Dipartimento di Scienze Biomediche, Sezione di Microbiologia, Università G. DAnnunzio, 66100 Chieti, Italy1
Dipartimento di Biologia, Università Roma Tre, 00146 Rome, Italy2
Istituto Superiore di Sanità, 00161 Rome, Italy3
Dipartimento di Genitica e Biologia Molecolare4, Dipartimento di Scienze di Sanità Pubblica, Sezione di Microbiologia5 and Dipartimento di Biologia Cellulare e dello Sviluppo, Sezione di Scienze Microbiologiche6, Università di Roma La Sapienza, 00185 Rome, Italy
Author for correspondence: Mauro Nicoletti. Tel: +39 06 4991 4657. Fax: +39 06 4991 4626. e-mail: mauro.nicoletti{at}uniroma1.it
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ABSTRACT |
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Keywords: ATP diphosphohydrolase, mRNA processing, polycistronic mRNA
Abbreviations: EIEC, enteroinvasive Escherichia coli; pINV, virulence plasmid; RT, reverse transcriptase
a This paper is dedicated to the memory of our dear friends and colleagues Giuseppe Carruba and Franco Tatò, who died prematurely.
b The GenBank accession number for the sequence reported in this paper is AJ315184.
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INTRODUCTION |
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In a previous study (Berlutti et al., 1998 ), we have shown that the pINV-carried apy gene, which encodes apyrase (ATP diphosphohydrolase), is regulated at the transcriptional level like other virulence genes by temperature, H-NS and the VirF/VirB regulatory cascade. Even though a specific role has not been assigned to apy, the finding that its transcription is controlled by the same regulatory network that governs the expression of virulence genes raises the possibility that apy might be involved in the pathogenicity of enteroinvasive bacteria. In this respect, it is worth mentioning that apyrase has been implicated in the decrease of dNTP levels occurring in host cells during intracellular multiplication as well as in the Shigella-induced process of actin polymerization (Mantis et al., 1996
; Babu et al., 2002
). Furthermore, it has also been suggested that apyrase could be considered either as a general cytotoxin (possibly involved in damaging cellular metabolism and eventually in cell death, and in the dephosphorylation of exogenous nucleotides to nucleosides necessary to cross the impermeable cytoplasmic membrane) or as an effector playing a role in some as yet unknown metabolic pathway not directly involved in pathogenicity (Berlutti et al., 1998
; Fernandez-Prada et al., 1997
; Zalkin & Nygaard, 1996
; Zychlinsky et al., 1996
).
In our previous study (Berlutti et al., 1998 ), Northern-hybridization analysis showed that apy is probably transcribed as part of a polycistronic, temperature-regulated mRNA. This indicated that unknown genes, located adjacent to apy, could be co-transcribed under the control of the same regulatory network. To address this issue and to better characterize the transcriptional organization of apy, we have cloned and sequenced an 8023 bp PstI fragment of the pINV of the O135:K-:H- EIEC strain HN280, encompassing apy as well as its adjacent genes. Analyses of sequence, Northern hybridization, RT-PCR and primer extension data and transcriptional fusions indicated that apy is co-transcribed on a bicistronic, temperature-regulated mRNA together with a gene located upstream of apy and identical to ospB, a gene with unknown function that encodes a secreted protein (Buchrieser et al., 2000
). These data also showed that transcription starts from a promoter element located upstream of ospB. The bicistronic mRNA is processed in the intercistronic ospBapy mRNA region, leading to the considerable accumulation of a smaller, more stable, apy-specific mRNA.
The implications of the results presented in this work with regard to the origin and role of the ospBapy operon within enteroinvasive micro-organisms are also discussed.
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METHODS |
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Overlapping DNA fragments, generated after digestion with different restriction enzymes of the 8·3 kb PstI apy-containing fragment of pHN290 (Table 1
) cloned into pACYC177, were subcloned into pUC18. Double-stranded DNA inserts were sequenced by the dideoxy-chain-termination method using QIA-express forward and reverse sequencing primers (Diagen) and a commercial T7 sequencing kit (Pharmacia Biotech). Primers were 5'-end-labelled with carbocyanin (Pharmacia Biotech) and sequencing products were analysed with an ALF-express automated DNA sequencer (Pharmacia Biotech). The sequences of individual fragments were assembled to obtain the nucleotide sequence of an 8023 bp PstI fragment that contained the entire apy locus. Sequence data were compared to known sequences by using the BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/).
PCR analysis.
The primers used throughout this study are listed in Table 2. Thermal cycling conditions were as described previously (Berlutti et al., 1998
). Specific annealing temperatures for the individual primers are reported in Table 2
.
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For Northern (RNA) analysis, total RNA (10 µg samples) was denatured at 65 °C for 15 min in the presence of 2 M formaldehyde and 50% formamide. It was then separated on a formaldehyde/MOPS/agarose gel and transferred to Hybond-C extra membranes (Amersham). DNA probes were prepared by PCR. PCR-generated DNA fragments were recovered from low-melting-agarose gels; they were then 32P-labelled by random priming (Sambrook et al., 1989 ). After hybridization, dried membranes were analysed in an Instant Imager electronic autoradiographer (Canberra Packard), to quantify the amount of bound probe. Results were adjusted by subtracting the background level for the filters and were normalized by probing duplicate filters with the 32P-labelled rrnB probe (7·5 kb BamHI fragment) from pKK3535 (Brosius et al., 1981
).
For RT-PCR experiments, total RNA was extracted from cultures of EIEC strain HN280 that had been grown overnight at 30 °C and then diluted 1:200 in fresh LB medium, incubated at 30 °C up to an OD600 of 0·2 and shifted rapidly to 37 °C until an OD600 of
1·1 was reached. RNA preparations were subjected to an additional treatment with RNase-free DNase. Reverse transcriptase (RT) assays were performed using SuperScript II RT (Gibco-BRL), according to the manufacturers instructions, with the 3'-reverse oligonucleotide AGZ18 (Table 2
) complementary to the 3' extremity of the apy gene. PCR amplifications were done using cDNA as template in 30 cycles of reaction using the primer pair INTXR1 (5'-sense)/INTXL1 (3'-reverse), which amplified a specific ospB 501 bp fragment, or the primer pair AGZ17 (5'-sense)/AGZ18 (3'-reverse), which amplified a specific 672 bp apy fragment (Table 2
). As controls, we performed RT assays without adding RT, without adding RNA or by adding pHN290 DNA instead of RNA. These controls were then subjected to PCR amplification as described above.
In the primer-extension experiments, oligonucleotide primers were 5'-end-labelled with [-32P]-dATP by using T4 polynucleotide kinase. Each labelled oligonucleotide was hybridized with 10 µg of total RNA isolated from strain HN280 grown at 30 or 37 °C to an OD600 of
0·6. Reverse transcription experiments were carried out at 43 °C with avian myeloblastosis virus RT (US Biochemicals), according to the manufacturers instructions. The 5'-labelled primers used for detecting the start sites were INTX2, complementary to nucleotides +104 to +85 of the ospBapy bicistronic mRNA (the ospBapy 5' end marks position +1), and INTAPY1 and INTAPY2 (Table 2
), complementary to nucleotides +187 to +169 and +116 to +97, respectively, of the apy mRNA (the apy 5' end marks position +1). The resulting cDNAs were analysed on denaturing 7% polyacrylamide gels, along with a sequencing ladder that was generated by using the same oligonucleotides and pHNEH2.2 (Table 1
) as template. Manual double-stranded DNA sequencing reactions were performed with the Sequenase DNA Sequencing Kit (version 2.0; US Biochemicals) and [35S]-dATP.
The determination of mRNA half-lives was carried out using bacterial cells that had been grown in LB medium at 30 °C to an OD600 of 0·2 and then shifted rapidly to 37 °C. When cultures reached OD600
1·1, rifampicin was added to a final concentration of 250 µg ml-1 and samples were taken at various time intervals. RNA was extracted as described above. Equal amounts (10 µg) of each RNA sample were separated on a formaldehyde/agarose gel and the resulting bands were transferred to a nitrocellulose membrane. RNAs were analysed by Northern-blot assay and hybridization with a 32P-labelled apy probe. To normalize the quantity of RNA in each lane, duplicate filters were hybridized with the labelled oligonucleotide 5'-ACTACCATCGGCGCTACGGC-3', which was used as a probe for 5S rRNA. The relative half-lives of the bicistronic and apy transcripts were obtained by determining (phosphorimager quantification) and plotting the normalized amounts of bound probe for each transcript at each time point. Half-lives were calculated from the slope of each plot, and the errors of these half-lives were estimated from the SD of the slopes.
Determination of promoter activity in vivo.
Fragments encompassing the DNA regions containing putative transcriptional signals of the apy operon were generated by PCR using pHN290 DNA as template and primers carrying restriction sites at their ends. Plasmid pHNAP1 (carrying an apylacZ fusion) was constructed by cloning a 376 bp PCR-generated fragment [primers AP1 (5'-sense) and AP2 (3'-reverse); Table 2] into the reporter plasmid pMP220 (carrying a promoterless lacZ gene downstream of a multiple-cloning site; Spaink et al., 1987
), which was used to generate transcriptional fusions. Plasmid pHNOR1 (carrying an ospBlacZ fusion) was constructed by cloning a 378 bp fragment [primers OR1 (5'-sense) and OR2 (3'-reverse); Table 2
] into pMP220. To precisely assess promoter activity, the same DNA regions but in the opposite orientation were also cloned into pMP220 using primers AP1-1 (5'-sense) and AP2-1 (3'-reverse) for the apy gene and primers OR1-1 (5'-sense) and OR2-1 (3'-reverse) for the ospB gene (Table 2
), thus generating recombinant plasmids pHNAP1-1 and pHNOR1-1, respectively. The recombinant plasmids were separately transformed into the
lac mutant invasive EIEC strain HN570 (Table 1
). ß-Galactosidase expression was assayed on SDS/chloroform-permeabilized cells, as described by Miller (1972)
.
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RESULTS |
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RT-PCR analysis was used to assess whether ospB and apy are transcribed as a single message. Oligonucleotide AGZ18 (3'-reverse primer; Table 2), which is complementary to the apy mRNA, was annealed to total RNA isolated from HN280 grown at 30 or 37 °C, and RT was used to generate cDNA. PCR amplifications were carried out using RT products as templates and using two pairs of oligonucleotide primers (Table 2
) designed to amplify fragments internal to the ospB coding region [INTXL1 (5'-sense) and INTXR1 (3'-reverse)] or to the apy coding region [AGZ17 (5'-sense) and AGZ18 (3'-reverse)]. Thus, if the mRNA was bicistronic, the cDNA would contain ospB and apy sequences, and 501 and 672 bp PCR products, respectively, would be amplified. Using this approach, we amplified the two expected DNA fragments from the RNA of HN280 grown at 37 °C and confirmed (by Southern hybridizations with the ospB- and apy-specific probes) that these fragments were, indeed, ospB and apy (Fig. 3a
, b
). As expected, no amplification products were detected from RNA preparations from HN280 grown at 30 °C. As a negative control, RNA annealed to primer AGZ18 and incubated without RT was used as the template for the PCR described above. Other positive and negative control mixtures contained pHN290 DNA or distilled water as templates. These results allowed us to unequivocally confirm that the ospB and apy genes are organized as a single bicistronic operon.
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Analysis of the sequence immediately downstream of apy reveals two inverted repeats, designated IR2 (G°, -8·7 kcal mol-1) and IR3 (
G°, -3·3 kcal mol-1) (Fig. 5c
). Although they must be considered imperfect terminators (because of the spacing or the span of the U nucleotides distal to the bases of the hairpin structures) they could nevertheless function as
-independent termination signals (Fig. 5c
). The distance between the 5' ends of the large ospBapy bicistronic transcript and the small apy transcript, and the putative terminators located just downstream of apy, agrees very well with the sizes of the identified transcripts (2 and 1 kb) as determined by Northern-hybridization experiments (Fig. 2
).
To confirm and further analyse the transcriptional organization of the ospB and apy genes and to determine promoter activity in vivo, two reporter gene constructs were prepared. ospB and apy putative promoter regions (a 378 bp DNA fragment encompassing 310 bp upstream of the ospB start codon and a 376 fragment encompassing 305 bp upstream of the apy start codon) were cloned upstream of the lacZ reporter gene of the promoter-probe plasmid pMP220, generating recombinant plasmids pHNOR1 and pHNAP1, respectively. Plasmids pHNOR1-1 and pHNAP1-1, which contained the same inserts as pHNOR1 and pHNAP1, respectively, but in an inverted orientation, were used as controls (see Methods for details). Since the expression of apy is regulated by the same regulatory network that governs the expression of virulence genes (Berlutti et al., 1998 ), recombinant plasmids were separately transformed into the
lac EIEC strain HN570 (Table 1
) and ß-galactosidase activity was used as a measure of promoter expression (Miller, 1972
). A comparison of the expression of the transcriptional fusions in HN570 grown at 37 °C revealed that pHNOR1 (ospBlacZ fusion) produces about 32-fold more ß-galactosidase activity than pHNAP1 (apylacZ fusion) (2689 versus 84 units, respectively). The results are expressed as the means of four independent experiments; the SE was less than 10% of the mean for all results. Since a level of ß-galactosidase activity similar to that obtained with pHNAP1 was detected with both pHNOR1-1 (92 units) and pHNAP1-1 (87 units), and since pMP220 alone scored 3 units, the low but detectable ß-galactosidase activity of pHNAP1 may reflect unspecific basal transcriptional initiation, probably due to the high A+T content of the cloned DNA regions (data not shown). These results are consistent with the hypothesis that the expression of the ospBapy operon is under the control of a promoter located upstream of ospB.
Time-course of ospBapy operon expression upon induction by temperature
To better define the relative levels of the two transcripts as well as the extent to which ospBapy transcription is influenced by temperature, we analysed the induction of transcription by Northern-blot hybridization. To this end, HN280 was grown at the non-permissive temperature of 30 °C, up to an OD600 of 0·2, and then shifted rapidly to 37 °C. Total RNA was extracted at various time intervals and hybridized with the 32P-labelled apy-specific probe I. As shown in Fig. 6a
, the expression of the ospBapy operon was strongly induced and peak expression was achieved when HN280 passed through the late-exponential to early-stationary phases of growth [i.e. 65 min (OD600
1·1) and 145 min (OD600
2·2), respectively, after the temperature shift]. At every time point the 1 kb transcript was more abundant than the 2 kb transcript (the ratios of the 2 and 1 kb transcripts at 65 and 145 min were 1:3 and 1:20, respectively) (Fig. 6b
). These results indicated that a difference in stability might be responsible for the different levels of the two transcripts.
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DISCUSSION |
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In this work, we present evidence that ospB and apy are part of a bicistronic operon and that they are co-transcribed on a temperature-repressible mRNA. Two transcripts, a large one (approx. 2 kb) encoding ospB and apy, and a smaller one (about 1 kb) encoding apy, were clearly detected in HN280 grown at 37 °C (Figs 2 and 3
). Based on the finding that ospB is co-transcribed with apy (this study), and that we have previously shown that the transcription of apy is under the control of the same regulatory network that governs the expression of the virulence genes ipa, mxi and spa (Berlutti et al., 1998
), we hypothesize that the ospBapy operon might play a role in the pathogenicity of enteroinvasive micro-organisms, even though a specific biological role for the two genes has not yet been assigned.
Primer-extension analysis identified the 5' ends of the 2 and 1 kb transcripts (35 and 68 nt, respectively) to be upstream of the ATG translational start codons of ospB and apy. Inspection of the DNA region upstream of the 5' end of ospB reveals sequences resembling E. coli 70 -10 and -35 consensus promoter hexamers, although the span of the spacing between the -35 and the -10 (12 nt) consensus sequences and between the -10 and +1 site (12 nt) is not canonical (Fig. 4b
).
Sequences resembling promoter elements were not found in the DNA region upstream of the 5' end of apy, and the expression of the transcriptional fusions indicated the presence of promoter activity for ospBlacZ fusions but not for apylacZ fusions. Moreover, the size of the 2 kb transcript is consistent with the transcription of the ospBapy operon starting at the identified 5' end (+1) of ospB and terminating at putative transcriptional terminators located downstream of apy. These data are consistent with the hypothesis of an ospBapy bicistronic transcript starting from a promoter located upstream of ospB.
Thus, the 1 kb transcript encoding apy alone must result from post-transcriptional processing of the 2 kb transcript at the identified 5' end, within the 328 bp ospBapy intercistronic region. That this could be the case is further supported by the following observations: (i) probes II (the ospB-specific probe) and III (the intercistronic-specific probe), which both recognized the 2 kb ospBapy transcript, also bound smaller RNA species that were not resolved into discrete bands (Fig. 2) and which are likely to represent degradation products of the processed 2·0 kb transcript at the newly formed 3' end; (ii) the 5' end of the 1 kb apy transcript maps within a sequence (GUUUU) (Fig. 5b
) homologous to the consensus recognition sequence (A/GAUUA/U) proposed for RNase E (Grunberg-Manago, 1999
); and (iii) computer analysis (not shown) revealed that the 5' end of the 1 kb apy transcript lies within a single-stranded segment in a secondary structure, with a predicted
G° value of -9·4 kcal mol-1, as expected for RNase E cleavage sites (Grunberg-Manago, 1999
). RNase E is a key enzyme that has been implicated in the cleavage and in the decay of several different RNA molecules in E. coli. mRNA decay is a process often initiated by specific RNase E cleavage followed by 3' to 5' exonucleolytic degradation at the newly formed 3' ends (Grunberg-Manago, 1999
). Further experiments are needed to precisely assess whether RNase E is involved in post-transcriptional processing of the 2 kb transcript.
Post-transcriptional processing of mRNA, rather than simply initiating mRNA decay, may also modulate gene expression, since the amount of a given gene product is also dependent on the stability of its own messenger. Segmental differences in mRNA stability have been demonstrated to be a mechanism of post-transcriptional regulation of gene expression in several operons of prokaryotic micro-organisms, including E. coli (Alifano et al., 1994 ; Baga et al., 1988
; McCarthy et al., 1991
; Naureckiene & Uhlin, 1996
; Nilsson & Uhlin, 1991
; Owolaby & Rosen, 1990
; Ruiz-Echevarria et al., 1995
), Salmonella typhimurium (Newbury et al., 1987
) and Rhodobacter capsulatus (Alifano et al., 1994
; Belasco et al., 1985
). In this work, we have quantified the relative amounts and stability of the two transcripts from HN280 (i.e. the 1 and 2 kb transcripts) and have found that they are not present in equivalent amounts and that they have considerably different half-lives [the 1 kb apy mRNA shows an half-life approximately 4·9-fold higher than that of the 2 kb ospBapy transcript (2·2±0·3 min versus 27±4 s, respectively)]. Stemloop structures have been reported to act as protective barriers against the degradation of upstream mRNA segments by 3' exonucleases (Alifano et al., 1994
; Grunberg-Manago, 1999
). The predominance and higher stability of the 1 kb transcript over the 2 kb transcript might reflect a stabilizing effect possibly mediated by the stemloop sequences located downstream of the apy stop codon (IR2 and IR3; see Fig. 5c
).
Although differences in the transcript levels may or may not be reflected in differences in the relative amounts of OspB and apyrase expressed, depending upon whether subsequent translation regulation occurs, the generation of a separate, more stable and more abundant apy transcript suggests that apyrase might stoichiometrically outnumber the amount of OspB present in virulent enteroinvasive micro-organisms. Moreover, peak expression of the ospBapy operon was shown to occur when HN280 entered into the late phases of bacterial growth (Fig. 6), as has been reported for the virulence genes of S. flexneri (Bahrani et al., 1997
; Day & Maurelli, 2001
). These results are in agreement with our previous findings which indicate that S. flexneri and EIEC strains display their highest levels of apyrase activity upon entry into the late phases of bacterial growth (Berlutti et al., 1998
).
Finally, the complete DNA sequence of pWR100 has been reported recently (Buchrieser et al., 2000 ; Venkatesan et al., 2001
). Based on an unexpected proportion of known and putative insertion sequences (IS) and on the G+C content of the genes and of their flanking regions (used as a marker to trace the phylogenetic origin of the genes), it has been proposed that the pINV is composed of a mosaic of blocks of genes which have different origins, probably resulting from IS-mediated acquisition and assembly of DNA across bacterial species (Buchrieser et al., 2000
; Venkatesan et al., 2001
). In the case of ospB and apy, their G+C contents (33 mol% for ospB, similar to that of virulence genes, and 42 mol% for apy) suggest that these genes may have a different origin and that they have probably been acquired separately in the evolutionary assembly of the pINVs of enteroinvasive micro-organisms. Additional experiments are required to assess precisely whether these two genes play a role in virulence or a role in some unknown bacterial metabolic function not directly involved in pathogenesis. However, the findings that ospB and apy were probably acquired independently, are expressed from a single transcript and are located in a highly conserved DNA region in both Shigella and EIEC (Berlutti et al., 1998
; Bhargava et al., 1995
; Buchrieser et al., 2000
; Venkatesan et al., 2001
) indicate that such a gene organization must provide some selective advantage to enteroinvasive micro-organisms.
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ACKNOWLEDGEMENTS |
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Received 18 February 2002;
revised 25 March 2002;
accepted 16 April 2002.
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