Max von Pettenkofer Institut für Medizinische Mikrobiologie und Hygiene, Ludwig Maximilians Universität, Pettenkoferstraße 9a, 80336 München, Germany1
Author for correspondence: J. Heesemann. Tel: +49 89 5160 5200. Fax: +49 89 5160 5202. e-mail: heesemann{at}m3401.mpk.med.uni-muenchen.de
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ABSTRACT |
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Keywords: cpdB gene, 2',3'-cAMP, ERIC, cAMPCRP-binding site
Abbreviations: cAMPCRP, cyclic AMPcyclic AMP receptor protein; ERIC sequence, enterobacterial repetitive intergenic consensus sequence; NPPC, p-nitrophenyl phosphorylcholine; PNPP, p-nitrophenyl phosphate
The GenBank accession number for the cpdB gene fragment of Yersinia enterocolitica O:8 strain WA-314 reported in this paper is X85742.
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INTRODUCTION |
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METHODS |
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The cpdB gene was isolated from the gene bank by screening with NPPC. Restriction enzyme digests of cosmids with Sau3AI and religation in pACYC177 restricted with BamHI resulted in pKT2. After digestion with EcoRI and religation with pACYC177, a 3 kb EcoRI fragment, which was still positive in the NPPC assay, was isolated (pKT2.1). For sequencing, this 3 kb EcoRI fragment was subcloned in pBluescript KS(-), resulting in pKT3. The nucleotide sequence of the 3 kb EcoRI fragment was determined using the Taq DyeDideoxy terminator method with a 373A DNA Sequencer (Applied Biosystems). Nested deletions were constructed using the Double-stranded Nested Deletions Kit (Pharmacia). Universal primers used to sequence the cpdB gene were as follows: forward, 5'-GTAAAACGACGGCCAGT-3'; reverse, 5'-CAGGAAACAGCTATGAC-3'. Non-overlapping regions were sequenced using specific oligonucleotides. Sequences were analysed and aligned with the HIBIO Macintosh DNASIS program (Hitachi Software Engineering) and with the Genetics Computer Group sequence-analysis software package (University of Wisconsin, Madison, WI, USA).
A cpdB mutant of Y. enterocolitica was constructed by introducing a 1·2 kb Km-GenBlock derived from pUC4K (Pharmacia LKB) into the BalI site of pKT3, resulting in pKT3.K. The BalI site is located inside the cpdB gene, 779 bp downstream of the start codon. The cpdB::Km gene fragment was transferred to the suicide vector pGPCAT (Roggenkamp et al., 1995 ) by using the SalI and SacI restriction sites. The resulting plasmid, pKT4.K, was mobilized into WA-314. Exconjugants resistant to nalidixic acid and kanamycin but sensitive to chloramphenicol were further characterized. The allelic exchange [disruption of cpdB by the insertion of a kanamycin-resistance (Km®) cassette] resulting in the WA-314 cpdB mutant (WA-314cpdB) was confirmed by Southern hybridization with digoxigenin (DIG)-labelled PCR probes as specified by Boehringer Mannheim Biochemica.
To study cpdB gene expression, translational fusions between cpdB promoter and luciferase were constructed by using PCR cloning procedures. Primers were designed not to produce frameshifts. The cpdB promoter region was amplified using the forward primer 5'-CCCAAGCTTCTTCTCAATAAAATAAGGGAA-3', preceded by a HindIII restriction site, and the reverse primer 5'-CGCGGATCCCAGTACTCGCAAATC-3', followed by a BamHI restriction site. Plasmid pCJYE138-L was digested with BamHI and HindIII and ligated with the HindIII- and BamHI-restricted PCR fragment, resulting in pKT5.
An ERIC deletion in the cpdB promoter region was generated by amplification of pKT5 with primers 5'-ATTAAATTTACATATTCTTTTGCGATACAGGTCGA-3' and 5'-GGTC G T C A T A A C A A A G T G T G A A G T TTGGCAGAAAAT- 3', which have a corresponding gap between their 5'-ends. This was accomplished using TaKaRa LA Taq polymerase (TAKARA Biotechnology) and a GenAmp system 2400 cycler (Perkin-Elmer) with the following thermal profile: denaturation at 94 °C for 15 min, 30 cycles of 98 °C for 20 s and 68 °C for 15 min, followed by 72 °C for 10 min. The amplified DNA fragment was treated with T4 DNA polymerase, to generate blunt-ended DNA, self-ligated and transformed into E. coli DH5 and Y. enterocolitica WA-314. Sequence analysis of the resulting pKT6 confirmed the deletion of bases 581708. To integrate the reporter system into the chromosome, the cpdBluciferase constructs in pKT5 and pKT6 were subcloned into the suicide vector pKAS 32, using the XbaI and SalI restriction sites after partial digestion (due to the XbaI restriction site in the luciferase gene). The resulting plasmids (pKT7, pKT8) were transferred, by conjugation, into WA-C. PCR analysis with primer 5'-CCATCGATTAGCGCTGCCAGTGCT-3', lying upstream of the cloned cpdB fragment, and a reverse primer within the luciferase gene (5'-AGTATTCCGCGTACGTGA-3') confirmed the expected integration resulting from homologous recombination via the cpdB promoter region. Primers used to screen for the presence of the cpdB gene and the ERIC element in other Yersinia spp. were 5'-ATCAGGTCGCCATTATCTAG-3' and 5'-TTCTGCCAAACTTCACACTT-3'.
Quantification of cpdBluc gene expression.
To study cpdBluc gene expression, a luciferase reporter gene assay was performed as described by the manufacturer (Roche Molecular Biochemicals). Bacteria were grown overnight at 28 °C, diluted 1:40 and then grown to exponential phase. The amount of bacteria was standardized by measuring the OD600 and plating the bacteria. The yersiniae were lysed and centrifuged, and the supernatant was transferred to a microtitre plate (Dynatech). Luciferin substrate was added and the emitted photons were counted for 10 min by a CCD camera. Activity was measured in a darkbox with a microtitre plate chemiluminometer (CCD camera C2400-77; Hamamatsu Photonics).
CpdB activity assays.
CpdB activity was determined by measuring absorbance at 410 nm after incubation with 5 mM bis(PNPP) or 20 mM NPPC for 20 min at 37 °C, in substrate buffer: (5 mM CoCl, 1 mM MgCl2, 50 mM Tris/maleate, pH 7·8) or (250 mM Tris/HCl, 1 mM Zn2+, 10 mM NaF, 45% sorbitol), respectively.
Overexpression of cpdB.
cpdB was overexpressed in E. coli using the temperature-sensitive T7 RNA polymerase/promoter system of Tabor & Richardson (Ausubel et al., 1989 ). The 3 kb EcoRI insert from pKT2.1 was cloned into pT7-6, restricted with EcoRI, in 3'5' and 5'3' orientation, resulting in pKT4.1 and pKT4.2, respectively. These plasmids were transformed into E. coli HB101 harbouring pGP1-2. T7 RNA polymerase was induced by incubating the bacteria at 42 °C and whole-cell lysates were analysed by SDS-PAGE.
Isolation of subcellular fractions.
Bacterial cells were fractionated as described by Ölschläger & Braun (1987) . The sediment from 10 ml bacterial culture was resuspended in 1·3 ml 3 mM Tris/HCl, 30 mM sucrose/0·03 mM EDTA solution, pH 8·0, containing 10 µg lysosyme. The suspension was frozen and thawed twice. After 45 min at room temperature, it was centrifuged. The supernatant contained periplasmic and cytoplasmic proteins. The sediment was resuspended in 1·2 ml 20 mM MgCl2 containing 5 µg bovine DNase. After 1 h incubation, the suspension was centrifuged at 30000 g for 60 min. Cytoplasmic proteins present in the supernatant were precipitated with ethanol. Membrane proteins were located in the sediment. Periplasmic proteins were released using the chloroform-shock method described by Ferro-Luzzi Ames et al. (1984)
.
Mouse virulence tests.
Virulence was tested in the intravenous and orogastric mouse infection models as described previously (Roggenkamp et al., 1995 ). For the intravenous and oral challenge, BALB/c mice (6- to 8-week-old females; Charles River WIGA) were infected with 4x104 (40 times the minimal lethal dose) (Vogel & Thompson, 1988
) and 2x108 bacteria, respectively. Mice were killed 2 and 4 d after the intavenous challenge and 5 d after the oral challenge. The number of bacteria in each organ was determined by plating serial dilutions of homogenized tissue.
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RESULTS |
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Sequence comparison
We compared the deduced amino acid sequence of the open reading frame on pKT3 with sequences in the EMBL and SWISS-PROT protein-sequence databases. Alignment by standard computerized methods revealed identities of 76% with E. coli (accession no. P08331) and S. typhimurium (accession no. P26265) CpdB (Liu et al., 1986 ; Liu & Beacham, 1990
), 60% with H. influenzae CpdB (accession no. P44764), 42% with the Bacillus subtilis CpdB homologue (accession no. BAA08981.1) (Yamamoto et al., 1996
), 33% with the putative nucleotidase of Streptomyces coelicolor (accession no. CAB52071.1), 26% with Helicobacter pylori CpdB (accession no. AAD05687), 25% with Clostridium perfringens CpdB (accession no. BAA81646.1), 57% with a fragment of the Aeromonas hydrophila CpdB homologue (accession no. s57941), as well as slightly more than 20% with 5'-nucleotidases and UDP-sugar hydrolases from a wide spectrum of bacterial as well as vertebrate sources. A search in the Sanger Centre BLAST server revealed a gene of Yersinia pestis that is 81% identical to the cpdB gene of Y. enterocolitica. No other 2',3'-cyclic phosphodiesterases (human, eukaryotic) had significant similarity to the Y. enterocolitica sequence, as determined by the BLAST program. Bacterial 2',3'-cyclic phosphodiesterases (EC 3.1.4.16), 5'-nucleotidases (EC 3.1.3.5) and mosquito apyrase (EC 3.6.1.5) belong to a group of related proteins that have several highly conserved regions in common. Two of these conserved regions, which could have considerable functional significance, are located in the N-terminal ends of these enzymes. These signature patterns (LIVM)-x-(LIVM)-(LIVM)-(HEA)-(TI)-x-D-x-H-(GSA)-x-(LIVMF) and (FYP)-x-x-x-x-(LIVM)-GNHEF-(DN) (Zimmermann, 1992
), the second of which contains a perfectly conserved pentapeptide, were also found in CpdB of Y. enterocolitica.
Analysis of nucleotide sequence and leader peptide
Analysis of the promoter region revealed a ribosome-binding site (GGAGA) 6 bp upstream of the start codon in position 779. Furthermore, the nucleotide sequence revealed two putative -35 (position 712 TTTACA, 725 TTGCGA) and -10 regions (737 TCGAAT, 749 CTTAAT). In position 2767, a palindromic sequence, which could form a stemloop structure consisting of a 10-base stem and a 5-base loop was identified downstream of the potential stop codon (TAA in position 2735). This structure could act as a rho-factor-independent transcriptional terminator. The free energy calculated by the method of Tinoco is -21·5 kcal mol-1 (-89·9 kJ mol-1) (Tinoco et al., 1973 ), which is in the range of a stable stem. Another characteristic of rho-independent terminators is the TCGT consensus sequence (Brendel & Trifono, 1984
), which is located downstream of the terminator. Three sequences starting in positions 2791, 2800 and 2865 have similarities to this sequence.
The deduced amino acid sequence beginning with the ATG in position 779 shows an N-terminal region of 24 amino acids (MFKRPLTLSLLASLIALTTSTAQAA) with features of a typical prokaryotic signal sequence (Pugsley, 1993 ). The N-terminal region contains three positively charged amino acids (lysine, arginine and proline) followed by a hydrophobic span that is rich in alanine and leucine. Furthermore, the two alanine residues in positions 24 and 25 represent the cleavage site of a signal peptidase (Ratnam et al., 1982
). The most likely initiation and termination codons in positions 779 and 2735 encode a protein with a molecular mass of 71958 kDa, which includes the signal sequence with a molecular mass of 2548 kDa. This is in agreement with a molecular mass of 68 kDa determined for the mature peptide by using SDS-PAGE (Fig. 1
). A second open reading frame was identified upstream of the CRP-binding site, from position 288 to the 5'-end of the sequenced fragment, which shows high homology to the E. coli cysQ gene, which is needed for cysteine synthesis (Neuwald et al., 1992
). A methionine codon was found in position 286 and a ShineDalgarno (SD) sequence in position 295, indicating that this gene is transcribed in the opposite direction with respect to the cpdB gene.
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DISCUSSION |
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The cpdB gene was cloned by screening a Y. enterocolitica O:8 gene bank with the chromogen NPPC. A 3 kb fragment was isolated and subsequently sequenced. This fragment showed an open reading frame with a good ShineDalgarno sequence encoding a protein of 68 kDa. The first 24 deduced amino acid residues showed features of a typical prokaryotic signal sequence, as is expected of a secreted protein. The cpdB gene of Y. enterocolitica showed the highest amino acid homology to cpdB genes of E. coli and S. typhimurium (Liu et al., 1986 ; Liu & Beacham, 1990
). Furthermore, significant homology to 5'-nucleotidases and mosquito apyrase was detected. These enzymes are evolutionarily related, as indicated by several highly conserved amino acid clusters in all three enzymes (Champagne et al., 1995
; Zimmermann, 1992
). Two of these clusters, which are located in the N-terminal ends of these enzymes, are also present in Y. enterocolitica CpdB. It is to be expected that these two regions have a significant role in enzyme function, possibly representing substrate-binding sites. 5'-Nucleotidases have been characterized from different cellular locations and from a wide variety of species ranging from bacteria to vertebrates. These enzymes hydrolyse 5'-ribo- and 5'-deoxyribonucleotides. Periplasmic bacterial 5'-nucleotidase, also known as UDP-sugar hydrolase, degrades UDP-glucose and other nucleotide diphosphate sugars, producing uridine monophosphate and sugar 1-phosphate. The physiological function of bacterial 5'-nucleotidases is probably to provide a carbon source for the cell (Zimmermann, 1992
). Mosquito apyrase (ATP-diphosphohydrolase) catalyses the hydrolysis of ATP into AMP, preventing ADP-dependent platelet aggregation in the host and thereby facilitating haematophagy (Champagne et al., 1995
).
The finding that Y. enterocolitica, which has high CpdB activity, harbours an ERIC sequence in its promoter region led us to investigate a possible function of ERIC in regulating cpdB gene expression. ERIC sequences are 126 bp repeat elements that have been found exclusively in transcribed regions of genomes, upstream or downstream of open reading frames. ERIC sequences are highly conserved, but locations differ among species. This is also the case for the ERIC element of the cpdB gene, which is present only in Y. enterocolitica strains, and absent in the closely related strains Y. pestis and Y. pseudotuberculosis. Most ERIC sequences have been found in E. coli and S. typhimurium, but their discovery in Yersinia, Klebsiella, Vibrio, Erwinia and Xenorhabdus suggests that they are more widely distributed (Hulton et al., 1991 ). The ERIC sequence in the Y. enterocolitica cpdB promoter region, like all ERIC sequences, contains core inverted repeats which can potentially form a stemloop structure when transcribed. In places where the Y. enterocolitica ERIC sequence differs from the consensus sequence, complementary base changes are seen in the stemloop structure. This, together with the fact that ERIC sequences can be found in both orientations relative to the direction of transcription, indicates that the function is dependent on the secondary structure and not the primary sequence. To date, there have been no reports demonstrating a specific function for ERIC sequences. However, a related repetitive sequence element of E. coli and S. typhimurium, the REP sequence, has been shown to have specific functions, stabilizing upstream mRNA and influencing gene expression (Newbury et al., 1987a
, b
), terminating transcription (Gilson et al., 1986
) or affecting translational coupling (Stern et al., 1988
). We were unable to demonstrate a specific effect on gene expression for the ERIC element in the cpdB promoter region.
The cyclic phosphodiesterase of S. typhimurium and E. coli was shown to be regulated by carbon-source availability. E. coli and S. typhimurium showed an increase in CpdB activity when grown with glucose, glycerol or succinate as the sole carbohydrate source. Mutants that were unable to synthesize cAMP or CRP showed reduced CpdB activity. CpdB of Y. enterocolitica could also be regulated by the cAMP catabolite repression system, since sequences with high similarity to the cAMPCRP complex were identified between positions 513 and 533. The essential binding element of the cAMPCRP complex is the TGTGA motif, which is completely conserved. Furthermore, cultures grown with added glucose downregulated cpdB expression, indicating that cpdB is catabolite-repressed and regulated by the cAMPCRP complex. The cpdB promoters of S. typhimurium and E. coli belong to a group of promoters that are only weakly modulated by cAMPCRP. These promoters have a cAMPCRP-binding site that is very close to the RNA-polymerase-binding site (512 bp upstream of the -35 region). This is not the case for Y. enterocolitica, in which the TGTGA sequence is located 37 bp upstream of the closest putative -35 hexamer. Downstream of the cAMPCRP complex, we found sequences with good agreement with the 28 consensus of E. coli, which directs transcription of flagellar genes (including flagellin), motility and chemotaxis genes. The
28 factor has been shown to promote RNA polymerase binding to this sequence (Helman, 1991
). In Y. enterocolitica, the
28 factor was required for motility but not for fibrillar synthesis or Yop secretion. The promoter regions of the Y. enterocolitica lcrD and myfA genes, which are responsible for the latter two functions, have regions that strongly resemble the
28 consensus sequence. However, these were not recognized by the
28 factor (Iriarte et al., 1995
).
Insertional inactivation of the cpdB gene of Y. enterocolitica showed only a marginal effect on virulence in the orogastric and intravenous mouse infection models. Y. enterocolitica strains of biotype 1B are highly pathogenic for mice. These enteric pathogens use Peyers patches as the port of entry and then disseminate to the spleen and the liver, where they form abscesses. The colonization of Peyers patches and the initiation of infection, as well as the ability to generate systemic infection, were comparable for wild-type and cpdB mutant strains. CpdB does, however, enable Y. enterocolitica to metabolize 2',3'-cAMP. This could give yersiniae and other enterobacteria a selective advantage over bacteria deficient in this enzyme activity, in certain ecological niches or in the infection process. The ubiquitous nature of this enzyme further suggests a scavenging role. We therefore propose that CpdB, like other periplasmic phosphatases, is a fitness factor involved in the salvage of nucleotides from the environment.
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ACKNOWLEDGEMENTS |
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Received 18 September 2000;
accepted 5 October 2000.