Abstract
A diazotrophic, acidophilic, iron-oxidizing bacterium, Leptospirillum ferrooxidans, known to be difficult to cultivate, was isolated from a fresh volcanic ash deposit on the island of Miyake, Japan. Here, we report the complete genome sequence of a cultured strain, C2-3.
GENOME ANNOUNCEMENT
Leptospirillum ferrooxidans is an acidophilic, obligate chemolithoautotrophic ferrous-iron-oxidizing bacterium, known to have nitrogenase-encoding genes (9). Our recent molecular ecological study showed that L. ferrooxidans was abundant in volcanic ash deposits derived from the Mount Oyama eruption in the year 2000 on the island of Miyake (Miyake-jima), Japan, suggesting that L. ferrooxidans is a pioneer microbe involved in the fixation of carbon and nitrogen in early soil ecosystems (1, 11). L. ferrooxidans strain C2-3 was isolated from a 7.6-year-old volcanic ash deposit at Mount Oyama by chemostat enrichment, and its acetylene reduction activity was characterized (10). The isolation of the strain is highly significant because L. ferrooxidans is poorly characterized due to the difficulty of maintaining pure cultures. Here, we determined the whole-genome sequence of L. ferrooxidans C2-3 to obtain its genomic information.
Genomic DNA was isolated from L. ferrooxidans C2-3 using phenol-chloroform extraction as described previously (12) and sequenced by the whole-genome shotgun sequencing method with ABI 3730xl DNA analyzer (Applied Biosystems). Approximately 7.3-fold coverage reads of the genome were assembled by Phred/Phrap/Consed software (3). The following annotation programs were used: MetaGeneAnnotator (7) for the identification of protein-coding sequences, tRNAscan-SE (6, 8) for tRNA, and RNAmmer (4) for rRNA.
The circular chromosome of L. ferrooxidans C2-3 contains 2,559,538 bp, for which 2,421 open reading frames, 51 tRNA genes, and 3 rRNA operons were predicted. Average G+C content of the genome was 50.1%. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes for the Calvin-Benson cycle were not found, but two copies of RubisCO-like protein were predicted. L. ferrooxidans C2-3 has genes for the reductive tricarboxylic acid (TCA) cycle, except for 2-oxoglutarate:ferredoxin oxidoreductase and ATP-citrate lyase. Instead, two copies of pyruvate ferredoxin oxidoreductase genes and succinyl-CoA synthetase were found, consistent with the near-complete genome sequences of “Leptospirillum rubarum” and “Leptospirillum ferrodiazotrophum” that were obtained by community genomic and proteomic analyses of acidic mine drainage environments (2). The L. ferrooxidans C2-3 genome contained all genes encoding the components of the pentose phosphate pathway and some of the genes for the Embden-Meyerhof-Parnas pathway; genes for the TCA cycle were found to be incomplete. A single operon of nitrogenase was found to contain the nifH, -D, -K, -E, -N, -X, -B, -S, -Z, -U, -T, -W, and -A genes, but the nifL gene was not predicted. This result suggested that L. ferrooxidans might have an NifL-independent nitrogenase regulation system. Furthermore, genes of the two-component nitrogen regulatory system in L. ferrooxidans C2-3 were identified: not only ntrCB but also nrtXY genes. ntrXY genes were also found in the complete genome sequence of Leptospirillum ferriphilum, which has not been deposited (5). Additionally, L. ferrooxidans C2-3 has some hydrogenase-encoding genes, which are considered to have potential for hydrogen oxidation.
The complete genome sequence of L. ferrooxidans C2-3 will provide significant genetic information of acidophilic, iron-oxidizing bacteria and be useful for further studies to obtain a clear understanding of the ecology of difficult-to-culture microbes.
Nucleotide sequence accession number.
The complete genome sequence of L. ferrooxidans C2-3 has been deposited in the DDBJ/EMBL/GenBank databases under accession number AP012342.
ACKNOWLEDGMENTS
This study was supported in part by Grants-in-Aid (17310018 and 22248038) for Scientific Research from the Japan Society for the Promotion of Science and from the Yuuji Ushiba Research Fund.
We thank the staff of the laboratory of M.H. for technical assistance.
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