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J Bacteriol. Mar 2007; 189(5): 2077–2085.
Published online Dec 28, 2006. doi:  10.1128/JB.01408-06
PMCID: PMC1855724

New Functional Identity for the DNA Uptake Sequence in Transformation and Its Presence in Transcriptional Terminators[down-pointing small open triangle]

Abstract

The frequently occurring DNA uptake sequence (DUS), recognized as a 10-bp repeat, is required for efficient genetic transformation in the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae. Genome scanning for DUS occurrences in three different species of Neisseria demonstrated that 76% of the nearly 2,000 neisserial DUS were found to have two semiconserved base pairs extending from the 5′ end of DUS to constitute a 12-mer repeat. Plasmids containing sequential variants of the neisserial DUS were tested for their ability to transform N. meningitidis and N. gonorrhoeae, and the 12-mer was found to outperform the 10-mer DUS in transformation efficiency. Assessment of meningococcal uptake of DNA confirmed the enhanced performance of the 12-mer compared to the 10-mer DUS. An inverted repeat DUS was not more efficient in transformation than DNA species containing a single or direct repeat DUS. Genome-wide analysis revealed that half of the nearly 1,500 12-mer DUS are arranged as inverted repeats predicted to be involved in rho-independent transcriptional termination or attenuation. The distribution of the uptake signal sequence required for transformation in the Pasteurellaceae was also biased towards transcriptional terminators, although to a lesser extent. In addition to assessing the intergenic location of DUS, we propose that the 10-mer identity of DUS should be extended and recognized as a 12-mer DUS. The dual role of DUS in transformation and as a structural component on RNA affecting transcription makes this a relevant model system for assessing significant roles of repeat sequences in biology.

Repeat sequences are a prominent feature of all genomes, promoting recombination and other forms of genetic variability (4, 17). The neisserial DNA uptake sequence (DUS), 5′-GCCGTCTGAA-3′, and the Haemophilus influenzae uptake signal sequence (USS), 5′-AAGTGCGGT-3′, are required for efficient transformation (12, 23). The recognition of homo-specific DNA by means of DUS and USS is a hallmark of transformation in the genus Neisseria (5, 15, 23, 25) and several species in the family Pasteurellaceae (12, 48). A potential DUS- or USS-specific DNA-binding receptor is, however, yet to be identified. All neisserial genome sequences available to date accommodate approximately 2,000 copies of DUS per genome, occupying as much as 1% of the chromosomes (32, 42). The extensive analysis on the abundance and distribution of DUS in Neisseria meningitidis Z2491 by Smith and coworkers (36) documented that sequence conservation exists in positions −1 and −2 of the 10-mer DUS. The T at −1 and A at −2 were found at high frequencies in all DUS. This particular finding has since then been overlooked in neisserial research and also in affinity and transformation studies, and the biological impact of this extended DUS, here termed the 12-mer DUS, has remained unexplored. We show that the two semiconserved residues 5′ of the 10-mer DUS are both present in 76% of all DUS occurrences in every neisserial genome sequence available, and we demonstrate a positive functional effect on transformation by the stepwise addition of these two nucleotides. Enhanced DNA uptake of the 12-mer DUS relative to the 10-mer was confirmed in DNA uptake assays in a meningococcal strain.

Preference for the uptake of DUS- or USS-containing DNA in genetic transformation is well documented, and uptake sequences have routinely been utilized as efficient DNA signals in experiments involving transformation (1, 18, 24). Even though transformation in neisserial species is particularly important for genetic exchange and creating diversity, recent evidence suggests that transforming DNA also can have a role in genome conservation (13). The pathogenic Neisseria are also naturally competent for transformation throughout their growth cycle (10, 29, 38). It has been demonstrated that the presence of a single DUS in the donor DNA is sufficient for the competitive inhibition of transformation (18, 23). Competitive inhibition of transformation has been measured by the ability of plasmid constructs with cloned restriction fragments of gonococcal origin to inhibit transformation of N. gonorrhoeae by genomic DNA encoding antibiotic resistance (23). Furthermore, the quantitative effect on transformation has been shown to be linearly related to the number of DUS, at least for fragments in the range of 3 to 17.5 kb (24). It has been inferred from previous studies that DUS arranged as an inverted repeat (IR) may function more efficiently in transformation than a single DUS species (18, 24). Upon testing this in a clear-cut assay, we have rejected this hypothesis and explain previous interpretations to be due to the comparison of different donor DNA species, which also varied in the extended sequential identity of DUS reported here.

Previously, we have assessed the genome-wide role of DUS/USS inside coding sequences (CDS) (13). In that study, we found that intragenic DUS/USS have a distribution which is biased towards genome maintenance genes. Intergenic DUS/USS, however, often occur in pairs arranged as inverted repeats and are likely to function as rho-independent transcriptional terminators or attenuators by forming loop structures in mRNA (9, 23, 36). The localization of DUS/USS in transcriptional terminators was first suggested based on the identification of nonrandom distribution of the spacer lengths between two such signals commonly found downstream of stop codons in N. meningitidis Z2491 and H. influenzae Rd/KW20, respectively (36). The involvement of DUS/USS in genomic instability and genetic flux motivated the investigation of the genomic distribution and abundance of these repeats. The extensive presence of DUS/USS in neisserial genomes and two Pasteurellaceae genomes was therefore addressed, this time with a focus on intergenic occurrences. We investigated the cooccurrence of DUS/USS and transcriptional terminators by genome-wide screens to identify all potential terminators followed by the definition of their DUS/USS content. By employing the genome scanner for terminators, GeSTer (46), we found that 50% of all 2,000 10-mer DUS and 50% of all 1,500 12-mer DUS in Neisseria spp. constituted parts of predicted transcriptional terminators, either singularly or as inverted pairs. The data further imply that a quarter of all neisserial genes are likely to be terminated or attenuated by means of a DUS-containing inverted repeat. In the Pasteurellaceae genomes, containing around 30% intergenic USS, between 13% and 21% of the USS colocalize with predicted transcriptional terminators, suggesting convergent evolution of DUS and USS as structural components of transcriptional terminators. The dual roles of DUS/USS make these relevant models for assessing the nature, evolution, and biological function of significant repeat sequences.

MATERIALS AND METHODS

Genome sequences.

The following genome sequences (with accession numbers) were downloaded from http://www.ncbi.nlm.nih.gov/: Neisseria gonorrhoeae FA1090 (NC_002946); Neisseria meningitidis MC58 serogroup B (NC_03112); Neisseria meningitidis Z2491 serogroup A (NC_003116), Neisseria meningitidis FAM18 serogroup C (NC_03221); Haemophilus influenzae Rd/KW20 (NC_000907); Pasteurella multocida PM70 (NC_002663). Data from the temporary sequence data obtained for Neisseria lactamica ST-640 are presented here with kind permission from Julian Parkhill, and the primary sequences were produced by the Pathogen Sequencing Unit at the Sanger Institute and were obtained from the website ftp://ftp.sanger.ac.uk/pub/pathogens/nl/ on 3 March 2006. The unannotated temporary genome sequence fragments of N. lactamica ST-640 larger than 91,894 nucleotides were concatenated in numerical order and annotated using the BASys web service available at http://wishart.biology.ualberta.ca/basys/cgi/submit.pl. The annotation data generated by BASys were used to create a conventional GenBank file. Access to the genome sequence of Neisseria meningitidis 8013 serogroup C was provided by Eduardo Rocha, ABI/Institut Pasteur, Paris, France, with kind permission from Vladimir Pelicic, Necker Hospital, Paris/Imperial College London. DNA strings containing all DUS occurrences, inverted repeats, and surrounding areas in the genomes of Neisseria were collected with customized PERL scripts.

Bacterial strains, plasmids, and DNA manipulations.

Plasmids and strains employed in the study are listed in Table Table1,1, and primers are listed in Table Table 2. 2. Escherichia coli and the neisserial strains were grown essentially as described previously (45). A DUS-negative hybrid plasmid, p0-DUS, was designed using the neisserial pilus biogenesis gene pilG (45) harboring a mini transposon (mTnErm) (35) in the position 888 nucleotides from the start of the pilG gene and with the cloning vector pBluescript II SK/KS (pBSK+; Stratagene). p0-DUS was constructed by inserting a PCR product from primers pilG5′XhoI and pilG3′SacII on an N. meningitidis H44/76 pilG::mTnErm DNA template (45) into the multiple cloning site of pBSK+. The 8-, 9-, 10-, 11-, and 12-mer DUS elements (Table (Table3)3) were inserted into p0-DUS by replacing a PsyI/XhoI fragment inside pilG outside mTnErm following their synthesis by PCR splicing by overlap extension (PCR-SOE) (28). Overlapping fragments were made in separate reactions using the primer pilG3′PsyI in combination with A primers (primer names starting with letter A) and primer pilG5′XhoI in combination with B primers (primer names starting with letter B). Fragments containing DUS inserts were then spliced and amplified in PCRs using primers pilG5′XhoI and pilG3′PsyI on corresponding overlapping A and B fragments as templates. Plasmids pSingle, pIR, and pDR were constructed by hybridizing complementary primers, F and R primers in Table Table2,2, creating overhangs at both termini, followed by ligation into PsyI-digested p0-DUS following the manufacturer's instructions. The plasmids were transformed into E. coli XL-1 Blue and purified by standard protocols. All strains employed and constructs generated are listed in Table Table1.1. recA6 (tetM) is an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible allele of recA, and this construct has previously been described (22, 34, 45). All constructs were sequenced using an ABI BigDye Terminator v.3.1 DNA sequencing kit (Applied Biosystems) with primers 2488, 2487, S1, S2, and S3. The sequences of the DUS-containing inserts are given in Table Table33.

TABLE 1.
Plasmids and strains employed in the study
TABLE 2.
Primers employed in the studya
TABLE 3.
Sequence variations in the transforming/competing donor DNA containing different variants and numbers of DUS in this and previous studies

Quantitative transformation and DNA uptake.

The quantitative transformation assays were performed essentially as described previously (40) with 1 μg plasmid DNA in 50 μl 10 mM Tris buffer (pH 8) mixed with 0.5 ml piliated (6) cells (typically 20 colonies in 5 ml GC broth to an optical density at 600 nm of <0.1) supplemented with 7 mM MgCl2 (and 1 mM IPTG for strains M400 and N400) and incubated for 30 min in 5% CO2-saturated GC broth at 37°C. Meningococcal strain M400 and gonococcal strain N400 have RecA expression under the control of an IPTG-inducible tetM promoter, ensuring stable pilin expression without phase or antigenic variation (34). Following the incubations, the samples were diluted 10-fold in GC broth and grown for 4.5 h (15 to 24 h for gonococci in 20-fold GC dilutions) before appropriate dilutions were plated onto agar medium with and without 8 μg ml−1 erythromycin (Erm). Experiments were conducted both with and without DNase I treatment prior to dilution of the cell cultures. Transformation frequencies were determined by dividing the number of Erm-resistant CFU by the total CFU. Each experiment was repeated at least three times with donor DNA from different plasmid preparations (QIAGEN, Germany). The DNA concentrations were adjusted after quantification on an ND-1000 spectrophotometer (NanoDrop Technologies). DNA saturation experiments were performed with 0.2, 0.5, 1, 2, and 3.3 μg/ml final DNA concentrations of p12-DUS DNA to eliminate the possibility that the 2-μg/ml final concentration was an insufficient amount for complete saturation of the bacterial suspensions (data not shown).

DNA uptake was tested in meningococci essentially as described for gonococci (2). Plasmids p0-DUS, p10-DUS, and p12-DUS were linearized with restriction endonuclease XhoI (New England BioLabs), purified on a GFX mini column (GE Healthcare, United Kingdom), and treated with exonuclease III (Fermentas, Canada) before heat inactivation at 70°C for 20 min. The partly digested plasmids were purified as before, and the three eluates were adjusted to identical concentrations with water based on readings from an ND-1000 spectrophotometer (NanoDrop Technologies). The DNA fragments were labeled in a reaction with Klenow Exo (Fermentas, Canada) in the presence of 25 μM dGTP-dTTP-dATP and 1 μM [α-32P]dCTP (3,000 Ci/mmol; GE Healthcare, United Kingdom) and purified as previously described. The resulting DNA substrates had specific activities of 6 × 106 to 7 × 106 CPM μg−1. A fresh lawn of meningococci on GC plates (5 h, 37°C, 5% CO2) was resuspended in liquid GC medium supplemented with 7 mM MgCl2 and adjusted to an optical density at 660 nm of 0.2. 2 μg, and 32P-labeled DNA was mixed with 1 ml of cells, split into two 500-μl samples, and incubated for 30 min at 37°C. DNase I treatment, washing of cells, and scintillation counting were conducted as previously described (2).

DUS/USS in terminators as identified by GeSTer.

The GenBank files of the different genomes were scanned for the presence of transcriptional terminators with a genome scanner for terminators (GeSTer; available at ftp://ftp.bork.embl-heidelberg.de/pub/users/suyama) (46, 47). The parameters used were the following: minimum stem length of 4, maximum stem length of 30, minimum bulb length of 3, maximum bulb length of 9, maximum mismatches allowed (i.e., internal loops plus bulges) of 3, maximum distance from open reading frame of 270. The generated files contain putative L-shaped (with a U-trail) and I-shaped (without a U-trail) terminators with the lowest ΔG values and exclude convergent/bidirectional X-shaped terminators. The up stem, bulb, down stem, and trail sequences were concatenated in a spreadsheet and analyzed with respect to DUS/USS content.

RESULTS

Sequence conservation at positions −1 and −2 of the 10-mer DUS and the effect on transformation.

Based on our compilation of DNA sequence strings containing the neisserial DUS and surrounding sequences, the conservation in positions −1 (T) and −2 (A) flanking the 5′ end of the established 10-mer DUS was evident in all three species of Neisseria (Fig. (Fig.1).1). Irrespective of the total number of 10-mers in the different Neisseria species, approximately 76% of all DUS were 12-mers (Table (Table4).4). Importantly, the DUS presence and abundance as well as the conservation of the extended 12-mer DUS were shared by the genomes of all neisserial species investigated, including the commensal, N. lactamica.

FIG. 1.
Nucleotide conservation outside DUS in three species of Neisseria. The DUS of N. gonorrhoeae FA1090, N. lactamica ST640, and N. menigitidis Z2491, MC58, FAM18, and 8013 all show conservation of nucleotides T in position −1 and A in position −2. ...
TABLE 4.
Selected genome and DUS/USS characteristics

The constructed plasmids p8-DUS, p9-DUS, p10-DUS, p11-DUS, and p12-DUS differ only in the size of the DUS element they carry, ranging from a 5′-shortened 8-mer DUS up to a 5′-extended 12-mer DUS, respectively (Table (Table3).3). All constructs were tested for their ability to transform meningococcal and gonococcal cells, and the transformation frequencies (Tf) are shown in Fig. Fig.2.2. The constructs p11-DUS and p12-DUS transformed all neisserial strains at substantially higher rates than p10-DUS. N. meningitidis H44/76 and M400 and N. gonorrhoeae N400 were transformed at about threefold-higher rates with p12-DUS compared to p10-DUS. In N. meningitidis M400, exhibiting the highest Tfs overall, the average frequency achieved with p12-DUS was 29 × 10−6, compared to 9.8 × 10−6 obtained with p10-DUS. The enhanced effect of the 12-mer DUS upon transformation was even more pronounced in the meningococcal wild-type strain, MC58, where a more than 30-fold increase in transformation rate was observed with the 12-mer (Tf, 71 × 10−7) compared to the 10-mer DUS (Tf, 2 × 10−7). Strain MC58 was only barely transformed with the p10-DUS, an observation which questions the ability of the 10-mer to aid transformation in this widely used strain whose annotated genome sequence is available (42). The significance of the 12-mer preference seems variable (ranging from a 3-fold to a >30-fold increase) in the different strains tested, but this preference was notably present for each strain. The significant increase in transformation rates produced by both the 11- and 12-mer DUS, a tendency observed in all four strains of two different species, suggests that these two residues contribute to increasing the DUS binding affinity with the putative DUS-specific receptor.

FIG. 2.
Elongated DUS improves efficacy of transformation. The graphs show transformation frequencies of various DNA substrates on three different strains of Neisseria meningitidis and one strain of Neisseria gonorrhoeae. Transformation frequencies were calculated ...

All the strains tested gave rise to a few transformants with p0-DUS (in contrast to the negative controls without DNA), showing a minimal level of DUS-independent DNA uptake, as described for the gonococci (8). DUS-independent DNA uptake was, however, in general very sparse. Constructs p8-DUS and p9-DUS exhibited minimal transformation rates close to the DUS-independent rates of p0-DUS. The negative effect on the performance in transformation by the incomplete DUS constructs signified the importance of DUS integrity with regard to transformation, as has been observed previously in both Neisseria spp. (DUS) and in H. influenzae (USS) (18, 19, 24).

Preference for the 12-mer DUS was further tested by using radiolabeled DNA in DNA uptake assays (Fig. (Fig.3).3). We tested the parent strain of M400, N. meningitidis M1080, and a nontransformable negative control, the M1080 pilQ null mutant, for their ability to take up p0-DUS, p10-DUS, and p12-DUS into a DNase I- protected state (Fig. (Fig.3).3). The maximum uptake was found for N. meningitidis M1080 cells, with labeled DNA containing the 12-mer DUS. The uptake of the 12-mer construct was considerably higher than the uptake of p0-DUS and p10-DUS, confirming the high efficiency of the 12-mer DUS as shown in the transformation experiments. The differential uptake of the 10-mer and the 12-mer suggests that the qualitative selection of DNA happens in the initial steps of the transformation process.

FIG. 3.
Higher DNA uptake of p12-DUS relative to p10-DUS and p0-DUS in N. meningitidis M1080. The graph shows DNA uptake as a percentage of maximum DNA uptake in strain M1080 and the noncompetent negative control, M1080ΔpilQ. Values are means plus standard ...

Local arrangement of DUS and its effect on transformation.

An enhanced effect on transformation by the inverted repeat of DUS has been implied in previous reports (18, 23, 24). The effect of a single, direct, and inverted repeat DUS was investigated by constructing and applying the plasmids pSingle, pDR, and pIR, which only differ in the number and orientation of 12-mer DUS as shown in Table Table3.3. Their individual contributions to meningococcal transformation were tested in two different strains, and the results are shown in Fig. Fig.4.4. Surprisingly, having suspected a higher transformation rate for the construct with an inverted DUS pair as indicated in previous studies, all three plasmids performed equally well. These experiments suggest that the inverted arrangement of DUS, at least when localized with the most common distance of 5 nucleotides (36), does not contribute to elevated transformation rates in any of the strains tested. This further indicates that the linear relationship between the number of DUS and the probability of making a sequence-specific interaction, as it has been demonstrated (24), does not apply to two DUS appearing in close proximity of each other but rather when DUS occur singly and are dispersed over large fragments of DNA.

FIG. 4.
Plasmids pSingle, pDR, and pIR transform two strains of N. meningitidis equally efficiently. The graphs show transformation frequencies (10−7) of four different DNA substrates containing none (p0), a single (pSingle), a direct repeat (pDR), and ...

Presence of DUS and USS in predicted rho-independent transcriptional terminators.

The numbers of DUS and USS and predicted terminators in five genomes of the Neisseria spp. and two Pasteurellaceae family members are listed in Tables Tables44 and and5.5. We found that out of the approximately 950 predicted rho-independent terminators in the Neisseria spp., nearly 400 contained an inverted repeat of DUS and approximately 130 contained a single DUS, and these values are only marginally and proportionally lower when assessing the 12-mer DUS. As for the inverted pairs of DUS (IR-DUS), manual inspection of the approximately 130 terminator sequences containing the single DUS revealed that they almost exclusively constituted parts of the stem in the stem-loop structure and paired with a degenerate DUS (allowing one or more mismatches in the DUS). As with the genomic 10-mer DUS distribution, showing a global chromosome coverage without a strand bias or particular clustering with respect to the origin of replication (32, 36, 42), the 12-mer and the IR-DUS colocalize with the 10-mer in this global spread. Furthermore, these findings showed that nearly 50% of all DUS in the Neisseria spp. are arranged as inverted repeats and that nearly all of these (>90%) can be predicted to constitute parts of transcriptional terminators. These results contrasted with the commonly held viewpoint that a quarter of all neisserial IRs are located entirely inside genes (36), an adjustment only made possible with the new genome sequences and annotations, particularly those annotations involving new start site identifications, in combination with the applied terminator-predicting algorithm. These results suggested that Neisseria spp. have adopted DUS-containing stem-loop structures for at least some level of transcriptional termination of more than 25% of the total number of genes in the genome. Only around 30% of USS are located intergenically in H. influenzae and P. multocida, as opposed to more than 60% of neisserial DUS having an intergenic location, and 13% and 21% of the USS in H. influenzae and P. multocida, respectively, were identified inside predicted terminators (Table (Table4)4) (13). Cooccurrence is consequently less pronounced in the Pasteurellaceae than in Neisseria, although there is a clear bias also in these species.

TABLE 5.
Predicted rho-independent terminators and colocalization with the DUS or USS in the genomes of Neisseria and Pasteurellaceae family members, respectively

Notably, when assessing DUS-IRs that are located entirely inside CDS, we identified only four DUS-IRs confidently located inside CDS by manual inspection of the MC58 genome sequence. In this context, this includes IRs with a spacer less than 20 nucleotides that are located completely inside a nonhypothetical CDS (excluding two open reading frames with no putative conserved domains) and not overlapping start or stop codons. The four CDS containing an IR encode proteins that are all involved in genome maintenance: MutY (NMB1396), exodeoxyribonuclease V 135-kDa polypeptide (NMB0785), DNA ligase (NMB0666), and DNA polymerase III γ/τ subunits (NMB1443). We do not exclude the possibility that these IRs might have a regulatory function, perhaps by allowing partial read-through or attenuation under certain conditions, for instance, stress, although this remains to be investigated. We have previously documented a bias of DUS inside CDS towards genome maintenance genes and proposed a role in genome stability by means of facilitated genetic flux and restoration of damaged genes containing DUS (13). We further identified the number of DUS/USS-like sequences with a single mismatch in any position as previously described (13) (Table (Table4).4). For the yet-unpublished or incompletely annotated sequences from N. meningitidis 8013 and FAM18 and N. lactamica ST-640, we found the number of DUS-like sequences (allowing one degenerate position) to be similar to the expected values we previously have predicted by the Markov model (Table (Table4)4) (13). The wide distribution of DUS in the Neisseria spp., combined with phylogenetic data based on 16S rRNA gene sequences (43), suggests that a whole range of Neisseriaceae family members share common ancestry with N. lactamica and N. meningitidis and contain an overrepresentation of DUS.

DISCUSSION

The characterization of unique genomic signatures and frequently occurring repeat sequences can provide novel insight into basic functions and evolutionary processes. The association between the DUS and transformation was investigated in two pathogenic representatives of Neisseria spp. A range of plasmids with sequential and locational variants of DUS were employed as tools to specify the DUS requirements for neisserial transformation. Thereby, the relative contribution of individual nucleotide residues comprising DUS at the 5′ end, the DUS length, and the local arrangement of DUS in transformation were defined. This is the first report defining the functional role of the 12-mer DUS, which may contribute to elucidation of the enigma of the transformation process. This report further ruled out an inferred influence on transformation by the inverted DUS pair in assays also assessing the influence of the 12-mer DUS. The results may also prove to be an important tool in the ongoing search for DUS-specific DNA-binding partners among neisserial components where binding affinity is a pertinent issue. Finally, these data will be helpful in facilitating the execution of any experiment involving neisserial transformation.

The finding that the 12-mer DUS enhanced transformation rates in all gonococcal and meningococcal strains tested encourages reconsideration of the DUS identity per se. Our data suggest that the 10-mer DUS compared to the 12-mer would interact more weakly in DUS affinity studies. A longer recognition signal will also increase specificity and will, as a consequence, facilitate the discrimination of foreign DNA entering the neisserial cell. A high discrimination threshold towards foreign DNA may contribute to the stabilization of a highly dynamic genome and maintenance of species integrity. In particular, the explicit 12-mer DUS preference demonstrated for MC58, which displayed an extreme transformation enhancement when exposed to the 12-mer DUS and a near lack of affinity for the 10-mer, encourages caution when selecting strains for investigations of transformation and perhaps as sources of proteins for DNA binding experiments. We suggest that the established and commonly applied 10-mer DUS should be replaced by the 12-mer DUS when possible for synthetically produced fragments for use in transformation, at least if optimal transformation rates are desirable.

Although it has been demonstrated that a single DUS is sufficient for DNA uptake in Neisseria (18, 24), most workers have included an inverted pair of DUS in constructs made for transformation experiments. We wanted to test in a clear-cut assay if paired inverted DUS targets were more efficient in transformation than a single or tandem repeat DUS, as has previously been suggested (24). We found no enhanced effect of the construct containing the inverted pair of DUS compared to the direct repeat and the single target constructs. Our assays have also considered the impact of the 12-mer DUS. Previous studies compared the affinities of several plasmids containing a range of different 10-, 11-, and 12-mers, and these findings are likely to explain the observed differences (Table (Table3).3). In the studies of Goodman and Scocca (23, 24) and Elkins et al. (18), the best-performing plasmids (pGCU1 and derivatives and pUP1, respectively) consistently, and probably exclusively, contained the 12-mer DUS, whereas in plasmids with which they were compared, the DUS identity was either unknown or contained less than 12 residues (Table (Table3).3). The best performing plasmids also contain the inverted DUS pair and enlighten how the uncertainty concerning the influence of the inverted DUS repeat in transformation came to appear. Both of the previous studies did, however, clearly demonstrate that a single DUS is sufficient for efficient uptake, and the unresolved matter was concerned solely with the suspected additional contribution of an inverted complement DUS copy in the near vicinity of DUS.

The transformation rates in this study were often lower than those observed in previous studies conducted with chromosomal DNA (7, 29, 38) or other plasmid DNA templates (1, 18, 45). Provided that the recipient strain is piliated, as they have been in our assays, one possible explanation of this result is that transformability may vary with the sequence identity, lengths, and/or ratios between homologous and heterologous sequences in the donor DNA. Our system measured the relative transforming activities of plasmids differing only in their DUS characteristics at a single site (in position 641 or 808 downstream from the start codon) inside an interrupted (mTnErm in position 888 downstream from the start codon) pilG gene of approximately 1.2 kb in length which originally contained no DUS. The pSY6 plasmid DNA (39), in comparison, contains a large 10-kb PstI fragment of gonococcal origin and, due to its agility in transformation of the gonococcus, has been adopted by several research groups as a standard test of competence. pSY6 contains an inverted repeat of DUS composed of one 12-mer and one 10-mer. For plasmids as donor DNAs in transformation, the lengths of homologous stretches of DNA in both the donor plasmid and the receiving chromosome would have an effect on transformation rates (14, 18). The assays in this study were designed with the aim of discriminating between differing DNA species with subtle DUS-related differences and not to construct vectors with the aim of maximizing transformation frequencies per se.

Most neisserial DUS are located between genes (65%), and a striking cooccurrence of DUS and predicted terminators in Neisseria sp. was found. The extent of this locational bias is much higher than has been previously estimated. Smith and coworkers (36) stated that a quarter of DUS-IRs are located entirely within predicted CDS, a result we failed to confirm, possibly due to the new annotation of the Z2491 genome (dated 2001/09/27). Importantly, we found that more than 90% of all inverted DUS repeats with a spacer smaller than or equal to 20 nucleotides are identified as potential terminators in all the neisserial genome sequences. The GeSTer algorithm may identify terminators residing partly inside annotated CDS, but only if at least 50% of the structure lies in the noncoding region, i.e., downstream of the stop codon and not overlapping into another gene. This locational bias downstream of genes was also present in the genomes of H. influenzae and P. multocida, with 13% and 21% of the USS identified inside predicted terminators. The frequent intergenic location of DUS and USS may reflect the cost of harboring such elements inside CDS. The reading frames in which the intragenic DUS appear are indeed weighted toward common relatively neutral amino acids, and the least-represented reading frames encode basic or aromatic amino acids, cysteine or, in one case, a stop codon (36). It is tempting to speculate that single DUS frequently found an abundant intergenic location early in evolution due to this constraint. Ultimately, the appearance of DUS itself may have been driven by the advantage of selective DNA rescue from the extracellular environment (16). Furthermore, it has been proposed that the mechanism behind the appearance of USS (and supposedly DUS) is mutation (33). It could then be envisaged that the inverted DUS pair would occur secondary to this event, also by mutation, driven by another selective advantage, namely, that of employing transcriptional control. The presence of DUS/USS as inverted repeats intergenically in the phylogenetically distant groups of Neisseria and Pasteurellaceae is the likely result of convergent evolution of parallel systems and, consequently, suggests that similar selective pressures have been exerted.

The efficacy of a given terminator may be influenced by the folding energy of the stem and the identity of the poly(U) trail known to follow the stem-loop structure of transcriptional terminators (9). The GeSTer algorithm identifies both terminators as adopting an L-shape, i.e., containing a poly(U) trail following the stem-loop structure, and those with an I-shape without the poly(U) trail (46). Unniraman and coworkers argue that the significance of the U trail, at least in E. coli, still remains unclear and points to the thr (30) and crp (3) terminators with demonstrable contradistinct dependency of the U-run in transcriptional termination. Compared to E. coli, much less is known about the modes and specificities of transcription terminators in Neisseria. However, intrinsic transcriptional termination by means of an inverted DUS repeat was first demonstrated for the groESL operon in N. gonorrhoeae (41), and this terminator was correctly identified here. Furthermore, the involvement of DUS repeats in transcription termination has been exemplified in the dcw cluster in the gonococcal genome (20, 37). Francis and coworkers (20) found five dcw terminators and assessed their functionality by reverse transcription-PCR; DUS was found to constitute part of the stem in four of these terminators. These structures were also identified in our genome scans. However, a follow-up study by Snyder and coworkers (37) produced and detected with higher specificity a reverse transcription-PCR product spanning one of the same intergenic regions in the dcw cluster containing an IR-DUS (located in the region between mraY and dcaC) and showing read-through, which consequently questions the previous findings. In the N. gonorrhoeae FA1090 genome sequence, we found by inspecting the dcw cluster that three of the DUS-containing terminators may form L-shaped terminators, whereas the terminator whose efficacy was disputed in the region between mraY and dcaC was predicted to be of the I shape, without a convincing U trail. Perhaps the inverted DUS pair, or indeed any inverted repeat, in some instances may act as a partial terminator, allowing variable levels of read-through and resulting in the synthesis of fragments of different lengths. This may also influence expression levels of various proteins and provide a means of fine tuning such. Indeed, a recent report by Gunesekere and coworkers suggests that an inverted repeat sequence inside a gene cluster in N. gonorrhoeae allows partial read-through, resulting in two transcripts of different lengths (26). Although this particular inverted repeat found downstream of the gene of NGO1947 does not contain a DUS, it is comparable in length and has an identical G+C content to a DUS-IR (up-stem sequence, 5′-AAGCCGCGUUUU-3′). The increasing amount of microarray data and transcriptional analyses that are becoming available may shed further light on this and may contribute to our understanding of the mechanisms affecting transcriptional termination and the specific requirements of the stem-loop structure and the trail sequence.

The large numbers of these highly similar DUS-containing structures, their common location downstream of genes, and the demonstrated lack of effect of the inverted repeat on transformation compared to the single DUS target firmly suggest that DUS containing inverted repeats are involved in at least some level of transcriptional termination.

Acknowledgments

We gratefully acknowledge Eduardo P. Rocha for customized scripts, assembly of GenBank files used in terminator predictions, and for helpful discussions. The preliminary genome sequence of N. lactamica ST-640 is presented here with kind permission from J. Parkhill, Sanger Institute Wellcome Trust Genome Campus, Hinxton, United Kingdom. The genome sequence of N. meningitidis 8013 was kindly provided by E. P. Rocha and Vladimir Pelicic. We are grateful to Tonje Davidsen and E. P. Rocha for critical reading of the manuscript and Einar A. Rødland for helpful discussions on the statistical analysis.

This work was financed by FUGE/CAMST funding provided by the Research Council of Norway and EMBIO, University of Oslo.

Footnotes

[down-pointing small open triangle]Published ahead of print on 28 December 2006.

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