CO2 assimilation strategies in stratified lakes: Diversity and distribution patterns of chemolithoautotrophs

Summary While mechanisms of different carbon dioxide (CO2) assimilation pathways in chemolithoautotrohic prokaryotes are well understood for many isolates under laboratory conditions, the ecological significance of diverse CO2 fixation strategies in the environment is mostly unexplored. Six stratified freshwater lakes were chosen to study the distribution and diversity of the Calvin-Benson-Bassham (CBB) cycle, the reductive tricarboxylic acid (rTCA) cycle, and the recently discovered archaeal 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) pathway. Eleven primer sets were used to amplify and sequence genes coding for selected key enzymes in the three pathways. Whereas the CBB pathway with different forms of RubisCO (IA, IC and II) was ubiquitous and related to diverse bacterial taxa, encompassing a wide range of potential physiologies, the rTCA cycle in Epsilonproteobacteria and Chloribi was exclusively detected in anoxic water layers. Nitrifiying Nitrosospira and Thaumarchaeota, using the rTCA and HP/HB cycle respectively, are important residents in the aphotic and (micro-)oxic zone of deep lakes. Both taxa were of minor importance in surface waters and in smaller lakes characterized by an anoxic hypolimnion. Overall, this study provides a first insight on how different CO2 fixation strategies and chemical gradients in lakes are associated to the distribution of chemoautotrophic prokaryotes with different functional traits.


Table S1
Specification of primers applied for the detection of different CO2 fixation pathways

Table S2
Summary of physico-chemical parameters in the studied lakes

Fig. S2
Coverage of two different primer sets targeting Form II RubisCO sequences

Fig. S3
Coverage of two different primer sets targeting Form IC RubisCO sequences

Fig. S4
Evaluation of different annealing temperatures for thaumarchaeal hcd qPCR primers

Fig. S5
Evaluation of the specificity of hcd qPCR primers

Fig. S6
Vertical profiles of ammonium and nitrate in the studied lakes

Fig. S7
Vertical profile of autotrophic cell numbers in Lake Egelsee

Optimization of the functional gene approach
In this study, a major effort was undertaken to (re)evaluate already published PCR-based marker systems and to design and test new primer pairs before they were applied on a routine basis at our study sites. Table S1 summarizes the specification of primers that were used. Not included in this table are other published primer pairs that were tested (in silico and/or in vitro), but were not appropriate for the investigation of lake water samples from our study sites.
Three sets of oligonucleotide primers were designed and successfully used for PCR and RT-PCR amplification of RubisCO form IA, form IC and form II gene fragments as applied in groundwater samples by Alfreider et al. (2003Alfreider et al. ( , 2009Alfreider et al. ( , 2012 and in exploratory studies in selected lakes (data not shown). Nonetheless, the specificity and coverage of these primers was evaluated based on new RubisCO sequences available in public sequence databases and recently published primer sets. For example, Kato et al. (2012) designed a PCR primer set targeting RubisCO form II gene where the coverage (in silico) was reported to be superior compared with the primers designed by Alfreider et al. (2003). However, in vivo validation of both primers systems revealed a different picture (Fig. S2) and individual primer sets covered only a part of the cbbM diversity observed in our study sites. In order to increase the coverage of cbbM genes, selected lake water samples were amplified with both primers sets for sequence analysis. For the same reason, two primers pairs were also used to study the diversity of the form IC RubisCO (Table S1. Fig. S3). Form IA genes were targeted with one primer pair because other primer pairs tested were not specific (resulting in multiple banding of PCR products).
Autotrophy in Thaumarchaeota was investigated by the analysis of genes coding for 4hydroxybutyryl-CoA dehydratase (hcd), a key protein in the HP/HB and dicarboxylate/4hydroxybutyrate (DC/4-HB) cycle (Berg, 2011). Several PCR based protocols were developed, and we selected two primers sets that produced specific amplification products in our samples (Offre et al. 2010;Yakimov et al. 2011, Table S1). For the quantification of hcd genes, a real time PCR primer pair (qPCR_hcd_f/qPCR_hcd_r, Table 1) was designed that specifically covers the sequence diversity of hcd genes obtained in this study. This primer was specifically designed on basis of the hcd sequences derived from PCR products with primers hcd-465F/hcd-1267R and 4HBD312F/4HBD1360R (Table   S1) of lakes ACH, STA and ZUR (Table S1, Fig. 2), and sequences of representatives within the marine Group I (MGI) Thaumarchaeota lineage. Gel electrophoresis and sequence analysis of selected PCR-amplificates produced with the qPCR primer from different samples of lakes ACH, STA and ZUR support the appropriateness of the qPCR assay (Fig. S3 and S4).
CO2 fixation based on the rTCA cycle is generally detected by targeting genes coding for the alpha or beta subunit of the ATP citrate lyase (aclAB genes), and/or the alpha subunit of 2oxoglutarate:ferredoxin oxidoreductase enzymes (oorA genes). Published primer sets, which were tested on samples from our study sites, mostly produced multiple bands or faint amplification products. Furthermore, sequence analysis of randomly selected PCR amplicons of aclAB genes produced nucleotide sequences that were mostly not affiliated with the target genes (data not shown).
In fact, most published primers were originally designed to study marine hydrothermal environments; therefore, these primers specifically target thermophilic Aquificales and Epsilonproteobacteria (reviewed by Hügler and Sievert, 2011). Further primer optimization was necessary and we developed a broad range detection system, based on a nested PCR approach, targeting genes coding for ATP citrate lyase alpha subunit (aclA) within different clades of Epsilonproteobacteria, Deltaproteobacteria, Acidobacteria, and Nitrospirae. A second primer set was developed to specifically target aclA genes in Nitrospirae. Finally, eleven primer systems were used to amplify different functional genes coding for key enzymes in the Calvin cycle, the HP/HB cycle, and the rTCA cycle in samples from six stratified lakes (Table S1).        Table S1) retrieved from different samples of the studied lakes. The green color indicates cbbM lineages that are covered by both primer pairs (Alfreider et al., 2003 andKato et al., 2012).
Overlapping regions of PCR products    Table S1) retrieved from different samples of the studied lakes. The green color indicates cbbL IC lineages that are covered by both primer pairs (Alfreider et al., 2009 andSelesi et al., 2009).
Overlapping regions of PCR products  Fig. S5. Evaluation of the specificity of hcd qPCR primers (annealing temperature 56°C) by sequencing of cloned PCR products derived from samples of lakes ACH, STA and ZUR.