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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Photosynthesis | The Heliobacteria

The heliobacteria are the only phototrophs in the Gram-positive group of bacteria. They are strict anaerobes that grow in a primarily fermentative mode in the dark. In the light, they use a homodimeric Type I photochemical reaction center to drive cyclic electron transport. They are unique in using the pigment bacteriochlorophyll g. All heliobacteria are unable to fix CO 2 but able to fix N2. Their core metabolism includes a complete TCA cycle with several reversible enzymes in common with the reverse (reductive) TCA cycle, but they lack an (ATP) citrate lyase that would allow CO 2 fixation. There are now several sequenced genomes available for different heliobacterial species, allowing hypotheses to be made concerning their metabolic potential. Here, techniques to introduce replicating plasmids into Heliobacterium modesticaldum, as well as leveraging its endogenous CRISPR/Cas system to edit its chromosome, have recently been pioneered, contributing to making this species a model system for this family.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Deletion of the cytochrome bc complex from Heliobacterium modesticaldum results in viable but non-phototrophic cells

The heliobacteria, a family of anoxygenic phototrophs, possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria can also grow chemotrophically via pyruvate metabolism in the dark. In the heliobacteria, the cytochrome bc complex is responsible for oxidizing menaquinol and reducing cytochrome c 553 in the electron flow cycle used for phototrophy. However, there is no known electron acceptor for the mobile cytochrome c 553 other than the photochemical reaction center. We have, therefore, hypothesized that the cytochrome bc complex is necessary for phototrophy, but unnecessary for chemotrophic growth in the dark. Here, we used a two-step method for CRISPR-based genome editing in Heliobacterium modesticaldum to delete the genes encoding the four major subunits of the cytochrome bc complex. Genotypic analysis verified the deletion of the petCBDA gene cluster encoding the catalytic components of the complex. Spectroscopic studies revealed that re-reduction of cytochrome c 553 after flash-induced photo-oxidation was over 100 times slower in the petCBDA mutant compared to the wild-type. Steady-state levels of oxidized P 800 (the primary donor of the photochemical reaction center) were much higher in the petCBDA mutant at every light level, consistent with a limitation in electron flow to the reaction center. The petCBDA mutant was unable to grow phototrophically on acetate plus CO 2 but could grow chemotrophically on pyruvate as a carbon source similar to the wild-type strain in the dark. The mutants could be complemented by reintroduction of the petCBDA gene cluster on a plasmid expressed from the clostridial eno promoter.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Examination of Genetic Control Elements in the Phototrophic Firmicute Heliomicrobium modesticaldum

Heliomicrobium modesticaldum has been used as a model organism for the Heliobacteria, the only phototrophic family in the Firmicutes. It is a moderately thermophilic anoxygenic phototrophic bacterium that is capable of fermentative growth in the dark. The genetic manipulation of H. modesticaldum is still in its infancy. Methods to introduce genes through the use of exogenous plasmids and to delete genes from the chromosome through the use of the native CRISPR/Cas system have been developed in the last several years. To expand our genetic toolkit, it was necessary to control gene expression. In this study, we analyzed constitutive and inducible promoters developed for clostridia for their use in H. modesticaldum and further tested two reporters, adhB and lacZ, as indicators of promoter strength. Alcohol dehydrogenase (AdhB) was unsuitable as a reporter in this species due to high endogenous activity and/or low activity of the reporter, but a thermostable LacZ worked well as a reporter. A set of constitutive promoters previously reported to work in Clostridium thermocellum was found to be reliable for controlling the expression of the lacZ reporter gene in H. modesticaldum at a range of activities spanning an order of magnitude. An anhydrotetracycline-inducible promoter was created by inserting tetO operators into a strong constitutive promoter, but it was not fully repressible. The implementation of a xylose-inducible promoter resulted in complete repression of β-gal in the absence of xylose, and reliable expression tunable through the concentration of xylose added to the culture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Differential sensitivity to oxygen among the bacteriochlorophylls g in the type-I reaction centers of Heliobacterium modesticaldum

The type-I, homodimeric photosynthetic reaction center (RC) of Heliobacteria (HbRC) is the only known RC in which bacteriochlorophyll g (BChl g) is found. It is also simpler than other RCs, having the smallest number of protein subunits and bound chromophores of any type-I RC. In the presence of oxygen, BChl g isomerizes to 8 1 -hydroxychlorophyll aF (Chl a F ). This naturally occurring process provides a way of altering the chlorophylls and studying the efect of these changes on energy and electron transfer. Transient absorbance diference spectroscopy reveals that triplet-state formation occurs in the antenna chlorophylls of HbRCs but does not provide site-specifc information. Herein, we report on an extended optically detected magnetic resonance (ODMR) study of the antenna triplet states in HbRCs with difering levels of conversion of BChl g to Chl a F . The data reveal pools of BChl g molecules with diferent triplet zero-feld splitting parameters and diferent susceptibilities to chemical oxidation. Finally, by relating the detailed spectroscopic characteristics derived from the ODMR data to the recently solved crystallographic structure, we have tentatively identifed BChl g molecules in which the probability of triplet formation is high and sites at which BChl g conversion is more likely, providing useful information about the fate of the excitation in the complex.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Excitonic structure and charge separation in the heliobacterial reaction center probed by multispectral multidimensional spectroscopy

Photochemical reaction centers are the engines that drive photosynthesis. The reaction center from heliobacteria (HbRC) has been proposed to most closely resemble the common ancestor of photosynthetic reaction centers, motivating a detailed understanding of its structure-function relationship. The recent elucidation of the HbRC crystal structure motivates advanced spectroscopic studies of its excitonic structure and charge separation mechanism. We perform multispectral two-dimensional electronic spectroscopy of the HbRC and corresponding numerical simulations, resolving the electronic structure and testing and refining recent excitonic models. Through extensive examination of the kinetic data by lifetime density analysis and global target analysis, we reveal that charge separation proceeds via a single pathway in which the distinct A 0 chlorophyll a pigment is the primary electron acceptor. In addition, we find strong delocalization of the charge separation intermediate. Our findings have general implications for the understanding of photosynthetic charge separation mechanisms, and how they might be tuned to achieve different functional goals.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Electronic structure and energetics of a heterodimeric BChl g ′/Chl a ′ special pair generated by exposure of Heliomicrobium modesticaldum to dioxygen

Heliobacteria are anoxygenic phototrophs that have a Type I homodimeric reaction center containing bacteriochlorophyll g (BChl g). Previous experimental studies have shown that in the presence of light and dioxygen, BChl g is converted into 8 1 -OH-chlorophyll a F (hereafter Chl a F ), with an accompanying loss of light-driven charge separation. These studies suggest that the reaction center only loses the ability to transfer electrons once both BChl g′ molecules of the P 800 special pair have been converted to Chl a F ′. Here, the present work confirms that the partially converted BChl g′/Chl a F ′ special pair remains functional in samples exposed to dioxygen by demonstrating its presence using hyperfine couplings obtained from Q-band 1 H ENDOR, 2D 14 N HYSCORE and DFT methods. The DFT calculations of the BChl g′/BChl g′ homodimeric primary donor, which are based on the recently published X-ray crystal structure, predict that the unpaired electron spin is equally delocalized over both BChl g′ molecules and provide an excellent match to the experimental hyperfine couplings of the anaerobic samples. Exposure to dioxygen leads to substantial changes in the hyperfine interactions, indicative of greater localization of the unpaired electron spin. The measured hyperfine couplings are reproduced in the DFT calculations by replacing one of the BChl g′ molecules of the primary donor with a Chl a F ′ molecule. The calculations reveal that the spin density becomes localized on BChl g′ in the heterodimeric primary donor. Time-dependent DFT calculations demonstrate that conversion of either or both of the accessory BChl g molecules and/or one of the BChl g′ molecules of P800 to Chl a F ′ results in minor effects on the energy of the charge-separated states. In contrast, if both of the BChl g′ molecules of P 800 are converted a large increase in the energy of the charge-separated state occurs. This suggests that the reaction center remains functional when only one half of the dimer is converted, however, conversion of both halves of the P 800 dimer leads to loss of function

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Deletion of Re -citrate synthase allows for analysis of contributions of tricarboxylic acid cycle directionality to the growth of Heliomicrobium modesticaldum

ABSTRACT Heliomicrobium modesticaldum,a phototrophic member of the phylum Firmicutes and family Clostridiales, possesses most of the enzymes specific to the reductive tricarboxylic acid (rTCA) cycle, except for the key enzyme, ATP-citrate lyase. It is thought to utilize a split TCA cycle when growing on pyruvate as a carbon source, in which the oxidative TCA (oTCA) direction generates most of the 2-ketoglutarate, but some can be produced in the reductive direction. Although a typicalSi-citrate synthase gene is not found in the genome, it was suggested that gene HM1_2993, annotated as homocitrate synthase, actually encodesRe-citrate synthase, which would function as the initial enzyme of the oTCA cycle. We deleted this gene to test this hypothesis and, if true, see what effect severing access to the oTCA cycle would have on this organism. The endogenous CRISPR-Cas system was used to replace the open reading frame with a selectable marker. The deletion mutants could grow on pyruvate but were unable to grow phototrophically on acetate + CO 2 as carbon source. Growth on acetate could be rescued by the addition of different electron sources (formate or ascorbate), suggesting that the oTCA cycle is used to oxidize acetate to generate electrons required to drive the carboxylation of acetyl-CoA. The deletion mutants were capable of growing in acetate minimal media without additional organic supplements beyond formate, demonstrating that the rTCA cycle can be employed to support sufficient 2-ketoglutarate production in this organism, unlike citrate synthase mutants in several chemoheterotrophic organisms utilizing the oTCA cycle. IMPORTANCE Heliobacteria are a unique group of phototrophic bacteria that are obligate anaerobes and possess a rudimentary system to use light as a source of energy. They do not make oxygen or fix carbon dioxide. Here, we explore their fundamental carbon metabolism to understand the role and operation of the central TCA cycle. This work shows both the role and operation of this cycle under different growth modes and explains how these organisms can obtain electrons to drive their biosynthetic metabolism. This foundational knowledge will be crucial in the future when attempts are made to use this organism as a platform for oxygen-sensitive synthesis of compounds in an anaerobe that can use light as its energy source.

Biotechnology & Applied Microbiology↗
DOE OSTI2021

The PshX subunit of the photochemical reaction center from Heliobacterium modesticaldum acts as a low-energy antenna

The anoxygenic phototrophic bacterium Heliobacterium modesticaldum contains a photochemical reaction center protein complex (called the HbRC) consisting of a homodimer of the PshA polypeptide and two copies of a newly discovered polypeptide called PshX, which is a single transmembrane helix that binds two bacteriochlorophyll g molecules. Here, to assess the function of PshX, we produced a ΔpshX strain of Hbt. modesticaldum by leveraging the endogenous Hbt. modesticaldum Type I-A CRISPR-Cas system to aid in mutant selection. We optimized this system by separating the homologous recombination and CRISPR-based selection steps into two plasmid transformations, allowing for markerless gene replacement. Fluorescence and low-temperature absorbance of the purified HbRC from the wild-type and ΔpshX strains showed that the bacteriochlorophylls bound by PshX have the lowest site energies in the entire HbRC. This indicates that PshX acts as a low-energy antenna subunit, participating in entropy-assisted uphill energy transfer towards the P 800 special bacteriochlorophyll g pair. We further discuss the role that PshX may play in stability of the HbRC, its conservation in other heliobacterial species, and the evolutionary pressure to produce and maintain single-TMH subunits in similar locations in other reaction centers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Identification and characterization of the low molecular mass ferredoxins involved in central metabolism in Heliomicrobium modesticaldum

The homodimeric Type I reaction center (RC) from Heliomicrobium modesticaldum lacks the PsaC subunit found in Photosystem I and instead uses the interpolypeptide [4Fe–4S] cluster F X as the terminal electron acceptor. Our goal was to identify which of the small mobile dicluster ferredoxins encoded by the H. modesticaldum genome are capable of accepting electrons from the heliobacterial RC (HbRC) and pyruvate:ferredoxin oxidoreductase (PFOR), a key metabolic enzyme. Analysis of the genome revealed seven candidates: HM1_1462 (PshB1), HM1_1461 (PshB2), HM1_2505 (Fdx3), HM1_0869 (FdxB), HM1_1043, HM1_0357, and HM1_2767. Heterologous expression in Escherichia coli and studies using time-resolved optical spectroscopy revealed that only PshB1, PshB2, and Fdx3 are capable of accepting electrons from the HbRC and PFOR. Modeling studies using AlphaFold show that only PshB1, PshB2, and Fdx3 should be capable of docking on PFOR at a positively charged patch that overlays a surface-proximal [4Fe–4S] cluster. Proteomic analysis of wild-type and gene deletion strains ΔpshB1, ΔpshB2, ΔpshB1pshB2, and Δfdx3 grown under nitrogen-replete conditions revealed that Fdx3 is undetectable in the wild-type, ΔpshB1, and Δfdx3 strains, but it is present in the ΔpshB2 and ΔpshB1pshB2 strains, implying that Fdx3 may substitute for PshB2. When grown under nitrogen-deplete conditions, Fdx3 is present in the wild-type and all deletion strains except for Δfdx3. None of the knockout strains demonstrated significant impairment during chemotrophic dark growth on pyruvate, photoheterotrophic light growth on pyruvate, or phototrophic growth on acetate+CO 2 , indicating a high degree of redundancy among these three electron transfer proteins. As a result, loss of both PshB1 and PshB2, but not FdxB, resulted in poor growth under N 2 -fixing conditions.

59 BASIC BIOLOGICAL SCIENCES↗