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J Bacteriol. Dec 2006; 188(23): 8307–8312.
Published online Sep 22, 2006. doi:  10.1128/JB.00850-06
PMCID: PMC1698192

BOX Elements Modulate Gene Expression in Streptococcus pneumoniae: Impact on the Fine-Tuning of Competence Development[down-pointing small open triangle]

Abstract

More than 100 BOX elements are randomly distributed in intergenic regions of the pneumococcal genome. Here we demonstrate that these elements can affect expression of neighboring genes and present evidence that they are mobile. Together, our findings show that BOX elements enhance genetic diversity and genomic plasticity in Streptococcus pneumoniae.

The BOX elements of Streptococcus pneumoniae are short repeated sequences whose function and origin are unknown. They were first discovered more than a decade ago in the vicinity of genes involved in virulence and natural competence, and it was therefore speculated that BOX elements could be involved in regulating the expression of these genes (13). A total of 127 BOX elements are randomly distributed in intergenic regions of the TIGR4 strain (15, 20), whereas 115 elements are present in strain R6 (7). Most of these elements consist of three different modules (boxA, boxB, and boxC), and boxB (45 bp) is flanked by boxA (59 bp) and boxC (50 bp) (13). Most often, one to four copies of boxB are located between boxA and boxC, but occasionally up to eight copies have been found. Some BOX elements, however, consist of only boxA and boxC or, in a few cases, of single or tandem boxB sequences. The mechanism behind this heterogeneity and the possible biological significance of it remain to be elucidated. BOX elements containing a boxA module and a boxC module have the potential to form a stable stem-loop structure. This structure appears to be functionally important as compensating base changes have been observed in several cases where the sequences of boxA-boxC pairs differ from the consensus sequence (13). The presence of such stem-loop structures in transcribed regions could modulate the expression of neighboring genes. We therefore decided to investigate whether the BOX elements associated with the early competence (com) genes qsrAB and comAB affect their level of expression. Competence for genetic transformation in S. pneumoniae is regulated by competence-stimulating peptide (CSP) (2, 5), a secreted peptide pheromone, through a signal transduction pathway consisting of the histidine kinase ComD and the cognate response regulator ComE (6, 17). When the external concentration of CSP in a pneumococcal culture reaches about 10 ng/ml, early and late com genes are expressed, resulting in development of competence (5). The early com genes are regulated by ComE, which initiates transcription from promoters (PE) containing a conserved direct-repeat motif (21). The comAB genes encode an ABC transporter (ComA) and its accessory protein (ComB), which together constitute the CSP secretion apparatus (8), whereas the qsrAB genes encode an ABC transporter having an unknown function.

The BOX elements associated with the qsrAB and comAB genes are located in the promoter regions of these operons, between PE and the first downstream gene (Fig. 1A and B), and they are therefore present at the 5′ end of transcripts originating from PE. We have previously shown that expression of qsrAB, which in noncompetent cells depends on a housekeeping σA promoter (PA) situated between boxC and the qsrA gene (Fig. 1A and B), increases about twofold when competence is induced (10, 21). Interestingly, insertion of a lacZ reporter gene immediately downstream of PE strongly impaired CSP-induced expression of the reporter compared with a location of lacZ in the qsrB gene (10). This observation suggested that the presence of a BOX element at the 5′ end of transcripts somehow enhances expression of downstream genes. To further explore this possibility, we reintroduced boxAB2C at its original position between PE and lacZ. This was done by using PCR to specifically amplify the qsr boxAB2C element with primers containing BamHI sites at their 5′ ends. Subsequently, the PCR fragment was ligated in either orientation into plasmid pOE4144 (10) at a unique BamHI site located exactly at the original position of the qsr boxAB2C (see Tables S1 and S2 in the supplemental material). Both constructs were transformed into strain EK100 and integrated into its genome by single-crossover insertion-duplication at the homologous target site upstream of boxAB2C (see the supplemental material for a more detailed description). The resulting strains, OE4144-AB2C and OE4144-CB2A, were assayed for CSP-induced production of β-galactosidase (β-Gal) as described previously (10). Strain OE4144-AB2C exhibited a fourfold increase in β-Gal activity compared to OE4144, demonstrating that the presence of the BOX element had a strong positive effect on the expression of lacZ (Fig. (Fig.2).2). The level of CSP-induced expression of lacZ was even higher with the inverted BOX element (strain OE4144-CB2A), indicating that the proposed secondary structure rather than the orientation of the BOX element is important. We then investigated the effects of different combinations and orientations of the boxA, boxB, and boxC modules (Fig. (Fig.2).2). The constructs shown in Fig. Fig.22 were made by using plasmid pCR2.1-TOPO::boxAB2C as the template in PCRs with specific primer pairs annealing to regions flanking the box modules to be deleted (see Table S2 in the supplemental material). As the primers were complementary to opposite strands and contained unique restriction sites at their 5′ ends, copies of pCR2.1-TOPO::boxAB2C containing different permutations of the box modules were created. The various box combinations were excised from their pCR2.1-TOPO plasmids with BamHI and ligated in either orientation into the corresponding site downstream the PE promoter in pOE4144. The resulting constructs were introduced into the genome of EK100 as described above (see the supplemental material for details). The constructs lacking boxB modules, OE4144-AC and OE4144-CA, both exhibited a fourfold increase in lacZ expression compared to OE4144 (Fig. (Fig.2),2), demonstrating that the presence of the boxA-boxC combination alone is sufficient to stimulate expression of downstream genes and that boxAC works equally well in both orientations. This finding supports the idea that the stimulatory effect of boxAB2C is due mainly to the formation of a stem-loop structure in the qsrAB transcript. The level of expression of lacZ was significantly higher in mutant OE4144-CB2A than in mutant OE4144-AB2C, indicating that the boxB modules might have an inhibitory effect that depends on their orientation. The properties of a series of mutants having various orientations and numbers of boxB modules support this view. The level of expression of the reporter gene in the OE4144-B2 inverted mutant, which contained two boxB modules in the inverted orientation, was not significantly different from the level of expression in the strain lacking the boxAB2C element (OE4144). In contrast, in mutants OE4144-B2, OE4144-AB2, and OE4144-B2C, in which the boxB modules were incorporated in the forward orientation, there was a clear reduction in lacZ expression compared to the expression in the OE4144 parental strain (Fig. (Fig.2).2). The number of boxB modules inserted in tandem between boxA and boxC in BOX elements dispersed throughout the genome was found to range from zero to eight. To determine whether the inhibitory effect observed with forward-oriented boxB modules increased with a larger number of tandem repeats, we constructed mutants OE4144-AB7C and OE4144-CB7A containing seven boxB modules in the forward and inverted orientations, respectively. This was done by amplifying a boxAB7C motif located upstream of the spr1604 gene by PCR, ligating it in either orientation into the BamHI site of pOE4144, and transforming the resulting constructs into the EK100 strain as described above. Our results showed that the level of β-Gal produced was reduced more than threefold in the mutant containing boxAB7C, strongly indicating that forward-oriented boxB modules residing in the 5′ end of transcripts downregulate expression of cotranscribed genes. In accordance with this finding, a comparison of mutants OE4144-AB2C and OE4144-AB7C showed that considerably less β-Gal was produced in the mutant containing the highest number of boxB modules (Fig. (Fig.2).2). Together, these data indicate that forward-oriented boxB modules have an inhibitory effect on the expression of downstream genes and that the level of inhibition increases with the number of tandemly repeated boxB units. Inverted boxB modules, on the other hand, apparently had no such effect.

FIG. 1.
Map of early competence genes qsrAB and comAB (A) and their promoter regions (B). The CSP-inducible promoter, PE, consists of two binding sites for ComE, designated DR1 and DR2, and the cognate −10 hexamer. PA is a strong constitutive housekeeping ...
FIG. 2.
Effects of the 5′-proximal qsr BOX element and various derivatives of this element on the level of expression of a cotranscribed lacZ reporter gene. The complete boxAB2C element and downstream PA promoter were removed in the OE4144 parental strain, ...

To determine whether the boxABC element residing in the comAB promoter (Fig. (Fig.1)1) affected the expression level of these genes, a mutant strain lacking this element was constructed. In brief, a PCR fragment generated by amplifying a 1,302-bp fragment encompassing the 5′ end of comA, the comAB promoter, and its upstream region was ligated into pLitmus 28 (New England Biolabs), producing recombinant plasmid pLicom1. boxABC was removed from pLicom1 by the PCR procedure described previously using primers flanking the boxABC element (see Table S2 in the supplemental material). The resulting plasmid, pLicom2, and parental plasmid pLicom1 were digested with HindIII and BamHI to excise the cloned inserts. Then the excised fragments were ligated into the corresponding restriction sites upstream of the promoterless lacZ gene of the nonreplicating pEVP3 vector (see Table S1 in the supplemental material). Finally, the constructs were introduced into the genome of the EK100 strain by natural transformation, followed by single-crossover homologous recombination (see the supplemental material for details). Deletion of the boxABC element (strain OE4151) resulted in a modest but significant reduction (26%) in CSP-induced β-Gal production compared to the production in the strain in which the lacZ reporter gene was placed behind the wild-type comAB promoter (strain OE4150) (data not shown). Even though the reduction was small compared to that observed for the qsrAB locus, we thought that it was worthwhile to investigate whether deletion of the comAB BOX element affected spontaneous competence development. Since it has been demonstrated previously that the CSP export capacity of wild-type cells is rate limiting for competence development (14), we hypothesized that competence would be delayed (i.e., would develop at a higher cell density) in cultures of the mutant lacking a BOX element compared to that in the wild-type parent. To check this hypothesis, we compared spontaneous competence induction in strains OE4171 (comAB BOX element deleted), OE4170 (identical to OE4171 but BOX element present), and OE4180 (wild-type comAB locus) by monitoring luciferase activity from a transcriptional fusion between the luc gene and the late com gene ssbB. To generate the OE4170 and OE4171 strains, we first constructed strains OE4061 (comAB BOX element deleted) and OE4060 (identical to OE4061 but comAB BOX element intact) by using insertion-duplication mutagenesis. The procedure used was essentially the same procedure that was used for construction of strains OE4150 and OE4151 (see above), except that a 1-kb PCR fragment containing the complete comAB promoter and the 5′ half of the comA gene was used as a starting point. The luc reporter gene was inserted behind the ssbB promoter by transforming OE4160, OE4161, and CP1200 with a derivative (pR459) of plasmid pR424 (1), resulting in strains OE4170, OE4171, and OE4180, respectively (see the supplemental material for details). Luciferase activity was detected as described previously (1, 18). The results showed that the OE4171 mutant strain developed competence at a higher cell density than the positive controls developed competence (Fig. (Fig.3),3), strongly suggesting that the BOX element plays an important role in the fine-tuning of development of spontaneous competence in S. pneumoniae.

FIG. 3.
Comparison of spontaneous competence development in pneumococcal strains containing (OE4170) or lacking (OE4171) a BOX element in the comAB promoter region. The OE4180 parental strain, which contains a wild-type comAB locus with an intact boxABC element, ...

Altogether, the data described above clearly show that BOX elements situated at the 5′ end of mRNA transcripts have the potential to change the expression pattern of operons (e.g., qsrAB) and even entire regulons (the competence regulon in the case of comAB). It has been firmly established for both gram-negative and gram-positive bacteria that the 5′ end of transcripts is a major determinant of mRNA stability and that mRNAs are stabilized by 5′-proximal stem-loop structures (3, 4, 9, 19). It is therefore highly plausible that the 5′-proximal BOX elements studied here stimulate expression of downstream genes by increasing the half-lives of their mRNAs. As exemplified by the qsrAB operon, BOX elements do not need to be located immediately upstream of an open reading frame in order to be part of the cognate mRNA transcript. It is therefore difficult to estimate the prevalence of pneumococcal mRNA transcripts with BOX elements in their 5′ ends. However, it is unlikely that the qsrAB and comAB mRNAs are unique. A comparison of 13 loci in the genomes of four different pneumococcal strains and Streptococcus mitis NCTC12261 demonstrated that BOX elements are only partially conserved at each specific site (Table (Table1).1). A typical example is the inverted boxABC element located 50 bp downstream of the SP0095 gene. This element is conserved in three pneumococcal strains but is missing in S. pneumoniae R6 and the S. mitis NCTC12261 strain. This implies that the BOX element was deleted from the R6 and S. mitis strains or that it was recently inserted into the genome of the common ancestor of the TIGR4, 670, and 23F strains. Interestingly, the boxABC element located upstream of the comA gene is conserved in all four pneumococcal strains, as well as in S. mitis. It is tempting to speculate that its stability is due to increased fitness of the cells carrying it and therefore that highly conserved BOX elements have acquired a function giving the host cell a selective advantage. In the examples listed in Table Table1,1, all boxB modules are flanked by boxA and boxC modules. Curiously, the number of boxB modules sandwiched between boxA and boxC modules varies for different BOX elements in the genome of a particular strain and also for corresponding elements in different strains (Table (Table1).1). This variation in the number of sandwiched boxB modules might not necessarily result from mobilization of boxB modules but could be generated by a gene conversion mechanism (e.g., through homology-dependent repair of a damaged boxB motif using intact tandemly repeated boxB modules as the template). However, the presence of elements consisting of only single or tandem boxB modules (data not shown) suggests that the boxB module is able to move independent of the boxA and boxC modules.

TABLE 1.
Comparison of BOX elements at 13 different loci

The availability of BOX element-free and BOX element-occupied sites (Table (Table1)1) enabled us to compare sequences flanking BOX element insertion sites with the aim of identifying possible nucleotide duplications. Duplications are very frequently generated during transposition (12). A dinucleotide (TA) was previously detected at the borders of another repeated element of S. pneumoniae, RUP (16). This dinucleotide was suggested to result from duplication of the target generated by the transposase of IS630-Spn1, an insertion sequence (IS) belonging to the IS630 family identified in the pneumococcal genome and proposed to be responsible for formation and mobilization-amplification of RUP elements (16). Interestingly, comparison of BOX element-free and BOX element-occupied sites revealed the presence of a single additional nucleotide (A, T, or C) in every case examined (Fig. (Fig.4A).4A). It is noteworthy that the same nucleotide flanks the other extremity of the BOX element (Fig. (Fig.4A),4A), strongly suggesting that BOX element insertion generates a single-nucleotide duplication. This duplication event readily accounts for the previous observation that identical nucleotide pairs (either AA or TT) were present at the base of the predicted stem-loop structure of a BOX element (13). The detection of a nucleotide duplication accompanying BOX element insertion led us to hypothesize that a BOX element could be derived from a mobile genetic element, as previously proposed for RUP (16). We therefore searched the pneumococcal genome for a candidate IS with inverted terminal repeats (IRs) homologous to BOX element extremities. We identified a new putative IS of S. pneumoniae, ISSpn2 (Fig. (Fig.4B).4B). ISSpn2 is a 1,091-bp element (which has been deposited in the ISFinder database [http://www-is.biotoul.fr]) which harbors potential IRs (Fig. (Fig.4B)4B) and has a calculated G+C content close to that of S. pneumoniae (37.7%). It appears to contain two potential open reading frames, orf1 and orf2 (Fig. (Fig.4B).4B). A BLASTN search of streptococcal genomes carried out with the ISSpn2 sequence revealed the presence of an iso-IS only in Streptococcus sobrinus (70% identity in the last 825 nucleotides). This 1,095-bp element (G+C content, 39%), designated ISStso1, is organized like ISSpn2. A BLASTP search carried out with an ISStso1 orf1-orf2 (arbitrary) fusion product revealed that the protein belongs to the IS630 family of transposases, and the closest homologue is the transposase of ISC1048 from Sulfolobus solfataricus P2 (data not shown). A transposase sequence similarly assembled from ISSpn2 orf1-orf2 sequences exhibited similar clustering (~28% identity between ISSpn2 and ISC1048 transposases [see Fig. S1 in the supplemental material]). The comparison of the IRs of ISSpn2 with BOX element extremities revealed significant homology only between the left inverted repeat and boxC (Fig. (Fig.4C).4C). However, significant homology was detected between both IRs of ISStso1 and boxA and boxB extremities (Fig. (Fig.4C).4C). Interestingly, the level of identity between both BOX element extremities and the IRs of ISStso1 was very similar to the level of identity between the left and right inverted repeats of the IS. As these IRs are presumably both recognized by the putative ISStso1 transposase, this finding strongly suggests that the transposase readily recognizes the termini of the BOX element as well. These observations led us to propose that the BOX element could be transactivated by the ISStso1 transposase and possibly also by ISSpn2, similar to the RUP elements in S. pneumoniae (16) and the Correia elements in Neisseria (11). We propose that through their mobilization BOX elements have contributed to shaping the genomes and transcriptomes of S. pneumoniae and S. mitis and could even have played a role in the evolution of S. pneumoniae as a human pathogen.

FIG. 4.
(A) BOX element insertion generates a single-nucleotide duplication. Comparison of chromosomal sequences flanking BOX elements in the TIGR4 genome (open reading frames identified by designations beginning with SP) with the corresponding BOX element-free ...

Acknowledgments

This work was supported by grants from the Research Council of Norway.

Footnotes

[down-pointing small open triangle]Published ahead of print on 22 September 2006.

Supplemental material for this article may be found at http://jb.asm.org/.

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