A novel N(alpha)-acetyl alanine aminopeptidase from Allomyces arbuscula.

An N(alpha)-acetyl alanine aminopeptidase has been purified from the aquatic fungus Allomyces arbuscula. The apparent molecular mass of the enzyme was estimated to be 280 kDa by gel filtration through calibrated Sephacryl S300 column. In SDS-PAGE, the purified enzyme appeared as a single band of M(r) 80 kDa. Catalytic activity of the enzyme was inhibited by specific serine protease inhibitors, 3,4-DCI and APMSF, as well as SH reacting compounds, HgCl(2) and iodoacetate, indicating that the enzyme is a serine protease with some functional SH group(s) involved in the catalytic reaction. 3H-DFP was used to label the reactive serine of the enzyme. When the labeled protein was analyzed in SDS-PAGE, most of the label appeared in the M(r) 80 kDa band, however, a few additional faster migrating minor bands were also seen, probably representing a minor degradation product of the enzyme. The enzyme cleaved mainly N(alpha)-acetlylated alanine, although a small but negligible activity was also obtained with acetylated leucine and phenylalanine. The role of the enzyme in N-end rule proteolysis is discussed.

Geodermatophilus tzadiensis sp. nov., a UV radiation-resistant bacterium isolated from sand of the Saharan desert.

Three novel Gram-positive, aerobic, actinobacterial strains, CF5/2(T), CF5/1 and CF7/1, were isolated in 2007 during environmental screening of arid desert soil in the Sahara desert, Chad. Results from riboprinting, MALDI-TOF protein spectra and 16S rRNA sequence analysis confirmed that all three strains belonged to the same species. Phylogenetic analysis of 16S rRNA sequences with the strains' closest relatives indicated that they represented a distinct species. The three novel strains also shared a number of physiological and biochemical characteristics distinct from previously named Geodermatophilus species. The novel strains' peptidoglycan contained meso-diaminopimelic acid; their main phospholipids were phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and a small amount of phosphatidylglycerol; MK-9(H4) was the dominant menaquinone. The major cellular fatty acids were the branched-chain saturated acids iso-C16:0 and iso-C15:0. Galactose was detected as diagnostic sugar. Based on these chemotaxonomic results, 16S rRNA gene sequence analysis and DNA-DNA hybridization between strain CF5/2(T) and the type strains of Geodermatophilus saharensis, Geodermatophilus arenarius, Geodermatophilus nigrescens, Geodermatophilus telluris and Geodermatophilus siccatus, the isolates CF5/2(T), CF5/1 and CF7/1 are proposed to represent a novel species, Geodermatophilus tzadiensis, with type strain CF5/2(T)=DSM 45416=MTCC 11411 and two reference strains, CF5/1 (DSM 45415) and CF7/1 (DSM 45420).

Microbial hitchhikers on intercontinental dust: catching a lift in Chad.

Ancient mariners knew that dust whipped up from deserts by strong winds travelled long distances, including over oceans. Satellite remote sensing revealed major dust sources across the Sahara. Indeed, the Bodélé Depression in the Republic of Chad has been called the dustiest place on earth. We analysed desert sand from various locations in Chad and dust that had blown to the Cape Verde Islands. High throughput sequencing techniques combined with classical microbiological methods showed that the samples contained a large variety of microbes well adapted to the harsh desert conditions. The most abundant bacterial groupings in four different phyla included: (a) Firmicutes-Bacillaceae, (b) Actinobacteria-Geodermatophilaceae, Nocardiodaceae and Solirubrobacteraceae, (c) Proteobacteria-Oxalobacteraceae, Rhizobiales and Sphingomonadaceae, and (d) Bacteroidetes-Cytophagaceae. Ascomycota was the overwhelmingly dominant fungal group followed by Basidiomycota and traces of Chytridiomycota, Microsporidia and Glomeromycota. Two freshwater algae (Trebouxiophyceae) were isolated. Most predominant taxa are widely distributed land inhabitants that are common in soil and on the surfaces of plants. Examples include Bradyrhizobium spp. that nodulate and fix nitrogen in Acacia species, the predominant trees of the Sahara as well as Herbaspirillum (Oxalobacteraceae), a group of chemoorganotrophic free-living soil inhabitants that fix nitrogen in association with Gramineae roots. Few pathogenic strains were found, suggesting that African dust is not a large threat to public health.

Structure, expression and function of Allomyces arbuscula CDP II (metacaspase) gene.

Allomyces arbuscula, a primitive chytridiomycete fungus, has two Ca(2+)-dependent cysteine proteases, the CDP I and CDP II. We have cloned and analyzed the nucleotide sequence of CDP II gene and domain structure of the protein. Blast analysis of the sequence has shown that the protein belongs to a newly described member of caspase superfamily protein, the metacaspase, a CD clan of C14 family cysteine protease, we hence-forth name it as AMca 2 (Allomyces metacaspase 2). Southern hybridization studies have shown that the gene exists in a single copy per genome. The transcriptional analysis by Northern hybridization has confirmed our previous results that the protein is developmentally regulated, i.e. present in active growth phase but disappears during nutritional stress which also induces reproductive differentiation, indicating that the protein promotes cell growth, not death. The recombinant gene product expressed in Escherichiacoli has all the catalytic properties of native enzyme, i.e. sensitivity to protease inhibitors and substrate specificity. There is an absolute requirement of Ca(2+) for the activation of catalytic activity and the presence of R residue at the cleavage site (P1 position) in the substrate. The presence of a second basic residue, either R or K, in the P2 position strongly inhibits the catalytic activity which is stimulated by the presence of P and to a lesser extent G at this site. Peptide substrates with D at the cleavage site are not recognised and therefore not cleaved. The enzyme activity is inhibited by EDTA-EGTA, cysteine protease inhibitors and a specific peptide inhibitor Ac GVRCHCL TFA, but not by E64, although a potent inhibitor of cysteine proteases.