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Attenuated virulence and genomic reductive evolution in the entomopathogenic bacterial symbiont species, Xenorhabdus poinarii - PubMed

️Wed Jan 01 2014

Attenuated virulence and genomic reductive evolution in the entomopathogenic bacterial symbiont species, Xenorhabdus poinarii

Jean-Claude Ogier et al. Genome Biol Evol. 2014.

Abstract

Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent X. doucetiae strain FRM16. G6 had a smaller genome (500-700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (hemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, and toxin-antitoxin systems). The genomes of all the X. poinarii strains analyzed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host.

Keywords: Lepidoptera; Steinernema; comparative genomics; entomopathogenic bacteria; genomic deletion; regions of genomic plasticity.

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Figures

F<sc>ig</sc>. 1.— — **Fig. 1.—**
Maximum-likelihood phylogenetic tree showing the positions of *Xenorhabdus poinarii* strains within the genus *Xenorhabdus*. The analysis is based on five concatenated protein-coding sequences (*recA*, *gyrB*, *dnaN*, *gltX*, and *infB*). It was carried out with the GTR model of substitution, with a gamma-distributed rate heterogeneity and a proportion of invariant sites. *Photorhabdus* and *Proteus* sequences were used as outgroups. Bootstrap values (Felsenstein 1988) of more than 80% (from 100 replicates) are indicated at the nodes. Clade C_I, which includes all the *X. poinarii* strains, is as previously described (Tailliez et al. 2010). The names of strains for which genomes have previously been sequenced or were sequenced in this study are indicated in bold italic and bold normal typescript, respectively. Bar: 10% divergence.

F<sc>ig</sc>. 2.— — **Fig. 2.—**
Venn diagram showing numbers of orthologous genes in the genomes of *Xenorhabdus nematophila* ATCC19061, *X. bovienii* SS-2004, *X. doucetiae* FRM16 and *X. poinarii* G6. The Xcg (1,904 gene families) is framed by red dashes, and includes the Ecg (1,547 gene families common to *Escherichia coli* K12) and the specific Xcg (357 gene families).

F<sc>ig</sc>. 3.— — **Fig. 3.—**
Whole-genome sequence alignments between *Xenorhabdus* genomes. The line plots were obtained with the results for synteny between (A) *X. poinarii* G6 (*Xp_G6*) and *X. doucetiae* FRM16 (Xd); (B) *Xp_G6*, Xn, and Xb; (C) Xd, Xn, and Xb. Matches between synteny groups occurring on the same strand are shown in purple; matches between synteny groups occuring on the opposite strand are shown in blue. Numbers in brackets indicate the percent of CDS in synteny for each whole-genome alignment.

F<sc>ig</sc>. 4.— — **Fig. 4.—**
Estimation of *Xenorhabdus poinarii* strains genome size by PFGE of I-*Ceu*I-hydrolyzed genomic DNA. The separation of I-*Ceu*I fragments was optimized by using different electrophoresis conditions for fragments of different sizes: (A) a pulse ramp from 150 to 400 s for 45 h for I-*Ceu*I fragments between 500 and 4,000 kb in size; (B) a pulse ramp from 5 to 35 s for 24 h for fragments of less than 500 kb in size. Schematic representations of the I-*Ceu*I PFGE patterns under two sets of migration conditions, making it possible to separate fragments from 500 to 4,000 kb in size (C) and fragments from 10 to 500 kb in size (D), were also shown. Lane 1: *Saccharomyces cerevisiae* (strain 972h); lane 2: *X. bovienii* SS-2004; lane 3: *X. poinarii* AZ26; lane 4: *X. poinarii* G6; lane 5: *X. poinarii* SK72; lane 6: *X. poinarii* CU01; lane 7: *X. poinarii* NC33; lane 8: *X. doucetiae* FRM16; lane 9: *Hansenula wingei* (strain YB-4662-VIA). Dashed bands around 120 kb in strains *Xp_AZ26* (lane 3) and *Xp_SK72* (lane 4) correspond to fragments with a lower staining intensity, probably plasmids. *Although these bands are difficult to see on the gel photography, there were directly distinguishable on the gel and their sizes were confirmed by the theorical I-*Ceu*I pattern of the genome sequences of *X. bovienii* SS-2004 and *X. poinarii* G6. Fragment and genome sizes of the four unsequenced *X. poinarii* strains were evaluated with the *X. poinarii G6*, *X. bovienii* SS-2004, and *X. doucetiae* FRM16 genomes used as a reference (lanes 2, 4, and 8) and molecular weight ladders (lanes 1 and 9).

F<sc>ig</sc>. 5.— — **Fig. 5.—**
The *xaxAB* locus, its genomic context and its shuffling point *exbD*/*rdgC* in the *X. doucetiae* FRM16 (Xd), *X. nematophila* ATCC19061 (Xn), *X. bovienii* SS-2004 (Xb), *X. poinarii* G6 (*Xp_G6*), AZ26 (*Xp_AZ26*), NC33 (*Xp_NC33*), SK72 (*Xp_SK72*), and CU01 (*Xp_CU01*) genomes. The large arrows represent individual ORFs, and the names of the genes are indicated above the arrows. Genes encoding proteins of unknown function are marked with an asterisk. Orthologous genes are indicated by arrows in the same color. Black and chequered arrows represent core-genome genes and transposase genes, respectively. The thin arrows indicate the binding sites of the primers used for PCR amplification. The vertical parallel lines indicate the end of the sequenced area and the dotted lines represent an unsequenced genomic region. The cladogram was obtained by the maximum-likelihood phylogenetic analysis of five concatenated protein-coding sequences (*recA*, *gyrB*, *dnaN*, *gltX*, and *infB*), as already described in figure 1. The accession numbers of the sequences of the subsequent amplicons are HG934736 (strain AZ26), HG934737 (strain NC33), HG934738 (strain SK72), HG934739 and HG934740 (strain CU01).

F<sc>ig</sc>. 6.— — **Fig. 6.—**
Analysis of the evolutionary history of the *xaxAB* locus by a comparison of topology between an Enterobacteriaceae tree and a *xaxA* gene tree. (A) Enterobacteriaceae phylogenetic tree based on a maximum-likelihood (ML) analysis of 12 core concatenated protein-coding sequences (*infB, nusA, polA, pyrD, rpoB, valS, cysS, metK, purA, tpiA, smpB, secY*). *Vibrio cholerae* sequences were used as the outgroup. Nodes are supported by bootstrap values of more than 93%, unless marked with an asterisk. (B) Phylogenetic tree based on ML analysis of the *xaxA* gene. Nodes are supported by bootstrap values of more than 86%, unless marked with an asterisk. Node A, the bacterial ancestor of the *Providencia–Proteus–Photorhabdus–Xenorhabdus* clade, which probably contained the *xaxA* gene. Node B, bacterial ancestor of the *Yersinia kristensenii* and *Y. enterocolitica* species, to which the *xaxA* gene was probably transferred horizontally. Crosses, probable deletions of the xaxA gene. *Vibrio cholerae* 16961: NC_002501; *Prot. penneri* ATCC35198: PRJNA54897; *Prot. mirabilis* HI4320: NC_010554; *Arsenophonus nasoniae* DSM15247: PRJNA185551; *Prov. stuartii* ATCC25827: PRJNA54899; *Prov. rettgeri* DSM1131: PRJNA55119; *Prov. rustigianii* DSM 4541: PRJNA55071; *Prov. alcalifaciens* DSM30120: PRJNA55119; *Ph. luminescens* TT01: NC_005126.1; *Ph. asymbiotica* ATCC43949: NC_012962; *X. cabanillasii* JM26: CBXE010000001-CBXE010000496; *X. bovienii* SS-2004: NC_013892; *X. szentirmaii* DSM16638: CBXF010000001-CBXF010000164; *X. nematophila* ATCC19061: NC_014228.1; *X. poinarii* G6: FO704551; *X. doucetiae* FRM16: FO704550; *Y. ruckeri* ATCC297473: PRJNA55249; *Y. pseudotuberculosis* IP31758: NC_009708; *Y. pestis* CO92: NC_003143; *Y. intermedia* ATCC29909: PRJNA54349; *Y. aldovae* ATCC35236: PRNJA35243; *Y. mollaretii* ATCC43969: PRJNA54345; *Y. bercovieri* ATCC43970: PRJNA54343; *Y. rohdei* ATCC43380: PRJNA55247; *Y. frederiksenii* ATCC33641: PRJNA54347; *Y. kristensenii* ATCC33638: PRJNA55245; *Y. enterocolitica* 8081: NC_008800; *Serratia proteamaculans* 568: NC_0098332; *Se. odorifera* DSM4582: PRJNA40087; *Dickeya zeae* 1591: NC_012912; *Dickeya dadantii* 586: NC_013592; *Pectobacterium carotovorum* PC1: NC_012917; *Pe. wasabiae* WPP163: NC_013421; *Pe. atrosepticum* SCRI1043: NC_004547; *Edwarsiella tarda* EIB202: NC_013508; *Edwarsiella ictulari* 93-146: NC_012779.2; *Pantoea ananatis* LMG20103: NC_013956; *Erwinia billingiae* At-9b: NC_014837; *Er. tasmaniensis* Eb661: NC_014306; *Er. pyrifoliae* Ep1/96: NC_02214; *Er. amylovora* ATCC49946: NC_013971; *Klebsiella variicola* At-22: NC_013850; *K. pneumoniae* 342: NC_011283; *Salmonella enterica* Typhimurium LT2: NC_003197; *Sal. enterica* Typhi CT18: AL513382; *Escherichia albertii* TW07627: PRJNA55089; *Es. fergusonii* ATCC35469T: NC_011740; *Es. coli* K12: NC_000913).

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Attenuated virulence and genomic reductive evolution in the entomopathogenic bacterial symbiont species, Xenorhabdus poinarii - PubMed