Identification and characterization of a minisatellite contained within a novel miniature inverted-repeat transposable element (MITE) of Porphyromonas gingivalis
© Klein et al. 2015
Received: 11 June 2015
Accepted: 23 September 2015
Published: 6 October 2015
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.
We identified a minisatellite contained within a miniature inverted-repeat transposable element (MITE) in Porphyromonas gingivalis. The P. 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 the genome of P. 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.
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.
KeywordsSpecies-specific repeat DNA structure Miniature Inverted-repeat Transposable Element BrickBuilt Transcriptional regulation Porphyromonas
Porphyromonas gingivalis, a gram-negative, anaerobic, asaccharolytic, black-pigmenting bacterium, is a keystone pathogen in the development and progression of periodontal disease [1, 2]. Multiple repetitive and transposable elements were previously identified in the P. gingivalis genomes [3–12]. Genome sequences are now available for multiple strains of P. gingivalis which has greatly facilitated genetic and genomic analyses of the species [9–16]. Each of the sequenced P. gingivalis genomes has contained multiple repetitive and transposable elements, an aspect that makes sequencing and alignment difficult.
Repetitive Elements (REs) are DNA sequences present in multiple copies throughout a genome, chromosome or vector. They are broadly classified into ‘terminal’, ‘tandem’ and ‘interspersed’ repeats, however, each of these classifications encompasses several sub-types of REs. Tandem repeats are classified as either identical or non-identical based on the level of nucleic acid matching. They are then further classified as either micro, mini or macro satellites based on size of the repeat. Repetitive elements can either be localized at a single site where a motif is recurrent sequentially adjacent to each other or at many loci as reiteration [17–19].
Transposable Elements (TEs) are ‘mobile’ DNA sequences that can change locus or multiply and insert into new loci within a genome or between genomes via excision/replication and insertion. They can insert into chromosomes, plasmids and bacteriophages. Class I TEs are retrotransposons, which require reverse-transcriptase activity to transpose. Class II TEs are DNA transposons, which unlike reverse transcriptase-utilizing Class I elements, require a transposase or a replicase to transpose [19–21]. Class II elements can either be autonomous or non-autonomous, the latter [canonically] having undergone mutations involving the transposase such that they can no longer duplicate or excise without the assistance of a parent element that utilizes a similar transposase. Within the non-autonomous element sub-class are miniature inverted-repeat transposable elements, or MITEs [22–25].
MITEs have a distinct structure relative to other TEs. They are between 50–1000 bp in length and are often present in high copy numbers per genome. MITEs are typically AT-nucleotide (nt) rich and frequently contain terminal inverted repeats (TIRs) and target site duplications (TSDs), but they lack the capacity to code for functional transposases [22–25]. Transposable elements, in particular MITEs, can be found in all taxa, varying in number and type between species and can account for greater than half of a genome. Bacteria typically carry between 10–20 copies of a MITE per genome, while plants may have up to 20,000 copies of a given MITE. Copy numbers are suggested to depend on non-coding region availability, polyploidy, the presence of a fully-functional autonomous version of a transposase, evolutionary ‘burst’ opportunities and regulatory potential of the given element [26–29]. Eukaryotic MITEs are frequently found in or closely associated with the coding region while prokaryotic MITEs are almost exclusively found intergenically [26, 30–36]. Intergenically located MITEs in prokaryotes have been shown to be able to affect gene expression [23, 25].
Several studies have demonstrated potential interactions of repetitive elements with transposable elements, which are generally thought to work independently and be mutually exclusive. In the wedge clam (Donax trunculu) genome as well as the butterfly and moth (Lepidoptera) genomes, ‘hitchhiking’ microsatellites were found within transposable elements [37, 38]. Microsatellites and simple sequence repeats have also been found closely associated with transposable elements in Neisseria meningitidis .
Here we describe ‘BrickBuilt’, a miniature inverted-repeat transposable element containing a minisatellite, in P. gingivalis. The sequences, location, copy number, prevalence throughout the species, as well as implications on genome (in)stability and transcriptional regulation are described. Similarities to other autonomous and non-autonomous P. gingivalis transposable elements are addressed with the goal of defining a potential network for the biogenesis of these elements in P. gingivalis and their effects on the P. gingivalis genome.
Results and discussion
Identification of a repetitive element in Porphyromonas gingivalis
Genes/Coding Sequences located 5′ and 3′ to BrickBuilt elements. Gene numbers and characterizations correspond to strain ATCC 33277. Loci of BrickBuilt elements across four sequenced and annotated P. gingivalis strains. BrickBuilt elements are situated intergenically between the genes noted. Grayed-out boxes represent loci at which BrickBuilt is aberrant
RmuC domain (DUF 805)
AraC family transcriptional regulator
Zinc protease (Peptidase M16)
PF05656 family protein (DUF 805)
Immunoreactive antigen/PorSS CTD
Peptidase S41/PorSS CTD
Glycosyl transferase family 2
TonB-dependent receptor Cna protein
Glycine cleavage system subunit T
Conserved hypothetical (DUF2149)
Serine protease (Peptidase C10)
Aspartyl-tRNA amidotransferase B
Membrane protein putative ion channel
GTP cyclohydrolase I/PorSS CTD
RNA-binding protein/PorSS CTD
N utilization substance/PorSS CTD
Gliding motility protein/PorSS CTD
DNA-binding protein (PF00216)
Peptidylarginine deiminase/PorSS CTD
Peptidase C10/PorSS CTD
Transport multidrug efflux
ClpB chaperone and protease
PorSS C-terminal sorting domain
Peptidase S10/PorSS CTD
Cysteine protease (Peptidase C1)
DNA-binding protein from starved cells
The genome sequence of strain ATCC 33277 contains 2,345,886 bases. When complied together all 19 BrickBuilt elements in strain ATCC 33277 make up 10,276 bases, or 0.44 percent of the overall genome; the equivalent of 9 protein coding sequences in this strain on average.
Conservation of BrickBuilt elements in other strains of P. gingivalis
Of the 19 versions of BrickBuilt found within strain ATCC 33277, 16 are conserved between the analogous coding sequences within strains W83, TDC60 and HG66 (Table 1 and Additional file 2: Table S1). Strains HG66 and TDC60 contain 19 versions of BrickBuilt, equivalent to the number in strain ATCC 33277. However, strain W83 only contains 18 versions of the element. Strains ATCC 33277 and HG66 share the exact same 19 loci for BrickBuilt elements. One locus in strain TDC60 (BrickBuilt_4) is deviant and is encompassed by two ISPg1 elements. Strain W83 has three sites that differ from the other strains, all which are located adjacent to other types of IS or repetitive elements. In this strain BrickBuilt_4 is completely lacking, while BrickBuilt_7 and BrickBuilt_18 are aberrant with respect to size having only maintained 23 nt repeats.
BrickBuilt elements can be identified in the genome of P. gingivalis strain JCVI SC001, which is not yet included in the default NCBI BLAST nucleotide database settings. Genome searches of the FASTA files from the JCVI SC001 revealed that most BrickBuilt loci in the JCVI SC001 genome contained strings of undetermined bases, which can be attributed to the manner of isolation, sequencing and assembly. Eight other P. gingivalis genomes have since been sequenced and deposited in NCBI, yet they are not completely assembled. Assembly gaps are located at sites where the corresponding surrounding CDS from ATCC 33277 contain BrickBuilt elements, suggesting BrickBuilt is present in those genomes as well and potentially capable of causing assembly difficulties (Additional file 3: Figure S2). Additionally, a degenerate version of the 23 nt repeat consensus sequence (AGAYCATARTATCCTCTCRTRTG) was searched against all 13 (8 unfinished) P. gingivalis genomes, each giving positive hits (data not shown). Because of assembly gaps and undetermined bases around BrickBuilt sites in the unfinished sequencing projects they were not included in multiple alignments.
The repetitive nature of the BrickBuilt elements, both the internal repeats and that they are found multiple times through the genome, can lead to sequencing, assembly and annotation issues. Because the strains W83, ATCC 33277, TDC60, HG66 and JCVI SC001 are unique strains, were sequenced independently, and were de novo assembled, placement of BrickBuilt elements at the same locus across genomes is unlikely to be due to use of a shared scaffold. However, care should be taken when aligning newly-sequenced P. gingivalis genomes to a scaffold.
Homology to MITEs and other repetitive elements
The 23 nt repeats and the leader and tail segments of the element were analyzed using BLAST (NCBI server) to determine whether the element is present in genomes other than the species P. gingivalis . With default BLAST nucleotide settings, a full-length BrickBuilt and each of the three distinct parts of the element match solely to P. gingivalis. All four sequenced and annotated strains of P. gingivalis available for BLAST searching harbored hits for BrickBuilt. Through querying discontiguous megablast as well as using less stringent search constraints within megablast with ‘max target sequences’, ‘expect threshold’, ‘word size’ and ‘filter low complexity regions’, low-homology hits were obtained with the terminal inverted repeat regions. However, matches identified in this manner were only homologous in the TIR sections. Of note, when BLASTx searches (protein database search using a translated nucleotide query) were performed with the leader and tail sequences under default settings several Bacteroidetes species contained tail hits and one species contained a leader hit. Porphyromonas gulae contained strong hits with both leader and tail, while Prevotella tannerae and P. dentalis contained weak tail hits only. All of the BLASTx hits were either part of a predicted transposase/partial transposase or a hypothetical protein. If BrickBuilt were indeed a non-autonomous transposable element, homology to sections of related transposons through BLASTx would not be unexpected. As such, low BLASTx homology within Prevotella tannerae and P. dentalis does not point to these hits being potential parent or identical elements of BrickBuilt.
Genome analysis of recently-uploaded Porphyromonas gulae strains was carried out using the NCBI-deposited WGS shotgun sequencing data, from which we determined that P. gulae strains do in fact carry BrickBuilt homologues (Additional file 4: Figure S3) [44, 45]. Some of the P. gingivalis BrickBuilt element locations are conserved within P. gulae strains. However, greater strain variation seems evident in P. gulae at certain BrickBuilt loci than between P. gingivalis strains (Additioal file 5: Figure S3). Of note, the original P. gulae genome was obtained from a wolf and the subsequent strains were obtained from domesticated dogs. The original strain (DSM 15663) only contains 4 BrickBuilt homologues within the genome, and importantly lacks the BrickBuilt_5 homologue that was used for the majority of BLAST database queries.
Within P. gingivalis there have been three previously identified groups of MITEs or non-autonomous transposable elements; named the 239, 464 (PgRS) and 700 groups [10, 13]. These numbers are the names of three types of MITEs already noted in P. gingivalis genomics publications. The numbers were initially related to the overall length of the elements, however, the 464 type was renamed in subsequent publications and in NCBI genome graphics annotations. General copy numbers of the four MITE versions are similar, holding around 10–20. The number of full copies and partial or fragment copies of each element differs slightly between genomes within the species. During our examination of BrickBuilt we analyzed whether any sequence overlap was apparent between the elements and found that the terminal inverted repeats are similar, yet the rest of the elements do not bear similarity. 464/PgRS elements were previously identified as containing 41 nucleotide tandem direct repeats [10, 13]. The 23 and 41 nucleotide internal tandem direct repeats of the elements do not share homology with each other and neither have non-P. gingivalis BLAST matches within the NCBI database. The segments of the 464/PgRS elements flanking the 41 nt tandem direct repeats are themselves repetitive, which is unlike the non-repetitive leader and tail segments of BrickBuilt. Although not related by sequence, similarities in copy number between 464/PgRS and BrickBuilt elements are evident. With P. gingivalis harboring four types of MITE-like elements it is interesting that two types, BrickBuilt and 464/PgRS, contain microsatellite repeats. Although several 236 and 700-type elements are located near repeats or other repetitive elements, they seem not to have encompassed any mini- or microsatellites from analyses of the currently available genomes.
In addition to Tn and IS elements, multiple groups have described repetitive sequences within P. gingivalis genomes ranging from single nucleotide tracts to mini- and microsatellites [13, 46]. Several 41, 23 and 22 nucleotide tandem direct repeats were described in P. gingivalis strain W83, yet the exact locations of such repeats were not identified, nor were comparative genomics an option at the time of the report . Some of the 23 nt tandem direct repeats noted are presumably the direct repeat portions of BrickBuilt.
Terminal Inverted Repeats (TIRs) of BrickBuilt elements from strain ATCC 33277. Terminal Inverted Repeats and family of selected IS and MITE-like elements in Porphyromonas gingivalis as well as ISNme1 from Neisseria meningitidis
TIR 5′ (nt)
TIR 3′ (nt)
Only 4 of the 19 BrickBuilt copies contain both an intact leader and tail associated TIR (Table 2). In order to determine whether BrickBuilt makes target site duplications (TSD), the DNA sequence immediately adjacent to the proposed TIRs were examined of these 4 copies. Three of the four copies of BrickBuilt do carry TSDs, however, they are not the same length or sequence for each element. BrickBuilt_3 has a ‘CT’ dinucleotide flanking its TIRs; BrickBuilt_4 has a ‘GAAA’ tetranucleotide flanking its TIRs; BrickBuilt_5 has an ‘AAAAA’ heptanucelotide; and BrickBuilt_18 does not contain a putative TSD because one of its two TIRs is shared by a MITE293 element. These duplications on either side of the elements may not reflect canonical TSDs, however, if these elements are mobilized by multiple transposases that each make different restrictions to the target DNA this could potentially occur. Within P. gingvialis, ISPg elements generate TSDs varying from 2–9 bp; some can lack TSDs completely and may frequently nest into other mobile elements and thus eliminate TSD identification. Additional TSD data related to element mobility will be presented below.
After determining the IS5 family-like TIRs of BrickBuilt other IS5 elements were scanned for potential similarity. Identical TIRs to that of BrickBuilt were found in the Neisseria meningitidis ISNmeI and its derivatives (Table 2). ISNmeI is the proposed (based on TIRs) parent element for the type II MITE ATR (AT-rich Repeat) in Neisseria meningitidis genomes . ATR elements are found 19 times within N. meningitidis genomes, which is similar to that of BrickBuilt’s distribution. Additionally, ATR elements are frequently associated with direct repeat elements of N. meningitidis known as REP2.
From initial characterizations of the configuration and locations of BrickBuilt elements, they can be classified within the large group of non-autonomous transposable elements, potentially best fitting within the MITE subcategory. A caveat must be placed, however, given that MITE elements are typically described as being comprised of two homologous flanking regions, and we have determined that BrickBuilt elements contain distinct ‘leader’ and ‘tail’ segments. Since all accessible genome sequences of P. gingivalis strains contain BrickBuilt elements, the parent element or first version of BrickBuilt probably occurred early within the phylogeny of the P. gingivalis species. Insertion of the 23 nt repeat(s) into the original parent element may be the event that catalyzed the inactivation of an autonomous transposable element. Alternatively, a version of BrickBuilt already containing the 23 nt repeats could have been laterally-transferred via plasmid or horizontally-transferred via phage. Given that no full-length (TIR-containing) leader or tail regions are present without a 23 nt repeat it may be deleterious to maintain a full leader or tail region on the chromosome, or the 23 nt repeat is required by the autonomous element.
The limited host range nature of BrickBuilt identified through NCBI BLAST is intriguing yet not uncommon for non-autonomous transposable elements [49–52]. Once a non-autonomous element occurs within a genome, potentially by deletions of an autonomous transposable element, as well as via conjugation-based horizontal transfer of a plasmid or transduction via a bacteriophage, movement between species will become less likely. Additionally, few bacterial species have multiple genome assemblies available for intraspecies comparisons, which could lead to missed elements due to strain variation. Furthermore, it is possible that repetitive sequences could be mis-sequenced or left out of genome assemblies due to repeat region sequencing difficulties or unassigned bases.
Predicted secondary structure of BrickBuilt
Predicted structures of BrickBuilt vary slightly between strains at a given conserved locus. The BrickBuilt_5 Mfold entropy predictions for strains ATCC 33277 and W83 are −127.96 and −102.50, respectively (Fig. 6). BrickBuilt_5 in ATCC 33277 is 991 nucleotides long and the analogous W83 version is 807 nucleotides. In this case the length difference is due to W83 BrickBuilt_5 having fewer 23 nt internal direct repeats; the leader and tails are of the same length. Within a multiple alignment of the four P. gingivalis strains at the BrickBuilt_5 locus there are 18 single nucleotide polymorphisms (SNPs) that separate the lineages (Fig. 6). Substituting SNPs between strains at the BrickBuilt_5 locus into the ATCC 33277 model changes the predicted entropy of the element by −7.25, or 5.7 %, to −135.17.
Although no BLASTn matches for BrickBuilt were found outside of the P. gingivalis species, MITEs from other species have been shown or are predicted to be of similar modular makeup and form long stem loops [23, 35, 38, 57–59]. In addition, repetitive sequences that are not MITE-associated also frequently form stem loop structures [58, 60–62]. Stem loop structures, especially long stem loops, are capable of modulating transcript half-life, modulating translational efficiency as well as serving as docking/receptor sites for proteins [12, 58, 61]. The Rho-independent terminator upstream of BrickBuilt_5 is located 111–148 nt from the 5′ CDS, with the leader region of BrickBuilt_5 located 182 nt from the 5′ CDS (Fig. 6). Thus, in this case, the BrickBuilt element has not disrupted the ‘natural’ terminator for the 5′ CDS. However, given the proximity to the Rho-independent terminator, this BrickBuilt element may be able to modulate the stability or accessibility of the terminator. The long stem loop structures of BrickBuilt_5 start 257 nt from the Rho-independent terminator and end 965 nt away.
Genome locations and surroundings
All BrickBuilt elements are located intergenically; no direct overlap or interruptions of genuine protein coding sequences are apparent in the complete genomes available to date (Table 1 and Additional file 2: Table S1). Several ‘hypothetical proteins’ are annotated to be within BrickBuilt elements, however expression of these proteins has not been confirmed experimentally [40–42]. Several of the predicted hypothetical proteins are part of repeated/overlapping probes on P. gingivalis microarrays. Additionally, the 23 nt repeats within BrickBuilt elements are predicted to cause frequent translational stops (data not shown). Lack of experimental confirmation of protein products, nonunique microarray probes and abundant translational stops suggests that translation of these regions is unlikely, and even if translation were to occur it would probably be truncated versions of a repetitive or mobile element.
Of the 38 genes surrounding the BrickBuilt elements in the ATCC 33277 genome there are several functional clusters (Table 1). Six genes encode proteases of the C1, (2) C10, (2) M16 and S41 families. Five genes are predicted to encode DNA/RNA-binding proteins, and another four are involved in tRNA metabolism. Noticeably, of the 19 BrickBuilt elements in strain ATCC 33277, 5 are located adjacent to a gene/protein containing a Por Secretion System C-Terminal Domain (PorSS CTD)  (Table 1). Likewise, 5 of the previously identified P. gingivalis MITEs within the W83 genome are also located next to PorSS CTDs. A total of 34 PorSS CTDs have been predicted within the W83 genome (only 22 annotated on NCBI with TIGR); 29 % of PorSS CTDs are associated with MITEs . The PorSS is connected to pigmentation and haem acquisition in P. gingivalis. Apart from those associated with PorSS, other genes surrounding BrickBuilt elements are hemG, dps, trx, and hmuY, which are involved in haem biosynthesis, acquisition and detoxification, respectively. Additionally, two separate DUF 805 motifs are found in genes surrounding BrickBuilt elements, which are associated with phage integrases. The locations relative to CDS raise the possibility that BrickBuilt could be acting as or similar to a Putative Mobile Promoter (PMP); a secondary regulatory circuit or mechanism to canonical transcription and translation modulation .
Expansion and contraction of the 23 nt repeats between strains is evident at the conserved BrickBuilt loci. Entire 23 nt repeat segments have been removed or added. Full and/or partial deletions of the leader and tail regions are also apparent. Deletions of the leader and tail regions occur from the distal ends of each segment with respect to the 23 nt internal repeats.
Pairwise and multiple alignments of a respective BrickBuilt locus across the four strains of P. gingivalis revealed SNPs that potentially suggest lineages or selected and compensatory mutations (Fig. 3). Whether the SNPs are generated de novo at each site, occur in stages and are distributed, or occur through site-to-site recombination cannot be determined definitively from currently published genome assemblies alone. However, multiple alignments of the conserved BrickBuilt loci within a given strain show patterns of non-random mutation. For sites at which SNPs have occurred, the SNP is frequently distributed at several positions within the element, yet this occurs at intervals. Additionally, SNPs appear localized around a 2–4 nt site when compared in multiple alignments. Long Term Evolution Experiments (LTEE) and plasmid-based recombination systems could be employed to determine mutation rates within BrickBuilt in comparison to the rest of the genome, whether 23 nt repeats expand and contract at a given locus, and how recombinogenic the elements are.
With respect to P. gulae strains, BrickBuilt_5 demonstrates the possibility or history of mobility. At this site, 4 of the 11 P. gulae strains contain (and 7 lack) BrickBuilt copies. In the strains that contain an element a ‘AAAA’ TSD can be seen (‘AAAAA’ in P. gingivalis at that site). The P. gulae strains lacking an element at that site only have one ‘AAAA’ tetranucelotide. This site is completely conserved in the published P. gingivalis strains.
BrickBuilt_18 in the strains ATCC 33277 and HG66 contain/encompass MITE239_11 between nucleotides 659–904 of the sequence. Strain TDC60 has a gap where MITE239_11 occurs in the other two strains, while the flanking portions of the BrickBuilt match (Fig. 7). The strain W83 version at this site is diminutive, having been reduced to 1.5 copies of the 23 nt internal repeat. While strain W83 doesn’t harbor a MITE-within-a-MITE configuration at any locus, BrickBuilt_11 in strain W83 contains an ‘extra’ gene adjacent to the 5′ region of the element unlike any other strain (Fig. 7).
Transcriptional expression of BrickBuilt
Repetitive and transposable elements are capable of modulating the genome stability and evolution of species [17, 19, 58, 66]. Interestingly, no endogenous plasmids have been found for P. gingivalis to date. The presence of many copies and types of repetitive and transposable elements could serve a quick way by which P. gingivalis could recombine/adapt to external stimuli beyond traditional host-directed transcriptional and translational controls [17, 18, 67–69].
Analysis of previously published data
Within the transcriptome transcript levels of individual BrickBuilt elements vary markedly and also vary according to growth medium, e.g. in/on minimal, tryptic soy, and blood media [9, 70]. Generally, transcripts from BrickBuilt regions are lowest on the blood-containing media. Transcript levels and distribution of transcripts of BrickBuilt elements, using strain W83 RNAseq data, can be grouped generally into three categories. Group one, displaying relatively high transcript levels throughout the element on only one strand bridging the entire CDS-to-CDS gap, includes BrickBuilt_1, 3, and 13. BrickBuilt_13, comprised only of the internal 23 nt repeats. The element’s expression correlates directly with that of the upstream gene, thus, the expression of BrickBuilt_13 could be completely due to transcriptional read-through from adjacent genes. Consistent with this, BrickBuilt_13-associated transcripts are all on the negative strand. Group two, displaying low to medium intermittent transcript in tryptic soy and minimal media but none on blood agar, includes BrickBuilt_2, 5, 6, 8, 10, 11, 12, 14, 15, 16, 17, and 19. Group three, displaying no transcript yet adjacent to upstream transcript that is well beyond an annotated CDS, includes only BrickBuilt_9.
Additional information about BrickBuilt elements and their surrounding regions can be garnered from the above microarrays and RNAseq studies as well as additional studies that have been carried out with P. gingivalis under defined conditions. High-density tiling microarray of P. gingivalis strain W83 by Chen et al. showed differential expression of BrickBuilt elements at several loci . Using a W83 strain based microarray, genes PG0626 and PG0089 were found to be aberrant in strain ATCC 33277, corresponding to BrickBuilt_ 11 and BrickBuilt_ 19 loci. The area in and around BrickBuilt_10 was identified as a potential sRNA (sRNA35) by Philips et al. . The highest expression of the putative sRNA35 occurred during mid-log cultures grown under hemin excess conditions after an initial period of hemin starvation. Under the experimental methods used by Philips et al., no other BrickBuilt loci were determined to be or be part of putative sRNAs expressed in response to hemin-variable growth conditions. BrickBuilt elements are not directly affected by FimR or LuxS regulation [72, 73]. However, genes surrounding BrickBuilt elements are regulated by LuxS. Lack of expression as well as partial expression of annotated genes surrounding BrickBuilt elements is evident from P. gingivalis strain W83 transcriptomic analyses by Hovik et al. . Five of the conserved 30 genes flanking BrickBuilt elements in the W83 genome are predicted to not be expressed and 11 (including 3 of the 5 ‘not expressed’) give partial or abortive transcripts in blood, tryptic soy or minimal media.
The genomic association with haem biosynthesis and pigmentation-associated genes in conjunction with transcriptional data from RNAseq and microarray studies may point to regulation of BrickBuilt regions by haem or iron. DNA tandem repeats have been shown previously to affect transcription of iron and haem-associated genes [18, 74].
E. coli expression vectors
We identified and provided preliminary characterization of a genetic element, ‘BrickBuilt’, in the genome of Porphyromonas gingivalis. BrickBuilt appears to be a MITE-like element that has trapped a 23 nt direct repeat; propagating itself and the direct repeat throughout the genome. From promoter-less lacZ assays and analyses of previous microarray and RNAseq data we determined certain BrickBuilt elements contain promoter elements capable of bi-directional transcription. Given the element’s exclusively intergenic locations and surrounding gene directionality, these transcripts may serve to regulate expression of surrounding genes. Relative stability of locations, overall copy number and expression levels of the elements throughout the sequenced P. gingivalis genomes point to neutral or advantageous maintenance of BrickBuilt.
Further sequencing projects and phylogenomics will be necessary to determine which other species and strains contain the BrickBuilt element and at what evolutionary point these species and/or strains diverged. Additionally, strain-specific experimental evolution and plasmid-based recombination systems could be employed to determine mutation rates within BrickBuilt in comparison to the rest of the genome, whether and how 23 nt repeats expand and contract at a given locus, and how recombinogenic the elements are.
With respect to ‘mobility’ of a whole or partial BrickBuilt element, several experimental setups could be considered. First, inducing expression of endogenous transposases in order to mobilize BrickBuilt elements. One would need to initially determine under what conditions each transposase type in P. gingivalis is expressed, then induce expression and either PCR or sequence target BrickBuilt locations. Additionally, whole genome sequence could be employed to find the locations of element movement or duplication. Second, exogenous transposases could be expressed in P. gingivalis. Given the specificity of some transposases, a panel may need to be tested. Third, BrickBuilt elements could be introduced on plasmids into other bacterial species, specifically other Bacteroidetes, in order to try to obtain insertion into the heterologous host.
Adding BrickBuilt to the list of transposable and repetitive elements types in P. gingivalis brings the current total to 4 MITEs (or MITE-like elements), 11 ISs, 2 Ctn and 1 Tn. The ORFs and total base pairs encompassed by these elements constitute an impressive proportion of the genome. When compiled, the total percent of the P. gingivalis genome encoded by MITEs is 1 %; 0.44 % from BrickBuilt elements, 0.39 % from MITEPgRS elements, and the remaining 0.17 % from MITE700 and MITE239 family elements.
The ability of several of these elements being involved in genome evolution has been established [47, 48]. However, the full effects of these elements on genome stability and evolution as well as transcriptional, translational and post-translational response to stimuli remains to be experimentally determined.
Genome sequence FASTA and GenBank files were downloaded from the NCBI database. At the time of this research, strains ATCC 33277, TDC60, W83, HG66 and JCVI SC001 were available as completed sequencing and assembly projects (ATCC 33277, TDC60 and W83 as ‘gapless chromosome’ status, HG66 as a single contig, and JCVI SC001 as a draft of many stitched contigs) [10, 13–16]. The five sequenced wild-type strains are disparate based on origin or lineage: W83 isolated in Germany (1950’s) from an oral lesion; ATCC 33277 was isolated in the USA (1980’s) from subgingival plaque; TDC60 was isolated in Japan (2011) from an oral lesion; HG66 isolated in the USA (1989) from a dental school patient; and JCVI SC001 was isolated in the USA (2013) from a hospital sink. The sequencing projects utilized different sequencing and assembly methods; each providing a de novo assembly. The JCVI SC001 genome sequence contained unidentified bases and residual gaps in the sequencing after the completed project.
Sequence analysis, clustering, alignment, phylogeneics/phylogenomics
NCBI BLAST suites were utilized to determine locations, structure and potential protein-coding capacity of the MITEs . Query inputs were FASTA sequences taken directly from NCBI genome sequencing projects. For initial characterizations prior to determining species-specificity of the elements, the entire NCBI sequence database was queried. Following determination that the elements were only found (as of 11/2013) in the genomes of P. gingivalis strains, subsequence queries were focused to either the P. gingivalis species as a whole or specific P. gingivalis strains. Megablast, discontiguous megablast and BLASTn program selections for search optimization were all used in determining species-specificity as well as genome localizations.
MultAlin, Clustal Omega and the MEME suite were used to perform DNA-based and amino acid-based multiple alignments of the MITEs to determine conserved nucleotides and the start and stop points of the elements as well as proteins surrounding the MITEs. Amino acid-based alignments were used to determine whether the surrounding genes had structural domains at either the 5′ or 3′ ends that could potentially account for or facilitate MITE localization [75–77].
The BioCyc sequence pattern search tool ‘PatMatch’ was used to determine the number and genomic location of P. gingivalis MITEs in strain ATCC 33277 . PatMatch indentifies potential sites given variations in the consensus sequences of the MITE direct repeats, TIRs, ‘leader’ and ‘tail’ regions because different mismatch numbers are allowed. Query inputs were nucleotide consensus sequences determined for each of the given parts of the MITE. Both DNA strands, as well as intergenic and coding sequences, were queried separately. Mismatches of ‘0’ through ‘3’ were allowed, with the constraint of the ‘mismatch type’ being a substitution.
The Tandem Repeats Database software was used for determining all types of tandem repeat elements in the P. gingivalis genomes (strains ATCC 33277 and W83 hosted on the server as of 12/2013), and to determine whether the tandem repeats or MITE as a whole was conserved in other sequenced species . BLAST query of the entire bacterial and viral tandem repeat database was carried out using the FASTA sequence downloaded from NCBI for P. gingivalis strain ATCC 33277 MITE. The Tandem Repeats Finder software was used to determine the composition of the P. gingivalis tandem repeat element . ‘Basic’ sequence analysis was selected for queries. Tandem Repeats Finder was also used to determine repeat conservation within and between loci as well as where a given element started and ended.
The Geneious software platform (version R8) was used to download, store, deposit, manipulate and query P. gingivalis genomes and BrickBuilt MITEs .
MITE and surrounding coding Sequences’ nucleic acid and protein motif analysis
The Pfam and InterProScan databases and programs software were used to determine the presence and characteristics of nucleic acid and protein motifs [82, 83]. Query inputs were FASTA sequences from NCBI download files. For Pfam, an E-value of 1.0 and checking Pfam-B motifs were selected options prior to submission.
ExPASy Translate Tool software was used to determine whether the MITEs potentially encoded proteins, and thus are not strictly nucleic acid elements . Genetic code option ‘standard’ was used for all queries. All six possible frames of translation were considered.
Modeling and structure prediction programs Mfold, RNAstructure and RegRNA2.0 were used to predict potential 2-D structures formed by MITE DNA and RNA [53–55]. Default options for programs in regard to structure prediction were chosen.
Cloning and reporter strains, media and growth conditions
Escherichia coli DH5α and TOP10 were used for cloning, plasmid maintenance and transcriptional assays. Ampicillin (100 μg/ml) was used when appropriate for prevention of contamination as well as isolation and maintenance of transformants containing plasmids. Strains were grown and maintained on LB [Lennox] agar or in LB [Lennox] broth (Invitrogen).
PCR primers containing BamHI and XmaI (NEB) restriction sites were designed immediately flanking the BrickBuilt_5 MITE associated with PGN_0361 (Additional file 2: Table S2). PCR products were generated using GoTaqLong Master Mix (Promega) and resultant bands were cloned into vector pCR TOPO-XL (Invitrogen), which was transformed into E. coli DH5α. Transformants were selected for kanamycin resistance and clones were confirmed by restriction digest and sequencing.
To generate constructs using the promoter probe vectors pCB182 and pCB192, pCR2.1 and pCR-TOPO-XL cloned BrickBuilt MITE constructs and pCB182/pCB192 were double-digested with BamHI (NEB) and XbaI (NEB) or BamHI and XmaI (NEB) and then transformed into E. coli TOP10. Transformants were selected for ampicillin resistance generated by an insertion event. Clones were confirmed by restriction digest and sequencing.
BROP, specifically the ‘Genomics Tools for Oral Pathogens’ and ‘Microbial Transcriptome Database’ sections of the resource, were used to determine genome location, characteristics of coding sequencings surrounding MITEs, differences between strains as well as transcriptome data . Over the course of the research, two different variations of the RNAseq data for P. gingivalis strain W83 were supported, one directly on BROP and then a later form on JBrowse. The JBrowse form gives greater functionality in displaying data and visualization . Under the ‘Genomics Tools for Oral Pathogens’ subset, the ‘GenomeViewer’ function was used to compare genome arrangements of P. gingivalis strains ATCC 33277 and W83 with relation to MITEs, as well as display the previously performed microarray data (under the strain W83 section) for MITE-associated genome areas under the three different nutrient conditions (the same conditions performed in the RNAseq) [9, 70].
β –galactosidase assays
Escherichia coli strains were grown and maintained in Luria-Bertani (LB) media supplemented with ampicillin (100 μg/l) as required. PCR primers and synthesized oligos used for strain constructions are listed in Additional file 2: Table S2. The pCB182 and pCB192 vectors lack promoters but contain translational start codons . As such, gene expression of lacZ, and in turn protein expression of LacZ read out through β –galactosidase activity, should be the result of promoter activity from fragments cloned into the vector. β -galactosidase assays were performed under plate-based (X-Gal) and broth-based (ONPG) setups. For plate-based assays, frozen stock cultures of the BrickBuilt MITE derivatives transcriptionally fused to lacZ in their respective E. coli strains were plated onto LB agar containing X-gal and ampicillin. For broth-based assays, cultures of the BrickBuilt MITE derivatives transcriptionally fused to lacZ in their respective E. coli strains were grown in LB broth for 3 h with shaking at 37 °C. An aliquot of each culture (500 μl) was added to a lysis and assay solution mixture (500 μl), vortexed, and then incubated at 28 °C for 3 h. Color development was measured spectrophotometrically at OD420 nm and cell debris at OD550 nm. Respective Miller units were calculated as previously described .
All data, genome sequences as well as RNAseq and microarray, are currently available in public repositories and publications related to these data have been referenced. Locations of the MITE sequences in P. gingivalis and P. gulae strains will be deposited to NCBI such that identifiers and notes can be amended to the graphic outputs of sequence files.
Miniature Inverted-repeat Transposable Element
Terminal Inverted Repeat
Target Site Duplication
Polymerase Chain Reaction
Bioinformatics Resource Oral Pathogens
Microbial Transcriptome Database
Basic Local Alignment Search Tool
Drs. Andrea Pauli and Eivind Valen working in the laboratory of Dr. Alexander Schier at Harvard University for their help and knowledge in non-coding RNA biology and RNA regulation. Dr. Michael Malamy at Tufts University for his expertise in mobile genetic elements and plasmid biology. Dr. Carla Cugini at Rutgers School of Dental Medicine for her expertise P. gingivalis biology.
This project was supported by grants from the National Institute of Dental & Craniofacial Research, F31 DE022491 (BAK), R01 DE024308 (LTH and MJD) and R01 DE015931 (MJD). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Dental & Craniofacial Research or the National Institutes of Health.
Open AccessThis 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.
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