Previously unrecognized stages of species-specific colonization in the mutualism betweenXenorhabdusbacteria andSteinernemanematodes

Abstract

Summary The specificity of a horizontally transmitted microbial symbiosis is often defined by molecular communication between host and microbe during initial engagement, which can occur in discrete stages. In the symbiosis between Steinernema nematodes and Xenorhabdus bacteria, previous investigations focused on bacterial colonization of the intestinal lumen (receptacle) of the nematode infective juvenile (IJ), as this was the only known persistent, intimate, and species-specific contact between the two. Here we show that bacteria colonize the anterior intestinal cells of other nematode developmental stages in a species-specific manner. Also, we describe three processes that only occur in juveniles that are destined to become IJs. First, a few bacterial cells colonize the nematode pharyngeal-intestinal valve (PIV) anterior to the intestinal epithelium. Second, the nematode intestine constricts while bacteria initially remain in the PIV. Third, anterior intestinal constriction relaxes and colonizing bacteria occupy the receptacle. At each stage, colonization requires X. nematophila symbiosis region 1 (SR1) genes and is species-specific: X. szentirmaii, which naturally lacks SR1, does not colonize unless SR1 is ectopically expressed. These findings reveal new aspects of Xenorhabdus bacteria interactions with and transmission by their Steinernema nematode hosts, and demonstrate that bacterial SR1 genes aid in colonizing nematode epithelial surfaces.

High Levels of the Xenorhabdus nematophila Transcription Factor Lrp Promote Mutualism with the Steinernema carpocapsae Nematode Host

“…The processes of X. nematophila colonization of both reproductive-stage and IJstage nematodes were described previously (4,16), and each process involves visible bacterial attachment to specific internal nematode surfaces. To directly test the influence of Lrp levels on the general ability of X. nematophila to attach to a surface, we used a well-established laboratory biofilm formation assay.…”

“…To create low-and high-Lrp-expressing X. nematophila strains, pMYC4 (low-lrp donor plasmid) and pMYC5 (high-lrp donor plasmid) were inserted into the kefA gene of an lrp-2::kan mutant via biparental conjugation and homologous recombination, respectively. Plasmid integration at the kefA gene locus does not interfere with nematode colonization (4,16,56,57). The Lrp-dependent fluorescence reporter pMYC1 (PfliC-gfp/P lac -rfp) was introduced into the attTn7 site of the genomes in both of the low-Lrp-and high-Lrp-expressing X. nematophila strains described above by triparental conjugation and Tn7 transposition (58,59), creating low-lrp in-genome (low-lrp/Tn7-PfliC-gfp/P lac -rfp) and high-lrp in-genome (high-lrp/Tn7-PfliC-gfp/P lac -rfp) strains.…”

“…We developed a simplified mathematical model to assess the relative contributions of development, reproduction, and emergence to our observed counts of nematodes from in vivo assays. The model was constructed under the assumptions that (i) all 50 aposymbiotic IJs injected into the insect survived and developed into adults; (ii) reproductive cycles were consecutive, with no overlaps; (iii) insect cadavers were not infected by competitive microbes such as fungi; (iv) carcass of dead nematodes did not decay; and (v) among different reproductive cycles, population expansion (parameter a), the percentage of the population that forms IJs (parameter b), and the percentage of IJs that emerged out of the insect cadaver (parameter c) were constant, except for the first reproduction cycle, in which no IJs are formed (b 1 ϭ 0; otherwise, b Ͼ 0) (16).…”

“…During infection of a living insect, X. nematophila encounters both constitutive and inducible immune factors such as melanin and antimicrobial peptides (12)(13)(14) and localizes to iron-and collagen-rich connective tissue in the midgut extracellular matrix (15). X. nematophila bacteria, but not other bacteria, adhere to the anterior intestinal region of developing and reproducing nematodes within the insect cadaver (16,17), and in emerging IJs, X. nematophila cells are physically associated with a host-derived anucleate structure called the IVS (intravesicular structure), which is coated with wheat germ-reactive mucus-like material containing either N-acetylglucosamine or N-acetylneuraminic acid moieties (17). Although biofilm formation in X. nematophila has yet to be fully characterized, the species-specific association of X. nematophila with distinctive nematode tissues may involve biofilm-mediated attachment, similar to that which occurs in other mutualisms such as that between Vibrio fischeri bacteria and Euprymna scolopes squid during initiation of colonization (18).…”

Ready or Not: Microbial Adaptive Responses in Dynamic Symbiosis Environments

“…In adult and juvenile stages of nematodes, multiple Xenorhabdus cells attach to the epithelial surface of the anterior intestinal cecum (AIC) (Fig. 2B) (51). The consequences of this colonization for either Xenorhabdus or Steinernema have not been elucidated, but these observations remind us that the insect cadaver does not represent a single homogenous environment but rather that different X. nematophila cells within the population may be encountering various challenges and various levels of nutritional content, depending on whether they are free-living in the liquefied insect cadaver, associated with insect tissues, attaching to the nematode AIC region, or passing through the nematode intestinal lumen while the nematode is actively digesting.…”