Form I RubisCO
Phylogenetic analyses based on large subunit genes and deduced amino acid sequences divide form I RubisCO into two groups (‘green’ and ‘red’), which may be further subdivided into types IA, IB, IC and ID. Whereas type IB RubisCO enzymes are mostly found in cyanobacteria, green algae and type ID in non-green algae, chemolithoautotrophic bacteria are known to contain types IA and IC of RubisCO. Specific information on metabolic and ecological properties affecting the occurrence and distribution of both bacterial forms in the environment are limited (Badger & Bek, 2008). Generally, bacteria containing form IC RubisCO are known for facile genetic transfer (Horken & Tabita 1999). It has also been suggested that obligate chemolithoautotrophs often possess form IA RubisCO, and form IC enzymes are often associated with facultative autotrophs (Badger & Bek, 2008). Exceptions are several ammonium-oxidizing bacteria affiliated with different Nitrospira species and the Gammaproteobacteria Nitrosococcus oceani, which are obligate autotrophs oxidizing ammonium.
In the current study, amplification of DNA (by PCR) and mRNA (by RT-PCR) coding for RubisCO genes revealed a distinct pattern in the individual samples (Table 1). Form IA RubisCO PCR and RT-PCR products were obtained from the original (inflowing) groundwater (LR) and groundwater amended with oxygen (LO). In groundwater amended with nitrate (LN) and active coal, RubisCO genes were amplified only from DNA extracts. Form IC RubisCO genes were exclusively detected in DNA and RNA extracted in samples obtained from the oxygenated groundwater. To evaluate the specificity of the PCR approach and to obtain information on the diversity and phylogenetic affiliation of putative RubisCO genes, amplification products obtained from all three groundwater sampling stations were cloned and selected clones were sequenced. Initial analysis of all sequenced clone inserts was accomplished by comparison with public databases based on blast search algorithm (see 'Materials and methods'), which confirmed the specificity of our approach because all sequences were found to be affiliated with the targeted genes. Phylogenetic trees based on deduced amino acid sequences of RubisCO form IA and IC sequences derived from this study and public databases are presented in Figs 1 and 2.
Figure 1. Neighbour-joining tree calculated from deduced amino acid sequences of form IA RubisCO genes obtained from sampling stations LO, LR and LN and sequences retrieved from NCBI database. DNA-based sequences obtained from this study are indicated in bold; transcripts are indicated in bold and are underlined. Consecutive numbers in parentheses following the clone sequences refer to information provided in Supporting Information Table S1, including all clone designations and their corresponding accession numbers. Accession numbers of reference sequences are also given in parentheses. The bootstrap consensus tree is inferred from 1000 replicates. Bootstrap values below 50% are not shown.
Download figure to PowerPoint
Figure 2. Neighbour-joining tree calculated from deduced amino acid sequences of form IC RubisCO genes obtained from sampling station LO and sequences retrieved from NCBI database. DNA-based sequences obtained from this study are indicated in bold; transcripts are indicated in bold and are underlined. Consecutive numbers in parentheses following the clone sequences refer to information provided in Table S1, including all clone designations and their corresponding accession numbers. Accession numbers of reference sequences are also given in parentheses. The bootstrap consensus tree was inferred from 1000 replicates. Bootstrap values below 50% are not shown.
Download figure to PowerPoint
The original groundwater sample revealed two phylogenetically clearly separated clusters of RubisCO form IA sequences, which were numerically dominated by (DNA and RNA based) sequences closely related to environmental RubisCO sequences obtained from anoxic and BTEX-contaminated groundwater aquifer sediments analyzed in a former study (groundwater environmental clones 9BSED C2 and C3; Alfreider et al., 2003). The closest RubisCO sequence (96% amino acid similarity) from cultivated bacteria is Sideroxydans lithotrophicus ES-1, a microaerobic ferrous iron-oxidizing Betaproteobacterium that was isolated from groundwater and grows at circumneutral pH (Emerson & Moyer, 1997; Druschel et al., 2008). Two clones (LR-RNA-C22 and LR-DNA-C14) were related to a DNA sequence obtained from groundwater supplied with nitrate in the current study and to RubisCO genes present in bacterial ectosymbionts of the shallow-water marine worm Tubificoides benedii (Ruehland & Dubilier, 2010). Another Tubifex-associated clone sequence (clone 76I2), obtained from the same study, was the closest relative of several sequences (from DNA as well as RNA extracts) from oxygenated groundwater of sampling station LO (Fig. 1). In this sample, a major cluster of highly similar sequences obtained from DNA (nine sequences) and transcripts (14 sequences) was affiliated with form IA RubisCO identified in S. lithotrophicus ES-1 (97% amino acid sequence similarity). Twenty-nine closely related sequences retrieved from DNA extracts of sampling station LN formed a clearly separated cluster in the phylogenetic tree (Fig. 1). Based on amino acid sequence identity, the closest relatives are the obligately chemolithoautotrophic and facultatively anaerobic Thiobacillus denitrificans (96% identity) and the haloalkaliphilic sulphur-oxidizing bacteria Thioalkalivibrio sp. HL-EbGR7 (95% similarity); the latter is also closely related to T. denitrificans. Other RubisCO form IA sequences were distantly related to cultivated representatives deposited in public databases. Therefore there is a lack for inferring ecophysiological characteristics from these sequences.
Form IC RubisCO sequences were exclusively detected in groundwater supplied with oxygen (LO, Fig. 2). The majority of the sequences obtained from RNA- and DNA-extracts clustered with clone sequences (S6C36) obtained from a non-contaminated shallow aquifer investigated in a former study (Alfreider et al., 2009). The addition of oxygen in an ammonium-rich environment is an ideal habitat for nitrifying bacteria. Sequence analysis of form IC transcripts revealed their affiliation to members of the Nitrosospira lineage and N. oceani (Fig. 2), indicating their potential role in the nitrification process at this sampling station. In a study of Utåker et al. (2002) it was ascertained that the majority of ammonium-oxidizing bacteria possess form IC RubisCO; therefore, the absence of RubisCO form IA sequences affiliated with ammonium-oxidizing bacteria at sampling station LO is not peculiar. All other form IC RubisCO clone sequences obtained from sample LO originated from DNA extracts and showed the highest amino acid similarities with sequences obtained by cultivation-independent studies from agricultural soils and a tar oil-contaminated aquifer (Selesi et al., 2005; Kellermann, 2008).
Nigro & King (2007) suggested that the distribution of form IA- and IC-containing chemolithoautotrophic bacteria corresponds to functional distinctions of both forms and is associated with the relative distribution of the availability of sulphide. In fact, the ability to use sulphide as electron donor is known for a number of form IA-containing bacteria – a physiological feature never observed in RubisCO form IC chemoautotrophs. This concept was supported by the results of this study. Form IC RubisCO DNA and mRNA were exclusively detected in groundwater samples supplied with oxygen (sampling station LO) but not in the original groundwater or the groundwater supplied with nitrate, which were characterized by very low sulphide concentrations.
Form II RubisCO
The form II RubisCO enzyme in Proteobacteria is markedly different from that of form I with regard to sequence similarity and kinetic properties. An essential biochemical characteristic of form II enzymes is the poor affinity for CO2 and a low discrimination against O2 (Tabita, 1999). From the viewpoint of RubisCO ecology, it has been suggested that form II enzymes are adapted to low-O2 and high-CO2 environments (Badger & Bek, 2008). Form II RubisCOs are found in two gene arrangements, which are well correlated with the metabolic functioning of the organisms in which they occur (Badger & Bek, 2008). An interesting feature of form II RubisCO is that it is often found in organisms that also contain form I. Chemoautotrophic bacteria that have acquired the genes encoding both forms of RubisCO may have an advantage in ecosystems where O2 and CO2 concentrations vary considerably, because the dissimilar kinetic properties of the enzymes would allow efficient CO2 assimilation under both aerobic and anaerobic conditions (Alfreider et al., 2003).
Form II RubisCO from RNA and DNA were successfully amplified from samples LR and LN but not in oxygen-amended groundwater (sampling station LO), where cbbM genes detected in bacterial DNA were not expressed (Table 1). This distribution pattern corresponds well with the kinetic properties known for RubisCO form II (see above). Sequence analysis of clone libraries revealed that cbbM sequences were widely distributed in the phylogenetic tree (Fig. 3). In the original groundwater sample LR, a single cbbM sequence (clone C10) was detected from RNA extracts, which was identical with RubisCO from DNA extracts obtained from sampling station LN. Form II RubisCO transcripts from LN were represented by two phylotypes: a single cbbM sequence (L6-RNA-C21) and a cluster of 10 almost identical sequences. Both phylotypes were closely related or identical to RubisCO clone sequences obtained from a tar oil-contaminated aquifer (Kellermann, 2008).
Figure 3. Neighbour-joining tree calculated from deduced amino acid sequences of form II RubisCO genes obtained from sampling stations LO, LR and LN and sequences retrieved from IMG and NCBI database. DNA-based sequences obtained from this study are indicated in bold, transcripts are indicated in bold and are underlined Consecutive numbers in parentheses following the clone sequences refer to information provided in Table S1, including all clone designations and their corresponding accession numbers. Accession numbers of reference sequences are also given in parentheses. The bootstrap consensus tree was inferred from 1000 replicates. Bootstrap values below 50% are not shown.
Download figure to PowerPoint
The affiliation of sequences obtained from groundwater samples to known cbbM sequences of cultured bacterial strains was wide ranging, including numerous obligate and facultative chemolithoautotrophs (Fig. 3). The closest relatives for cbbM transcripts obtained from sampling stations LN and LR include RubisCO cbbM analyzed for T. denitrificans, S. lithotrophicus ES-1 and Accumulibacter phosphatis clade IIA with amino acid sequence similarities ranging between 92% and 96%. In contrast to the distribution and diversity patterns observed with form IA RubisCO sequences, cbbM sequences were often represented by identical or closely related form II sequences retrieved from all three sampling stations, LR, LO and LN. For example, one distinct cluster with DNA sequences obtained from all samples showed a high degree of sequence similarity; these sequences were affiliated to different environmental clones obtained from polluted and pristine groundwater or soils (Fig. 3, sequences at the top of the tree).
The original groundwater (LR) was characterized by virtually anoxic conditions with elevated concentrations of ammonium and ferrous iron (Table 1). Nitrate was below the detection limit; in contrast, sulphate was present in significant amounts. Bacteria using form IC RubisCO for CO2 fixation, which are often represented by facultative autotrophs or mixotrophs, were not detected in the original sampling station. It has been suggested that facultative autotrophic bacteria can be found in environments where inorganic and organic compounds are available (Badger & Bek, 2008). In sample LR, DOC consists mainly of MTBE and tert-butyl alcohol (TBA), which is recalcitrant under the in situ conditions and therefore does not serve as an organic carbon source (Table 1).
Sample LO, which was obtained from the conditioning unit supplied with oxygen, showed in comparison with the reference site a decline in ammonium concentration and the occurrence of nitrate (Table 1), which can be explained by nitrification of ammonium to nitrate. RubisCO transcripts affiliated with ammonium-oxidizing bacteria confirm their active role in the nitrogen cycle at this sampling station. Recent studies suggest that aerobic ammonium oxidation by autotrophic Archaea is of major significance in marine and soil ecosystems (e.g. Zhang et al., 2010; Pratscher et al., 2011; Yakimov et al., 2011). CO2 fixation of autotrophic thaumarchaeal ammonium oxidizers is accomplished via the 3-hydroxypropionate/4-hydroxybutyrate cycle, which was not investigated in this study. Consequently, future studies should also include the analysis of genes coding for key enzymes in this pathway in order to assess whether inorganic carbon fixation by Archaea is also associated with ammonium oxidation in groundwater systems. The oxidation of ferrous iron is another source for the consumption of oxygen. The analysis of RubisCO genes and transcripts revealed sequences affiliated with the iron-oxidizing Betaproteobacteria S. lithotrophicus. The concentration of DOC (in the form of MTBE/TBA) was not affected by the supply with oxygen (Table 1), indicating that MTBE was not degraded under aerobic conditions.
The addition of nitrate and active coal at sampling station LN caused a significant reduction of ammonium and MTBE in the effluent (Table 1). Furthermore, nitrate was completely consumed, suggesting the importance of dentrification and/or anammox (anaerobic ammonium oxidation) activities. The ecological role of anammox bacteria was not covered by our investigations because autotrophy in these microorganisms is based on the reductive acetyl-CoA pathway for carbon fixation (Schouten et al., 2004; Strous et al., 2006). RubisCO form I obtained from DNA was closely related to several denitrifying bacteria including T. denitrificans, which is capable of oxidizing inorganic sulphur compounds or ferrous iron using nitrate as electron acceptor (Beller et al., 2006). Thiobacillus denitrificans is also able to use sulphur/iron minerals, for example pyrite, as electron donors, which is an important physiological trait for adaptation to groundwater systems. Some sulphate might be reduced to sulphide, as the sulphate concentrations slightly decreased (Table 1). However, on the one hand, the question is whether indeed sufficient amounts of reduced sulphur compounds or iron-sulphur minerals were available for reducing the added nitrate. On the other hand, the inflowing groundwater sample (LR) contained ferrous iron in significant amounts (Table 1). Thus, the denitrification process might be driven partly by autotrophic ferrous iron-oxidizing phylotypes related to T. denitrificans using nitrate as electron acceptor. The presence of cbbM transcripts affiliated with RubisCO genes hosted in dentrifiers, including A. phosphatis and T. denitrificans, was detected at sampling station LN (Fig. 3). Accumulibacter phosphatis is well known to be primarily responsible for biological phosphorus removal in waste water and sludge, suggesting the preference for a heterotrophic lifestyle in an environment with high amounts of readily available organic carbon. In a metagenomic study, the detection of key genes of the Calvin cycle including phosphoribulokinase and RubisCO is evidence of the ability of A. phosphatis to fix CO2 (Garcia Martin et al., 2006). These findings indicate that Accumulibacter clades are also adapted to carbon limited habitats which was verified in a recent study by Peterson et al. (2008), which included the investigation of lakes, rivers and springs. Although the denitrification capabilities in different clades of A. phosphatis strains remains to be clarified (Zeng et al., 2003; Flowers et al., 2009), the high similarity with A. phosphatis sequences of the majority of cbbM transcripts obtained from sample LN suggests the presence of autotrophic bacteria that are actively involved in dissimilatory nitrate reduction. In this context it should be noted that the groundwater at sampling stations LO and LN was supplied with the same amount of phosphate (27 g K2HPO4 m−3), but the actual phosphate concentration was lower in sampling station LN. Phosphate is probably metabolized by bacterial populations affiliated with Accumulibacter spp., which are well known to accumulate inorganic phosphate efficiently (Hesselmann et al., 1999; Flowers et al., 2009).
Besides ferrous iron, MTBE and the related DOC might be other important electron donors for nitrate reduction at sampling station LN, as MTBE and DOC were significantly reduced during the passage through the conditioning unit LN (Table 1). It cannot be excluded, however, that MTBE was almost completely adsorbed by the active coal used as a filling material of this channel. Indeed, MTBE oxidation with nitrate or ferric iron (which probably accumulated in the channel due to the constant oxidation of ferrous iron with nitrate) as electron acceptors is rarely observed in the environment (Bradley et al., 2001; Somsanak et al., 2001). Thus, the results suggest the existence of two main biological sinks for nitrate in the form of anoxic, nitrate-dependent microbial oxidation of ferrous iron and ammonium. Nitrite formed during the ferrous iron-dependent nitrate reduction might be channelled in the anammox process (Kuenen, 2008).
Although the role of facultative autotrophic prokaryotes for the degradation of MTBE was not particularly investigated in this study, their potential importance at the sampling station should be noted. For example the methylotrophic bacterium Methylibium petroleiphilum PM1 is known to play a key role for aerobic MTBE degradation in contaminated aquifers (Wilson et al., 2002; Smith et al., 2005). Methylotrophic autotrophy as an alternative type of nutrition based on RubisCO pathway was demonstrated for the methylotrophic bacterium Beijerinckia mobilis (Dedysh et al., 2005). Although an autotrophic metabolism for strain M. petroleiphilum PM1 has not yet been confirmed, a whole genome analysis study of strain PM1 revealed two sets of genes coding for form I RubisCO and associated enzymes necessary for CO2 assimilation via the Calvin cycle (Kane et al., 2007). Whereas the activity of PM1 at the Leuna site has not been proven yet, the closely related (95.6% 16S rRNA gene sequence similarity) MTBE-degrading bacterium Aquincola tertiaricarbonis str L108 has been isolated from Leuna groundwater (Rohwerder et al., 2006; Lechner et al., 2007) and its activity in aerated Leuna groundwater trenches was recently demonstrated (Jechalke et al., 2011). The genome of strain L108 has been partly sequenced and genes coding for the small and large subunit of RubisCO were identified (T. Rohwerder, pers. commun.). Preliminary sequence analysis showed that the cbbL gene of strain L108 is affiliated with RubisCO large subunit genes in M. petroleiphilum PM1 (93 and 83% amino acid identity; data not shown). An explanation as to why MTBE was not degraded at the sampling station supplied with oxygen (LO) is provided by the presence of a metabolically active nitrifying microbial community. The competitive effect of ammonium oxidizers, which are characterized by a higher growth rate than MTBE degraders, was shown in a model that was developed for an experimental packed bed reactor (Waul et al., 2008).