Abstract
Miniature inverted-repeat transposable elements (MITEs) are nonautonomous transposons (devoid of the transposase gene tps) that affect gene functions through insertion/deletion events. No transposon has yet been reported to occur in “Candidatus Liberibacter asiaticus,” an alphaproteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease). In this study, two MITEs, MCLas-A and MCLas-B, in “Ca. Liberibacter asiaticus” were detected, and the genome was characterized using 326 isolates collected in China and Florida. MCLas-A had three variants, ranging from 237 to 325 bp, and was inserted into a TTTAGG site of a prophage region. MCLas-A had a pair of 54-bp terminal inverted repeats (TIRs), which contained three tandem repeats of TGGTAACCAC. Both “filled” (with MITE) and “empty” (without MITE) states were detected, suggesting the MITE mobility. The empty sites of all bacterial isolates had TIR tandem repeat remnants (TRR). Frequencies of TRR types varied according to geographical origins. MCLas-B had four variants, ranging from 238 to 250 bp, and was inserted into a TA site of another “Ca. Liberibacter” prophage. The MITE, MCLas-B, had a pair of 23-bp TIRs containing no tandem repeats. No evidence of MCLas-B mobility was found. An identical open reading frame was found upstream of MCLas-A (229 bp) and MCLas-B (232 bp) and was predicted to be a putative tps, suggesting an in cis tps-MITE configuration. MCLas-A and MCLas-B were predominantly copresent in Florida isolates, whereas MCLas-A alone or MCLas-B alone was found in Chinese isolates.
INTRODUCTION
Miniature inverted repeat transposable elements (MITEs) are a type of nonautonomous transposons that involve insertion/deletion of DNA in the genomes of both prokaryotes and eukaryotes and influence gene functions (1, 2). MITEs are generally small (<400 bp) and contain a noncoding short central region (CR) flanked by a pair of terminal inverted repeats (TIRs). MITE sequences can form highly stable secondary structures at the transcriptional level. Direct repeats (DRs) are typically found outside the TIRs on the host bacterial genomic DNA (1, 3). MITEs were first described in Neisseria sp. (4) and are now reported in many other bacteria (5–14).
“Candidatus Liberibacter asiaticus” is a nonculturable, phloem-restricted alphaproteobacterium associated with citrus huanglongbing (HLB; yellow shoot disease, also known as greening disease). HLB is one of the most destructive diseases in citrus production worldwide (15). In China, HLB was observed over 100 years ago (16), and the association with “Ca. Liberibacter asiaticus” was confirmed in 1996 (17, 18). The bacterium was found in São Paulo, Brazil, in 2004 (19), and in Florida a year later (20). Following the reports of its occurrence in several southern states, “Ca. Liberibacter asiaticus” was detected in California in 2012 (21). Due to the lack of in vitro culture, much of the “Ca. Liberibacter asiaticus” biology, including the status of transposons, remains to be studied.
Analysis of the whole-genome sequence concluded that no transposon was identified in a Florida isolate of “Ca. Liberibacter asiaticus” (22). No transposon was annotated in the complete sequences of three phages/prophages of “Ca. Liberibacter asiaticus,” SC1, SC2, and FP2 (23, 24). In sequence analyses, two characteristics are highly indicative of transposons: presence of TIRs and identification of a transposase gene (tps). Since MITEs are devoid of tps, their detection is even more difficult, if not impossible, should a single bacterial genome sequence be studied. Analysis of a single genome sequence cannot simultaneously reveal the presence (“filled”) and absence (“empty”) states of a MITE. Detection of the two states is a direct evidence of transposon mobility.
Tettelin et al. (25) described the concept of the pangenome, with emphasis on genomic sequences from multiple isolates to reveal a “core genome” and a “dispensable genome” of a bacterial species. Mobile elements, such as phages and transposons, contribute to the “dispensable genome” and may be present in the genomes of some isolates and absent in others. Thus, analyses of a bacterial population may provide an opportunity for transposon detection. In the past few years, there have been several reports of DNA polymorphisms in “Ca. Liberibacter asiaticus” populations using single primer sets (24, 26–29). By analyzing two Chinese “Ca. Liberibacter asiaticus” populations, one with 120 isolates and the other with 39 isolates, Liu et al. (27) observed a significant difference in the frequency of a prophage gene that suggested the presence of an active phage. In this study, we applied the concept of population or pangenome analysis to search for transposons in “Ca. Liberibacter asiaticus.” A polymorphic genomic locus was identified and analyzed using a total of 326 “Ca. Liberibacter asiaticus” isolates from China and Florida. As a result, two MITEs, MCLas-A and MCLas-B, were discovered and are reported here.
MATERIALS AND METHODS
Sources of “Ca. Liberibacter asiaticus” DNA.
Because “Ca. Liberibacter asiaticus” is not culturable in vitro, pure bacterial DNA could not be obtained. Instead, “Ca. Liberibacter asiaticus” DNA was extracted from HLB-affected citrus trees along with host DNA. Leaves showing characteristic yellowing and/or mottling HLB symptoms (15, 16) were collected for DNA extraction. The presence of “Ca. Liberibacter asiaticus” was indicated by positive PCR detection with primer set OI1/OI2c (30). A bacterial isolate was represented by DNA from a “Ca. Liberibacter asiaticus”-infected tree. DNA extracted from leaves of non-“Ca. Liberibacter asiaticus”-infected citrus trees in both California, where only a single case of “Ca. Liberibacter asiaticus” was reported, and Chongqing, China, where “Ca. Liberibacter asiaticus” has not been reported, was used as negative control. “Ca. Liberibacter asiaticus” samples were collected from nine provinces in China and from Florida in the United States (Table 1). Samples in China were collected between December 2008 and October 2011 and extracted using a modified cetyltrimethylammonium bromide (CTAB) method (31). DNA preparations from Florida were kindly provided by X. Sun and M. Irey. Details of HLB sample collection and DNA extractions in Florida were described previously (26).
Table 1.
Distributions of three amplicon types with primer set Lap-PF1-f/Lap-PF1-r from “Candidatus Liberibacter asiaticus” isolates from China and Florida
| Origin | Total no. of isolates | No. of isolates with electrophoretic type(s): |
||||||
|---|---|---|---|---|---|---|---|---|
| B720 | B630 | B350 | B720, B350 | B630, B350 | B720, B630, B350 | None | ||
| China | ||||||||
| Yunnan | 63 | 25 | 1 | 3 | 23 | 4 | 3 | 4 |
| Guizhou | 10 | 7 | 0 | 0 | 3 | 0 | 0 | 0 |
| Sichuan | 20 | 12 | 0 | 2 | 6 | 0 | 0 | 0 |
| Guangdong | 67 | 7 | 0 | 31 | 0 | 29 | 0 | 0 |
| Guangxi | 39 | 5 | 0 | 21 | 1 | 12 | 0 | 0 |
| Fujian | 37 | 8 | 0 | 23 | 0 | 4 | 0 | 2 |
| Jiangxi | 13 | 3 | 0 | 10 | 0 | 0 | 0 | 0 |
| Zhejiang | 18 | 4 | 0 | 14 | 0 | 0 | 0 | 0 |
| Total | 267 | 71 | 1 | 104 | 33 | 49 | 3 | 6 |
| Florida, USA | 59 | 0 | 0 | 5 | 6 | 0 | 48 | 0 |
PCR primers and procedures.
DNA sequences of “Ca. Liberibacter asiaticus” strain Psy62 (CP001677), phage SC1 (HQ377372), phage SC2 (HQ377373), and phage FP2 (JF773396) were downloaded from the GenBank DNA database in the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/). Primers were designed using Primer 3 software (32). As an extension of a genomic diversity research (29), primer set LapPF1-f/LapPF1-r (5′-GCCACTTTGGGGTAGCAGTA-3′/5′-AAAACTTTCGTCACGGCTTT-3′) were found to yield multiple amplicons from some “Ca. Liberibacter asiaticus” isolates (Fig. 1) and therefore selected for this investigation.
Fig 1.
Electrophoretic profiles of representative “Candidatus Liberibacter asiaticus” isolates from PCR amplification with primer set LapPF-1f/LapPF-1r. Three amplicon types, B720, B630, and B350, are designated. CK represents DNA from healthy (non-“Ca. Liberibacter asiaticus”-infected) citrus. Lane M on the right shows molecular markers in bp.
A previously published procedure was followed for PCR (29). Briefly, a reaction mixture (25 μl) included 2 μl of template DNA, 0.3 μl of Taq DNA polymerase at 5 U/μl (TaKaRa Bio Inc., Shiga, Japan), 0.4 μl of each forward and reverse primer (10 μM), and 2.5 μl of 2.5 mM deoxynucleoside triphosphate. Thermal cycling comprised an initial denaturing of 94°C for 2 min, followed by 35 cycles of amplification (94°C for 30 s, 55°C for 30 s, and 72°C for 30 s) and a final extension for 7 min. PCR products were electrophoresed in a 1.5% agarose gel and visualized by ethidium bromide staining under UV light.
DNA sequencing and analysis.
PCR amplicons from “Ca. Liberibacter asiaticus” isolates were purified from agarose gels using a QIAquick Gel Extraction kit (Qiagen, Valencia, CA) and cloned with pGEM T-easy vector (Promega Corp., Fitchburg, WI). Four to seven clones were randomly selected from each isolate for sequencing using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Inc., Foster City, CA) in a 3130 × 1 Genetic Analyzer (Perkin-Elmer, Boston, MA). Sequences were aligned using the ClustalW program (Ver.1.74) (33) hosted by the European Bioinformatics Institute (www.ebi.ac.uk/Tools/msa/clustalw2) with the default parameters. Manual adjustment was made when appropriate.
Identification and characterization of MITEs.
A candidate MITE was identified for its small size (<400 bp) with TIRs, a noncoding CR, and DRs (1). Evidence of mobility and a putative tps further confirmed the MITE status. MITEs were classified based on TIR sequence and then subgrouped into variants based on CR similarity.
TIRs were identified by the einverted program in the EMBOSS package (34). DRs and internal TIR repeats were identified manually. The CR was checked for amino acid encoding by GeneMark web software (http://opal.biology.gatech.edu/GeneMark/) (35). Secondary structure and stabilities were predicted using Vienna RNA Websuite (36). MITE copy numbers were estimated based on BLASTn searches (37) using MITE sequences as queries against the whole-genome sequences of “Ca. Liberibacter asiaticus” strain Psy62 and phages SC1, SC2, and FP2 with a cutoff E value of <e−5. Phylogenetic trees of related MITE variants were generated using the neighbor-joining method implemented through MEGA 4.0 (38), and bootstrap analysis (1,000 replicates) was performed to assess the tree reliability.
To search for possible in cis tps, sequences of the open reading frames (ORFs) both up- and downstream of the identified MITEs in the SC1 and SC2 genomes (23) were used as queries for BLASTx searches in the ISfinder database (http://www-is.biotoul.fr/) (39) with a cutoff E value of 10−3.
MITE mobility and remnant types.
Based on a PCR result, a “Ca. Liberibacter asiaticus” isolate showed a larger amplicon that was filled (with a MITE), a smaller amplicon that was empty (without a MITE), or both large and small amplicons (a mixture of filled and empty). The presence of both filled and empty states was considered evidence of MITE mobility. In the MITE empty “Ca. Liberibacter asiaticus” sequences, various TIR remnant sequences were found. The remnant sequences were classified into remnant types. The frequency and distribution of remnant types of “Ca. Liberibacter asiaticus” isolates from different geographic populations were compared. Statistical significance was evaluated by chi-square analyses.
Nomenclature of MITE.
A synopsis of MCLas-Xi was used for MITE nomenclature, where MCLas indicated that MITE origined from “Ca. Liberibacter asiaticus,” X was an uppercase letter assigned to a MITE group based on TIR sequence similarity, and i was a number for a variant based on sequence similarity in CR.
RESULTS
Detection and characterization of MCLas-A.
Representative PCR results with primer set LapPF1-f/LapPF1-r for “Ca. Liberibacter asiaticus” isolates from China and Florida are shown in Fig. 1. Three DNA bands, B720 (∼720 bp), B630 (∼630 bp), and B350 (∼350 bp), were amplified. In silico analyses showed that B720 was present in phage SC1 (720 bp) and in a prophage region in the genome of “Ca. Liberibacter asiaticus” strain Psy62 (719 bp). Similarly, B630 was found in phages SC2 and FP2 (635 bp in both). In contrast, no identical sequence of the size of B350 was found in the published genomes of “Ca. Liberibacter asiaticus” strain Psy62 and phages SC1, SC2, and FP2. PCR results showed that B350 was present in 70.8% (189/267) of the isolates from China and in all isolates from Florida (59/59). Primer set LapPF1-f/LapPF1-r did not yield any PCR amplicon from DNA samples extracted from healthy (non-“Ca. Liberibacter asiaticus”-infected) citrus leaves collected in California or Chongqing. The distributions of the three DNA fragments in all isolates from China and Florida are listed in Table 1.
A Chinese isolate, YN-JS-835, harboring all three bands, B720, B630, and B350 (Fig. 2), was selected to demonstrate the relationships among the three amplicons. In this isolate, the exact sizes of B720, B630, and B350 were 731, 598, and 303 bp, respectively. The B630 sequence of isolate YN-JS-835 was identical to the B350 sequence except for an insertion of a 295-bp fragment (325 − 30 = 295, as shown in Fig. 2). The 325-bp sequence in the B630 was characterized by (i) a pair of 54-bp perfect TIRs, containing three tandem repeats of 10 bp (note that the single G/C was interspaced between the second and the third repeat units in Fig. 3 but the sequence was still considered to be in tandem for the convenience of discussion); (ii) a noncoding CR of 217 bp; (iii) an entire 325-bp sequence, which could form a highly stable secondary structure at the transcriptional level, i.e., RNA (ΔG = −160.80 kcal/mol); and(iv) a pair of 6-bp DRs outside the TIR pair with a 83.3% (5/6) similarity. All these are characteristic features of a MITE. Therefore, the 325-bp insert was designated MCLas-A1, a variant of MCLas-A (Fig. 3).
Fig 2.
Schematic comparison of MITE-related loci among the genomes of “Candidatus Liberibacter asiaticus” Psy62, phages SC1, SC2, and PF2, and “Ca. Liberibacter asiaticus” isolate YN-JS-835. Primer set LapPF1-f/LapPF1-r is marked with a pair of small arrowheads. Three amplicon types, B720, B630, and B350, are indicated. Dashed lines represent MCLas-As flanked by two dashed-line boxes indicating terminal inverted repeats (TIRs). Solid lines represent MCLas-B flanked by two solid-line boxes (TIRs). Open reading frames (ORFs) from SC1_gp120, CLIBASIA_05620, SC2_gp120, and Psy-FP2-gm125 are identical and encode a putative transposase. ORFs from SC1_gp125, CLIBASIA_05625, SC2_gp125, and Psy-FP2-gm130 encode a putative XRE family transcriptional regulator.
Fig 3.
Schematic representation of MCLas-A and comparison of MCLas-A1s, MCLas-A2, and MCLas-A3. TIR, terminal inverted repeat; DR, direct repeat (gray triangle); CR, central region. Black triangle, a tandem repeat unit. In sequence alignments, a dot represents nucleotide identity to the first sequence (YN-JS-835); a dash represents a missing base; a space flanked by two slashes represents omitted nucleotides. The relatedness of MITEs is summarized by a neighbor-joining tree on the left. Subgroups of MITEs are numbered on the right.
Amplicons of 36 representative isolates from China and Florida were sequenced (Table 2). Eight isolates yielded single B630 sequences ranging from 509 to 635 bp. One isolate, GD-HZ-D64, yielded two B630 sequences of 598 and 512 bp. MCLas-A was found in all B630 sequences and the genomes of phage SC2 and FP2 as mentioned above. Three MCLas-A variants were identified, with MCLas-A1 being dominant (10/12 or 83.3%) (Fig. 3). Among the 10 MCLas-A1s, 6 originated from China and 4 from Florida. The other two variants (GD-HZ-D64-C2 and YN-MG-4) had partially deleted left TIRs. A BLASTn search revealed that all MCLas-A sequences had no significant hits other than the single copies in genome sequences of phages SC2 and FP2 in the GenBank sequence database.
Table 2.
Comparative information of 36 representative isolates of “Candidatus Liberibacter asiaticus” in China and the United States selected for sequence analyses of three amplicon types
| Origin and designation of isolate | Yr | Location | Amplicon type (accession no.) |
||
|---|---|---|---|---|---|
| B720 | B630 | B350 | |||
| Yunnan | |||||
| YN-JS-835 | 2008 | Jianshui | 731 (KC478846) | 598 (KC478847) | 303 (KC478848); 292 (KC478849); 303 (KC478850) |
| YN-JS-4 | 2010 | Jianshui | 721 (KC478851) | 292 (KC478852); 303 (KC478853) | |
| YN-GJ-831 | 2008 | Gejiu | 731 (KC478854) | 291 (KC478855) | |
| YN-MG-7 | 2010 | Maguan | 719 (KC478856) | ||
| YN-MG-4 | 2010 | Maguan | 509 (KC478857) | ||
| YN-MLP-9 | 2010 | Malipo | 303 (KC478858); 313 (KC478859) | ||
| Guizhou | |||||
| GZ-CH-3 | 2009 | Ceheng | 731 (KC478860) | 303 (KC478861); 313 (KC478862) | |
| Sichuan | |||||
| SC-DC-D-2 | 2010 | Dechang | 719 (KC478863) | ||
| SC-NN-6 | 2011 | Ningnan | 722 (KC478864) | ||
| SC-NN-9 | 2011 | Ningnan | 303 (KC478865); | ||
| Guangdong | |||||
| GD-SH-2 | 2009 | Sihui | 719 (KC478867) | ||
| GD-QY-874 | 2008 | Qingyuan | 598 (KC478868) | 303 (KC478869) | |
| GD-QY-875 | 2008 | Qingyuan | 598 (KC478870) | 303 (KC478871) | |
| GD-QY-876 | 2008 | Qingyuan | 598 (KC478872) | 313 (KC478873) | |
| GD-HZ-D64 | 2008 | Huizhou | 598 (KC478874); 512 (KC478875) | 303 (KC478876) | |
| GD-SH-1 | 2009 | Sihui | 303 (KC478877); 292 (KC478878); 303 (KC478879) | ||
| GD-GZ-A4 | 2010 | Guangzhou | 313 (KC478880) | ||
| Guangxi | |||||
| GX-LP-2 | 2009 | Lipu | 722 (KC478881); 718 (KC478882) | ||
| GX-LZ-5 | 2009 | Luzai | 598 (KC478883) | 292 (KC478884); 303 (KC478885) | |
| GX-GL-3 | 2011 | Guiling | 313 (KC478886) | ||
| GX-GL-5 | 2011 | Guiling | 292 (KC478887); 303 (KC478888) | ||
| GX-FC-2 | 2009 | Fuchuan | 292 (KC478889); 303 (KC478890) | ||
| Fujian | |||||
| FJ-CT-2 | 2011 | Changtai | 731 (KC478891) | ||
| FJ-XM-1 | 2010 | Xiamen | 733 (KC478892) | ||
| FJ-CT-1 | 2011 | Changtai | 292 (KC478893); 303 (KC478894) | ||
| FJ-YC-3 | 2011 | Yongchun | 303 (KC478895); 292 (KC478896) | ||
| FJ-FZ-1 | 2010 | Fuzhou | 313 (KC478897) | ||
| Jiangxi | |||||
| JX-GZ-4 | 2011 | Ganzhou | 731 (KC478898) | ||
| Zhejiang | |||||
| ZJ-TZ-3 | 2011 | Taizhou | 731 (KC478899) | ||
| ZJ-TZ-2 | 2011 | Taizhou | 313 (KC478900) | ||
| Florida, USA | |||||
| FL-132701 | 2009 | Florida | 720 (KC478901) | 329 (KC478902); 340 (KC478903) | |
| FL-30286 | 2009 | Florida | 719 (KC478904) | 329 (KC478905) | |
| FL-61427 | 2008 | Florida | 720 (KC478906) | 635 (KC478907) | 329 (KC478908); 340 (KC478909) |
| FL-61437 | 2008 | Florida | 720 (KC478910) | 635 (KC478911) | 329 (KC478912); 340 (KC478913) |
| FL-132879 | 2009 | Florida | 302 (KC478914); 312 (KC478915); 302 (KC478916) | ||
| FL-132881 | 2009 | Florida | 302 (KC478917); 312 (KC478918); 302 (KC478919) | ||
Referenced to the complete genome sequence of phage SC2 (as well as phage FP2, which is identical to phage SC2), an ORF, SC2_gp120, annotated as a hypothetical protein, was 229 bp upstream of MCLas-A (Fig. 2). Evaluation of conserved domains showed that the hypothetical protein is a member of the HTH_Tnp_1 (PF01527) family, which can bind to DNA and have a transposase function (3, 40). The putative transposase belonged to the IS3 family based on BLASTx searching against the ISfinder database (39). Downstream 770 bp of MCLas-A in the genome of SC2/FP2 was an ORF annotated as a putative transcriptional regulator of the XRE family, not known to be associated with transposases.
Detection of B630 (MCLas-A filled) was mostly associated with B350 (MCLas-A empty). The only exception was in the case of MCLas-A3 (Fig. 3). The detection of this MITE was not associated with any empty form of the MITE in isolate YN-MG-4 (Table 1). On the other hand, 148 isolates (137 [104 + 33] from China and 11 [5 + 6] from Florida) of “Ca. Liberibacter asiaticus” were empty, with MCLas-A (B350) only, i.e., without the filled MCLas-A (B630) state (Table 1).
All MCLas-A empty sites in B350 were filled with TIR tandem repeat remnants (TRRs) (Fig. 4). A total of 132 B350 clones (96 from China and 36 from Florida) selected from 26 “Ca. Liberibacter asiaticus” isolates (20 from China and six from Florida) were sequenced and formed six TRR types (Fig. 4). GenBank accession numbers of 46 representative sequences are listed in Table 2. Among the 26 “Ca. Liberibacter asiaticus” isolates (Table 2), 3 isolates harbored three TRR types, 11 isolates had two TRR types, and 12 isolates had a single TRR type. There was no significant difference (P = 0.19) in TRR type distribution between Yunnan and Florida isolates. However, isolates from Guangdong were significantly different from those in Yunnan (P < 0.001) and in Florida (P < 0.001) (Fig. 4).
Fig 4.
Schematic illustration of terminal inverted repeats (TIRs) of MCLas-As and their relations to tandem repeat remnants (TRRs) among the six TRR types of “Candidatus Liberibacter asiaticus.” In MCLas, tandem repeat units are underlined, and central region is represented by in a solid rectangle. Each sequence begins and ends with a 6-bp direct repeat. Frequencies of each TRR type in Yunnan, China (YN), Guangdong, China (GD), and Florida (FL) are tabulated on the right.
Identification of MCLas-B.
A total of 161 isolates (107 from China and 54 from Florida) yielded B720 with primer set Lap-PF1-f/Lap-PF1-r (Table 1). As shown in Table 2, 16 (12 from China and 4 from Florida) B720 sequences, ranging from 718 to 733 bp, were obtained. Each sequence contained a MITE, ranging from 238 to 250 bp. The MITE shared no sequence similarity with MCLas-A and was, therefore, designated MCLas-B with four variants (Fig. 5). MCLas-B1 and MCLas-B2 were flanked by two 23-bp perfect TIRs with no internal tandem repeats. TIRs of MCLas-B3 were 78.3% similar to those of MCLas-B1 and MCLas-B2. MCLas-B4, represented in a single isolate, YN-JS-4, shared almost identical TIRs with MCLas-B1 and MCLas-B2 but exhibited significant variation in CR. MCLas-B1 was also present in phage SC1 and “Ca. Liberibacter asiaticus” strain Psy62 and was the dominant type (10/16, 62.5%). In the SC1 sequence, an ORF, SC1_gp120, identical to SC2_gp120 of phage SC2 was located 232 bp upstream of MCLas-B1 and therefore was a putative tps. Downstream 606 bp was an ORF not known to be related to any transposase in the genomes of both phage SC1 and “Ca. Liberibacter asiaticus” Psy62. MCLas-B was able to form a stable secondary structure at the RNA level (ΔG = −80.00 kcal/mol). No “Ca. Liberibacter asiaticus” isolates with empty MCLas-B were found, and the MITE mobility could not be evaluated. A BLASTn search revealed that all MCLas-B sequences were unique in the GenBank sequence database with the exception of the genomes of “Ca. Liberibacter asiaticus” Psy62 and phage SC1, where a single homolog was found in each.
Fig 5.
Schematic representations of MCLas-B and comparison of MCLas-B1, MCLas-B2, MCLas-B3, and MCLas-B4. TIR, terminal inverted repeat; DR, direct repeat (gray triangle); CR, central region. In sequence alignments, a dot represents nucleotide identity to the first sequence (SC-NN-6); a dash represents a missing base; a space flanked by two slashes represents omitted nucleotides. For demonstration of sequence variation, only nucleotide changes are listed; nucleotide insertions/deletions are not shown. The relatedness of MITEs is summarized by a neighbor-joining tree on the left. Subgroups of MITEs are numbered on the right.
Coexistence status of MCLas-A and MCLas-B.
In the Florida population of “Ca. Liberibacter asiaticus,” 48 of the 59 (81.4%) isolates were detected with both MCLas-A (B720) and MCLas-B (B630), indicating a high level of coexistence or mix of the two MITEs (Table 1). In the China population of “Ca. Liberibacter asiaticus,” separation of MCLas-A and MCLas-B was obvious. A total of 50 “Ca. Liberibacter asiaticus” isolates were MCLas-A alone (B630 alone or B630 + B350), which accounted for 18.5% (50/267) of the total collection. Among them, 58.0% (29/50) were from Guangdong, whereas only 8.0% (4/50) were from Yunnan. For MCLas-B, 71 MCLas-B alone isolates were detected, which accounted for 26.6% (71/267) of the total collection. Among them, 35.2% (25/71) were from Yunnan, in contrast to the 9.9% (7/71) from Guangdong.
DISCUSSION
One characteristic of transposons, including MITEs, is the presence of TIRs, which can be conveniently identified by a computer program such as the einverted program. However, not all sequences with TIRs are transposons. It was the mobility of a TIR-flanked sequence (both filled and empty states) that served as the strongest evidence of a transposon, or a MITE, in this study. A key contribution was the concept of pangenome or population genome, which encouraged the analyses of DNAs from multiple isolates. In this study, we selected a genomic locus with multiamplicons from a single primer set, analyzed the variations using a large collection of “Ca. Liberibacter asiaticus” isolates, and identified a MITE, MCLas-A, something that could not be achieved by single-genome analyses. It should be noted that evidence of MCLas-B mobility was not found in this study. However, the organization similarity to MCLas-A, i.e., the presence of DRs, TIRs, noncoding CR, and putative tps, strongly suggested that MCLas-B is, or was, a MITE.
A unique feature of MCLas-A is the presence of three units of tandem repeats in its TIRs. To our knowledge, this feature has not been reported in any MITE. Stavrinides et al. (13) recently reported a MITE, E622, in Pseudomonas syringae. The 168-bp TIRs of E622 contained two sets of interspaced repeats. The first set had two 12-bp units interspaced by 10 bp. The second set had two 15-bp units interspaced by 18 bp. This is still structurally very different from the TIRs of MCLas-A, where the repeats are basically in tandem (Fig. 3). Mahillon and Chandler (3) concluded that TIRs could be divided into two functional domains: the outer terminal 2 or 3 bp, involved in cleavages and strand transfer of transposition; and the inner part, involved in transposase binding. For MCLas-A, the tandem repeat regions seemed to be the sites of MITE cleavage. Examination of TIR remnants in the empty sites (Fig. 4) shows that all repeat units were almost intact, suggesting the entire 10 bp were used as a cleavage site during transposition. In fact, deletion within a tandem repeat seemed to prevent the mobility of MCLas-A3 in isolate YN-MG-4 (Fig. 3 and Table 1).
It is interesting that the distributions of TRR types are significantly different among the “Ca. Liberibacter asiaticus” populations from different geographical regions (Fig. 4). Isolates from Guangdong, China, are unique, while isolates from Yunnan, China, and Florida are similar. Based on this, it can be speculated that the “Ca. Liberibacter asiaticus” population in Yunnan is more related to the Florida population than that in Guangdong, China. The mechanism of how a specific remnant type was formed is unknown. Current information may reflect the imprints of MCLas-A behavior under different environmental conditions.
The mobility of MCLas-A strongly suggests the presence of a fully functional transposase in “Ca. Liberibacter asiaticus.” Currently, MITEs are considered to require a transposase acting in trans for transposition (1). With the close physical proximity, it is tempting to assume that the transposase upstream of MCLas-A (also MCLas-B) would mediate the transposition. Further substantiation of this notion is that both the putative transposase and MCLas-A belong to the IS3 family. A signature of IS3 transposase is that the majority of their transposons terminate with 5′-TG and CA-3′ (3). This matches with all the MCLas-A members in this study, but not with any MCLas-B members. A consequence of the in cis tps MITE configuration is that the movement of these MITEs might be limited to the vicinity of the tps. This could explain the observations that only a single copy of both MCLas-A and MCLas-B were found in the bacterial/prophage genome. Transposon Tn10 was reported to have more efficient transposition activity if the transposase was provided by a tps located close by on the same DNA molecule (41).
In contrast, no evidence of MCLas-B mobility was found in isolates from either China or Florida. This may be explained by the different structure of MCLas-B, which may be associated with the low or nonexistent transposition efficiency under the conditions where MCLas-A was active. In any case, further research is needed to provide direct proof of transposase activity. Related to MITE mobility, it should be noted that all MCLas-B coexisted or mixed with MCLas-A in the “Ca. Liberibacter asiaticus” population in Florida. Since the introduction of “Ca. Liberibacter asiaticus” into Florida was recent, the bacterium should have originated from a region dominated with both MCLas-A and MCLas-B. This intermediate location is currently unknown.
In summary, our knowledge about MITEs in bacteria is still limited. Studies of transposons/MITEs in “Ca. Liberibacter asiaticus” are further limited by the unavailability of in vitro culture, making genome sequence analyses a current major tool of research. This study applied the pangenome concept to analyze DNA polymorphisms of “Ca. Liberibacter asiaticus” detected by PCR, leading to the first detection and characterization of MITE or MITE type transposons in the bacterium. It is expected that future studies on MITEs will enrich our knowledge of the “Ca. Liberibacter asiaticus” biology, particularly the bacterial niche-specific adaptation, mechanisms of population diversity, and bacterial genomic evolution.
ACKNOWLEDGMENTS
This work was supported by Special Fund for Agro-scientific Research in the Public Interest (201003067-02), Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, IRT0976), Natural Science Foundation Project of CQ CSTC (cstc2012jjA80025), Chongqing Key Laboratory of Citrus (CKLC201108), and California Citrus Research Board (5300-151).
We thank X. Sun and M. Irey for providing “Ca. Liberibacter asiaticus” DNAs and E. Civerolo for editing the manuscript.
Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture (USDA).
Footnotes
Published ahead of print 28 June 2013
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