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Microbiology and Biotechnology Letters

Genome Report(Note)

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Genome Report  |  Genome Report

Microbiol. Biotechnol. Lett. 2024; 52(4): 500-504

https://doi.org/10.48022/mbl.2408.08014

Received: August 20, 2024; Revised: September 25, 2024; Accepted: October 3, 2024

Draft Genome Sequence of Acetobacter oryzifermentans AFY-4 Isolated from Rice Vinegar

Ha-Yeon Lee1,2, Jeong-Ah Yoon2, Jae-Geel Lim1, Hye-Jeong Kwon1, Eun-Hee Park3, and Myoung-Dong Kim3,4*

1Agro-Food Research Institute, Gangwon State Agricultural Research and Extension Services, Chuncheon 24203, Republic of Korea
2Department of Food Biotechnology and Environmental Science, Kangwon National University, Chuncheon 24341, Republic of Korea
3Department of Food Science and Biotechnology, Kangwon National University, Chuncheon 24341, Republic of Korea
4Institute of Fermentation and Brewing, Kangwon National University, Chuncheon 24341, Republic of Korea

Correspondence to :
Myoung-Dong Kim,        mdkim@kangwon.ac.kr

Abstract

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Acetobacter oryzifermentans AFY-4, a potent acetic acid producer, was isolated from traditional Korean rice vinegar. This study presents the draft genome sequence of A. oryzifermentans AFY-4. The genome of A. oryzifermentans AFY-4 is 3,222,280 bp in length, with a G+C content of 52.5%, including 2,962 protein-coding sequences (CDSs), 57 transfer RNA genes, and 15 ribosomal RNA genes. A. oryzifermentans AFY-4 exhibited high orthologous average nucleotide identity values with other A. oryzifermentans strains, including SLV-7, DmCS_004, and DM001. Pan- and core-genome analysis showed that the AFY-4 strain is more closely related to the SLV-7 strain than other A. oryzifermentans strains.

Keywords: Acetobacter oryzifermentans, genome, acetic acid bacteria, vinegar

Body

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Acetic acid bacteria are strictly aerobic, Gram-negative, and non-spore-forming bacteria belonging to the family Acetobacteraceae and typically exhibit optimal growth at pH 5−6.5, with the ability to grow at pH 3−4 [1]. Acetobacter is commonly found in alcoholic, acidic, sugary habitats, including traditional fermented foods and alcoholic beverages like vinegar, kombucha, sour beer, and water kefir [2, 3]. Acetobacter oryzifermentans was isolated from Korean traditional vinegar and proposed as a novel species due to its low average nucleotide identity (ANI) value (<91.0%) and in silico DNA-DNA hybridization (DDH) values (44.2%) with A. pasteurianus, despite its high 16S rRNA gene sequence similarity (>99.59%) to A. pasteurianus [4]. A. oryzifermentans AFY-4, known for its high acetic acid production and tolerance to low pH 3 [5], was previously reported as A. pasteurianus AFY-4 based on 16S rRNA sequences. This study confirmed that the AFY-4 strain is A. oryzifermentans based on genome sequence and present draft genome information.

A. oryzifermentans AFY-4 was incubated in YCM broth (0.5% yeast extract, 1.0% CaCO3, 2.5% mannitol, 3.0% ethanol) at 25℃ for 48 h. Genomic DNA was extracted using the FastDNATM spin kit (MP Biomedicals, USA) according to the manufacturer’s instructions. Size selection was performed using the BluePippinTM size selection system (Sage Science, USA) according to the PacBio 20-kb library protocol. The sequencing library was prepared using the SMRTbell template prep kit 1.0 (Pacific Biosciences, USA). It was sequenced using the PacBio RS II sequencer (Pacific Biosciences) based on a single molecule, real-time (SMRT) sequencing technology. The library was filtered for genome assembly using the hierarchical genome assembly process (HGAP, ver. 2) protocol with default parameter in SMRT analysis software (ver. 2.3.0) [6].

A phylogenetic tree and heat map were constructed based on the unweighted pair group method with arithmetic mean (UPGMA), and orthologous ANI (OrthoANI) were calculated from OAT software (ver. 0.93.1) [7]. EggNog (evolutionary genealogy of genes: non-supervised orthologous groups) classification was mapped to clusters of orthologous genes (COG) categories. Pan- and core-genome analysis was performed using all CDSs' gene presence and absence matrix and a Venn diagram [8] with the EzBioCloud comparative genomics database.

The total bases, mean length, and N50 value of the polymerase read in the draft genome of A. oryzifermentans AFY-4 were 3,220,280 bp, 941.5 bp, and 2,730,921 bp, respectively. The genomic information of A. oryzifermentans AFY-4 and other Acetobacter species are summarized in Table 1. The assembled draft genome of A. oryzifermentans AFY-4 consists of 5 contigs with a G+C content of 52.5% and includes 2,962 CDSs, 15 rRNA, and 57 tRNA genes. The numbers of rRNA and tRNA genes in A. oryzifermentans AFY-4 were like those in other Acetobacter strains.

Table 1 . Genome features of A. oryzifermentans AFY-4 and Acetobacter species.

StrainGenome size (bp)G+C content (%)CDSsrRNAstRNAsGenBank Accession No.
A. oryzifermentans AFY-43,222,28052.52,9625, 5, 5 (5S, 16S, 23S)57JBFNUA000000000
A. oryzifermentans SLV-7T2,799,48852.52,7895, 5, 5 (5S, 16S, 23S)57CP011120
A. oryzifermentans DmCS_0041)2,843,88452.52,6821, 1, 1 (5S, 16S, 23S)43NZ_JOKL00000000
A. oryzifermentans DM0011)2,883,42152.32,6881, 1, 1 (5S, 16S, 23S)43NZ_AEUP00000000
A. pasteurianus Ab32,806,14853.52,7865, 5, 5 (5S, 16S, 23S)58CP012111
A. oryzoeni B6T2,744,39353.02,9125, 5, 5 (5S, 16S, 23S)57CP042808
A. pomorum SH2,969,51452.52,9995, 5, 5 (5S, 16S, 23S)56CP023189
A. tropicalis BDGP13,988,64955.53,7135, 5, 5 (5S, 16S, 23S)58CP022699
A. vaccinii KACC 21233T2,814,61859.52,7325, 5, 5 (5S, 16S, 23S)57CP043506

T: Type strain

DmCS_004 and DM001 strains were reported as A. pomorum in GenBank. However, two strains were reclassified as A. oryzifermentans because these shared high ANI values (98.53-99.88%) with A. oryzifermentans [4].

The OrthoANI values were calculated based on comparisons with other Acetobacter genomes (Fig. 1). A. oryzifermentans AFY-4 exhibited a high OrthoANI value of 99.95% with A. oryzifermentans SLV-7, 98.75% with A. pomorum SH, and 90.24% with A. pasteurianus Ab3.

Figure 1.OrthoANI analysis heat map based on the UPGMA tree of A. oryzifermentans AFY-4 and other Acetobacter spp.

In eggNog analysis (Fig. 2), significant categories included genes related to replication, recombination, and repair (317 genes, 12.04%); amino acid transport and metabolism (189 genes, 7.18%); and inorganic ion transport and metabolism (169 genes, 6.42%), excluding genes of unknown function, which represented the largest group.

Figure 2.Histogram of eggNog classification of A. oryzifermentans AFY-4 based on COG functional categories.

Gene presence and absence analysis revealed numerous gene insertions and deletions distributed across the genome of A. oryzifermentans AFY-4 compared to the other four strains (Fig. 3A). The AFY-4 strain harbors more than 200 genes not found in different strains. As shown in Fig. 3B, the Venn diagram indicates that 2,237 core-pan-genome orthologous groups (POGs) were identified among the four A. oryzifermentans strains (cut off=100%). Furthermore, the AFY-4 strain has 248 additional genes, including those for aspartate racemase, which are absent in the other strains. Both gene content (presence/absence) and Venn diagram results suggest that the AFY-4 strain is more closely related to the SLV-7 strain isolated from vinegar than the DmCS_004 [9] and DM001 [10] strains, isolated from the gut of Drosophila melanogaster. This geographic distribution of core genome evolution may be related to the 2,237 core-genome genes shared among the four A. oryzifermentans strains.

Figure 3.Heat map based on gene content (presence/absence) of all CDSs among five Acetobacter species (A) and Venn diagram for specific and shared CDSs among four A. oryzifermentans strains (B).

In conclusion, genomic analysis of A. oryzifermentans AFY-4 may provide invaluable information for future research on acetic acid production.

Nucleotide Sequence Accession Numbers

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The draft A. oryzifermentans AFY-4 genome sequence has been deposited in the GenBank database under the accession number JBF-NUA000000000.

Acknowledgments

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This study was performed by the research program (Project No. R0003863) funded by the Ministry of Trade, Industry and Energy in the Republic of Korea.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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