Comparative Genomic Analysis of Closely Related Acetobacter pasteurianus Strains Provides Evidence of Horizontal Gene Transfer and Reveals Factors Necessary for Thermotolerance

Abstract

Acetobacter pasteurianus is an industrial strain used for the vinegar production. Many A. pasteurianus strains with different phenotypic characteristics have been isolated so far. To understand the genetic background underpinning these phenotypes, a comparative genomic analysis of A. pasteurianus strains was conducted. Based on bioinformatics and experimental results, we report the following. (i) The gene repertoire related to the respiratory chains showed that several horizontal gene transfer events occurred after the divergence of these strains, indicating that the respiratory chain in A. pasteurianus has the diversity to adapt to its environment. (ii) There is a clear difference in thermotolerance even between 12 closely related strains. NBRC 3279, NBRC 3284, and NBRC 3283, in particular, which have only 55 mutations in total, showed differences in thermotolerance. The Na⁺/H⁺ antiporter gene nhaK2 was mutated in the thermosensitive NBRC 3279 and NBRC 3284 strains and not in the thermotolerant NBRC 3283 strain. The Na⁺/H⁺ antiporter activity of the three strains and expression of nhaK2 gene from NBRC 3283 in the two thermosensitive strains showed that these mutations are critical for thermotolerance. These results suggested that horizontal gene transfer events and several mutations have affected the phenotypes of these closely related strains.IMPORTANCEAcetobacter pasteurianus, an industrial vinegar-producing strain, exhibits diverse phenotypic differences such as respiratory activity related to acetic acid production, acetic acid resistance, or thermotolerance. In this study, we investigated the correlations between genome sequences and phenotypes among closely related A. pasteurianus strains. The gene repertoire related to the respiratory chains showed that the respiratory components of A. pasteurianus has a diversity caused by several horizontal gene transfers and mutations. In three closely related strains with clear differences in their thermotolerances, we found that the insertion or deletion that occurred in the Na⁺/H⁺ antiporter gene nhaK2 is directly related to their thermotolerance. Our study suggests that a relatively quick mutation has occurred in the closely related A. pasteurianus due to its genetic instability and that this has largely affected its phenotype.

Keywords: acetic acid bacterium; comparative genomics; horizontal gene transfer; thermotolerant strain.

FIG 1

Maximum-likelihood genome-based phylogenetic tree calculated with MUSCLE from 1,579 orthologous nucleotide sequences of A. pasteurianus. The orthologous set was identified as previously reported (13). The gene-support frequency (GSF) is shown (55). The tree was largely separated into four groups (1 to 4), which also correspond to the differences in ANIb values (data not shown). Group 1 was further separated into four subgroups (A to D).

FIG 2

Gene content dendrogram constructed by hierarchical clustering based on the gene content (presence or absence of each protein family) for 22 Acetobacter genomes. Unique clades A and B were defined apart from the genome-based phylogenetic tree (Fig. 1). Other clades correspond to the phylogenetic tree (group 1-A, group 1-C, group 1-D, group 2, group 3, and group 4).

FIG 3

Core and pangenome analysis of 22 closely related A. pasteurianus strains (sequence overlap ≥ 70% and amino acid sequence identity ≥ 80%). (A) Estimate of core genome size with the Tettelin fit (48). (B) Estimate of pangenome size with the Tettelin fit.

FIG 4

Growth comparison of 12 strains determined by using dot spotting at various temperatures on potato medium. NBRC 3284, 3279, and 3283 (subgroup A), NBRC 3278, 3277, and 3280 (subgroup B), SKU1108 (subgroup C), and NBRC 3188, 3191, 106471, and 3222 (subgroup D) belonging to group 1 of A. pasteurianus were assessed (Fig. 1). In addition, NBRC 3299 (group 4) was also used as the outgroup strain. Precultures of these strains were diluted with potato medium to 10⁰, 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴. The diluted samples (7 μl) were dotted onto plates of YPGD medium, followed by incubation at 30, 39, and 41 for 40 h.

FIG 5

Sequence alignment of NhaK2 antiporter homologs of A. tropicalis SKU1100 with three A. pasteurianus strains, NBRC3283, NBRC 3284, and NBRC 3279. Mutated regions (gap 1 and gap 2) were closed up to show the nucleotide sequences.

FIG 6

Comparison of pH change measured at different pH values in ISO membrane vesicles prepared from three A. pasteurianus strains. The pH change was measured at pH 6.5, 7.5, or 8.5 in the presence of 60 mM NaCl (A) or 60 mM KCl (B) and compared with the ISO (40.6, 37.4, 117.8, and 28.5 μg of protein, respectively) prepared from A. tropicalis SKU1100 and A. pasteurianus NBRC 3283, 3284, and 3279.

FIG 7

Growth comparison determined by dot spot analysis at various temperatures on YPGD medium between A. pasteurianus NBRC 3283 and A. pasteurianus 3284 (A) or A. pasteurianus 3279 (B) harboring nhaK2 homolog of NBRC 3283. Precultures were diluted with YPGD medium to 10⁰, 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴. The diluted samples (7 μl) were dotted onto plates of YPGD medium and then incubated at 30, 37, and 39°C. pCM62 is a broad-host-range vector, and pCM83 is pCM62 harboring the nhaK2 gene of NBRC 3283.