The transcription of the cbb operon in Nitrosomonas europaea

Xueming Wei, Luis A. Sayavedra-Soto and Daniel J. Arp

Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331-2902, USA

Correspondence
Daniel J. Arp
arpd{at}science.oregonstate.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Nitrosomonas europaea is an aerobic ammonia-oxidizing bacterium that participates in the C and N cycles. N. europaea utilizes CO2 as its predominant carbon source, and is an obligate chemolithotroph, deriving all the reductant required for energy and biosynthesis from the oxidation of ammonia (NH3) to nitrite (). This bacterium fixes carbon via the Calvin–Benson–Bassham (CBB) cycle via a type I ribulose bisphosphate carboxylase/oxygenase (RubisCO). The RubisCO operon is composed of five genes, cbbLSQON. This gene organization is similar to that of the operon for ‘green-like’ type I RubisCOs in other organisms. The cbbR gene encoding the putative regulatory protein for RubisCO transcription was identified upstream of cbbL. This study showed that transcription of cbb genes was upregulated when the carbon source was limited, while amo, hao and other energy-harvesting-related genes were downregulated. N. europaea responds to carbon limitation by prioritizing resources towards key components for carbon assimilation. Unlike the situation for amo genes, NH3 was not required for the transcription of the cbb genes. All five cbb genes were only transcribed when an external energy source was provided. In actively growing cells, mRNAs from the five genes in the RubisCO operon were present at different levels, probably due to premature termination of transcription, rapid mRNA processing and mRNA degradation.


Abbreviations: AMO, ammonia monooxygenase; HAO, hydroxylamine oxidoreductase; PRK, phosphoribulokinase; RubisCO, ribulose bisphosphate carboxylase/oxygenase


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Nitrosomonas europaea is an aerobic ammonia-oxidizing bacterium that participates in the C and N cycles and hence is involved in events that affect the environment (Bock et al., 1986). As an obligate chemolithotroph N. europaea derives all the reductant it requires for energy and biosynthesis from the oxidation of ammonia (NH3) to nitrite () (Winogradsky, 1931). This bacterium is an autotroph that utilizes CO2 as its primary carbon source (Bock et al., 1991). Organic compounds such as acetate, 2-oxoglutarate, succinate, some amino acids, and possibly some sugars are also taken up and metabolized by N. europaea, albeit at low levels relative to heterotrophic bacteria, and typically at levels insufficient to fulfil the carbon requirement of the bacterium (Clark & Schmidt, 1966, 1967; Martiny & Koops, 1982; Wallace et al., 1970). However, in the absence of added CO2, N. europaea can grow with fructose or pyruvate as the sole C source (Hommes et al., 2003).

Autotrophic nitrifiers assimilate CO2 via the Calvin–Benson–Bassham (CBB) cycle. Genetic information about the enzyme that catalyses CO2 fixation in N. europaea was revealed by the sequence of its genome (Chain et al., 2003). The DNA sequence suggests that the enzyme is a type I ribulose bisphosphate carboxylase/oxygenase (RubisCO). Most of the genes encoding the enzymes for a complete CBB cycle are present in the genome. The two missing genes are those encoding sedoheptulose 1,7-bisphosphatase (EC 3.1.3.37) and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). However, fructose 1,6-bisphosphatase (EC 3.1.3.11) in N. europaea may function primarily for sedoheptulose 1,7-bisphosphate hydrolysis in the CBB cycle, rather than for fructose 1,6-bisphosphate hydrolysis in gluconeogenesis (Yoo & Bowien, 1995). NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase is apparently replaced by an NADH-dependent enzyme (EC 1.2.1.12) (Chain et al., 2003).

There are two recent reports on the cbb genes of ammonia-oxidizing bacteria. In one, Hirota et al. (2002) cloned and sequenced the cbbLS genes of Nitrosomonas ENI-11 and expressed functional RubisCO activity in Escherichia coli. In the other, Utåker et al. (2002) cloned and sequenced the cbbLS genes of Nitrosospira sp. isolate 40KI and showed its functionality in a cbb-deletion strain of Ralstonia eutropha. However, to date there are no reports describing the transcription patterns and regulation of the cbb genes in ammonia-oxidizing bacteria. Instead, most research has focused on the genetic makeup and the regulation for the utilization of ammonia in N. europaea and other ammonia-oxidizing bacteria (Arp et al., 2002; Hommes et al., 1998, 2001; Klotz & Norton, 1995, 1998; Norton et al., 1996). The two key enzymes in this process, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO), catalyse the sequential oxidation of NH3 to . The genetic loci for AMO and HAO are in multiple copies in nitrifiers. In N. europaea there are two gene copies for AMO and three gene copies for HAO (compared to a single gene copy for RubisCO). Mutation of the different gene copies of AMO and HAO indicated that no copy was indispensable, although some phenotypes were different from the wild-type (Hommes et al., 1996, 1998; Stein et al., 2000). Transcript analysis and mutagenesis studies suggested that the transcription of the amoCAB operon may be regulated by more than one promoter (Hommes et al., 2002; Stein et al., 2000). In this work, the transcription and regulation of cbb genes in response to major nutrients and environment factors were characterized.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Media, bacterial cultures, determination of enzyme activities and materials.
N. europaea (ATCC 19178) was grown in batch cultures as previously described (Ensign et al., 1993; Stein & Arp, 1998). Cells were harvested from 3-day-old or mid- to late-exponential-phase cultures by centrifugation. The cells were washed in -free, -free buffer to remove residual growth medium. The NH3 deprivation (starvation) treatments were prepared by incubating cells in -free, -free medium for 16 h in order to deplete most mRNAs and to avoid the toxic effects of accumulated metabolites (Stein & Arp, 1998). For the experiments where cells in stationary phase were necessary, the cells were harvested 3 days after reaching the maximum OD600 (~0·07). Because the pH of the medium in the cultures decreases as nitrite accumulates, NH3 becomes limiting in stationary phase (depriving the cells of the growth substrate). It is known that the mRNAs of AMO and HAO are induced upon incubation in and decrease to negligible amounts upon approximately 1 day of starvation (Sayavedra-Soto et al., 1996). The induction experiments involving gases were carried out in bottles sealed with grey butyl septa. In the O2 limitation experiments, a vacuum gas manifold was used to replace headspace air with N2. The intended O2 and CO2 levels were added to the bottles through septa by injecting pure O2 and CO2 as necessary. The headspace CO2 and O2 levels were determined using a Shimadzu GC8A gas chromatograph equipped with a thermal conductivity detector and a 4 ft (1·2 m) Porapak Q column. In experiments requiring the inactivation of AMO, acetylene was injected at 2 % (v/v) and allowed to equilibrate with the cell suspension in NH3-free medium for 1 h before an energy source was added. Induction of mRNA transcription and enzyme activities was commonly done by incubation with the inducing agent at 30 °C on a rotary shaker at 100 r.p.m. for 2 h. Nitrite concentration was determined colorimetrically using the Griess reagent (sulfanilamide and N-naphthylethylenediamine) (Hageman & Hucklesby, 1971).

Nucleic acid manipulation and hybridization.
RNA was isolated as described by Reddy & Gilman (1993) and Vangnai et al. (2002). Briefly, 250 µl acid phenol, 250 µl chloroform, SDS to 1 % and sodium acetate to 0·3 M were added to 500 µl cell suspension in buffer (2 mM MgCl2 and 50 mM NaH2PO4, pH 7·5). The cell suspension was mixed thoroughly and centrifuged for 5 min at 16 000 g. The total RNA was precipitated with ethanol and resuspended in diethylpyrocarbonate (DEPC)-treated water. When necessary, cells were resuspended in a solution of NaN3 (1 mM) and aurintricarboxylic acid (1 mM) (Reddy & Gilman, 1993), or in a commercial RNA stabilizer solution (RNAlater; Ambion), to prevent mRNA changes and degradation during sample preparation. For Northern hybridization analysis, total RNA was resolved in denaturing 1·2 % agarose gels (Sambrook et al., 1989). Prior to electrophoresis, RNA was stained with ~5 µg ethidium bromide ml–1 in the loading buffer. The RNA was blotted onto Nytran membranes (Schleicher & Schuell BioScience). Probes for hybridization were generated by PCR with primers specific for each gene and labelled by random priming (Prime-a-Gene Labelling System, Promega) with [{alpha}32P]dCTP (3000 Ci mmol–1, 110 TBq mmol–1; ICN). Hybridization was carried out as described by Sambrook et al. (1989) and Sayavedra-Soto et al. (1998). Images and relative signal densities were obtained by phosphorimaging and ImageQuant softwares as described by the manufacturer (Molecular Dynamics).

DNA preparation, restriction digestions and agarose gel electrophoresis were done as described by Sambrook et al. (1989). The recovery of DNA fragments was carried out with a commercial kit (Qiagen). PCR was performed with Taq DNA polymerase (Promega). RT-PCR was done with M-MLV reverse transcriptase (Promega) according to the manufacturer's instructions, with a 50 °C extension temperature. RNA templates for RT-PCR were treated with RQ1 DNase (Promega) or ‘DNA-free’ DNase (Ambion) multiple times until no DNA product was detected by Taq DNA polymerase in a PCR with any of the cbb primers used. The primers used in the PCR and RT-PCR experiments are listed in Table 1.


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Table 1. Primers used for the amplification of cbb and other genes and for RT-PCR

 
The start of transcription was determined using a commercial kit (GeneRacer; Invitrogen). Briefly, the RNA oligonucleotide provided in the kit was ligated with RNA ligase to the 5'-ends of the mRNA pool as directed by the manufacturer. The corresponding cDNA was then made with a cbbL-specific reverse primer, followed by PCR amplification of the chimeric DNA fragment with the cbbL reverse primer and the kit's forward primer. Twelve chimeric fragments were cloned and sequenced. The start of transcription was at the nucleotide where ligation of the 5'-end of the mRNA and the oligonucleotide occurred.

Total cell protein was estimated by the biuret method (Gornall et al., 1949) and the protein composition was analysed by PAGE as described by Hyman & Arp (1993).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
N. europaea cbb operon characterization
The putative RubisCO operon in this bacterium is composed of five genes, namely cbbLSQO and a fifth gene here designated cbbN. This composition was deduced from the nucleotide sequence of the genome of N. europaea and gene similarity comparisons. In this operon, cbbL and cbbS code for the RubisCO large and small subunits respectively. The genes cbbQ and cbbO encode proteins that are expected to be involved in the processing, folding, assembling, activation and regulation of the RubisCO complex enzyme as in other organisms (Baxter et al., 2002; Hayashi et al., 1997, 1999). cbbN has the same orientation as cbbLSQO. The intergenic region between cbbO and cbbN is only 20 bp, in which no apparent promoter could be inferred from the sequence, implying that it is transcribed from the same cbb promoter. cbbN encodes a hypothetical protein of 101 amino acids and has no similarity to known annotated genes. A putative regulatory gene, cbbR, was found 194 bases upstream of the start codon of cbbL and is transcribed in the opposite direction. The deduced amino acid sequence of N. europaea cbbR is most similar to that of Thiobacillus denitrificans (NCBI: AF307090) and Allochromatium vinosum (formerly Chromatium vinosum) (Viale et al., 1991). In other species, cbbR is identified as a LysR-type regulatory gene (Shively et al., 1998). The cbb genes, including cbbR, are contained in a 6581 nucleotide DNA fragment (Fig. 1A). A stem–loop structure was identified in the intergenic region between cbbS and cbbQ with a calculated free energy ({Delta}G0) of –155 kJ mol–1. The stem–loop is formed by 101 of the 119 nucleotides that form the intergenic region and may have a regulatory or processing role.



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Fig. 1. The RubisCO gene organization in N. europaea. (A) Arrows indicate the orientation of the genes. The numbers are DNA segment lengths in bp for genes (upper) and intergenic regions (lower). (B) Detection of intergenic regions by RT-PCR. The bars indicate the regions amplified by RT-PCR. The amplified products were resolved on an agarose gel. The ‘PCR only’ lane is a control reaction performed without reverse transcriptase to test for RNA purity. Lanes 1 to 5 correspond to the amplified cDNA fragments located in the areas indicated by the bars above. The numbers below the bars are the size of the fragments in kb. Lane 6 is a DNA size marker.

 
The organization of the N. europaea cbb operon is similar to that in All. vinosum (Viale et al., 1989), Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) (Kusano & Sugawara, 1993; Kusano et al., 1991), Methylococcus capsulatus (Tichi & Tabita, 2002) and Rhodobacter capsulatus (Baxter et al., 2002; Paoli et al., 1998). In these organisms, cbbLSQ is a common block, a feature of ‘green-like’ type I RubisCO genes. The term ‘green-like’ type I RubsiCO implies similarity to the RubisCOs in plants and green algae (Utåker et al., 2002). The N. europaea cbbLSQO block is similar to the Rb. capsulatus cbbI operon (Vichivanives et al., 2000) and Hydrogenophilus thermoluteolus (Terazono et al., 2001). A sequence database search revealed that N. europaea CbbL is most similar to that of Ac. ferrooxidans (91 % amino acid sequence) (Heinhorst et al., 2002), T. denitrificans (90 %) (Hernandez et al., 1996), All. vinosum (87 %) (Viale et al., 1989) and Hydrogenophaga pseudoflavas (86 %) (Lee & Kim, 1998). In contrast, the N. europaea CbbL protein shares only 85 % amino acid identity with that of Nitrosomonas sp. strain ENI-11 (Hirota et al., 2002), and has much lower identity to that in other nitrifying bacteria (e.g. <=83 % for Nitrobacter vulgaris; GenBank accession no. L2285) and some Nitrosospira species (Utåker et al., 2002). N. europaea CbbS is most similar to that of T. denitrificans (83 %) and Ac. ferrooxidans (79 %) (Pulgar et al., 1991). As with CbbL, N. europaea CbbS showed only 53 % amino acid sequence similarity to the CbbS of both Nitrosomonas sp. strain ENI-11 (Hirota et al., 2002) and Nitrobacter vulgaris. cbbQ is similar to nirQ, a denitrification gene in Pseudomonas species (Yokoyama et al., 1995). The N. europaea CbbO shares 50 % amino acid identity to the putative Ac. ferrooxidans CbbO-like protein (AJ133725.1), and 40 % to the H. thermoluteolus CbbO (Hayashi et al., 1997). CbbO has moderate similarity to the probable denitrification protein NorD (AE004489.1). In H. thermoluteolus, cbbY is downstream of cbbLSQO. However, H. thermoluteolus cbbY and N. europaea cbbN are different (771 bp vs 303 bp respectively) (Hayashi & Igarashi, 2002; Hayashi et al., 2000; Terazono et al., 2001). It is also worth noting that cbbY in other species is not immediately downstream of cbbQ and is in a different transcriptional unit (Gibson & Tabita, 1997).

The predicted molecular masses for N. europaea Cbb proteins are as follows (kDa): CbbR, 34·9; CbbL, 52·9; CbbS, 13·8; CbbQ, 29·8; CbbO, 88·4; CbbN, 11·4. The molecular masses of N. europaea CbbL and CbbS are typical of those RubisCOs in most autotrophic bacteria.

Analysis of the cbb promoter and intergenic regions
The intergenic region between cbbR and cbbL is 194 bp in N. europaea, compared to 213 bp in Nitrosomonas sp. strain ENI-11 (Hirota et al., 2002), 182 bp in Ral. eutropha (Kusian et al., 1995), 226 bp in All. vinosum (Viale et al., 1989) and 144 bp in Ac. ferrooxidans (Pulgar et al., 1991). The nucleotide sequence upstream of cbbL in N. europaea does not show significant similarity to those of the above-mentioned species. No putative promoter between other cbb genes could be inferred by visual inspection and promoter predicting programs (e.g. http://www.fruitfly.org/seq_tools/promoter.html). Furthermore, the intergenic spaces between cbbQ and cbbO (41 bp), and between cbbO and cbbN (20 bp), are smaller than the considered 50 bp minimum in most promoter-predicting programs (however, the possibility of a promoter overlapping the upstream gene cannot be discarded).

Since none of the alignments with other autotrophic bacteria provided convincing evidence for promoter and transcriptional start sites of the N. europaea cbb operon, we proceeded to determine experimentally the 5' end of the cbb transcript using a commercial kit (see Methods). Of the 12 chimeric clones sequenced, five did not contain any cbbL sequence and seven revealed two potential transcriptional start sites: two clones showed a thymidine, 79 bases upstream of the ATG start codon of cbbL, and five clones showed a guanine, 83 bases upstream of the ATG start codon of cbbL (Fig. 2). The putative promoter region at –10 (TATAGT) and –35 (TTTAAC) bases shows similarity to the E. coli {sigma}70 consensus sequence (Fig. 2). The –35 region shows similarity to that in other autotrophic bacteria such as Xanthobacter flavus and Ral. eutropha (TTTANN) (reviewed by Shively et al., 1998). Two possible start sites for the transcription of the cbb genes were also identified in Nitrosomonas sp. ENI-11 (Hirota et al., 2002). A feature of the regulatory regions of the RubisCO genes in other bacteria is the AT-rich boxes found upstream of the cbb operon (Schell, 1993). In N. europaea, an AT-rich element (50 ATs out of 56 bp) can be readily identified in the intergenic region of cbbR and L (Fig. 2). It is known that CbbR belongs to the LysR-type regulators. LysR regulators bind to DNA sequences with T/A-(N)11/12-A/T inverted repeats (Goethals et al., 1992; Schell, 1993; Xu & Tabita, 1994). In N. europaea several such symmetrical repeats exist in the intergenic region of cbbR and cbbL (Fig. 2).



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Fig. 2. The nucleotide sequence upstream of N. europaea cbbL. The two putative starts of transcription are in bold (+1/+1). The numbers –10 and –35 indicate the deduced promoter region. An AT-rich region with at least 89 % AT composition is boxed. The T/A-N11–14-A/T symmetry sequences match the inverted arrowheads in the lines next to the nucleotide sequence. The ribosome-binding site for cbbL is labelled as RBS. The cbbL and cbbR start codons (ATG) are indicated in bold italics at both ends of the sequence.

 
Gene transcription of cbb operon
We wanted to determine if all five genes for the RubisCO operon were transcribed in N. europaea under normal growing conditions. We detected their cDNAs using RT-PCR (Fig. 1B) and their mRNAs in Northern hybridizations (Fig. 3). The cbb mRNAs were detected at higher levels in cells in growth medium than in NH3-deprived cells (Fig. 3). Two transcripts were detected by the cbbL probe. One transcript was approximately 1600 bases in length, corresponding to the length of cbbL plus the putative untranslated regions. The other was approximately 2000 bases in length, corresponding to the total length of cbbL, cbbS and the intergenic regions. The cbbS probe detected a clear mRNA for cbbLS (~2000 bases) and a faint hybridization signal that could be attributed to cbbS (~350 bases). Probes for cbbQ, cbbO and cbbN detected lower levels of mRNA compared to the cbbLS signal. The cbbQ probe detected possible mRNAs of 3500, 2000, 1500 and 900 bases. The probes for cbbO and cbbN detected fragments smaller than 1500 bases, mostly in the form of a smear. In actively growing cells, the levels of transcript of the five genes in the RubisCO operon were not at equivalent levels. We were also able to detect cbbR mRNA by Northern hybridization (Fig. 3), demonstrating that it was transcribed during growth, but it was present at much lower levels.



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Fig. 3. Northern hybridizations showing the level of transcription of the cbb genes in (i) induced cells (actively growing) and (s) starved cells (ammonia-deprived). The far left lane is an RNA ladder. The experiment was repeated in triplicate with similar trends. Equivalent amounts of RNA were loaded in all lanes in the gel as estimated by staining with ethidium bromide.

 
We tested which of the five cbb genes were cotranscribed. We designed oligonucleotide primers to amplify the intergenic regions by RT-PCR. All four predicted fragments spanning the intergenic regions were obtained (Fig. 1B), suggesting that any given pair is linked in a transcript. Yet an mRNA long enough to include all five components of RubisCO was not detected consistently by Northern hybridization. Again, this result suggests that the cbb mRNA is actively processed or it degrades rapidly, if indeed it is transcribed from a single promoter.

The transcription of the genes for RubisCO was compared to the transcription of key genes for (a) energy-harvesting enzymes – AMO, HAO, glyceraldehyde 3-phosphate dehydrogenase and succinate dehydrogenase, (b) carbon metabolism – the anion transporter NE1927 (speculated to transport sulfate or ) and phosphoribulokinase (PRK), and (c) N metabolism – glutamate dehydrogenase and glutamate synthase. The genes for these enzymes were all transcribed in growing cells but not in NH3-deprived cells (data not shown). The mRNA for AMO was detected in low amounts in NH3-deprived cells (see below). This result differed from a previous observation where no amo transcript was detected in starved cells (Sayavedra-Soto et al., 1996). The preparation of NH3-deprived cells in this study (at 30 °C for 16 h) was different from the previous study, in which sedimented cells were incubated for a day at 4 °C to deplete mRNA. Apparently, incubation in -free medium allowed the cells to keep a detectable level of amo mRNA. In contrast, the mRNAs of cbb and all other genes we examined were not detected either in stationary phase cells or in NH3-deprived cells (data not shown).

Profiles of cbb mRNA induction and decay
The induction profiles of cbbL and cbbS were determined in NH3-deprived cells upon transfer to growing conditions, and were compared to the induction profile of hao. NH3-deprived cells had low levels of detectable hao and RubisCO mRNAs by Northern hybridization. These cells, upon transfer to fresh medium, produced the mRNAs for cbbL and cbbS within 0·5 h (Fig. 4A). The mRNA of hao increased as previously reported (Sayavedra-Soto et al., 1996). In these induction experiments, the levels of the mRNAs of cbbL and cbbS reached a maximum level at around 2 h and decreased by 4 h (Fig. 4A). To determine the biological half-life of the mRNAs, time-course depletion experiments were conducted (by following the net decrease in mRNA in cells deprived of energy source but with no RNA synthesis inhibitors). Messages of cbbL and cbbS declined much faster than those of amo and hao (Fig. 4B, and blots not shown). After 16 h starvation, messages from cbbL and cbbS were depleted to about 5 % of initial levels, while the mRNAs for amo and hao were more abundant compared to those of cbbL and cbbS (Fig. 4B).



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Fig. 4. Time-course for cbbL and cbbS induction and decay. (A) Northern hybridization showing the time-course for the induction of cbbL, cbbS and hao. Exponential-phase cells were washed and starved overnight, then induced in normal culture medium. (B) Time-course for the depletion of cbbL ({circ}), cbbS ({bullet}), amoA ({triangleup}) and hao ({blacktriangleup}) mRNAs. Mid-exponential-phase cells were transferred to ammonia-free medium and incubated at 30 °C. Transcript levels were determined by densitometry of the signals in the Northern blots.

 
Ammonia as a signal for gene transcription
NH3 is thought to be the main signal to induce the transcription of amo in addition to providing energy for all cellular functions (Hyman & Arp, 1995; Sayavedra-Soto et al., 1996). We wanted to determine whether it is a signal for RubisCO gene transcription as well. Exposure of N. europaea cells to acetylene (C2H2), a potent inactivator of AMO, prevents NH3 use as an energy source. Cells depleted of mRNA were transferred to growth medium in the presence of C2H2 and tested for cbbLS gene transcription. In the presence of NH3 and C2H2, the mRNAs of cbbL and cbbS were not transcribed (Fig. 5). To ensure that the cells had sufficient energy to carry out transcription, hydroxylamine (HA) was supplied to the cell in the presence of C2H2 with and without NH3. When NH3 oxidation is inhibited, HA, the product of NH3 oxidation, can be used as energy source by N. europaea cells. In the presence of HA, the cbbL and cbbS mRNAs were detected regardless of the absence or presence of NH3, as is evident in the C2H2 treatments (Fig. 5). In agreement with what previously was reported (Sayavedra-Soto et al., 1996), amo was transcribed in media containing NH3 and C2H2 (not shown). In these cells, NH3 served as a signal to turn on the transcription of amo, presumably at the expense of internally reserved energy sources. However, in our experiments with cbbL and cbbS, NH3 was not required as a signal and internally reserved energy sources were not sufficient for their transcription. The genes for carbon fixation were all transcribed at detectable levels as long as an energy supply was available. The levels of cbbL and cbbS mRNA in the HA treatment were higher than those in the treatments that contained NH3. In these treatments, faint hybridization signals were detected for transcripts long enough to contain up to five cbb genes (Fig. 5).



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Fig. 5. Dependence of cbbLS genes transcription on energy source. Late-exponential-phase N. europaea cells were washed and starved overnight, then transferred to NH3-free medium with or without C2H2 and allowed to equilibrate for 1 h. The medium was then supplemented with NH4+ (ammonium sulfate, 25 mM), or hydroxylamine (HA, 1 mM), or both. After incubation for 2 h, cells were harvested for RNA extraction and Northern analysis (see Methods). The bottom panel shows the rRNAs stained with ethidium bromide to show equivalent amounts in the samples in the analysis. The experiment was repeated three times and yielded similar trends.

 
Effect of CO2 levels on transcription
The transcription of cbbL, cbbS, amo and hao in response to gaseous CO2 (no Na2CO3 added to the medium) was studied. The cbbL and cbbS mRNAs were detected at higher levels in the treatments with low CO2. The cbbL message level was over five times higher at air (0·03 %) CO2 than at 3 % CO2 (Fig. 6). In contrast, the amo and hao mRNA levels increased as the CO2 levels increased; the message levels were about eight- and threefold higher, respectively, at 3 % CO2 than at 0·03 %. This response was similar when different levels of Na2CO3 were added to the medium (not shown). The transcription levels of cbbL and cbbS decreased as the carbonate (Na2CO3) levels in the medium increased. As with CO2, the amo and hao mRNA levels and those of the anion transporter (NE1927) and PRK, increased as the concentration of Na2CO3 increased (data not shown). In the absence of added Na2CO3 in the medium (i.e. air CO2 only), the levels of the cbbL and cbbS mRNAs were approximately sevenfold higher than that in the medium with full Na2CO3. These results suggest that a similar response would be observed in the environment regardless of whether CO2 or was the predominant carbon source.



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Fig. 6. Effect of CO2 levels on the transcription of amoA, hao, cbbL and cbbS in N. europaea. Exponential-phase cells were washed and incubated in N-free, carbonate-free medium overnight. The cells were then induced for 2 h in growth medium with various CO2 levels, and harvested for RNA isolation and Northern analysis (see Methods). The numbers at the bottom of the blot are the relative signal intensities. The images shown are representative of experiments done in triplicate with duplicate samples.

 
Effect of O2 levels on cbb mRNA levels
The presence of O2 is another major factor for the growth and metabolism of N. europaea, an aerobic chemoautotroph. Transcription of cbbL, cbbS, amoA and hao in response to three O2 levels (0·2 %, 2 % and air) was studied by transferring cells to fresh medium in sealed bottles with controlled O2 levels. When the O2 level was lower, all four genes, amo, hao, cbbL and cbbS, were transcribed at lower levels (blots not shown). The highest transcription was observed at 21 % O2 (air level). The transcription of these genes could be detected even at an O2 level as low as 0·2 %.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Six contiguous genes in N. europaea were identified as cbb genes based on similarity to the RubisCO genes in other organisms, of which cbbL and cbbS encode RubisCO (Baxter et al., 2002; Hayashi et al., 1997, 1999). Five of these genes (cbbLSQON) appear to form an operon. A cbbLSQON operon is suggested by the production of cDNAs containing the intergenic regions between all the five genes (Fig. 1B) and the observed mRNA fragments of appropriate sizes to contain any combination of contiguous genes in the cbb operon (Figs 5 and 6). Although we were not able to detect a transcript long enough to include all five messages, the presence of a single promoter for all five genes in the cbb operon is not unprecedented. For example, the transcription of the cbb operons in Ac. ferrooxidans, X. flavus, Ral. eutropha and Rhodobacter sphaeroides are transcribed from a single promoter (Kusano et al., 1991; Kusian et al., 1995; Schäferjohann et al., 1996). In Ral. eutropha, X. flavus (Meijer et al., 1991) and Rb. sphaeroides a single promoter was demonstrated by insertional mutations in their cbb genes. The mutations prevented the transcription of cbb genes downstream from the insertion, suggesting that the cbb operons in these bacteria are indeed large (e.g. in Ral. eutropha could be 15 kb) (Gibson & Tabita, 1997; Meijer et al., 1991; Schäferjohann et al., 1995; Windhovel & Bowien, 1990).

The mRNAs of cbbL and cbbS were the most abundant, while the other cbb mRNAs were detected consistently at low levels. Similar results were observed in other autotrophic bacteria (English et al., 1992; Kusano et al., 1991; Meijer et al., 1991). In Ral. eutropha this was interpreted as a premature transcriptional termination at a sequence resembling a terminator structure downstream of the cbbLS genes (Schäferjohann et al., 1996). Indeed in N. europaea premature termination of transcription is likely to occur, since a stem–loop structure could be formed in the intergenic region between cbbS and cbbQ with a calculated free energy ({Delta}G0) of –155 kJ mol–1 (not shown). This predicted stem–loop structure appears more stable than that in Ral. eutropha, in which the free energy is –102 kJ mol–1 (Schäferjohann et al., 1996). Potential hairpin structures were also identified downstream of the cbbLS genes in T. denitrificans and X. flavus (Hernandez et al., 1996; Pulgar et al., 1991). The spatial conformation of an mRNA is known to affect its stability or longevity (Grunberg-Manago, 1999). Different structures of N. europaea cbb mRNAs may have contributed to the different levels of abundance that we observed.

Although a transcription terminator immediately downstream cbbL was identified in some species (Valle et al., 1988), an examination of the intergenic sequence (63 bp) between cbbL and cbbS in N. europaea failed to identify a potential transcriptional terminator. This result with N. europaea is similar to what has been reported for Ac. ferrooxidans (Kusano et al., 1991). In support of an mRNA processing alternative, the consensus cleavage site sequence of RNase E, (G/A)ATT(A/T) (Ehretsmann et al., 1992), was identified in the first three intergenic regions in the cbb operon in N. europaea. A cleavage of the mRNA containing cbbL and cbbS may also occur. RNA processing in bacteria can occur by means other than RNase E (Calin-Jageman & Nicholson, 2003; Otsuka et al., 2003). In the N. europaea genome sequence, a number of endoribonuclease genes were identified (Chain et al., 2003), including RNase E, RNase G and RNase III. RNase III acts on double-stranded RNAs. These results and sequence analyses suggest that a complex processing of the cbb mRNA may be involved in the regulation of transcription and function, and is affected by either excision/cleavage or differential degradation.

The intergenic region between cbbR and cbbL should contain promoters in opposite directions, for both cbbR and cbbLSQON. CbbR is a LysR-type transcriptional regulator (Schell, 1993). CbbR is believed to be involved in autoregulation of its own transcription as in Ral. eutropha and X. flavus (Kusian & Bowien, 1995; Shively et al., 1998; van Keulen et al., 2003). Thus the unique features of the intergenic region (i.e. AT-rich region and T-(N)n-A inverted repeats) may allow binding of CbbR and other potential regulators in both orientations, possibly with different affinities (Fig. 2). The AT-rich element upstream of the rbc (RubisCO) gene in Synechococcus sp. PCC 7002 was required for CO2-dependent repression (Onizuka et al., 2002). In their mobility-shift assay, a strong signal of a repressor binding to the AT-box was observed in extracts from cells cultured at 15 % CO2, but only a weak signal from cells cultured at 1 % CO2. It was suggested that the AT-rich element was involved in the negative regulation of the rbc transcription in response to CO2 levels (Onizuka et al., 2002). We do not know whether the AT box plays a similar role in the regulation of the cbb gene transcription in N. europaea, but our results indicate that high CO2 can repress its transcription.

In the absence of an exogenous energy source, the presence of NH3 can turn on amo transcription by using energy reserves in the cells (Hyman & Arp, 1995; Sayavedra-Soto et al., 1996). N. europaea preferentially directs its internal energy reserves for the synthesis of amo mRNA in the presence of NH3 and C2H2 included experimentally to block oxidation of NH3 (Sayavedra-Soto et al., 1996). In contrast, NH3 was not a signal to turn on cbbLS gene transcription. Rather, an exogenous energy source was required (Fig. 5). Because NH3 is the sole energy source for ammonia-oxidizing bacteria, regulation of major pathways by NH3 is expected. However, the energy status of the cell appeared to be the key factor for the transcription of the cbb genes and NH3 itself was not required. N. europaea cells did not commit their internal energy reserves to the transcription of the cbb genes (Fig. 5). N. europaea uses limited internal energy for the transcription of amo and hao, which are directly involved in energy harvesting (Hyman & Arp, 1995; Sayavedra-Soto et al., 1996). In the absence of any exogenous energy source cbbL and cbbS mRNAs were depleted faster than amo and hao mRNAs (Fig. 4B), further indicating the dependence of cbb message levels on the cellular energy status. The CBB cycle is an intensely energy-consuming process; thus it is not surprising that transcription of the cbb operon is dependent upon the presence of an abundant energy source.

The RubisCO gene transcription in N. europaea is responsive to the available carbon levels, with low carbon levels resulting in the highest transcription (Fig. 6), perhaps through transcriptional derepression. Increases in the levels of RuBisCO synthesis and activity under CO2 limitation have also been documented for other micro-organisms. For example, in the facultative autotrophic bacteria Ralstonia oxalatica (formerly Pseudomonas oxalaticus) (Dijkhuizen & Harder, 1979) and Ral. eutropha (formerly Alcaligenes eutrophus) (Friedrich, 1982), the Calvin cycle was less repressed with limited carbon source (C1 compounds such as formate). Growth of the obligate autotroph T. neapolitanus in a chemostat under CO2 limitation caused increased activity of the Calvin cycle (Beudeker et al., 1980). However, complete carbon starvation did not induce the Calvin cycle in the facultative autotroph Ral. eutropha (Friedrich, 1982). In other organisms such as cyanobacteria and algae, carbon-concentrating mechanisms have been identified (Shibata et al., 2001; Xiang et al., 2001). In contrast, PRK mRNA in N. europaea increased with elevated /CO2, presumably due to the increase in the total carbon fixed.

In N. europaea, the highest cbb transcription levels were observed with atmospheric levels of CO2. Atmospheric CO2 content is about 0·03 %, which equilibrates to approximately 9 µM CO2 in water at 30 °C. In spite of the high cbb gene transcription, N. europaea grew poorly in media lacking Na2CO3 and where C was available only from the atmosphere (data not shown), and the cultures exhibited a long lag phase (~5 days, compared to 1 day in carbonate-containing medium). Cells incubated in such medium made much more RubisCO mRNA than cells grown in medium containing carbonate. Genes for ammonia metabolism (amo, hao) showed the opposite trend. T. neapolitanus had higher carbon-fixing capacity per unit of total cell protein in chemostat cultures under carbon-limiting conditions than under carbon-saturated conditions (Beudeker et al., 1980). As seen in our experiments, N. europaea cells appeared to respond to carbon limitation by prioritizing more resources for synthesizing more RubisCO mRNA, and presumably RubisCO enzyme as well. CO2 levels in natural habitats may fluctuate frequently, so the responsiveness of the transcription of genes for carbon fixation to CO2 levels may have adaptive value. The observed high cbb gene transcription with limited CO2 may be an adaptation of the cells to scavenge the limited CO2 (0·03 % in air). Regardless of the cbb gene transcription, more efficient carbon fixation appears to be correlated with the CO2 levels in the media, as is evidenced by higher growth rates of N. europaea in the presence of higher available carbon in the medium (data not shown). Carbonic anhydrase is required for Ral. eutropha to grow at atmospheric CO2 concentrations (Kusian et al., 2002). Genes for potential carbonic anhydrase and an anion transporter (NE1927) in N. europaea were transcribed similarly under low- and high-carbon conditions (blots not shown), suggesting that they may not be functioning as a CO2/-concentrating mechanism. The apparent lack of a CO2-concentrating mechanism is supported by the observation of poor growth of N. europaea cultures in -free medium exposed to air. The responsiveness of cbb gene transcription to carbon levels may also be a reflection of the lack of a specific CO2-concentrating mechanism in N. europaea. It will be interesting to see how other ammonia-oxidizing bacteria, such as those with carboxysomes, respond to low C levels.


   ACKNOWLEDGEMENTS
 
This research was supported by the Office of Science (BER), US Department of Energy grant no. DE-FG03-01ER63149 and the Oregon Agricultural Experimental Station.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 24 September 2003; revised 30 January 2004; accepted 5 March 2004.



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