Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus

Abstract

Acetobacter species have been used for brewing traditional vinegar and are known to have genetic instability. To clarify the mutability, Acetobacter pasteurianus NBRC 3283, which forms a multi-phenotype cell complex, was subjected to genome DNA sequencing. The genome analysis revealed that there are more than 280 transposons and five genes with hyper-mutable tandem repeats as common features in the genome consisting of a 2.9-Mb chromosome and six plasmids. There were three single nucleotide mutations and five transposon insertions in 32 isolates from the cell complex. The A. pasteurianus hyper-mutability was applied for breeding a temperature-resistant strain grown at an unviable high-temperature (42 degrees C). The genomic DNA sequence of a heritable mutant showing temperature resistance was analyzed by mutation mapping, illustrating that a 92-kb deletion and three single nucleotide mutations occurred in the genome during the adaptation. Alpha-proteobacteria including A. pasteurianus consists of many intracellular symbionts and parasites, and their genomes show increased evolution rates and intensive genome reduction. However, A. pasteurianus is assumed to be a free-living bacterium, it may have the potentiality to evolve to fit in natural niches of seasonal fruits and flowers with other organisms, such as yeasts and lactic acid bacteria.

Figure 1.

Mutation accumulation within maintenance of A. pasteurianus IFO 3283 (NBRC 3283) for 21 years. (a) A schematic illustration of the maintenance and generations from 1954 to 1974. (Bottom) Indicates cell multiplication for the first 5 days up to 6.7 generations (a solid line) and decreased cell viability during the slants storage at 5°C for 85 days (a broken line) in a passage of every 3 months. Gs in the panel mean generation numbers. (b) Phenotypic variations in the IFO 3283 (NBRC 3283) cell complex stored in 1974. The top photo shows at least two types of colonies from multi-phenotype cell complex. Two independent clones, (R; rough colony and S; smooth) are shown in the two sets of bottom photos, the transparency (upper) and texture of the surface (middle) of colonies. (Bottom) Shows that R type clones produce a pellicle (biofilm) on the surface of liquid culture, but not S clones.

Figure 2.

Genome map and structure of A. pasteurianus IFO 3283-01 chromosome. (a) The origin of replication was determined based on three values from cumulus of the GC (blue) and keto (green) skews, and transcription directions (red), and shown as ori (47). The locations of orthologs are shown by linking between A. pasteurianus and G. oxydans (11). (b) Genes in the chromosome are mapped after gene categorization into nine groups such as cell structure, replication and so on. Transcription and stress are indicated in black and red, respectively. Genes in translation are separated into protein coding in blue, and 52 tRNA in green and 15 rRNA in red. Numbers next to the categories are the number of genes in the chromosome and whole genome on the left and right, respectively. Genes in ‘common’ of metabolism means that its orthologs are found in both genomes of Gluconobacter oxydans (11) and Granulibacter bethesdensis (12). The mutation loci identified in this work are also mapped. Genome variation by three SNPs in red, four transposon insertions in blue and five HTRs in green, and terminal deletion in red of the 42°C evolved strain.

Figure 3.

Mutation accumulation within maintenance of A. pasteurianus IFO 3283 (NBRC 3283) for 21 years. (a) Genotype lineage (chromosome) and phenotypes of 32 substrains of the IFO 3283 multi-phenotype complex is summarized as an illustration based on Table 1. S and R indicate smooth and rough in the colony textures. (b) Profiles of neutral sugar content of the representing substrains measured in liquid and plate media. (Top and bottom) Show the amounts of neutral sugar in liquid and plate culture, respectively. White and gray bars indicate the amounts of soluble and insoluble neutral sugar, respectively. Vertical error bars show 1 SD calculated from at least four independent experiments and asterisk means significantly different in sugar production (P < 0.02).

Figure 4.

Features of A. pasteurianus hyper-mutability. Proportion of transposase genes in the total number of genes (vertical) and DNA fragment number of the genome including chromosomes and plasmids (horizontal) are plotted using each genome of 777 bacterial species with gene sequences in the public database (ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Bacteria). Blue and purple spots indicate the proportion for A. pasteurianus IFO 3283-01 and other AAB, respectively. Remarkable bacterial in this plotting, such as Borrelia (60), Shigera (61), Xanthomonas (62) and Orientia (63), are indicated in orange, red, light green and green. Profiles for other bacteria are shown in black, and those close to A. pasteurianus were indicated by bars, such as Aliivibrio, Acinetobacter and Lactococcus.

Figure 5.

A schematic illustration of the structures of gene products with hyper-mutable tandem repeat sequences. Products of the genes with AP-TR04 are shown on top. Similar DNA helicases in the largest plasmid (BABN01000001) of Gluconacetobacter xylinus NBRC 3288, Azotobacter vinelandii DJ (65), Pseudomonas syringae pv. tomato str. DC3000 (NC-004578) and Myxococcus xanthus DK 1622 (NC-008095) are shown below. N-terminal regions in gray indicate a conserved DNA helicase domain. Tandem repeat regions are shown in green with strips for A. pasteurianus and G. xylinus and in white stripes for an A. vinelandii homolog. Amino acid sequences in C-terminal regions indicated in blue, yellow and red are encoded by same DNA sequences but different frame based on repetitive number of the AP-TR04 in A. pasteurianus. Amino acid sequences in C-terminal region in blue from four species are similar to each other. Gene locations, chromosome and plasmid, are indicated as chr and pld, respectively.

Figure 6.

Adaptation to an unviable environment at high-temperatures. (a) Growth profiles of A. pasteurianus IFO 3283-01 in the successful case for adaptation. Green, blue and red curves indicate cell growth at 30, 40 and 42°C, respectively. Three arrows, 40C(3), 40C(5) and 42C(1), indicate the cell cultures analyzed for mutations. (b) A heritability analysis of growth at 42°C. The seed strain (IFO 3283-01), the bred substrain (IFO 3283-01/42°C) and a different cell type strain (IFO32383-02) were inoculated on same plate and tested for growth at 30°C and 42°C. Cells grown on a 30°C plate were then inoculated onto the next two plates for growth at 30°C and 42°C. (c) A mapping analysis of sequencing reads on the chromosome of IFO 3283-01/42°C (top). Vertical and horizontal axes indicate chromosome location and frequency of high-quality sequence reads in 1000 bases. PCR analyses were carried out for the large deletion region and positions of primer pairs for PCR analyses were shown in the middle. DNA was used from the clone IFO 3283-01-42C isolated from the culture 42C(1) indicated by the right arrow in the panel a. Gray zones and gray arrows in the middle panel indicate existence of the region in the genomic DNA, while white areas, no-mapped regions and white arrow show the region deleted from the genome. PCR products analyzed in agarose gel electrophoresis were shown in the bottom. O and A on the top of each lane designate DNA templates for PCR prepared from the original (IFO 3283-01) and the adapted strain (IFO 3283-01-42C), respectively. (d) A summary of the mutations found in the adapted strain (IFO 3283-01-42C). Original and mutated sequences are shown in capital and small letters, respectively.