Klein et al. Mobile DNA (2015) 6:18 DOI 10.1186/s13100-015-0049-1 RESEARCH Open Access Identification and characterization of a minisatellite contained within a novel miniature inverted-repeat transposable element (MITE) of Porphyromonas gingivalis Brian A. Klein1,2, Tsute Chen2, Jodie C. Scott2, Andrea L. Koenigsberg1, Margaret J. Duncan2 and Linden T. Hu1* Abstract Background: Repetitive regions of DNA and transposable elements have been found to constitute large percentages of eukaryotic and prokaryotic genomes. Such elements are known to be involved in transcriptional regulation, host-pathogen interactions and genome evolution. Results: We identified a minisatellite contained within a miniature inverted-repeat transposable element (MITE) in Porphyromonas gingivalis.TheP. gingivalis minisatellite and associated MITE, named ‘BrickBuilt’, comprises a tandemly repeating twenty-three nucleotide DNA sequence lacking spacer regions between repeats, and with flanking ‘leader’ and ‘tail’ subunits that include small inverted-repeat ends. Forms of the BrickBuilt MITE are found 19 times in thegenomeofP. gingivalis strain ATCC 33277, and also multiple times within the strains W83, TDC60, HG66 and JCVI SC001. BrickBuilt is always located intergenically ranging between 49 and 591 nucleotides from the nearest upstream and downstream coding sequences. Segments of BrickBuilt contain promoter elements with bidirectional transcription capabilities. Conclusions: We performed a bioinformatic analysis of BrickBuilt utilizing existing whole genome sequencing, microarray and RNAseq data, as well as performing in vitro promoter probe assays to determine potential roles, mechanisms and regulation of the expression of these elements and their affect on surrounding loci. The multiplicity, localization and limited host range nature of MITEs and MITE-like elements in P. gingivalis suggest that these elements may play an important role in facilitating genome evolution as well as modulating the transcriptional regulatory system. Keywords: Species-specific repeat, DNA structure, Miniature Inverted-repeat Transposable Element, BrickBuilt, Transcriptional regulation, Porphyromonas Background genetic and genomic analyses of the species [9–16]. Each Porphyromonas gingivalis, a gram-negative, anaerobic, of the sequenced P. gingivalis genomes has contained mul- asaccharolytic, black-pigmenting bacterium, is a keystone tiple repetitive and transposable elements, an aspect that pathogen in the development and progression of peri- makes sequencing and alignment difficult. odontal disease [1, 2]. Multiple repetitive and transposable Repetitive Elements (REs) are DNA sequences present elements were previously identified in the P. gingivalis in multiple copies throughout a genome, chromosome genomes [3–12]. Genome sequences are now available for or vector. They are broadly classified into ‘terminal’, multiple strains of P. gingivalis which has greatly facilitated ‘tandem’ and ‘interspersed’ repeats, however, each of these classifications encompasses several sub-types of REs. Tandem repeats are classified as either identical or non- * Correspondence: [email protected] identical based on the level of nucleic acid matching. They 1Department of Molecular Biology and Microbiology, Tufts University Sackler School of Biomedical Sciences, Boston, MA 02111, USA are then further classified as either micro, mini or macro Full list of author information is available at the end of the article © 2015 Klein et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Klein et al. Mobile DNA (2015) 6:18 Page 2 of 19 satellites based on size of the repeat. Repetitive elements prevalence throughout the species, as well as implica- can either be localized at a single site where a motif is tions on genome (in)stability and transcriptional regula- recurrent sequentially adjacent to each other or at many tion are described. Similarities to other autonomous and loci as reiteration [17–19]. non-autonomous P. gingivalis transposable elements are Transposable Elements (TEs) are ‘mobile’ DNA se- addressed with the goal of defining a potential network quences that can change locus or multiply and insert into for the biogenesis of these elements in P. gingivalis and new loci within a genome or between genomes via exci- their effects on the P. gingivalis genome. sion/replication and insertion. They can insert into chro- mosomes, plasmids and bacteriophages. Class I TEs are Results and discussion retrotransposons, which require reverse-transcriptase ac- Identification of a repetitive element in Porphyromonas tivity to transpose. Class II TEs are DNA transposons, gingivalis which unlike reverse transcriptase-utilizing Class I ele- We identified a DNA element, ‘BrickBuilt’, in the genome ments, require a transposase or a replicase to transpose of P. gingivalis strain ATCC 33277. The element was [19–21]. Class II elements can either be autonomous or initially identified as a tandemly-repeated sequence of non-autonomous, the latter [canonically] having under- 23 nt located intergenically at a single site (Additional file 1: gone mutations involving the transposase such that they Figure S1). A more thorough investigation of the genome can no longer duplicate or excise without the assistance of revealed 19 independent, non-identical segments of the a parent element that utilizes a similar transposase. Within element scattered throughout the genome of strain ATCC the non-autonomous element sub-class are miniature 33277 (Table 1). The smallest number of 23 nt direct re- inverted-repeat transposable elements, or MITEs [22–25]. peats is 1 (BrickBuilt_1) and the largest 22.8 (Brick- MITEs have a distinct structure relative to other TEs. Built_12). The 23 nt direct repeats are imperfect within a They are between 50–1000 bp in length and are often given element, imperfect bases vary from one element to present in high copy numbers per genome. MITEs are another and imperfections do not correlate with length or typically AT-nucleotide (nt) rich and frequently contain total number of repeats within a given element (Fig. 1). terminal inverted repeats (TIRs) and target site duplica- The percent of mismatches within a given element varies tions (TSDs), but they lack the capacity to code for func- from 0 to 11, and the percent of insertions and deletions tional transposases [22–25]. Transposable elements, in within an element varies from 0 to 6. Within the 23 nt re- particular MITEs, can be found in all taxa, varying in peats there are conserved and non-conserved nucleotide number and type between species and can account for sites, with the latter half of the element containing the greater than half of a genome. Bacteria typically carry majority of non-conserved sites (Fig. 1). Although similar between 10–20 copies of a MITE per genome, while in length to CRISPR element spacers and microRNAs, plants may have up to 20,000 copies of a given MITE. BrickBuilt elements are seemingly unrelated to these other Copy numbers are suggested to depend on non-coding entities. region availability, polyploidy, the presence of a fully- After determining the length and locations of each inde- functional autonomous version of a transposase, evolu- pendent direct repeat element we performed alignments tionary ‘burst’ opportunities and regulatory potential of of the sequences flanking the direct repeats to determine the given element [26–29]. Eukaryotic MITEs are fre- whether specific DNA sequences or motifs were necessary quently found in or closely associated with the coding for the presence of the element. Alignments of the se- region while prokaryotic MITEs are almost exclusively quences flanking the direct repeats revealed regions of found intergenically [26, 30–36]. Intergenically located homology, different for the two flanks of the repeat, which MITEs in prokaryotes have been shown to be able to were determined to be ‘leader’ and ‘tail’ regions that affect gene expression [23, 25]. encompassed the direct repeats (Fig. 2). Of the 19 ele- Several studies have demonstrated potential interactions ments, 11 are flanked by portions of both a leader and a of repetitive elements with transposable elements, which tail, 3 by just leader, 2 by just tail, and 3 by neither. When are generally thought to work independently and be mu- considered as a single whole element, all BrickBuilt ele- tually exclusive. In the wedge clam (Donax trunculu)gen- ments are intergenic, although some are within regions ome as well as the butterfly and moth (Lepidoptera) where annotation pipelines predicted hypothetical genes genomes, ‘hitchhiking’ microsatellites were found within that do not appear to be expressed based on proteomic transposable elements [37, 38]. Microsatellites and simple data [40–42]. Total length of the complete elements sequence repeats have also been found closely associated ranges from 991 nt (BrickBuilt_5) to 84 nt (BrickBuilt_14), with transposable elements in Neisseria meningitidis [39]. which is determined