Comparative Analysis of Xenorhabdus koppenhoeferi Gene Expression during Symbiotic Persistence in the Host Nematode

Abstract

Species of Xenorhabdus and Photorhabdus bacteria form mutualistic associations with Steinernema and Heterorhabditis nematodes, respectively and serve as model systems for studying microbe-animal symbioses. Here, we profiled gene expression of Xenorhabdus koppenhoeferi during their symbiotic persistence in the newly formed infective juveniles of the host nematode Steinernema scarabaei through the selective capture of transcribed sequences (SCOTS). The obtained gene expression profile was then compared with other nematode-bacteria partnerships represented by Steinernema carpocapsae-Xenorhabdus nematophila and Heterorhabditis bacteriophora-Photorhabdus temperata. A total of 29 distinct genes were identified to be up-regulated and 53 were down-regulated in X. koppenhoeferi while in S. scarabaei infective juveniles. Of the identified genes, 8 of the up-regulated and 14 of the down-regulated genes were similarly expressed in X. nematophila during persistence in its host nematode S. carpocapsae. However, only one from each of these up- and down-regulated genes was common to the mutualistic partnership between the bacterium P. temperata and the nematode H. bacteriophora. Interactive network analysis of the shared genes between X. koppenhoeferi and X. nematophila demonstrated that the up-regulated genes were mainly involved in bacterial survival and the down-regulated genes were more related to bacterial virulence and active growth. Disruption of two selected genes pta (coding phosphotransacetylase) and acnB (coding aconitate hydratase) in X. nematophila with shared expression signature with X. koppenhoeferi confirmed that these genes are important for bacterial persistence in the nematode host. The results of our comparative analyses show that the two Xenorhabdus species share a little more than a quarter of the transcriptional mechanisms during persistence in their nematode hosts but these features are quite different from those used by P. temperata bacteria in their nematode host H. bacteriophora.

Fig 1. The number and distribution of SCOTS identified genes in Xenorhabdus koppenhoeferi during persistence in Steinernema scarabaei.

The Venn diagram shows the proportion of up- (blue color) and down-regulated (olive green) genes unique to X. koppenhoeferi (X.k.) or common to the bacteria Xenorhabdus nematophila (X.n.) and Photorhbdus temperata (P.t.) during persistence in their nematode hosts.

Fig 2. Pathway diagram of genes with expression patterns shared by X. nematophila and X. koppenhoeferi during persistence in their respective host nematode S. carpocapsae and S. scarabaei.

Direct-connection networks for the genes with shared expression signatures were built in PathwayStudio by leveraging databases of currently published literatures. The genes are represented by red, blue or green ovals, where red ovals represent genes in the database of the PathwayStudio program with direct connection to the shared up- (green in panel A) and down-regulated (blue in panel B) genes identified in this study. Connecting lines between the gene symbols indicate interactions and different types of interactions are denoted by symbols on the lines. Purple square indicates binding; blue square, expression; grey square, regulation.

Fig 3. Growth and survival of Xenorhabdus nematophila wild-type, pta and acnB mutant cells in vitro.

The bacteria were incubated aerobically or anaerobically at 25°C in the BHI broth, M9 minimal medium, and M9 minimal medium with acetate for up to 14 days. The relative extents of growth (A and C: aerobic growth; B and D: anaerobic growth) were recorded at 48 h by measuring OD 600 and viability (E) at 14 d by plating on BHI agar. WT represents the wild-type strain of X. nematophila, Δpta denotes the mutant of the pta gene (encoding phosphotransacetylase), CΔpta is the genetically complemented pta⁺ strain, ΔacnB denotes the mutant of the acnB gene (encoding aconitate hydratase), CΔacnB is the genetically complemented acnB⁺ strain, and pJB861 indicates the mutants carrying the empty vector pJB861. Results (E) are shown as mean ± SEM from two independent experiments with three replicates per treatment, and the error bars (A, B, C, D) represent the standard error of the mean. Star symbol (*) indicates the statistical significance (p < 0.05) with comparison to the wild-type strain in each growth medium in the multiple t tests.

Fig 4. Persistence of the bacteria Xenorhabdus nematophila in the infective juvenile stage of nematode host Steinernema carpocapsae.

The data shown are the mean (± SEM) number of the pta mutant (A: Δpta denotes the pta mutant; CΔpta is the genetically complemented pta⁺ strain; pJB861 indicates the pta mutant with the control vector pJB861), acnB mutant (B: ΔacnB denotes the acnB mutant; CΔacnB is the genetically complemented acnB⁺ strain; pJB861 indicates the acnB mutant with the control vector pJB861) or wild-type (WT) bacterial cells persisting in an infective juvenile at 14, 30 and 45 days post-inoculation of the nematode eggs on the respective bacterial lawn. Star symbol (*) indicates the statistical significance (p < 0.05) with comparison to the wild-type strain in the multiple t tests.