Identification and functional analysis of recent IS transposition events in rhizobia

Abstract

Rhizobia are alpha- and beta- Proteobacteria that, through the establishment of symbiotic interactions with leguminous plants, are able to fix atmospheric nitrogen as ammonium. The successful establishment of a symbiotic interaction is highly dependent on the availability of nitrogen sources in the soil, and on the specific rhizobia strain. Insertion sequences (ISs) are simple transposable genetic elements that can move to different locations within the host genome and are known to play an important evolutionary role, contributing to genome plasticity by acting as recombination hot-spots, and disrupting coding and regulatory sequences. Disruption of coding sequences may have occurred either in a common ancestor of the species or more recently. By means of ISComapare, we identified Differentially Located ISs (DLISs) in nearly related rhizobial strains of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium. Our results revealed that recent IS transposition could have a role in adaptation by enabling the activation and inactivation of genes that could dynamically affect the competition and survival of rhizobia in the rhizosphere.

Keywords: DLIS; ISCompare; Insertion sequence; Rhizobia; Symbiosis; Transposition.

Fig. 1

Distribution of transposase and IS related genes. A. Normalized IS and pseudo IS counts. B. Normalized number of IS elements by replicon. C. Normalized numbers of ISs and pseudo ISs by replicon. D. Normalized number of IS elements by species and replicon. E. Normalized number of ISs and pseudo ISs by species. The number of transposases and IS related genes was estimated from the genbank files annotation using custom python scripts that looked for the terms ‘transposase’ and ‘insertion seq’ in the product descriptions of coding sequences and pseudo genes. Normalized counts were calculated as ISs / 100,000 bp. Significance was determined using the Mann-Whitney test and the normalized counts (*: 1.00e-02 < p < = 5.00e-02; **: 1.00e-03 < p < = 1.00e-02; ***: 1.00e-04 < p < = 1.00e-03; ****: p < = 1.00e-04)

Fig. 2

Distribution of DLISs. Chromosomes vs. plasmids. A. Total DLIS counts. B. DLIS counts and normalized DLIS counts discriminated by rhizobia and replicon type. C. Proportions of DLISs in chromosomal replicons, calculated from DLIS counts. D. Proportions of DLISs in chromosomal replicons, calculated from DLIS normalized counts. DLISs were identified with ISCompare, and those with a full match for an insertion sequence and confidently identified as DLIS were analyzed. Significance was determined either using Chi2 test and the total DLIS counts in each species, or the Mann-Whitney test and the normalized DLIS counts (DLIS counts / 100,000 bp). *: 1.00e-02 < p < = 5.00e-02; **: 1.00e-03 < p < = 1.00e-02; ***: 1.00e-04 < p < = 1.00e-03; ****: p < = 1.00e-04

Fig. 3

Proportion of recently active ISs. (A) Proportion of active ISs. The proportion of recently active ISs was estimated as the relation of DLISs over the total ISs count. Significance was determined using the Mann-Whitney test and the proportions of active ISs (DLISs / ISs). *: 1.00e-02 < p < = 5.00e-02; **: 1.00e-03 < p < = 1.00e-02; ***: 1.00e-04 < p < = 1.00e-03; ****: p < = 1.00e-04. (B) Pearson correlation of IS counts and DLIS counts

Fig. 4

Distribution of DLISs in coding and intergenic regions. A. Proportion of DLISs inserted within genes, intergenic regions and putative operons. B. Proportion of DLISs inserted within genes, intergenic regions and putative operons discriminated by rhizobial species. C. Absolute count numbers of DLISs within genes, intergenic regions, and putative promoter regions. The count of the promoter regions is a subset of the counts for intergenic regions. When a DLIS was inserted within a short intergenic region between two divergent genes it was counted twice since it could be affecting the transcription of both genes. The location of the DLIS was determined using custom python scripts and the bedtools software as described in material and methods. Significance was determined using the Mann-Whitney test and the normalized counts

Fig. 5

Distribution of COG functional categories. COGs functional categories for the genes interrupted by a DLIS were assigned with eggNOG-mapper. A. Distribution of DLIS interrupted genes by COG functional category. B. Heatmap of COG distribution per reference strain. COG categorías are as follows. -: Not assigned; A: RNA processing and modification; B: Chromatin Structure and dynamics; C: Energy production and conversion; D: Cell cycle control and mitosis; E: Amino Acid metabolism and transport, F: Nucleotide metabolism and transport, G: Carbohydrate metabolism and transport; H: Coenzyme metabolism; I: Lipid metabolism; J: Translation; K: Transcription; L: Replication and repair; M: Cell wall/membrane/envelope biogenesis; N: Cell motility; O: Post-translational modification, protein turnover, chaperone functions; P: Inorganic ion transport and metabolism; Q: Secondary metabolites biosynthesis, transport and catabolism; R: General Functional Prediction only; S: Function Unknown; T: Signal Transduction; U: Intracellular trafficking and secretion; V: Defense mechanisms: W: Extracellular structures: X: Mobilome: prophages, transposons; Y: Nuclear structure; Z: Cytoskeleton