ABSTRACT
Here, we report the complete genome sequence of Geobacter sulfurreducens strain YM35, which was isolated from biofilms formed on an anode in a bioelectrochemical system where river sediment was used as an inoculum. The chromosome is 3,745,223 bp with a G+C content of 60.9%. The chromosome contains 3,324 protein-coding genes.
ANNOUNCEMENT
Geobacter sulfurreducens is one of the high current-producing bacteria, which transfer electrons to extracellular electrodes in their respiration processes (1, 2). The mechanism of the extracellular electron transfer to electrodes in G. sulfurreducens has been extensively studied in the strain PCA, which is the type strain of G. sulfurreducens (3). G. sulfurreducens strains KN400 and YM18, which produce a higher current density than strain PCA, were also isolated, and their complete genome sequences have been determined (4–6). Here, we determined the complete genome sequence of G. sulfurreducens strain YM35, which is another high current-producing strain, isolated from biofilms formed on an anode poised at −0.2 V (versus standard electrode system [SHE]) in a bioelectrochemical system where river sediment was used as an inoculum (T. Fujikawa, Y. Ogura, K. Ishigami, Y. Kawano, M. Nagamine, T. Hayashi, and K. Inoue, submitted for publication).
A frozen stock of strain YM35 (Fujikawa et al., submitted) was inoculated into NBAF medium with acetate (10 mM) and fumarate (40 mM) as the sole electron donor and acceptor, respectively, and cultivated at 30°C, as described previously (7). The medium was bubbled with N2-CO2 mixed gas (80:20, vol/vol) and autoclaved at 121°C for 30 min. The sterilized anaerobic l-cysteine solution was added to the medium before use (final concentration, 1 mM). Genomic DNA was purified with the genomic-tip 100/G column and genomic DNA buffer set (Qiagen) according to the manufacturer’s instructions. A paired-end library was constructed using the Nextera XT DNA library preparation kit (Illumina). The library was sequenced using an Illumina MiSeq instrument with the MiSeq reagent kit version 2 (Illumina) to obtain 251-bp paired-end reads. An 8-kb mate-pair library was also prepared using the Nextera mate-pair sample preparation kit (Illumina) and sequenced using the MiSeq platform. Low-quality sequences and adapters were removed using Platanus_trim version 1.0.7 (http://platanus.bio.titech.ac.jp/pltanus_trim). Default parameters were used for all software unless otherwise specified. A total of 3,206,380 paired-end and 1,057,520 mate-pair reads were assembled by Platanus version 1.2.1 (8), yielding 1 scaffold containing 33 gaps. The gap-spanning regions were amplified by PCR using the genomic DNA extracted from strain YM35 cells cultured in NBAF medium at 30°C as the template and the amplification primers listed in Table 1. Amplicons were sequenced by Sanger sequencing using the sequencing primers listed in Table 1, and gap sequences (69,183 bp in total) were determined. The coverage of the whole-genome sequence was 137×. The complete genome of strain YM35 consists of a 3,745,223-bp circular chromosome with a G+C content of 60.9%. The genome was smaller than that of strain PCA (3,814,128 bp) and larger than those of strains KN400 (3,726,411 bp) and YM18 (3,714,272 bp). No plasmid was found. Annotation by NCBI Prokaryotic Genome Annotation Pipeline (PGAP) version 5.2 (https://www.ncbi.nlm.nih.gov/genome/annotation_prok) (9) identified 2 rRNA operons, 48 tRNA genes, and 3,324 protein-coding sequences (CDSs). The 3,324 CDSs included 102 genes for c-type cytochromes, which was comparable to those of strains PCA, KN400, and YM18.
TABLE 1.
Primers used for PCR and sequencing of the gaps in the assembled draft genome sequence of strain YM35
| Gap no. | Forward primer (5′ to 3′)a | Reverse primer (5′ to 3′)a | For use in: | Annealing temp (°C)b | Extension time (s)b | In the final closed chromosome |
||
|---|---|---|---|---|---|---|---|---|
| No. of Ns | Start position | End position | ||||||
| 1 | GGACGAACTACCGGTTTCTTTC | CATCCAGGCTGATGTAGACCAT | PCR amplification, sequencing | 60 | 30 | 287 | 225930 | 226216 |
| 2 | CCGGTATACGAGACAACTCCAT | CTGGCAGAGAATGATCAAGATG | PCR amplification, sequencing | 60 | 30 | 5,772 | 489062 | 494833 |
| CATGTCCGTTGCCGGATCGAAG | AGCAGTGCGGCCGTAGTGAAAC | Sequencing | ||||||
| TGTGGTCACAACGTGATCGC | TCGAGACGGTGACGAACTTC | Sequencing | ||||||
| TCGAAGATGTGCGGCAGATC | ACAAGGGGTCGTCGGATATC | Sequencing | ||||||
| CAACTCCGAATACCACAAAC | GCTGGACCTTTTCAAGGGTC | Sequencing | ||||||
| TTCCGCATGAAGTACTCGAG | Sequencing | |||||||
| 3 | CTGAAACGGTACACGGAGGT | GATGTCGCTCATGTGGATGC | PCR amplification, sequencing | 60 | 30 | 211 | 606728 | 606938 |
| 4 | TGGGGAATGATTTGGTAGTTTC | GATGCTATCCTTCCGACCATAC | PCR amplification, sequencing | 60 | 30 | 4,872 | 690253 | 695124 |
| AAGCACCGGCTAACTCCGTGCC | AGCTGCCCGTGCAGTATCATCG | Sequencing | ||||||
| AGCGCAACCCTTATCATCAG | ATGAGCCGACATCGAGGTGC | Sequencing | ||||||
| AGGAGGTCATCGGTTCGAAC | AGTCGCTACCGCCATTTCTC | Sequencing | ||||||
| CATCGTTTTCCACTTAGCATGG | Sequencing | |||||||
| AGGGTGAATCAAGGTTTCGC | Sequencing | |||||||
| 5 | CCACGATCCCCTTGATATAGTT | CGGTAACCGTAACTGACAATCC | PCR amplification, sequencing | 60 | 30 | 1,846 | 776037 | 777882 |
| TGTCGCGGATGAAGGCATTGAG | ACGTCCTCATTGCCCGGTTCAC | Sequencing | ||||||
| 6 | TTCCCCAGAACTTGATCTGATT | CAGATTGTATTGGCAGGAACAA | PCR amplification, sequencing | 60 | 30 | 6,166 | 812018 | 818183 |
| GATCACCTTCACGAGCCTTGAG | ACCCACCTGGAGGATATGTACG | Sequencing | ||||||
| TGGCGATCGTCTACATGCTG | ACAACTGGCAGCGGATTATC | Sequencing | ||||||
| GAACAAATGGCCACGGCCTC | TGATTCCGCCCTGGTAGAG | Sequencing | ||||||
| ACGTCACCAACCAAATCAGC | CCATAACTAAACCGGCCATG | Sequencing | ||||||
| 7 | CATATAGTCGAGCGTCTGTTCG | GGTTCTGCTCCAGGACTTTACC | PCR amplification, sequencing | 60 | 1 | 302 | 913748 | 914049 |
| 8 | ATACCGTGGTGCTCGTAGAAAT | GAATACTCCAGCAGCAGATCCT | PCR amplification, sequencing | 57 | 25 | 44 | 985548 | 985591 |
| 9 | GAAGCTACACCATCAGCGAGTA | GTAATATCAACGGTCGACACCA | PCR amplification, sequencing | 60 | 1 | 234 | 1007442 | 1007675 |
| 10 | ACGGCATCTGTTTTCAAATCTT | GGAACCTGTTTCGTACCTTTTG | PCR amplification, sequencing | 60 | 30 | 3,292 | 1045471 | 1048762 |
| GCCATAGATGACCTGTTCTTTG | TCCAGCTCGACTGCAAGCCTTG | Sequencing | ||||||
| TACACGGCTATAGAACGACG | ACGTTCTGACAGTGCGTTCG | Sequencing | ||||||
| TATCCTGTTCCGCTGGTTCC | GTGACTTGAAGCTCTTGATG | Sequencing | ||||||
| 11 | CAAGATAGGGATTCATTCTCACG | AAAGAGATCCATGGTCAGGGTA | PCR amplification, sequencing | 60 | 1 | 305 | 1070868 | 1071172 |
| 12 | TTGGTAGTTTCGGTGTTCCTTT | CACATATCTCATCGACTGAGAC | PCR amplification, sequencing | 58 | 30 | 4,872 | 1235830 | 1240701 |
| GAGAGGATGATCAGCCACAC | CTGCCCGTGCAGTATCATCG | Sequencing | ||||||
| AGCGCAACCCTTATCATCAG | AGTCGCTACCGCCATTTCTC | Sequencing | ||||||
| AGGAGGTCATCGGTTCGAAC | CATCGTTTTCCACTTAGCATGG | Sequencing | ||||||
| AGGGTGAATCAAGGTTTCGC | Sequencing | |||||||
| 13 | TTTCCTGGAGGACTATGCATTT | GGGCTATCTGCTTCTTTTCAGA | PCR amplification, sequencing | 60 | 1 | 157 | 1288215 | 1288371 |
| 14 | CTGGCGAATGTTTTCCATCTAT | TGAAAGATATTGAGCGGGAGAT | PCR amplification, sequencing | 60 | 1 | 279 | 1305843 | 1306121 |
| 15 | TTTCTGGCTCATCCTCAATCAT | ATCAAGGTCAAGATCCTGGAAA | PCR amplification, sequencing | 60 | 1 | 369 | 1386224 | 1386592 |
| 16 | CCGTAGTGAAGGGAGAGTATGC | GGGATATAGGTCCAGATGGTCA | PCR amplification, sequencing | 60 | 1 | 285 | 1606542 | 1606826 |
| 17 | GCTGATGAGACCTGTAGCACTG | AAAGGAAGCGCAAGTTTTACCT | PCR amplification, sequencing | 65 | 40 | 1,078 | 2268239 | 2269316 |
| AGGCTGCTGAGGCGCCATAG | AAAGGCCGTTGCCGCCAGCACGAT | Sequencing | ||||||
| TTGATGCCGAGGCCAGGCGGGG | Sequencing | |||||||
| 18 | CCGGAGTCGATAGTACATGAGA | GCAACGAGTGGTGGTACTTCAC | PCR amplification, sequencing | 60 | 1 | 315 | 2312066 | 2312380 |
| 19 | CTTGACGAAGAACGACATGAAC | CTTACGTTCGCCGTCTGGTACT | PCR amplification, sequencing | 60 | 30 | 5,412 | 2590955 | 2596366 |
| AATACCGATTCACCCACCGATG | GATAATCACCTGCATGATCACC | Sequencing | ||||||
| CAGCTCGTAGTAGATCTTGC | CAGTACGAGATCACCCGCTG | Sequencing | ||||||
| AACTCGGAGAGCATGCTGTC | CGGAGCCATCAAGGTCTGTC | Sequencing | ||||||
| ATCTTCGCCGTGGACGTGGAAC | CTCTCTCATCTATCGCTACC | Sequencing | ||||||
| TCGGCGAGGAAACGCTTCTCTC | Sequencing | |||||||
| 20 | GGACCACTTAACACAACCCTGT | CAACTATCCTGCGTGAATTGAG | PCR amplification, sequencing | 60 | 30 | 5,723 | 2663241 | 2668963 |
| CACAGGTGTTAAGGAACAGATG | GATCACGCTGGTGAACAGGCTG | Sequencing | ||||||
| ATCTCGTCCAGGAAGATCGTG | ACAGGGACGATCACCATGAG | Sequencing | ||||||
| TGAGGATGGCATCGTCCACG | GCCGACTGGCATGGATATTG | Sequencing | ||||||
| CCTTGATATCCAGGCGCTTG | ATAACCTCCTGGGCGATCTC | Sequencing | ||||||
| 21 | CCCAAATATACCGCACTATTCC | TTCAATGGTGAAATCGATGTGT | PCR amplification, sequencing | 60 | 30 | 1,895 | 2686577 | 2688471 |
| CTCGACTCTGGCGGAGATGAAC | ATACTCACCGAGGACACCAATG | Sequencing | ||||||
| 22 | CTTGATTCCCACTCTCAGGTTC | AACGTGTTTCCCTTGGTCATTC | PCR amplification, sequencing | 60 | 1 | 248 | 2742900 | 2743147 |
| 23 | AAATACTCCTGACTGGCCAAAA | TTATGAAACGTCGTGAAAATGC | PCR amplification, sequencing | 60 | 30 | 7,459 | 2756485 | 2763943 |
| AAGCGGATGCGCTCATTAAC | GTGCCATCAACACTGAAGTG | Sequencing | ||||||
| CAGATCGACATCTGTTGACG | GAAATCGAAAGACGAGCCGC | Sequencing | ||||||
| AACATCACCTGTCCGCTTTC | GACAAACACACCACACAAGG | Sequencing | ||||||
| CTTCTTCCAGGGTGAAGATC | AGGTTGTCCTGCGGTTGATG | Sequencing | ||||||
| CTTTCGAGTTGCAGCATCAG | AAGAGCATCCGGGAGATCTC | Sequencing | ||||||
| GTGCTGGATGTCGCTCATAG | GGGACACGGCAAAGCATTTC | Sequencing | ||||||
| 24 | CGGTAAATCCTGAATTCCATCC | ATTCCCTCGACTTCAATCAATC | PCR amplification, sequencing | 60 | 1 | 210 | 2777910 | 2778119 |
| 25 | CTCCATCATGTGTTCAGGGTTA | GGTTTCTGGTAGTGTTGGAAGC | PCR amplification, sequencing | 60 | 1 | 843 | 1874360 | 1875202 |
| 26 | ATCCACCTCGTAGTCATGAAAG | CCAGATTCTCTACTCGGTGGTG | PCR amplification, sequencing | 60 | 30 | 3,285 | 2898646 | 2901930 |
| TCTTCTGGCCGATCTGACGGAG | ATAACGTGGAGACCCTTGCGAC | Sequencing | ||||||
| TGTAGCCGTCCACCGGAATC | GCTACTTCCGGAATGAATACG | Sequencing | ||||||
| 27 | TCGTAAATATCGATTCGTGGTG | GTGACGGAGATAACGCTAAACC | PCR amplification, sequencing | 60 | 30 | 1,024 | 2949114 | 2950137 |
| GGCTTTGACGACAAGATCATC | Sequencing | |||||||
| GTCAGTCATGGCGGAACAAC | Sequencing | |||||||
| 28 | TCGTAAATATCGATTCGTGGTG | GTGACGGAGATAACGCTAAACC | PCR amplification, sequencing | 60 | 30 | 17 | 2954455 | 2954471 |
| 29 | GAACCGAAGACCTTCATCCATC | GAAATCGTCGCAAAGGTTAAAG | PCR amplification, sequencing | 60 | 1 | 893 | 3026979 | 3027871 |
| 30 | CCCGAACTATTCGATAATGGAG | GTCACTACTCACCGTGGTTGCT | PCR amplification, sequencing | 60 | 30 | 3,188 | 3043295 | 3046482 |
| GCTCGTCCTTGTCGCCATTGAG | GAACATGTACGGCGTGCACTGC | Sequencing | ||||||
| TTACCAAGGAAGTGCTCTAC | ATCGGGAGAAGAGGTTGCAG | Sequencing | ||||||
| CCAATGGAGGCAGAATCAAG | CGAGGGCATCGACATGGATG | Sequencing | ||||||
| 31 | CCCCAGAAACATAGGGTGAATA | GTGGTGCCGTTGTAGACATAGA | PCR amplification, sequencing | 60 | 30 | 3,554 | 3427838 | 3431391 |
| AGCTCGGTGCCAATGAAATTCG | TGATCTTCAGGGCGTCGTTCAC | Sequencing | ||||||
| GCCACTTTGAAGGTTATGAC | GATAGGTGCCGTTATAGAACC | Sequencing | ||||||
| 32 | CCTGAAGATCAATGAGGGAAAG | ATTGTACTCGGTCATCCACCTC | PCR amplification, sequencing | 60 | 1 | 173 | 3511982 | 3512154 |
| 33 | AACCACCAGCACATCAGAAG | GCGCCAACCACCAGCACATC | PCR amplification, sequencing | 57 | 25 | 4,408 | 3627984 | 3632391 |
| GTTGTCCATTGAACAGCTGG | CCAGCTGTTCAATGGACAAC | Sequencing | ||||||
| CAAGTAATTCCAAATGGAGCG | GATGATTACCTGCTGCATTGC | Sequencing | ||||||
| GCAATGCAGCAGGTAATCATC | GAAAGCTCCTTCCCATCCTTCG | Sequencing | ||||||
| CCGTGTTCCTAAATTCTTGAGG | AAGCCGCTACCTGAAGGCTTC | Sequencing | ||||||
| CGAAGGATGGGAAGGAGCTTTC | Sequencing | |||||||
| CTTCAACTTCAGCCGTTCAC | Sequencing | |||||||
| 34 | CACGTCCTTGGGATATTTGTAA | GAAGATTACGACCTCTGGCTGA | PCR amplification, sequencing | 60 | 1 | 165 | 3736120 | 3736284 |
The primer sets in bold were used for both PCR amplification and the sequence in each gap.
PCR conditions used were as follows: 25 cycles of 98°C for 10 s, annealing temperature indicated in the list for each reaction for 5 s, 68°C for extension time indicated in the list for each reaction, and 68°C for 5 min. For all reactions, KOD One PCR master mix (Toyobo) was used. The amplicons were gel purified by Monarch DNA gel extraction kit (New England BioLabs).
Data availability.
The complete genome sequence of strain YM35 was deposited under GenBank accession number CP074693. The raw reads were deposited in the Sequence Read Archive under BioProject accession number PRJNA742140.
ACKNOWLEDGMENTS
This work was financially supported by the Program to Disseminate Tenure Tracking System from the Japanese Ministry of Education, Culture, Sports, Science and Technology, a grant for Scientific Research on Priority Areas from the University of Miyazaki, and the Institute for Fermentation, Osaka (IFO).
Contributor Information
Kengo Inoue, Email: kinoue@cc.miyazaki-u.ac.jp.
J. Cameron Thrash, University of Southern California
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Associated Data
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Data Availability Statement
The complete genome sequence of strain YM35 was deposited under GenBank accession number CP074693. The raw reads were deposited in the Sequence Read Archive under BioProject accession number PRJNA742140.