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1 Cystic Fibrosis Laboratory, Medical Microbiology, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, UK
2 Department of Infection, Immunity and Inflammation, University of Leicester, Maurice Shock Building, University Road, PO Box 138, Leicester LE1 9HN, UK
3 Department of Medical Microbiology, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK
4 Laboratorium voor Microbiologie, Faculteit Wetenschappen, Universiteit Gent, Ledeganckstraat 35, B-9000, Gent, Belgium
Correspondence
Dervla T. Kenna
dkenna{at}staffmail.ed.ac.uk
Received 30 May 2005
Accepted 30 August 2005
Abbreviations: Bcc, Burkholderia cepacia complex; CF, cystic fibrosis; hINVPCR, hemi-nested inverse PCR; IS, insertion sequence.
Present address: Cystic Fibrosis Group, Centre for Infectious Diseases, University of Edinburgh College of Medicine and Veterinary Medicine, The Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK. 
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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The molecular and genetic basis for the flexibility of these bacteria has yet to be clarified but should be aided by the annotation of the genome of B. cenocepacia strain J2315 currently underway at the Sanger Centre (http://www.sanger.ac.uk/Projects/B_cenocepacia/). Recent annotation of the closely related species Burkholderia pseudomallei and Burkholderia mallei has emphasized the impact of insertion sequences (ISs) and bacteriophages on both the plasticity and adaptability of these organisms (Holden et al., 2004; Nierman et al., 2004). Furthermore, previous studies have shown that ISs are shared between these highly virulent species and B. cenocepacia (Mack & Titball, 1998). ISs are small, mobile genomic elements possessing only the genes that allow their transposition from one genomic site to another. In addition to their ability to cause genomic rearrangements such as deletions, duplications and recombination, these elements can also insert into genes and promoter regions and may hence affect gene transcription (Wood et al., 1991; Miché et al., 2001; Mahillon & Chandler, 1998). To date, at least 16 different types of IS have been discovered within the Bcc (Wood et al., 1991; Tyler et al., 1996; Lessie et al., 1996; Miché et al., 2001; Liu et al., 2003), supporting the hypothesis that ISs play a key role in the diversity of these organisms. Of particular interest are active ISs that can upregulate the transcription of genes close to their point of insertion. One of these ISs can affect the transcription of genes involved in adaptation of the Bcc to diverse environmental conditions, thus enabling them to utilize potentially toxic compounds as a carbon source (Hubner & Hendrickson, 1997). In addition, several studies have demonstrated the in vitro activation of Bcc genes by ISs (Scordilis et al., 1987; Wood et al., 1991; Miché et al., 2001).
To our knowledge, little work has been done on the potential contribution of active ISs to the evolution of Bcc species as opportunistic pathogens for plants, animals and humans, particularly studies that take account of recent developments in the complex taxonomy of these bacteria. In this study, we examined the distribution and copy number of three active ISs (IS402, IS407 and IS1416) in 66 isolates from clinical and environmental sources; the isolates represented all nine genomovars and included six strains with undetermined genomovar status. In addition, IS407 insertion sites of six strains were mapped to the genome and the impact of their insertion on downstream genes considered.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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DNA extraction and PCR. Bacteria were grown overnight at 37 °C on Columbia-base agar (Oxoid). DNA was extracted using a Puregene DNA isolation kit (Gentra systems), with a few modifications (Kenna et al., 2003). PCR to test for the presence of IS402, IS407 and IS1416 was conducted using the PCR primers and amplification cycling conditions described by Miché et al. (2001). A 25 µl reaction contained the following PCR reagents from Qiagen: 20 ng of DNA, 250 µM (each) deoxynucleoside triphosphate, 1·5 mM MgCl2, 1x PCR buffer, 20 pmol each primer (Invitrogen), 1 U Taq polymerase and sterile distilled water. Ten microlitres of PCR product was run alongside a 1 kb Plus DNA ladder (Invitrogen) on a 1·2 % pre-cast E-gel (Invitrogen). A positive control (the B. vietnamiensis type strain LMG 10929T) that had previously been found to be positive for all three ISs by Miché et al. (2001) was used, and negative controls consisted of PCR reaction mix without DNA.
Southern blotting. Southern blotting was conducted using a digoxigenin (DIG)-labelled probe. Briefly, approximately 2·5–4 µg DNA was digested at 37 °C for 4 h with 10 U BglII, 10 U HindIII or 12 U EcoRI (Promega). DNA was separated on a 0·8 % 0·5x TBE agarose gel for 16 h at 1·0 V cm–1 alongside 0·05 µg DIG-labelled DNA molecular mass marker II (Roche Diagnostics). DNA from the agarose gels underwent Southern blotting transfer via a capillary-transfer method onto a positively charged nylon membrane as described by Sambrook et al. (1989). The IS407 DIG-labelled probe was created using a PCR DIG probe synthesis kit (Roche Diagnostics) with the primers used by Miché et al. (2001) to amplify IS407. The membrane was pre-hybridized with pre-hybridization solution [5x SSC, 0·1 % N-lauroylsarcosine (w/v), 0·02 % SDS (w/v), 2 % blocking solution (v/v) (Roche Diagnostics)] in a hybridization bag (Roche Diagnostics) for 4 h at 68 °C. This step was followed by 12 h hybridization using 100 ml pre-hybridization buffer and 20 µl DIG-labelled probe. All post-hybridization washes were conducted using materials from Roche Diagnostics, unless otherwise stated. Post-hybridization washes consisted of two 5 min washes at room temperature with 2x SSC and 0·1 % SDS, followed by two 15 min washes at 68 °C with pre-heated 0·1x SSC and 0·1 % SDS. The membrane was then incubated for 30 min in blocking solution, followed by 30 min in blocking solution with anti-DIG AP Fab fragments before two 15 min washes in washing buffer and 3 min in detection buffer. To allow visual detection of the bound probe 20–25 drops of ‘CDP* ready-to-use’ was added. The membrane was sealed in a hybridization bag and left with Lumi-film chemiluminescent detection film to expose for 2–10 min. Film was processed using an Optimax film processor (Protec Medizintechnik).
Hemi-nested inverse PCR.
Hemi-nested inverse PCR (hINVPCR) was carried out, with some modifications, based on a method by Yesilkaya et al. (2003). The main steps of the method are illustrated in Fig. 1
. To reach as many insertion sites as possible, both sides of the IS were targeted using primers specific for both the 5'- and 3'-ends of the IS. Briefly 2·5–4 µg DNA was digested with 5 U of EaeI (New England BioLabs) for 2 h at 37 °C. Digested DNA was heat-inactivated at 65 °C, then self-ligated in a thermal cycler with a heated lid at 16 °C for 16 h. The ligation mixture consisted of 0·5–2 µg DNA in 20 µl 10x ligation buffer [stock: 50 mM Tris/HCl (pH 7·6), 10 mM dithioerythritol, 500 µg bovine serum albumen ml–1] and 4 U T4 ligase (Roche Diagnostics) made up to a final volume of 200 µl with sterile distilled water.
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) and 72 °C for 2 min and a final extension step of 1 cycle of 72 °C for 3 min. Two microlitres of primary PCR product was used to set up a 50 µl nested PCR reaction. All the other conditions remained the same apart from the primer concentration, which was increased to 60 pmol. The primers used in the nested PCR amplification were BC3 and BC2 (left side of IS407), and BC5 and BC6 (right side of IS407). The PCR amplification conditions were the same as for the primary PCR except that the number of cycles was increased from 15 to 25. Twelve microlitres of the nested PCR product was electrophoresed for 5 h at 1·3 V cm–1 in a 2 % MetaPhor agarose gel (Cambrex Biosciences). Agarose gels were examined using a UV transilluminator after staining with ethidium bromide. Bands of interest were separated from one another using a hypodermic needle (BD Microlance 3, Becton Dickinson) as a method of extracting the amplified product from the gel (Bjourson & Cooper, 1992). The DNA attached to the needle was washed into fresh nested PCR mix and reamplified. Bands were then purified using a QIAquick PCR purification kit (Qiagen) and sequenced. The sequencing programme was as follows: 95 °C for 1 min, and 25 cycles of 95 °C for 30 s, 45 °C for 15 s and 60 °C for 4 min. The sequencing mixture consisted of 4 µl ABI PRISM BigDye terminator v3.1 cycle sequencing mix (Applied Biosystems), 40 pmol either primer BC2 or primer BC6 and 0·35–0·8 µl DNA, depending on the concentration of DNA. The mixture was made up to 20 µl with sterile, distilled water.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). IS402 was absent from all 16 strains of B. anthina tested, IS407 was absent from all 10 B. pyrrocinia strains and IS1416 was absent from all eight strains of B. multivorans. IS1416 was more common in strains of B. pyrrocinia (8/10, 80 %) and B. anthina (7/16, 44 %) than in strains from other genomovars. To our knowledge this is the first time that the prevalence of IS1416 has been investigated across the whole Bcc. IS1416 was only recently found to be present in Burkholderia species other than Burkholderia glumae, the species with which it was first associated (Miché et al., 2001; Hasebe et al., 1998).
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suggested that IS407 might be uncommon or absent in B. cepacia (previously genomovar I). However, when an additional six B. cepacia strains were tested, five were positive for IS407. Likewise all eight strains of B. multivorans were negative for IS1416, but unlike the previous example, when an additional four B. multivorans strains were tested these were all negative also. Overall, our data support previous reports that different Burkholderia species possess different active IS elements (Hasebe et al., 1998; Miché et al., 2001). The only IS to show a different distribution between clinical and environmental strains was IS402, which was present in 41 % (17/41) of clinical isolates, but in only 17 % (4/24) of environmental isolates. However, it should be noted that there was no balance between clinical and environmental isolates in each of the Bcc species. Both IS407 and IS1416 were relatively evenly distributed between clinical and environmental strains, with IS407 present in 46 % (11/24) of environmental strains and 32 % (13/41) of clinical strains, while IS1416 was found in 50 % (12/24) of environmental strains and 27 % (11/41) of clinical strains. The only animal isolate in this study was from a sheep suffering from subclinical mastitis (Berriatua et al., 2001) and this strain was found to have only one of the three IS elements (IS402).
Copy-number variation of IS407
In our study, we decided that IS407 should be examined in more detail to determine copy-number variation amongst the different genomovars. IS407 was chosen because of its well-established mechanism for gene activation, which works on the basis of the outwardly directed 
70-like promoter within its IR-R (an inverted repeat at the right end of the IS) (Wood et al., 1991). In addition, of the three ISs that were tested by PCR, IS407 was found to be the most common IS in J2315. Initial BLAST analysis of the J2315 sequence from the Sanger centre revealed that there were 13 copies of IS407: 10 copies on chromosome 1, one copy on the plasmid and one each on chromosomes 2 and 3.
Genomic DNA from 23 IS407-positive strains was digested and the IS407 copy number determined using Southern blotting. Three restriction enzymes (BglII, HindIII and EcoRI) were used to allow accurate coverage of the genome of the Bcc, which has been shown to vary in size from 5 Mb to just over 9 Mb (Lessie et al., 1996). The copy number ranged from two to 16 with a mean of five, which was in agreement with the range noted by others (Mack & Titball, 1998; Miché et al., 2001). There seemed to be a greater number of IS407 copies in isolates of B. cenocepacia than in other species, especially B. anthina (a mean of 8·75, compared to 3·75, respectively). A disparity between the clonal ET12 B. cenocepacia strains J2315, BC7 and C5424 was found on Southern blotting. The ET12 lineage predominates among CF patients in Canada and the UK (Govan et al., 1993), and, though clonal, reports of morphological differences and variation in antibiotic sensitivities between these strains have been noted elsewhere (Winstanley et al., 2001; Nzula et al., 2002). In this study, BC7 was found to have 16 copies while BLAST analysis found J2315 to have 13 (although this laboratory found only 10) and C5424 had only six (Fig. 2). Likewise, the study by Mack & Titball (1998) found only eight copies for J2315. Perhaps restrictions with a wider variety of enzymes would be necessary before all copies could be successfully identified. Alternatively, perhaps IS407 copy-number variation does exist between these strains. K56-2 was only found to be positive for IS407 when this study was nearing completion, thus no blotting data exist for this strain.
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IS407 insertion site mapping
The second aim of this study was to map IS407 to the genome to establish putative links between insertion site and downstream gene transcription. IS407 insertion sites from six IS407-positive strains (B. cenocepacia LMG 18826, B. cenocepacia LMG 16656T, B. multivorans LMG 18823, B. stabilis LMG 14294T, B. vietnamiensis LMG 16232 and B. anthina J2944) were identified by hINVPCR and mapped to the genome. These results are summarized in Table 3. In some cases, only a few of the copies from each strain were successfully mapped.
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In general, flanking sequences could not be matched to one particular gene family but rather to genes with a range of different functions (Table 3). In some cases, matches were found to putative regulatory genes such as transcriptional regulators and methylases, but also to housekeeping genes such as those involved in amino acid synthesis, aromatic compound metabolism and disulphide oxidoreductase activity. Several matches were also found to putative outer membrane porin genes and also to genes encoding transmembrane proteins. In all, there did not seem to be a link to a particular gene family, suggesting that the presence of IS407 affects potential virulence factors but also upregulation of genes crucial to cell survival as noted elsewhere for the ß-lactamase genes of the Bcc (Scordilis et al., 1987). Indeed, it seems likely that IS407 inserts at random locations in the genome. Although it is hard to be certain without testing a greater number of strains, there did not seem to be any evidence of a preferential locus for insertion, as has been found for IS6110 in Mycobacterium tuberculosis (which, like IS407, is a member of the IS3 family of ISs) (Fang & Forbes, 1997).
Interestingly, from the perspective of flanking sequence matches to J2315, two strains, LMG 18826 (BC7) and LMG 14294T (Burkholderia stabilis) had identical sequence matches to the Sanger centre strain (five of the 16 BC7 IS407 copies that were successfully sequenced were mapped to J2315 and likewise four of the 10 IS407 copies from LMG 14294T). While it is understandable that both the clonal ET12 strains J2315 and BC7 would share sequence homology, it is interesting that a B. stabilis strain would have the same level of similarity to J2315. However, perhaps the flanking sequence for the six IS407 copies that were not successfully sequenced in this study would not exhibit a high level of similarity to J2315, thus giving a deceptive impression of overall genome similarity. It is, however, interesting that strain LMG 16232 (B. vietnamiensis), the strain most successfully amplified by hINVPCR (flanking sequence for all nine copies was sequenced), showed no matches to the J2315 genome. Likewise, neither did flanking sequences from strains LMG 18823 (B. multivorans) and J2944 (B. anthina), although for both these strains only one of each of the IS407 copies was successfully sequenced.
Conclusions
In conclusion, with some interesting exceptions, this study has shown that the three active ISs IS402, IS407 and IS1416 are distributed across the nine species of the Bcc. IS407 copies from six strains were mapped to the genome and were found to have inserted upstream of a wide range of putative housekeeping, informational and hypervariable genes including genes involved in amino acid synthesis, transcriptional regulation and outer-membrane proteins. A fuller analysis of the hemi-nested inverse PCR results will be made easier by the completed annotation of the J2315 genome sequence. A closer look at genomic regions of interest will also help to determine the impact of active ISs on the diversity and variable virulence within the Bcc.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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