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Ureases are multisubunit enzymes requiring Ni2+ for activity. The low pH-inducible urease gene cluster in Streptococcus salivarius 57.I is organized as an operon, beginning with ureI, followed by ureABC (structural genes), and ureEFGD (accessory genes). Urease biogenesis also requires a high-affinity Ni2+ uptake system. By searching the partial genome sequence of a closely related organism, Streptococcus thermophilus LMG18311, three open reading frame (ORFs) homologous to those encoding proteins involved in cobalamin biosynthesis and cobalt transport (cbiMQO) were identified immediately 3′ to the ure operon. To determine whether these genes were involved in urease biogenesis by catalyzing Ni2+ uptake in S. salivarius, regions 3′ to ureD were amplified by PCRs from S. salivarius by using primers identical to the S. thermophilus sequences. Sequence analysis of the products revealed three ORFs. Reverse transcriptase PCR was used to demonstrate that the ORFs are transcribed as part of the ure operon. Insertional inactivation of ORF1 with a polar kanamycin marker completely abolished urease activity and the ability to accumulate 63Ni2+ during growth. Supplementation of the growth medium with NiCl2 at concentrations as low as 2.5 μM partially restored urease activity in the mutant. Both wild-type and mutant strains showed enhanced urease activity when exogenous Ni2+ was provided at neutral pH. Enhancement of urease activity by adding nickel was regulated at the posttranslational level. Thus, ORF1, ORF2, and ORF3 are part of the ure operon, and these genes, designated ureM, ureQ, and ureO, respectively, likely encode a Ni2+-specific ATP-binding cassette transporter.
Ureases are Ni2+-requiring metallo-enzymes that have been isolated and characterized from a variety of prokaryotes and eukaryotes (21). The production of ammonia and CO2 from urea hydrolysis by urease has been shown to have a major impact on microbial pathogenesis (4). Genes required for the biogenesis of bacterial ureases are generally arranged as operons, with the structural genes: ureC, ureB, and ureA, encoding the α, β, and γ subunits, respectively, followed by the accessory genes: ureE, ureF, ureG, and ureD, encoding proteins essential for the incorporation of Ni2+ into the metallocenter. Other genes, such as ureI, encoding urea transporters, are found in the urease operons of Helicobacter pylori and Streptococcus salivarius (9, 29). Although Ni2+ is an essential cofactor for the catalytic activity of urease, most known ure operons do not contain genes encoding proteins for Ni2+ transportation. One possible exception is ureH of Bacillus sp. strain TB-90 (20), which shares homology with the high-affinity nickel-specific permease encoded by hoxN of Ralstonia eutropha (12).
Nickel is usually present in trace amounts in the natural environment and is crucial for a number of biological processes, such as hydrolysis of urea, consumption of molecular hydrogen, and methanogenesis. Free nickel is toxic (28); therefore, nickel-specific transporters usually display high affinity but low capacity, presumably to guard against potential toxic effects caused by high levels of intracellular nickel. Thus far, two distinct high-affinity nickel transport systems have been described in prokaryotes: the single-component Ni2+ permeases, which belong to the nickel/cobalt transporter (NiCoT) family, and the Nik systems, which belong to the ATP-dependent binding cassette (ABC) transporter family (14). The most studied, single-component Ni2+-specific permease is HoxN from R. eutropha (13, 15, 30). Similar systems have been identified in other bacteria, including HupN from Bradyrhizobium japonicum (16), NixA from H. pylori (17), and possibly UreH from Bacillus sp. strain TB-90 (20).
The Nik system was originally identified in Escherichia coli and is composed of one periplasmic Ni2+ binding protein (NikA), two hydrophobic transmembrane proteins, NikB and NikC, which are assumed to form the channel for Ni2+ uptake, and two membrane-associated components, NikD and NikE, which contain the conserved signature sequences of ATPases and are believed to be involved in the energy-coupling process for transport (22). The expression of the E. coli nik operon is negatively regulated at the transcriptional level by the NikR repressor protein when intracellular nickel levels are high (11). Similar Ni2+ uptake systems have been identified in Brucella suis (18), Vibrio parahaemolyticus (23), Actinobacillus pleuropneumoniae (2), and Yersinia pseudotuberculosis (25), and in all cases this Ni2+-specific transporter is associated with ureolytic activity of the microorganisms.
S. salivarius is one of the most abundant and highly ureolytic microorganisms in the oral cavity and can use urea as a primary source of nitrogen (5). The expression of urease in S. salivarius is subject to environmental signals, with higher levels of expression in cells grown under acidic conditions, and the induction at acidic pH values can be enhanced by growth in excess amounts of carbohydrate (6). It is believed that regulation by low pH and carbohydrate availability allows for maximal production of the enzyme when it is most needed for survival of the organisms against lethal acidification (5). The urease gene cluster (ure) of S. salivarius is arranged as an operon, beginning with ureI, followed by ureABCEFGD (9). Previous studies indicated that ureABCEFGD are required for assembly of a functional urease. However, a recombinant Streptococcus mutans strain (ACUS6) harboring only the 3′ portion of ureI, starting at the sequence encoding the 65th amino acid of the deduced UreI, and intact ureABCEFGD (10) (Fig. (Fig.1A)1A) requires supplementation with NiCl2 to produce an active urease enzyme. The purpose of the present study was to identify genes involved in nickel uptake by S. salivarius.
S. salivarius 57.I and its UreMQO-deficient derivative were routinely grown in brain heart infusion (BHI; Difco Laboratories) at 37°C in 5% CO2 and 95% air. Recombinant E. coli strains were routinely maintained in L broth. Kanamycin was included in the growth medium, when indicated, at 750 or 50 μg ml−1 for recombinant S. salivarius or E. coli strains, respectively. All chemical reagents and antibiotics were purchased from Sigma. 63NiCl2 (9.87 mCi of Ni mg−1) was purchased from Amersham Biosciences (Piscataway, N.J.). To obtain cultures grown under neutral or acidic conditions, cells were grown in BHI containing 50 mM potassium phosphate buffer (pH 7.4; BHI-KPB), BHI alone, or BHI that had been adjusted to pH 5.8 by the addition of 2 N HCl (BHI-HCl).
Genomic DNA from S. salivarius 57.I was isolated as previously described (8). Plasmid DNA from recombinant E. coli strains was purified by using the QIAprep spin plasmid kit (Qiagen, Inc.). Cloning, Southern blot analysis, and hybridizations were carried out by using an established protocol (1). Restriction endonucleases, DNA polymerases, and RNA reverse transcriptase (RT) were obtained from Invitrogen or New England Biolabs (NEB). Total cellular RNA from S. salivarius strains was isolated as described elsewhere (9). Levels of ure-specific mRNA were quantitated by densitometry using slot blot analysis, with hybridization and washes carried out at high stringency (6).
The region immediately 3′ to the ure cluster was amplified from S. salivarius 57.I by PCRs with primers derived from the Streptococcus thermophilus LMG18311 genome sequences (http://www.biol.ucl.ac.be/gene/genome/). PCRs were initiated with five cycles at a less stringent annealing temperature (50°C), followed by 20 cycles at a more stringent annealing temperature (55°C). All PCR products were cloned onto plasmid pCRII (Invitrogen) and the sequences were determined.
A HindIII-NcoI fragment, 2.9 kbp in size, containing the 3′ portion of ureG, ureD through ureM, and the 5′ portion of ureQ (Fig. (Fig.1A)1A) was initially subcloned from pMC11 (8) into pGEM3Zf(+) to generate plasmid pMC281. A DNA fragment containing a kanamycin resistance marker flanked by transcription/translation terminators (Ωkan) (24) was subsequently cloned into the unique NsiI site, located at the beginning of ureM, on plasmid pMC281. The resulting chimeric plasmid, pMC282, was transferred into strain 57.I by electroporation (9) to introduce a polar mutation in ureM by allelic exchange. The configuration of the double-crossover integration of the kanamycin resistance marker was confirmed by PCR and Southern blot analysis.
A nickel accumulation assay was adopted from Wolfram et al. (30) with minor modifications. Briefly, overnight cultures of S. salivarius strains in BHI were diluted 1:50 into fresh BHI-KPB or BHI containing 500 nM 63NiCl2. Unlabeled metal chlorides as competitors were added to a final concentration of 5 μM. All cultures were grown at 37°C for 5 h at which point the optical density at 600 nm (OD600) of the cultures was ca. 0.9. Cells were harvested, washed twice with an equal volume of ice-cold buffer A (50 mM Tris-HCl, 10 mM MgCl2 [pH 7.5]), and then concentrated 10-fold in the same buffer. The radioactivity of an aliquot of the cell suspension (100 μl) was determined by liquid scintillation counting. To monitor the amount of 63Ni2+ accumulated intracellularly over time, cells were grown in BHI at 37°C for 3 h and 45 min prior to addition of 63NiCl2. A 1-ml aliquot of the cell suspension was harvested every 15 min after addition and processed as described above. The CFU count in the same amount of cell suspension was determined by serial dilution and plating on BHI agar plates. Alternatively, the same amount of cell suspension was subjected to mechanical disruption in the presence of glass beads (0.1 mm in diameter) by homogenization in a Bead Beater (Biospec Products) for a total of 40 s at 4°C. The concentration of each cell lysate was determined by using the Bio-Rad protein assay based on the method of Bradford (3). The cellular content of 63NiCl2 was expressed in picomoles per 109 CFU or picomoles per milligram of protein.
Overnight cultures of the wild-type S. salivarius 57.I and its UreMQO-deficient derivative were diluted 1:20 into fresh BHI-KPB, BHI, or BHI-HCl containing 0, 2.5, 5, 10, 25, 50, 75, or 100 μM NiCl2 and grown to an OD600 of ~0.65. Cultures were harvested by centrifugation, washed once with an equal volume of 10 mM KPB (pH 7.0), and then concentrated 40-fold in the same buffer. Concentrated cell suspensions were subjected to mechanical disruption as described above. Urease activity was measured as described previously (6) and normalized to the protein concentration.
Urease biogenesis by bacteria requires a high-affinity Ni2+ uptake system. By searching the partial genome sequence of a closely related, ureolytic organism, S. thermophilus LMG18311, three open reading frames (ORFs) related to those encoding proteins similar to the ABC-type cobalt transport system found in cobalamin biosynthesis operons (cbiMQO) were identified immediately 3′ to the ure operon. To determine whether these genes were present in S. salivarius, a chromosome walking approach was used to obtain sequences 3′ to ureD by PCRs from S. salivarius with primers identical to the cbiM, cbiQ, and cbiO genes of S. thermophilus. Approximately 3 kbp of sequence immediately 3′ to ureD was obtained from the three overlapping PCR products (Fig. (Fig.1A).1A). Three complete ORFs and an additional partial ORF, all in the same orientation as the ure operon, could be identified within this 3-kbp region. ORF1, with its ribosome binding site embedded in ureD, is located three bases 3′ to the stop codon of ureD. ORF2 overlaps with ORF1 by two bases, and ORF3 is located one base 3′ to ORF2. The partial ORF is located 165 bp 3′ to ORF3. To determine whether these ORFs were cotranscribed with the ure operon, RT-PCR was used to detect the existence of contiguous transcripts between ureD, ORF1, ORF2, and ORF3. The results indicated the presence of contiguous transcript(s) between ureD, ORF1, ORF2, and ORF3, suggesting that all three ORFs could be cotranscribed with the urease genes and were part of the ure operon (Fig. (Fig.1B).1B). To determine whether ORF1, ORF2, and ORF3 could be transcribed independently, presumably via a promoter embedded somewhere within the ureI-D genes, total cellular RNA was isolated from a recombinant S. salivarius strain in which ureC had been insertionally inactivated by allelic exchange (5), and the presence of ORF1-specific mRNA was determined by RT-PCR (Fig. (Fig.1C).1C). When a polar mutation was introduced into ureC, no detectable ORF1-specific transcript was observed by RT-PCR, indicating ORF1, ORF2, and ORF3 were transcribed exclusively from the promoter 5′ to ureC. A polar insertion in ureI also resulted in no detectable ORF1 transcript by RT-PCR (data not shown). A stable stem-loop structure, with ΔG° = −10.3 kcal, followed by a stretch of six T residues, which could potentially function as a rho-independent terminator, was identified seven bases 3′ to the stop codon of ORF3, and no transcript could be detected by RT-PCR between ORF3 and the partial downstream ORF (data not shown), a finding indicating that this partial ORF is not part of the ure operon. The sequence of this partial ORF encoded a truncated peptide that shared significant homology (60 to 70% identity) with the N terminus of the substrate-binding proteins of amino acid ABC-type transporters (AtmA) from other streptococcal species. The lack of involvement of this ORF in urea metabolism was further confirmed by demonstrating that there was no change in urease activity in a recombinant strain in which this ORF was insertionally inactivated (data not shown). ORF1, ORF2, and ORF3 were designated ureM, ureQ, and ureO, respectively, and it was concluded that the ure operon of S. salivarius 57.I consists of 11 genes (ureIABCEFGDMQO). The complete sequence of the ure operon has been deposited with GenBank under accession number U35248.
Even though utilizing an ABC-type Ni uptake system for urease biogenesis is not unique to S. salivarius, the organization of the ure operon of this organism is different from that of other urease-producing bacteria. Specifically, all other known bacterial ABC-type nickel uptake systems that are associated with urease biogenesis are encoded by separate operons, e.g., the nik or cbi operons, and the expression of these operons is generally regulated independently from ure operons (2, 18).
Translation of S. salivarius ureM predicted a protein of 325 amino acids with a pI of 8.99 and a calculated mass of 35.2 kDa. UreM shared significant degrees of similarity with CbiM, which has been suggested to encode an integral membrane protein involved in cobalt transport for cobalamin biosynthesis, from Thermoanaerobacter tengcongensis (47% identity and 64% similarity) and Clostridium acetobutylicum (39% identity and 58% similarity). The conserved domain of the permease component of ABC-type Co2+ transport systems could also be identified within UreM, and three transmembrane helices were predicted by using CBS prediction services (Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark [http://www.cbs.dtu.dk/services/]). S. salivarius ureQ (777 nucleotides) encoded a 29.1-kDa protein with a pI of 9.63. The highest degree of similarity was observed between ureQ and genes predicted to encode cobalt permeases from Lactobacillus plantarum (28% identity and 43% similarity) and from C. acetobutylicum (24% identity and 46% similarity). Eight transmembrane helices were predicted in deduced UreQ sequence by using CBS prediction services. S. salivarius ureO (714 nucleotides) encoded a 27.6-kDa protein with a pI of 5.44. UreO shared 44, 40, and 37% identity to the ATPase components of ABC-type cobalt transport systems (CbiO) of L. plantarum, C. acetobutylicum, and T. tengcongensis, respectively. Significant levels of similarity to the ATP-binding proteins of cobalt ABC transporters from Methanosarcina species were also observed (35% identity). The linker peptide (LSGGEKKK), and the Walker A (GENGTGKS) and Walker B (VYILD) motifs commonly found in many ATP- and GTP-binding and hydrolyzing proteins were also observed in the correct respective positions in the protein.
To examine the impact of UreMQO in urease biosynthesis, a polar mutation (Ωkan) was introduced into ureM, four bases 3′ to the ATG start codon. To ensure that the transcription of the urease operon terminated at the position of Ωkan, total RNA was isolated from S. salivarius wild-type and UreMQO-deficient strains, and RT-PCR was used to detect ureM-specific mRNA (Fig. (Fig.1D).1D). No RT-PCR product could be detected 3′ to Ωkan.
Earlier studies demonstrated that urease expression in S. salivarius is predominantly regulated by growth pH (6, 9). At neutral pH, expression is almost completely repressed. Induction occurs and increases as the growth pH becomes more acidic. To determine whether the capacity to accumulate Ni2+ in both wild-type and UreMQO-deficient strains was also regulated by pH, cells were cultured in BHI-KPB or in BHI as described in Materials and Methods to late-exponential phase, at which point the cultures were at approximately pH 7.0 and 5.5, respectively. When cells were incubated with 500 nM 63NiCl2, it was found that 63Ni2+ accumulation was ca. 3.5-fold higher in the wild-type organisms that were cultured in acidic medium compared to those grown in neutral medium (Fig. (Fig.2).2). In contrast, 63Ni2+ accumulation in the UreMQO-deficient strain was abolished, indicating that UreMQO are essential for the uptake of 63Ni2+ from the environment. The accumulation of 63Ni2+ in wild-type S. salivarius during growth was further confirmed by incubating the cells with different amounts of 63Ni2+, and it was found that the amount of intracellular 63Ni2+ was a direct result of the concentrations of exogenous of 63Ni2+ (Fig. (Fig.33).
The substrate specificity of UreMQO in wild-type S. salivarius was examined by the addition of unlabeled Co2+, Zn2+, Mn2+, Mg2+, and Ni2+ at a 10-fold excess (5 μM) in growth medium containing 500 nM 63Ni2+. It was found that excess amounts of unlabeled Co2+, Zn2+, Mn2+, or Mg2+ chloride did not result in a significant decrease in the amount of 63Ni2+ accumulated (Fig. (Fig.4).4). However, the inclusion of 5 μM unlabeled NiCl2 in the growth medium inhibited the accumulation of 63Ni2+ by 85%, indicating that UreMQO have specificity for Ni2+ ion.
Initial attempts to determine the kinetic parameter of UreMQO in nickel transport by using standard transport assays were unsuccessful, presumably due to the low transport capacity of the system and a high level of nonspecific binding of the Ni2+ to the cell surface. To circumvent this limitation, we used a modified accumulation assay in a time course study, in which a larger size of cell suspension was collected by centrifugation at each time point, followed by two washes with buffer containing 10 mM Mg2+ to remove nonspecifically bound 63Ni2+. It was found that the expression of the S. salivarius urease operon, which is tightly regulated by growth pH (7), was repressed until the pH of the culture reached ~6.5, at which time the urease specific activity increased linearly and peaked during the late exponential phase of growth (Fig. (Fig.5A).5A). To ensure that nickel accumulation was monitored during biogenesis of an active urease, 63Ni2+ was added to the cultures at 3 h and 45 min postinoculation, and the amount of 63Ni2+ was monitored every 15 min afterward. A time-dependent accumulation was observed, and the accumulation reached maximal levels 60 min after the addition of 63Ni2+ (Fig. (Fig.5B).5B). No significant uptake of nickel occurred prior to the induction of the urease operon (data not shown).
It is known that the average concentration of nickel in the natural environment is in the nanomolar range and that the most commonly occurring oxidation state of nickel is Ni(II). When Ni2+ is present at higher concentrations, it can be transported by Mg2+ transport systems (19, 26). To determine the influence of UreMQO in overall urease biosynthesis and whether high concentrations of exogenous Ni2+ could compensate for the deficiency of the Ni2+-specific uptake system, possibly through a Mg2+ uptake system, urease activities were examined in wild-type and UreMQO-deficient strains at different growth pH values, with or without additional NiCl2 (Fig. (Fig.6).6). No detectable urease activity was observed in the UreMQO-deficient strain in the absence of supplemented NiCl2, regardless of the growth pH. When cells were grown at neutral pH, supplementation with as little as 2.5 μM NiCl2 was able to partially restore the ureolytic phenotype in the UreMQO-deficient strain, and the levels of urease activity increased in a NiCl2 concentration-dependent manner. NiCl2-dependent increases in urease activity were also observed in cells grown in BHI and BHI-HCl, and the highest levels of urease activity at each NiCl2 concentration supplied to the system were consistently observed in cells grown in BHI-HCl. In the absence of exogenous NiCl2, urease activity in the wild-type strain was solely regulated by growth pH, with the highest urease activity observed in cells grown in BHI-HCl.
Interestingly, when cells were grown at neutral pH (BHI-KPB), urease activity in the wild-type cells increased in response to increasing concentrations of NiCl2, and the enhancement by NiCl2 reached saturation at 25 μM. The enhancement of urease activity by NiCl2 was less prominent when cells were grown at acidic pH. When cells were grown in BHI without any buffer, the highest level of activation by NiCl2 occurred at 2.5 μM, but there was no significant increase in urease activity with higher concentrations of NiCl2 when cells were grown in BHI-HCl. Therefore, under conditions in which the ureMQO may not be fully induced, the addition of nickel can increase the amount of activated urease, suggesting that nickel uptake is a limiting factor in urease biogenesis at a neutral pH. Under acidic conditions, when expression of the operon is optimal, the accumulation of intracellular Ni2+ via the activity of UreMQO appears to be sufficient for the activation of all translated urease subunits, and additional nickel had no impact on the levels of urease activity. Utilizing the capacity of nickel accumulation as a limiting factor for total urease activity at neutral pH provides an additional level of control for urease expression in S. salivarius.
The expression of urease genes and urease activity in H. pylori is regulated by the availability of nickel (27) and supplementation the growth medium with micromolar levels of NiCl2 leads to higher levels of transcription of the operon. To investigate whether the upregulation of urease activity by NiCl2 in S. salivarius could be mediated at the level of urease gene transcription from pureI, the level of expression was monitored in the recombinant S. salivarius strain PureIcat, which carries a single copy of the pureI-cat fusion at the lacZ locus (7), by measuring chloramphenicol acetyltransferase (CAT) specific activity. No significant differences in CAT activity were observed in cells grown in different concentrations of NiCl2, regardless of the growth pH (data not shown), confirming that higher levels of urease activity in the presence of NiCl2 did not result from elevated levels of ure transcription.
To further confirm that urease gene transcription was not regulated by NiCl2 in S. salivarius, total cellular RNA was isolated from wild-type cells and the UreMQO-deficient strain grown in BHI-KPO4 that was supplemented with 0 to 100 μM NiCl2 and the amounts of ureC- and ureM-specific mRNA were quantitated by slot blot analysis. No significant difference in the levels of ureC-specific mRNA could be detected in the wild-type strain under all concentrations of NiCl2 tested (Fig. (Fig.7).7). Likewise, no differences in ureC-specific mRNA were detected between the wild-type and UreMQO-deficient strains in response to nickel concentrations, indicating that the nickel-responsive activation of urease activity is not mediated at the transcriptional level. As expected, the levels of ureM-specific message in the wild-type strain were not influenced by the amount of NiCl2 in the growth medium (Fig. (Fig.7),7), and there was no detectable ureM signal in the UreMQO-deficient strain (data not shown).
To determine whether posttranscriptional regulation is a factor in the enhancement of urease activity by exogenous NiCl2, we also examined the levels of UreC protein, the α subunit of the urease enzyme, by Western blot analysis with an anti-UreC polyclonal antibody in strains grown in different concentrations of NiCl2. No significant differences in the levels of UreC were noted in the wild-type or UreMQO-deficient strains in response to the amount of NiCl2 added to the medium (data not shown). These results indicate that the absence of detectable urease activity in the UreMQO-deficient strains in the absence of exogenous NiCl2 was due to the inability to transport NiCl2 in the mutant rather than the lack of production of urease subunits and, again, that NiCl2 does not regulate urease expression at the level of transcription or translation.
In conclusion, S. salivarius possess a multicomponent nickel transporter belonging to the ABC transporter superfamily. In contrast to most pathogenic microorganisms, which generally possess more than one nickel uptake systems for all nickel-requiring enzymes, UreMQO is likely to be the only nickel-specific uptake system in S. salivarius. Nevertheless, other metal transport systems, presumably magnesium transporters, could also transport nickel when this metal was supplied in excess.
This work was supported by Public Health Service grant DE10362 from the NIDCR to R.A.B.