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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Urea is an important nitrogen source for many microorganisms, but urea active transporters have not been characterized at a molecular level in any bacterium. Cells of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 exhibited the capacity to take up [14C]-urea from low-concentration (<1 μM) urea solutions. The Ks of Anabaena cells for urea was about 0.11 μM, and the observed uptake activity involved the transport and metabolism of urea. In contrast to urease, which was constitutively ex-pressed, expression of the high-affinity urea uptake activity was subjected to nitrogen control. In an Anabaena ureG (urease) mutant, a concentrative, active transport of urea could be demonstrated. We found that a mutant of open reading frame (ORF) sll0374 from the Synechocystis genomic sequence lacked urea transport activity. This ORF encoded a conserved component of an ABC-type transporter, but it is not clustered together with any other possible transporter-encoding gene. An Anabaena homologue of sll0374, urtE, was isolated and found to be part of a cluster of genes, urtABCDE, putatively encoding all the elements of an ABC-type permease. Although the longest transcript that we could detect only covered urtABC, the impairment of urea transport by inactivation of urtA, urtB or urtE suggested that the whole gene cluster is expressed producing the urea permease. Expression was induced under nitrogen-limiting conditions, and a complex promoter regulated by the cyanobacterial global nitrogen control transcription factor NtcA was found upstream from urtA. Our work adds urea to the known substrates of the versatile class of ABC-type transporters and suggests the involvement of a transporter of this superfamily in urea scavenging by some bacteria in natural environments.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Urea metabolism has been widely investigated, as the urea cycle is central to nitrogen metabolism in animals. Additionally, both higher plant (jack bean) and microbial ureases have been intensively characterized. In contrast, little is known about mechanisms of urea transport across biological membranes, probably reflecting the fact that diffusion of urea across lipid bilayers takes place readily, making it difficult to investigate urea transport. Nonetheless, urea channels have recently been characterized. The bacterium Helicobacter pylori expresses a urea-specific, H+-gated channel, the product of the ureI gene (Weeks et al., 2000), and channel proteins of the major intrinsic protein (MIP) family, which exhibit specificity for small neutral molecules including water, glycerol and urea, are ubiquitous (Saier, 1999). Flux through these channels, perhaps along with diffusion, may represent a method of urea translocation across biological membranes when this compound is present at relatively high concentrations (e.g. >1 mM) inside or outside the cells.

Using urea as a nutrient may pose a different problem as, in this case, urea has to be taken up from the environment where it may be found at low concentrations (e.g. in the micromolar range). Urea can serve as a nitrogen source for many microorganisms including some bacteria, yeast, fungi and algae. Although some yeasts and algae degrade urea by means of the ATP- and biotin-dependent enzyme urea amidolyase, most microorganisms use the Ni2+-dependent urea amidohydrolase or urease, an intracellular enzyme that catalyses the hydrolysis of urea, finally rendering CO2 and two molecules of ammonia, and exhibits a KM for urea generally above 1 mM (Mobley and Hausinger, 1989). Transporters with a relatively high affinity for urea (Ks in the range 10–40 μM) have been physiologically characterized in yeast (Cooper and Sumrada, 1975), fungi (Pateman et al., 1982), algae (McCarthy, 1972; Syrett and Bekheet, 1977) and a few bacteria (Healey, 1977; Jahns et al., 1988; Siewe et al., 1998). A common feature of the urea uptake activities reported for these organisms is that they are derepressed under nitrogen limitation. Only the urea active transporter of Saccharomyces cerevisiae has been characterized to date at the molecular level (ElBerry et al., 1993). This transporter is the product of the DUR3 gene and consists of a polypeptide that exhibits 14 putative transmembrane domains. In this report, we present the first molecular characterization of a bacterial urea active transporter, an ABC-type permease that we have identified in cyanobacteria.

The cyanobacteria are phototrophic eubacteria that perform oxygenic photosynthesis and fix CO2 through the Calvin cycle. The nitrogen sources most commonly used by these organisms are ammonium, nitrate and urea and, in some species, dinitrogen. Although the cyanobacterial assimilatory nitrogen metabolism has been widely investigated (Flores and Herrero, 1994), information on urea assimilation is scarce and mostly related to urease (Ge et al., 1989; Argall et al., 1992; Jahns et al., 1995; Collier et al., 1999; Palinska et al., 2000). In cyanobacteria, expression of genes encoding the assimilatory systems for sources of nitrogen alternative to ammonium, such as nitrate or N2, is subjected to positive regulation mediated by NtcA, a transcription factor of the CAP family (Herrero et al., 2001). In the absence of ammonium, NtcA activates transcription from promoters that bear a –10 box (TAN3T) and an NtcA binding site (GTAN8TAC), which is most frequently centred at –41.5 with regard to the transcription start point (Luque et al., 1994).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Assimilation of urea

Cells of Synechocystis sp. PCC 6803, a non-nitrogen-fixing unicellular cyanobacterium, and of Anabaena sp. EF116, a Fox (non-nitrogen-fixing under aerobic conditions) mutant of the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120, grew on urea at rates (μ, 0.84 and 0.82 day–1 respectively) close to those observed with nitrate (1.03 and 1.01 day–1 respectively) as the nitrogen source. However, no urea-dependent growth was detected with the non-nitrogen-fixing unicellular cyano-bacterium Synechococcus sp. PCC 7942. Whereas the latter strain showed no detectable urease activity (see also Berns et al., 1966), strains PCC 6803 and PCC 7120 exhibited urease activities, determined in permeabilized cells, of about 3.6 and 1.6 U mg–1 chlorophyll a (Chl) respectively. No significant differences in urease activity were observed when ammonium-grown Ana-baena or Synechocystis cells were incubated for 24 h with ammonium, urea, nitrate or no source of combined nitrogen. Constitutive urease expression was also observed for two other cyanobacteria that were tested, Calothrix sp. PCC 7601 and Pseudanabaena sp. PCC 6903 (not shown). Consistent with a lack of nitrogen control, urease activities in strain CSE2, an ntcA mutant of Anabaena sp. PCC 7120 (Frías et al., 1994), were similar to those observed with the wild type for the different nitrogen sources. The KM of urease for urea, determined in permeabilized Anabaena cells, was 2.5 mM, and the urease activity was inhibited by more than 95% by a 40 min incubation of the Anabaena cells with 100 μM acetohydroxamic acid, a nickel chelator that has been shown to inhibit urease (Mobley and Hausinger, 1989).

Urea uptake

Cells of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 took up [14C]-urea efficiently when this substrate was supplied at a low concentration (≤1 μM). In contrast to urease, uptake activity at a low (1 μM) concentration of [14C]-urea was subjected to nitrogen control. Activity was highest in cells incubated in the absence of a source of combined nitrogen and lowest in cells incubated with ammonium or urea as a nitrogen source, and it was about fivefold lower in the ntcA mutant strain CSE2 (Table 1). The rate of [14C]-urea uptake was determined for urea concentrations of 0.05–1 μM in Anabaena cells that had been induced by incubation for 10 h with no source of combined nitrogen. Uptake was linear for 3– 10 min, depending on the concentration of substrate, and a Ks value of 0.11 μM and a Vmax of about 4–5 nmol min–1 mg–1 Chl were obtained. Detection of a regulated high-affinity uptake activity and of a high-affinity kinetic component suggested the presence of a urea permease in these cells.

Table 1. High-affinity [14C]-urea uptake in Anabaena sp.
 [14C]-urea uptake rate (nmol min–1 mg–1 Chl)
Nitrogen sourceStrain PCC 7120Strain CSE2 (ntcA)
  1. Ammonium-grown cells were incubated for 10 h in BG110 medium with 17.6 mM NaNO3, 2 mM NH4Cl, 2 mM urea or no source of combined nitrogen. The cells were then harvested, washed and used in 10 min uptake assays with 1 μM [14C]-urea as described in Experimental procedures. Data are the mean of three independent determinations with similar results (standard deviation about 22% of the mean for the data for strain PCC 7120 and about 33% of the mean for the data for strain CSE2).

Urea1.240.16
NH4+1.300.23
NO32.960.66
–N4.960.97

In Anabaena cells treated for 1 h with 150 μM acetohydroxamic acid, a Ks (urea) similar to that obtained with untreated cells was found, but Vmax was about 50%, suggesting that, in addition to transport, urea uptake involved metabolism through urease. Consistent with this observation, the radioactivity in the soluble fraction of extracts of Anabaena cells that had been incubated for 15 min with 1.1 μM [14C]-urea, analysed by thin-layer chromatography (TLC), was distributed among several radioactive spots (not shown).

When urea concentrations of 10 and 50 μM, which are above the saturation level for the high-affinity permease (i.e. above about 5 μM), were tested, no further increase in the rate of uptake was observed for acetohydroxamic acid-treated cells. However, higher than expected rates of uptake were observed for untreated cells. These urease-dependent increased uptake levels might correspond to diffusion of urea, but we did not pursue investigation of this phenomenon. Because some metabolism of urea was still observed in the acetohydroxamic acid-treated cells (not shown), we sought to isolate an Anabaena urease mutant to facilitate study of the high-affinity urea transporter.

Urea transport in a ureG mutant

[14C]-Urea uptake experiments (2 h incubation, 1 μM [14C]-urea as substrate) carried out with a collection of Tn-1063a-induced Anabaena mutants (see Experimental procedures) identified a clone, strain CSAV56, which exhibited <2% of the wild-type urea uptake activity, lacked urease activity and carried the transposon inserted into a gene encoding a protein homologous to UreG, an accessory protein required for the production of an active urease (Mobley et al., 1995). To corroborate that the impairment in [14C]-urea uptake resulted from inactivation of ureG, the Anabaena sp. PCC 7120 genomic region carrying this gene was cloned and mutated. A plasmid, pCSAV3, bearing a 4.6 kb insert of Anabaena DNA that carried the ureC, ureE, ureF and ureG genes was isolated, gene cassette C.S3 was introduced into pCSAV3 substituting for part of the ureG gene, and the resulting construct was used to generate an Anabaena mutant, strain CSAV1, which was homozygous for the mutant ureG::C.S3 chromosomes (see Experimental procedures). Strain CSAV1 lacked any detectable urease activity and was impaired in the uptake of 0.1 μM [14C]-urea (2.8% of wild-type activity). These results confirmed that the ureG mutation causes an impairment in [14C]-urea uptake.

The time course of uptake of 0.1 μM [14C]-urea was investigated in strain CSAV56 (ureG::Tn1063a) and compared with that of the wild type (Fig. 1). Whereas uptake in the wild type proceeded for at least 45 min, the capacity for uptake in the mutant was saturated after 1 min. (In order to observe retention of radioactivity from [14C]-urea in the mutant, the step of washing the cells on the filter after the uptake assays was avoided in these experiments.) We suggest that, whereas urea metabolism permits a high level of urea uptake in the wild type, in the mutant, net [14C]-urea uptake will proceed only until the urea accumulated inside the cells reaches the highest level permitted by the capability of concentration of the transport system.

image

Figure 1. Time course of [14C]-urea uptake in Anabaena sp. strains PCC 7120 and CSAV56 (ureG::Tn1063a). Nitrate-grown cells were incubated in BG110 medium for 10 h, harvested, washed and used in assays of uptake of 0.1 μM [14C]-urea as described in Experimental procedures. The cell suspensions contained 1 μg ·ml–1 Chl, and the cells were not washed after the filtration step. Note the different scales for the two graphs.

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To characterize this high-affinity [14C]-urea transport activity further, we tested the effect of the FoF1ATPase inhibitor DCCD on the transport activity and analysed the fate of the [14C]-urea taken up. In these experiments, nitrate-grown cells incubated for 10 h in BG110 medium (which lacks any source of combined nitrogen) were used, the washing of the cells after the filtration step was avoided, and retention of radioactivity by boiled cells was used as a blank. Uptake of [14C]-urea by Synechococcus sp. PCC 7942, a strain unable to assimilate urea (see above), was also tested as a negative control. Using 1 μM [14C]-urea as a substrate in 4 min uptake assays, no transport activity was detected for strain PCC 7942, whereas activities of 1.52 and 17.5 nmol mg–1 Chl were observed for strains CSAV56 and PCC 7120 respectively. Incubation of cells of strain CSAV56 for 10 min with 50 μM DCCD led to a 97% inhibition of urea transport (tested with 1.1 μM [14C]-urea for 4 min). The fate of [14C]-urea taken up by strain CSAV56 was analysed by TLC. Only one spot that could be identified as [14C]-urea was detected in the soluble fraction of cells that had been incubated with 1.1 μM [14C]-urea for 15 min (not shown). The ratio of intracellular to extracellular concentration of [14C]-urea at the end of the incubation was close to 20. These results demonstrated that Anabaena sp. PCC 7120 and its derivative strain CSAV56 bear a concentrative, active, high-affinity urea transporter that is not present in Synechococcus sp. PCC 7942.

Cloning of the urt genes

We have reported previously the inactivation of some open reading frames (ORFs) from the genome of Synechocystis sp. PCC 6803 whose putative products are homologous to elements of amino acid transporters from other biological sources (Montesinos et al., 1997). Inactivation was achieved by insertion of the C.K3 gene cassette (encoding resistance to kanamycin–neomycin) and, whereas some of the generated Synechocystis mutants were indeed impaired in amino acid transport, some others were not (Montesinos et al., 1997). We checked the latter mutants for transport of other possible substrates and found that a Synechocystis strain mutated in ORF sll0374 exhibited <2% of the wild-type urea transport activity (tested with 1.6 μM [14C]-urea in 10 min assays). This ORF would encode a conserved component of an ABC-type transporter, but it is not clustered together with other possible transporter-encoding ORFs in the Synechocystis genome (Kaneko et al., 1996).

The Anabaena sp. PCC 7120 genomic region carrying a homologue of Synechocystis sll0374 was identified by hybridization, cloned and sequenced. The insert of plasmid pCSAV8 carried a whole ORF homologous to sll0374 and part of an ORF that would encode another ABC-type transporter conserved component (Fig. 2). Plasmids pCSAV90 and pCSAV98 were then isolated as described in Experimental procedures, and their inserts were sequenced and found to cover a region of the Anabaena genome including five ORFs that would encode the elements of an ABC-type permease. Because they are involved in urea transport (see below), we have named these ORFs as urt genes (Fig. 2; nucleotide sequence accession number AJ271599). The proteins of known function that show highest similarities to the Anabaena urt gene products are the products of the Pseudomonas aeruginosa bra genes, which encode an ABC-type permease for branched-chain amino acids (Hoshino and Kose, 1989; 1990). UrtA is homologous to the periplasmic substrate-binding protein BraC (25% identity), UrtB to the transmembrane protein BraD (26% identity), UrtC to the transmembrane protein BraE (25% identity), UrtD to the ATP-binding subunit BraF (31% identity) and UrtE to the ATP-binding subunit BraG (38% identity). The P. aeruginosa bra genes constitute an operon with a structure similar to that of the Anabaena urt gene cluster. With regard to Synechocystis sp. PCC 6803, the product of sll0374, the ORF that was used as a probe to identify the Anabaena urt gene cluster, would be homologous to UrtE (72% identity), the product of slr0447 to UrtA (65% identity), that of slr1200 to UrtB (62% identity), that of slr1201 to UrtC (59%) and that of sll0764 to UrtD (61% identity). Of these ORFs, only slr1200 and slr1201 are clustered together in the Synechocystis genome. Gene clusters that would encode proteins homologous to the Urt proteins are also present in the genomes of the heterocyst-forming cyanobacterium Nostoc punctiforme and the unicellular marine cyanobacteria Syne-chococcus sp. WH8102 and Prochlorococcus sp. strains MED4 and MIT9313 (http://www.jgi.doe.gov/). Interestingly, in these marine strains, the putative urt genes are clustered together with urease-encoding genes.

image

Figure 2. The urt gene cluster of Anabaena sp. PCC 7120 (nucleotide sequence accession number AJ271599). The parts of the genomic region covered by the inserts of plasmids pCSAV8, pCSAV90 and pCSAV98 are indicated. The location of the urtA/urtB probe used to identify a clone carrying pCSAV98, the restriction sites used to insert the C.S3 gene cassette and the place where a HindIII site was created in urtE to insert the C.K3 gene cassette are also indicated. The urtC and urtD genes show a 26 bp overlap.

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urt and ureG urtE mutants

To test the involvement of the urt gene cluster in urea transport in Anabaena sp. PCC 7120, some of the urt genes were inactivated. The C.S3 gene cassette, encoding resistance to Sm and Sp and carrying transcriptional terminators, was inserted into urtA (with the concomitant deletion of a 241 bp fragment) and urtB, and the C.K3 gene cassette, encoding resistance to Km and Nm, was inserted into urtE. The resulting constructs were transferred to Anabaena sp. PCC 7120, and clones homozygous for urtA::C.S3, urtB::C.S3 and urtE::C.K3 mutant chromosomes were obtained and named strain CSAV4, CSAV5 and CSAV2 respectively. The three mutant strains showed very low [14C]-urea uptake activities, confirming that the urt genes encode a high-affinity urea transporter (Table 2).

Table 2. Uptake of [14C]-urea in Anabaena urtA, urtB, urtE and ureG urtE mutants.
StrainGenotype[14C]-Urea uptake (nmol min–1 mg–1 Chl)
  1. Nitrate-grown cells were incubated for 10 h in BG110 medium and used in 7 min uptake assays with 0.1 μM [14C]-urea. The cells were not washed after the filtration step. Data are the mean of three independent experiments with similar results (standard deviation 13% of the mean for strain PCC 7120 and about 33% of the mean for the mutants).

PCC 7120Wild type1.96
CSAV4 urtA 0.06
CSAV5 urtB 0.05
CSAV2 urtE 0.05
CSAV3 ureG, urtE<0.01

However, some radioactivity from [14C]-urea was still incorporated by the urt mutants. To test whether this incorporation was the result of some uptake, perhaps unspecific, pulled by urease, we generated a urt ure double mutant. Plasmid pCSAV12 (ureG::C.S3; see above) was conjugated to strain CSAV2 (urtE::C.K3), and a clone homozygous for the chromosomes carrying the second mutation (ureG::C.S3) was obtained and named strain CSAV3. This strain showed negligible urea uptake activity (Table 2), suggesting that the identified urt and ure genes are the only genes specifically involved in urea assimilation in Anabaena sp. PCC 7120.

Expression of urt genes

The expression of the urt genes was investigated by Northern analysis using probes of each of the urt genes and RNA isolated from Anabaena cells grown with ammonium or grown with ammonium and incubated for 3 or 6 h with no combined nitrogen, nitrate or urea. Hybridization was observed only with the urtA probe (Fig. 3), and the size of the hybridizing band, 1.6 kb, could correspond to a transcript covering the urtA gene (1305 bp). However, the effect of the urtB and urtE mutations (see Table 2) suggests that the whole gene cluster is expressed. We therefore tried to identify a longer transcript by reverse transcriptase–polymerase chain reaction (RT-PCR). Retrotranscription was performed with oligonucleotides corresponding to the urtC (URT6 primer), urtD (URT2 primer) and urtE (URT28 primer) genes, and PCR of the generated cDNAs was carried out with an oligonucleotide corresponding to urtA (URT15, forward primer) and the same primers used for retrotranscription (see Fig. 4). We only observed amplification of the retrotranscription product generated with the urtC primer, for which an amplified fragment of 1.7 kb was expected (Fig. 4). Amplification was strictly dependent on the presence of RNA in the reaction (see Fig. 4, sample 2) and therefore could not be attributed to any contaminating DNA. The results indicate co-transcription of at least urtA, urtB and urtC.

image

Figure 3. Northern analysis of the expression of the urtA gene.

A. RNA was isolated from cells of Anabaena sp. PCC 7120 grown with ammonium (NH4+) or grown with ammonium and incubated for 3 or 6 h with no source of combined nitrogen (–N), nitrate (NO3) or urea.

B. RNA was isolated from cells of Anabaena sp. PCC 7120 and the ntcA mutant, strain CSE2, grown with ammonium (0) or grown with ammonium and incubated for 3 or 6 h in a medium lacking combined nitrogen (3, 6). The urtA probe was generated by PCR using pCSAV98 as a template and oligonucleotides URT11 and URT18 as primers. The size of the hybridization band is indicated in kb. The filter used in panel B was also hybridized with a probe of the RNase P RNA gene, rnpB, as a loading and transfer control.

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image

Figure 4. RT–PCR analysis of expression of the urt gene cluster. RNA isolated from cells of Anabaena sp. PCC 7120 grown with ammonium and incubated for 3 h without any source of combined nitrogen was used for retrotranscription with primer URT6, URT2 or URT28, and the products of these reactions were subjected to PCR with oligonucleotide URT15 (forward primer) and the same primer used for retrotranscription as reverse primer. The PCR products were then subjected to Southern analysis using an urtC probe. Amplification was only observed for the reaction with URT6, which gave rise to a DNA fragment of the expected size, 1.7 kb (sample 1). Control assays in which the RNA preparation was treated with RNase I (sample 2) or in which the PCR reaction was carried out with the same primers and strain PCC 7120 DNA (sample 3) are also presented.

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The results in Fig. 3A also show that the expression of urtA is subjected to nitrogen control. Quantification of the hybridization signals indicated a five- and twofold induction by incubation for 3 h with no combined nitrogen and nitrate respectively. To test whether this induction was dependent on the NtcA transcriptional regulator, Northern analysis was carried out with RNA isolated from an Anabaena ntcA mutant, strain CSE2. Induction was impaired in the mutant, which showed only a basal level of urtA expression (Fig. 3B).

Promoter structure

The putative transcription start point (tsp) of the urt operon was investigated by primer extension using primers URT17 and URT22, which are located close to the 5′ end of urtA (see Fig. 5A). Similar results were obtained with the two primers, and those obtained with primer URT17 are presented in Fig. 5B. Two RNA 5′ ends were detected at –67 and –77 (RNA1 and RNA2 respectively) with respect to the urtA start. RNA1 was observed with RNA from wild-type cells incubated without combined nitrogen and, at a much lower level, with RNA from ammonium-grown cells, but was undetectable with RNA isolated from strain CSE2 (ntcA). RNA2 was preferentially observed with RNA from CSE2 cells and from ammonium-grown wild-type cells. These results suggested that NtcA activates the promoter originating RNA1 and represses that originating RNA2.

image

Figure 5. Analysis of the urtA promoter.

A. Nucleotide sequence of the 5′ end and sequences upstream of urtA. The location of primers URT17, URT19 and URT22 as well as the possible tsps and promoter elements for tsp1 (–10 box and NtcA binding site, underlined) and for tsp2 (–10 and –35 hexamers, double overlined) are indicated.

B. Primer extension using the URT17 primer and RNA isolated from Anabaena sp. PCC 7120 or strain CSE2 (ntcA) cells grown with ammonium (0) or grown with ammonium and incubated for 3 or 6 h without combined nitrogen.

C. Binding of NtcA to a 330 bp DNA fragment, containing the promoter region of urtA, generated by PCR using oligonucleotides URT17 and URT19 as primers and plasmid pCSAV98 as template. The 32P-labelled DNA fragment (lane 1) was incubated with histidine-tagged Anabaena NtcA protein (lanes 2–4). A 25-fold excess of the same unlabelled DNA fragment (lane 3) or of an unlabelled fragment carrying the PI promoter of the Anabaena glnA gene (Frías et al., 1994; lane 4) was also added.

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To corroborate direct regulation by NtcA of urtA expression, in vitro binding of purified NtcA protein to a DNA fragment carrying the urtA promoter region was tested by bandshift assays. NtcA effected retardation of this DNA fragment (Fig. 5C, lane 2), and this retardation was inhibited by sequestering of NtcA by the addition of a 25-fold excess of the same unlabelled fragment (Fig. 5C, lane 3) or of a fragment carrying the NtcA-regulated PI promoter of the Anabaena glnA gene (Fig. 5C, lane 4). These data show that NtcA can bind to the urtA promoter region.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Some cyanobacteria, such as Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, are capable of taking up urea even when it is present at a low concentration (e.g. <1 μM) in the extracellular medium. We have shown in this work that an ABC-type permease, encoded by the urt genes, is essential for this uptake activity. The urt genes are clustered together in the Anabaena genome and exhibit the structure urtABCDE. Sequence homologies suggest that urtA encodes the periplasmic-binding protein, urtB and urtC the transmembrane proteins and urtD and urtE the ATP-binding subunits. This gene arrangement is similar to that commonly found for ABC-type permeases that are encoded by operons (Boos and Lucht, 1996). Although we have observed an abundant transcript only for urtA and have been able to detect by RT–PCR a transcript that covers only urtABC, the physiological effect of inactivation of urtE indicates that the last genes of the gene cluster are also expressed. Expression of this gene cluster appears therefore to decrease 5′ to 3′, probably rendering higher levels of the substrate-binding protein than of the other permease subunits. This is a common feature for ABC-type uptake permeases (Boos and Lucht, 1996).

Inactivation of urtA, urtB or urtE in Anabaena sp. PCC 7120 leads to a 97–98% reduction in the activity of uptake observed in the wild type for a urea concentration of 0.1 μM. The activity remaining in the urt mutants is similar to that observed for DCCD-treated Anabaena cells, suggesting that it corresponds to passive uptake that could take place by either diffusion of urea through the membrane or flux through a channel. Lack of any de-tectable uptake in an ureG urtE double mutant (Table 2) indicates that the uptake remaining in the urt single mutants is a consequence of metabolism of urea via urease. The radioactivity detected inside the cells would thus correspond to products of the fixation of the 14C-labelled CO2 released in the urease reaction. On the other hand, in Urt+ureG cells, an intracellular accumulation of urea was observed, consistent with the operation of an active transport system. The fact that the accumulation ratio observed for urea (up to about 20) was relatively low is probably a consequence of the easy diffusion of urea through bacterial membranes (Siewe et al., 1998). Thus, a high level of urea uptake is dependent on urea metabolism (Fig. 1).

The ABC-type transporters commonly exhibit a higher affinity for their substrates than many other types of transporters. Consistently, the Urt system of Anabaena sp. PCC 7120 exhibits a lower Ks for urea (about 0.1 μM) than the urea permease of Saccharomyces cerevisiae (Ks, 14 μM) (Cooper and Sumrada, 1975; ElBerry et al., 1993) or the urea carrier of Corynebacterium glutamicum (Ks, 9 μM) that has been suggested to be a proton motive force-dependent secondary transport system (Siewe et al., 1998). A binding protein with high affinity for urea and short-chain amides that may be a component of an ABC-type transporter has also been characterized in Methylophilus methylotrophus (Mills et al., 1998). The gene encoding this binding protein is clustered together with genes that would encode proteins homologous to the BraD and BraE transmembrane proteins, respectively, but none of these genes has been mutated and, therefore, their physiological function has not been established. The presence of transporters with a high affinity for urea suggests that some cyanobacteria can take up the urea that may be found at very low concentrations in their natural environments. Recent determinations in some natural water bodies have indicated the presence of urea at 0.1–3 μM concentrations (Cho et al., 1996; Mitamura et al., 2000a,b), and it has been suggested that cyano-bacterial phytoplankton use urea as a nitrogen source in a lake in which this substrate is found at 0.1–0.6 μM (Mitamura et al., 2000a).

Urease expression is subjected to nitrogen control in some bacteria (Bender, 1991; Collins et al., 1993; Goss and Bender, 1995), but it is constitutive in the cyanobacteria investigated in this work. In contrast, an induction of about two- and fivefold is observed by incubation of the Anabaena cells in medium with nitrate or lacking combined nitrogen, respectively, for both urea transport activity and urtA expression (Table 1 and Fig. 3). In an Anabaena ntcA mutant, transport activity was about fivefold lower and urtA expression was impaired, indicating that NtcA is involved in this regulation. NtcA is known to be capable of acting as a transcriptional activator or a transcriptional repressor in different promoters, although there have been more reports of NtcA-dependent activation (Herrero et al., 2001). For urtA, NtcA appears to activate promoter P1, which has a structure (GTAN8AACN23TAN3T; see Fig. 5A) very close to that of the canonical NtcA-activated promoter, and to repress promoter P2, which has a structure (GTATCAN18TTAAAT) similar to the σ70 canonical promoters and with which the NtcA bind-ing site overlaps (see Fig. 5A). When activated, P1 appears to be stronger than the derepressed P2 (Fig. 5B), thus explaining the increased expression of urtA under N-limiting conditions. On the other hand, a nitrogen-dependent regulation of urea uptake is evident in the ntcA mutant (Table 1), but the molecular mechanism for this NtcA-independent regulation is currently unknown.

Nitrogen regulation of the Urt permease is consistent with the use of urea as a nutrient and would ensure expression of the urt gene cluster when ammonium or urea itself is not available at high concentrations. In contrast, constitutive expression of urease suggests a role for this enzyme in nitrogen metabolism independently of the presence of urea in the extracellular medium. The cyanobacterial strains for which we have described a constitutive urease can accumulate cyanophycin, a co-polymer of arginine and aspartate unique to some cyanobacteria (Allen, 1984), and we have found recently that arginine catabolism involves the urea cycle and arginase pathways (Quintero et al., 2000). It is therefore possible that the constitutive expression of urease is related to a role in arginine catabolism and cyano-phycin mobilization in cyanobacteria that synthesize cyanophycin.

As a group, the ABC-type transporters are known to translocate quite diverse substrates bidirectionally across the cytoplasmic membrane, from simple inorganic ions to proteins (Saier, 1998). The bacterial ABC uptake systems (TC no. 3.A.1 in the transporter classification of Saier, 2000) constitute a phylogenetically well-defined group of permeases within the ABC superfamily, but different phylogenetic clusters are observed within these ABC transporters (Saier, 1998). Permeases in each cluster transport chemically related or similar substrates, i.e. sugars, inorganic anions, amino acids, etc. Although most similar to transporters in cluster 4 of the ABC-type uptake permeases that have hydrophobic amino acids as substrates (TC no. 3.A.1.4; Saier, 2000), the cyanobacterial Urt transporters appear to represent a new family of ABC uptake permeases, as the percentage identity of Urt proteins to cluster 4 proteins is relatively low, and urea represents a new type of substrate for ABC transporters.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bacterial strains and growth conditions

Anabaena sp. PCC 7120, Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 were grown axenically in BG11 medium, which contains 17.6 mM NaNO3 (Rippka et al., 1979), or in BG110 (nitrogen-free) medium supplemented with 2 mM NH4Cl and 4 mM TES-NaOH buffer (pH 7.5) or 2 mM urea and 4 mM TES-NaOH buffer (pH 7.5). For plates, the medium was solidified with 1% separately autoclaved agar (Difco). Cultures were grown at 30°C in the light (75 μE m–2 s–1), with shaking (80–90 r.p.m.) for liquid cultures. Anabaena sp. PCC 7120 mutants carrying gene cassette C.K3 (Elhai and Wolk, 1988) were grown routinely in medium supplemented with 25 μg ml–1 Nm, and mutants carrying gene cassette C.S3 (Prentki and Krisch, 1984) were grown in medium supplemented with 2–5 μg ml–1 Sp and Sm. The cultures used for RNA isolation were grown ex-ponentially in BG110C (BG110 supplemented with 0.84 g l–1 NaHCO3) supplemented with 5–8 mM NH4Cl and 10–16 mM TES-NaOH buffer (pH 7.5) and bubbled with a mixture of CO2 (1.5% v/v) and air. To analyse the effect of different nitrogen sources on the expression of urt genes, ammonium-grown cells were harvested at room temperature, washed with BG110 and resuspended in BG110C medium supplemented with nitrate, urea or without combined nitrogen and incubated under culture conditions.

Growth rates were estimated from the increase in protein concentration in the cultures. Protein concentration was determined by a modified Lowry procedure (Markwell et al., 1978) in 0.2 ml aliquots periodically withdrawn from the cultures. The growth rate constant (μ) corresponds to ln2/td, where td represents the doubling time. The Chl content of the cultures was determined in methanolic extracts of the cells (Mackinney, 1941). The amount of cells that contains 1 μg of Chl contains about 20–25 μg of protein.

Escherichia coli strains DH5α, HB101, MC1061 and GM48 were grown in LB medium with 50 μg ml–1 Ap, 50 μg ml–1 Km, 30 μg ml–1 Cm, 25 μg ml–1 Sm or 100 μg ml–1 Sp when necessary.

Urease activity

For the determination of urease activity, an amount of cells containing 17–20 μg of Chl was mixed with 40 mM tricine-NaOH buffer (pH 8.1), 25 mM urea and 0.1 mg ml–1 MTA and incubated at 30°C. (MTA is mixed alkyltrimethylammonium bromide, a detergent added to make the cells permeable to small molecules.) Samples were taken after different incubation times, and the cells were removed by centrifugation at room temperature. To determine the ammonium produced, 0.1 ml of supernatant was supplemented with water and mixed with 0.5 ml of a commercial reagent (Sigma ammonia colour reagent) in a final volume of 1.5 ml, and the A430 was measured. The reaction was linear for at least 60 min. One activity unit corresponds to 1 μmol of ammonium produced min–1. For the determination of the KM of urease for urea, different urea concentrations were tested up to 0–100 mM.

Urea uptake assays

Wild-type or mutant cells grown in nitrate-containing medium and, unless indicated otherwise, incubated in the absence of combined nitrogen for 10–12 h were harvested by centrifugation at room temperature, washed with 5 mM tricine-NaOH buffer (pH 8.1) and resuspended in the same buffer. After preincubation at 30°C in the light (180 μE m–2 s–1) for 5– 30 min, the assays were started by mixing the suspension of cells (1–15 μg ml–1 Chl) with a solution of [14C]-CO(NH2)2 (200–2000 Bq μmol–1) in tricine-NaOH buffer. After different incubation times, 0.1–1 ml samples were filtered (0.45 μm pore size Millipore HA filters were used) and washed with 2– 5 ml of tricine-NaOH buffer. In some experiments, as indicated, the washing step was omitted in order to avoid the exit of urea from the cells. The filters carrying the cells were then immersed in scintillation cocktail, and their radioactivity was measured. Retention of radioactivity by boiled cells was used as a blank. For the determination of the Ks of the cells for urea, rates from the linear parts of the uptake assays with different urea concentrations were used to derive kinetic parameters from a Lineweaver–Burk representation.

Intracellular accumulation of labelled urea

Filters containing cells that had been used in the uptake assays described above were immediately immersed in 2– 3 ml of boiling water and incubated at 100°C for 5 min. The filters were removed, and the resulting suspensions were centrifuged. Samples from the supernatants were lyophilized and dissolved in a small volume of water. Metabolites present in these samples were resolved by two-dimensional TLC using 0.1 mm cellulose plates (20 cm by 20 cm; Merck). The solvents used were n-butanol–pyridine–water (6:4:3) and n-butanol–ethanol–water (2:1:1). The resulting radioactive areas were quantified in an InstantImager scanner for β particles (Packard). To calculate the intracellular concentration of [14C]-urea, an intracellular volume of 125 μl mg–1 Chl was assumed (see references in Quintero et al., 2000).

Transposon mutagenesis

Mutagenesis of Anabaena sp. PCC 7120 by Tn5-derived transposons was performed as described by Wolk et al. (1991), generating a collection of 426 clones carrying the transposon. Transposon Tn1063a and DNA contiguous with it were recovered from Anabaena strain CSAV56 by digestion with EcoRV and transformation of E. coli. The plasmid recovered was called pCSAV56. The DNA contiguous with each side of Tn1063a was sequenced using primers that were homologous to the corresponding ends of the transposon (LTn1063a: 5′-CAATGCTATCAATGAG-3′; RTn1063a: 5′-CACATGGAATATCAG-3′).

Plasmids

Plasmid pCSAV3 contains a 4.6 kb DNA fragment from the ure region of Anabaena sp. PCC 7120 cloned into ClaI-digested vector pIC20R. This DNA fragment was identified by hybridization using pCSAV56 as a probe and extends from nucleotide –246 with respect to the translation start of ureC to the first ClaI site located downstream of ureG (see http://www.Kazusa.or.jp/cyano/anabaena/). For the generation of a ureG mutant, a 0.7 kb HindIII fragment from pCSAV3 containing the last 540 bp of ureG was substituted by HindIII-ended gene cassette C.S3 from plasmid pRL463 (Elhai and Wolk, 1988) rendering plasmid pCSAV11. Then, the EcoRI fragment from pCSAV11 filled with Klenow enzyme was cloned into the sacB-bearing vector pRL278 (Cai and Wolk, 1990) digested with NruI, rendering pCSAV12. The sacB gene determines sensitivity to sucrose and can be counter-selected for in Anabaena sp., allowing positive selection for double recombinants (Cai and Wolk, 1990).

To clone urtE from Anabaena sp. PCC 7120, a 0.5 kb DNA fragment containing part of the ORF sll0374 from Synechocystis sp. PCC 6803 was used as a probe in heterologous hybridization with Southern blots of total DNA from strain PCC 7120. Hybridization was ascribed to a DraI fragment of ≈ 1.4 kb from the Anabaena genome. A subgenomic gene library from strain PCC 7120 was constructed by cloning DraI restriction fragments of 1–2 kb in cloning vector pIC20R. From this subgenomic library, a clone exhibiting hybridization to the 0.5 kb Synechocystis fragment was identified and named pCSAV8. The insert of pCSAV8 extends from a DraI site located 189 bp downstream from the translation start codon of urtD to the first DraI site located downstream of urtE. For inactivation of urtE, a HindIII restriction site was generated at position 493 with respect to the translation start of urtE, as described previously (Ausubel et al., 2001). The mutagenic oligonucleotides used were URT25 (5′-CGACGGACGGAAGCTTCAATT-3′) and URT26 (5′-GAAATTGAAGCTTCCGTCCGTCG-3′), and the flanking ones were M13 forward and reverse primers; pCSAV8 was used as template. The plasmid generated, pCSAV14, was digested with HindIII and ligated to the 1.1 kb KmR gene cassette C.K3, excised with BamHI and filled with Klenow enzyme, generating plasmid pCSAV15. The PvuII fragment from pCSAV15, containing the interrupted urtE gene, was ligated to the sacB vector pRL277 digested with SpeI and filled with Klenow enzyme rendering pCSAV16.

Plasmid pCSAV90 contains a 7 kb DNA fragment from the urt region of Anabaena sp. PCC 7120 that was cloned by transformation of E. coli MC1061 with a library of HindIII DNA fragments from Anabaena strain CSAV2 and selection for KmR. This fragment, which was cloned into HindIII-digested vector pIC20R, extends from a HindIII site located 640 bp downstream of the translation start of urtA to the first HindIII site located downstream of urtE. pCSAV93 was generated to inactivate urtB. This plasmid contains a 1.9 kb DNA fragment amplified by PCR using the oligonucleotides URT6 (complementary to nucleotides 275–253 with respect to the translation start of the urtC gene) and M13 forward primer; pCSAV90 was used as template. PCR products were cloned in pGEM-T vector (Promega). The 2 kb SmR SpR gene cassette C.S3 excised with EcoRV and SmaI was inserted into a unique Eco47III site that is present in the Anabaena DNA insert of pCSAV93 to generate plasmid pCSAV94. The PvuII fragment from pCSAV94, containing the interrupted urtB gene, was ligated to the sacB vector pRL278 digested with NruI rendering pCSAV96.

Plasmid pCSAV98 contains a 2.3 kb DNA fragment from the urt region of Anabaena sp. PCC 7120 isolated from a library of Eco47III DNA fragments cloned into EcoRV-digested low-copy-number vector pDUCA7RV. This fragment extends from an Eco47III site located 251 bp upstream from the translation start codon of urtA to the Eco47III site located 754 bp downstream of the translation start of urtB. pCSAV120, which was generated to inactivate urtA, contains a 1.84 kb DNA fragment amplified by PCR using the oligonucleotides URT9 (complementary to nucleotides 245–222 with respect to the translation start of urtB) and URT19 (cor-responding to nucleotides –235 to –216 with respect to the translation start of urtA) and genomic DNA from strain PCC 7120 as template. PCR products were cloned in pGEM-T vector (Promega). The 2 kb SmR SpR gene cassette C.S3 excised with EcoRV and SmaI was inserted into the BclI, EcoRI sites that are present in the Anabaena DNA insert of pCSAV120 to generate plasmid pCSAV121. The PvuII fragment from pCSAV121, containing the interrupted urtA gene, was ligated to the sacB vector pRL278 digested with BglII and filled in with Klenow enzyme rendering pCSAV122.

Generation of Anabaena mutants

In vitro-generated constructs bearing a gene cassette inserted into ureG, urtA, urtB and urtE of Anabaena sp. PCC 7120 and cloned in sacB vectors were transferred by conjugation (Elhai and Wolk, 1988) to Anabaena sp. to generate strains bearing mutations in the ure and/or urt genomic regions. For the generation of strains CSAV1, CSAV2, CSAV3, CSAV4 and CSAV5, E. coli HB101 containing plasmid pCSAV12, pCSAV16, pCSAV12, pCSAV122 or pCSAV96, respectively, and helper plasmids pRL528 (Elhai and Wolk, 1988) and pRL591-W45 (Elhai et al., 1994) was mixed with E. coli ED8654 carrying the conjugative plasmid pRL443 and thereafter with Anabaena sp. Exconjugants were isolated (Elhai and Wolk, 1988), and double recombinants were identified as clones resistant to the antibiotic for which resistance was encoded in the inserted gene cassette, resistant to sucrose and sensitive to the antibiotic for which the resistant determinant was present in the vector portion of the transferred plasmid and were confirmed by Southern or PCR analysis.

DNA and RNA isolation and manipulation

Total DNA (Cai and Wolk, 1990) and RNA (García-Domínguez and Florencio, 1997; based on Golden et al., 1987) from Anabaena sp. PCC 7120 and its derivatives was isolated as described previously. Sequencing was carried out by the dideoxy chain termination method, using a T7Sequencing kit (Amersham Pharmacia Biotech) and [α-35S]-thio-dATP. DNA fragments were purified from agarose gels with the Geneclean II kit (Bio101). Plasmid isolation from E. coli, transformation of E. coli, digestion of DNA with restriction endonucleases, ligation with T4 ligase and PCR were performed by standard procedures (Sambrook et al., 1989; Ausubel et al., 2001).

Southern blotting and hybridization

Southern analysis was carried out as described previously (Montesinos et al., 1997) using GeneScreen Plus membranes (Dupont). DNA probes used in the hybridizations were obtained by restriction of plasmids or by PCR and were labelled with a DNA labelling kit (Ready to Go; Amersham Pharmacia Biotech) and [α-32P]-dCTP.

Northern blotting and hybridization

For Northern analysis, 70 μg of RNA was loaded per lane and electrophoresed in 1% agarose denaturing formaldehyde gels. Transfer and fixation to Hybond-N+ membranes (Amersham Pharmacia Biotech) were carried out using 0.1 N NaOH. Hybridization was performed at 65°C according to the recommendations of the manufacturers of the membranes. The urtA probe was amplified by PCR using oligonucleotides URT11 (complementary to positions +1260 to +1235 relative to the translation start of urtA) and URT18 (corresponding to positions +38 to +59 relative to the translation start of urtA) and pCSAV98 as template. This DNA fragment was 32P labelled with a Ready to Go DNA labelling kit (Amersham Pharmacia Biotech) using [α-32P]-dCTP. Images of radio-active filters were obtained and quantified using a Cyclone storage phosphor system and the OPTIQUANT image analysis software (Packard).

Primer extension analysis

Primer extension analysis was carried out as described previously (Muro-Pastor et al., 1999). Oligonucleotides used for analysis of the urtA transcript were URT17 (complementary to positions +94 to +74 relative to the translation start of urtA) and URT22 (complementary to positions +61 to +40 relative to the translation start of urtA). Plasmid pCSAV98, which contains the upstream region of the urtA gene, was used to generate dideoxy sequencing ladders using the same primers.

RT–PCR

For RT–PCR experiments, 10 μg of strain PCC 7120 total RNA was mixed with 40 pmol of oligonucleotide URT6 (see above), URT2 (complementary to positions +821 to +793 relative to the translation start codon of urtD) or URT28 (complementary to positions +146 to +125 relative to the translation start of urtE) in the presence of 10 mM Tris-HCl, pH 8.0, 150 mM KCl and 1 mM EDTA, heated for 2 min at 85°C and then at 50°C for 1 h for annealing. The extension reactions were carried out at 47°C for 1 h in a final volume of 45 μl containing the whole annealing reaction, 0.25 mM each deoxynucleoside triphosphate, 200 U of reverse transcriptase (Superscript II; Gibco BRL) and the buffer recommended by the transcriptase provider. To control for the presence of contaminating DNA, samples containing 10 μg of RNA, 40 pmol of oligonucleotides and 1 μg of RNase A (DNase free; Boehringer) were incubated in a 45 μl reaction volume at 37°C for 1 h. PCR was carried out with 3–5 μl of retrotranscription mixture or RNase-treated sample as the template and oligonucleotides URT6, URT2 or URT28 (see above) and URT15 (corresponding to nucleotides +1084 to +1102 with respect to the urtA translation start) as primers. Samples containing the same oligonucleotides and strain PCC 7120 genomic DNA as the template were run in parallel and used as standards. PCR was performed by standard procedures. Half of each sample was resolved by electrophoresis in a 0.7% agarose gel and transferred to membranes for Southern blot analysis. The urtC probe was amplified by PCR using oligonucleotides URT23 (corresponding to positions +45 to +60 relative to the translation start of urtC) and URT24 (complementary to positions +872 to +855 relative to the translation start of urtC) and pCSAV90 as template.

Bandshift assays

DNA fragments to be used in electrophoretic mobility shift assays (EMSAs) were obtained by PCR amplification. Oligonucleotides URT17 and URT19 (see above) and plasmid pCSAV98 were used for PCR amplification of the urtA upstream region. In the case of the glnA upstream region, oligonucleotides GA3 (corresponding to positions –238 to –215 relative to the translation start of glnA) and GA6 (complementary to positions –70 to –87 relative to the translation start of glnA) and plasmid pAN503 (Tumer et al., 1983) were used. The same unlabelled DNA fragments were added in a 25-fold molar excess as competitors in some assays. DNA fragments were end-labelled with T4 polynucleotide kinase (Boehringer) and [γ-32P]-dATP as described previously (Ausubel et al., 2001). Assays were carried out as described previously (Luque et al., 1994) with 0.05 pmol of labelled fragment and 5 pmol of histidine-tagged NtcA purified from an extract of E. coli strain BL21 (pCSAM70, pREP4) carrying the Anabaena sp. PCC 7120 ntcA gene (Muro-Pastor et al., 1999). Images of radioactive gels were obtained using a Cyclone storage phosphor system (Packard).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank A. M. Muro-Pastor and J. E. Frías for their generous help throughout this work, and F. Jüttner for useful discussions. Preliminary sequence data obtained from the Kazusa DNA Research Institute (Japan) and the DOE Joint Genome Institute (USA) are acknowledged. This work was supported by grants PB97-1137 and PB98-0481 from the Ministerio de Ciencia y Tecnología (Spain).

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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