Phylogenomic analysis and predicted physiological role of the proton-translocating NADH:quinone oxidoreductase (complex I) across bacteria

Abstract

The proton-translocating NADH:quinone oxidoreductase (complex I) is a multisubunit integral membrane enzyme found in the respiratory chains of both bacteria and eukaryotic organelles. Although much research has focused on the enzyme's central role in the mitochondrial respiratory chain, comparatively little is known about its role in the diverse energetic lifestyles of different bacteria. Here, we used a phylogenomic approach to better understand the distribution of complex I across bacteria, the evolution of this enzyme, and its potential roles in shaping the physiology of different bacterial groups. By surveying 970 representative bacterial genomes, we predict complex I to be present in ~50% of bacteria. While this includes bacteria with a wide range of energetic schemes, the presence of complex I is associated with specific lifestyles, including aerobic respiration and specific types of phototrophy (bacteria with only a type II reaction center). A phylogeny of bacterial complex I revealed five main clades of enzymes whose evolution is largely congruent with the evolution of the bacterial groups that encode complex I. A notable exception includes the gammaproteobacteria, whose members encode one of two distantly related complex I enzymes predicted to participate in different types of respiratory chains (aerobic versus anaerobic). Comparative genomic analyses suggest a broad role for complex I in reoxidizing NADH produced from various catabolic reactions, including the tricarboxylic acid (TCA) cycle and fatty acid beta-oxidation. Together, these findings suggest diverse roles for complex I across bacteria and highlight the importance of this enzyme in shaping diverse physiologies across the bacterial domain.

Importance: Living systems use conserved energy currencies, including a proton motive force (PMF), NADH, and ATP. The respiratory chain enzyme, complex I, connects these energy currencies by using NADH produced during nutrient breakdown to generate a PMF, which is subsequently used for ATP synthesis. Our goal is to better understand the role of complex I in bacteria, whose energetic diversity allows us to view its function in a range of biological contexts. We analyzed sequenced bacterial genomes to predict the presence, evolution, and function of complex I in bacteria. We identified five main classes of bacterial complex I and predict that different classes participate in different types of respiratory chains (aerobic and anaerobic). We also predict that complex I helps maintain a cellular redox state by reoxidizing NADH produced from central metabolism. Our findings suggest diverse roles for complex I in bacterial physiology, highlighting the need for future laboratory-based studies.

FIG 1

The distribution of complex I across bacteria. The distribution of predicted complex I enzymes in sequenced bacterial genomes. (A) The number of bacterial genomes predicted to encode 0, 1, or 2 complex I enzymes, organized by phylum or class. The x axis phylogeny was modeled using data from Ciccarelli et al. (77). (B) The percentage of bacterial genomes predicted to encode 0, 1, or 2 complex I enzymes, organized by trait. An asterisk (*) indicates that the distribution of complex I (0, 1, or 2 or more enzymes) for a specific trait is significantly different from the distribution of complex I in all bacteria (fist bar) at P values of <0.0005 (chi-square test). “N/A” indicates there were not enough genomes in the category to perform the statistical analysis.

FIG 2

Phylogeny of bacterial complex I. A phylogeny of predicted complex I enzymes from 508 sequenced bacterial genomes was generated using amino acid sequences from all 14 concatenated complex I subunits. There are five main clades of bacterial complex I (clades A to E), which can be distinguished by specific subunit features. Genome names and most of the support values were omitted for clarity.

FIG 3

The extended C terminus of clade A NuoE is poorly conserved. Protein alignment of the first 300 amino acids of the complex I subunit NuoE from clade E E. coli and clade A R. sphaeroides, R. capsulatus, and Agrobacterium tumefaciens. The first ~170 amino acids of NuoE are well conserved between all organisms. Most clade A NuoE subunits have an extended C terminus, which is poorly conserved even between closely related bacteria.

FIG 4

Inheritance pattern of complex I in specific bacterial groups. A species phylogeny of representative members of Bacteroidetes, Actinobacteria, Deltaproteobacteria, and Gammaproteobacteria was generated using the amino acid sequences of 9 highly conserved housekeeping genes. The absence or presence of complex I (by complex I clade) was mapped onto the phylogeny to show the patchwork inheritance pattern of complex I within specific groups of bacteria. For visualization purposes, Bacillus cereus was used to root the tree, as the Firmicutes phylum represents a distantly related phylum in which no complex I enzymes were identified. Support values were omitted for clarity, but the full phylogeny is available in Fig. S8 in the supplemental material. Abbreviations: C. Amoebophilus asiaticus, “Candidatus Amoebophilus asiaticus”; Blattabacterium sp. B. germ, Blattabacterium sp. Blattella germanica; Anaeromyxo. sp. Fw109-5, Anaeromyxobacter sp. Fw109-5; Syntrophobact. fumaroxidans, Syntrophobacter fumaroxidans; Pseudoxanthomonas s., Pseudoxanthomonas suwonensis; Pseuodoaltero. sp. SM9913, Pseudoalteromonas sp. SM9913; Actinobacillus pleuro., Actinobacillus pleuropneumoniae.

FIG 5

Biochemical pathways enriched in bacterial genomes that encode complex I. (A) Heat map showing the biochemical pathways (KEGG modules) that are enriched in bacterial genomes predicted to encode complex I compared to members of the same class/phylum that are predicted to lack complex I. We present pathways that show significant enrichment with a P value of <0.01 (see Data Set S2 in the supplemental materials for full results). Pathways are colored according to their level of significance, which was calculated by −log₁₀(P value), so that the most significantly enriched pathways have a higher value on the color key and are darker shades of blue. For visual purposes, the value of the most significant pathway (“NADH:quinone oxidoreductase, prokaryotes” in Actinobacteria) was adjusted from 44 to 25, which still represents the upper limit of the scale, while improving the resolution of other enriched pathways found in the analysis. Pathway names highlighted in pink produce NADH as a byproduct (72). (B) Schematic of biochemical pathways that are enriched across different groups of bacteria predicted to encode complex I. Beta-oxidation produces NADH and acetyl-CoA, the latter of which feeds into the TCA cycle to produce additional NADH. The glyoxylate cycle and ethylmalonyl pathway (not shown) assimilate acetyl-CoA by bypassing the CO₂-generating reactions of the TCA cycle.