2015 Annual Science Report

University of Wisconsin Reporting  |  JAN 2015 – DEC 2015

Project 1D: Comparative Genomic Analysis of Chemolithotrophic Fe(II)-Oxidizing Bacteria

Project Summary

A comparative genomic analysis was performed to identify candidate genes involved in extracellular electron transfer (EET) by Fe(II)-oxidizing bacteria (FeOB). The analysis included a variety of publically-available FeOB genomes, together with genomes from FeOB isolated from subsurface sediments, previously-isolated marine basalt-associated FeOB, and metagenomes from chemolithoautotrophic aerobic pyrite-oxidizing and nitrate-reducing Fe(II)-oxidizing enrichment cultures. We identified outer membrane multi-copper oxidase (MCO) genes homologous to proteins known to be involved in EET in several of the FeOB genomes, as well as homologs to the outer membrane c-type cytochrome (ctyc) Cyc2 known to be involved in bacterial Fe(II) oxidation by Acidithiobacillus ferrooxidans under acidic conditions. Further, we found gene clusters that may potentially encode novel “porin-cytochrome-c protein complex” (PCC) in the well-known neutral-pH FeOB S. lithotrophicus ES-1, and homologous operons were found in other recognized FeOB (Leptothrix cholodnii SP-6 and Leptothrix ochracea L12. Another gene cluster consisting of a porin and three periplasmic multiheme cytc was identified in Hyphomicrobium sp. genome retrieved from a pyrite-oxidizing enrichment culture, and its homologous gene clusters are also present in five marine Zetaproteobacterial FeOB genomes. Overall, this analysis, which is based on our current understanding of bacterial EET in Fe redox reactions, provides a list of candidate genes for further experimental and genomic studies.

4 Institutions
3 Teams
1 Publication
0 Field Sites
Field Sites

Project Progress

The goal of this project is to better understand electron transfer and genes involved in bacterial Fe(II) oxidation at circumneutral pH through comparative genomic analysis. Identification of the functional biological components (i.e. genes and proteins) involved in these chemolithotrophic processes is critical for making a concrete linkage between biological metabolism and the generation of mineralogical and isotopic biosignatures in relation to redox gradients on Earth and other rocky planets.

We have obtained genomes from Fe(II)-oxidizing bacteria (FeOB) isolated from subsurface sediments (Benzine et al., 2013) and previously-isolated marine basalt-associated FeOB, as well as metagenomes of a chemolithoautotrophic pyrite-oxidizing enrichment culture (Percak-Dennett et al., 2016) and a chemolithoautotrophic nitrate-dependent Fe(II)-oxidizing enrichment cultures (Straub et al., 1996; He et al., 2016). Together with the increasing number of publically available FeOB genomes, we performed a comparative analysis to search for candidate Fe(II) oxidase genes involved in extracellular electron transfer (EET) by FeOB.

The redox active enzymes in iron oxidation and reduction are often c-type cytochromes (cytc) (Richter et al., 2012; Shi et al., 2012) and to a lesser extent, multicopper oxidases (MCO) (Mehta et al., 2006). Based on current knowledge of EET, we generalized the Gram-negative bacterial iron oxidase (and reductase) systems into two models:

In one model, the redox active enzyme is secreted as an outer membrane or extracellular protein in order to directly interact with extracellular iron. Examples include the outer membrane c-type cytochrome (cytc) Cyc2 in acidiphilic Fe(II)-oxidizer Acidithiobacillus ferroxidans (Castelle et al., 2008), and outer membrane multiheme cytc (e.g. OmcB, OmcC, OmcE and OmcS) (Leang et al., 2003; Mehta et al., 2005) and MCO (e.g. OmpB and OmpC) (Mehta et al., 2006; Holmes et al., 2008) in Fe(III)-reducing Geobacter sulfurreducens.

In the other model, the redox active enzyme, which is often a multiheme cytc, is secreted to the periplasm, and embedded into a porin on the outer membrane to directly or indirectly interact with extracellular iron, and this system was referred to as the “porin-cytochrome c protein complex” (PCC) (Richardson et al., 2012). Examples include the homologous MtrAB in Shewanella oneidensis (Beliaev and Saffarini, 1998), PioAB in Rhodopseudomonas palustris TIE-1 (Jiao and Newman, 2007) and MtoAB in Sideroxydans lithotrophicus ES-1 (Liu et al., 2012; Shi et al., 2012; Emerson et al., 2013), as well as the more recently discovered novel PCC in G. sulfurreducens (Liu et al., 2014).

We therefore searched for genes that encode outer-membrane/extracellular cytc or MCO, and gene clusters that potentially encode porin-periplasmic cytc complex (i.e. PCC) or porin-periplasmic MCO complex in neutrophilic FeOB, including microaerophilic, anaerobic (nitrate-reducing), and phototrophic FeOB. We identified outer membrane MCO homologous to OmpB in several FeOB, and outer membrane Cyc2 in many microaerophilic FeOB and several anaerobic or phototrophic FeOB (Figure 1). Notably, these Cyc2 proteins are distant homologs of Cyc2 in A. ferroxidans (Figure 2). PCC genes homologous to MtrAB/MtoAB/PioAB (tentatively referred to as “PCC1”) were identified in a few microaerophilic, anaerobic, or phototrophic FeOB, while PCC homologous to G. sulfurreducens (Liu et al., 2014) (tentatively “PCC2”) is not present in any of the FeOB.

Figure 1. Occurrence of candidate Fe(II) oxidase genes in FeOB genomes. FeOB genomes in which we did not find candidate Fe(II) oxidase genes are excluded in this summary figure.
Figure 2. Phylogenetic tree reconstructed with Cyc2 homologs. Cyc2 proteins from A. ferroxidans are labeled in blue, Cyc2 homolog from Bradyrhizobium sp. recovered from a pyrite-oxidizing enrichment culture is labeled in green, and Cyc2 homologs from Gallionellaceae sp. and Rhodanobacter sp. recovered from nitrate-dependent Fe(II) oxidation enrichment cultures are labeled in red. IMG Gene Object IDs are indicated in the brackets.

Further, we found gene clusters that may potentially encode novel PCCs. For example, two operons, each consisting of a porin, an extracellular multiheme cytc and a periplasmic multiheme cytc (tentatively “PCC3”) were identified in S. lithotrophicus ES-1, and homologous operons were found in Leptothrix cholodnii SP-6 and Leptothrix ochracea L12 (Figure 3a). Another gene cluster consisting of a porin and three periplasmic multiheme cytc (tentatively “PCC4”) was identified in hyphomicrobium sp. genome retrieved from a pyrite-oxidizing enrichment culture (Figure 3b), and its homologous gene clusters are also present in five marine Zetaproteobacterial FeOB genomes (Figure 1).

Figure 3. Examples of gene clusters that potentially encode novel porin-cytochrome c protein complex (PCC). Porins are labeled in green; periplasmic cytc are labeled in red, and extracellular cytc are labeled in orange. The number in the cytc indicates the number of heme-binding sites, and the number in the porin indicates the number of transmembrane regions. Predicted cellular locations of porin and cytc are indicated by different line types: thick solid black lines for outer membrane, thick solid gray lines for extracellular, and dash lines for periplasmic, respectively.

In addition to putative PCC, operons consisting of porin and periplasmic MCO that are homologous to PcoAB genes were also identified in a number of FeOB (Figure 1). Notably, we did not find any obvious candidate Fe(II) oxidase genes in about half of anaerobic FeOB genomes searched, probably suggesting that their electron transfer mechanisms in Fe(II) oxidation might be different from the two general models described above. Overall, this analysis, which is based on our current understanding of bacterial EET in iron redox reactions, provides a list of candidate genes for further experimental and genomic studies.

References
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  • PROJECT INVESTIGATORS:
    Eric Roden Eric Roden
    Project Investigator
  • PROJECT MEMBERS:
    Eric Boyd
    Co-Investigator

    Shaomei He
    Collaborator

  • RELATED OBJECTIVES:
    Objective 2.1
    Mars exploration.

    Objective 3.2
    Origins and evolution of functional biomolecules

    Objective 4.1
    Earth's early biosphere.

    Objective 5.1
    Environment-dependent, molecular evolution in microorganisms

    Objective 5.3
    Biochemical adaptation to extreme environments