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Acetobacter pasteurianus IFO 3283-32 (= NBRC 105190)

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close this sectionAbout this Microorganism


Photo by Dr. Muramatsu (NBRC, NITE)

Acetic acid bacteria (AAB) are a group of widely diverged bacteria within alpha-proteobacteria and also known for their genetic instability. Acetobacter pasteurianus is one of the popular AAB, which has been used for traditional vinegar production. A. pasteurianus IFO 3283 (= NBRC 3283) was isolated from a pellicle formed on the surface of fermentation of traditional Japanese rice vinegar. Since the deposit of the pure culture in IFO (Institute for Fermentation, Osaka) in 1954, the strain had been maintained by a series of passages at three-months intervals until 1974 when freeze-dry preservation method was established. As a result, a multi-phenotype cell complex with non-homogeneous colony textures was formed.

To avoid the loss of genetic information pertaining to useful features maintained in the cell complex, whole-genome shotgun libraries were constructed from genomic DNA extracted from the cell complex without any single colony isolation. After sequencing and assembly, 38 assembling gaps and sequence discrepancies remained as candidate loci of genomic variations. A total of 32 substrains were then isolated from the cell complex and their sequence variations were confirmed by resequencing of the candidate loci. In total, 4 transposon insertions, 3 SNPs and 3 Hyper-mutable Tandem Repeats (HTRs) were identified as chromosome variations, while a transposon insertion and a HTR were observed in the largest plasmid. This strategy allowed us to establish whole genome sequences, each being composed of a single chromosome and 6 plasmids, of 7 representative substrains (IFO 3283-01, 03, 07, 12, 22, 26 and 32). Three SNPs and 4 transposon insertions on the chromosome were used to deduce the generation order of genomic variations as shown in the Figure presented below. In addition, a temperature resistant mutant was obtained from one of the isolates, IFO 3283-01, after prolonged cultivation at 40 C. Genome sequencing of the temperature resistant mutant revealed 3 SNPs and a 92 kb deletion. Genome analysis of A. pasteurianus thus provided important insights into the genetic instability and flexibility of hyper-mutabe AAB.

For simplicity, this database deals only with one of the 7 representative substrains, IFO 3283-32, which is thought to bear the original phenotype of IFO 3283. Genomic variations found in other 6 substrains are summarized in and can be accessed from the following figures.

lineage
Fig. Genotype lineage (chromosome) of 32 substrains of the IFO 3283 cell complex

Seven representative substrains isolated from IFO 3283 cell comlex (IFO 3283-01, 03, 07, 12, 22, 26, 32) and the high temperature-resistant strain (IFO 3283-01-42C) were redeposited in NBRC with catalogue numbers shown in the "General Procedure" head. The original cell complex is also available as NBRC 3283.

Variation observed in IFO 3283 cell complex
Chromosome variation
1 type: Hyper mutable Tandem Repeats (HTR)
releated ORFs: APA32_02730
product: Beta-Ig-H3/fasciclin repeat containing protein
TR01 Show Annotation
2 type: Single Nucleotide Polymorphism (SNP)
releated ORFs: APA32_09550
product: DNA-directed RNA polymerase alpha subunit
gene: rpoA
SNP01 Show Annotation
3 type: Insertion Sequence (IS)
releated ORFs: APA32_10210
product: Two component sensor histidine kinase
Gap14 Show Annotation
4 type: Single Nucleotide Polymorphism (SNP)
releated ORFs: APA32_12550
product: Glycerol kinase
SNP02 Show Annotation
5 type: Hyper mutable Tandem Repeats (HTR)
releated ORFs: APA32_16984
product: Transcriptional regulator MerR
TR03 Show Annotation
6 type: Single Nucleotide Polymorphism (SNP)
releated ORFs: APA32_17930
product: Hypothetical protein
SNP03 Show Annotation
7 type: Insertion Sequence (IS)
releated ORFs: APA32_18050
product: Transposase
Gap18 Show Annotation
8 type: Hyper mutable Tandem Repeats (HTR)
releated ORFs: APA32_18746, APA32_18748
product: DNA helicase restriction enzyme Type III R subunit
TR04 Show Annotation 1
Show Annotation 2
9 type: Hyper mutable Tandem Repeats (HTR)
releated ORFs: APA32_20310
product: DNA polymerase III exonuclease epsilon subunit like protein
TR06 Show Annotation
10 type: Insertion Sequence (IS)
releated ORFs: APA32_21760
product: Protein translocase subunit SecB
Gap03 Show Annotation
11 type: Insertion Sequence (IS)
releated ORFs: APA32_24440
product: Glycosyl transferase
Gap22 Show Annotation

Plasmid variation
12 type: Insertion Sequence (IS)
releated ORFs: none (Intergenic region)
Gap107
13 type: Hyper mutable Tandem Repeats (HTR)
releated ORFs: APA32_40324
product: Hypothetical protein
TR07 Show Annotation

close this sectionProject history

close this date 2009-10-20 ..... 1
2009-10-20 Release of the Acetobacter pasteurianus IFO 3283 genomic sequence data
imageWe published the genome data of Acetobacter pasteurianus IFO 3283 including the information of DNA clones distributed from the NBRC

close this sectionSummary of the genomic data

AS1
Genomic size 3,337,040 bp
G+C content 53.10 %
Number of ORFs assigned 3,046
Percentage of the coding regions 89.27 %
Percentage of the intronic regions 0.00 %
Number of rRNA genes 15
5S16S23S
555
Number of tRNA genes 57
AlaArgAsnAspCysGln
731112
GluGlyHisIleLeuLys
231552
MetPheProSerThrTrp
713431
TyrVal
14
Number of other features
(misc_RNA,misc_feature,repeat)
0

close this sectionGeneral Procedure

Shotgun DNA sequencing was carried out using mixed genomic DNA extracted from the cell complex of A. pasteurianus IFO 3283.


General Procedure
  • DNA shotgun library
    DNA shotgun libraries with inserts of 1.5 and 5.0 kb in pUC118 vector (TAKARA) was constructed.

  • Cosmid library
    A Cosmid library with inserts of 35 kb in the pCC1FOS fosmid vector was constructed (Epicentre).

  • Nucleotide sequencing
    Plasmid clones were end-sequenced using dye-terminator chemistry on an Applied Biosystems 3730xl DNA Analyzer.
    Fosmid DNA was extracted from E. coli transformants using the Montage BAC96 MiniPrep Kit (Millipore) and end-sequencing was carried out using dye-terminator chemistry on Applied Biosystems 3730xl DNA Analyzer.
    Raw sequence data corresponding to approximately 9.3-fold coverage were assembled using PHRED/PHRAP/CONSED software (http://www.phrap.org).

  • Gap closing and substrain isolation
    30 gaps and 84 low quality regions under 40 in Phrap score were remained after construction of seven supercontigs corresponding to a chromosome and 6 plasmids.
    DNA fragments were produced to fill all the gaps by PCR from genomic DNA of the 32 isolates from the IFO 3283 cell complex. All regions with lower quality sequences than 25 in Phred value were re-sequenced by primer walking.
    As a result, 13 variable loci were identified in the 32 isolates of A. pasteurianus IFO 3283 (see About this microbe page).

  • Validation of the assembled sequence data
    The assembled sequence was validated by Pulse field gel electrophoresis (Bio-Rad), restriction enzyme digestion of fosmid clone and Optical Mapping system (OpGen).
    When any inconsistency in assembling and co-existence of different nucleotides were detected, the sequence variations were confirmed by re-sequencing all 32 isolated clones.

Genome analysis and annotation
  • rRNA and tRNA genes were predicted using programs, BLASTN and tRNAscan-SE, respectively.

  • For the identification of protein-coding genes, candidates of ORFs were extracted by using GenomeGambler, GeneHackerplus and Glimmer 3.0 with length longer than 90 bp and manually inspected.

  • The translated sequences of the predicted protein-coding genes were searched against non-redundant protein database using BLASTP and FASTA3.

  • Transmembrane protein genes were predicted using SOSUI.

  • The software, JSTRING, was used for the extraction of hyper-mutable tandem repeats (HTR) from genomic DNA sequences of IFO 3283 substrains.


Summary of the A. pasteurianus IFO 3283 substrains Genomic Data

Chromosome
Strain name IFO 3283-32 IFO 3283-01 IFO 3283-07 IFO 3283-12 IFO 3283-22 IFO 3283-26 IFO 3283-03 IFO 3283-01-42C
Genomic size: 2,904,642 bp 2,907,495 bp 2,906,044 bp 2,904,624 bp 2,907,267 bp 2,907,309 bp 2,907,287 bp 2,815,241 bp
GC content: 53.03% 53.04% 53.04% 53.03% 53.04% 53.04% 53.04% 53.09%
Coding region coverage: 90.31% 90.17% 90.17% 90.31% 90.31% 90.32% 90.26% 90.25%
Protein coding genes: 2,625 2,628 2,626 2,625 2,627 2,627 2,627 2,562
5S, 16S and 23S rRNA genes: 5 each 5 each 5 each 5 each 5 each 5 each 5 each 4 each
tRNA genes: 57 57 57 57 57 57 57 54

Plasmid 1
Strain name: IFO 3283-32 IFO 3283-01 IFO 3283-07 IFO 3283-12 IFO 3283-22 IFO 3283-26 IFO 3283-03 IFO 3283-01-42C
Plasmid name: pAPA32-012 pAPA01-011 pAPA07-010 pAPA12-014 pAPA22-010 pAPA26-013 pAPA03-010 pAPA42C-011
Genomic size: 191,443 bp 191,799 bp 191,427 bp 191,411 bp 191,427 bp 191,419 bp 191,427 bp 191,799 bp
GC content: 53.37% 53.36% 53.37% 53.37% 53.37% 53.37% 53.37% 53.36%
Coding region coverage: 80.53% 80.54% 80.54% 80.56% 80.54% 80.52% 80.54% 80.54%
Protein coding genes: 177 178 177 177 177 177 177 178

Plasmid 2
Genomic size: 182,940 bp
GC content: 53.50%
Coding region coverage: 85.10%
Protein coding genes: 174

Plasmid 3
Genomic size: 49,961 bp
GC content: 53.88%
Coding region coverage: 82.62%
Protein coding genes: 63

Plasmid 4
Genomic size: 3,204 bp
GC content: 53.75%
Coding region coverage: 80.81%
Protein coding genes: 3

Plasmid 5
Genomic size: 3,035 bp
GC content: 55.78%
Coding region coverage: 36.38%
Protein coding genes: 2

Plasmid 6
Genomic size: 1,815 bp
GC content: 58.57%
Coding region coverage: 53.55%
Protein coding genes: 2

Related links of A. pasteurianus IFO 3283 substrains data

Strain name
IFO 3283-32 IFO 3283-01 IFO 3283-07 IFO 3283-12 IFO 3283-22 IFO 3283-26 IFO 3283-03 IFO 3283-01-42C
NBRC culture catalogue
NBRC No. NBRC 105190 NBRC 105184 NBRC 105187 NBRC 105191 NBRC 105188 NBRC 105189 NBRC 105186 NBRC 105185
INSD, RefSeq
Chromosome DDBJ AP011156 AP011121 AP011135 AP011170 AP011142 AP011149 AP011128 AP011163
RefSeq NC_013209

Plasmid 1 DDBJ AP011157 AP011122 AP011136 AP011171 AP011143 AP011150 AP011129 AP011164
RefSeq NC_013210

Plasmid 2 DDBJ AP011158 AP011123 AP011137 AP011172 AP011144 AP011151 AP011130 AP011165
RefSeq NC_013211

Plasmid 3 DDBJ AP011159 AP011124 AP011138 AP011173 AP011145 AP011152 AP011131 AP011166
RefSeq NC_013212

Plasmid 4 DDBJ AP011160 AP011125 AP011139 AP011174 AP011146 AP011153 AP011132 AP011167
RefSeq NC_013213

Plasmid 5 DDBJ AP011161 AP011126 AP011140 AP011175 AP011147 AP011154 AP011133 AP011167
RefSeq NC_013214

Plasmid 6 DDBJ AP011162 AP011127 AP011141 AP011176 AP011148 AP011155 AP011134 AP011169
RefSeq NC_013215

close this sectionRelated links to external databases