Ruben Ortega Perez

Purification and identification of Calmodulin from Neurospora crassa

The calcium-dependent regulatory protein calmodulin first described [ 1,2] as an activator of the CAMP-phosphodiesterase (PDE) in the bovine brain has been found in a variety of eucaryotic organisms. Calmodulin isolated from some fungi [3-61 and plants [7-91 seems to be similar to mammalian calmodulin [lo]. 1 mM 2-mercaptoethanol, pH 7.0) was used at 1 ml/g mycelia wet wt. The homogenate was filtered through a single layer of nylon cloth, the remaining fragments of mycelia were recovered and retreated in half a volume of extraction buffer. This homogenate was pooled with the first filtrate. The pooled material was centrifuged at 48 000 X g for 30 min in the fixedangle rotor SS-34 with a Sorvall RC-SB centrifuge. All steps were carried out at 4°C. Calmodulin has not yet been reported in Ascomycetes and calmodulin-like activity could not be detected in the yeast Saccharomyces cerevisiae [5]. Here, we describe the use of an affinity chromatography column with an immobilized chlorpromazine derivative [ 1 l] which enabled us to isolate Neurospora crassa calmodulin. It was identified on the basis of its electrophoretic properties, its activation of bovine heart PDE and its absorption spectrum. The resulting supernatant was mixed with DEAESephacel and filtered on a sintered glass funnel by gravity. The filtrate was passed again on the resin and recovered by suction. The bulk of loaded proteins was released by subsequent washings with 100 mM NaCl in the extraction buffer. Further washings with 500 mM NaCl in the same buffer released another mixture of proteins (-15% of the loaded proteins) containing calmodulin. CaClz was added to the eluate to a final concentration of 5 mM. The suspension was clarified by centrifugation at 48 000 X g for 30 min. 2. Materials and methods

Calcium, calmodulin-dependent protein phosphorylation in Neurospora crassa

A calcium, calmodulin‐dependent protein kinase activity has been partially purified by calmodulin‐Sepharose affinity chromatography from the soluble fraction of Neurospora crassa. The phosphorylated peptide has an apparent molecular mass on SDS‐polyacrylamide gel of 47 kDa. The apparent half maximal phosphorylation is obtained after 1.5 min at 30° C in the presence of calcium and calmodulin. The apparent half maximal activation of the phosphorylation is obtained at 1 μM calcium, and 0.1 or 0.2 μM calmodulin from bovine brain or Neurospora, respectively. The 32P incorporation is enhanced about 10‐fold by calmodulin.

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.

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.

Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurosproa crassa

Cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-Sepharose affinity chromatography from a soluble extract of Neurospora crassa. The phosphodiesterase activity remained bound to the affinity column even in the presence of 6 M urea and could only be eluted by calcium chelation. The enzyme exhibits cAMP and cGMP phosphodiesterase activities. Both activities can be enhanced by calmodulin in a Ca2+-dependent manner. Stimulation of cyclic nucleotide phosphodiesterase by calmodulin can be inhibited by calmodulin antagonists such as pimozide, trifluoperazine and chlorpromazine.

Molecular cloning of a cDNA encoding calmodulin from Neurospora crassa

A full‐length cDNA encoding Neurospora crassa calmodulin was isolated from λZAP II cDNA expression library. The open reading frame encodes a protein of 148 amino acid residues with a calculated M r, of 16,865 Da. Using site‐directed mutagenesis, the complete cDNA was ligated into a trc promoter‐regulated bacterial expression vector to allow expression of N. crassa calmodulin in E. coli. The expressed protein was found to be identical to the native protein on the basis of some of its biochemical properties. Finally, Southern analysis of restriction digests of genomic DNA indicates that calmodulin is encoded by a single‐copy gene.

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.

Characterization of Neurospora crassa α-actinin.

Abstractα-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.

Specificity of commercial anti-spectrin antibody in the study of fungal and Oomycete spectrin: cross-reaction with proteins other than spectrin.

Spectrin was first described in erythrocytes where it forms a filamentous network in the cytoplasmic face of the plasma membrane and participates in the membranes structural integrity in addition to controlling the lateral mobility of integral membrane proteins. In fungi, spectrin-like proteins have been described in the plasma membrane, concentrated mainly in the region of maximum apical expansion. This localization led to the idea of a spectrin based membrane skeleton in fungi participating in mechanical integrity of the plasma membrane, generating and maintaining cell polarity. The occurrence of spectrin-like proteins in filamentous fungi, yeasts and Oomycetes, however, is questionable since the presence of such proteins has only been demonstrated with immunochemical methods using antibodies whose specificity is unclear. There is no evidence of a gene coding for the high molecular weight alphabeta-spectrin in the genome of these organisms. Mass spectrometric analysis of the anti alphabeta-spectrin immunoreacting peptides from Neurospora crassa and Phytophthora infestans identified them as elongation factor 2 (NCU07700.4) and Hsp70 (PITG_13237.1), respectively. An attempt was made to correlate the reactivity of anti-spectrin antibody to a common feature of these three proteins i.e., spectrin, elongation factor 2 and heat shock protein 70, in that they all have a hydrophobic region implicated in chaperon activity.