Protein Engineering of the 4-Methyl-5-Nitrocatechol Monooxygenase from Burkholderia sp. Strain DNT for Enhanced Degradation of Nitroaromatics

4-Methyl-5-nitrocatechol (4M5NC) monooxygenase (DntB) from Burkholderia sp. strain DNT catalyzes the second step of 2,4-dinitrotoluene degradation by converting 4M5NC to 2-hydroxy-5-methylquinone with the concomitant removal of the nitro group. DntB is a flavoprotein that has a very narrow substrate range. Here, error-prone PCR was used to create variant DntB M22L/L380I, which accepts the two new substrates 4-nitrophenol (4NP) and 3-methyl-4-nitrophenol (3M4NP). At 300 μM of 4NP, the initial rate of the variant expressing M22L/L380I enzyme (39 ± 6 nmol/min/mg protein) was 10-fold higher than that of the wild-type enzyme (4 ± 2 nmol/min/mg protein). The values of k_cat/K_m of the purified wild-type DntB enzyme and purified variant M22L/L380I were 40 and 450 (s⁻¹ M⁻¹), respectively, which corroborates that the variant M22L/L380I enzyme has 11-fold-higher efficiency than the wild-type enzyme for 4NP degradation. In addition, the variant M22L/L380I enzyme has fourfold-higher activity toward 3M4NP; at 300 μM, the initial nitrite release rate of M22L/L380I enzyme was 17 ± 4 nmol/min/mg protein, while that of the wild-type enzyme was 4.4 ± 0.7 nmol/min/mg protein. Saturation mutagenesis was also used to further investigate the role of the individual amino acid residues at positions M22, L380, and M22/L380 simultaneously. Mutagenesis at the individual positions M22L and L380I did not show appreciable enhancement in 4NP activity, which suggested that these two sites should be mutated together; simultaneous saturation mutagenesis led to the identification of the variant M22S/L380V, with 20% enhanced degradation of 4NP compared to the variant M22L/L380I. This is the first report of protein engineering for nitrite removal by a flavoprotein.     

A Cytochrome P-450 Monooxygenase Catalyzes the First Step in the Conversion of Tabersonine to Vindoline in Catharanthus roseus

Hydroxylation at the C-16 position of the indole alkaloid tabersonine has been suggested as the first step toward vindoline biosynthesis in Catharanthus roseus. Tabersonine 16-hydroxylase (16-OH) activity was detected in total protein extracts from young leaves of C. roseus using a novel coupled assay system. Enzyme activity was dependent on NADPH and molecular oxygen and was inhibited by CO, clotrimazole, miconazole, and cytochrome c. 16-OH was localized to the endoplasmic reticulum by linear sucrose density gradient centrifugation. These data suggest that 16-OH is a cytochrome P-450-dependent monooxygenase. The activity of 16-OH reached a maximum in seedlings 9 d postimbibition and was induced by light. The leaf-specific distribution of 16-OH in the mature plant is consistent with the localization of other enzymes in the tabersonine to vindoline pathway. However, in contrast to enzymes that catalyze the last four steps of vindoline biosynthesis, enzymes responsible for the first two steps from tabersonine (16-OH and 16-O-methyltransfersase) were detected in C. roseus cell-suspension cultures. These data complement the complex model of vindoline biosynthesis that has evolved with respect to enzyme compartmentalization, metabolic transport, and control mechanisms.     

Hydroxylation of the triterpenoid dipterocarpol with CYP106A2 from Bacillus megaterium

The bacterial steroid-hydroxylase CYP106A2 from Bacillus megaterium ATCC 13368 hydroxylates a variety of 3-oxo-Δ-4-steroids and has recently been shown to catalyse regioselective hydroxylation of the diterpene abietic acid, as well as the pentacyclic triterpene 11-keto-β-boswellic acid. The broad substrate spectrum of this enzyme makes it an excellent candidate for biotechnological application. Because the natural substrate of this enzyme is not known, we assumed that the whole substrate spectrum might not yet be fully discovered. The difference spectroscopy method was used to screen a natural product library of 502 compounds. Screening of the library resulted in the identification of twelve hits, among them eight potential and four known substrates for CYP106A2. Interestingly, when testing the potential substrates, product formation was obtained only with triterpenes, namely dipterocarpol and betulin. Dipterocarpol is the most promising compound for biotechnological application because it is a dammarane-type triterpenoid, as are the major bioactive compounds of ginseng. The dipterocarpol hydroxylation products were analysed by NMR and identified as 7β-hydroxydipterocarpol and 7β,11α-dihydroxydipterocarpol. To investigate the putative bioactive properties of these novel compounds, in vitro cytotoxicity assays with HeLa and COS-1 cells were performed. The substrate dipterocarpol and the dihydroxylated product did not show cytotoxic activity in our study. By contrast, the 7β-hydroxylated product was found to be cytotoxic to both tested cell lines. This study highlights the potency of CYP106A2 as a versatile biocatalyst for the bioconversion of natural products into pharmaceutically relevant bioactive products.     

Purification and partial characterization of the gamma-D-glutamyl-L-di-amino acid endopeptidase II from Bacillus sphaericus.

1. A gamma-D-glutamyl-L-di-amino acid endopeptidase II (EC3.4.-.-) active on the peptide moieties of some bacterial peptidoglycans has been purified to homogeneity from the sporulation medium and from the spores of Bacillus sphaericus. 2. Enzyme from both sources showed a single protein band (Mr 28,000) by polyacrylamide gel electrophoresis under denaturing conditions. It is an acidic protein (pI 4.1). Kinetic studies have shown a Km value of 0.24 mM and an apparent Vmax of 8.3 mumol min-1 mg-1 with the pentapeptide L-Ala-gamma-D-Glu-L-Lys-D-[14C]Ala-D-[14C]Ala as substrate. 3. The enzyme was inhibited by p-hydroxymercuribenzoate, a sulfhydryl inhibitor. 4. The 38-residue N-terminal region was sequenced. It may be useful to construct a nucleotide probe for the research of the gene encoding this enzyme.     

Sequencing and heterologous expression of the gene encoding penicillin V amidase from Bacillus sphaericus

The Bacillus sphaericus gene encoding penicillin V amidase, which catalyzes the hydrolysis of penicillin V, has been characterized. The entire nucleotide sequence of the coding region, as well as 5'- and 3'-flanking regions, was determined using an improved sequencing strategy. The deduced amino acid sequence suggests a protein consisting of 338 residues with an Mr of 37,500. The ATG initiator codon is preceded by a putative ribosome-binding site, typical for genes of Gram-positive origin. High expression of the gene was obtained in Escherichia coli using an inducible promoter, showing that the gene product is stable in this heterologous host.     

Cloning of the biotin synthetase gene from Bacillus sphaericus and expression in Escherichia coli and Bacilli

Biotin synthetase (BS) catalyses the biotransformation of dethiobiotin (DTB) to biotin. Here we report the cloning, characterization and expression of the gene encoding BS of Bacillus sphaericus. A recombinant plasmid pSB01, containing an 8.2-kb DNA fragment from B. sphaericus, was isolated by phenotypic complementation of an Escherichia coli bioB strain. Nucleotide sequence analysis of this fragment and N-terminal sequence determination of the recombinant protein product revealed that the bioB gene of B. sphaericus consists of a 996-bp open reading frame which is closely associated with at least one other gene. E. coli cells transformed with a bioB expression vector performed efficient bioconversion of DTB to biotin under defined culture conditions. Biotin production from transformed Bacillus subtilis and B. sphaericus recombinant strains was also demonstrated. Comparison of the amino acid sequences of BS from E. coli and B. sphaericus revealed extensive similarity.     

Recycling potential and selective retrieval of Bacillus sphaericus from soil in a mosquito habitat

A selective medium containing a combination of starch, milk proteins, and streptomycin was used as a reliable indicator for the presence of Bacillus sphaericus in soil samples collected from a mosquito habitat where this pathogen was previously applied. The medium can also be used as an indicator substrate to retrieve B. sphaericus from infected mosquito larvae. Results show that B. sphaericus remains viable and infective 9 months after application as a larvacidal agent of mosquitoes in a roadside ditch.     

Evolution of resistance toward Bacillus sphaericus or a mixture of B. sphaericus+Cyt1A from Bacillus thuringiensis, in the mosquito, Culex quinquefasciatus (Diptera: Culicidae)

The 2362 strain of Bacillus sphaericus (Bs) Neide is a highly mosquitocidal bacterium used in commercial bacterial larvicides primarily to control mosquitoes of the genus Culex. Unfortunately, Bs is at high risk for selecting resistance in mosquito populations, because its binary toxin apparently only binds to a single receptor type on midgut microvilli. A potential key strategy for delaying resistance to insecticidal proteins is to use mixtures of toxins that act at different targets within the insect, especially mixtures that interact synergistically. We tested this hypothesis for delaying the phenotypic expression of resistance by exposing Culex quinquefasciatus Say larvae to Bs alone or in combination with Cyt1A from Bacillus thuringiensis subsp. israelensis. Two laboratory lines of Cx. quinquefasciatus, one sensitive to Bs and the other containing Bs resistance alleles, were subjected to intensive selection pressure for 20 generations with either Bs 2362 or a 3:1 mixture of Bs 2362+Cyt1A. At the end of the study, the sensitive line had evolved >1000-fold resistance when selected with Bs alone, whereas the parallel line selected with Bs+Cyt1A exhibited only low resistance toward this mixture (RR95, 1.4). Similar results were observed in the lines containing Bs resistance alleles. Both lines selected with Bs+Cyt1A exhibited substantial resistance to Bs in the absence of Cyt1A. Although selection with Bs+Cyt1A did not prevent the underlying evolution of resistance to Bs, these results suggest that a mixture of Bs with other endotoxins, particularly one like Bs+Cyt1A in which the components interact synergistically, will provide longer lasting and more effective mosquito control than Bs alone.     

Comparison of the cellular fatty acid composition of a bacterium isolated from a human and alleged to be Bacillus sphaericus with that of Bacillus sphaericus isolated from a mosquito larvicide.

The cellular fatty acid (CFA) composition of the cytoplasmic membrane of a bacillus isolated from a human lung and deposited in the National Collection of Type Cultures as Bacillus sphaericus NCTC 11025 was determined by gas-liquid chromatography. The CFA composition of B. sphaericus 2362, isolated from a microbial larvicide, and those of B. sphaericus reference strains obtained from public collections were also determined. Samples were grouped by hierarchical cluster analysis based on the unpaired-group method using arithmetic averages. Samples that linked at a Euclidean distance of < or = 2.0 U were considered to belong to the same strain. NCTC 11025 and the type strain of B. sphaericus, ATCC 14577, were mixed; all other isolates were monotypic. The predominant fatty acid in NCTC 11025 was 12-methyltetradecanoic acid, while the predominant fatty acid in the remaining isolates was 13-methyltetradecanoic acid. NCTC 11025 linked to the other isolates at a Euclidean distance of 83.8 U, and we concluded that it belongs to a different species that we could not identify. We could distinguish among six DNA homology groups of B. sphaericus by using fatty acids. Within DNA homology group IIA, strain 2362 could be distinguished from other strains belonging to serotype H5a, 5b. We concluded that CFA analysis is a useful technique to determine if future human isolates identified as B. sphaericus in fact belong to other species of bacteria or whether the isolates originated from commercial products.     

Cloning and Expression of the Binary Toxin Gene from Bacillus sphaericus IAB872 in a Crystal-Minus Bacillus thuringiensis subsp. israelensis

Acidity is an important environmental condition encountered by lactobacilli during food fermentation. In this report we show that triggering the stationary-phase acid tolerance response (ATR) in L. acidophilus CRL 639 depends on the final growth pH. In free-pH fermentation runs (final pH = 4.5), the cells were completely resistant to acid stress, whereas cells from cultures under controlled pH (pH = 6.0) were very sensitive. The relationship between the final pH and the development of cross-resistance to different kinds of environmental stress was also evaluated. The study of protein profiles showed the overexpression of 16 proteins from 6.5 to 70.9 kDa in stationary phase cells. Seven of these proteins (26.3, 41.4, 48.7, 49.3, 54.5, 56.1, and 70.9 kDa) were expressed as result of the stationary phase itself, while nine proteins (14.1, 18.6, 21.5, 26.9, 29.3, 41.9, 42.6, 49.6, and 56.2 kDa) were exclusively induced as a result of the drop in culture pH during free fermentation runs. These results strongly suggest the involvement of these proteins in cell adaptation to environmental changes. Received: 5 June 2000 / Accepted: 5 July 2000     

Cloning and sequence analysis of the cyclomaltodextrinase gene from Bacillus sphaericus and expression in Escherichia coli cells

The gene for cyclomaltodextrinase (CDase; EC 3.2.1.54) from Bacillus sphaericus E-244 was cloned in the recombinant plasmid pCD629. Sequencing a portion of pCD629 revealed a unique open reading frame of 1,773 nucleotides coding for a 591-amino-acid polypeptide. The deduced polypeptide sequence showed about 50% homology with that of a neopullulanase, and was slightly homologous to those of the cyclodextrin glucanotransferases and the -amylases. The optimum pH, specific activity and K _m value for -cyclodextrin of the CDase that has been produced in Escherichia coli cells were 8.0, 16.4 units/mg protein, and 0.41 mm, respectively. These values were almost identical to those from B. sphaericus E-244. Correspondence to: T. Oguma     

Influence of heat and sodium dodecyl sulfate on the endopeptidase I from Bacillus sphaericus 9602

Endopeptidase I from Bacillus sphaericus is a stable enzyme which retains its activity at 37 degrees C in the presence of sodium dodecyl sulfate. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed two forms of the enzyme: an active, fast-running form, for the enzyme preheated at 37 degrees C and a denatured, slow-running form, for the enzyme preheated at 100 degrees C. Such behavior is similar to that of the "heat-modifiable" outer membrane proteins from gram-negative bacteria. In the absence of sodium dodecyl sulfate, endopeptidase I aggregated in an enzymatically active dimer, with an apparent molecular weight of 90,000 daltons, which could be the native form of the enzyme.     

Expression in Bacillus subtilis of the 51- and 42-kilodalton mosquitocidal toxin genes of Bacillus sphaericus

A 3,080-base-pair KpnI-HindIII DNA fragment from Bacillus sphaericus 2362 coding for 51- and 42-kilodalton mosquitocidal proteins was cloned into Bacillus subtilis DB104 by using the vector pUB18. In B. subtilis these proteins were not detected during vegetative growth but were expressed during sporulation at levels comparable to those found in B. sphaericus.     

Mosquito active strains of Bacillus sphaericus isolated from soil and mud samples collected in Israel

Bacteria of typical Bacillus sphaericus appearance were isolated from mud and soil samples taken from mosquito breeding sites in Israel. Five isolates, 2613, 2615, 2619, 2620, and 2631, all belonging to Phage Group 3, were highly active against Culex pipiens larvae. The most toxic isolates recovered, 2615 and 2631, had calculated ITU values of approximately 1500 ITU/mg, compared with 1000 ITU/mg for the B. sphaericus RB-80 reference standard. Isolates belonging to Phage Group 4 were of significantly lower toxicity when assayed against Culex larvae and exhibited a high variability in their toxicity. In this survey, B. sphaericus strains toxic to mosquito larvae were recovered only from the desert regions of southern Israel. The isolates in Phage Group 3 were all recovered from the central Negev region of Israel. Material taken from sources close to the Dead Sea produced isolates belonging to Phage Group 4.     

Association of the components of the binary toxin from Bacillus sphaericus in solution and with model lipid bilayers

We show herein that interaction in aqueous solution of the two components of binary toxin from Bacillus sphaericus, BinA and BinB, leads to a dramatic conformational change, from beta turns or random coil, to beta structure. Also, either BinA or BinB separately or their equimolar mixture, interact with lipid bilayers resulting in further conformational changes. Upon membrane association, the change in conformation observed for BinA or BinB separately is different from that observed when the proteins are combined, indicating that proper folding depends on the presence of the complementary subunit. We also show, in contrast to previous reports, that BinB, but not BinA, is able to insert in model neutral lipid monolayers.     

Analysis of Expression of the Binary Toxin Genes from Bacillus sphaericus in Anabaena and the Potential in Mosquito Control

Anabaena strains expressing the binary toxin genes of Bacillus sphaericus produce high larvicidal activity with living cells. Western blot analysis showed that the 51-kDa and 42-kDa toxin proteins were stable in Anabaena. When a DNA fragment upstream of the 51-kDa protein gene was deleted, the toxicity was reduced by over a hundred-fold, whereas deletions at the coding regions showed that the cooperation of the two proteins expressed in Anabaena is essential for the larvicidal activity. Outdoor tests showed that the genetically altered Anabaena could keep containers with natural water from being inhabited by Culex larvae for over 2 months. Received: 8 May 2000 / Accepted: 13 June 2000     

Recombinant Strain of Bacillus thuringiensis Producing Cyt1A, Cry11B, and the Bacillus sphaericus Binary Toxin

A novel recombinant Bacillus thuringiensis subsp. israelensis strain that produces the B. sphaericus binary toxin, Cyt1Aa, and Cry11Ba is described. The toxicity of this strain (50% lethal concentration [LC₅₀] = 1.7 ng/ml) against fourth-instar Culex quinquefasciatus was higher than that of B. thuringiensis subsp. israelensis IPS-82 (LC₅₀ = 7.9 ng/ml) or B. sphaericus 2362 (LC₅₀ = 12.6 ng/ml).     

Recombinant strain of Bacillus thuringiensis producing Cyt1A,Cry11B,and the Bacillus sphaericus binary toxin

A novel recombinant Bacillus thuringiensis subsp. israelensis strain that produces the B. sphaericus binary toxin, Cyt1Aa, and Cry11Ba is described. The toxicity of this strain (50% lethal concentration [LC(50)] = 1.7 ng/ml) against fourth-instar Culex quinquefasciatus was higher than that of B. thuringiensis subsp. israelensis IPS-82 (LC(50) = 7.9 ng/ml) or B. sphaericus 2362 (LC(50) = 12.6 ng/ml).