Evolution of rhizobia by acquisition of a 500-kb symbiosis island that integrates into a phe-tRNA gene

Abstract

Nodulation and nitrogen fixation genes of Mesorhizobium loti are encoded on the chromosome of the bacterium. Nevertheless, there is strong evidence that these genes can be transferred from an inoculant strain to nonsymbiotic mesorhizobia in the field environment. Here we report that the chromosomal symbiotic element of M. loti strain ICMP3153 is transmissible in laboratory matings to at least three genomic species of nonsymbiotic mesorhizobia. The element is 500 kb in size, integrates into a phe-tRNA gene, and encodes an integrase of the phage P4 family just within its left end. The entire phe-tRNA gene is reconstructed at the left end of the element upon integration, whereas the 3' 17 nucleotides of the tRNA gene are present as a direct repeat at the right end. We termed the element a symbiosis island on the basis of its many similarities to pathogenicity islands. It may represent a class of genetic element that contributes to microbial evolution by acquisition.

Figure 1

Southern blot analysis demonstrating transfer of the symbiotic element to three species of nonsymbiotic mesorhizobia. Genomic DNA from the donor strain ICMP3153 and pairings of nonsymbiotic and symbiotic strains was digested with EcoRI and hybridized (A) with three random genomic HindIII fragments of ICMP3153 (18) and (B) with pRLnodAC containing M. loti nodAC genes (18).

Figure 2

(A) Alignment of sequences of two nonsymbiotic strains CJ4 (AF049244) and CJ7 (AF049245) at the site of insertion. The phe-tRNA gene is marked in yellow and homologous flanking sequences are marked in blue. A phe-tRNA sequence from Neisseria gonorrhoeae (U82700) is included for comparison. (B) Alignment of sequences of six symbiotic strains at the left junction. Identical nucleotides upstream of the element are marked in blue, the phe-tRNA gene is marked in yellow, and the adjoining element DNA is in brown. (C) Alignment of sequences of six symbiotic strains at the right junction. Element sequence is marked in brown and the 17 nucleotides of the 3′ end of the phe-tRNA are marked in yellow. The three homology groups beyond the junction are indicated by like colors.

Figure 3

(A) Linking of SpeI fragments of symbiotic element DNA by hybridization. R12C and R16C are diverse symbiotic strains that received the element from ICMP3153 in the field (17). Cosmids used for linking the fragments are named below the panels and are indicated together with other cosmids used for walking above the map in B. (B) Physical map of the element and location of features identified by sequencing of ICMP3153 DNA. Sizes of SpeI (S) fragments are shown in kilobases. There is 1.6 kb between the left junction of the element and the first SpeI site and 52 kb between the last SpeI site and the right junction. The sizes of the sequenced regions from ICMP3153 at the left junction (GenBank accession no. AF049242) and right junction (AF049243) are shown in base pairs. The region upstream of the element showed homology to the Escherichia coli melibiose regulatory gene melR (U14003), and the sugar-binding protein gene lacE from Agrobacterium radiobacter (X66596). The sequence at the right end homologous to fmet-tRNA is shown in comparison to sequence from Bartonella bacilliformis (L10238).

Figure 4

Alignment of the predicted amino acid sequence of the ICMP3153 symbiotic element integrase (3153 int) with sequences of some other members of the P4 integrase family: φR73 int, retronphage phi-R73 integrase (A42465); φSF6 int, Shigella flexneri phage Sf6 integrase (X59553); SlpA int, SlpA integrase from cryptic E. coli phage CP-57 (U36840); V. chol int, tcp-associated integrase from V. cholerae (U39068); D. nodo int, vap-associated integrase IntA from Dichelobacter nodosus (L31763). Identical amino acid residues are shaded.