Biology subjects

Fixen, K. R.

Publications and source records attributed to Fixen, K. R..

5 recordsLinked to original sources

bioRxiv2026

Leveraging a genetically tractable alphaproteobacterium reveals molecular determinants of bacterial growth in fungal-decayed wood

Brown rot wood-degrading fungi release carbon (C) from deadwood but leave behind a large fraction of C sequestered in lignin residues or as fungal metabolites. The strength of sequestration in these C residuals remains unclear, but proteobacteria-dominated bacterial communities have been implicated in metabolizing C from decay residues, possibly erasing the C sequestration potential assumed for brown rot. Here, we paired a model brown rot fungus (Rhodonia placenta) with a model Alphaproteobacterium (Rhodopseudomonas palustris) to track fungal release and bacterial utilization of C derived from decaying wood. We found that fungal decay products generated by R. placenta could be used by R. palustris for growth, and later decay stages contained more usable substrates than early stages. High performance liquid chromatography with mass spectrometry identified a range of aromatic and non-aromatic compounds in the fungal-decayed wood, but after 95 days of bacterial growth, R. palustris preferentially consumed non-aromatic acids over aromatic lignin monomers. Genes involved with aromatic compound degradation were unimportant for bacterial growth, and RNA sequencing revealed that aromatic compound degradation genes were repressed on decayed wood extract. Randomly barcoded transposon sequencing failed to identify a solitary catabolic pathway used by R. palustris, suggestive of substrate co-utilization, and surprisingly, showed that genes involved with copper toxicity were essential. Finally, we found that genes involved with biosynthesis of certain cofactors and amino acids were no longer essential on decayed wood extract, suggesting these nutrients were readily accessible. This study helps lay the foundation to understand potential bacterial-fungal interactions in decayed wood. Graphical abstractTo explore how brown rot fungi support and compete with bacterial partners in the wood decay environment, the model brown rot fungus Rhodonia placenta was used to degrade aspen wafers which were then infused into bacterial growth medium. By leveraging the range of molecular biology tools available for the model Alphaproteobacterium Rhodopseudomonas palustris, we discovered that R. palustris preferentially consumes short organic acids instead of aromatic lignin monomers which it would otherwise consume if provided in isolation. Additionally, R. palustris scavenged certain amino acids (AAs) and enzyme cofactors including methionine, biotin, and PLP from the decayed wood extract, highlighting these as key shared resources for bacterial-fungal partnerships. We found that R. placenta increased the concentration of certain metals (Cu and Al) inducing a metal stress response in R. palustris, indicating that metal toxicity could be an important mode of competition between fungi and bacteria in the wood decay environment. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/723453v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@16f31fcorg.highwire.dtl.DTLVardef@13a9b34org.highwire.dtl.DTLVardef@a37dcforg.highwire.dtl.DTLVardef@198bf1c_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Predicting compatibility between ferredoxins and the Fe protein of nitrogenase using in silico protein modeling

Biological nitrogen fixation is the process by which certain bacteria and archaea use the enzyme nitrogenase to reduce atmospheric nitrogen into bioavailable ammonium. Engineering non-nitrogen-fixing organisms, like plants, to use nitrogenase could reduce dependency on synthetic fertilizer and mitigate the environmental impacts of industrial fertilizer production. However, nitrogenase activity requires delivery of reducing power by small electron carrying proteins known as ferredoxins and flavodoxins, and successfully engineering nitrogenase into new systems will require a mechanistic understanding of electron delivery by these proteins. Most organisms often have multiple ferredoxins, raising the question of which ferredoxin can support nitrogenase activity. The purpose of this study is to gain insight into how we can predict which ferredoxin is compatible with the Fe protein, the component of nitrogenase that interacts with ferredoxin or flavodoxin. Our in silico protein-protein docking simulations reveal that ferredoxins and flavodoxins involved in nitrogen fixation have a distance [≤] 10 [A] between their redox cofactor and the [4Fe-4S] cluster of the Fe protein. We found shorter cofactor distance contributes to faster intermolecular electron tunneling rates (> 106 sec-1). These nitrogen-fixing bacterial ferredoxins also form more complementary interactions with the Fe protein compared to non-nitrogen-fixing bacterial and plant ferredoxins. Heterologous expression of a set of ferredoxins from both nitrogen-fixing and non-nitrogen-fixing bacteria in the diazotroph Rhodopseudomonas palustris support our model-derived prediction that cofactor distances of [≤] 10 [A] favor nitrogenase compatibility. These findings offer a framework to predict and potentially enhance ferredoxin-nitrogenase compatibility, which will help to improve our ability to engineer nitrogen fixation into non-nitrogen-fixing organisms like plants.

microbiology↗

bioRxiv2023

Evolution of Rhodopseudomonas palustris to degrade halogenated aromatic compounds involves changes in pathway regulation and enzyme specificity

Halogenated aromatic compounds are used in a variety of industrial applications but can be harmful to humans and animals when released into the environment. Microorganisms that degrade halogenated aromatic compounds anaerobically have been isolated but the evolutionary path that they may have taken to acquire this ability is not well understood. A strain of the purple nonsulfur bacterium, Rhodopseudomonas palustris, RCB100, can use 3-chlorobenzoate (3-CBA) as a carbon source whereas a closely related strain, CGA009, cannot. To reconstruct the evolutionary events that enabled RCB100 to degrade 3-CBA, we selected for and isolated a CGA009 strain capable of growing on 3-CBA, although not as well as RCB100. Comparative whole-genome sequencing of the evolved strain and RCB100 revealed large deletions encompassing badM, a transcriptional repressor of genes for anaerobic benzoate degradation. It was previously shown that in strain RCB100, a single nucleotide change in an alicyclic acid coenzyme A ligase gene, named aliA, gives rise to a variant AliA enzyme that has high activity with 3-CBA. When we introduced the RCB100 aliA allele and a badM deletion into R. palustris CGA009, it grew on 3-CBA at a similar rate as RCB100. This work provides an example of pathway evolution that includes a variant of a promiscuous enzyme with enhanced substrate specificity and a regulatory mutation that leads to constitutive expression of a pathway that does not regulate the promiscuous enzyme. ImportanceBiodegradation of man-made compounds often involves the activity of promiscuous enzymes whose native substrate is structurally similar to the man-made compound. Based on the enzymes involved, it is possible to predict what microorganisms are likely involved in biodegradation of anthropogenic compounds. However, there are examples of organisms that contain the required enzyme(s) and yet cannot metabolize these compounds. We found that even when the purple nonsulfur bacterium, Rhodopseudomonas palustris, encodes all the enzymes required for degradation of a halogenated aromatic compound, it is unable to metabolize that compound. Using adaptive evolution, we found a regulatory mutation and a variant of promiscuous enzyme with increased substrate specificity were required, but the ability to metabolize a halogenated aromatic compound also resulted in reduced fitness on another aromatic compound. This work provides insight into how an environmental isolated evolved to use halogenated aromatic compounds and the potential ecological trade-offs associated with this adaptation.

microbiology↗

bioRxiv2022

Selecting a new electron transfer pathway for nitrogen fixation uncovers an electron bifurcating-like enzyme involved in anaerobic aromatic compound degradation

Nitrogenase is the key enzyme involved in nitrogen fixation and uses low potential electrons delivered by ferredoxin (Fd) or flavodoxin (Fld) to reduce dinitrogen gas (N2) to produce ammonia and hydrogen. Although the phototrophic alphaproteobacterium Rhodopseudomonas palustris encodes multiple proteins that can reduce Fd, the FixABCX complex is the only one shown to support nitrogen fixation, and R. palustris Fix- mutants grow poorly in nitrogen-fixing conditions. To investigate how native electron transfer chains (ETCs) can be redirected towards nitrogen fixation, we leveraged the strong selective pressure of nitrogen limitation to isolate a suppressor of R. palustris {Delta}fixC that grows under nitrogen-fixing conditions. We found two mutations were required to restore growth under nitrogen-fixing conditions in the absence of functional FixABCX. One mutation was in the gene encoding the primary Fd involved in nitrogen fixation, fer1, and the other mutation was in aadN, which encodes a homolog of NAD+-dependent Fd:NADPH oxidoreductase (Nfn). We present evidence that AadN plays a role in electron transfer to benzoyl-CoA reductase, the key enzyme involved in anaerobic aromatic compound degradation. Our data support a model where the ETC for anaerobic aromatic compound degradation was re-purposed to support nitrogen fixation in the suppressor strain. ImportanceThere is increasing evidence that protein electron carriers like Fd have evolved to form specific partnerships with select electron donors and acceptors to keep native electron transfer pathways insulated from one another. This makes it challenging to integrate a Fd-dependent pathway like biological nitrogen fixation into non-nitrogen-fixing organisms and provide the high-energy reducing power needed to fix nitrogen. Here we show that amino acid substitutions in an electron donor for anaerobic aromatic compound degradation and a Fd involved in nitrogen fixation enabled electron transfer to nitrogenase. This work provides a model system to understand electron transfer chain specificity and how new electron transfer pathways can be evolved for biotechnologically valuable pathways like nitrogen fixation.

microbiology↗

bioRxiv2021

Iron oxidation is regulated by the two-component system, RegSR, and plays a role in photolithoheterotrophic growth in Rhodopseudomonas palustris

Purple nonsulfur bacteria (PNSB) are metabolically versatile organisms generate energy through both aerobic and anaerobic respiration as well as anoxygenic photosynthesis. In many PNSB, the redox-sensing, two-component system RegBA is a global regulator of energy generating and consuming pathways, such as photosynthesis, carbon fixation, and nitrogen fixation, when cells are shifted from an aerobic to an anaerobic environment. However, in the PNSB Rhodopseudomonas palustris, the role of the RegBA homolog, RegSR, was unclear since global regulation of these same pathways involves the oxygen-sensing signal transduction system, FixJL-K, in R. palustris. Using RNA-seq analysis, we found that RegSR plays a role in regulating the operon pioABC, which encodes genes required for Fe(II) oxidation. We found that transcript levels of pioABC under photoheterotrophic conditions was dependent on the oxidation state of the carbon substrate and whether the cells were fixing nitrogen. We also found that R. palustris can carry out photolithoheterotrophic growth using Fe(II) oxidation when grown with the oxidized carbon substrate, malate, requiring regSR and pioABC. We present a model in which RegSR regulates pioABC in response to a cellular redox signal, allowing R. palustris to use Fe(II) oxidation to access more electrons when there is an increased cellular demand for reducing equivalents. SignificanceMixotrophy is thought to be widespread in aquatic environments, yet little is understood about how mixotrophy affects biogeochemical cycles. Fe(II)-oxidizing anoxygenic phototrophs likely play an important role in iron cycling since they are thought to have thrived in the anoxic, iron-rich oceans of early Earth and can be found in both freshwater and marine environments. Although Fe(II) oxidation by anoxygenic phototrophs is largely studied in the context of photoautotrophic growth, these organisms can also grow photoheterotrophically. We present the first evidence linking photolithoheterotrophic growth using Fe(II) to the pathway required for photoautotrophic Fe(II) oxidation in an anoxygenic phototroph. Understanding this metabolism will be important for understanding how mixotrophic metabolism contributes to iron cycling in anoxic environments.

microbiology↗