Characterization of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> inhibitory lipopeptide from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> IB

Bacillus subtilis strain IB exhibiting inhibitory activity against the Fusarium head blight disease fungus Fusarium graminearum was isolated and identified. The major inhibitory compound was purified from the culture broth through anion exchange, hydrophobic interaction, and reverse phase high-performance liquid chromatography (RP-HPLC) steps. It was a 1,463-Da lipopeptide and had an amino acid composition consisting of Ala, Glx, Ile, Orn, Pro, Thr, and Tyr at a molar ratio of 1:3:1:1:1:1:2. Electrospray ionization mass spectrometry/mass spectrometry (ESI MS/MS) analyses of the natural and the ring-opened peptides showed the antagonist was fengycin, a kind of macrolactone molecule with antifungal activity produced by several Bacillus strains. Fluorescence microscopic analysis indicated this peptide permeabilized and disrupted F. graminearum hyphae.     

Comparative structural bioinformatics analysis of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> chemotaxis proteins within <Emphasis Type="Italic">Bacillus subtilis</Emphasis> group

Chemotaxis is a process in which bacteria sense their chemical environment and move towards more favorable conditions. Since plant colonization by bacteria is a multifaceted process which requires a response to the complex chemical environment, a finely tuned and sensitive chemotaxis system is needed. Members of the Bacillus subtilis group including Bacillus amyloliquefaciens are industrially important, for example, as bio-pesticides. The group exhibits plant growth-promoting characteristics, with different specificity towards certain host plants. Therefore, we hypothesize that while the principal molecular mechanisms of bacterial chemotaxis may be conserved, the bacterial chemotaxis system may need an evolutionary tweaking to adapt it to specific requirements, particularly in the process of evolution of free-living soil organisms, towards plant colonization behaviour. To date, almost nothing is known about what parts of the chemotaxis proteins are subjected to positive amino acid substitutions, involved in adjusting the chemotaxis system of bacteria during speciation. In this novel study, positively selected and purified sites of chemotaxis proteins were calculated, and these residues were mapped onto homology models that were built for the chemotaxis proteins, in an attempt to understand the spatial evolution of the chemotaxis proteins. Various positively selected amino acids were identified in semi-conserved regions of the proteins away from the known active sites.     

Characterization of the <Emphasis Type="Italic">codY</Emphasis> gene and its influence on biofilm formation in <Emphasis Type="Italic">Bacillus </Emphasis><Emphasis Type="Italic">cereus</Emphasis>

The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant (M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation. Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type. Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels CodY represses production of an unknown protease and is involved in biofilm formation.     

Artificial induction of genetic competence in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> isolates

Objectives

To induce natural genetic competence in Bacillus amyloliquefaciens isolates through overexpression of the master regulator, ComK, from B. subtilis (ComK _Bsu ).

Results

Plasmid pUBXC carrying the xylose-inducible comK expression cassette was constructed using plasmid pUB110 as a backbone. Plasmid pUBXC could be transferred from B. subtilis to B. amyloliquefaciens through plasmid pLS20-mediated biparental conjugation. After being induced by xylose, four B. amyloliquefaciens strains harbouring plasmid pUBXC developed genetic competence. Under optimal conditions, the transformation efficiencies of plasmid DNA ranged from 129 ± 20.6 to 1.7 ± 0.1 × 10⁵ cfu (colony-forming units) per μg DNA, and the transformation efficiencies of PCR-assembled deletion constructs ranged from 3.2 ± 0.76 to 3.5 ± 0.42 × 10⁴ cfu per μg DNA in the four tested strains.

Conclusion

Artificial induction of genetic competence through overexpressing ComK _Bsu in B. amyloliquefaciens completed the tasks of replicative plasmid delivery and gene knockout via direct transformation of PCR-generated deletion cassettes.

     

Genetic mapping of the <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> avirulence gene corresponding to the <Emphasis Type="Italic">LepR1</Emphasis> resistance gene of <Emphasis Type="Italic">Brassica napus</Emphasis>

AvrLepR1 of the fungal pathogen Leptosphaeria maculans is the avirulence gene that corresponds to Brassica LepR1, a plant gene controlling dominant, race-specific resistance to this pathogen. An in vitro cross between the virulent L. maculans isolate, 87-41, and the avirulent isolate, 99-56, was performed in order to map the AvrLepR1 gene. The disease reactions of the 94 of the resulting F₁ progenies were tested on the canola line ddm-12-6s-1, which carries LepR1. There were 44 avirulent progenies and 50 virulent progenies suggesting a 1:1 segregation ratio and that the avirulence of 99-56 on ddm-12-6s-1 is controlled by a single gene. Tetrad analysis also indicated a 1:1 segregation ratio. The AvrLepR1 gene was positioned on a genetic map of L. maculans relative to 259 sequence-related amplified polymorphism (SRAP) markers, two cloned avirulence genes (AvrLm1 and AvrLm4-7) and the mating type locus (MAT1). The genetic map consisted of 36 linkage groups, ranging in size from 13.1 to 163.7 cM, and spanned a total of 2,076.4 cM. The AvrLepR1 locus was mapped to linkage group 4, in the 13.1 cM interval flanked by the SRAP markers SBG49-110 and FT161-223. The AvrLm4-7 locus was also positioned on linkage group 4, close to but distinct from the AvrLepR1 locus, in the 5.4 cM interval flanked by FT161-223 and P1314-300. This work will make possible the further characterization and map-based cloning of AvrLepR1. A combination of genetic mapping and pathogenicity tests demonstrated that AvrLepR1 is different from each of the L. maculans avirulence genes that have been characterized previously.     

Isolation and characterization of an AHL lactonase gene from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>

An AHL lactonase gene (aiiA) was PCR amplified from the genomic DNA of Bacillus amyloliquefaciens, with the intact open reading frame of 753 base pair. The gene shares high identity to its homologues present in different Bacillus species. The expression plasmid carrying a tact aiiA-PEBA gene was constructed and the gene was overproduced in Escherichia coli BL21 (DE3). The product expressed resulted in attenuation and suspension of the infection of Pectobacterium carotovorum subsp. carotovorum on carrot. This study verified the existence of the aiiA gene in B. amyloliquefaciens and provided a prospect of the strain as biocontrol agents with quorum quenching property on bacterial disease control.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Mutational analysis of the <Emphasis Type="Italic">ribC</Emphasis> gene of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The nucleotide sequence of the ribC gene encoding the synthesis of bifunctional flavokinase/flavine adenine nucleotide (FAD) synthetase in Bacillus subtilis have been determined in a family of riboflavinconstitutive mutants. Two mutations have been found in the proximal region of the gene, which controls the transferase (FAD synthase) activity. Three point mutations and one double mutation have been found (in addition to the two mutations that were detected earlier) in the distal region of the gene, which controls the flavokinase (flavin mononucleotide (FMN) synthase) activity. On the basis of all data known to date, it has been concluded that the identified mutations affect riboflavin and ATP binding sites. No mutations have been found in the PTAN conserved sequence, which forms the magnesium and ATP common binding site and is identical for organisms of all organizational levels, from bacteria too humans.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Inhibition of biofilm in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> Q-426 by diketopiperazines

Biofilm formation can make significant effects on bacteria habits and biological functions. In this study, diketopiperazines (DKPs) produced by strain of Bacillus amyloliquefaciens Q-426 was found to inhibit biofilm formed in the gas–liquid interface. Four kinds of DKPs were extracted from B. amyloliquefaciens Q-426, and we found that 0.04 mg ml^?1 DKPs could obviously inhibit the biofilm formation of the strain. DKPs produced by B. amyloliquefaciens Q-426 made a reduction on extracellular polymeric substance (EPS) components, polysaccharides, proteins, DNAs, etc. Real-time PCR was performed to determine that whether DKPs could make an obvious effect on the expression level for genes related to biofilm formation in the strain. The relative expression level of genes tasA, epsH, epsG and remB which related to proteins, extracellular matrix, and polysaccharides, were downregulated with 0.04 mg ml^?1 DKPs, while the expression level of nuclease gene nuc was significantly upregulated. The quantitative results of the mRNA expression level for these genes concerted with the quantitative results on EPS levels. All of the experimental results ultimately indicated that DKPs could inhibit the biofilm formation of the strain B. amyloliquefaciens Q-426.     

Antimicrobial factor from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> inhibits <Emphasis Type="Italic">Paenibacillus larvae</Emphasis>, the causative agent of American foulbrood

Bacillus amyloliquefaciens LBM 5006 produces an antimicrobial factor active against Paenibacillus larvae, a major honeybee pathogen. The antagonistic effect and the mode of action of the antimicrobial factor were investigated. The antibacterial activity was produced starting at mid-logarithmic growth phase, reaching its maximum during the stationary phase. Exposure of cell suspensions of P. larvae to this antimicrobial resulted in loss of cell viability and reduction in optical density associated with cell lysis. Scanning electron microscopy showed damaged cell envelope and loss of protoplasmic material. The antimicrobial factor was stable for up to 80°C, but it was sensitive to proteinase K and trypsin. Mass spectrometry analysis indicates that the antimicrobial activity is associated with iturin-like peptides. The antimicrobial factor from B. amyloliquefaciens LBM 5006 showed a bactericidal effect against P. larvae cells and spores. This is the first report on iturin activity against P. larvae. This antimicrobial presents potential for use in the control of American foulbrood disease.     

Cyclic lipopeptide profile of three <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains; antagonists of <Emphasis Type="Italic">Fusarium</Emphasis> head blight

The objective of the study was to identify the lipopetides associated with three Bacillus subtilis strains. The strains are antagonists of Gibberella zeae, and have been shown to be effective in reducing Fusarium head blight in wheat. The lipopeptide profile of three B. subtilis strains (AS43.3, AS43.4, and OH131.1) was determined using mass spectroscopy. Strains AS43.3 and AS43.4 produced the anti-fungal lipopeptides from the iturin and fengycin family during the stationary growth phase. All three strains produced the lipopeptide surfactin at different growth times. Strain OH131.1 only produced surfactin under these conditions. The antifungal activity of the culture supernatant and individual lipopeptides was determined by the inhibition of G. zeae. Cell-free supernatant from strains AS43.3 and AS43.4 demonstrated strong antibiosis of G. zeae, while strain OH131.1 had no antibiosis activity. These results suggest a different mechanism of antagonism for strain OH131.1, relative to AS43.3 and AS43.4.     

Bio-resolution of glycidyl (<Emphasis Type="Italic">o</Emphasis>, <Emphasis Type="Italic">m</Emphasis>, <Emphasis Type="Italic">p</Emphasis>)-methylphenyl ethers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84–99% enantiomeric excess and with 21–73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.