Positive and negative cis-acting regulatory sequences control expression of leukotoxin in Actinobacillus actinomycetemcomitans 652

Abstract

Integration of IS1301 into an AT-rich inverted repeat located upstream of the ltx operon was previously shown to confer a hyperleukotoxic phenotype in Actinobacillus actinomycetemcomitans IS1 (T. He, T. Nishihara, D. R. Demuth, and I. Ishikawa, J. Periodontol. 70:1261-1268, 1999), but the mechanism leading to increased leukotoxin production was not determined. We show that an IS1 ltx promoter::lacZ reporter construct expresses 12-fold higher levels of beta-galactosidase activity than a reporter containing the ltx promoter from A. actinomycetemcomitans 652, suggesting that IS1301 increases transcription of the ltx operon. Examination of the IS1301 sequence identified a potential outwardly directed promoter. However, site-specific mutagenesis of the -35 element of the putative promoter had no effect on the transcriptional activity of the IS1 reporter construct. Furthermore, reverse transcriptase PCR and real-time PCR experiments did not detect a transcript that was initiated within IS1301. These results suggest that increased expression of leukotoxin in strain IS1 does not arise from an outwardly directed IS1301 promoter. To determine how IS1301 alters transcriptional regulation of the ltx operon, cis-acting sequences that regulate leukotoxin expression were identified. The AT-rich sequence that resides downstream from the site of IS1301 insertion was shown to function as a positive cis-acting regulator of leukotoxin expression. This sequence resembles an UP element in its location, AT-rich content, and activity and is homologous to the consensus UP element sequence. In addition, a negative cis-acting sequence was identified upstream from the site of IS1301 insertion, and deletion of this region increased promoter activity by fourfold. Mobility shift experiments showed that this region bound to a protein(s) in extracts from A. actinomycetemcomitans 652. The specific sequences required for this interaction were localized to a 26-nucleotide segment of the ltx promoter that resides 17 bp upstream from the site of IS1301 insertion. Together, these results suggest that positive and negative cis-acting sequences regulate leukotoxin expression and that IS1301 may increase transcription of the ltx operon in A. actinomycetemcomitans IS1 by displacing a negative cis-acting regulator approximately 900 bp upstream from the basal elements of the ltx promoter.

FIG. 1.

IS1301 increases transcription of the ltx operon. (A) Schematic representation of the A. actinomycetemcomitans 652 and A. actinomycetemcomitans IS1 leukotoxin operons and upstream sequences. ltxA encodes the toxin polypeptide, and ltxC, ltxB, and ltxD encode toxin-activating and secretion proteins. orfA represents an open reading frame encoding a hypothetical protein of unknown function that is cotranscribed with ltxCABD. The upstream gene, glyA, encodes serine hydroxymethyltransferase and is not a part of the leukotoxin transcriptional unit. The locations of an AT-rich imperfect IR and −10 and −35 promoter elements of the orfA promoter (P_orf) are shown. In A. actinomycetemcomitans IS1, a copy of IS1301 is integrated into the IR sequence. IS1301 carries a single gene, trnA, encoding a transposase (15). The arrows represent the annealing sites for oligonucleotide primers 16 to 20 used for RT-PCR and real-time PCRs. (B) Transcriptional activity of ltx promoter::lacZ reporter constructs. The glyA-ltxC intergenic regions of A. actinomycetemcomitans 652 and A. actinomycetemcomitans IS1 were fused to lacZ to generate constructs 652lacZ and ISlacZ, respectively. With the exception of the IS1301 sequence, the nucleotide sequences of the IS1 and 652 promoter fragments are >99% identical. For each construct, β-galactosidase activity is shown on the right and was measured from cell extracts obtained from the recombinant strains by using ONPG as the substrate. (C) Real-time PCR results obtained by using total RNA from A. actinomycetemcomitans strains 652 and IS1. Reactions were carried out as described in Materials and Methods by using the primer pairs indicated. The cytolethal distending toxin B gene (cdtB) is transcribed independently of the ltx operon and was used in a control reaction in these experiments.

FIG. 2.

IS1301 does not possess an outwardly directed promoter. (A) The nucleotide sequence from the stop codon of trnA to the transcriptional initiation site of P_orf is shown. The bold italic sequence is the terminal repeat of IS1301. The terminal repeat contains a putative −35 element (TTGTAC; underlined), indicated by the larger font, that is 18 nucleotides upstream from a potential −10 sequence (AATAAT; underlined). The −35 and −10 promoter elements of P_orf are also labeled and underlined. The nucleotides that were substituted by site-specific mutagenesis are shown above the sequence. (B) Activities of the IS1 promoter::lacZ reporter construct (IS1) and the construct carrying site-specific mutations (−35M) were determined as described in Materials and Methods. The activity of the unaltered IS1 reporter was normalized to 1.0. (C) Results of RT-PCR with total IS1 RNA. The annealing sites for the primers used in the RT-PCR for each lane are shown in Fig. 1A. Lane 1, primers 16 and 17; lane 2, primers 16 and 20; lane 3, primers 19 and 17; lane 4, primers 18 and 17; lane 5, no-RT control with primers 16 and 17; lane 6, 100-bp DNA size standard ladder.

FIG. 3.

(A) Identification of cis-acting sequences that influence ltx operon expression in A. actinomycetemcomitans 652. The control construct, 652lacZ, contains the entire glyA-ltxC intergenic region fused to lacZ. Constructs IRL and IR20 were derived from 652lacZ and contain 20 random nucleotides separating the two segments of the IR and a deletion of the upstream segment of the IR, respectively. Deletion construct 229lacZ lacks the AT-rich IR and all additional sequences that reside upstream from the −35 element of P_orf; construct 191lacZ contains the AT-rich sequence but lacks all other upstream sequences; construct 197lacZ contains only the portion of IR that resides downstream from the site of IS1301 insertion (see the text). The negative control, YGKlacZ, contains only a promoterless lacZ in the shuttle vector pYGK. For each construct, β-galactosidase activity is shown on the right and was measured from cell extracts obtained from the recombinant strains by using ONPG as the substrate. (B) The AT-rich IR sequence resembles an UP element. The upper line represents the consensus UP element sequence (9), where W is A or T and N is any nucleotide; the lower sequence is the AT-rich IR element of the ltx promoter from A. actinomycetemcomitans 652 (Aa652). Homologous residues are indicated by colons; nucleotides that are analogous to the nonspecific residues in the consensus are indicated with X.

FIG. 4.

(A) Schematic representation of the glyA-orfA intergenic region showing the probes used for mobility shift experiments. Probe B101IR contains the entire AT-rich IR sequence and extends from nucleotides −39 to −166, where +1 is the transcriptional initiation site of P_orf. Probe B101 lacks the IR region and extends from −77 to −166. Probe B180 extends from −87 to −218, and probe B154 extends from −111 to −218. (B and C) For mobility shift experiments, 2 ng of biotinylated ltx promoter fragments B101 (B) and B101IR (C) was incubated with 7.5 μg of a protein extract derived from A. actinomycetemcomitans 652 and electrophoresed in 5% polyacrylamide gels. Competition experiments were carried out as described above in the presence of 2 to 10 ng of unlabeled B101 probe. Lanes 1, labeled probe; lanes 2, labeled probe and protein extract; lanes 3, labeled probe, extract, and 2 ng of unlabeled B101; lanes 4, labeled probe, extract, and 5 ng of unlabeled B101; lanes 5, labeled probe, extract, and 10 ng of unlabeled B101. (D) DNA probes B101IRL and B101IR20 correspond to B101IR but contain altered IR sequences. These probes were amplified from the recombinant plasmid constructs IRL and IR20 (Fig. 3). Biotinylated probes B101IR (lanes 1 to 4), B101IRL (lanes 5 to 8), and B101IR20 (lanes 9 to 12) were incubated with 7.5 μg of A. actinomycetemcomitans protein extract in the absence and presence of unlabeled B101IR probe (2 and 5 ng) and electrophoresed in 5% polyacrylamide gels. Lane 1, labeled B101IR; lane 2, B101IR and extract; lane 3, B101IR, extract, and 2 ng of unlabeled B101IR; lane 4, B101, extract, and 5 ng of unlabeled B101IR; lane 5, labeled B101IRL alone; lane 6, B101IRL and extract; lane 7, B101IRL, extract, and 2 ng of unlabeled B101IR; lane 8, B101IRL, extract, and 5 ng of unlabeled B101IR; lane 9, labeled B101IR20 alone; lane 10, B101IR20 and extract; lane 11, B101IR20, extract, and 2 ng of unlabeled B101IR; lane 12, B101IR20, extract, and 5 ng of unlabeled B101IR. (E) Probes B180 and B154 lack 10 and 36 bp, respectively, from the 3′ end of probe B101. Biotinylated B180 and B154 were incubated with 30 μg of A. actinomycetemcomitans protein extract and electrophoresed in 5% polyacrylamide gels. Lane 1, labeled B154; lane 2, B154 and extract; lane 3, labeled B180; lane 4, B180 and extract.

FIG. 4.

(A) Schematic representation of the glyA-orfA intergenic region showing the probes used for mobility shift experiments. Probe B101IR contains the entire AT-rich IR sequence and extends from nucleotides −39 to −166, where +1 is the transcriptional initiation site of P_orf. Probe B101 lacks the IR region and extends from −77 to −166. Probe B180 extends from −87 to −218, and probe B154 extends from −111 to −218. (B and C) For mobility shift experiments, 2 ng of biotinylated ltx promoter fragments B101 (B) and B101IR (C) was incubated with 7.5 μg of a protein extract derived from A. actinomycetemcomitans 652 and electrophoresed in 5% polyacrylamide gels. Competition experiments were carried out as described above in the presence of 2 to 10 ng of unlabeled B101 probe. Lanes 1, labeled probe; lanes 2, labeled probe and protein extract; lanes 3, labeled probe, extract, and 2 ng of unlabeled B101; lanes 4, labeled probe, extract, and 5 ng of unlabeled B101; lanes 5, labeled probe, extract, and 10 ng of unlabeled B101. (D) DNA probes B101IRL and B101IR20 correspond to B101IR but contain altered IR sequences. These probes were amplified from the recombinant plasmid constructs IRL and IR20 (Fig. 3). Biotinylated probes B101IR (lanes 1 to 4), B101IRL (lanes 5 to 8), and B101IR20 (lanes 9 to 12) were incubated with 7.5 μg of A. actinomycetemcomitans protein extract in the absence and presence of unlabeled B101IR probe (2 and 5 ng) and electrophoresed in 5% polyacrylamide gels. Lane 1, labeled B101IR; lane 2, B101IR and extract; lane 3, B101IR, extract, and 2 ng of unlabeled B101IR; lane 4, B101, extract, and 5 ng of unlabeled B101IR; lane 5, labeled B101IRL alone; lane 6, B101IRL and extract; lane 7, B101IRL, extract, and 2 ng of unlabeled B101IR; lane 8, B101IRL, extract, and 5 ng of unlabeled B101IR; lane 9, labeled B101IR20 alone; lane 10, B101IR20 and extract; lane 11, B101IR20, extract, and 2 ng of unlabeled B101IR; lane 12, B101IR20, extract, and 5 ng of unlabeled B101IR. (E) Probes B180 and B154 lack 10 and 36 bp, respectively, from the 3′ end of probe B101. Biotinylated B180 and B154 were incubated with 30 μg of A. actinomycetemcomitans protein extract and electrophoresed in 5% polyacrylamide gels. Lane 1, labeled B154; lane 2, B154 and extract; lane 3, labeled B180; lane 4, B180 and extract.