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Announcement
9 January 2020

Draft Genome Sequence of Sideroxydans sp. Strain CL21, an Fe(II)-Oxidizing Bacterium

ABSTRACT

Sideroxydans sp. strain CL21 is an aerobic Fe(II)-oxidizing bacterium isolated from peat sediment from the Fe-rich, moderately acidic Schlöppnerbrunnen fen (northern Bavaria, Germany). Here, we report the draft genome sequence of strain CL21, highlighting genes involved in Fe(II), sulfur, and H2 oxidation.

ANNOUNCEMENT

Sideroxydans sp. strain CL21 is an Fe(II)-oxidizing Gram-negative bacterium that belongs to the Gallionellaceae family within the class Betaproteobacteria. Isolated from a moderately acidic minerotrophic fen, CL21 can oxidize Fe(II) at pH 4.0 to 6.0 under microaerobic conditions (1). Like its close relative, Sideroxydans lithotrophicus ES-1, it was isolated as a chemolithoautotrophic Fe(II) oxidizer, though strain CL21 growth can be augmented with organics, including lactate. There are relatively few terrestrial Fe(II)-oxidizer isolate genomes, so we sequenced and analyzed the CL21 genome.
Sideroxydans sp. CL21 stock cultures were first cultivated at room temperature in the dark in semisolid gradient tubes containing 1% agarose-stabilized modified Wolfe’s minimal medium (MWMM), a defined freshwater medium, as previously described (1, 2), with Fe0 as the Fe source and 1 mM Na-lactate. Cultures were transferred to 250-ml serum bottles containing 100 ml MWMM amended with 10 ml liter−1 Wolfe’s vitamin solution, 10 ml liter−1 trace mineral solution, 10 mM MES (2-[N-morpholino]ethanesulfonic acid) buffer (pH 5.5), 1 mM lactate, and an Fe0 bottom-layer plug (10 ml MWMM, 3% agarose [PanReac Applichem agarose low EEO {electroendoosmosis} {agarose standard}], 100 mg 10 ml−1 Fe0). To maintain microaerobic conditions, the headspace was continuously flushed with N2:CO2:O2 at a ratio of 78:20:2 (flow rate, 300 ml min−1). Biomass was harvested by centrifugation (10 min, 10,000 × g, 4°C), and genomic DNA was extracted using a standard phenol-chloroform-based protocol (3). Whole-genome sequencing was performed on the PacBio RS II platform (Menlo Park, CA) according to the standard manufacturer’s protocol. Briefly, a 10- to 20-kb library was prepared and sequenced on the PacBio RS II sequencer using C4-P6 chemistry on single-molecule real-time (SMRT) cells, with a 180-min collection protocol. Sequence reads were filtered and assembled de novo with Hierarchical Genome Assembly Process v4 (HGAP4) using default parameters, except for the seed coverage (25×), seed length cutoff (15,000 bp), and estimated genome size (3.0 Mbp) (4), and annotated with RASTtk (v2.0) using default parameters (57). After HGAP4 assembly, the subread count was 129,903, comprising 1,021,527,933 bp. The mean subread length and N50 value were 5,649 bp and 7,803 bp, respectively.
The draft genome of CL21 was assembled into 1 contig with 263-fold average coverage, a total sequence length of 3.77 Mbp, and a GC content of 54.9%. The sequence quality was assessed with CheckM (v1.0.13) using default parameters (8), which detected 411 of 418 single-copy marker genes only once and 3 single-copy genes twice, equating to 99.37% completeness and 0.79% redundancy. The draft genome contains 3,795 coding sequences (CDS), 52 RNA-coding genes, and 2 16S rRNA genes. Homologs of the Fe(II) oxidation genes mtoAB and cyc2 were identified in Sideroxydans sp. CL21 (9). Additionally, genes involved in O2 reduction, CO2 fixation (RuBisCO), organic C utilization, sulfate respiration, sulfur oxidation, and hydrogen utilization were identified. Taken together, this genome sequence analysis shows that Sideroxydans sp. CL21 couples Fe(II) oxidation to assimilation of either inorganic or organic carbon compounds, which are particularly important metabolic processes in organic matter-rich environments like the Schlöppnerbrunnen fen.

Data availability.

The sequencing reads and assemblies for this whole-genome shotgun project are available in the European Nucleotide Archive (ENA) repository under the BioProject accession number PRJEB33828. The version described in this paper is the first version. The individual genome assembly is available under the accession number LR699166.

ACKNOWLEDGMENTS

This study was part of the Collaborative Research Centre Chemical Mediators in Complex Biosystems (SFB 1127 ChemBioSys) of the Friedrich Schiller University Jena, funded by the Deutsche Forschungsgemeinschaft. Additional funding was provided by a National Science Foundation grant (EAR-1833525).
We thank Jens D. Wurlitzer (Friedrich Schiller University Jena) for technical assistance in the laboratory and Karol Miaskiewicz from the University of Delaware DNA Sequencing and Genotyping Center at the Delaware Biotechnology Institute for assistance with HGAP4 assembly of raw sequencing data.

REFERENCES

1.
Lüdecke C, Reiche M, Eusterhues K, Nietzsche S, Küsel K. 2010. Acid-tolerant microaerophilic Fe(II)-oxidizing bacteria promote Fe(III)-accumulation in a fen. Environ Microbiol 12:2814–2825.
2.
Emerson D, Floyd MM. 2005. Enrichment and isolation of iron-oxidizing bacteria at neutral pH. Methods Enzymol 397:112–123.
3.
Wegner C-E, Gaspar M, Geesink P, Herrmann M, Marz M, Küsel K. 2019. Biogeochemical regimes in shallow aquifers reflect the metabolic coupling of the elements nitrogen, sulfur, and carbon. Appl Environ Microbiol 85:e02346-18.
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Chin C-S, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J. 2013. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569.
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Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75.
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Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M, Vonstein V, Wattam AR, Xia F, Stevens R. 2014. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res 42:D206–D214.
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Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ, Olson R, Overbeek R, Parrello B, Pusch GD, Shukla M, Thomason JA, 3rd, Stevens R, Vonstein V, Wattam AR, Xia F. 2015. RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep 5:8365.
8.
Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25:1043–1055.
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Liu J, Wang Z, Belchik SM, Edwards MJ, Liu C, Kennedy DW, Merkley ED, Lipton MS, Butt JN, Richardson DJ, Zachara JM, Fredrickson JK, Rosso KM, Shi L. 2012. Identification and characterization of MtoA: a decaheme c-type cytochrome of the neutrophilic Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1. Front Microbiol 3:37.

Information & Contributors

Information

Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 9Number 29 January 2020
eLocator: 10.1128/mra.01444-19
Editor: Frank J. Stewart, Georgia Institute of Technology

History

Received: 17 November 2019
Accepted: 6 December 2019
Published online: 9 January 2020

Contributors

Authors

Rebecca E. Cooper
Aquatic Geomicrobiology, Institute of Biodiversity, Friedrich Schiller University Jena, Jena, Germany
Aquatic Geomicrobiology, Institute of Biodiversity, Friedrich Schiller University Jena, Jena, Germany
Sean M. McAllister
School of Marine Science and Policy, University of Delaware, Newark, Delaware, USA
Olga Shevchenko
DNA Sequencing and Genotyping Center, University of Delaware, Newark, Delaware, USA
Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, USA
School of Marine Science and Policy, University of Delaware, Newark, Delaware, USA
Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, USA
Department of Earth Sciences, University of Delaware, Newark, Delaware, USA
Kirsten Küsel
Aquatic Geomicrobiology, Institute of Biodiversity, Friedrich Schiller University Jena, Jena, Germany
The German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Leipzig, Germany

Editor

Frank J. Stewart
Editor
Georgia Institute of Technology

Notes

Address correspondence to Kirsten Küsel, [email protected].

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