Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis

Aims:  Antibiotics from Bacillus subtilis JA show strong pathogen inhibition ability, which has potential market application; yet, the composition of these antibiotics has not been elucidated. The aim of this paper is to isolate and identify these antibiotics.
Methods and Results:  The antagonistic activity of JA was tested in vitro ; it exhibited strong inhibition against some important phytopathogens and postharvest pathogens. Crude antibiotic production was extracted with methanol from the precipitate by adding 6 mol l⁻¹ HCl to the bacillus-free culture broth. The crude extract was run on Diamonsil C₁₈ column (5  μ m, 250 × 4·6 mm) in HPLC system to separate the antibiotics. Major antibiotics were classified into three lipopeptide families according to electrospray ionization–mass spectrometry analysis. Subsequently, the classification of antibiotics was confirmed with typical collision-induced dissociation fragments.
Conclusions:  Three kinds of antibiotics were isolated from B. subtilis JA and were identified to the lipopeptide families, surfactin, iturin and fengycin. These compounds could function as biocontrol agents against a large spectrum of pathogens.
Significance and Impact of the Study:  This study provided a reliable and rapid method for isolation and structural characterization of lipopeptide antibiotics from B. subtilis .     

Endophytic Bacillus sp. Isolated from the Interior of Balloon Flower Root

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C_15:0 (37%), C_17:0 (5.1%), and iso-C_15:0 (27.7%). DNA G + C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.     

A Simple and Rapid Extraction of High Molecular Weight Chromosomal DNA from Bacillus subtilis Protoplasts for Cosmid Cloning and Interspecific Transformation

After conversion of Bacillus subtilis vegetative cells to protoplasts, a simple and rapid method for extracting high-molecular-weight chromosomal DNA was devised with the inclusion of bovine serum albumin and phenol-chloroform treatments. The DNA sample thus prepared was the size of 100-450 kb and could be used for cosmid cloning and interspecific transformation.     

Restriction on Conjugational Transfer of pLS20 in Bacillus subtilis 168

Conjugational transfer of pLS20 in Bacillus subtilis Marburg 168 is restricted by the BsuM restriction-modification system. Restriction efficiency was measured using pLS20 derivatives possessing various numbers of XhoI sites, which are known to be recognized by BsuM. An increase in XhoI sites clearly reduced the conjugational efficiency of pLS20 as compared with that of pUB110 plasmid lacking XhoI.     

Transfer and expression of the cryIVB and cryIVD genes of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus 2297

Abstract The genes encoding the CryIVB and CryIVD crystal polypeptides of B. thuringiensis subsp. israelensis were cloned indepently on a stable shuttle vector, and transfered into B. sphaericus 2297. Recombinant cells expressed the B. thuringiensis toxins during sporulation and were shown to be toxic to Aedes aegypti fourth instar larvae, whereas the parental strain was not.     

Effect of nonionic detergents on the activity of a thermostable lipase from Bacillus stearothermophilus MC7

The influence of nonionic surfactants on the activity of a novel thermostable lipase from Bacillus stearothermophilus MC7 was investigated with a view to its potential for synthesis of structured lipids. A large number of modifiers within a broad concentration range were applied. The activity of the enzyme was measured at a relatively high reaction temperature. Highest degree of activation was observed when PEG₆₀₀₀ was applied (up to 2.3-fold increase). Modification essentially changed the performance of the lyophilised preparations—they keep up to 80% of the activity of the native enzyme in the presence of a detergent against 30% in its absence. The effect of sorbitan esters (spans) and polyoxyethylene derivatives of sorbitan esters (tweens) on lipase MC7 was estimated, their HLB value varying within the interval 2.1–16.7. Tweens were strong inhibitors at higher concentrations. For all spans, excepting span 60, an increase of enzyme activity with concentration was observed. All studied additives slow down the process of thermal denaturation. Lipase preparations preserve more than 60% of their activity after 30-min incubation at 75 °C in the presence of tween 60 or PEG₄₀₀₀.     

Dispersal capacity in the Mediterranean corn borer, Sesamia nonagrioides

Corn (Zea mays L.) borers are the primary target of Bacillus thuringiensis Berliner (Bt) transgenic maize. Management of corn borer resistance to Bt requires information on larval and adult dispersal capacities, a feature that is particularly unknown in Sesamia nonagrioides Lefèbvre (Lepidoptera: Noctuidae), the most damaging corn borer in Spain. Larval dispersal was studied over a 3 year period by infesting plants with egg masses and dissecting the neighbouring plants 7, 14, and 32 days later to measure larval dispersal at several ages. The number and age of larvae were recorded in the dissected plants. Only mature larvae dispersed in significant numbers; they moved at least to rows adjacent to those containing the infested plant, and down the row five plants. The percentage of larvae that dispersed from the infested plant was density‐dependent. Adult dispersal was studied with directional light and pheromone uni‐traps over 5 and 3 year periods, respectively. Directional light traps were placed in the margins between Bt and non‐Bt maize fields, half oriented towards each of the two kinds of maize field. Pheromone traps were placed in the Bt and non‐Bt fields at increasing distances (0–100 m) from the border. The numbers of males and females caught in directional light traps were not different in traps oriented towards Bt or non‐Bt fields, but the number of males caught in the third flight in Bt fields was lower than in non‐Bt fields. These results suggest that males from adjacent Bt and non‐Bt fields mate indiscriminately with females emerging in any of the two kinds of maize fields. However, male movement in the third flight may not be sufficient to randomly distribute males between the two fields.