Metaproteomics and ultrastructure characterization of Komagataeibacter spp. involved in high-acid spirit vinegar production.

Acetic acid bacteria (AAB) are widespread microorganisms in nature, extensively used in food industry to transform alcohols and sugar alcohols into their corresponding organic acids. Specialized strains are used in the production of vinegar through the oxidative transformation of ethanol into acetic acid. The main AAB involved in the production of high-acid vinegars using the submerged fermentation method belong to the genus Komagataeibacter, characterized by their higher ADH stability and activity, and higher acetic acid resistance (15-20%), compared to other AAB. In this work, the bacteria involved in the production of high-acid spirit vinegar through a spontaneous acetic acid fermentation process was studied. The analysis using a culture-independent approach revealed a homogeneous bacterial population involved in the process, identified as Komagataeibacter spp. Differentially expressed proteins during acetic acid fermentation were investigated by using 2D-DIGE and mass spectrometry. Most of these proteins were functionally related to stress response, the TCA cycle and different metabolic processes. In addition, scanning and transmission electron microscopy and specific staining of polysaccharide SDS-PAGE gels confirmed that Komagataeibacter spp. lacked the characteristic polysaccharide layer surrounding the outer membrane that has been previously reported to have an important role in acetic acid resistance in the genus Acetobacter.

Genome sequences of the high-acetic acid-resistant bacteria Gluconacetobacter europaeus LMG 18890T and G. europaeus LMG 18494 (reference strains), G. europaeus 5P3, and Gluconacetobacter oboediens 174Bp2 (isolated from vinegar).

Bacteria of the genus Gluconacetobacter are usually involved in the industrial production of vinegars with high acetic acid concentrations. We describe here the genome sequence of three Gluconacetobacter europaeus strains, a very common bacterial species from industrial fermentors, as well as of a Gluconacetobacter oboediens strain.

Characterization of Neurospora crassa α-actinin.

α-Actinin, an actin-binding protein of the spectrin superfamily, is present in most eukaryotes except plants. It is composed of three domains: N-terminal CH-domains, C-terminal calcium-binding domain (with EF-hand motifs), and a central rod domain. We have cloned and expressed Neurospora crassa α-actinin as GST and GFP fusion proteins for biochemical characterization and in vivo localization, respectively. The intracellular localization pattern of α-actinin suggests that this protein is intimately associated with actin filaments and plays an important role in the processes of germination, hyphal elongation, septum formation, and conidiation. These functions were confirmed by the experiments on the effect of α-actinin gene deletion in N. crassa.

Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting.

Acetic acid bacteria (AAB) are widespread microorganisms characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. The suitability of matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) for the identification of cultured AAB involved in the industrial production of vinegar was evaluated on 64 reference strains from the genera Acetobacter, Gluconacetobacter and Gluconobacter. Analysis of MS spectra obtained from single colonies of these strains confirmed their basic classification based on comparative 16S rRNA gene sequence analysis. MALDI-TOF analyses of isolates from vinegar cross-checked by comparative sequence analysis of 16S rRNA gene fragments allowed AAB to be identified, and it was possible to differentiate them from mixed cultures and non-AAB. The results showed that MALDI-TOF MS analysis was a rapid and reliable method for the clustering and identification of AAB species.

Proteome analysis of Acetobacter pasteurianus during acetic acid fermentation.

Acetic acid bacteria (AAB) are Gram-negative, strictly aerobic microorganisms that show a unique resistance to ethanol (EtOH) and acetic acid (AcH). Members of the Acetobacter and Gluconacetobacter genera are capable of transforming EtOH into AcH via the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes and are used for the industrial production of vinegar. Several mechanisms have been proposed to explain how AAB resist high concentrations of AcH, such as the assimilation of acetate through the tricarboxylic acid (TCA) cycle, the export of acetate by various transporters and modifications of the outer membrane. However, except for a few acetate-specific proteins, little is known about the global proteome responses to AcH. In this study, we used 2D-DIGE to compare the proteome of Acetobacter pasteurianus LMG 1262(T) when growing in glucose or ethanol and in the presence of acetic acid. Interesting protein spots were selected using the ANOVA p-value of 0.05 as threshold and 1.5-fold as the minimal level of differential expression, and a total of 53 proteins were successfully identified. Additionally, the size of AAB was reduced by approximately 30% in length as a consequence of the acidity. A modification in the membrane polysaccharides was also revealed by PATAg specific staining.