Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita, Osaka, 565-0871, Japan
Correspondence
Tetsuya Iida
iida{at}biken.osaka-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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The genome sequences of two EHEC strains isolated from patients in the USA and Japan have been reported (Hayashi et al., 2001; Perna et al., 2001
). The sequences revealed that the urease gene cluster is present on the chromosome of both strains. Urease genes from both strains are identical in genetic organization. The urease genes are composed of three structural genes, ureA, ureB and ureC, and four accessory genes, ureD, ureE, ureF and ureG (Hayashi et al., 2001
; Perna et al., 2001
). Based on the genome sequence, the urease genes are most similar to those of Klebsiella aerogenes (Mulrooney & Hausinger, 1990
; Lee et al., 1992
).
Recently, we reported that, among pathogenic E. coli strains, the urease gene is specifically associated with EHEC strains (Nakano et al., 2001). This finding suggests that the urease gene could be a useful genetic marker for the detection of EHEC strains. In that study, we also demonstrated that most EHEC strains showed no urease activity, despite the presence of the urease gene. Although other studies have shown that urease genes are regulated by environmental signals in various species (Mobley et al., 1995
), the regulation of the urease genes and the urease activity of EHEC remain unclear. Recently, Heimer et al. (2002)
demonstrated that the urease genes of EHEC could be regulated both by Fur and by an unidentified trans-acting factor. However, it is not known why most EHEC strains do not express urease activity.
Here, we analysed the nucleotide sequence of the entire urease gene cluster derived from a urease-activity-positive EHEC strain and compared it with that of the urease-activity-negative EHEC O157 : H7 strain RIMD0509952, the genome sequence of which has been reported (Hayashi et al., 2001; Watanabe et al., 1996
). We demonstrate that the exhibition of urease activity by EHEC strains depends on a specific one-base substitution in the ureD gene.
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METHODS |
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Oligonucleotide primers and PCR conditions.
The sequences of oligonucleotide primers used in this study are described in Table 2. The oligonucleotide primers were constructed from the O157 Sakai genome sequence (Hayashi et al., 2001
). Standard PCR conditions were as follows: after 3 min denaturation, a cycle of denaturation at 94 °C for 30 s, annealing at appropriate temperature for 30 s and extension at 72 °C for 1 min was repeated 30 times; PCR was performed using Gene Taq DNA polymerase (Wako Pure Chemical Industries) and Takara Ex Taq (Takara Bio). PCR conditions for determining the length of the urease gene cluster using primers LA1 and LA2 were as follows: after 3 min denaturation, a cycle of denaturation at 94 °C for 1 min, annealing at 63 °C for 1 min and extension at 68 °C for 10 min was repeated 30 times; PCR was performed using LA-PCR Kit (version 2; Takara Bio). PCR conditions for the amplification of the entire urease gene cluster using primers LA3 and LA4 have been described by Heimer et al. (2002)
. PCR reactions were performed in a total reaction volume of 50 µl.
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Nucleotide sequence analysis.
Nucleotide sequences were determined by using the BigDye Terminator Cycle Sequencing Kit version 3.1 and an Avant-3100 automatic DNA sequencer (Applied Biosystems). DNASIS Software (version 3.7, Hitachi Software Engineering) was used to analyse the DNA sequence data. The sequences were analysed using the BLAST program available on the National Center for Biotechnology Information website.
RT-PCR.
Bacterial strains were grown in M9 medium with 1 µM NiCl2 until mid-exponential phase (OD600 0·6) and total RNA was extracted using a RNeasy Mini Kit (Qiagen). To remove the genomic DNA in extracted RNA, total RNA was treated with DNase I according to the manufacturer's recommendations (Takara Bio). The RNA LA-PCR kit (AMV) version 1.1 (Takara Bio) was used for RT-PCR detection of ureD mRNA, according to the manufacturer's recommendations. The oligonucleotide primers RT1 and RT2, used to amplify ureD, are described in Table 2.
Test for urease activity.
To test for the expression of urease activity, we used urease agar base (Merck). In brief, EHEC strains were cultured in 2 ml LB broth at 37 °C with shaking for 1216 h. Cells were inoculated onto urease agar and incubated overnight at 37 °C. EHEC strains with urease activity cause the medium to turn red. To calculate the urease activity, we used an Ammonia Test Kit (Wako Pure Chemical Industries) as described by Park et al. (2000). In brief, bacterial strains were cultured in 5 ml M9 medium with 1 µM NiCl2 at 37 °C for 18 h, and cells were then disrupted by sonication. The supernatant was used in the urease assay, and also to determine the total protein content using the Coomassie Plus Protein Assay Reagent kit (Pierce). Urease activity was expressed as nmol urea hydrolysed min1 (mg protein)1.
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RESULTS |
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Cloning of the entire urease gene cluster from the Ure+-1 strain
To investigate why O157 Sakai does not express urease activity despite the possession of the urease genes, we first examined the size of the entire urease gene cluster (from ureD to ureG) of 120 EHEC strains out of 158 isolates from various sources. Among these strains, we found two urease-activity-positive EHEC strains. We used PCR with primers LA1 and LA2, which hybridize with the ureD and ureG genes, respectively, to estimate the length of the entire urease gene cluster. The PCR products of all strains, including the two urease-activity-positive strains, were identical in size (4940 bp) to that of O157 Sakai (data not shown). These results indicate that the 120 EHEC strains have urease gene clusters of similar size, irrespective of their urease activity.
To compare the nucleotide sequence of the urease gene cluster derived from a urease-activity-positive EHEC strain, Ure+-1, with that of O157 Sakai (GenBank accession no. BA000007; Hayashi et al., 2001), we cloned the
7 kb KpnIEcoRI fragment containing the entire urease gene cluster from the Ure+-1 strain (pUreAG, Table 1
). The nucleotide sequence of the entire urease gene cluster revealed that its genetic organization was the same as that of O157 Sakai, containing seven genes in the order ureD, ureA, ureB, ureC, ureE, ureF and ureG, transcribed in the same direction. Except for ureD, the length of each gene from ureA to ureG in the Ure+-1 strain was identical to that of the corresponding gene of O157 Sakai, and the nucleotide and amino acid sequences were highly conserved between the two strains (over 99 % identity). However, it was revealed that the ureD gene of the Ure+-1 strain had a longer ORF compared with that of O157 Sakai (Fig. 1
a).
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We investigated the nucleotide sequences of ureD in 158 EHEC strains (see Methods). We used PCR with primers D1 and D2 (Table 2, Fig. 1a
), which hybridized on either side of the premature stop codon in ureD of O157 Sakai, and analysed the nucleotide sequences of the amplified products. All the tested urease-activity-negative strains isolated from different sources (human, bovine, deer, sheep and seagull), had sequences identical to that of O157 Sakai (data not shown). We also analysed the nucleotide sequences of ureD in another urease-activity-positive EHEC strain (RIMD05091546) isolated from a human patient. This strain had an identical sequence to that of the Ure+-1 strain (data not shown). Furthermore, the nucleotide sequence of ureD in EHEC strain EDL933 (GenBank accession no. AE00517H; Perna et al., 2001
) was identical to that of O157 Sakai. These results indicate that ureD containing a premature stop codon, as found in O157 Sakai, is common among urease-activity-negative EHEC strains. Therefore, we speculated that the presence or absence of the premature stop codon could be relevant to the expression of urease activity in EHEC strains.
ureD is transcribed in O157 Sakai in vitro
To investigate whether ureD of O157 Sakai was transcribed in vitro, we carried out RT-PCR specific for ureD, using primers RT1 and RT2 (Table 2) to detect ureD mRNA. The mRNA was detected in O157 Sakai, as it was in the Ure+-1 strain (Fig. 2
). This result indicates that the ureD gene is transcribed in O157 Sakai irrespective of the lack of urease activity.
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DISCUSSION |
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Although we detected substantial urease activity in MS-1 [700±90 nmol NH3 min1 (mg protein)1], we detected only low activity in MS-2 [65±20 nmol NH3 min1 (mg protein)1]. These results indicate that the urease activity observed in MS-1 was due to the amber suppressor phenotype of the host strain. In this strain, the amber stop codon at nt742 of ureD could be read through and translation could proceed to next ochre or opal stop codon (Garen, 1968). This resulted in the production of a full-length form of UreD, leading to the formation of active urease.
Regulation of the urease genes in EHEC is not well understood. Recently, Heimer et al. (2002) reported that the putative Fur boxes are present upstream of the urease operon, and that Fur is involved in the regulation of expression of the urease activity. Except for the Fur boxes, no other regulatory elements are known in the upstream region of the EHEC urease operon. From the results showing that the EHEC ure operon introduced in E. coli DH5
produced active urease, Heimer et al. (2002)
postulated that an unidentified trans-acting factor also regulates ure expression. The phenomenon observed by those authors can be explained by our finding in the present study, i.e. the production of active urease in E. coli DH5
is due to the amber suppressor phenotype of the E. coli DH5
strain.
Previous studies showed that very few urease-activity-positive EHEC strains are isolated from patients or cattle (Nakano et al., 2001; Tutenel et al., 2003
). Nevertheless, the present study revealed that EHEC strains retain the complete urease gene cluster, irrespective of their urease activities. The nucleotide sequence at nt742 of ureD in O157 Sakai is well conserved in the urease-activity-negative EHEC strains tested. At present, it is unclear why most EHEC strains carry the inactive form of ureD. Synthesized bacterial urease accumulates in the cytoplasm (Mobley et al., 1995
). If EHEC strains were to continuously synthesize the active form of urease, ammonia produced by hydrolysis of urea could accumulate in the cytoplasm, leading to the elevation of pH in bacterial cells. Thus, most EHEC strains might retain the short form of ureD to prevent the damage of bacterial cells by alkaline conditions caused by ammonia. Since it is known that urease is essential for some bacteria to use urea as the nitrogen source (Chen et al., 2000
), producing active urease could be beneficial for EHEC strains under certain circumstances in natural environments. One possible explanation is that active urease would be produced by EHEC strains by a mutation at the nt742 site of ureD. The putative mutation at nt742 might be spontaneous or caused by a so far unknown genetic mechanism in the organism. Alternatively, under certain circumstances, the amber stop codon at nt742 could be somehow read through in EHEC strains. These possibilities are to be explored in the future.
Phenomena which turn on/off the ureolytic activity of bacteria by a one-base alteration have been reported in Yersinia pestis and Helicobacter pylori (Hansen & Solnick, 2001; Sebbane et al., 2001
). In Y. pestis, ureolytic activity depends on the number of guanine (G) residues (poly G tract) in ureD, and in H. pylori it depends on a one-base insertion in ureA. It is not known whether a specific nucleotide substitution at nt742 of ureD occurs in EHEC strains. If it does occur, the mechanism should differ from those of Y. pestis and H. pylori because the type of sequence alteration is obviously different.
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ACKNOWLEDGEMENTS |
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Received 28 April 2004;
revised 6 July 2004;
accepted 14 July 2004.
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