Anaerobic Bacteria: Solving a shuttle mystery

Shewanella oneidensis bacteria use an abiotic reaction to help shuttle electrons outside of the cell.
  1. Bridget Conley  Is a corresponding author
  2. Jeffrey Gralnick  Is a corresponding author
  1. University of Minnesota, United States

Life is powered by ‘redox’ reactions: electrons are released from a source (oxidation) and then transferred from one molecule to another until they are captured by a terminal electron acceptor (reduction). Humans, for example, oxidize glucose and reduce oxygen. However, many places on our planet lack glucose and oxygen, so the microorganisms in these ecosystems must rely on other molecules for their redox reactions. In particular, they must find other compounds to act as terminal electron acceptors.

In 1988, it was reported that the bacterium known as Shewanella oneidensis was able to use minerals such as manganese and iron oxides as terminal electron acceptors (Myers and Nealson, 1988). Known as extracellular electron transfer, this metabolism is unique because manganese and iron oxide cannot enter the cell, so the electrons must go outside to meet their acceptor. A diverse range of microorganisms can transfer electrons outside of the cell, suggesting this form of metabolism may be widespread in many ecosystems (Koch and Harnisch, 2016). Transferring electrons extracellularly means these species can change the solubility of important metal nutrients, but also that these bacteria can be harnessed in a variety of technologies including bioremediation, generating electricity in microbial fuel cells, and biosynthesis of valuable chemicals (Kappler and Bryce, 2017; Kato, 2015).

The major components of the extracellular electron transfer pathway in S. oneidensis have already been described: a donor compound gives electrons, which are then transferred to molecules such as menaquinone and c-type cytochromes, before they are delivered to the extracellular electron acceptor (Shi et al., 2016). Yet, how does an electron cross membrane barriers so it can get out of a bacterium and reach an extracellular acceptor? Electrons can be directly captured by the acceptor when in contact with electron-carrying proteins on the surface of the bacterium, but Shewanella species also secrete flavin molecules that can shuttle electrons to external compounds (Brutinel and Gralnick, 2012).

The first evidence showcasing that S. oneidensis may produce extracellular electron shuttles came from a mutant that was unable to synthetize a molecule known as menaquinone: this strain could not reduce a compound called AQDS unless it was physically close to wild-type cells (Newman and Kolter, 2000; Figure 1A). Analyzing the medium in which the wild-type bacteria were growing revealed the presence of a quinone-like molecule that could be ‘borrowed’ by the mutant bacteria. The role of this unidentified molecule has been the subject of debate: is it shuttling electrons to acceptors outside of the bacteria, or is it an intermediate in menaquinone synthesis that can fix the defect in mutant S. oneidensis? Now, in eLife, and nearly twenty years after its initial description, Jon Clardy and colleagues – including Emily Mevers and Lin Su as joint first authors – report that they have identified this molecule as ACNQ (2-amino-3-carboxyl-1,4-napthoquinone), a soluble analogue of menaquinone that can work as an electron shuttle (Mevers et al., 2019).

An abiotic reaction produces ACNQ, a molecule that serves as an electron shuttle in Shewanella oneidensis.

(A) Redox reactions take place when electrons released from a molecule (oxidation) are accepted by another compound (reduction). Wild-type S. oneidensis can reduce AQDS, a molecule present in the environment (reduced AQDS is shown in red and oxidized AQDS in yellow); they also produce the newly identified compound called ACNQ, which shuttles electrons from the cells into the extracellular environment. When grown alone, mutant S. oneidensis bacteria that cannot produce menaquinone fail to reduce AQDS (upper right); however, when they are grown close to a wild-type colony, they can use the ACNQ molecules present in the milieu to complete the redox reaction (lower left). This diagram summarizes the AQDS reduction assays performed by Newman and Kolter as well as Mevers et al. (B) The work by Mevers et al. reveals how S. oneidensis can produce ACNQ. The enzymes MenA and UbiE convert the molecular precursor DHNA into menaquinone-7 (MQ), its dominant product. Menaquinone is found in the inner membrane (IM) of the bacterium, where it serves as a lipid-soluble electron carrier in the electron transport chain (blue structure in right inset). About 2% of the DHNA pool can also chemically react with an ammonia source (NH3+) to form ACNQ. AQDS: anthraquinone-2,6-disulfonate; ACNQ: 2-amino-3-carboxyl-1,4-napthoquinone; DHNA: 1,4-dihydroxy-2-naphthoic acid.

The team, which is based at Harvard Medical School, Lawrence Berkeley National Laboratory and Southeast University, confirmed that S. oneidensis is capable of producing ACNQ in culture, but at concentrations ten times higher than those observed in other microorganisms. Mevers et al. then focused on how this molecule is produced. Analysis of mutants defective in menaquinone synthesis suggested it is created from a molecule called DHNA, by a reaction that should be catalyzed via a transaminase. However, genetic analysis of S. oneidensis and E. coli did not yield any candidate enzymes that could produce ACNQ. This led to the intriguing hypothesis that ACNQ is synthetized from DHNA without the help of enzymes (Mevers et al., 2019). And indeed, Mevers et al. showed that, in a sterile medium, DHNA and an ammonia source could react to form ACNQ, demonstrating that the molecule could be created through an abiotic mechanism (Figure 1B).

Mevers et al. also explored whether ACNQ acts as an electron shuttle or as a menaquinone synthesis intermediate in S. oneidensis, as was debated previously (Newman and Kolter, 2000; Myers and Myers, 2004). Adding purified ACNQ to an S. oneidensis menaquinone mutant did not replenish the menaquinone pool, but it did restore the ability to reduce AQDS. A large dose of 1μM of ACNQ increased the rate at which the bacteria could transfer electrons to an electrode, but that mechanism did not require the cytochromes usually involved in extracellular electron transfer. These data support the hypothesis that ACNQ can work as an electron shuttle at micromolar concentrations in S. oneidensis. However, this activity may involve redox reactions that occur inside the cell (Yamazaki et al., 2002b) rather than the cytochrome-mediated mechanisms that are already known to participate in extracellular electron transfer (Shi et al., 2016).

The biological relevance of ACNQ remains an open question. In certain organisms, it can act as an electron sink and accept electrons, shifting the redox balance of the cell and directing the metabolism towards fermentation end products which are more energetically favorable (Freguia et al., 2009; Yamazaki et al., 2002a; Yamazaki et al., 2002b).

A variety of organisms that do not transfer their electrons outside still produce low levels of ACNQ, which may suggest other roles for the compound. For instance, it may be involved in cell-to-cell communication as well as in unconventional cellular warfare where redox balance is disrupted, or act as a nutrient for surrounding organisms. Regardless of the biological role(s) of ACNQ, Mevers et al. highlight that abiotic processes can add to gene-encoded activities to enhance metabolic diversity.

References

Article and author information

Author details

  1. Bridget Conley

    Bridget Conley is in the BioTechnology Institute, University of Minnesota, Minneapolis and Saint Paul, United States

    For correspondence
    conle215@umn.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2986-764X
  2. Jeffrey Gralnick

    Jeffrey Gralnick is in the Department of Plant and Microbial Biology and the BioTechnology Institute, University of Minnesota, Minneapolis and Saint Paul, United States

    For correspondence
    gralnick@umn.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9250-7770

Publication history

  1. Version of Record published: August 8, 2019 (version 1)

Copyright

© 2019, Conley and Gralnick

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,550
    Page views
  • 140
    Downloads
  • 4
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Bridget Conley
  2. Jeffrey Gralnick
(2019)
Anaerobic Bacteria: Solving a shuttle mystery
eLife 8:e49831.
https://doi.org/10.7554/eLife.49831
  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    2. Plant Biology
    Dietmar Funck, Malte Sinn ... Jörg S Hartig
    Research Article

    Metabolism and biological functions of the nitrogen-rich compound guanidine have long been neglected. The discovery of four classes of guanidine-sensing riboswitches and two pathways for guanidine degradation in bacteria hint at widespread sources of unconjugated guanidine in nature. So far, only three enzymes from a narrow range of bacteria and fungi have been shown to produce guanidine, with the ethylene-forming enzyme (EFE) as the most prominent example. Here, we show that a related class of Fe2+- and 2-oxoglutarate-dependent dioxygenases (2-ODD-C23) highly conserved among plants and algae catalyze the hydroxylation of homoarginine at the C6-position. Spontaneous decay of 6-hydroxyhomoarginine yields guanidine and 2-aminoadipate-6-semialdehyde. The latter can be reduced to pipecolate by pyrroline-5-carboxylate reductase but more likely is oxidized to aminoadipate by aldehyde dehydrogenase ALDH7B in vivo. Arabidopsis has three 2-ODD-C23 isoforms, among which Din11 is unusual because it also accepted arginine as substrate, which was not the case for the other 2-ODD-C23 isoforms from Arabidopsis or other plants. In contrast to EFE, none of the three Arabidopsis enzymes produced ethylene. Guanidine contents were typically between 10 and 20 nmol*(g fresh weight)-1 in Arabidopsis but increased to 100 or 300 nmol*(g fresh weight)-1 after homoarginine feeding or treatment with Din11-inducing methyljasmonate, respectively. In 2-ODD-C23 triple mutants, the guanidine content was strongly reduced, whereas it increased in overexpression plants. We discuss the implications of the finding of widespread guanidine-producing enzymes in photosynthetic eukaryotes as a so far underestimated branch of the bio-geochemical nitrogen cycle and propose possible functions of natural guanidine production.

    1. Biochemistry and Chemical Biology
    2. Medicine
    Giulia Leanza, Francesca Cannata ... Nicola Napoli
    Research Article

    Type 2 diabetes (T2D) is associated with higher fracture risk, despite normal or high bone mineral density. We reported that bone formation genes (SOST and RUNX2) and advanced glycation end-products (AGEs) were impaired in T2D. We investigated Wnt signaling regulation and its association with AGEs accumulation and bone strength in T2D from bone tissue of 15 T2D and 21 non-diabetic postmenopausal women undergoing hip arthroplasty. Bone histomorphometry revealed a trend of low mineralized volume in T2D (T2D 0.249% [0.156–0.366]) vs non-diabetic subjects 0.352% [0.269–0.454]; p=0.053, as well as reduced bone strength (T2D 21.60 MPa [13.46–30.10] vs non-diabetic subjects 76.24 MPa [26.81–132.9]; p=0.002). We also showed that gene expression of Wnt agonists LEF-1 (p=0.0136) and WNT10B (p=0.0302) were lower in T2D. Conversely, gene expression of WNT5A (p=0.0232), SOST (p<0.0001), and GSK3B (p=0.0456) were higher, while collagen (COL1A1) was lower in T2D (p=0.0482). AGEs content was associated with SOST and WNT5A (r=0.9231, p<0.0001; r=0.6751, p=0.0322), but inversely correlated with LEF-1 and COL1A1 (r=–0.7500, p=0.0255; r=–0.9762, p=0.0004). SOST was associated with glycemic control and disease duration (r=0.4846, p=0.0043; r=0.7107, p=0.00174), whereas WNT5A and GSK3B were only correlated with glycemic control (r=0.5589, p=0.0037; r=0.4901, p=0.0051). Finally, Young’s modulus was negatively correlated with SOST (r=−0.5675, p=0.0011), AXIN2 (r=−0.5523, p=0.0042), and SFRP5 (r=−0.4442, p=0.0437), while positively correlated with LEF-1 (r=0.4116, p=0.0295) and WNT10B (r=0.6697, p=0.0001). These findings suggest that Wnt signaling and AGEs could be the main determinants of bone fragility in T2D.