolerant to ciprofloxacin in the L/M form than in the L/P form.

Fig. 4

Single-cell reporter studies of the madswitch during the development of symbiosis in nematodes. (A) A gfp gene was inserted between madA and madB so as not to disrupt expression of downstream mad genes. The madswitch ON cells are small, green fluorescent cells capable of maternal adhesion, and the madswitch OFF cells are large, non-fluorescent cells that are the majority of cells transiently present in the maternal nematode intestine. (B) Few cells (white arrows) have the madswitch oriented ON in culture. (C) Small colonies have the madswitch oriented ON, and large colonies have the madswitch oriented OFF. (D) An isolated colony of the M form (green) develops dark, opaque sectors that are madswitch oriented OFF P form. (E) madswitch-ON cells adhere to the posterior maternal nematode intestine, and most cells transiently present are not fluorescent, with the madswitch OFF. (F) Most adherent cells that invade and grow inside vacuoles of the rectal gland cells have the madswitch oriented ON. (G) One or two cells on or inside the pharyngeal intestinal valve cells have the madswitch oriented ON. (H) Seven days after the symbionts fully colonize the IJs, all cells are not fluorescent with the madswitch OFF, and again, the insect pathogenic P form is arming the nematode for insect infection. i, intestine; r rectum; p, pharynx.