Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331-2902, USA
Correspondence
Daniel J. Arp
arpd{at}science.oregonstate.edu
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ABSTRACT |
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INTRODUCTION |
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Autotrophic nitrifiers assimilate CO2 via the CalvinBensonBassham (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.
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METHODS |
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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 ml1 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 [
32P]dCTP (3000 Ci mmol1, 110 TBq mmol1; 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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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)
.
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RESULTS |
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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
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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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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DISCUSSION |
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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 stemloop structure could be formed in the intergenic region between cbbS and cbbQ with a calculated free energy (
G0) of 155 kJ mol1 (not shown). This predicted stemloop structure appears more stable than that in Ral. eutropha, in which the free energy is 102 kJ mol1 (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.
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ACKNOWLEDGEMENTS |
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Received 24 September 2003;
revised 30 January 2004;
accepted 5 March 2004.
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