Different Functions of Phylogenetically Distinct Bacterial Complex I Isozymes

Abstract

NADH:quinone oxidoreductase (complex I) is a bioenergetic enzyme that transfers electrons from NADH to quinone, conserving the energy of this reaction by contributing to the proton motive force. While the importance of NADH oxidation to mitochondrial aerobic respiration is well documented, the contribution of complex I to bacterial electron transport chains has been tested in only a few species. Here, we analyze the function of two phylogenetically distinct complex I isozymes in Rhodobacter sphaeroides, an alphaproteobacterium that contains well-characterized electron transport chains. We found that R. sphaeroides complex I activity is important for aerobic respiration and required for anaerobic dimethyl sulfoxide (DMSO) respiration (in the absence of light), photoautotrophic growth, and photoheterotrophic growth (in the absence of an external electron acceptor). Our data also provide insight into the functions of the phylogenetically distinct R. sphaeroidescomplex I enzymes (complex IA and complex IE) in maintaining a cellular redox state during photoheterotrophic growth. We propose that the function of each isozyme during photoheterotrophic growth is either NADH synthesis (complex IA) or NADH oxidation (complex IE). The canonical alphaproteobacterial complex I isozyme (complex IA) was also shown to be important for routing electrons to nitrogenase-mediated H2 production, while the horizontally acquired enzyme (complex IE) was dispensable in this process. Unlike the singular role of complex I in mitochondria, we predict that the phylogenetically distinct complex I enzymes found across bacterial species have evolved to enhance the functions of their respective electron transport chains.

Importance: Cells use a proton motive force (PMF), NADH, and ATP to support numerous processes. In mitochondria, complex I uses NADH oxidation to generate a PMF, which can drive ATP synthesis. This study analyzed the function of complex I in bacteria, which contain more-diverse and more-flexible electron transport chains than mitochondria. We tested complex I function in Rhodobacter sphaeroides, a bacterium predicted to encode two phylogenetically distinct complex I isozymes. R. sphaeroides cells lacking both isozymes had growth defects during all tested modes of growth, illustrating the important function of this enzyme under diverse conditions. We conclude that the two isozymes are not functionally redundant and predict that phylogenetically distinct complex I enzymes have evolved to support the diverse lifestyles of bacteria.

FIG 1

Anaerobic growth of R. sphaeroides wild-type and complex I mutant strains. (A) The two complex I-encoding operons of R. sphaeroides. (B) Cells growing by anaerobic DMSO respiration in the dark (with succinate as the carbon source); (C) cells growing photoautotrophically (anaerobic in the light, with CO₂ and H₂ as the carbon and electron sources); (D) cells growing photoheterotrophically (anaerobic in the light, with succinate as the carbon source). Each quadrant contains a different strain: 1, wild type; 2, Δcomplex I_E strain; 3, Δcomplex I_A strain; 4, Δcomplex I_A/Δcomplex I_E strain.

FIG 2

Photoheterotrophic growth of wild-type and complex I mutant strains. (A) Membrane-associated electron transfer reactions in photoheterotrophic R. sphaeroides cells. Dashed lines indicate electron transfer reactions. During cyclic phototrophic electron transfer, the light-excited reaction center (RC) produces quinol (QH₂), which carries electrons to the cytochrome bc₁ complex (bc₁). Electrons are transferred to the cytochrome (cyt_red), producing a proton motive force (H⁺), and these electrons are carried back to the reaction center. Quinol is also produced via carbon catabolism by enzymes like succinate or lactate dehydrogenase (succ dehy and lact dehy, respectively). Electrons from the quinol pool can be used to synthesize NADH (via complex I [CI]) or to reduce DMSO (via DMSO reductase [DMSO red]). (B) Photoheterotrophic growth of wild-type and complex I mutant strains with lactate, succinate, or fumarate as the carbon source. Shown are representative curves for each strain from ≥3 replicates. (C) Strains were grown photoheterotrophically with the indicated carbon sources in the presence of the external electron acceptor DMSO (100 mM). Shown are representative curves for each strain from ≥3 replicates. (D) Summary of photoheterotrophic growth on different carbon sources. Superscripts: a, calculated doubling times (from ≥3 replicates), including standard error; b, NG (no growth) indicates that the average maximum cell density was <20% of the average maximum cell density of wild-type cultures under the same conditions; c, PG (poor growth) indicates that the average maximum cell density was 20 to 40% of the average maximum cell density of wild-type cultures under the same conditions; d, the relative quinol/quinone ratio of wild-type cells grown on the indicated carbon source as predicted by the R. sphaeroides metabolic model (6, 18) (see Table S2 in the supplemental material). The last column shows NAD⁺/NADH ratios measured in wild-type cells grown photoheterotrophically on the indicated carbon source (shown are the averages of ≥10 replicate measurements, including standard error).

FIG 3

Complex I nuoA transcript levels in wild-type cells. Transcript levels of the nuoA gene from the complex I_A or complex I_E operon in wild-type cells grown photoheterotrophically with the indicated carbon and nitrogen sources, and in the presence or absence of 100 mM DMSO. Conditions under which glutamate is the provided nitrogen source are H₂-producing conditions. Fold change values represent expressions relative to wild-type cells grown photoheterotrophically with succinate, ammonia, and in the absence of DMSO, where the relative expression of the nuoA transcript from the complex I_A or complex I_E operon was set to 1 (transcript levels were normalized to the “housekeeping gene,” rpoZ). Relative expression levels were quantified from 3 replicates and include standard errors.

FIG 4

H₂ production by wild-type and complex I mutant strains grown photoheterotrophically with the indicated carbon sources. All cultures were grown with glutamate as the nitrogen source and the external electron acceptor DMSO (100 mM). Total gas production by each culture was measured and was assumed to be 90% H₂, as has been previously described (26). The specific H₂ composition for measurable amounts of gas can be viewed in Table S3 in the supplemental material. The bar graph shows data from ≥4 replicates and includes standard errors. The table below reports the doubling time of each strain (3 replicates, including standard errors) during H₂-producing conditions (anaerobic in the light, with glutamate as the nitrogen source).

FIG 5

Loss of complex I_A increases bacteriochlorophyll levels. (A) Photoheterotrophic cultures (succinate at the carbon source, supplemented with 100 mM DMSO) show different pigmentation in the lanes: 1, wild type; 2, Δcomplex I_A strain; 3, Δcomplex I_E strain; 4, Δcomplex I_A/Δcomplex I_E strains. (B) Quantification of bacteriochlorophyll (Bchl) in wild type and complex I mutant strains. Data are from 3 replicates, and standard errors are shown. (C) Transcript abundance of the bacteriochlorophyll synthesis gene, bchM, in wild-type and complex I mutant strains grown photoheterotrophically (succinate at the carbon source, supplemented with 100 mM DMSO). Data are from 3 replicates, and standard errors are shown.

FIG 6

Increased biomass production in the complex I_A mutant. (A) Maximum cell density of wild-type and complex I mutant strains grown photoheterotrophically with succinate as the carbon source and supplemented with DMSO. Data are from 5 replicates, and standard errors are shown. (B) Dry weight of wild-type and complex I mutant strains grown photoheterotrophically (succinate as the carbon source, supplemented with DMSO) harvested at maximum cell density (see panel A). Data are from 3 replicates, and standard errors are shown.

FIG 7

Aerobic growth rates of wild-type and complex I mutant strains. Doubling times of wild-type and complex I mutant strains grown aerobically by shaking in 96-well plates with the indicated carbon sources. Data are from 3 replicates, and standard errors are shown.

FIG 8

The proposed contribution of complex I isozymes during photoheterotrophic growth. We predict that lactate catabolism produces high levels of both quinol (49) and NADH (Fig. 4), succinate catabolism produces high levels of quinol (6, 18), and fumarate catabolism produces high levels of NADH (Fig. 4). To explain the properties of complex I mutants, we propose that complex I_E is important for growth on carbon sources that produce high levels of NADH, where it oxidizes NADH to maintain redox state. Alternatively, we propose that complex I_A is important for growth on carbon sources that produce quinol, where it functions to synthesize NADH, thereby preventing overreduction of the quinone pool and producing the cellular reducing power that is shuttled into nitrogenase (N₂ase)-mediated H₂ production.