Identification of a New Serine Alkaline Peptidase from the Moderately Halophilic Virgibacillus natechei sp. nov., Strain FarDT and its Application as Bioadditive for Peptide Synthesis and Laundry Detergent Formulations.

A new peptidase designated as SAPV produced from a moderately halophilic Virgibacillus natechei sp. nov., strain FarDT was investigated by purification to homogeneity followed by biochemical and molecular characterization purposes. Through optimization, it was determined that the optimum peptidase activity was 16,000 U/mL. It was achieved after 36 h incubation at 35°C in the optimized enzyme liquid medium (ELM) at pH 7.4 that contains only white shrimp shell by-product (60 g/L) as sole energy and carbon sources. The SAPV enzyme is a monomer protein with a molecular mass of 31 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) gel filtration chromatography. The sequence of its NH2-terminal amino-acid residues showed homology with those of Bacillus peptidases S8/S53 superfamily. The SAPV showed optimal activity at pH 9 and 60°C. Irreversible inhibition of enzyme activity by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine peptidases. Considering its interesting biochemical characterization, the sapV gene was cloned, sequenced, and heterologously overexpressed in the extracellular fraction of E. coli BL21(DE3)pLysS. The biochemical properties of the recombinant peptidase (rSAPV) were similar to those of the native one. The highest sequence identity value (97.66%) of SAPV was obtained with peptidase S8 from Virgibacillus massiliensis DSM 28587, with 9 amino-acid residues of difference. Interestingly, rSAPV showed an outstanding and high resistance to several organic solvents than SPVP from Aeribacillus pallidus VP3 and Thermolysin type X. Furthermore, rSAPV exhibited an excellent detergent stability and compatibility than Alcalase 2.4 L FG and Bioprotease N100L. Considering all these remarkable properties, rSAPV has attracted the interest of industrialists.

Multiresistant opportunistic pathogenic bacteria isolated from polluted rivers and first detection of nontuberculous mycobacteria in the Algerian aquatic environment.

Opportunistic infections constitute a major challenge for modern medicine mainly because the involved bacteria are usually multiresistant to antibiotics. Most of these bacteria possess remarkable ability to adapt to various ecosystems, including those exposed to anthropogenic activities. This study isolated and identified 21 multiresistant opportunistic bacteria from two polluted rivers, located in Algiers. Cadmium, lead, and copper concentrations were determined for both water samples to evaluate heavy metal pollution. High prevalence of Enterobacteria and non-fermentative Gram-negative rods was found and a nontuberculous Mycobacterium (NTM) strain was isolated. To the best of our knowledge, this is the first detection of NTM in the Algerian environment. The strains were tested for their resistance against 34 antibiotics and 8 heavy metals. Multiple antibiotics and heavy metals resistance was observed in all isolates. The two most resistant strains, identified as Acinetobacter sp. and Citrobacter freundii, were submitted to plasmid curing to determine if resistance genes were plasmid or chromosome encoded. Citrobacter freundii strain P18 showed a high molecular weight plasmid which seems to code for resistance to zinc, lead, and tetracycline, at the same time. These findings strongly suggest that anthropized environments constitute a reservoir for multiresistant opportunistic bacteria and for circulating resistance genes.

Assessment of the Antimicrobial Activity and the Entomocidal Potential of Bacillus thuringiensis Isolates from Algeria.

This work represents the first initiative to analyze the distribution of B. thuringiensis in Algeria and to evaluate the biological potential of the isolates. A total of 157 isolates were recovered, with at least one isolate in 94.4% of the samples. The highest Bt index was found in samples from rhizospheric soil (0.48) and from the Mediterranean area (0.44). Most isolates showed antifungal activity (98.5%), in contrast to the few that had antibacterial activity (29.9%). A high genetic diversity was made evident by the finding of many different crystal shapes and various combinations of shapes within a single isolate (in 58.4% of the isolates). Also, over 50% of the isolates harbored cry1, cry2, or cry9 genes, and 69.3% contained a vip3 gene. A good correlation between the presence of chitinase genes and antifungal activity was observed. More than half of the isolates with a broad spectrum of antifungal activity harbored both endochitinase and exochitinase genes. Interestingly, 15 isolates contained the two chitinase genes and all of the above cry family genes, with some of them harboring a vip3 gene as well. The combination of this large number of genes coding for entomopathogenic proteins suggests a putative wide range of entomotoxic activity.

Virgibacillus ainsalahensis sp. nov., a Moderately Halophilic Bacterium Isolated from Sediment of a Saline Lake in South of Algeria.

A Gram-positive, moderately halophilic, endospore-forming bacterium, designated MerVT, was isolated from a sediment sample of a saline lake located in Ain Salah, south of Algeria. The cells were rod shaped and motile. Isolate MerVT grew at salinity interval of 0.5-25% NaCl (optimum, 5-10%), pH 6.0-12.0 (optimum, 8.0), and temperature between 10 and 40 °C (optimum, 30 °C).The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, a glycolipid, a phospholipid, and two lipids, and MK-7 is the predominant menaquinone. The predominant cellular fatty acids were anteiso C15:0 and anteiso C17:0. The DNA G+C content was 45.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain MerVT was most closely related to Virgibacillus halodenitrificans (gene sequence similarity of 97.0%). On the basis of phenotypic, chemotaxonomic properties, and phylogenetic analyses, strain MerVT (=DSM = 28944T) should be placed in the genus Virgibacillus as a novel species, for which the name Virgibacillus ainsalahensis is proposed.

Virgibacillus natechei sp. nov., a moderately halophilic bacterium isolated from sediment of a saline lake in southwest of Algeria.

A novel, Gram-positive, moderately halophilic bacterium, oxidase- and catalase-positive designated FarD(T) was isolated from sediments of a saline lake located in Taghit, 93 km from Bechar, southwest of Algeria. Cells were rod-shaped, endospore forming, and motile. Growth occurred at 15-40 °C (optimum, 35 °C), pH 6.0-12.0 (optimum, 7.0) and in the presence of 1-20 % NaCl (optimum, 10 %). Strain FarD(T) used glucose, mannitol, melibiose, D-mannose, and 5 ketogluconate. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, and three phospholipids; MK-7 is the predominant menaquinone. The predominant cellular fatty acids were anteiso C15:0, anteiso C17:0, C20:0, and anteiso C19:0. The DNA G+C content was 42.1 mol%. Phylogenetic analysis of the small-subunit ribosomal RNA gene sequence indicated that strain FarD(T) had as its closest relative Virgibacillus salinus (similarity of 96.3 %). Based on phenotypic, phylogenetic, and taxonomic characteristics, strain FarD(T) is proposed as a novel species of the genus Virgibacillus within the order Clostridiales, for which the name V. natechei is proposed. The type strain is FarD(T) (=DSM 25609(T) = CCUG 62224(T)).