Use of rpoB gene phylogenetic marker-based distinction of abiotic stress tolerant and plant-growth promoting Bacillus paralicheniformis isolates from their closely related Bacillus licheniformis

Bacillus paralicheniformis is a new identified species, which was distinguished from Bacillus licheniformis in 2015 through extensive phylogenomic and phylogenetic analyses. In this context, this study aimed to achieve a clear identification of the active plant-growth promoting rhizobacteria (PGPR) B. paralicheniformis isolates among the closely related B. licheniformis through molecular typing, helping for the development of clearly-identified PGPR isolates to be used as biofertilizers. A total of 15 rhizobacteria were isolated from the olive rhizosphere soils. These bacterial isolates exhibited various proprieties in terms of abiotic stress tolerance, biofilm formation under stress conditions, and enzyme activities (i.e., lipases, cellulases, and proteases). In addition, several PGP traits such as phosphate solubilization and the production of siderophores and indol-3 acetic acid were also observed. Molecular identification through 16S rRNA sequencing initially identified all the isolates as Bacillus spp. The multi-locus sequence typing (MLST) scheme, using six housekeeping genes (adk, ccpA, recF, rpoB, sucC, and spo0A) had unveiled distinct allelic profiles resulting in 13 unique sequence types (ST). Notably, a comprehensive analysis indicated no exact allele matches existed between the examined isolates and those documented in the PubMLST database. Among the six housekeeping genes, we noticed that rpoB gene (RNA polymerase, subunit beta) had multiple polymorphic sites that were bordered by conserved sequences. Furthermore, the phylogenetic analysis based on rpoB had identified clusters indicating distinct phylogenetic relationships between B. paralicheniformis and B. licheniformis, and successfully differentiated the two species from a pool of 90 strains. The rpoB partial sequence proved to be effective for accurate species discrimination

Quantification of thermo-halotolerant alkaline protease activity derived from Bacillus licheniformis strains isolated from extreme environments in Morocco

Proteases; especially alkaline proteases, constitute the most used group of enzymes in industry. The wide scope of their application requires varying properties; most commonly stability throughout the conditional changes that would occur during the various industrial processes. This study aimed to identify the potentially interesting bacterial strains obtained from Moroccan extreme environment, and evaluate their proteolytic activity under the various growing conditions. In this study, the impact of temperature and salinity on the alkaline protease activity (pH 11) of 33 Bacillus strains deposited in the Moroccan Coordinated Culture Collection, which originated from the various extreme environments from Morocco, was studied. Bacterial identification to the species level was performed using 16S rRNA gene sequencing and MultiLocus Sequence Typing (MLST), a technique that is much more precise and can identify the bacteria to the strain level. Strain B950 showed a relatively stable protease activity at the tested extreme conditions (i.e., 130.8 U/ ml at 60 °C and 10 % NaCl). On the other hand, strain B961 displayed a better overall activity at either high temperature (140.8 U/ ml) or salinity (137 U/ ml); however, it was not as stable as when grown under both extreme conditions. Regarding identity of the bacterial strains, they were all representatives of Bacillus licheniformis; although B950 and B961 strains belonged to Sequence Types 3 and 5, respectively

Hemicellulosic biomass conversion by Moroccan hot spring Bacillus paralicheniformis CCMM B940 evidenced by glycoside hydrolase activities and whole genome sequencing

International audienceAbstract Thermophilic bacteria, especially from the genus Bacillus , constitute a huge potential source of novel enzymes that could be relevant for biotechnological applications. In this work, we described the cellulose and hemicellulose-related enzymatic activities of the hot spring Bacillus aerius CCMM B940 from the Moroccan Coordinated Collections of Microorganisms (CCMM), and revealed its potential for hemicellulosic biomass utilization. Indeed, B940 was able to degrade complex polysaccharides such as xylan and lichenan and exhibited activity towards carboxymethylcellulose. The strain was also able to grow on agriculture waste such as orange and apple peels as the sole carbon source. Whole-genome sequencing allowed the reclassification of CCMM B940 previously known as B. aerius into Bacillus paralicheniformis since the former species name has been rejected. The draft genome reported here is composed of 38 contigs resulting in a genome of 4,315,004 bp and an average G + C content of 45.87%, and is an important resource for illuminating the molecular mechanisms of carbohydrate metabolism. The annotated genomic sequences evidenced more than 52 genes encoding glycoside hydrolases and pectate lyases belonging to 27 different families of CAZymes that are involved in the degradation of plant cell wall carbohydrates. Genomic predictions in addition to in vitro experiments have revealed broad hydrolytic capabilities of the strain, thus reinforcing its relevance for biotechnology applications

Exploring the hemicellulolytic properties and safety of<i> Bacillus paralicheniformis</i> as stepping stone in the use of new fibrolytic beneficial microbes

Bacillus strains from the Moroccan Coordinated Collections of Microorganisms (CCMM) were characterised and tested for fibrolytic function and safety properties that would be beneficial for maintaining intestinal homeostasis, and recommend beneficial microbes in the field of health promotion research. Forty strains were investigated for their fibrolytic activities towards complex purified polysaccharides and natural fibres representative of dietary fibres (DFs) entering the colon for digestion. We demonstrated hemicellulolytic activities for nine strains of Bacillus aerius, re-identified as Bacillus paralicheniformis and Bacillus licheniformis, using xylan, xyloglucan or lichenan as purified polysaccharides, and orange, apple and carrot natural fibres, with strain- and substrate-dependent production of glycoside hydrolases (GHs). Our combined methods, based on enzymatic assays, secretome, and genome analyses, highlighted the hemicellulolytic activities of B. paralicheniformis and the secretion of specific glycoside hydrolases, in particular xylanases, compared to B. licheniformis. Genomic features of these strains revealed a complete set of GH genes dedicated to the degradation of various polysaccharides from DFs, including cellulose, hemicellulose and pectin, which may confer on the strains the ability to digest a variety of DFs. Preliminary experiments on the safety and immunomodulatory properties of B. paralicheniformis fibrolytic strains were evaluated in light of applications as beneficial microbes' candidates for health improvement. B. paralicheniformis CCMM B969 was therefore proposed as a new fibrolytic beneficial microbe candidate

Exploring the hemicellulolytic properties and safety of Bacillus paralicheniformis as stepping stone in the use of new fibrolytic beneficial microbes

Abstract Bacillus strains from the Moroccan Coordinated Collections of Microorganisms (CCMM) were characterised and tested for fibrolytic function and safety properties that would be beneficial for maintaining intestinal homeostasis, and recommend beneficial microbes in the field of health promotion research. Forty strains were investigated for their fibrolytic activities towards complex purified polysaccharides and natural fibres representative of dietary fibres (DFs) entering the colon for digestion. We demonstrated hemicellulolytic activities for nine strains of Bacillus aerius, re-identified as Bacillus paralicheniformis and Bacillus licheniformis, using xylan, xyloglucan or lichenan as purified polysaccharides, and orange, apple and carrot natural fibres, with strain- and substrate-dependent production of glycoside hydrolases (GHs). Our combined methods, based on enzymatic assays, secretome, and genome analyses, highlighted the hemicellulolytic activities of B. paralicheniformis and the secretion of specific glycoside hydrolases, in particular xylanases, compared to B. licheniformis. Genomic features of these strains revealed a complete set of GH genes dedicated to the degradation of various polysaccharides from DFs, including cellulose, hemicellulose and pectin, which may confer on the strains the ability to digest a variety of DFs. Preliminary experiments on the safety and immunomodulatory properties of B. paralicheniformis fibrolytic strains were evaluated in light of applications as beneficial microbes' candidates for health improvement. B. paralicheniformis CCMM B969 was therefore proposed as a new fibrolytic beneficial microbe candidate