Departments of Microbiology1, Preventive Dentistry2 and Periodontology and Endodontology3, Okayama University Dental School, Shikata-cho 2-chome, Okayama 700-8525, Japan
Laboratory of Microbial Ecology, Department of Bioresource Science, Ibaraki University School of Agriculture, Ami-machi, Ibaraki 300-0393, Japan4
Author for correspondence: Tetsuyoshi Inoue. Tel: +81 86 235 6656. Fax: +81 86 235 6659. e-mail: inouet{at}cc.okayama-u.ac.jp
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ABSTRACT |
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Keywords: RTX toxin, periodontopathogen, chemostat culture, catabolite repression
The GenBank/EMBL/DDBJ accession number for the sequence data in this paper is AB054839.
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INTRODUCTION |
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Although the leukotoxin gene operon (ltxC, ltxA, ltxB and ltxD) appears to be present in all A. actinomycetemcomitans strains (Poulsen et al., 1994 ), the level of toxin expression varies considerably between different strains (Baehni et al., 1981
; Spitznagel et al., 1991
). The strains are roughly classified as being highly leukotoxic (represented by strain JP2), moderately toxic (strain 652), minimally toxic (strain ATCC 33384) or variably toxic (ATCC 29523). The differences in leukotoxin expression appear to be due to transcriptional regulation, since there is a direct correlation between the level of leukotoxin and the amount of ltx RNA in a given strain (Spitznagel et al., 1991
). The ltx promoter sequences of a highly toxic (JP2) and a moderately toxic (652) strain of A. actinomycetemcomitans were analysed by Brogan et al. (1994)
. In strain JP2, two promoters at 350 bp (P1) and 50 bp (P2) upstream of ltxC were identified, whereas there was an insertion of 530 bp between the P1 and P2 promoters in strain 652. Recently, Kolodrubetz et al. (1996)
performed cis/trans analyses to assess the relative contributions of transcription factors and promoter sequences in determining the different leukotoxin levels in different strains. They concluded that strain-specific trans factors and the promoter sequence difference were equally significant in determining the levels of ltx expression in A. actinomycetemcomitans.
Previously, we reported that a variably toxic strain of A. actinomycetemcomitans (strain 301-b) did not produce detectable amounts of leukotoxin in batch cultures but stably produced high amounts of toxin in anaerobic fermentable-sugar-limited chemostat cultures (Ohta et al., 1991 , 1993
). In further experiments (Mizoguchi et al., 1997
), toxin production in the chemostat culture of strain 301-b immediately stopped when the culture was shifted from sugar-limited to sugar-excess conditions, suggesting that leukotoxin expression is regulated by fermentable sugar levels in the culture. In this study, we have sequenced the promoter region for the 301-b ltx operon and determined how the external sugar level influenced the transcription of the ltx operon by using Northern blot analysis. In addition, we present some data which support the idea that the production of leukotoxin by the variably toxic strain 301-b is subjected to catabolite repression.
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METHODS |
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Escherichia coli JM109 (Takara Shuzo), E. coli XL-1 Blue MRA (Stratagene), replacement vector EMBL3 (Promega) and plasmid vectors pUC18 (Takara Shuzo) and pSPT19 (Boehringer Mannheim) were used for the cloning experiments. E. coli strains were grown in LuriaBertani (LB) broth or on LB agar plates (Sambrook et al., 1989
) at 37 °C. When necessary, 100 µg ampicillin ml-1 was added to the medium.
Chemostat culture.
A. actinomycetemcomitans 301-b was grown in an anaerobic chemostat system as described previously (Ohta et al., 1989 ). The chemostat system was kept under a stream (200300 ml h-1) of N2 which was freed of traces of oxygen by passage over a gas-purifying column (Gas Clean GC-RX; Nikka Seiko). The temperature of culture was set at 37 °C and the pH was maintained at 7·0 with 2 M NaOH or 2 M HCl. Anaerobic conditions were checked by monitoring culture redox potential using a platinum electrode with an Ag/AgCl reference cell. In this system the culture redox potential (without bicarbonate, pH 7·0) was maintained at below -400 mV (Ohta et al., 1996
). The OD660 of cultures was measured in a 1 cm light path cuvette to determine the cell densities by the averaged coefficient of the dry cell weight at OD660 (0·852 mg dry wt cells ml-1 per OD660 unit): the relationship was linear up to 0·6 units of OD660 (Ohta et al., 1989
).
DNA manipulations.
Chromosomal DNA from A. actinomycetemcomitans was isolated by the method of Saito & Miura (1963) . Plasmid DNA was purified from E. coli hosts using a commercial Qiagen plasmid purification kit according to the manufacturers instructions. Digestion of DNA with restriction enzymes, ligation, transformation by electroporation, agarose gel electrophoresis of digested DNA and PCR were carried out according to the suppliers instructions and/or by standard procedures (Sambrook et al., 1989
).
Cloning the ltx promoter region and structural gene.
A genomic library of A. actinomycetemcomitans 301-b DNA fragments, partially digested with Sau3AI, was prepared in E. coli JM109 using plasmid pUC18 by the same procedure as described previously (Abo et al., 1991 ). The genomic bank was screened for LtxA expression by using a rabbit antiserum raised against the purified leukotoxin from A. actinomycetemcomitans 301-b (Ohta et al., 1991
). Through the immunological screening of approximately 5x104 recombinant clones, two immunopositive clones were obtained. One positive clone, designated TO1 (pTO1 when referring to plasmid), produced an Mr 55000 polypeptide that reacted with the anti-leukotoxin serum. Analysis of the nucleotide sequence of the insert from pTO1 identified the 1·6 kbp fragment of ltxCA genes (positions 3491956 in the ltxCA genes of strain JP2) (Kraig et al., 1990
). For the cloning of the ltx promoter region, a partial Sau3AI digest of strain 301-b DNA was ligated with
replacement vector EMBL3 previously digested with BamHI. The resultant preparations were packaged into phage
heads using an in vitro packaging kit (Amersham Pharmacia Biotech) and transduced into E. coli XL-1 Blue MRA cells. Approximately 4x105 plaques were screened by plaque hybridization using a probe prepared from plasmid pTO1. Probe labelling and hybridized probe detection were conducted by the Photo ChemiProbe 1-Step Kit (Orgenics) according to the manufacturers instructions. The screening resulted in the isolation of four recombinant
phages. Phage DNAs of the positive clones were isolated and digested with various restriction enzymes and further analysed by Southern hybridization with the pTO1 probe. One positive clone, designated
TO3, contained an insert of approximately 18 kbp and was selected for further study. The 4·0 kbp EcoRI fragment containing the ltx promoter region was subcloned from
TO3 into the pUC18 vector, and the resulting plasmid, designated pTD01, was used for DNA sequence analysis.
Preparation of antisense RNA probe.
The fragment of ltxCA from A. actinomycetemcomitans 301-b was obtained by PCR amplification with oligonucleotide primers corresponding to bp -756 to -740 (5'-GTTTTATTCAGTTCCCA-3') of the 301-b ltx promoter region and bp 3402 to 3421 (5'-CCCTCTAAGTGGTCGTCGCC-3') of ltxCA and strain 301-b genomic DNA as a template. The amplified fragment was digested with PstI and HindIII, which yielded a 600 bp fragment from the middle of ltxA. The resulting fragment was ligated to PstI- and HindIII-digested plasmid pSPT19. This ligation was used to transform E. coli JM109 to ampicillin resistance, resulting in plasmid pPA10. Plasmid pPA10 was linearized and used as a template for in vitro generation of full-length runoff transcripts. RNA probe syntheses were carried out by a digoxigenin (DIG) RNA Labelling kit (Boehringer Mannheim) according to the manufacturers instructions.
Northern blot analysis.
Total cellular RNA was prepared from 20 ml culture sampled from the chemostat by the hot phenol extraction method of Emory & Belasco (1990) . Northern blot analysis was performed basically according to the protocol described by Inoue et al. (2000)
. Briefly, RNA samples were electrophoresed on 1% (w/v) agarose gels containing 6·7% (v/v) formaldehyde and transferred onto nylon filters (Hybond-N membrane; Amersham) by capillary blotting. Prehybridizations and hybridizations were done at 65 °C in a solution containing 50% (v/v) formamide. The hybridized RNA was visualized with a DIG detection kit (Boehringer Mannheim).
Nucleotide sequencing and sequence analysis.
Sequences of double-stranded plasmid DNA were determined using the Taq Dye Deoxy Terminator Cycle Sequencing kit (Perkin Elmer) and read on an Applied Biosystems 373A DNA sequencer. To sequence the ltx promoter region, an ordered set of deletion plasmids was prepared from the pTD01 plasmid by using the Kilo-Sequence Deletion kit (Takara Shuzo). These plasmids were sequenced using a primer based on the M13 site presented on the pUC18 vector. The pPA10 plasmid was sequenced using the SP6 and T7 sequencing primers (Promega). The accession number of the sequence data in GenBank/EMBL/DDBJ is AB054839. Sequence data were compiled and analysed with GENETYX-MAC genetic information processing software (Software Development).
Determination of leukotoxin levels.
Bacterial cultures were removed from the chemostat and cells were harvested by centrifugation at 10000 g for 10 min at 4 °C. Cellular and extracellular leukotoxin concentrations were quantitated by Western (immunoblot) analysis using rabbit antiserum against the purified leukotoxin from A. actinomycetemcomitans 301-b. For the measurement of total cellular leukotoxin, cell suspensions (1·9 mg protein ml-1) or cell sonicates (3·5 mg protein ml-1) were mixed 11:9 with SDS-containing sample buffer [0·05 M Tris/HCl (pH 6·8), 1% SDS, 8·8% glycerol, 2% 2-mercaptoethanol, 0·0063% bromphenol blue]. The cell sonicate was prepared as described previously (Ohta et al., 1989 ). For the determination of extracellular leukotoxin, the culture supernatants resulting from the centrifugation of bacterial cultures were passed through cellulose acetate membrane filters (Advantec Toyo; pore size, 0·22 µm), then the filtrates were dialysed against deionized water, lyophilized and dissolved in distilled water (1/20 the original volume). The extracellular samples were also mixed 11:9 with the SDS-containing sample buffer. These samples were boiled for 3 min and separated by SDS-PAGE with a 7·5% (w/v) acrylamide gel as described by Laemmli (1970)
. Proteins were electrophoretically transferred to PVDF membranes (Bio-Rad) according to the method of Towbin et al. (1979)
. Reactivity with the anti-leukotoxin serum was detected with swine anti-rabbit immunoglobulin G conjugated to peroxidase (DAKO Japan) and then visualized by adding 4-methoxy-1-naphthol and hydrogen peroxide. Leukotoxin band intensities were analysed by using a Foto/Analyst Image Analysis System (Fotodyne). To construct a standard curve of leukotoxin concentration, serial dilutions of the purified leukotoxin (0100 µg ml-1) (Ohta et al., 1991
) were processed essentially as above and analysed together with the samples on the same gel.
Assay of cAMP.
The intracellular and extracellular concentrations of cAMP were determined with an enzyme-linked immunoassay kit (Cayman Chemical). To determine the intracellular cAMP concentration, 2·5 ml culture was sampled from the chemostat and filtered immediately through a cellulose acetate membrane filter (pore size, 0·45 µm; Advantec Toyo). The filter was immediately placed in 5 ml ice-cold ethanol and stored at -20 °C. Prior to the assay, the extracts were dried under a stream of N2 at room temperature and the residue was resuspended in 0·5 ml phosphate buffer (supplied with kit). To determine the extracellular cAMP concentration, the culture filtrate was collected as described above and stored at -20 °C prior to the assay. The enzyme-linked immunoassay procedure was in accordance with instructions provided by the manufacturer. Since the extracellular fluid was found to contain a large amount of cAMP as in E. coli (Wayne & Rosen, 1974 ; Matin & Matin, 1982
), we estimated the amount of extracellular fluid retained on the filters under these conditions. This was done by filtering a known amount (2·5 ml) of cAMP solution (1·27 µM) and assaying cAMP on the filter as above. The filters retained about 1·7% of the total filtered amount of cAMP and, accordingly, the corresponding amount of cAMP in the extracellular fluid was subtracted from the amount of cAMP recovered from the filters.
Chemical analysis.
Fructose concentrations in the reservoir medium and culture supernatants were determined by using an enzyme system composed of hexokinase, glucose-6-phosphate dehydrogenase and glucose-6-phosphate isomerase (Boehringer Mannheim). Protein concentrations were determined by the method of Lowry using bovine albumin as a standard.
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RESULTS |
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Regulation of leukotoxin gene expression by the fructose level
We previously reported that strain 301-b stably produced significant amounts of leukotoxin in anaerobic fermentable-sugar-limited chemostat cultures, but did not do so under sugar-excess conditions, suggesting that leukotoxin gene expression is regulated by fermentable sugar levels in the culture (Mizoguchi et al., 1997 ). In the present study, we conducted a fructose pulse experiment to investigate the effect of external fructose level on leukotoxin gene expression. The steady-state chemostat culture of strain 301-b was first established at a dilution rate of 0·15 h-1 under anaerobic fructose-limited conditions and then shifted to fructose-excess conditions by pulsing the culture with 150 mM fructose. Bacterial cells and culture supernatant were taken out at several time points before and after fructose pulsing and the amounts of cell-bound and cell-free leukotoxin were determined by densitometric analysis of the Western blot using anti-leukotoxin serum. As shown in Fig. 1(a)
, leukotoxin production was clearly repressed by fructose pulsing. Fig. 2
illustrates the relationship between leukotoxin production and residual fructose level. When fructose was added to the culture, toxin production was immediately stopped and the level of leukotoxin decreased with increasing dilution of culture. The slightly faster than theoretically expected decrease in the cellular leukotoxin level suggests that there was a slight decomposition of leukotoxin, as we observed previously (Ohta et al., 1996
; Mizoguchi et al., 1997
). With respect to the leukotoxin gene expression, total RNA was simultaneously isolated at each time point and subjected to Northern blot analysis using an ltxA-specific RNA probe. As shown in Fig. 1(b)
, an intense band was clearly observed before pulsing and seemed to correspond to a ltxCA transcript that has mainly been detected in A. actinomycetemcomitans Y4 by Kolodrubetz et al. (1996)
. Pulsing of fructose into the culture resulted in strong repression of ltx transcription, which continued as long as residual fructose in the culture was detected. When the residual fructose level decreased to less than 1 mM at 11·7 h after the pulse, the ltx transcription was partially restored. After 24 h, leukotoxin production and the ltxA mRNA level returned to the same level as before pulsing. These results led us to conclude that change in toxin production in response to external fructose level was due to regulation of ltx operon transcription.
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DISCUSSION |
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Based on the present results, we propose that the production of leukotoxin by variably toxic strain 301-b is regulated by a mechanism mediated by cAMP. Our findings supporting this idea are as follows: (1) addition of fructose into the fructose-limited culture resulted in the repression of the leukotoxin mRNA level and in concomitant significant reduction of intracellular cAMP level; (2) as fructose added to the medium was diluted and exhausted, both leukotoxin mRNA level and cAMP level were recovered to the levels before the fructose pulsing; (3) addition of cAMP apparently derepressed leukotoxin production under the fructose-excess conditions. In addition to these results, our previous study showed that leukotoxin gene expression was repressed by glucose pulsing, but not by pulsing with a non-fermentable glucose analogue, methyl -D-glucoside (Mizoguchi et al., 1997
). All these observations are consistent with the characteristics of catabolite repression, a well-known mechanism for regulation of gene expression in E. coli. In E. coli, the intracellular cAMP level in sugar-limited chemostat culture is more than 10-fold higher than that under sugar-excess conditions (Matin & Matin, 1982
; Villarejo et al., 1978
; Wright et al., 1979
) and the accumulated cAMP activates cAMP-dependent gene expression. A comparable increase in intracellular cAMP level in response to fermentable sugar limitation was also observed in A. actinomycetemcomitans chemostat cultures; hence it is likely that catabolite repression mediated by cAMP occurs in this bacterium.
The molecular mechanisms for regulation of gene expression by catabolite repression have been extensively studied in E. coli. In the regulatory model, binding of the cAMPcAMP receptor protein (CRP) complex to the consensus sequence, TGTGAnnnnnnTCACA, just upstream of the promoter, is needed for the transcriptional activation of catabolite-repressible genes (Stormo & Hartzell, 1989 ). However, no sequence significantly similar to the consensus was found in the vicinity of the A. actinomycetemcomitans leukotoxin promoter, suggesting that the cAMPCRP complex may not directly affect the expression of the leukotoxin gene. Recently, Kolodrubetz et al. (1996)
reported data indicating that a positive or negative transcriptional regulator(s) is involved in the regulation of leukotoxin gene expression. Based on their data, it may be possible that the cAMPCRP complex controls the transcriptional level of such a regulator gene(s) in 301-b, resulting in the alteration of leukotoxin gene expression in response to cellular cAMP level.
A. actinomycetemcomitans leukotoxin belongs to a family of RTX (repeats in toxin) cytotoxins which have tandemly repeated nonapeptides with the consensus sequence of GGXGXDX[L/I/V/W/Y/F]X (Lally et al., 1999 ). RTX toxins are important virulence factors produced by a wide range of Gram-negative bacteria (Coote, 1992
). However, there has been no report that catabolite repression is involved in regulation of RTX toxin production. On the other hand, there are several virulence factors whose expression is regulated by catabolite repression: colonization factor antigen II, enterotoxins, Pap pilus in E. coli and haemolysin in Vibrio vulnificus (Evans et al., 1991
; Alderette & Robertson, 1977
; Busque et al., 1995
; Goransson et al., 1989
; Bang et al., 1999
). These examples might indicate that fermentable sugar limitation is a condition that these bacteria encounter frequently in vivo prior to expansion of infection. With respect to the periodontal environment, a report by Hara & Loe (1969)
is noteworthy. These authors showed that glucose concentration in the human periodontal pocket varies from 6·9 mM to 39 mM. In our previous growth analyses of A. actinomycetemcomitans using chemostat cultures, a sugar concentration over 30 mM was considered an excess condition and one below 10 mM was considered a limited condition for A. actinomycetemcomitans 301-b cells (Mizoguchi et al., 1997
). Therefore, it seems reasonable to expect that A. actinomycetemcomitans switches leukotoxin gene expression on and off in response to fermentable sugar concentration in the periodontal environment, which in turn determines whether this bacterium is in a virulent or less-virulent state.
In summary, we here cloned and sequenced the leukotoxin promoter region from variably toxic strain 301-b of A. actinomycetemcomitans, demonstrating that this strain has a promoter region almost identical to that of moderately toxic strain 652. Pulsing of fructose into the fructose-limited chemostat culture of 301-b greatly reduced the intracellular cAMP level and, concomitantly, the leukotoxin mRNA level. When the culture was returned to fructose-limited conditions, the levels of both intracellular cAMP and leukotoxin mRNA were restored to those before pulsing. Addition of external cAMP into the fructose-excess culture resulted in an apparent recovery of leukotoxin production. From these findings, we conclude that a cAMP-dependent mechanism resembling catabolite repression is involved in the regulation of leukotoxin production in A. actinomycetemcomitans. Further genetic and molecular biological approaches will be needed to elucidate the regulatory mechanism for leukotoxin gene expression at the molecular level.
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Received 9 February 2001;
revised 21 May 2001;
accepted 27 June 2001.
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