Novel cloning vectors for Bacillus thuringiensis.

Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The cryIA(c) and cryIIA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned cryIA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides.     

Construction of cloning vectors for Bacillus thuringiensis.

The replication region of the Bacillus thuringiensis plasmid, pHT1030, was treated with hydroxylamine. Various copy-number mutants were selected and subsequently used to construct shuttle vectors with multiple cloning sites. These recombinant plasmids are very stable and allowed the cloning of a delta-endotoxin-encoding gene in B. thuringiensis. Comparison between gene expression level and vector copy-number indicated that a plateau in delta-endotoxin production is reached with a copy-number of about fifteen per equivalent chromosome.     

Structurally stable Bacillus subtilis cloning vectors

Cloning of long DNA segments (greater than 5 kb) in Bacillus subtilis is often unsuccessful when naturally occurring small (less than 10 kb) plasmids are used as vectors. In this work we show that vectors derived from the large (26.5 kb) plasmids pAM beta 1 and pTB19 allow efficient cloning and stable maintenance of long DNA segments (up to 33 kb). The two large plasmids differ from the small ones in several ways. First, replication of the large plasmids does not lead to accumulation of detectable amounts of ss DNA, whereas the rolling-circle replication typical for small plasmids does. In addition, the replication regions of the two large plasmids share no sequence homology with the corresponding regions of the known small plasmids, which are highly conserved. Taken together, these observations suggest that the mode of replication of the large plasmids is different from that of small plasmids. Second, short repeated sequences recombine much less frequently when carried on large than on small plasmids. This indicates that large plasmids are structurally much more stable than small ones. We suggest that the high structural stability of large plasmids is a consequence of their mode of replication and that plasmids which do not replicate as rolling circles should be used whenever it is necessary to clone and maintain long DNA segments in any organism.     

Novel non-toxic isolates of Bacillus thuringiensis

Argentinean isolates INTA Mo14–4 and INTA 33–5 of Bacillus thuringiensis were characterized. INTA 33–5 (serovar kenyae ) had an amorphous crystal containing proteins of 200 and 130 kDa. INTA Mo14–4 (serovar darmstadiensis ) had a bipyramidal crystal and a bar-shaped inclusion, containing proteins of 130, 60 and 40 kDa. Crystals of both strains showed no toxicity to lepidopteran, dipteran and coleopteran targets. Trypsin digestion of solubilized crystal proteins of INTA 33–5 produced four peptides (≈65 kDa). No putative δ–endotoxin was detected in Mo14–4. Both isolates showed unique plasmid patterns. Southern analyses showed no homology to four known cursive genes. These results indicate the uniqueness of two novel strains of B. thuringiensis which, in turn, confirm the great diversity of this species.     

Novel plasmid vectors for gene cloning in Pseudomonas.

Novel host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. We found that a new Pseudomonas strain, Pseudomonas flavida IF-4, isolated from soil, carried two small cryptic plasmids, named pNI10 and pNI20. They were multi-copy, but not self-transmissible, and the genome size was 3.7 kb for pNI10 and 2.9 kb for pNI20. Several types of cloning vectors containing a kanamycin or streptomycin resistance (Kmr or Smr) gene were constructed from pNI10 and pNI20. These plasmid vectors were efficiently transformed into several strains of Pseudomonas at a frequency up to 4 x 10(5) transformants per 1 microgram plasmid DNA by the usual competent cell method. The vectors derived from pNI10 replicated not only in Pseudomonas but also in some other Gram-negative enteric bacteria such as Escherichia coli, Enterobacter aerogenes, and Proteus mirabilis.     

Novel narrow-host-range vectors for direct cloning of foreign DNA inPseudomonas

Narrow-host-range vectors, based on an indigenous replicon and containing a multiple cloning site, have been constructed in aPseudomonas host capable of growth on unusual substrates. The new cloning vectors yield sufficient amounts of DNA for preparative purposes and belong to an incompatibility group different from that of the incP and incQ broad-host-range vectors. One of these vectors, named pDB47F, was used to clone, directly inPseudomonas, DNA fragments fromAgrobacterium, Pseudomonas, andRhizobium. A clone containingAgrobacterium and Km^R gene sequences was transformed with a higher efficiency than an RSF1010-derived vector (by as much as 1250-fold) in four out of fivePseudomonas strains tested. The considerable efficiency obtained with this system makes possible the direct cloning and phenotypic selection of foreign DNA inPseudomonas.     

Novel streptococcal-integration shuttle vectors for gene cloning and inactivation.

Seven new streptococcal integration shuttle vectors have been constructed which contain different antibiotic-resistance-encoding genes capable of expression in both Streptococcus sp. and Escherichia coli. These plasmids can replicate in E. coli, but not in streptococci because of the absence of a streptococcal origin of replication. The size, antibiotic resistance, and number of unique restriction sites available for cloning for each plasmid are as follows: pSF141 (7.6 kb, CmR and KmR, 7 sites), pSF143 (5.7 kb, TcR, 6 sites), pSF148 (7.3 kb, CmR and SpR, 7 sites), pDL285 (3.4 kb, KmR, 3 sites), pDL286 (3.1 kb, SpR, 4 sites), pSF151 (3.5 kb, KmR, 10 sites), pSF152 (3.2 kb, SpR, 9 sites). If these plasmids carry a fragment of streptococcal DNA they can specifically integrate into the chromosome via Campbell-like, homologous recombination. Therefore, they should be useful for gene inactivation, cloning, chromosomal walking, or linkage analysis in streptococci. The availability of these integration plasmids resistant to different antibiotics, along with the previously described plasmid, pVA891 (ErR), should also allow the construction of mutants possessing multiple insertionally inactivated genes useful for a variety of genetic studies.     

Novel Vip3-related protein from Bacillus thuringiensis

A novel vip3-related gene was identified in Bacillus thuringiensis. This novel gene is 2,406 bp long and codes for a 91-kDa protein (801 amino acids). This novel protein exhibits between 61 and 62% similarity with Vip3A proteins and is designated Vip3Ba1. Vip3Ba1 has several specific features. Differences between Vip3Ba1 and the Vip3A proteins are spread throughout the sequence but are more frequent in the C-terminal part from amino acid 456 onward. The regions containing the two proteolytic processing sites, which are highly conserved among the Vip3A toxins, are markedly different in Vip3Ba1. The pattern DCCEE (Asp Cys Cys Glu Glu) is repeated four times between position 463 and 483 in Vip3Ba1, generating the sequence 463-DCCEEDCCEEDCCEEDCCEE-483. This sequence, which is rich in negatively charged amino acids, also contains 73% of the cysteines present in Vip3Ba1. This repeated sequence is not present in Vip3A proteins. The Vip3Ba1protein was produced in Escherichia coli and tested against Ostrinia nubilalis and Plutella xylostella, and it generated significant growth delays but had no larvicidal effect, indicating that its host range might be different than that of Vip3A proteins.     

Novel Vip3-Related Protein from Bacillus thuringiensis

A novel vip3-related gene was identified in Bacillus thuringiensis. This novel gene is 2,406 bp long and codes for a 91-kDa protein (801 amino acids). This novel protein exhibits between 61 and 62% similarity with Vip3A proteins and is designated Vip3Ba1. Vip3Ba1 has several specific features. Differences between Vip3Ba1 and the Vip3A proteins are spread throughout the sequence but are more frequent in the C-terminal part from amino acid 456 onward. The regions containing the two proteolytic processing sites, which are highly conserved among the Vip3A toxins, are markedly different in Vip3Ba1. The pattern DCCEE (Asp Cys Cys Glu Glu) is repeated four times between position 463 and 483 in Vip3Ba1, generating the sequence 463-DCCEEDCCEEDCCEEDCCEE-483. This sequence, which is rich in negatively charged amino acids, also contains 73% of the cysteines present in Vip3Ba1. This repeated sequence is not present in Vip3A proteins. The Vip3Ba1protein was produced in Escherichia coli and tested against Ostrinia nubilalis and Plutella xylostella, and it generated significant growth delays but had no larvicidal effect, indicating that its host range might be different than that of Vip3A proteins.     

Characterization of a Novel Strain of Bacillus thuringiensis

Bacillus thuringiensis is a well-known species of entomopathogenic bacteria that is widely used as a biopesticide against many insect pests. Insecticidal proteins, coded for by genes located in plasmids, form typical parasporal, crystalline inclusions during sporulation. In this report, an unusual strain of B. thuringiensis subserovar oyamensis (LBIT-113), isolated from living larvae of Anopheles pseudopunctipennis in Mexico, was characterized by its ultrastructure, the protein composition of its parasporal crystal, plasmid pattern, and toxicological properties against several insect and noninsect targets. The parasporal crystal is enclosed within the spore's outermost envelope (exosporium), as determined by transmission electron microscopy, and exhibits a square, flat shape. Its main components are two proteins with sizes of 88 and 54 kDa. Despite some crystal morphology resemblance, both proteins are immunologically unrelated to the Cry IIIA protein, as shown by immunoblot analysis, when probed with antisera raised against the 88-kDa protein and the Cry IIIA protein. Partial N-terminal sequence of the 88-kDa protein revealed a unique amino acid arrangement among the Cry proteins. Solubilization of the crystal proteins was achieved at 3.3 M NaBr, and its digestion with trypsin showed only one ca. 60-kDa peptide, as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The patterns of three plasmids of strain LBIT-113 were considerably different from those of B. thuringiensis subspp. kurstaki, tenebrionis, and israelensis. Parasporal crystals showed no toxicity to larvae of four species of caterpillar, three species of mosquito, two species of beetle, one species of cricket, one species of ant, one species of aphid, one species of nematode, one species of ostracod, one species of ameba, and one species of rotifer.     

Novel fermentation media for production of Bacillus thuringiensis subsp. israelensis

The production of Bacillus thuringiensis subsp. israelensis (deBarjac) (Bti) as a biopesticide is not cost-effective using existing fermentation technology. In this study, we explored the use of several less expensive alternative culture media (potato, common sugar, and Bengal gram) for the growth and production of Bti. Growth was obtained in all tested media and was comparable to that obtained in conventional medium (Luria-Bertani). Toxicity assays showed that the toxin produced from the novel growth media were effective in killing larvae of Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti and toxicity was comparable to that produced from Luria-Bertani medium. These observations suggest that potato can be used as a cheap source of culture medium for the production of Bti toxin in mosquito control programs.     

Distribution of cryV-type insecticidal protein genes in Bacillus thuringiensis and cloning of cryV-type genes from Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. entomocidus.

DNA dot blot hybridizations with a cryV-specific probe and a cryI-specific probe were performed to screen 24 Bacillus thuringiensis strains for their cryV-type (lepidopteran- and coleopteran-specific) and cryI-type (lepidopteran-specific) insecticidal crystal protein gene contents, respectively. The cryV-specific probe hybridized to 12 of the B. thuringiensis strains examined. Most of the cryV-positive strains also hybridized to the cryI-specific probe, indicating that the cryV genes are closely related to cryI genes. Two cryV-type genes, cryV1 and cryV465, were cloned from B. thuringiensis subsp. kurstaki HD-1 and B. thuringiensis subsp. entomocidus BP465, respectively, and their nucleotide sequences were determined. The CryV1 protein was toxic to Plutella xylostella and Bombyx mori, whereas the CryV465 protein was toxic only to Plutella xylostella.     

Construction of plasmid vectors for gene cloning in Escherichia coli and Bacillus subtilis

The construction and some properties of new hybrid plasmids which are able to replicate in both Escherichia coli and Bacillus subtilis are presented. A 5.5 Md hybrid plasmid pJP9 was constructed from pBR322 (Tc, Ap) and pUB110 (Nm) plasmids. pIM1 (7.0 Md) and pIM3 (7.7 Md) plasmids are its different erythromycin resistant derivatives. Tetracycline, ampicillin, neomycin and possibly erythromycin resistance genes are expressed in E. coli while neomycin and erythromycin resistance genes are expressed in B. subtilis. Insertional inactivation of only one gene is possible using the pJP9 plasmid as a vector in B. subtilis. However, insertional inactivation of at least two different genes can be achieved and monitored in E. coli and B. subtilis transformants in cloning experiments with PIM1 and pIM3 plasmids. Insertional inactivation of antibiotic resistance genes present in pJP9 plasmid was achieved by cloning of Streptococcus sanguis DNA fragments generated by appropriate restriction endonucleases. The pJP9 plasmid and its derivatives were found to be stable in both hosts cells.     

New Strategy for Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.     

Molecular cloning of the delta-endotoxin gene of Bacillus thuringiensis var. israelensis

A transformant of Bacillus megaterium, VB131, was isolated which carries a 6.3-kb XbaI segment of the crystal toxin gene of Bacillus thuringiensis var. israelensis (BTI) cloned in a vector plasmid pBC16 to yield pVB131. The chimeric plasmid DNA from VB131 was introduced into a transformable Bacillus subtilis strain by competence transformation. Both the B. megaterium VB131 strain and the B. subtilis strain harboring the chimeric plasmid produced irregular, parasporal, phase-refractile, crystalline inclusions (Cry+) during sporulation. The sporulated cells as well as the isolated crystal inclusions of the pVB131-containing B. megaterium and B. subtilis strains were highly toxic to the larvae of Aedes aegypti. Also, the solubilized crystal protein preparation from VB131[pVB131] showed clear immuno cross-reaction with antiserum to the BTI crystal toxin. 32P-labeled pVB131 plasmid DNA showed specific hybridization with a 112-kb plasmid DNA of Cry+ strains of BTI, and no hybridization with other plasmid or chromosomal DNA of either Cry+ or Cry- variants. These results are in agreement with our previous findings (González and Carlton, 1984) that the 112-kb plasmid of BTI is associated with the production of the crystal toxin.     

Construction of improved bacteriophage phi 105 vectors for cloning by transfection in Bacillus subtilis

A series of improved phage vectors have been constructed, based on Bacillus subtilis bacteriophage phi 105, which can be used to clone genes in B. subtilis by direct transfection of protoplasts. The new vectors, designated phi 105J23, phi 105J24, phi 105J27 and phi 105J28, show frequencies of plaque formation that are equal to those of wild-type phi 105. This represents at least a 10-fold improvement over phi 105J9, the vector used in previous cloning experiments. Two of the new vectors phi 105J27 and phi 105J28 incorporate a mutation, cts-52, that renders the prophage temperature inducible. This has made it possible to devise a rapid small-scale procedure for screening progeny phage for the presence of inserted DNA. The usefulness of the new vectors is illustrated in the accompanying paper by cloning more than 20 B. subtilis sporulation genes.     

含质粒复制起始区ori44的苏云金芽胞杆菌解离载体的构建

将苏云金芽胞杆菌转座子Tn4430的解离酶识别位点res分别插入克隆载体pRSET B和pUC19得到质粒pBMB1201和pBMB1202。这两个质粒分别经BamHI/Hin dⅢ和EcoRI/HindⅢ双酶切回收含res位点的小DNA片段,与穿梭载体pHT3101经EcoRI/HindⅢ双酶切后加收的含大肠杆菌复制起始区、氨苄青霉素抗性基因和红霉素抗性基因的3．3kb片段连接,获得重组质粒pBMB1203。封闭pBMB1203两res位点外的BamHI和EcoRI位点后,得到解离载体pBMB1204。将来源于苏云金芽胞杆菌库斯塔克亚种YBT－1520的质粒复制起始区ori44片段插入pBMB1204的两res位点之间,得到解离穿梭载体pBMB1205。该解离载体插入壮观霉素抗性基因后电转化无晶体突变株,在辅助质粒所提供的解离酶作用下可发生解离消除抗性基因,解离频率为100％,解离后的质粒稳定性为93％。利用解离穿梭载体pBMB1205可在用抗性筛选到转化子后特定消除抗性标记基因和其它非苏云金芽胞杆菌DNA片段。     

Molecular Characterization of Novel Serovars of Bacillus thuringiensis Isolates from India

Novel Bacillus thuringiensis isolates GS4, GN24 and UP1 were isolated and characterized by determination of serotyping, insecticidal protein by SDS-PAGE, plasmid composition, cry gene content and insect toxicity. Serologically two isolates GS4 and UP1 were allocated to the H3abce which is a new serovar while isolate GN24 was of H3ab type. Isolate GS4 produced flat crystal inclusions while UP1 produced cuboidal crystals. PCR analysis found that both isolates contained cry1 and cry1Ac genes. The major protein bands found of isolate GS4 were of molecular weights 175, 135, 97, 88, 66, 54 and 27 kDa, isolate UP1 were of 85, 60 and 40 kDa and isolate GN24 were of 130, 90, 66 and 45 kDa. Though isolates GS4 and UP1 belonged to a new serovar H3abce, they showed different crystal inclusions and cry gene content. Isolate GS4 was toxic to lepidopteran insect larvae of Helicoverpa armigera but UP1 did not showed any toxicity.     

A micromethod for serotyping Bacillus thuringiensis

P. LAURENT, H. RIPOUTEAU, V. COSMAO DUMANOIR, E. FRACHON AND M.-M. LECADET. 1996. Serotyping of Bacillus thuringiensis is possible using 96-well microplates instead of tubes. The advantages are a reduction on the incubation time from 120 to 75 min and the amounts of antisera and bacterial suspensions needed 10-fold.