Identification of catabolic pathway for 1-deoxy-D-sorbitol in Bacillus licheniformis.

1-Deoxy-D-sorbitol, the 1-deoxy analogue of D-sorbitol, has been detected in human urine as well as in natural herbs and spices. Although there are sporadic reports about 1-deoxy-D-sorbitol dehydrogenase, the complete catabolic pathway of 1-deoxy-D-sorbitol remains unsolved. Informed by the promiscuous activities of fructose-6-phosphate aldolase (FSA) which is involved in the sorbitol (glucitol) utilization (gut) operon and guided by the large scale bioinformatics analysis, we predicted and then experimentally verified the gut operon encoded by Bacillus licheniformis ATCC14580 is responsible for the catabolism of both D-sorbitol and 1-deoxy-D-sorbitol by in vitro activity assays of pathway enzymes, in vivo growth phenotypes, and transcriptomic studies. Moreover, the phylogenetic distribution analysis suggests that the D-sorbitol and 1-deoxy-D-sorbitol catabolic gene cluster is mostly conserved in members of Firmicutes phylum.

A comparative GC-MS analysis of bioactive secondary metabolites produced by halotolerant Bacillus spp. isolated from the Great Sebkha of Oran.

The reemergence of infectious diseases and resistant pathogens represents a serious problem for human life. Hence, the screening for new or alternative antimicrobial compounds is still urgent. Unusual ecosystems such as saline habitats are considered promising environments for the purposes of isolating bacterial strains able to produce potent natural products. The aim of this study is the identification of bioactive compounds biosynthesized by three halotolerant strains isolated from the Sebkha of Oran (Algeria) using gas chromatography coupled to mass spectrometry. Primary screening investigation of antimicrobial activities were performed against reference bacterial and fungal strains and revealed a broad-spectrum activity. Secondary metabolite extraction was carried out using ethyl acetate and chloroform. Crude extracts were tested for bioactivity using the disc diffusion method and subjected to GC-MS analysis. The extracts showed an important inhibitory effect against all tested strains. Fifty-six compounds were identified; they include tert-butyl phenol compounds, fatty acid methyl esters due to the methylation procedure, hydrocarbons, aldehydes, benzoquinones, pyrrols, and terpenes. Literature reports such compounds to have wide biological and pharmaceutical applications. The molecular identification of the three isolates was achieved using the 16S-23S rRNA gene intergenic spacer region (ITS) and 16S rRNA sequencing. Partial 16S rRNA gene sequencing showed very high similarity with many species of Bacillus. This study provided insights on the potential of halotolerant Bacillus as drug research target for bioactive metabolites. The findings suggest that the Great Sebkha of Oran is a valuable source of strains exhibiting variety of beneficial attributes that can be utilized in the development of biological antibiotics.

Fructanogenic traits in halotolerant Bacillus licheniformis OK12 and their predicted functional significance.

AIMS: Isolating a novel bacterial source of fructan from a saltern and analysis of its genome to better understand the possible roles of fructans in hypersaline environments. METHODS AND RESULTS: Bacteria were isolated from crude salt samples originating from Çamalti Saltern in Western Turkey and screened for fructanogenic traits in high-salt and sucrose-rich selective medium. Exopolysaccharide accumulated in the presence of sucrose by isolate OK12 was purified and chemically characterized via HPLC, FT-IR and NMR, which revealed that it was a levan-type fructan (ß-2,6 linked homopolymer of fructose). The isolate was taxonomically classified as Bacillus licheniformis OK12 through 16S rRNA gene and whole-genome sequencing methods. Strain OK12 harbours one levansucrase and two different levanase genes, which altogether were predicted to significantly contribute to intracellular glucose and fructose pools. The isolate could withstand 15% NaCl, and thus classified as a halotolerant. CONCLUSIONS: Fructanogenic traits in halotolerant B. licheniformis OK12 are significant due to predicted influx of glucose and fructose as a result of levan biosynthesis and levan hydrolysis, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Fructans from the residents of hypersaline habitats are underexplored compounds and are expected to demonstrate physicochemical properties different from their non-halophilic counterparts. Revealing fructanogenic traits in the genome of a halotolerant bacterium brings up a new perspective in physiological roles of fructans.

A new maltogenic amylase from Bacillus licheniformis R-53 significantly improves bread quality and extends shelf life.

Maltogenic amylase suppressed starch retrogradation in baked products. Here, a maltogenic amylase-producing strain of bacteria was screened and identified as Bacillus licheniformis R-53. Its coding gene was cloned and over-expressed in Bacillus subtilis WB600. Recombinant maltogenic amylase BLMA exhibited activity of 3235 U/mg under optimal conditions (60 °C and pH 6.5), with a good thermostability and pH stability. Mixolab experiment showed that a concentration of 60 ppm BLMA significantly improved the operating characteristics of dough. Baking test indicated the recombinant BLMA reduced bread hardness by 2.12 times compared with the control. Compared with maltogenic amylase from Novozymes (Novamyl 3D BG) and Angel Yeast Co. Ltd. (MAM100), BLMA has better effect on improving the bread volume, and almost the same effect on reducing hardness, improving elasticity and maintaining sensory as Novamyl 3D BG. Adding BLMA improved bread quality, increased bread volume and decreased hardness during storage, thus extending its shelf life.

Two genes involved in clindamycin resistance of Bacillus licheniformis and Bacillus paralicheniformis identified by comparative genomic analysis.

We evaluated the minimum inhibitory concentrations of clindamycin and erythromycin toward 98 Bacillus licheniformis strains isolated from several types of fermented soybean foods manufactured in several districts of Korea. First, based on recent taxonomic standards for bacteria, the 98 strains were separated into 74 B. licheniformis strains and 24 B. paralicheniformis strains. Both species exhibited profiles of erythromycin resistance as an acquired characteristic. B. licheniformis strains exhibited acquired clindamycin resistance, while B. paralicheniformis strains showed unimodal clindamycin resistance, indicating an intrinsic characteristic. Comparative genomic analysis of five strains showing three different patterns of clindamycin and erythromycin resistance identified 23S rRNA (adenine 2058-N6)-dimethyltransferase gene ermC and spermidine acetyltransferase gene speG as candidates potentially involved in clindamycin resistance. Functional analysis of these genes using B. subtilis as a host showed that ermC contributes to cross-resistance to clindamycin and erythromycin, and speG confers resistance to clindamycin. ermC is located in the chromosomes of strains showing clindamycin and erythromycin resistance and no transposable element was identified in its flanking regions. The acquisition of ermC might be attributable to a homologous recombination. speG was identified in not only the five genome-analyzed strains but also eight strains randomly selected from the 98 test strains, and deletions in the structural gene or putative promoter region caused clindamycin sensitivity, which supports the finding that the clindamycin resistance of Bacillus species is an intrinsic property.

Algicidal characterization and mechanism of Bacillus licheniformis Sp34 against Microcystis aeruginosa in Dianchi Lake.

Microcystis aeruginosa blooms are a worldwide serious environmental problem and bloom control with bacteria is promising. In this study, a Bacillus licheniformis strain Sp34 with potent algicidal and inhibitory effects on the microcystins synthesis against fast-growing M. aeruginosa was isolated from Dianchi Lake. Sp34 killed the bloom-causing algal strain M. aeruginosa DCM4 of Dianchi Lake with an initial Chlorophyll-a concentration of 2.0 mg/L at a cell density of no less than 1.35 × 105 CFU/ml. It can also efficiently kill some other harmful algal species, such as M. wesenbergii and Phormidium sp. The algicidal activity of Sp34 relied on the release of algicidal substances, which had good heat (-20°C to 121°C) and acid-base (pH 3-11) resistance. In addition, the high algicidal activity depended on the good growth of algae indicated by the significantly positive correlations between algal growth and algicidal ratio (p < .001). The algicidal effect of Sp34 involved causing oxidative stress, lipid peroxidation, and morphological injury of algal cells, along with DNA damage and dysfunction of DNA-repair function, weakening the photosynthesis system, and inhibiting microcystin synthesis. In general, Sp34 can kill fast-growing M. aeruginosa and inhibit algal microcystin synthesis efficiently, so, it is a promising biocontrol agent to mitigate cyanobacterial blooms.

Production and Anticancer Activity of an L-Asparaginase from Bacillus licheniformis Isolated from the Red Sea, Saudi Arabia.

Microbial L-asparaginase (ASNase) is an important anticancer agent that is used extensively worldwide. In this study, 40 bacterial isolates were obtained from the Red Sea of Saudi Arabia and screened for ASNase production using a qualitative rapid plate assay, 28 of which were producing large L-asparagine hydrolysis zones. The ASNase production of the immobilized bacterial cells was more favorable than that of freely suspended cells. A promising isolate, KKU-KH14, was identified by 16S rRNA gene sequencing as Bacillus licheniformis. Maximal ASNase production was achieved using an incubation period of 72 h, with an optimum of pH 6.5, an incubation temperature of 37 °C, an agitation rate 250 rpm, and with glucose and (NH4)2SO4 used as the carbon and nitrogen sources, respectively. The glutaminase activity was not detected in the ASNase preparations. The purified ASNase showed a final specific activity of 36.08 U/mg, and the molecular weight was found to be 37 kDa by SDS-PAGE analysis. The maximum activity and stability of the purified enzyme occurred at pH values of 7.5 and 8.5, respectively, with maximum activity at 37 °C and complete thermal stability at 70 °C for 1 h. The Km and Vmax values of the purified enzyme were 0.049995 M and of 45.45 µmol/ml/min, respectively. The anticancer activity of the purified ASNase showed significant toxic activity toward HepG-2 cells (IC50 11.66 µg/mL), which was greater than that observed against MCF-7 (IC50 14.55 µg/mL) and HCT-116 cells (IC50 17.02 µg/mL). The results demonstrated that the Red Sea is a promising biological reservoir, as shown by the isolation of B. licheniformis, which produces a glutaminase free ASNase and may be a potential candidate for further pharmaceutical use as an anticancer drug.

Biosurfactant-Producing Capability and Prediction of Functional Genes Potentially Beneficial to Microbial Enhanced Oil Recovery in Indigenous Bacterial Communities of an Onshore Oil Reservoir.

Microbial enhanced oil recovery (MEOR) is a bio-based technology with economic and environmental benefits. The success of MEOR depends greatly on the types and characteristics of indigenous microbes. The aim of this study was to evaluate the feasibility of applying MEOR at Mae Soon Reservoir, an onshore oil reservoir experiencing a decline in its production rate. We investigated the capability of the reservoir's bacteria to produce biosurfactants, and evaluated the potentials of uncultured indigenous bacteria to support MEOR by means of prediction of MEOR-related functional genes, based on a set of metagenomic 16s rRNA gene data. The biosurfactant-producing bacteria isolated from the oil-bearing sandstones from the reservoir belonged to one species: Bacillus licheniformis, with one having the ability to decrease surface tension from 72 to 32 mN/m. Gene sequences responsible for biosurfactant (licA3), lipase (lipP1) and catechol 2,3-dioxygenase (C23O) were detected in these isolates. The latter two, and other genes encoding MEOR-related functional proteins such as enoyl-CoA hydratase and alkane 1-monooxygenase, were predicted in the bacterial communities residing the reservoir's sandstones. Exposure of these sandstones to nutrients, consisting of KNO3 and NaH2PO4, resulted in an increase in the proportions of some predicted functional genes. These results indicated the potentials of MEOR application at Mae Soon site. Using the approaches demonstrated in this study would also assist evaluation of the feasibility of applying MEOR in oil reservoirs, which may be enhanced by an appropriate nutrient treatment.

Putative antibiotic resistance genes present in extant Bacillus licheniformis and Bacillus paralicheniformis strains are probably intrinsic and part of the ancient resistome.

Whole-genome sequencing and phenotypic testing of 104 strains of Bacillus licheniformis and Bacillus paralicheniformis from a variety of sources and time periods was used to characterize the genetic background and evolution of (putative) antimicrobial resistance mechanisms. Core proteins were identified in draft genomes and a phylogenetic analysis based on single amino acid polymorphisms allowed the species to be separated into two phylogenetically distinct clades with one outlier. Putative antimicrobial resistance genes were identified and mapped. A chromosomal ermD gene was found at the same location in all B. paralichenformis and in 27% of B. licheniformis genomes. Erythromycin resistance correlated very well with the presence of ermD. The putative streptomycin resistance genes, aph and aadK, were found in the chromosome of all strains as adjacent loci. Variations in amino acid sequence did not correlate with streptomycin susceptibility although the species were less susceptible than other Bacillus species. A putative chloramphenicol resistance gene (cat), encoding a novel chloramphenicol acetyltransferase protein was also found in the chromosome of all strains. Strains encoding a truncated CAT protein were sensitive to chloramphenicol. For all four resistance genes, the diversity and genetic context followed the overall phylogenetic relationship. No potentially mobile genetic elements were detected in their vicinity. Moreover, the genes were only distantly related to previously-described cat, aph, aad and erm genes present on mobile genetic elements or in other species. Thus, these genes are suggested to be intrinsic to B. licheniformis and B. paralicheniformis and part of their ancient resistomes. Since there is no evidence supporting horizontal transmission, these genes are not expected to add to the pool of antibiotic resistance elements considered to pose a risk to human or animal health. Whole-genome based phylogenetic and sequence analysis, combined with phenotypic testing, is proposed to be suitable for determining intrinsic resistance and evolutionary relationships.

Industrially relevant cellulase production by indigenous thermophilic Bacillus licheniformis TLW-3 strain: Isolation-molecular identification and enzyme yield optimization.

Cellulases are the third largest single industrial bio-robots. These enzymes are employed in industries like pharmaceutical, textile, food processing, paper recycling and detergent manufacturing. In order to produce broadly diversified cellulases, microbes (both bacteria and fungi) have been exploited. Different ecological niches have already been explored for the isolation of cellulolytic microbes. However, there have been no remarkable reports viz a viz to the hot oven ash (for cellulolytic bacterial flora). In this regard, a Bacillus strainTLW-3 was isolated and selected for CMCase production and optimization. The strain was identified as B. licheniformis TLW-3 through 16S rDNA sequencing that was submitted to Gen Bank with accession numberKY440432. The isolate growth and CMCase production conditions were optimized to get the maximum CMCase yield. The highest growth and maximum CMCase production by B. licheniformis TLW-3 were recorded at pH 7 and 50ºC, after the incubation period of 72 (hour) at 150rpm. Studies on the various nitrogen and carbon sources on CMCase production showed that the medium having 1% peptone, 0.5% yeast extract and 1% CMC can significantly enhance the enzymatic yield as compared to other (studied) sources. EDTA, Tween-20 and Tween-80 acted as inhibitors for the enzyme production. The present study holds the conviction that the (reported) organism could directly be applied to produce industrial thermophilic CMCase.

Biodegradation of chlorpropham and its major products by Bacillus licheniformis NKC-1.

Chlorpropham [isopropyl N-(3-chlorophenyl) carbamate] (CIPC), an important phenyl carbamate herbicide, has been used as a plant growth regulator and potato sprout suppressant (Solanum tuberosum L) during long-term storage. A bacterium capable of utilizing the residual herbicide CIPC as a sole source of carbon and energy was isolated from herbicide-contaminated soil samples employing selective enrichment method. The isolated bacterial strain was identified as Bacillus licheniformis NKC-1 on the basis of its morphological, cultural, biochemical characteristics and also by phylogenetic analysis based on 16S rRNA gene sequences. The organism degraded CIPC through its initial hydrolysis by CIPC hydrolase enzyme to yield 3-chloroaniline (3-CA) as a major metabolic product. An inducible 3-CA dioxygenase not only catalyzes the incorporation of molecular oxygen but also removes the amino group by the deamination yielding a monochlorinated catechol. Further, degradation of 4-chlorocatechol proceeded via ortho- ring cleavage through the maleylacetate process. 3-Chloroaniline and 4-chlorocatechol are the intermediates in the CIPC degradation which suggested that dechlorination had occurred after the aromatic ring cleavage. The presence of these metabolites has been confirmed by using ultra-violet (UV), high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), Fourier transmission-infrared (FT-IR), proton nuclear magnetic resonance (1H NMR) and gas chromatography-mass (GC-MS) spectral analysis. Enzyme activities of CIPC hydrolase, 3-CA dioxygenase and chlorocatechol 1, 2-dioxygenase were detected in the cell-free-extract of the CIPC culture and are induced by cells of NKC-1 strain. These results demonstrate the biodegradation pathways of herbicide CIPC and promote the potential use of NKC-1 strain to bioremediate CIPC-contaminated environment with subsequent release of ammonia, chloride ions and carbon dioxide.

Purification and biochemical characterization of a thermostable and acid-stable alpha-amylase from Bacillus licheniformis B4-423.

Novel thermostable amylase need to be continuously explored with the improvement of industrial requirements. A new acidophilic and thermostable amylase producing bacterium isolated from spring was identified as Bacillus strain on the basis of 16S rDNA. The amylase was purified by ammonium sulphate precipitation, gel chromatography and anion exchange chromatography. SDS-PAGE revealed that the enzyme was monomeric with a molecular weight of 58 kDa. The amylase exhibited optimal activity at pH 5.0 and temperature 100 °C. Then the enzyme showed high stability in pH ranges 4.0-10.0 and more than 90% of maximal activity was found from 20 °C to 80 °C. Apart from good stability toward SDS and non-ionic detergent, the purified enzyme exhibited high compatibility with some inhibitors such as urea and EDTA. The results demonstrated the stability of the enzyme in different organic solvents. Moreover, we determined the amylase gene, compared the structure with α-amylase BAA and BLA and found some thermostability determinants in our enzyme. Overall, presenting various properties were including high thermostability, Ca2+-independency, broad temperature and pH profiles, organic-solvent tolerance as well as excellent stability with detergents. Such characteristics have not been reported for this type of enzyme, and the α-amylase will be a suitable candidate in industrial fields.

Biochemical characterization of a novel cold-active, halophilic and organic solvent-tolerant lipase from B. licheniformis KM12 with potential application for biodiesel production.

Nowadays, finding a talented lipase with high potency in biodiesel production has been attracted to researchers. In this study, an extracellular salt tolerant, cold-active and organic solvent-stable lipase producing bacterium was isolated from oil-contaminated environments located in Kerman province (Iran) and identified as Bacillus licheniformis KM12. Lipase was purified by 36.0% recovery and molecular weight was estimated about 33 kDa by SDS-PAGE. The optimal pH and temperature for lipase activity were found to be 8.0 and 35 °C, respectively. Lipase activity was remarkably increased about 37% after 20 min incubation at 20 °C. Km value of KM12 lipase was 0.53 mM for p-nitrophenyl palmitate (p-NPP) and maximum activity was detected for pNP-decanoate and glyceryl-decanoate substrates. KM12 lipase displayed significant stability in different organic solvents after 7 and 21 days of incubation, especially in polar organic solvents. Biodiesel production with 78% yields was achieved with the one-step addition of methanol at around 18 h by using Myrtus oil (as a non-edible oils feedstocks). These unique properties of KM12 lipase make it talented as a potential biocatalyst for biodiesel production.

Screening and identification of lignin-degrading bacteria in termite gut and the construction of LiP-expressing recombinant Lactococcus lactis.

Lignin, a common natural polymers, is abundant and complex, and termites can break down and utilize the lignin in their food. In this study an attempt was made to isolate and characterize the lignolytic bacteria from termite (Reticulitermes chinensis Snyder) gut. Two strains (PY12 and MX5) with high lignin peroxidase (LiP) activity were screened using the azure B method. By analyzing their 16S rRNA, the strain PY12 was classified as Enterobacter hormaechei; MX5, as Bacillus licheniformis. We then optimized the different conditions of liquid fermentation medium, and obtained LiP activities of 278 U/L and 256 U/L for PY12 and MX5, respectively. Subsequently, we confirmed the LiP activities of the strains by evaluating their decolorizing effects on various dyes. Finally, we cloned the LiP gene of strain PY12 and successfully transferred it to Lactococcus lactis. We believe that our results provide the theoretical and practical basis for the production of genetically engineered bacteria that produce LiP, thus allowing for the utilization of naturally available lignin as an energy resource.

Enhancement of Exochitinase Production by Bacillus licheniformis AT6 Strain and Improvement of N-Acetylglucosamine Production.

A strain producing chitinase, isolated from potato stem tissue, was identified as Bacillus licheniformis by biochemical properties and 16S RNA sequence analysis. Statistical experimental designs were used to optimize nine independent variables for chitinase production by B. licheniformis AT6 strain in submerged fermentation. Using Plackett-Burman design, (NH4)2SO4, MgSO4.7H2O, colloidal chitin, MnCl2 2H2O, and temperature were found to influence chitinase production significantly. According to Box-Behnken response surface methodology, the optimal fermentation conditions allowing maximum chitinase production were (in gram per liter): (NH4)2SO4, 7; K2HPO4, 1; NaCl, 1; MgSO4.7H2O, 0.1; yeast extract, 0.5; colloidal chitin, 7.5; MnCl2.2H2O, 0.2; temperature 35 °C; pH medium 7. The optimization strategy led to a 10-fold increase in chitinase activity (505.26 ± 22.223 mU/mL versus 50.35 ± 19.62 mU/mL for control basal medium). A major protein band with a molecular weight of 61.9 kDa corresponding to chitinase activity was clearly detected under optimized conditions. Chitinase activity produced in optimized medium mainly releases N-acetyl glucosamine (GlcNAc) monomer from colloidal chitin. This enzyme also acts as an exochitinase with ß-N-acetylglucosaminidase. These results suggest that B. licheniformis AT6 secreting exochitinase is highly efficient in GlcNAc production which could in turn be envisaged as a therapeutic agent or as a conservator against the alteration of several ailments.