Watanalai Panbangred

WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line

Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2-3 days to provide confirmatory results. In this study we standardized a one-step cytotoxicity assay using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and compared with the traditional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay for rapid detection of cytotoxic Bacillus spp. using Chinese hamster ovary (CHO) cell line. Crude toxin preparations from 50 isolates of Bacillus spp. were exposed to CHO cell line for 1 h or 24 h and the cytotoxicity was determined by using WST-1 and MTT-based methods. Most B. cereus strains and some strains of other Bacillus species from our collection or from food sources showed comparably high cytotoxicity using either of the methods (P=0.81); however, WST-1 assay provided results in only 3 h while MTT assay in 44-52 h. A positive correlation (R2=0.93) between WST-1 and MTT assays strongly suggests that the WST-1-based cytotoxicity assay could be used as an alternative method to MTT assay for rapid (3 h) confirmation of toxigenic Bacillus species in foods prior to their retail distribution or consumption.

Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase

Abstract Various concentrations of isopropyl β-d-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recom binant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacI q. At low IPTG concentrations (0.025 – 0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations (up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme (i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation of proenzyme (i.e., precursor polypeptide lacking a signal peptide).

Inheritance, expression, and silencing of a chitinase transgene in rice

Abstract The inheritance and expression of a transgene locus consisting of multiple copies of a rice chitinase gene under the control of the CaMV 35S promoter was studied in the T3 and T4 generations of a transformed line that expressed the chitinase at a high level. All T3 progeny of a homozygous T2 parent expressed the chitinase constitutively at 3 weeks after germination, but a proportion of the progeny had undetectable levels of chitinase 8 weeks after germination, indicating silencing of the transgene. Transgene silencing was also observed among progeny of a hemizygous parent. However, we did not observe chitinase gene silencing among progeny of another homozygous line that expressed the transgenic chitinase at a five- to tenfold lower level. Thus, expression level, rather than copy number, of the transgene appears to be critical for silencing. Silencing was observed in the leaf, sheath, and root tissues of the plant, indicating that it is not restricted to specific tissues. Silencing was first observed in the youngest leaves and only later in the oldest leaves of the same plant. There was co-silencing of the selectable marker gene, hpt, which is also driven by the CaMV 35S promoter. Unlike the two transgenes (chitinase and marker), the resident homologous chitinase gene with seed-specific expression and two nonhomologous chitinase genes induced in the leaves upon pathogen infection were not silenced. The silent phenotype was inherited in the T4 generation plants, while progeny of expressing plants exhibited silencing. The chitinase transgene appeared intact, and no evidence for gross alterations or methylation of CCGG sites was found. The silent phenotype could not be reversed by treatment with 5-azacytidine. Northern blot analysis and nuclear run-on transcription studies indicated that silencing occurred at the transcriptional level. The implications of transgene silencing in genetic engineering of monocot plants for disease resistance are discussed.

Rakicidin D, an inhibitor of tumor cell invasion from marine-derived Streptomyces sp.

Tumor metastasis is the leading cause of death in cancer patients. It is the process by which a tumor cell leaves the primary tumor, disseminates to a distant site through the circulatory system and establishes a secondary tumor.1 During the metastatic cascade, tumor cells must pass through the extracellular matrix barriers to accomplish the metastasis. Although the genetic basis of tumorigenesis can vary greatly, the steps required for metastasis are similar for all tumor cells. Therefore, interruption of metastasis is a promising approach to the treatment of cancers of various genetic origins. In our continuing search for anti-invasive compounds from microbial secondary metabolites,2–5 rakicidin D (1) was isolated from the culture broth of an actinomycete strain of the genus Streptomyces. Rakicidins are the 15-membered depsipeptides consisting of three amino acids and a 3-hydroxyfatty acid (Figure 1). To date, three congeners, rakicidins A (2) and B (3) from Micromonospora and rakicidin C (4) from Streptomyces, have been reported.6,7 Rakicidins contain a rare unusual amino acid, 4-amino-2,4-pentadienoate, which has been found only in the secondary metabolites from actinomycetes. Except for rakicidins, only two classes of cyclic peptides, BE435478 and vinylamycin,9 are reported to date to contain this unusual amino acid. Herein, we describe the isolation, structure elucidation and biological properties of rakicidin D (1). The producing strain Streptomyces sp. MWW064 was isolated from a marine sediment sample collected in Samut Sakhon province, Thailand. The strain was cultured in our standard medium for actinomycetes and the whole culture broth was extracted with 1-butanol. The extract showed inhibitory activity toward tumor cell invasion into Matrigel, the reconstituted extracellular matrix proteins.10 Bioassay-guided fractionation of the extract led to the isolation of a new compound, rakicidin D (1). Compound 1 was obtained as a colorless amorphous powder. The high-resolution ESITOFMS indicated a molecular formula of C24H38N4O7, which was consistent with the 1H and 13C NMR data. The IR spectrum of 1 indicated the presence of OH or NH (3356 cm 1) and carbonyl (1688 and 1648 cm 1) functionalities. The UV spectrum and the 1H and 13C NMR spectra of 1 showed high similarity to those for rakicidins A and B.7 The 13C NMR and HMQC analysis confirmed the presence of 24 carbons attributable to six deshielded signals including carbonyl carbons, two sp2 methines, one sp2 methylene, five sp3 methines, six sp3 methylenes, four methyl carbons and five exchangeable protons. 2-Amino-2,4-pentadienoate moiety was elucidated by a COSY correlation for H-9/H-10 and a series of HMBC correlations from H-12 to C-10 and C-11, from H-10 to C-8, C-11 and C-12, and from H-9 to C-8 and C-10 (Figure 2). An NOE between H-10 and an exomethylene proton at dH 5.41 indicated that the latter proton and C-10 were located on the same side of the C-11/C-12 double bond. E configuration for the double bond between C-9 and C-10 was confirmed by a large coupling constant for H-9 and H-10 (JHH1⁄415.0 Hz). HMBC correlations from H-7 to C-6 and C-8, and from H-6 to C-5 and C-8 established the connectivity of N-methylglycine and the pentadienoate. COSY correlations for 2-NH/H-2/H-3 and HMBC correlations from H-2 to C-1, H-3 to C-4 and 4-NH2 to C-3 confirmed the presence of a b-hydroxyasparagine moiety. Connection of this amino acid to the glycine moiety was established by an HMBC correlation from 2-NH to C-5. Three additional fragments, H-23/H-14/H-15, H-24/H-16 and H-21/H-22, were recognized from the COSY spectrum. HMBC correlations from H-23 to C-13, C-14 and C-15, and from H-24 to C-15, C-16 and C-17, established the 2,4-dimethyl-3-hydroxyalkanoate substructure. Connectivity of this fragment to the peptide unit was elucidated by HMBC correlations from 11-NH to C-13 and from H-15 to C-1. Finally, the fragment C-22/C-21 containing the triplet methyl group, the methylene fragment C-20 that had an HMBC correlation from H-22 and

Multiple chitinase enzymes from a single gene of Bacillus licheniformis TP-1

Three chitinase activity bands in a culture supernatant of Bacillus licheniformis TP-1 were detected by non-denaturing PAGE. They were designated chitinase bands 1, 2, and 3 in order from the gel origin. Analysis by immunodiffusion and immunoelectrophoresis using polyclonal antibody raised against chitinase band 3 indicated that these chitinases were antigenically similar. B. licheniformis TP-1 and Escherichia coli harboring the cloned chitinase gene (pCHIL3) from strain TP-1 expressed 3 chitinase bands by non-denaturing PAGE and SDS-PAGE which gave estimated molecular masses of 68, 62, and 50 kDa (named Chi68, Chi62, and Chi50, respectively). All three chitinases had the same N-terminal amino acid sequences, strongly suggesting that Chi62 and Chi50 were derived from Chi68 by a processing step(s) at the C-terminus. The deduced C-terminal amino acid sequence of Chi68 showed homology to amino acid sequences of known chitin and cellulose binding domains. Chi62 and Chi50 lacked this domain (judging from their deduced amino acid sequences and calculated molecular masses) and they were unable to bind chitin, suggesting that they were generated from Chi68 by cleavage of the chitin binding domain at the C-terminus. Comparison of chitinase activities indicated that this binding domain was important for complete hydrolytic activity towards colloidal chitin.

Endophytic Actinomycetes: A Novel Source of Potential Acyl Homoserine Lactone Degrading Enzymes

Several Gram-negative pathogenic bacteria employ N-acyl-L-homoserine lactone (HSL) quorum sensing (QS) system to control their virulence traits. Degradation of acyl-HSL signal molecules by quorum quenching enzyme (QQE) results in a loss of pathogenicity in QS-dependent organisms. The QQE activity of actinomycetes in rhizospheric soil and inside plant tissue was explored in order to obtain novel strains with high HSL-degrading activity. Among 344 rhizospheric and 132 endophytic isolates, 127 (36.9%) and 68 (51.5%) of them, respectively, possessed the QQE activity. The highest HSL-degrading activity was at 151.30 ± 3.1 nmole/h/mL from an endophytic actinomycetes isolate, LPC029. The isolate was identified as Streptomyces based on 16S   rRNA gene sequence similarity. The QQE from LPC029 revealed HSL-acylase activity that was able to cleave an amide bond of acyl-side chain in HSL substrate as determined by HPLC. LPC029 HSL-acylase showed broad substrate specificity from C6- to C12-HSL in which C10HSL is the most favorable substrate for this enzyme. In an in vitro pathogenicity assay, the partially purified HSL-acylase efficiently suppressed soft rot of potato caused by Pectobacterium carotovorum ssp. carotovorum as demonstrated. To our knowledge, this is the first report of HSL-acylase activity derived from an endophytic Streptomyces.

High expression of the penicillin G acylase gene (pac) from Bacillus megaterium UN1 in its own pac minus mutant

By marker exchange mutagenesis, Bacillus megaterium strain UN‐1 (Bm‐UN1) was used to prepare a mutant strain B. megaterium UN‐cat (Bm‐UNcat) lacking the penicillin G acylase gene (pac). The pac gene from Bm‐UN1 was subcloned into pTF6 and the resultant plasmid, pBA402, was introduced into Bm‐UNcat and Bacillus subtilis. Bm‐UNcat harbouring pBA402 produced high penicillin G acylase (PAC) activity of 13·7, 19·5 and 20·4 U ml−1 at 24, 36 and 48 h of culture, respectively. This was two‐ to fivefold higher than PAC produced by B. subtilis harbouring pBA402 and about 20‐fold higher than PAC produced by the parent strain, Bm‐UN1.

Toxicity of Bacillus thuringiensis toward Aedes aegypti larvae.

Abstract Among six strains of Bacillus thuringiensis and five other species of Bacillus, only two strains of B. thuringiensis, strains HD-1 and BA-068, were toxic to Aedes aegypti larvae within 24 hr. The LC50s were 5.6 × 104 and 2.4 × 105 spores/ml for strains HD-1 and BA-068, respectively. The toxic factor(s) was heat sensitive and γ ray resistant and preliminary evidences indicated that it was associated with the crystalline body of B. thuringiensis.

Actinoallolides A–E, New Anti-trypanosomal Macrolides, Produced by an Endophytic Actinomycete, Actinoallomurus fulvus MK10-036

Five new anti-trypanosomal macrolides, actinoallolides A-E (1-5), were discovered from the cultured broth of Actinoallomurus fulvus MK10-036. The structures of the actinoallolides, including absolute stereochemistry, were elucidated by a combination of spectroscopic analyses and a series of chemical derivatization studies. Actinoallolide A showed the most potent and selective in vitro anti-trypanosomal activity without cytotoxicity. A new class of promising lead compounds was identified for the development of drugs for both sleeping sickness and Chagas disease.

Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange

BackgroundAcellular Pertussis vaccines against whooping cough caused by Bordetella pertussis present a much-improved safety profile compared to the original vaccine of killed whole cells. The principal antigen of acellular Pertussis vaccine, Pertussis Toxin (PT), must be chemically inactivated to obtain the corresponding toxoid (PTd). This process, however, results in extensive denaturation of the antigen. The development of acellular Pertussis vaccines containing PTd or recombinant PT (rPT) with inactivated S1, Filamentous Hemagglutinin (FHA), and Pertactin (PRN) has shown that the yield of PRN was limiting, whereas FHA was overproduced. To improve antigen yields and process economics, we have constructed strains of Bordetella pertussis that produce enhanced levels of both rPT and PRN.ResultsThree recombinant strains of Bordetella pertussis were obtained by homologous recombination using an allelic exchange vector, pSS4245. In the first construct, the segment encoding PT subunit S1 was replaced by two mutations (R9K and E129G) that removed PT toxicity and Bp-WWC strain was obtained. In the second construct, a second copy of the whole cluster of PT structural genes containing the above mutations was inserted elsewhere into the chromosome of Bp-WWC and the Bp-WWD strain was obtained. This strain generated increased amounts of rPT (3.77 ± 0.53 μg/mL) compared to Bp-WWC (2.61 ± 0.16 μg/mL) and wild type strain (2.2 μg/mL). In the third construct, a second copy of the prn gene was inserted into the chromosome of Bp-WWD to obtain Bp-WWE. Strain Bp-WWE produced PRN at 4.18 ± 1.02 μg/mL in the cell extract which was about two-fold higher than Bp-WWC (2.48 ± 0.10 μg/mL) and Bp-WWD (2.31 ± 0.17 μg/mL). Purified PTd from Bp-WWD at 0.8-1.6 μg/well did not show any toxicity against Chinese hamster ovary (CHO) cell whereas purified PT from WT demonstrated a cell clustering endpoint at 2.6 pg/well.ConclusionsWe have constructed Bordetella pertussis strains expressing increased amounts of the antigens, rPT or rPT and PRN. Expression of the third antigen, FHA was unchanged (always in excess). These strains will be useful for the manufacture of affordable acellular Pertussis vaccines.