Identification and distribution of insertion sequences of Paracoccus solventivorans

Abstract

Three novel insertion sequences (ISs) (ISPso1, ISPso2, and ISPso3) of the soil bacterium Paracoccus solventivorans DSM 11592 were identified by transposition into entrapment vector pMEC1. ISPso1 (1,400 bp) carries one large open reading frame (ORF) encoding a putative basic protein (with a DDE motif conserved among transposases [Tnps] of elements belonging to the IS256 family) with the highest levels of similarity with the hypothetical Tnps of Rhodospirillum rubrum and Sphingopyxis macrogoltabida. ISPso2 (832 bp) appeared to be closely related to ISPpa2 of Paracoccus pantotrophus DSM 11072 and IS1248 of Paracoccus denitrificans PdX22, both of which belong to the IS427 group (IS5 family). These elements contain two overlapping ORFs and a putative frameshift motif (AAAAG) responsible for production of a putative transframe Tnp. ISPso3 (1,286 bp) contains a single ORF, whose putative product showed homology with Tnps of ISs classified as members of a distinct subgroup of the IS5 group of the IS5 family. The highest levels of similarity were observed with ISSsp126 of Sphingomonas sp. and IS1169 of Bacteroides fragilis. Analysis of the distribution of ISs of P. solventivorans revealed that ISPso2-like elements are the most widely spread of the elements in nine species of the genus PARACOCCUS: ISPso1 and ISPso3 are present in only a few paracoccal strains, which suggests that they were acquired by lateral transfer. Phylogenetic analysis of Tnps of the novel ISs and their closest relatives showed their evolutionary relationships and possible directions of lateral transfer between various bacterial hosts.

FIG. 1.

Comparison of the amino acid sequences of the predicted DDE motifs of the putative Tnps encoded by ISPso1, ISPso2, and ISPso3 with the appropriate family- and group-specific consensus sequences (23). In the consensus sequences uppercase letters indicate conservation within the family, lowercase letters indicate predominant amino acids, and dashes indicate nonconserved residues. Residues forming the DDE motif are indicated by a black background. The N2, N3, and C1 domains are enclosed in boxes and labeled. The residues conserved in the domains of the Tnps analyzed and the consensus sequences are indicated by boldface type. The numbers in parentheses are the distances (in amino acids [aa]) between the residues forming the DDE motif.

FIG. 2.

Alignment of the terminal nucleotide sequences of ISPso1 (A), ISPso2 (B), and ISPso3 (C) and their relatives. The identical residues of the termini of an IS of P. solventivorans are indicated by a black background. The putative IRs are indicated by bars. The nucleotides of other ISs identical to those of ISPso1 (IS1194 [accession no. Y13626] and ISBli2 [accession no. AF195203]), ISPso2 (ISPpa2 [accession no. AY179508] and IS1248 [accession no. PDU08856]), or ISPso3 (IS1246 [accession no. NC 003350] and ISSsp126 [accession no. SSP277295]) are indicated by boldface type and a gray background. The consensus sequence (Cons) of the IRs compared is included in each panel. L, sequences at the 5′ (left) end; R, complementary sequences at the 3′ (right) end of the elements.

FIG. 3.

Analysis of the distribution of ISPso1-like (A), ISPso2-like (B), and ISPso3-like (C) elements by DNA hybridization. The lanes contained EcoRI-PstI-digested DNAs of P. solventivorans DSM 11592 (lane 1), P. alkenifer DSM 11593 (lane 2), P. aminophilus JMC 7686 (lane 3), P. aminovorans JCM 7685 (lane 4), P. denitrificans DSM 413 (lane 5), P. denitrificans LMD 22.21 (lane 6), P. methylutens DM12 (lane 7), P. pantotrophus DSM 65 (lane 8), P. pantotrophus LMD 82.5 (lane 9), P. pantotrophus DSM 11072 (lane 10), P. pantotrophus DSM 11073 (lane 11), P. alcaliphilus JCM 7364 (lane 12), P. thiocyanatus IAM 12816 (lane 13), P. versutus UW1 (lane 14), and pSOV1 of P. solventivorans DSM 11592 (lane 15). The positions of the markers are indicated on the left.

FIG. 4.

Unrooted bootstrap tree (1,000 replicates) for putative Tnps encoded by ISPso1, ISPso2, and ISPso3 and their closest relatives (identified in available databases), constructed by the parsimony method. The tree was constructed by using SEQBOOT, DNAPARS, CONSENSUS, and DRAWTREE in the software package PHYLIP (14). The numbers at the nodes indicate bootstrap values for the nodes based on 1,000 bootstrap resamplings (values less than 80 are not shown). The taxonomic groups (as described by Garrity and Holt [15]) are indicated in brackets, as follows: A, Actinobacteria; B, Bacteroidetes; αP, alpha subgroup of the Proteobacteria; βP, beta subgroup of the Proteobacteria; γP, gamma subgroup of the Proteobacteria; Bl, bacilli. The sequences used for this analysis were sequences of Agrobacterium tumefaciens (accession no. X53945), Azotobacter vinelandii (ZP 00089333 [superscript 1] and ZP 00088382[superscript 2]), Bacteroides fragilis (S44507 [superscript 1] and I40184[superscript 2]), Bacteroides vulgatus (I40597), Bradyrhizobium japonicum (NP 769117 [superscript 1], NP 769117 [superscript 2], NP 771828 [superscript 3], and NP 774822 [superscript 4]), Brevibacterium linens (AAF09242), Brucella melitensis (NP 540341 [superscript 1] and NP 540319 plus NP 540320 [superscript 2]), Brucella ovis (Q08082 [superscript 1] and M94960 [superscript 2]), Corynebacterium efficiens (NP 737799 [superscript 1], NP 737110 [superscript 2], and NP 702969 [superscript 3]), Corynebacterium jeikeium (NP 848205), Corynebacterium striatum (AAG03374), Mesorhizobium loti (NP 109452 plus NP 109453 [superscript 1] and NP 108276 [superscript 2]), Micrococcus sp. (AAK62483), Mycobacterium avium (AAA69904 [superscript 1], CAA11709 [superscript 2], and AAC71696 [superscript 3]), Mycobacterium celatum (CAA65977), Mycobacterium gordonae (AAB54010), Mycobacterium tuberculosis (NP 218157 [superscript 1], NP 338287 [superscript 2], and NP 337078 [superscript 3]), Neisseria meningitidis (Z49092), Novosphingobium aromaticivorans (ZP 00093225 plus ZP 00093229), P. denitrificans (AAC43507 plus AAC43509), P. pantotrophus (AAO21198 plus AAO21199), P. solventivorans (AAO84921 [superscript 1], AAP76386 [superscript 2], and AAO48787 plus AAO48788 [superscript 3]), Porphyromonas gingivalis (CAA10225), Ralstonia solanacearum (BAA97979 plus BAA97980), Rhodococcus opacus (AAB57888), Rhodococcus rhodochrous (BAA11042), Rhodospirillum rubrum (ZP 00016443), Sphingomonas sp. (CAB87573), Sphingopyxis macrogoltabida (BAB07803), and Streptococcus thermophilus (AF454495). Some of the Tnps of the IS427 group are encoded by two overlapping ORFs; therefore, two accession numbers are given above. In these cases the sequences of the transframe fusion Tnps (generated in silico within the putative frameshift motif) were used for the analysis. The designations of the defined ISs are the GenBank and ISDatabase designations.