High ratio of bacteriochlorophyll biosynthesis genes to chlorophyll biosynthesis genes in bacteria of humic lakes

Abstract

Recent studies highlight the diversity and significance of marine phototrophic microorganisms such as picocyanobacteria, phototrophic picoeukaryotes, and bacteriochlorophyll- and rhodopsin-holding phototrophic bacteria. To assess if freshwater ecosystems also harbor similar phototroph diversity, genes involved in the biosynthesis of bacteriochlorophyll and chlorophyll were targeted to explore oxygenic and aerobic anoxygenic phototroph composition in a wide range of lakes. Partial dark-operative protochlorophyllide oxidoreductase (DPOR) and chlorophyllide oxidoreductase (COR) genes in bacteria of seven lakes with contrasting trophic statuses were PCR amplified, cloned, and sequenced. Out of 61 sequences encoding the L subunit of DPOR (L-DPOR), 22 clustered with aerobic anoxygenic photosynthetic bacteria, whereas 39 L-DPOR sequences related to oxygenic phototrophs, like cyanobacteria, were observed. Phylogenetic analysis revealed clear separation of these freshwater L-DPOR genes as well as 11 COR gene sequences from their marine counterparts. Terminal restriction fragment length analysis of L-DPOR genes was used to characterize oxygenic aerobic and anoxygenic photosynthesizing populations in 20 lakes differing in physical and chemical characteristics. Significant differences in L-DPOR community composition were observed between dystrophic lakes and all other systems, where a higher proportion of genes affiliated with aerobic anoxygenic photosynthetic bacteria was observed than in other systems. Our results reveal a significant diversity of phototrophic microorganisms in lakes and suggest niche partitioning of oxygenic and aerobic anoxygenic phototrophs in these systems in response to trophic status and coupled differences in light regime.

FIG. 1.

Phylogenetic analysis including L-DPOR involved in bacteriochlorophyll (bchL) and chlorophyll (chlL) biosynthesis, COR (bchX), and nitrogenase reductase (nifH). (A) Partial protein sequences, including the ATP-binding domain of the iron protein and the cysteine residues that function as a ligand for the 4Fe-4S cluster, were used for phylogenetic reconstruction. Consensus trees based on reference sequences with phylogenetic information, Global Ocean Survey sequences (39, 47) and clone libraries from seven lakes (Bodsjön, Gevsjön, Nedre Björntjärn, Vättern, Vallentunasjön, Torneträsk, and Lilla Ullfjärden) were constructed by maximum likelihood analysis (RaxML). Reference sequences from bacterial isolates used for panel B are indicated by numbers (1, Acidiphilium rubrum AB017351; 2, Rhodospirillum rubrum CP000230; 3, Rhodopseudomonas palustris CP000301; 4, Rhodopseudomonas palustris CP000283; 5, Erythrobacter sp. strain AY672000; 6, Citromicrobium sp. strain AY671998; 7, Erythrobacter sp. strain AY672002; and 8, Rhodobacter capsulatus Z11165). nifH genes were used as outgroups. Outer rings indicate habitat type (marine or freshwater), molecular approach (source), and combined mismatches to PCR primer pairs used in this study. (B) Subtree of L-DPOR sequences with bootstrap values for main branches. The sources of sequences are indicated by habitat type and lake system. The collapsed branch includes 34 marine sequences from a global ocean survey (39).

FIG. 1.

Phylogenetic analysis including L-DPOR involved in bacteriochlorophyll (bchL) and chlorophyll (chlL) biosynthesis, COR (bchX), and nitrogenase reductase (nifH). (A) Partial protein sequences, including the ATP-binding domain of the iron protein and the cysteine residues that function as a ligand for the 4Fe-4S cluster, were used for phylogenetic reconstruction. Consensus trees based on reference sequences with phylogenetic information, Global Ocean Survey sequences (39, 47) and clone libraries from seven lakes (Bodsjön, Gevsjön, Nedre Björntjärn, Vättern, Vallentunasjön, Torneträsk, and Lilla Ullfjärden) were constructed by maximum likelihood analysis (RaxML). Reference sequences from bacterial isolates used for panel B are indicated by numbers (1, Acidiphilium rubrum AB017351; 2, Rhodospirillum rubrum CP000230; 3, Rhodopseudomonas palustris CP000301; 4, Rhodopseudomonas palustris CP000283; 5, Erythrobacter sp. strain AY672000; 6, Citromicrobium sp. strain AY671998; 7, Erythrobacter sp. strain AY672002; and 8, Rhodobacter capsulatus Z11165). nifH genes were used as outgroups. Outer rings indicate habitat type (marine or freshwater), molecular approach (source), and combined mismatches to PCR primer pairs used in this study. (B) Subtree of L-DPOR sequences with bootstrap values for main branches. The sources of sequences are indicated by habitat type and lake system. The collapsed branch includes 34 marine sequences from a global ocean survey (39).

FIG. 2.

Occurrence patterns of genes encoding L-DPOR involved in bacteriochlorophyll (bchL) and chlorophyll (chlL) synthesis, COR (bchX) genes, and nitrogenase reductase (nifH) genes, represented by peaks in T-RFLP profiles, as assessed using relative fluorescence. A higher proportion of occurrence in a given sample is represented by black to dark gray, and lower levels of occurrence are represented by light gray to white. E, epilimnion; H, hypolimnion.

FIG. 3.

Two-dimensional nonparametric MDS scaling plot of T-RFLP fingerprints of gene amplicons from L-DPOR. Black symbols indicate samples from humic/dystrophic lakes, dark gray symbols samples from eutrophic lakes, light gray symbols samples from mesotrophic lakes, and white symbols samples from oligotrophic lakes. Samples from mountain lakes in Lappland are labeled with a hexagon, samples from lowland lakes in Norrland are labeled with a triangle (an upward triangle indicates epilimnion samples, and a downward triangle indicates hypolimnion samples), squares indicate samples from lakes in Jämtland, circles indicate samples from lakes in Uppland, and stars indicate samples from Lake Vättern (the gradation from gray to black indicates increasing depth). The stress value was 0.09.