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1 Division of Primary Oral Health Care, University of Southern California School of Dentistry, 925 W 34th Street, Room 4107, Los Angeles, CA 90089-0641, USA
2 Department of Microbiology and Immunology, University of Oklahoma, Oklahoma City, OK 73190, USA
Correspondence
Casey Chen
ccchen{at}usc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Division of Infectious Diseases, Childrens Hospital Los Angeles, University of Southern California, CA 90027, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Lorenz & Wackernagel, 1994). Bacteria are the only organisms known to have this capability. Natural transformation can be found in phylogenetically distant bacteria, including both Gram-positive and Gram-negative bacteria.
The mechanisms used by competent bacterial species are also distinct. Several transformation systems have been well characterized, including those of Gram-positive Streptococcus–Bacillus, Gram-negative Haemophilus–Neisseria (Dubnau, 1999; Lorenz & Wackernagel, 1994; Smeets & Kusters, 2002) and type IV secretion-dependent Helicobacter pylori (Smeets & Kusters, 2002).
In competent bacterial species employing the Haemophilus–Neisseria system, the transformation efficiency is greatly enhanced if the donor DNA contains specific oligonucleotides designated uptake signal sequences (USS). Furthermore, USS sites are highly represented in the genomes of these bacteria. The USS of Haemophilus influenzae have been defined experimentally as both a 9-base core of 5'-AAGTGCGGT and its complementary sequence. Genomic sequence analysis of the USS in the Rd strain of H. influenzae revealed an extended 29-base consensus sequence (Smith et al., 1995, 1999). For the purpose of this study this extended USS will be described as comprising 3 regions: the 1st region (10-base, 5'-aAAGTGCGGT), which contains a semi-conserved adenosine followed by the originally defined 9-base USS core, followed by the 2nd (9-base, 5'-nrwwwwwnn. n: any nucleotide, r: A or G, w: A or T) and 3rd (10-base, 5'-nnnnrwwwww) semi-conserved AT-rich regions. The role of these AT-rich regions in facilitating transformation remains to be determined.
The Gram-negative, capnophilic bacterium Actinobacillus actinomycetemcomitans, a member of the family Pasteurellaceae, is implicated as a major causative agent of localized aggressive periodontitis and other forms of periodontitis (Asikainen & Chen, 1999; Slots, 1999). A. actinomycetemcomitans is naturally competent for transformation (Fujise et al., 2004; Tønjum et al., 1990; Wang et al., 2002, 2003). The transformation system of A. actinomycetemcomitans is remarkably similar to that of H. influenzae (Fujise et al., 2004; Thomson et al., 1999; Wang et al., 2002, 2003). A. actinomycetemcomitans also displays preferential uptake of donor DNA with the same 9-base USS core as H. influenzae.
The objective of this study was to further examine the requirements for the extended 29-base USS, in particular the 2nd and the 3rd semi-conserved regions, in transformation of A. actinomycetemcomitans. Transformation frequencies and relative transformation efficiencies were determined. The frequency and distribution pattern of USS sites in the genome of A. actinomycetemcomitans were also examined and compared to those in H. influenzae.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A naturally transformable A. actinomycetemcomitans strain D7S-smooth was selected for this study (Wang et al., 2002). The strain was a nonfimbriated variant of strain D7S that was recovered from a patient with aggressive periodontitis. The nonfimbriation was the result of a spontaneous point mutation in the promoter region of the fimbria/Tad locus (Wang et al., 2005). A. actinomycetemcomitans was routinely grown in sTSB medium [3 % trypticase soy broth (TSB), 0.3 % yeast extract, 1.5 % agar and 5 % horse serum], or mTSB broth (3 % TSB and 0.6 % yeast extract) at 37 °C in 5 % CO2. Escherichia coli plasmids were propagated in E. coli DH5
by standard methods (Sambrook et al., 1989). When required, the media were supplemented with spectinomycin (Spe, 50 µg ml–1).
Plasmids.
The recombinant plasmid pDpilB870-770 was constructed previously (Wang et al., 2003). It contains a cloned recombinant DNA (pilA-SpeR-pilC) that was derived from the pilABC locus of A. actinomycetemcomitans strain D7S. The internal 846 bp of pilB was replaced with a 1.2 kb SpeR cassette leaving fragments of 870 bp (pilA) and 770 bp (pilC) flanking the cassette. There are two naturally occurring USS sites in pDpilB870-770 (Figs 1 and 2). The first USS site is located upstream of pilA. The second USS site is located at the 5' end of pilC next to the SpeR cassette.
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) is a derivative of pDpilB870-770. It was constructed by removing a 76 bp SalI–MluI fragment containing the USS site nearest the SpeR cassette from pDpilB870-770.
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). The genomic DNA of the resultant 
hmsFR mutant was used as a template to amplify the SpeR marker and its flanking sequences with primers HMpre1, 5'-CGCGGTTATGTGACATCCGA (710 bases upstream of SpeR), and HM-USS, 5'-GGAAAATCCAAATGTTTTTTA (407 bases downstream of SpeR). The 2.3 kb amplicon was then cloned in pBluescript KS (Stratagene) at the EcoRV site to generate pD-HM.
Several recombinant plasmids were constructed and used directly as donor DNA in transformation assays. These plasmids were constructed by cloning into pBluescript KS the PCR products amplified from pDpilB870-694 using appropriate primers that incorporated base substitutions in USS regions (see Fig. 4 for primer sequences).
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, or provided with the results (Figs 3 and 5). Each PCR reaction of a 60 µl mixture contained 25–50 ng pre-boiled plasmid, 30 pmol each of primers, 1.6 mM MgCl2, and 3 units Taq polymerase (Promega), and was amplified under the following conditions: 30 cycles at 94 °C for 30s, 56 °C for 30s, 72 °C for 3.5 min, and a final extension of 5 min at 72 °C. After amplification, the DNA was digested with DpnI (New England Biolabs) at 37 °C for 1 h to remove template DNA. Our preliminary studies have shown that digestion of amplicons with DpnI removes methylated DNA and destroys 80–90 % of the transforming activity of the template DNA, but has no apparent effect on the transforming ability of the unmethylated amplicons. If the PCR products exhibited a single strong band on an agarose gel, the DNAs were directly used in transformation assays. Occasionally, when the qualities of amplicons were not satisfactory, the PCR reactions were repeated with slightly modified conditions, or PCR products were purified through columns (Qiagen) and quantified before use in a transformation.
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). Briefly, bacteria were grown on sTSB agar for 20 h, collected and suspended in TSB to 
5x109 c.f.u. ml–1. The bacterial suspension (20 µl) was spotted on a prewarmed sTSB plate and incubated for 2 h until cells reached optimum competency. Ten microlitres of donor DNA (0.2–0.5 µg in TE buffer) was added to the recipient bacteria and mixed with an inoculation loop. The bacteria were further incubated for 5 h and then plated on the selective medium (sTSB agar containing 50 µg spectinomycin ml–1) and incubated for 2–3 days to enumerate transformants. Transforming DNA was always used at more than the saturation level (0.1 µg DNA). The results are reported as transformation frequency (transformants per c.f.u.) or as relative transformation efficiency.
Sequence analysis.
Computational analyses were performed on the genome of A. actinomycetemcomitans strain HK1651. Detection and distribution of USS sites were determined using a series of Perl scripts, which have been compiled into a publicly available, web-based application called SeqSeek (unpublished), which is available at http://microgen.ouhsc.edu/software/seqseek.htm. Utilizing the 9-base USS core sequence as a query, SeqSeek displays count data for the number of USS sites found on each strand, along with the distribution of USS sites in intragenic and intergenic spaces. SeqSeek also reports large regions of the genome that are devoid of USS sites, listing the genes contained within each of these regions. Repeat regions were discovered using the European Molecular Biology Open Software Suite (EMBOSS) package of sequence analysis tools. Specifically, einverted and etandem were used to find inverted and tandem repeats, respectively.
Statistical analysis.
The relative differences in the transformation efficiencies of donor DNA were evaluated by ANOVA and Tukey-HSD multiple range test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and (ii) the number of USS sites of the donor DNA (Fig. 2). The transformation frequency for donor DNA with a right arm 766 bp in length was 2x10–3 per cell; a frequency that was similar to the optimum transformation frequency in standard transformation assays (Fujise et al., 2004; Wang et al., 2002, 2003). There was a trend of a gradual decrease of transformation frequency with decreasing length of homologous regions from 66 % (500 bp) to 5 % (160 bp) of the transformation frequency with the full-length DNA.
Fig. 2 (bottom) shows that transformation frequencies were similar with donor DNA containing one or two USS sites, and with donor DNA having a USS site in the middle or at the 5' end of the DNA (no significant differences; Tukey-HSD test, P>0.05).
Transformation by linear DNA with base substitutions in the 1st region of the USS
Fig. 3 shows the results of the transformation assays with amplicons (2.7 kb) that contained a full USS site or a USS site with either a single-base substitution or missing the first 5 bases. The amplicons with single-site transition mutations of the 1st region retained 28–67 % of the relative transformation efficiency of the positive control (Tukey-HSD test, P<0.05). The deletion of the first 5 bases in the 1st region reduced the relative transformation efficiency to 1 %.
Transformation with recombinant plasmids
The use of linear DNA for transformation as described above did not address the question whether optimum transformation of DNA requires the presence of DNA 5' to the USS in the donor DNA. Therefore, we designed a series of recombinant plasmids as donor DNA for transformation assays. Fig. 4 shows that the relative transformation efficiencies of plasmids pUSS4, pUSS8A and pUSS23 (all with a base substitution in the 1st region) were 45–58 % and were significantly different from both the positive control pUSS1 and the negative control pUSS-0 (P<0.05; Tukey-HSD test). The relative transformation efficiencies of pUSS-core (containing a perfect 1st region but with non-consensus 2nd and 3rd regions), pRegions23 (with a non-consensus 1st region and intact 2nd and 3rd regions) and the negative control pUSS-0 (without USS) were severely reduced: 21 %, 6 %, and 6 %, respectively. The results again suggested that single-base mutations in the 1st region affected transformation efficiency, even in donor DNA with an extended DNA region 5' to the USS site. The results were also consistent with our experience that the 1st region was more important than the 2nd–3rd region in transformation.
Involvement of the 2nd and 3rd regions of the USS in transformation
The results from the above experiments did not distinguish between the individual contributions from the 2nd or 3rd regions of USS in transformation. We took advantage of a native USS site occurring at the 3' end of the hmsD gene to design amplicon donor DNA with a perfect USS, a non-consensus 2nd region, a non-consensus 3rd region, or a truncated 3rd region to test their relative transformation efficiencies (Fig. 5). The first three amplicons were identical in length and contained a tail of 3 nucleotides 3' to the USS. The last amplicon DNA was shorter and lacked the last 6 nucleotides of the 3rd region of the USS. The relative transformation efficiencies of donor DNA with non-consensus 2nd or non-consensus 3rd regions were 47 % and 73 %, respectively. The results showed that the 2nd and 3rd regions were both involved in transformation, and further suggested that the 2nd region was more critical than the 3rd region in transformation. Truncation of the 3rd region of the USS severely reduced the transformation efficiency to 3 %, suggesting that the presence of DNA beyond the 2nd region (irrespective of its sequence) was also critical for transformation.
Frequency of USS sites in the genome of strain HK1651
We searched the genome of A. actinomycetemcomitans strain HK1651 for the highly conserved 9-base core sequence of the USS and found a total of 1759 copies, identified as 881 copies on one strand and 878 copies on the complementary strand. Fewer than 14 copies of the USS would be expected by random chance. The frequency of the USS in A. actinomycetemcomitans is similar to that in H. influenzae (both occur at 0.8 copy of USS per kb genome). We also confirmed the presence of the extended 29-base full USS in A. actinomycetemcomitans, as noted previously (Wang et al., 2002).
Distribution patterns of USS sites in the genome of strain HK1651
The location of USS sites in relation to ORFs in the genome of HK1651 was further examined. There were 1402 copies of the 9-base USS core sequence located within coding regions, while the remaining 357 copies were found to be intergenic. The large-scale distribution pattern of the USS appears to be random in the genome. There are several large genomic regions (5 kb or greater) without USS in the HK1651 genome. Some USS-free regions were found to harbour rRNA operons and loci for ribosomal proteins (Smith et al., 1999) and genomic islands (Chen et al., 2005) (see also http://www.oralgen.lanl.gov/), whereas others contain putative virulence factors of A. actinomycetemcomitans [e.g. matrix binding protein EmaA (Mintz, 2004), cytolethal distending toxin (Mayer et al., 1999; Shenker et al., 2000; Sugai et al., 1998), fimbria/Tad locus (Haase et al., 1999; Inouye et al., 1990; Ishihara et al., 1997; Kachlany et al., 2000; Planet et al., 2003) and leukotoxin (Kolodrubetz et al., 1989; Kraig et al., 1990; Lally et al., 1989)].
The distribution pattern of USS within a close distance to a second USS was not random. We found 162 pairs of USS sites separated by a distance of 35 bp or less. Among these sites, 160 are inverted repeats (96 +/– pairs, and 64 –/+ pairs), and 2 are direct repeats. The 160 inverted repeats have the potential to form stem–loop structures when transcribed into RNA. Of these, 92 (53 +/– pairs and 39 –/+ pairs) are located completely in intergenic regions. A high number of these USS sites were observed to cluster within 50 bp of the 3' end of ORFs. No such clustering in relationship to the 5' end of the coding regions was found. The remaining 68 inverted repeats (43 +/– pairs and 25 –/+ pairs) were located within coding regions. The potential regulatory functions of these USS sites remain to be determined.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The function of A. actinomycetemcomitans USS may be inferred from studies of transformation of H. influenzae. However, while the 1st USS region in the donor DNA was required for DNA uptake in H. influenzae, the involvement of the 2nd and the 3rd USS regions in transformation has not been fully tested (Danner et al., 1980). In this study, we examined the involvement of three regions of the USS in the transformation of A. actinomycetemcomitans. We also examined the frequency and distribution patterns of USS in the A. actinomycetemcomitans genome. The results showed that all three USS regions were required to achieve optimum transformation efficiency. The relative contributions to transformation among individual USS regions were 1st region > 2nd region > 3rd region. We further noted that the frequency and distribution patterns of USS were similar in A. actinomycetemcomitans and H. influenzae.
Several large genomic regions (>5 kb) of A. actinomycetemcomitans strain HK1651 were found to contain no USS sites. Some of these regions also exhibited low G+C content. It is worthy of note that several of these USS-free regions contain putative virulence genes of A. actinomycetemcomitans. Perhaps these USS-free regions were DNA blocks acquired by horizontal gene transfer and constitute part of the flexible gene pool that enhances the fitness or virulence of bacteria (Hacker et al., 1997; Hacker & Carniel, 2001).
The USS of A. actinomycetemcomitans is prevalent in the genomes of some Pasteurellaceae species. Albritton et al. (1984, 1986) observed that the genomic DNA from Haemophilus parainfluenzae, Haemophilus aphrophilus, Haemophilus paraphrophilus, Pasteurella pneumotrophica, Pasteurella multocida and A. actinomycetemcomitans competed for homospecific transformation in H. influenzae. Hong & Dewhirst (2002) found preliminary evidence for the frequent occurrences of the USS in Haemophilus parahaemolyticus, H. parainfluenzae, Actinobacillus suis, P. multocida, Pasteurella canis and several additional Pasteurella species.
The evolutionary basis for the high prevalence of USS in the genomes of A. actinomycetemcomitans and H. influenzae is not completely understood. Since the identical USS core sequence is found at high frequencies in the genomes of diverse Pasteurellaceae species, USS may not function as a species barrier to prevent the uptake of non-self DNA as suggested previously. Bakkali et al. (2004) examined the occurrence of USS sites in P. multocida, H. influenzae and A. actinomycetemcomitans. An identical 9-base USS sequence was identified in 927, 1205 and 1760 copies, respectively in these three species. Moreover, the locations of USS sites were conserved in homologous genes in the three species. It was postulated that the USS were selected for and maintained by a biased-DNA-uptake system (Bakkali et al., 2004). It remains to be determined whether the shared USS promote genetic exchange among these bacterial species.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 23 March 2006;
revised 3 August 2006;
accepted 6 August 2006.
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