Purification and Characterization of an Extracellular Alkaline Serine Protease with Dehairing Function from <Emphasis Type="Italic">Bacillus pumilus</Emphasis>

An extracellular alkaline serine protease (called DHAP), produced by a Bacillus pumilus strain, demonstrates significant dehairing function. This protease is purified by hydrophobic interaction chromatography, ion exchange, and gel filtration. DHAP had a pI of 9.0 and a molecular weight of approximately 32,000 Dalton. It shows maximal activity at pH 10 and with a temperature of 55°C; the enzyme activity can be completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP). The first 20 amino acid residues of the purified DHAP have been determined with a sequence of AQTVPYGIPQIKAPAVHAQG. Alignment of this sequence with other alkaline protease demonstrates its high homology with protease from another B. pumilus strain. Received: 17 April 2002 / Accepted: 24 May 2002     

Cloning and Expression of <Emphasis Type="Italic">cyt</Emphasis>2Ba7 Gene from a Soil-Isolated <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Bacillus thuringiensis (Bt) cyt genes coding hemolytic and cytolytic toxins constitute a gene family, which are divided into two groups: cyt1 and cyt2. A novel cyt2 gene was detected from a soil-isolated Bt strain T301, which was highly homologous to cyt2Ba1 and finally designated cyt2Ba7. Until now, Cyt2Ba has not been expressed alone in Bt or other hosts. In this study, the cyt2Ba7 gene was cloned into the vector pQE30 and expressed as a fusion protein with 6×Histidine residues in Escherichia coli. Unlike cyt1A, cyt2Ba7 was freely expressed and formed cytoplasmic inclusions without the need for a “helper” protein. The 6×His-tagged Cyt2Ba7 was purified in one step by Ni-NTA affinity chromatography, examined cytolytic activity on Sf9 cells, and developed as an antigen to obtain the antiserum against Cyt2Ba by subcutaneous injection into rabbits. This gene was also cloned into the Bt–E. coli shuttle vector pHT3101 and expressed in Bt strain 4Q7. Immunoblotting analysis revealed that the antiserum was remarkably selective and specific to Cyt2Ba. Received: 21 December 2001 / Accepted: 28 January 2002     

Chitinolytic and antifungal activity of a <Emphasis Type="Italic">Bacillus pumilus</Emphasis> chitinase expressed in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Bacillus pumilus SG2 chitinase gene (ChiS) and its truncated form lacking chitin binding (ChBD) and fibronectin type III (FnIII) domains were transformed to Arabidopsis plants and the expression, functionality and antifungal activity of the recombinant proteins were investigated. Results showed that while the two enzyme forms showed almost equal hydrolytic activity toward colloidal chitin, they exhibited a significant difference in antifungal activity. Recombinant ChiS in plant protein extracts displayed a high inhibitory effect on spore germination and radial growth of hyphae in Alternaria brassicicola, Fusarium graminearum and Botrytis cinerea, while the activity of the truncated enzyme was strongly abolished. These findings demonstrate that ChBD and FnIII domains are not necessary for hydrolysis of colloidal chitin but play an important role in hydrolysis of chitin–glucan complex of fungal cell walls. Twenty microgram aliquots of protein extracts from ChiS transgenic lines displayed strong antifungal activity causing up to 80% decrease in fungal spore germination. This is the first report of a Bacillus pumilus chitinase expressed in plant system.     

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.     

Characterization of an Extracellular Serine Protease Gene from the Nematophagous Fungus <Emphasis Type="Italic">Lecanicillium psalliotae</Emphasis>

The gene encoding a cuticle-degrading serine protease was cloned from three isolates of Lecanicillium psalliotae (syn. Verticillium psalliotae) by 3′ and 5′ RACE (rapid amplification of cDNA ends) method. The gene encodes for 382 amino acids and the protein shares conserved motifs with subtilisin N and peptidase S8. Comparison of translated cDNA sequences of three isolates revealed one amino acid polymorphism at position 230. The deduced protease sequence shared high degree of similarities to other cuticle-degrading proteases from other nematophagous fungi.     

Biofilm formation by different serological variants of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in association with <Emphasis Type="Italic">Bacillus pumilus</Emphasis>

Ability of Listeria monocytogenes serological variants belonging to phylogenetic lines I and II to form biofilms on glass, both in monoculture and in a consortium with Bacillus pumilus, was shown. After 72 h, both L. monocytogenes serological variants formed mature biofilms both in monoculture and in association with bacilli. The presence of B. pumilus in Listeria biofilms resulted in alteration of L. monocytogenes colony morphology, decrease in their enzymatic activity and aghesive capacity, enhanced virulence and hemolytic activity, and cell motility observed at 37°С. Importantly, all of these modifications of the biological characteristics were of a phenotypic nature and were restored when joint incubation of bacteria was terminated.     

Antifungal chitinases from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> SG2: preliminary report

A halotolerant bacterium capable of chitinase production was previously isolated from saline soils of Gavkhooni marsh, Iran. The strain was identified as Bacillus pumilus strain SG2. This strain secretes two chitinases into the medium in response to the presence of chitin in the medium. The two chitinases, ChiS and ChiL, were active on both polymeric as well as oligosaccharide substrates. In this study, these two extracellular chitinases were purified from the supernatant of the Bacillus culture by ammonium sulfate precipitation. The optimum pH and temperature for both of these chitinases were pH 6 and 37°C, respectively. According to the conserved domain analysis, ChiS and ChiL were categorized in family 18 of the glycosylhydrolases. Three essential conserved amino acid residues (Asp-194, Asp-196 and Glu-198) in ChiS and (Asp-228, Asp-230 and Glu-232) in ChiL were found within the active site. Antifungal activities of the two purified chitinases were assessed on Rhizoctonia solani, Verticillium sp., Nigrospora sp., Stemphyllium botryosum and Bipolaris sp. demonstrating inhibition of all the tested strains. However, these chitinases did not inhibit cell wall formation of the non-chitin-containing fungi (oomycetes) Phytophthora citricola and Phytophthora capsici. This is the first investigation demonstrating antifungal activity of chitinases purified from B. pumilus SG2.     

Cloning and Expression of <Emphasis Type="Italic">H. influenzae</Emphasis> 49247 IgA Protease in <Emphasis Type="Italic">E. coli</Emphasis>

IgA protease is secreted by various mucosal pathogenic bacteria which can cleave human immunoglobulin A1 (IgA1) in its hinge region. In addition to be considered as a virulence factor, it's reported that IgA protease can also be used for IgA nephropathy (IgAN) treatment. Our previous study identified bacteria H. influenzae 49247 expressed high activity of IgA protease with promised application in IgAN therapy. In this study, we cloned the IgA protease gene of H. influenzae 49247 with degenerate primers. Alignment analysis indicated that H. influenzae 49247 IgA protease showed unique DNA and amino acid sequence but with typical endopeptidase domain and beta transporter domain compared with known IgA proteases from the same species. To facilitate expression and purification, the H. influenzae 49247 IgA protease gene was sub-cloned into the pET28-A(+) vector with insertion of a 6xHis tag downstream of the endopeptidase domain and upstream of the potential autocleavage site. The recombined IgA protease can be constitutively expressed in E. coli and secreted into the culture medium. With a simple nickel affinity binding, the secreted IgA protease can be purified with high purity (95%) and a molecular weight of about 130 kDa. The identity of the IgA protease was validated by the presence of 6xHis tag in the purified protein by western blotting and its ability to cleave human IgA1 molecule. Collectively, the successful cloning, expression and purification of H. influenzae 49247 IgA protease will augment its therapeutic study in IgAN treatment.     

Cloning of <Emphasis Type="Italic">srfA</Emphasis> operon from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> C9 and its expression in <Emphasis Type="Italic">E. coli</Emphasis>

The srfA operon is required for the nonribosomal biosynthesis of the cyclic lipopeptide, surfactin. The srfA operon is composed of the four genes, srfAA, srfAB, srfAC, and srfAD, encoding the surfactin synthetase subunits, plus the sfp gene that encodes phosphopantetheinyl transferase. In the present study, 32 kb of the srfA operon was amplified from Bacillus subtilis C9 using a long and accurate PCR (LA-PCR), and ligated into a pIndigoBAC536 vector. The ligated plasmid was then transformed into Escherichia coli DH10B. The transformant ET2 showed positive signals to all the probes for each open reading frame (ORF) region of the srfA operon in southern hybridization, and a reduced surface tension in a culture broth. Even though the surface-active compound extracted from the E. coli transformant exhibited a different R _f value of 0.52 from B. subtilis C9 or authentic surfactin (R _f = 0.63) in a thin layer chromatography (TLC) analysis, the transformant exhibited a much higher surface-tension-reducing activity than the wild-type strain E. coli DH10B. Thus, it would appear that an intermediate metabolite of surfactin was expressed in the E. coli transformant harboring the srfA operon.     

Function of the <Emphasis Type="Italic">rel</Emphasis> Gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Sokawa et al. suggest that rel^- strains of Escherichia coli possess abnormal protein synthesizing machinery, which cannot carry out normal protein synthesis when the supply of amino-acids is limited.     

Gene analysis,optimized production and property of marine lipase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> B106 associated with South China Sea sponge <Emphasis Type="Italic">Halichondria rugosa</Emphasis>

Gene cloning, optimized production and property of marine lipase from Bacillus pumilus B106 associated with South China Sea sponge Halichondria rugosa were investigated in this paper. A lipase gene with whole ORF encoding 215 amino acids was obtained by PCR, protein domain prediction suggested that the deduced lipase belongs to α/β hydrolases family. Based on single factor Seriatim-Factorial test and Plackett–Burman experimental design, the optimal medium consisted of (per l) 12.5 ml maize oil, 5.0 g beef extract, 2.0 g PO₄ ³⁻ (0.6 g KH₂PO₄, 1.4 g K₂HPO₄), 17.15 g Mg²⁺, 5.0 g yeast extract, 2.282 g CaCl₂ and 5.0 ml Tween80 with artificial sea water. Using this optimum medium, lipase activity and cell concentration were increased by 3.54- and 1.31-fold over that of the basal medium, respectively. This lipase showed tolerance to high salinity, pH and temperature. About 10–20% methanol exhibited a stimulatory effect on the lipase activity, while activity was inhibited by 30–40% methanol, 2-propanol, DMSO, and ethanol. This study provides a valuable resource for marine lipase production and extends our understanding of the possible role of sponge-associated bacteria in the biotransformation of chemical compounds for the sponge host.     

<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.     

Efficient Expression of the Mosquito Larvicidal Binary Toxin Gene from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The binary toxin gene encoding BinA (42 kDa) and BinB (51 kDa) from Bacillus sphaericus strain 2297 was cloned and expressed in E. coli. Low expression level was found when both proteins were expressed from a single operon. High expression was observed when the gene encoding an individual protein was placed downstream of the T7 promoter. The expression level of BinB was not different when expressed alone (non-fusion) or as a fusion form with T7 peptide (T7-BinB). Both forms of BinB were equally stable. Unlike BinB, the non-fusion form of BinA was less stable than T7-BinA. The mosquito larvicidal test showed that BinA or BinB alone was not toxic to mosquito larvae, but high toxicity was found when both BinA and BinB were applied. The results suggest that a short peptide of T7 linked to the N-terminus of either BinA or BinB does not affect their toxicity, but may make the toxin, especially BinA, more stable.