Identification and distribution of new insertion sequences in the genome of alkaliphilic Bacillus halodurans C-125

Abstract

Fifteen kinds of new insertion sequences (ISs), IS641 to IS643, IS650 to IS658, IS660, IS662, and IS663, and a group II intron (Bh.Int) were identified in the 4,202,352-bp genome of alkaliphilic Bacillus halodurans C-125. Out of 120 ISs identified in the C-125 genome, 29 were truncated, indicating the occurrence of internal rearrangements of the genome. The ISs other than IS650, IS653, IS660, and IS663 generated a 2- to 9-bp duplication of the target site sequence, and the ISs other than IS650, IS653, and IS657 carry 14- to 64-bp inverted repeats. Sequence analysis revealed that six kinds of ISs (IS642, IS643, IS654, IS655, IS657, and IS658) belong to a separate IS family (IS630, IS21, IS256, IS3, IS200/IS605, and IS30, respectively) as a new member. Also, IS651 and IS652 were characterized as new members of the ISL3 family. Significant similarity was found between the transposase (Tpase) sequences between IS650 and IS653 (78.2%), IS651 and IS652 (56.3%), IS656 and IS662 (71.0%), and IS660 and IS663 (44.5%), but the others showed no similarity to one another. Tpases in 28 members of IS651 in the C-125 genome were found to have become diversified. Most of the IS elements widely distributed throughout the genome were inserted in noncoding regions, although some genes, such as those coding for an ATP-binding cassette transporter/permease, a response regulator, and L-indole 2-dehydrogenase, have been mutated through the insertion of IS elements. It is evident, however, that not all IS elements have transposed and caused rearrangements of the genome in the past 17 years during which strain C-125 was subcultured under neutral and alkaline conditions.

FIG. 1

Distribution of IS elements and group II intron in the B. halodurans C-125 genome. Arrows indicate the direction of the ISs, and the number in parentheses is the copy number of each element.

FIG. 2

Terminal IRs and TSD of each element identified in the B. halodurans genome. IRs are shown in blue capitals and TSDs are boxed. Red letters indicate the sequence of the B. halodurans genome.

FIG. 3

Structure of each IS element and group II intron identified in the B. halodurans genome. The box shows the Tpase of each element, and the numbers beside each box indicate the position of the Tpase in the element. The gray bars indicate the elements identified in the genome. The gray and black dashed lines indicate deleted and inserted parts, respectively, in the element. The small vertical bar at the end of the element denotes IRs. The black upside-down triangle denotes insertion of another element. The partial IS element without a terminal sequence shorter than 100 bp is not shown.

FIG. 4

Evolutionary relationships among Tpases in the IS elements identified in the B. halodurans genome. (A) Similarity matrix based on percent similarity among 15 Tpases of IS elements identified in the C-125 genome. The Tpases of the IS elements (IS642, IS655, IS657, and IS658) showed very low similarity to one another and to those of the other IS elements, and therefore they are not shown here. (B) Unrooted phylogenetic tree of the Tpases of the copies of IS651 identified in the C-125 genome. Amino acid sequences of the IS651 Tpases were aligned using the Clustal multiple-alignment program (Clustal X) (49). Sites involving gaps were excluded from all analyses. Consensus sequence segment alignment of the whole region was used to construct a phylogenetic tree for the various IS651 Tpases. A phylogenetic tree was constructed by the neighbor-joining method (39) using the Clustal X program, version 1.64b, and drawn by means of Tree view. Bar = 0.01 Knuc unit.

FIG. 5

Pattern of insertion of IS elements into protein-coding regions of the genome. (A) The case in which CDS 1 and CDS 2 identified on both sides of the Tpase coding region in the IS element merge as one CDS upon elimination of the IS element. (B) The case in which an alternation occurred in the C-terminal region of the CDS upon insertion of the IS element.

FIG. 6

Comparison of the IS elements amplified by PCR from the chromosomes of the strains C-125-83 and C-125-00. The primer sets used are shown by short solid arrows. Lanes O, strain C-125-83; lanes N, strain C-125-00. The positions of molecular size (in kilobases) markers are on the left.