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

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.

Genes regulated by branched-chain polyamine in the hyperthermophilic archaeon Thermococcus kodakarensis.

Branched-chain polyamine (BCPA) synthase (BpsA), encoded by the bpsA gene, is responsible for the biosynthesis of BCPA in the hyperthermophilic archaeon Thermococcus kodakarensis, which produces N4-bis(aminopropyl)spermidine and spermidine. Here, next-generation DNA sequencing and liquid chromatography-mass spectrometry (LC-MS) were used to perform transcriptomic and proteomic analyses of a T. kodakarensis strain (DBP1) lacking bpsA. Subsequently, the contributions of BCPA to gene transcription (or transcript stabilization) and translation (or protein stabilization) were analyzed. Compared with those in the wild-type strain (KU216) cultivated at 90 °C, the transcript levels of 424 and 21 genes were up- and downregulated in the DBP1 strain, respectively. The expression levels of 12 frequently-used tRNAs were lower in DBP1 cells than KU216 cells, suggesting that BCPA affects translation efficiency in T. kodakarensis. LC-MS analyses of cells grown at 90 °C detected 50 proteins in KU216 cells only, 109 proteins in DBP1 cells only, and 499 proteins in both strains. Notably, the transcript levels of some genes did not correlate with those of the proteins. RNA-seq and RT-qPCR analyses of ten proteins that were detected in KU216 cells only, including three flagellin-related proteins (FlaB2-4) and cytosolic NiFe-hydrogenase subunit alpha (HyhL), revealed that the corresponding transcripts were expressed at higher levels in DBP1 cells than KU216 cells. Electron microscopy analyses showed that flagella formation was disrupted in DBP1 cells at 90 °C, and western blotting confirmed that HyhL expression was eliminated in the DBP1 strain. These results suggest that BCPA plays a regulatory role in gene expression in T. kodakarensis.

Alkalibacterium gilvum sp. nov., slightly halophilic and alkaliphilic lactic acid bacterium isolated from soft and semi-hard cheeses.

Nine novel strains of halophilic and alkaliphilic lactic acid bacteria isolated from European soft and semi-hard cheeses by using a saline, alkaline medium (7 % NaCl, pH 9.5) were taxonomically characterized. The isolates were Gram-stain-positive, non-sporulating and non-motile. They lacked catalase and quinones. Under anaerobic cultivation conditions, lactate was produced from D-glucose with the production of formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1. Under aerobic cultivation conditions, acetate and lactate were produced from D-glucose. The isolates were slightly halophilic, highly halotolerant and alkaliphilic. The optimum NaCl concentration for growth ranged between 2.0 % and 5.0 % (w/v), with a growth range of 0-1 % to 15-17.5 %. The optimum pH for growth ranged between 8.5 and 9.5, with a growth range of 7.0-7.5 to 9.5-10.0. Comparative sequence analysis of the 16S rRNA genes revealed that the isolates occupied a phylogenetic position within the genus Alkalibacterium, showing the highest sequence similarity (98.2 %) to Alkalibacterium kapii T22-1-2(T). The isolates constituted a single genomic species with DNA-DNA hybridization values of 79-100 % among the isolates and <29 % between the isolates and other members of the genus Alkalibacterium, from which the isolates were different in motility and flagellation, growth responses to NaCl concentrations and pH, and profiles of sugar fermentation. The DNA G+C contents were between 36.0 and 37.6 mol%. The cell-wall peptidoglycan was type A4ß, Orn-D-Asp. The major components of cellular fatty acids were C14 : 0, C16 : 0 and C16 : 1ω9c. Based on the phenotypic characteristics and genetic distinctness, the isolates are classified as a novel species within the genus Alkalibacterium, for which the name Alkalibacterium gilvum sp. nov. is proposed. The type strain is 3AD-1(T) ( = DSM 25751(T) = JCM 18271(T)).

Alkalibacterium subtropicum sp. nov., a slightly halophilic and alkaliphilic marine lactic acid bacterium isolated from decaying marine algae.

Two novel strains of marine lactic acid bacteria, isolated from decaying marine algae collected from a subtropical area of Japan, are described. The isolates, designated O24-2(T) and O25-2, were Gram-positive, non-sporulating and non-motile. They lacked catalase and quinones. Under anaerobic cultivation conditions, lactate was produced from glucose with the production of formate, acetate and ethanol in a molar ratio of approximately 2:1:1. Under aerobic cultivation conditions, acetate and lactate were produced from carbohydrates and related compounds. The isolates were slightly halophilic, highly halotolerant and alkaliphilic. They were able to grow in 0-17.0% (w/v) NaCl, with optimum growth of strains O24-2(T) and O25-2 at 1.0-3.0 and 1.0-2.0% (w/v) NaCl, respectively. Growth of strain O24-2(T) was observed at pH 7.5-9.5, with optimum growth at pH 8.0-8.5. Comparative 16S rRNA gene sequence analysis revealed that the isolates occupied a phylogenetic position within the genus Alkalibacterium, showing highest similarity (99.6%) to Alkalibacterium putridalgicola T129-2-1(T). Although sequence similarity was high, the DNA-DNA relatedness value between strain O24-2(T) and A. putridalgicola T129-2-1(T) was 27%, indicating that they are members of distinct species. The DNA G+C contents of O24-2(T) and O25-2 were 43.7 and 44.4 mol%, respectively, and DNA-DNA relatedness between the isolates was 89%. The cell-wall peptidoglycan was type A4ß, Orn-d-Asp. The major cellular fatty acid components were C(14:0), C(16:0) and C(16:1)ω9c. Based on phenotypic characteristics and genetic distinctiveness, the isolates were classified as representatives of a novel species within the genus Alkalibacterium, for which the name Alkalibacterium subtropicum sp. nov. is proposed; the type strain is O24-2(T) (=DSM 23664(T)=NBRC 107172(T)).

Hydrogen peroxide resistance of Acetobacter pasteurianus NBRC3283 and its relationship to acetic acid fermentation.

The bacterium Acetobacter pasteurianus can ferment acetic acid, a process that proceeds at the risk of oxidative stress. To understand the stress response, we investigated catalase and OxyR in A. pasteurianus NBRC3283. This strain expresses only a KatE homolog as catalase, which is monofunctional and growth dependent. Disruption of the oxyR gene increased KatE activity, but both the katE and oxyR mutant strains showed greater sensitivity to hydrogen peroxide as compared to the parental strain. These mutant strains showed growth similar to the parental strain in the ethanol oxidizing phase, but their growth was delayed when cultured in the presence of acetic acid and of glycerol and during the acetic acid peroxidation phase. The results suggest that A. pasteurianus cells show different oxidative stress responses between the metabolism via the membrane oxidizing pathway and that via the general aerobic pathway during acetic acid fermentation.

Cloning and characterization of the dnaKJ operon in Acetobacter aceti.

The dnaKJ operon of Acetobacter aceti was cloned and sequenced. The profile of the gene configuration was similar to that of other alpha-proteobacteria. In the DnaK and DnaJ proteins of A. aceti, the characteristic domains/motifs reported in other organisms were well conserved. This operon was transcribed in response to a temperature shift and exposure to ethanol/acetic acid. The overexpression of this operon in A. aceti resulted in improved growth compared to the control strain at high temperature or in the presence of ethanol, suggesting a correlation to resistance against stressors present during fermentation, although the overexpression did not increase the resistance to acetic acid.

Cloning and characterization of groESL operon in Acetobacter aceti.

The groESL operon of Acetobacter aceti was cloned and sequenced. We observed that GroES and GroEL of A. aceti had high amino acid sequence homologies to GroES and GroEL of Escherichia coli and Bacillus subtilis. The upstream region of the groESL operon contained the heat-shock promoter, which was previously reported in alpha-purple proteobacteria, and the highly conserved inverted repeat sequence. Phylogenetic analysis revealed that the A. aceti GroES and GroEL are very closely related to those of other alpha-purple proteobacteria. Transcription of this operon in A. aceti was induced by heat shock as well as by exposure to ethanol and acetic acid, which are present during fermentation of acetic acid. A. aceti that overexpressed the groESL was more resistant than the control strain to Stressors such as heat, ethanol, or acetic acid, indicating that GroES and GroEL are closely associated with the characteristic nature of Acetobacter and play an important role in acetic acid fermentation.

Characterization of rpoH in Acetobacter pasteurianus NBRC3283.

The RpoH in Acetobacter pasteurianus NBRC3283 was characterized. It was revealed that the rpoH controls the expression of groEL, dnaKJ, grpE, and clpB to different extents. In addition, the rpoH disruption mutant became apt to be affected by heat, ethanol, and acetic acid, indicating its importance in acetic acid fermentation.

Cloning and characterization of clpB in Acetobacter pasteurianus NBRC 3283.

The clpB gene in Acetobacter pasteurianus was cloned and characterized. Although the clpB gene was transcribed in response to a temperature shift and exposure to ethanol, the clpB disruption mutant was only affected by high temperature, suggesting that the ClpB protein is closely associated with heat resistance in A. pasteurianus.

Cloning and characterization of grpE in Acetobacter pasteurianus NBRC 3283.

The grpE gene in Acetobacter pasteurianus NBRC 3283 was cloned and characterized, to elucidate the mechanism underlying the resistance of acetic acid bacteria to the stressors existing during acetic acid fermentation. This gene was found to be located in tandem with two related genes, appearing on the genome in the order grpE-dnaK-dnaJ. A sigma(32)-type promoter sequence was found in the upstream region of grpE. The relative transcription levels of grpE, dnaK, and dnaJ mRNA were in the ratio of approximately 1:2:0.1, and the genes were transcribed as grpE-dnaK, dnaK, and dnaJ. The transcription level of grpE was elevated by heat shock and treatment with ethanol. Co-overexpression of GrpE with DnaK/J in cells resulted in improved growth compared to the single overexpression of DnaK/J in high temperature or ethanol-containing conditions, suggesting that GrpE acts cooperatively with DnaK/J for expressing resistance to those stressors considered to exist during acetic acid fermentation. Our findings indicate that GrpE is closely associated with adaptation to stressors in A. pasteurianus and may play an important role in acetic acid fermentation.

Alkalibacterium thalassium sp. nov., Alkalibacterium pelagium sp. nov., Alkalibacterium putridalgicola sp. nov. and Alkalibacterium kapii sp. nov., slightly halophilic and alkaliphilic marine lactic acid bacteria isolated from marine organisms and salted foods collected in Japan and Thailand.

We describe 10 new strains of marine lactic acid bacteria isolated from decaying marine algae, decaying seagrass, raw fish, salted fish and salted and fermented shrimp paste ('ka-pi') collected from a temperate area of Japan and Thailand. The isolates are Gram-positive and non-sporulating. They have motility with peritrichous flagella depending on the strains. They lack catalase and quinones. Under anaerobic conditions, lactate yields were 64-93 % of the glucose consumed; residual products were formate, acetate and ethanol with a molar ratio of approximately 2 : 1 : 1. The pH of the fermentation medium markedly affected the product composition; at higher pH, the yield of lactate decreased (15-48 % at pH 9.0) and yields of other products increased, retaining the molar ratio. Under aerobic conditions, acetate and lactate were produced from carbohydrates and related compounds. The isolates were slightly halophilic, highly halotolerant and alkaliphilic. The optimum NaCl concentration for growth ranged between 0.5 and 4.0 % (w/v), depending on the strain, with a growth range of between 0 and 17-21 % (11 % for one isolate). The optimum pH for growth ranged between 8.0 and 9.5, with a growth range of 6.0-11.0, depending on the strains. Comparative sequence analysis of the 16S rRNA genes revealed that the isolates occupied three phylogenetic positions within the genus Alkalibacterium, showing 97.1-99.8 % similarity to Alkalibacterium indicireducens. DNA-DNA hybridization values (<46 %) among the 10 isolates and phylogenetically related taxa resulted in the identification of four genomic species (designated groups GS1-GS4). The G+C contents of the DNA were 41.7 mol% (group GS1), 42.2 mol% (group GS2), 41.0-43.0 mol% (group GS3) and 38.4-39.4 mol% (group GS4). The cell-wall peptidoglycan was type A4beta, Orn-d-Asp, for three genomic species (groups GS1, GS2 and GS3), and type A4beta, Orn-d-Glu, for the remaining species (group GS4). The major components of cellular fatty acids were C(16 : 0), C(16 : 1)omega9c and C(18 : 1)omega9c (oleic acid). On the bases of phenotypic characteristics, genetic distinctiveness and phylogenetic affiliations, the four genomic species, groups GS1, GS2, GS3 and GS4, were classified as four novel species within the genus Alkalibacterium, for which the names Alkalibacterium thalassium sp. nov., Alkalibacterium pelagium sp. nov., Alkalibacterium putridalgicola sp. nov. and Alkalibacterium kapii sp. nov., respectively, are proposed. The respective type strains are T117-1-2(T) (=DSM 19181(T)=NBRC 103241(T)=NRIC 0718(T)), T143-1-1(T) (=DSM 19183(T)=NBRC 103242(T)=NRIC 0719(T)), T129-2-1(T) (=DSM 19182(T)=NBRC 103243(T)=NRIC 0720(T)) and T22-1-2(T) (=DSM 19180(T)=NBRC 103247(T)=NRIC 0724(T)).