Morio Ishikawa

Presence of halophilic and alkaliphilic lactic acid bacteria in various cheeses.

Aim:  We sought to confirm the presence of halophilic and alkaliphilic lactic acid bacteria (HALAB) of marine origin in cheeses and thus contribute to the understanding of the roles of LAB flora in cheese ripening.

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.

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 A4β, 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)).

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.

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.

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.

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 A4β, 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.

Physiology of Acetobacter spp.: Involvement of Molecular Chaperones During Acetic Acid Fermentation

Acetic acid bacteria produce acetic acid from ethanol to acquire energy through a process called acetic acid fermentation. These bacteria are inevitably exposed to various stressors during fermentation but have developed resistance to these stressors. These resistance properties have been attributed to the combination of several kinds of mechanisms, including the role of molecular chaperones. In this chapter, we outline the involvement of major molecular chaperones (GroESL, DnaKJ, GrpE, and ClpB) in the stress-resistant abilities of Acetobacter pasteurianus NBRC3283, in addition to the role of the regulatory factor RpoH, with reference to our recent studies using proteomic analyses, RNA-seq analyses, and the mutants of these chaperones.