Distribution and genome structures of temperate phages in acetic acid bacteria

Abstract

Acetic acid bacteria (AAB) are industrial microorganisms used for vinegar fermentation. Herein, we investigated the distribution and genome structures of mitomycin C-inducible temperate phages in AAB. Transmission electron microscopy analysis revealed phage-like particles in 15 out of a total 177 acetic acid bacterial strains, all of which showed morphology similar to myoviridae-type phage. The complete genome sequences of the six phages derived from three strains each of Acetobacter and Komagataeibacter strains were determined, harboring a genome size ranging from 34,100 to 53,798 bp. A phage AP1 from A. pasteurianus NBRC 109446 was predicted as an active phage based on the genomic information, and actually had the ability to infect its phiAP1-cured strain. The attachment sites for phiAP1 were located in the 3'-end region of the tRNA^ser gene. We also developed a chromosome-integrative vector, p2096int, based on the integrase function of phiAP1, and it was successfully integrated into the attachment site of the phiAP1-cured strain, which may be used as a valuable tool for the genetic engineering. Overall, this study showed the distribution of mitomycin C-inducible temperate phages in AAB, and identified the active temperate phage o f A. pasteurianus.

Figure 1

TEM photographs of temperate phages produced by acetic acid bacteria. Phage particles of acetic acid bacteria (AAB) were photographed with TEM. Fractions were prepared from AAB grown in YPG-1% glucose medium containing mitomycin C (MMC) with shaking culture for 48 h. Bars, 50 nm.

Figure 2

Schematic representation of temperate phage genomes in AAB. (A) Genome structure and gene organization of the temperate phages determined in this study. Number above arrow represent ORF number in each temperate phage (annotations for each gene product are shown in Table S4). Numbers in parentheses indicate the genome size and structure of the phages. (B) Distribution of phiAP1-like prophage elements in the genomes of A. pasteurianus strains. Downward arrowheads indicate the putative attB-L and attB-R sites.

Figure 3

Nucleotide sequences of att sites in A. pasteurianus NBRC 109446 and phiAP1. (A) Bacterial host att sites (attB-L and attB-R) underlined are located in the genomic boundary region between the host and phiAP1. The tRNA^ser gene is shown in red letters. (B) The attB site in the phiAP1-cured C-27 strain is underlined. The tRNA^ser gene is shown in red letters. (C) Comparison of the nucleotide sequences of the att sites with a length of 24 bp.

Figure 4

Gene organization of tRNA^ser gene locus in Acetobacter spp. The gene compositions and arrangements located in the tRNA^ser locus of Acetobacter spp. are shown. The upstream region of tRNA^ser was highly conserved among the genus of Acetobacter. Arrowheads indicate the putative attB sites. Genes for tRNA^ser and integrase are indicated by red and blue-colored arrowheads, respectively.

Figure 5

Multiple alignments of attB sites in Acetobacter spp. Nucleotide sequences of the tRNA^ser gene and its 3’ flanking region were compared with CLUSTAL W (see Materials and Methods). Anticodon (TGA) was located in the position 35 to 37 bp. The coding sequences of the tRNA^ser gene are shown by the line on top of the alignment. Putative attB-L and attB-R are shown in red text. N.D., not detected. Asterisks, colons, and periods are indicative of completely, highly, and semi-conserved nucleotide sequences, respectively.

Figure 6

Infective ability of phiAP1 to the A. pasteurianus C-27 strain. The phiAP1 and phiAP1-Amp-containing fractions were prepared from A. pasteurianus NBRC 109446 wild-type and GMS3 strains cultured with YPG-1% glucose containing MMC. As a recipient cell, the C-27 strain grown in YPG-1% glucose was used. The phage-infected C-27 strains were selected on YPG-1% glucose containing ampicillin at 30 °C for 3 days.

Figure 7

Plasmid map of a chromosome-integrative vector p2096int. p2096int vector contains attP, the integrase gene, multiple cloning sites (MCS), lac promoter, and an ampicillin-resistance gene. The vector is a pUC-based multicopy number plasmid propagated in E. coli, while it is a chromosome-integrative vector in the A. pasteurianus C-27 strain.