Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
    • JB Special Collection
    • JB Classic Spotlights
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JB
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Bacteriology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
    • JB Special Collection
    • JB Classic Spotlights
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JB
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
GENE REGULATION

Complex Regulation of Urease Formation from the Two Promoters of the ure Operon of Klebsiella pneumoniae

Qiong Liu, Robert A. Bender
Qiong Liu
Department of Molecular, Cellular, and Developmental Biology, The University of Michigan, Ann Arbor, Michigan 48109-1048
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Robert A. Bender
Department of Molecular, Cellular, and Developmental Biology, The University of Michigan, Ann Arbor, Michigan 48109-1048
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: rbender@umich.edu
DOI: 10.1128/JB.01096-06
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Klebsiella pneumoniae can use urea as the sole source of nitrogen, thanks to a urease encoded by the ureDABCEFG operon. Expression of this operon is independent of urea and is regulated by the supply of nitrogen in the growth medium. When cells were growth rate limited for nitrogen, the specific activity of urease was about 70 times higher than that in cells grown under conditions of excess nitrogen. Much of this nitrogen regulation of urease formation depended on the nitrogen regulatory system acting through the nitrogen assimilation control protein, NAC. In a strain deleted for the nac gene, nitrogen limitation resulted in only a 7-fold increase in the specific activity of urease, in contrast to the 70-fold increase seen in that of the wild type. The ure operon was transcribed from two promoters. The proximal promoter (P1) had an absolute requirement for NAC; little or no transcription was seen in the absence of NAC. The distal promoter (P2) was independent of NAC, but its activity increased about threefold when the growth rate of the cells was limited by the nitrogen source. Transcriptional regulation of P1 and P2 accounted for most of the changes in urease activity seen under various nitrogen conditions. However, when transcription of ureDABCEFG was less than 20% of its maximum, the amount of active urease formed per transcript of ure decreased almost linearly with decreasing transcription. This may reflect a defect in the assembly of active urease and accounted for as much as a threefold activity difference under the conditions tested here. Thus, the ure operon was transcribed from a NAC-independent promoter (P2) and the most strongly NAC-dependent promoter known (P1). Most of the regulation of urease formation was transcriptional, but when ure transcription was low, assembly of active urease also was defective.

Klebsiella pneumoniae is capable of using a large number of organic and inorganic compounds as sole sources of nitrogen. In almost every case, the production of the machinery for this assimilatory process is regulated by the general nitrogen regulatory (Ntr) system (13). In some cases, this regulation is direct and uses the phosphorylated form of NtrC to activate transcription from promoters that use σ54-dependent RNA polymerase (13). In other cases, including the regulation of urease formation from the ureDABCEFG operon (2, 11), this regulation is mediated by the nitrogen assimilation control protein (NAC), which is itself under the control of NtrC phosphate (3, 11).

NAC has been shown to be an activator of transcription by the σ70-dependent RNA polymerase, thus bringing σ70-dependent operons under the control of the σ54-dependent Ntr system (1). In the best-characterized example of NAC-mediated regulation, the histidine utilization (hut) operon, NAC binds to a site centered at −64 relative to the start of transcription and activates transcription independent of any small molecule or known modification (5). NAC also has been shown to activate from other positions, but in those cases the increase in transcription was less the 10-fold that seen at hut (7, 16, 17).

Macaluso et al. noted an apparent Ntr-independent regulation of urease formation in response to nitrogen limitation (11). At the time, this was the only known example of Ntr-independent nitrogen regulation, and this led us to explore the regulation of urease formation further.

The promoter region of the ure operon (ureDp region) was sequenced (8), and a NAC binding site in this region was identified both by gel mobility shift assay and by DNase I footprinting (5). NAC binds to a region that includes the sequence ATAA-N5-TGNTAT, located 90 bp upstream of the start of ureD translation. If this sequence were centered at −64, then a possible match to the −10 consensus sequence (TATCTT) would provide an arrangement similar to that seen in the hut operon. In a preliminary study of the ure operon from the related species K. oxytoca, Collins and her coworkers (2) used primer extension to search for a transcriptional start site. The transcriptional start site that they reported was only 5 or 6 bp upstream of the start of ureD translation, and this start did not correspond to the putative −10 sequence identified by them by assuming that NAC bound at −64. In fact, the putative −10 sequence would have been located at −30 relative to the start of transcription. Since the DNA sequence of this region from K. oxytoca is the same as that from K. pneumoniae, it was unclear where the promoter lay, and it seemed likely that the start of transcription was farther upstream than the site identified by Collins et al. (2). Moreover, their primer extension suggested that the principal ureD promoter was about 10-fold regulated, whereas their assays of ureDp-lacZ operon fusions suggested that the regulation was 1,000-fold or more.

In their study, larger ureDp fragments, extending 0.7 kb farther upstream, showed considerable transcriptional activity even in the presence of excess ammonium, in which the Ntr system (and NAC) would be nonfunctional (2). It was not determined whether (or how) this activity was regulated. Thus, there were several questions regarding the regulation of urease formation that seemed to suggest that there might be features unique to this NAC-regulated promoter. Where is the principle ureD promoter, and how strong is its regulation by NAC? Where is the upstream promoter, and is it regulated by nitrogen? Is expression from the upstream promoter sufficient to explain the Ntr-independent nitrogen regulation of urease formation?

MATERIALS AND METHODS

Strains and plasmids.All K. pneumoniae strains used in this study were derived from strain W70, which originally was called K. aerogenes (12). Strains used in this study are listed in Table 1. Isolation of single-copy ureDp-lacZ operon fusions was done by a two-step process. First, the fusions were generated in pRS415 (19) using standard methods involving primers, PCR, and cloning. These fusions next were transferred to a pir-dependent plasmid, pCB1583, which is derived from pKAS46 (20), to allow selection of integrants via the landing pad method, similar to that of Wu et al. (22). The specific details of the adaptation of this method to K. pneumoniae strain W70 will be described elsewhere (T. J. Goss and R. A. Bender, unpublished data). The transcriptional fusions then were integrated into strain KC2653 in single copies at the rbs locus.

View this table:
  • View inline
  • View popup
TABLE 1.

List of strains

Media.Minimal medium was W4 salts (W salts adjusted to an initial pH of 7.4) (11) supplemented with glucose (0.4%, wt/vol) as the sole carbon source. Nitrogen sources for nitrogen-limited growth were monosodium glutamate (0.2%, wt/vol), glutamine (0.1% or 0.04%, wt/vol; Calbiochem grade A, freshly made), or potassium nitrate (0.2%, wt/vol). A mixture of ammonium sulfate (0.2%, wt/vol) and glutamine (0.1%, wt/vol) was used as a nitrogen excess condition. Rich medium was L broth (15). When present, isopropyl-β-d-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside were added at final concentrations of 1 mM and 40 μg/ml, respectively. Ampicillin (100 μg/ml), kanamycin sulfate (50 μg/ml), and streptomycin (50 μg/ml) were added to the medium when appropriate. For cultures in which urease activity was measured, 1 μM nickel sulfate was included in the growth medium.

Genetic techniques.Recombinant DNA techniques were carried out as described by Maniatis et al. (14). Transduction was performed as described previously (4) using the phage P1vir.

Enzyme assays.Cultures were grown at 30°C to a density of about 50 Klett units. All assays were performed with cells that had been washed in 1% KCl and suspended at a concentration that contained 1 to 1.5 mg of protein per ml. Assays for β-galactosidase were performed on detergent-treated whole cells as described previously (11). Urease was assayed in the presence of 40 mM potassium phosphate, pH 7.5, 16 μg/ml cetyltrimethylammonium bromide, and 100 mM urea at 30°C. Reactions were initiated by the addition of urea. Portions of the assay mixtures were withdrawn at intervals, and the amount of ammonia released was determined by conversion to indolephenol (21), which was quantified by comparison to a standard curve and by measuring the absorbance at 546 nm. The protein content of whole cells was measured by the method of Lowry et al. (10) with bovine serum albumin as the standard. Specific activities are expressed as nanomoles of product formed (β-galactosidase) or substrate consumed (urease) per minute per milligram of cell protein at 30°C. All data are the results of three or more independent assays with standard errors of the means of less than 10% in every case.

Primer extension.Primer extension analysis was carried out as described by Yu and DiRita (23). Total RNA was isolated from DH5α/pCB1612 (for the full ureDp region), pCB634, or DH5α/pCB1630 (for UreP2) grown to mid-log phase in L broth. A template of 50 μg of total RNA was used in each reaction mixture. Two ureD open-reading-frame-specific primers, ure240 and ure200 (5′-CCGGCCTGGTGAAAGCGGAG-3′ and 5′-CCTGCCAGCCTTTTTTGAGTGGTGGTAAC-3′, respectively), were used for primer extension and for DNA sequencing. The DNA nucleotide sequence was determined for the primer extension by using the chain termination method with the Thermo sequenase radiolabeled terminator cycle sequencing kit (USB) supplied by the manufacturer.

RESULTS

Nitrogen regulation of urease expression: NAC-dependent and NAC-independent components.We cloned a short fragment containing 197 bp of upstream sequence and the first 64 bp of ureD and fused this fragment to a promoterless lacZ gene (see the sequence shown in Fig. 1). Using a landing pad technique similar to that described by Stewart et al. (22; Goss and Bender, unpublished), this operon fusion was transferred to the chromosome at the rbs locus of three K. pneumoniae strains. In the first strain (KC2653), the nac locus was wild type and NAC expression was driven by the native nac promoter and, thus, was regulated by the quality of the nitrogen source. In the second strain (KC6122), the nac locus was deleted and the strain produced no NAC at all. In the third strain (KC6146), the nac locus contained an insertion of Tn5-tac between the nac promoter and the nac coding sequence. In this strain, NAC expression was constitutive (NACConst) when IPTG was included in the growth medium. All three strains contained a wild-type ure operon at the normal chromosomal locus and a ureDp-lacZ fusion at the rbs locus, both in single copies.

FIG. 1.
  • Open in new tab
  • Download powerpoint
FIG. 1.

Sequence of the ureD promoter region. Bent arrowheads indicate the start of transcription from the two promoters (P2 and P1, respectively) in the region. Underlined regions indicate the presumed −35 and −10 regions corresponding to P2. The boxed nucleotides indicate the NAC binding site. The double-underlined nucleotides are the beginning of the ureD coding region. The vertical bars with numbers indicate the endpoints of the deletions and fusions used. Primer 200 and Primer 240 indicate the positions of the 5′ end of the 29-mer and 20-mer primers, respectively, used in primer extension experiments.

As expected, urease formation in the Nac+ strain was about 75-fold higher under conditions of nitrogen limitation than under nitrogen excess (Table 2). In the Nac− strain, most of this regulation was lost, but the Nac− strain still showed a sevenfold increase in urease activity under conditions of severe nitrogen limitation. This suggests that there is a NAC-independent activation of urease expression in response to nitrogen limitation. In the NacConst strain, urease activity was expressed at high levels even under nitrogen excess conditions; however, under nitrogen-limiting conditions the activity was about twofold higher than that under nitrogen excess, again suggesting that nitrogen limitation can increase urease expression even in the presence of NAC.

View this table:
  • View inline
  • View popup
TABLE 2.

NAC dependence of urease activity and ure-lac expression under nitrogen limitation and nitrogen excess growth conditions

In order to determine whether this regulation of urease formation is at the transcriptional or posttranscriptional level, we next analyzed the expression of β-galactosidase from the ure-lac fusion in these strains. In the Nac+ strain, β-galactosidase expression was about 12-fold higher under conditions of nitrogen limitation than under nitrogen excess. In the Nac− strain, most of this regulation was lost, but the Nac− strain still showed a two- to threefold increase in β-galactosidase expression under conditions of nitrogen limitation, again suggesting a NAC-independent expression of β-galactosidase from this promoter region. In the NacConst strain, β-galactosidase was expressed at high levels even under nitrogen excess conditions; however, under nitrogen-limiting conditions it was somewhat higher than that under nitrogen excess, again suggesting that nitrogen limitation can increase expression from this promoter even in the presence of NAC.

In other words, the pattern of β-galactosidase regulation was qualitatively similar to the pattern of urease regulation. However, there were significant quantitative differences. For example, in the Nac+ strain, urease expression was 75-fold higher under nitrogen-limiting conditions than under excess conditions, whereas β-galactosidase expression was only 12-fold higher. Since there appeared to be both a NAC-dependent and a NAC-independent regulation affecting expression, we suspected that there might be more than one promoter in this region, as was found for K. oxytoca (2). We therefore generated a deletion fragment (Fig. 2, P1) that lacked 77 bp of the upstream region. This fragment retains the NAC binding site and all the material between the NAC binding site and the start of the ureD coding region. In the Nac+ strain there was a very strong NAC-dependent regulation of β-galactosidase expression, with at least 370 times more β-galactosidase formed under nitrogen limitation than under nitrogen excess. All of this regulation was NAC dependent, since there was no detectable β-galactosidase expression in the Nac− strain. In the NacConst strain, β-galactosidase expression was constitutive at high levels, and there was no increase under nitrogen limitation.

FIG. 2.
  • Open in new tab
  • Download powerpoint
FIG. 2.

The ureD-lacZ transcriptional fusions used. Solid lines indicate DNA sequences present, and the numbers on the lines indicate the last nucleotide remaining, using the numbering of Fig. 1. P1 and P2 indicate the approximate positions of the two promoters, and SD indicates the approximate position of the Shine-Dalgarno sequence in front of the ureD coding sequence. Full refers to the 262-bp fragment illustrated in Fig. 1. The negative numbers indicate constructs that contain K. pneumoniae DNA upstream of the sequence shown in Fig. 1. lacZ indicates the promoterless lacZ sequence in plasmid pRS415 (19).

A NAC-independent urease promoter, ureDp2 .In an attempt to identify the NAC-independent promoter, we generated another fusion (Fig. 2, P2) that contained only the 77 bp of the upstream region fused to the ureD coding sequence and lacZ. β-Galactosidase was expressed at high levels from P2 in all three strains, and this expression was independent of NAC. However, there was a two- to threefold increase in β-galactosidase expression under nitrogen-limiting conditions not only in the Nac+ strain but also in the Nac− strain and the NacConst strain. Thus, there appears to be two promoters that drive ure operon expression, an operon-proximal promoter, P1, that is very strongly dependent on NAC, and an operon-distal one, P2, that is independent of NAC but still responds to nitrogen limitation by an unknown mechanism.

If the activities of the two promoters were independent of each other, we would expect that β-galactosidase expression from the full-length promoter should be the sum of the expression from the two promoters in isolation. This clearly was not the case. For example, in the Nac− strain, all expression comes from P2 (P1 is inactive in the absence of NAC). However, the isolated P2 promoter gave significantly higher levels of β-galactosidase than did the full promoter (Table 2). One explanation for this discrepancy might be that the long leader between P2 and the start of translation might be responsible for the reduction in expression. To test this, we examined a variant of the P1 construct (P1Δ) (Fig. 1) in which 50 bp of sequence between P1 and the start of translation had been deleted. In this construct, only NAC-dependent transcription should yield β-galactosidase. Again, expression of β-galactosidase was significantly higher in the deleted version than when the intervening sequence was present. Therefore, our working model is that some feature of the sequence between the promoters and the coding sequence is responsible for attenuating the effectiveness of the transcription.

The data in Table 2 suggest that there is a NAC-independent promoter within the 77-bp region present in construct P2 and a NAC-dependent promoter located in the 72-bp region between bp 78 and 149. To determine their locations precisely, we next carried out a primer extension analysis to identify the start of transcription in these regions. The data in Fig. 3 suggest that the transcript coming from P2 begins with the G at bp 48, and the transcript from P1 begins at bp 155 or 156 (T and G, respectively). An analysis of the sequence upstream of bp 48 reveals the sequence AATAAT about 10 bp upstream of the start of transcription (of P2), a reasonable match to the consensus −10 region of σ70-dependent promoters. Eighteen base pairs upstream of that putative −10 region we found the sequence TTGACG, a good match to the consensus −35 region for σ70-dependent promoters. We were concerned that the sequence of the promoter extends only to −47 in our constructs and that we might be missing regulatory information upstream of P2. However, similar constructs with an additional 30 or 253 bp of upstream sequence gave the same pattern of transcription (data not shown).

FIG. 3.
  • Open in new tab
  • Download powerpoint
FIG. 3.

Primer extension of mRNA from the ureD promoter region. Primers extending back to bp 200 and 240 were used to determine the start of transcription. The same primers were used to generate a DNA sequencing ladder. Arrows indicate the start of transcription, and the DNA sequence (complementary to the sequencing ladder but matching the sequence shown in Fig. 1) is indicated. GATC indicates the DNA sequencing ladder. RT indicates the experimental lane with reverse transcriptase present during the reaction. Primer indicates a control reaction with no reverse transcriptase. Note that Primer 200 gave an artifactual signal, somewhat shorter than the actual runoff transcript generated by reverse transcriptase. (A) Primer extension of mRNA isolated from strain DH5α carrying a plasmid with the complete ureDp region (designated Full in Fig. 2) and a plasmid expressing K. pneumoniae nac constitutively. (B) Primer extension of mRNA from DH5α carrying only a plasmid with ureDp2 (designated P2 in Fig. 2).

Upstream from the P1 transcription start site, we found no sequences resembling either a −10 or −35 consensus sequence for σ70-dependent promoters. At best, we noted a sequence, AATCAT, which might represent a −10 consensus sequence, but this was located at −13, a bit farther from the start of transcription than would be expected. The absence of an obvious promoter consensus for P1 is not surprising because, in the absence of NAC, no expression of the promoter was detectable.

Posttranscriptional effects on urease activity.If there were no posttranscriptional effects, the ratio of urease to β-galactosidase (enzyme activity to transcriptional activity) should be constant, at least for a given construct. However, the data in Table 2 contradict this assumption. For example, if we compare the ratio of enzymatic activity from the chromosomally encoded ure operon to transcriptional activity from the full-length fusion, we find that the ratio varies from a low of 0.04 to a high of 0.26. Two features became immediately obvious from the data in Table 2. First, even under conditions in which only P2 was expressed (the Nac− strain), the ratio of urease to β-galactosidase was not constant. Second, in general, the lower the amount of transcription, the lower the ratio of urease to β-galactosidase. Therefore, we examined this result in more detail.

A wild-type strain carrying the full-length ure-lac fusion (to measure transcription) and the wild-type ure operon was grown with three different nac alleles in a variety of nitrogen sources, as described in Materials and Methods, to give a range of transcriptional activities for the ure promoters (Fig. 4). The results of these 20 experiments show clearly that, below 2,000 U of transcription (measured as β-galactosidase), the enzyme activity per transcript (measured as the urease/β-galactosidase ratio) fell almost linearly with transcriptional activity. At amounts of transcription corresponding to 2,000 U of β-galactosidase or more, the value of urease/β-galactosidase more or less leveled off, though the actual value of that ratio was slightly dependent on the growth medium, with the most nitrogen-limited cultures giving the highest ratios (above 0.2). The raw data for this figure are given in Table S1 in the supplemental material. There was considerable scatter in the ratios for cells with high levels of transcriptional activity, but the linearity of the data at lower levels of transcription was clear.

FIG. 4.
  • Open in new tab
  • Download powerpoint
FIG. 4.

Amount of urease activity formed per transcript as a function of transcription amount. Three strains differing only in their nac allele were used, KC2653 (Nac+), KC6122 (Nac−), and KC6146 (Nacconst) (which was grown both with and without IPTG present), and each carried a wild-type ure operon and a ure-lacZ fusion (designated Full in Fig. 2) inserted at the rbs locus. The strains were grown in glucose minimal medium with five different nitrogen sources, resulting in a variety of levels of ure transcription. The specific activity of the urease formed by the cells was determined by direct assay of urease, and the amount of transcription from the ure promoter was determined indirectly by measuring β-galactosidase from the ure-lac fusion. The ratio of urease activity to β-galactosidase activity (urease activity per transcript) was plotted against β-galactosidase activity (amount of transcription). The raw data for the 20 different conditions are given in Table S1 in the supplemental material. All data were the result of three or more assays of independent cultures with standard errors of the means of less than 10% in all cases.

DISCUSSION

Collins et al. (2) showed that ureDp1 is activated as much as 1,000-fold by nitrogen starvation. We show here that this regulation is mediated entirely by NAC. Since there was no detectable transcription from ureDp1 in the absence of NAC, it was not surprising that we were unable to identify a good match to the consensus for −10 or −35 regions. The best we found was a sequence, AATACT, located at −13 rather than −10. Whatever the actual promoter sequence may be, its absolute dependence on NAC is unusual. For all other promoters studied to date, NAC activates expression only 3- to 10-fold, reflecting the fact that those promoters had substantial basal expression in the absence of positive regulators. The ureDp1 promoter also is atypical in the relative positions of RNA polymerase and NAC. When a NAC binding site was placed in front of the lacZ promoter, it activated transcription strongly (10-fold) when the site was centered at −64 and weakly when it was centered at −42, −52, and −54. It was not able to activate transcription when it was positioned at −47, −49, −59, −69, or −74 (17). Thus, the activation from a site in ureDp1 at −47 was unexpected both for its strength and its location.

The earlier work with K. oxytoca (2) strongly suggested the existence of an Ntr-independent promoter for ureD, but its location and regulation were not identified. The primer extension data that identify the ureP2 transcription start site at bp 48 agree well with the presence of a rather good match to a consensus σ70-dependent promoter (TTGACG-N18-AATAAT; located at bp 10 to 40 in Fig. 1). Growth rate limitation by poor nitrogen sources results in a slight (two- to threefold) increase in ureDp2 activity, but our data define only the quantity of the effect (and the fact that it increases with increasing limitation, up to the limit of two- to threefold), not the molecular mechanism. In other words, we are unable to distinguish among models such as growth rate control, stringent response (caused by nitrogen starvation), or a variety of other alternatives. In any event, this transcriptional effect on ureDp2 accounts for only a portion of the Ntr-independent nitrogen regulation of urease formation.

Another portion of that regulation is an indirect effect of the observation that, when transcription of the ure operon is low, the amount of active urease formed per transcript is low. Again, our data define the magnitude of the effect (the amount of active urease formed per transcript is at least sevenfold lower when transcription is 8% of the maximum) but do not address the molecular mechanism of the effect. The ure operon encodes not only the structural genes for the enzyme (ureABC) but also the genes required for nickel incorporation, ureD and ureEFG (21). It is tempting to speculate that it is a limitation for the nickel-incorporating apparatus that restricts the amount of active urease that can be formed. However, our data also are consistent with the hypothesis that at low concentrations of UreA, UreB, and UreC subunits, apoenzyme assembly is defective (9).

What is clear is that the two- to threefold activation of ureDp2 in response to nitrogen limitation is not mediated by NAC or the Ntr system. Strains lacking NAC (Table 2) or NtrC (data not shown) both show the elevation in response to growth-rate-limiting nitrogen sources. However, ureDp1 remains the focus of our future work. Its absolute dependence on NAC for expression is unique, and the position of the NAC binding site relative to the polymerase suggests a very different mechanism for the activation of ureDp1 than for hutP.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant GM47156 from the National Institutes of Health to R.A.B.

FOOTNOTES

    • Received 24 July 2006.
    • Accepted 15 August 2007.
  • Copyright © 2007 American Society for Microbiology

REFERENCES

  1. 1.↵
    Bender, R. A. 1991. Role of the NAC protein in the nitrogen regulation of Klebsiella aerogenes. Mol. Microbiol. 5 : 2575-2580.
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    Collins, C. M., D. M. Gutman, and H. Laman. 1993. Identification of a nitrogen-regulated promoter controlling expression of Klebsiella pneumoniae urease genes. Mol. Microbiol. 8 : 187-198.
    OpenUrlCrossRefPubMed
  3. 3.↵
    Feng, J., T. J. Goss, R. A. Bender, and A. J. Ninfa. 1995. Activation of transcription initiation from the nac promoter of Klebsiella aerogenes. J. Bacteriol. 177 : 5523-5534.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    Goldberg, R. B., R. A. Bender, and S. L. Streicher. 1974. Direct selection for P1-sensitive mutants of enteric bacteria. J. Bacteriol. 118 : 810-814.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    Goss, T. J., and R. A. Bender. 1995. The nitrogen assimilation control protein, NAC, is a DNA binding transcription activator in Klebsiella aerogenes. J. Bacteriol. 177 : 3546-3555.
    OpenUrlAbstract/FREE Full Text
  6. 6.
    Goss, T. J., B. K. Janes, and R. A. Bender. 2002. Repression of glutamate dehydrogenase formation in Klebsiella aerogenes requires two binding sites for the nitrogen assimilation control protein, NAC. J. Bacteriol. 184 : 6966-6975.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    Janes, B. K., and R. A. Bender. 1998. Alanine catabolism in Klebsiella aerogenes: molecular characterization of the dadAB operon and its regulation by the nitrogen assimilation control protein. J. Bacteriol. 180 : 563-570.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    Lee, M. H., S. B. Mulrooney, M. J. Renner, Y. Markowicz, and R. P. Hausinger. 1992. Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. J. Bacteriol. 174 : 4324-4330.
    OpenUrlAbstract/FREE Full Text
  9. 9.↵
    Lee, M., S. B. Mulrooney, and R. P. Hausinger. 1990. Purification, characterization, and in vivo reconstitution of Klebsiella aerogenes urease apoenzyme. J. Bacteriol. 172 : 4427-4431.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193 : 265-275.
    OpenUrlFREE Full Text
  11. 11.↵
    Macaluso, A., E. A. Best, and R. A. Bender. 1990. Role of the nac gene product in the nitrogen regulation of some NTR-regulated operons of Klebsiella aerogenes. J. Bacteriol. 172 : 7249-7255.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    MacPhee, D. G., I. W. Sutherland, and J. F. Wilkinson. 1969. Transduction in Klebsiella. Nature 221 : 475-476.
    OpenUrlCrossRefPubMed
  13. 13.↵
    Magasanik, B. 1996. Regulation of nitrogen utilization, p. 1344-1356. In F. C. Neidhardt, R. Curtis III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, DC.
  14. 14.↵
    Maniatis, T., E. F. Frisch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  15. 15.↵
    Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  16. 16.↵
    Muse, W. B., C. J. Rosario, and R. A. Bender. 2003. Nitrogen regulation of the codBA (cytosine deaminase) operon from Escherichia coli by the nitrogen assimilation control protein, NAC. J. Bacteriol. 185 : 2920-2926.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    Pomposiello, P. J., and R. A. Bender. 1995. Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor. J. Bacteriol. 177 : 4820-4824.
    OpenUrlAbstract/FREE Full Text
  18. 18.
    Schwacha, A., and R. A. Bender. 1993. The nac (nitrogen assimilation control) gene from Klebsiella aerogenes. J. Bacteriol. 175 : 2107-2115.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    Simons, R. W., F. Houman, and N. Kleckner. 1987. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53 : 85-96.
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    Skorupski, K., and R. K. Taylor. 1996. Positive selection vectors for allelic exchange. Gene 169 : 47-52.
    OpenUrlCrossRefPubMedWeb of Science
  21. 21.↵
    Weatherburn, M. W. 1967. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 39 : 971-974.
    OpenUrlCrossRef
  22. 22.↵
    Wu, S. Q., W. Chai, J. T. Lin, and V. Stewart. 1999. General nitrogen regulation of nitrate assimilation regulatory gene nasR expression in Klebsiella oxytoca M5a1. J. Bacteriol. 181 : 7274-7284.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    Yu, R. R., and V. J. DiRita. 1999. Analysis of an autoregulatory loop controlling ToxT, cholera toxin, and toxin-coregulated pilus production in Vibrio cholerae. J. Bacteriol. 181 : 2584-2592.
    OpenUrlAbstract/FREE Full Text
PreviousNext
Back to top
Download PDF
Citation Tools
Complex Regulation of Urease Formation from the Two Promoters of the ure Operon of Klebsiella pneumoniae
Qiong Liu, Robert A. Bender
Journal of Bacteriology Oct 2007, 189 (21) 7593-7599; DOI: 10.1128/JB.01096-06

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Bacteriology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Complex Regulation of Urease Formation from the Two Promoters of the ure Operon of Klebsiella pneumoniae
(Your Name) has forwarded a page to you from Journal of Bacteriology
(Your Name) thought you would be interested in this article in Journal of Bacteriology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Complex Regulation of Urease Formation from the Two Promoters of the ure Operon of Klebsiella pneumoniae
Qiong Liu, Robert A. Bender
Journal of Bacteriology Oct 2007, 189 (21) 7593-7599; DOI: 10.1128/JB.01096-06
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Gene Expression Regulation, Bacterial
Klebsiella pneumoniae
operon
Promoter Regions, Genetic
urease

Related Articles

Cited By...

About

  • About JB
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #Jbacteriology

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0021-9193; Online ISSN: 1098-5530