Enhanced expression of a second mosquito larvicidal gene fromB.sphaericus 1593M inE.coli

Summary Enhanced expression of a second mosquito larvicidal gene fromB.sphaericus 1593M inE.coli has been achieved by the recloning of the DNA fragment encoding for larvicidal activity previously reported by us, in a pMal vector system. The potency of this recombinant strain was only 10 fold lower than the parentalB.sphaericus 1593M strain. The protein encoded was different from the previously reported larvicidal gene products ofB.sphaericus. Neverthelesss, this protein is recognized by the antiserum raised against crystal proteins. This result has indicated the presence of multiple mosquito larvicidal genes inB.sphaericus, a situation similar to that encountered withB.thuringiensis toxins.     

Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis.

Integrative plasmids were constructed to enable integration of foreign DNA into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Integration of the aphA3 kanamycin resistance gene by a two-step procedure demonstrated that this strategy was applicable with antibiotic resistance selection. Hybridization experiments evidenced two copies of the operon encoding the binary toxin from B. sphaericus in the recipient strain. The Bacillus thuringiensis subsp. israelensis cry11Aal gene (referred to as cry11A), encoding a delta-endotoxin with toxicity against Culex, Aedes, and Anopheles larvae, was integrated either by a single crossover event [strain 2297 (::pHT5601), harboring the entire recombinant plasmid] or by two successive crossover events [strain 2297 (::cry11A)]. The level of the Cry11A production in B. sphaericus was high; two crystalline inclusions were produced in strain 2297 (::pHT5601). Synthesis of the Cry11A toxin conferred toxicity to the recombinant strains against Aedes aegypti larvae, for which the parental strain was not toxic. Interestingly, the level of larvicidal activity of strain 2297 (::pHT5601) against Anopheles stephensi was as high as that of B. thuringiensis subsp. israelensis and suggested synergy between the B. thuringiensis and B. sphaericus toxins. The toxicities of parental and recombinant B. sphaericus strains against Culex quinquefasciatus were similar, but the recombinant strains killed the larvae more rapidly. The production of the Cry11A toxin in B. sphaericus also partially restored toxicity for C. quinquefasciatus larvae from a population resistant to B. sphaericus 1593. In vivo recombination therefore appears to be a promising approach to the creation of new B. sphaericus strains for vector control.     

Interpreting the SDS-PAGE protein patterns with self-organizing maps: application for the characterization of mosquito-pathogenic Bacillus strains

Aims: To present the pairwise comparison of potential mosquito‐pathogenic Bacillus strains based on their SDS‐PAGE protein patterns and to evaluate their characteristic toxicity patterns. Methods and Results: In this work, 20 Bacillus strains were subjected to qualitative toxicity tests against Aedes aegypti and Culex quinquefasciatus larvae. The selected strains were then characterized by SDS‐PAGE protein profiles. The highly heterogeneous multiple protein components of protein patterns were analysed using self‐organizing map (SOM), a ‘visualization and clustering’ tool. Members of mosquitocidal Bacillus species were classified in four distinct clusters, and then toxicity patterns were examined. Cluster (1, 1) comprised of three highly toxic strains of Bacillus sphaericus: SPH88, 1593 and KSD‐4; cluster (1, 2) consisted of two B. sphaericus strains: SSII‐1 and Bsp‐R that showed weak larvicidal activity; cluster (2, 1) constituted two B. sphaericus strains: WHO2297 and ISPC‐5 that possessed moderate toxicity; and cluster (2, 2) contained four B. thuringiensis ssp. israelensis strains: ONR‐60A, HD500, IPS70 and IPS82 belonging to serotype H14 but exhibited moderate to high mosquito larvicidal toxicity. Conclusions: SOM served as a colour‐coded alternate for easy visualization of similarities or dissimilarities between the strains even at the infra subspecies level. Furthermore, characteristic toxicity patterns of Bacillus strains of different clusters were determined. Significance and Impact of the Study: Analysis of electrophoretic protein patterns using SOM provides a better insight into the inter‐relationships of bacterial strains through similarity‐based clustering and pairwise comparison of two strains.     

Initial studies of the use of aminopeptidases for the differentiation ofBacillus sphaericus strains

Summary Analysis of aminopeptidase activity in commercially available enzyme panels (SAP-II and SAP-III) indicated qualitative differences among the eight strains ofBacillus sphaericus tested. These differences involved specific substrates utilized, the importance of sampling time, as well as differences between whole cells and cell-free extracts. Further detailed analysis of four of thel-amino acid--naphthylamide substrates using polyacrylamide gel electrophoresis (PAGE) demonstrated a series of allelic differences in the multilocus patterns observed. The resulting phenogram constructed from the enzyme and PAGE information showed a marked separation of the noninsecticidal strains from the insecticidal strains (58% similarity). In addition, there was a separation within the insecticidal strains of strain SSII-1, phage type 2 from strain 2297, phage type 4, and of both of these strains from the remaining highly insecticidal members of phage type 3 (strains 1593, 2013-4, 2362, 2500).     

Growth,sporulation and toxin production byBacillus thuringiensis subsp.israelensis andB. sphaericus in media based on mustard-seed meal

Bacillus thuringiensis subsp.israelensis andB. sphaericus strains 2362 and 1593 were grown in media based on defatted mustard-seed meal (MSM). The meal contains 40% (w/w) protein, with glutamic acid and arginine as the major amino acids. The toxic potencies of the final bacterial powders towardsCulex pipens quinquefasciatus Say, compared with those of the respective international reference standards, were 46% forB. thuringiensis subsp.israelensis, 62% forB. sphaericus 2362 and 88% forB. sphaericus 1593 when 2% (w/v) MSM was used for growth. With 4% (w/v) MSM,B. thuringiensis subsp.israelensis grew better but had undetectable larvicidal activity, whereas theB. sphaericus strains not only grew better but gave a higher degree of sporulation and toxicity. The potencies ofB. sphaericus in medium with 4% MSM were comparable with those of international reference standards.The authors are with the Department of Life Sciences, University of Bombay, Bombay 400 098, India.     

Effects of Bacillus sphaericus 1593 and 2362 spore/crystal toxin on cultured mosquito cells

An in vitro assay system for the toxin of Bacillus sphaericus strains 1593 and 2362 has been developed utilizing cultured Culex quinquefasciatus cells. The cytotoxic activity of extracts of B. sphaericus strain 1593 did not necessarily correlate with insecticidal activity. Cytotoxicity and larvicidal activity were neutralized by immune rabbit serum prepared against crude toxin extracts as well as by serum prepared against purified toxin from strain 2362. This purified toxin was also found to be cytotoxic. Activation with mosquito larval gut homogenates enhanced cytotoxicity of both 1593 extracts and purified toxin from 2362. The activity of cytotoxic preparations against three mosquito cell lines paralleled the activity of B. sphaericus spores against larvae of these mosquito species. The results suggest the presence of a protoxin and one or more cytotoxic proteins derived from it.     

Metabolic response of Bacillus sphaericus 1593M for dual-substrate limitation in continuous and total-cell-retention cultures

A chemically defined medium has been developed to support the growth and the production of mosquito larvicidal factor(s) (MLF) of Bacillus sphaericus 1593M. On the basis of the data of steady-state continuous cultures, it has been understood that acetate can serve as a sole carbon and energy source for B. sphaericus 1593M. Utilization of acetate by B. sphaer-icus 1593M and the production of MLF are further enhanced by the addition of glutamate at low concentrations, both in steady-state continuous as well as in total-cell-retention cultures (TCRC). A two-step TCRC procedure resulted in better biomass and MLF production by B. sphaericus 1593M. It was also found that glutamate can serve as a carbon source as well as a growth factor in the presence of acetate and hence is a partially substitutable carbon source. Received: 3 January 1997 / Accepted: 31 January 1997     

Efficacy of dry powders from Bacillus sphaericus: RB 80, a potent reference preparation for biological titration

Larvicidal potency of three primary powders based on Bacillus sphaericus strains 1593 and 1881 was studied on mosquito larvae. Two acetone powders, P 1593 and P 1881, were very toxic for Anopheles stephensi larvae. The potency of a third lyophilized powder RB 80 made from 1593 strain compared even better when tested against Anopheles stephensi and Culex pipiens pipiens larvae. LC₅₀'s after 48 hr were 0.15 and 0.003 mg/ml, respectively. After storage of RB 80 aqueous suspensions over 2 years or after heat exposure of RB 80 powder, larvicidal potency was still high, indicating an excellent stability. The use of RB 80, because of all its qualities, is suggested as a first experimental standard for titration of B. sphaericus preparations.     

Various Levels of Cross-Resistance to Bacillus sphaericus Strains in Culex pipiens (Diptera: Culicidae) Colonies Resistant to B. sphaericus Strain 2362

We studied the cross-resistance to three highly toxic Bacillus sphaericus strains, IAB-59 (serotype H6), IAB-881 (serotype H3), and IAB-872 (serotype H48), of four colonies of the Culex pipiens complex resistant to B. sphaericus 2362 and 1593, both of which are serotype H5a5b strains. Two field-selected highly resistant colonies originating from India (KOCHI, 17,000-fold resistance) and France (SPHAE, 23,000-fold resistance) and a highly resistant laboratory-selected colony from California (GeoR, 36,000-fold resistance) showed strong cross-resistance to strains IAB-881 and IAB-872 but significantly weaker cross-resistance to IAB-59 (3- to 43-fold resistance). In contrast, a laboratory-selected California colony with low-level resistance (JRMM-R, 5-fold resistance) displayed similar levels of resistance (5- to 10-fold) to all of the B. sphaericus strains tested. Thus, among the mosquitocidal strains of B. sphaericus we identified a strain, IAB-59, which was toxic to several Culex colonies that were highly resistant to commercial strains 2362 and 1593. Our analysis also indicated that strain IAB-59 may possess other larvicidal factors. These results could have important implications for the development of resistance management strategies for area-wide mosquito control programs based on the use of B. sphaericus preparations.     

The comparative persistence of toxicity of Bacillus sphaericus strain 1593 and Bacillus thuringiensis serotype H-14 against mosquito larvae in different kinds of environments

Bacillus sphaericus strain 1593 and B. thuringiensis serotype H-14 were evaluated for persistence of toxicity against two species of mosquito larvae, Culex quinquefasciatus and Aedes aegypti, in a selected simulating plot in Bangkok. Both strains of bacteria demonstrated larvicidal activity towards both species of mosquito larvae. In tap water, the toxicity of B. sphaericus strain 1593 was found to be greater towards C. quinquefasciatus larvae than A. aegypti larvae, whereas the toxicity of B. thuringiensis serotype H-14 was found to be greater towards A. aegypti larvae than C. quinquefasciatus larvae. The persistence of toxicity of these two bacteria was found to be different. The lethal concentration of B. thuriengiensis H-14 against A. aegypti decreased from LC₉₀ to below LC₅₀ in about 15 weeks when tested in tap water. The decrease was faster in polluted water. The toxicity of B. sphaericus 1593 towards C. quinquefasciatus larvae persisted for at least 9 months in tap water and 6 months in polluted water. The multiplication of bacteria was indicated only in populations of B. sphaericus 1593 tested with C. quinquefasciatus larvae.     

Biocide gene(s) and biocidal activity in different strains of Bacillus sphaericus. Expression of the gene(s) in E. coli maxicells

Summary The recently cloned biocidal determinant of the highly toxic strain of B. sphaericus 1593M (Ganesan et al. 1983) was used as probe to investigate homologous sequences in different toxic and non-toxic strains of B. sphaericus. It was found that the potent strains we have analysed are characterised by the presence of DNA sequences (6.6, 6.4, 5.8, 1.6, 1.3 and 0.6 Kb) not found in the non-toxic strains. These results further show that one of the two weakly toxic strains analysed presents a hybridisation pattern completely different from that observed with the highly potent strains of B. sphaericus. When the DNA of the two non-toxic strains was analysed, SSII-I failed to hybridise to the probe and Rem4 exhibited mainly one hybridisable sequence of 2.3 Kb not detectable in the toxic strains.No region of homology to the probe was found in the DNA of two strains of B. thuringiensis (var. berliner and var. israeliensis) analysed.By dot blot hybridisation experiments it was estimated that the larvicidal determinant might be present in about one to three copies per genome.With the use of E. coli maxicells we have shown further that the toxin gene(s) encoded four polypeptides with molecular weights of 21, 19, 15, and 12 Kd. The significance of these findings is discussed.     

Production of Cry11A and Cry11Ba Toxins in Bacillus sphaericus Confers Toxicity towards Aedes aegypti and Resistant Culex Populations

Cry11A from Bacillus thuringiensis subsp. israelensis and Cry11Ba from Bacillus thuringiensis subsp. jegathesan were introduced, separately and in combination, into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Two loci on the B. sphaericus chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against Aedes aegypti and Culex pipiens. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to A. aegypti larvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible C. pipiens larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of C. pipiens SPHAE and Culex quinquefasciatus GeoR to B. sphaericus strain 2297.     

Effect of corn-steep liquor on growth and mosquito larvicidal activity of Bacillus thuringiensis var israelensis de Barjac 1978 and B. sphaericus Neide 1904.

Influence of corn steep liquor on the cell yield and toxicity of three strains of B. thuringiensis var israelensis and two strains of B. sphaericus was studied and compared with peptone-yeast extract using a laboratory fermentor. Large increase in the cell yield of all the three strains of B. thuringiensis var israelensis was observed when cornsteep liquor was used as the sole nitrogen source. Significant increase in toxicity was also observed in B. thuringiensis var israelensis strains B17 and B113. Among the two B. sphaericus strains tested, the strain 1593 showed no significant change in cell yield and toxicity, whereas the strain VCRC B42 showed increased cell yield and toxicity in this medium. The results indicate that cornsteep liquor can effectively replace both peptone and yeast extract in the media presently used for large scale multiplication of the two larvicidal bacilli.     

Hyper-Production of Insecticidal Crystal Protein (δ-Endotoxin)by Bacillus thuringiensis var. israelensis Is Not Related toSporulation-Specific Biochemical Functions

Hypertoxic mutant strains of Bacillus thuringiensis var. israelensis were isolated by mutagenesis of the parent strain. The correlation, if any, between hyper-production of insecticidal crystal protein (δ-endotoxin) by hypertoxic mutant strains of Bacillus thuringiensis var. israelensis and sporulation-specific biochemical functions was studied. No increase in sporulation-specific biochemical markers was observed in the hypertoxic mutant strains. Asporogenous mutants of hypertoxic mutant strains blocked at different stages of sporulation were isolated, and larvicidal activity was studied. The hypertoxic parent strains and the sporulation-deficient, hypertoxic mutant strains showed almost identical larvicidal activity. Therefore, the increased production of toxin is not related to sporulation-specific biochemical changes. Received: 14 February 2000 / Accepted: 21 March 2000     

Evaluation of <Emphasis Type="Italic">Volvariella volvacea</Emphasis> Strains for Yield and Diseases/Insect-Pests Resistance Using Composted Substrate of Paddy Straw and Cotton Mill Wastes

Out of the 3 parent strains and 4 single spore isolates of Volvariella volvacea evaluated, strain, OE-274 gave earliest yield in 11.25–11.50 days post-spawning in all 4 trials. The yield varied in different strains in different trials and it was highest in strain, OE-272 in trial 1, SSI, OE-55-08 in trial 2, and strain, OE-274 in trial 3 and 4. In overall average, highest yield was in strain, OE-272, closely followed by strain, OE-274. The number of fruiting bodies per q substrate also varied in different strains in different trials. Highest numbers were in strain, OE-272, SSIs, OE-55-08 and OE-12-22, and strain, OE-210 in trial 1, 2, 3 and 4, respectively. Highest fruiting body wt was in strain, OE-274 in all 4 trials. The yield during different weeks of cropping varied in different strains but invariably it was highest in first week, which accounted for 60–70% of the total yield. The fruiting bodies of strain, OE-274 were of bigger size, brownish, toughest and with least tendency of veil opening, while that of strain, OE-272 and SSI, OE-55-08 were whitish to grayish-white, oblong, medium size, delicate and lesser tendency of veil opening. The strain, OE-274 and SSI, OE-55-08 exhibited higher resistance against the growth of competitor moulds and infestations of insect-pests, while strain, OE-272 exhibited highest susceptibility to insect-pests infestation.     

Characterization of Six Highly Mosquitocidal Bacillus thuringiensis Strains That Do Not Belong to H-14 Serotype

Four strains belonging to Bacillus thuringiensis serovars thompsoni, malaysiensis, canadensis, jegathesan and two auto-agglutinating B.t. strains were identified as being highly toxic to the mosquito larvae of the species Aedes aegypti, Anopheles stephensi, and Culex pipiens. Their larvicidal and hemolytic activities were determined and compared with those of strains known to be highly mosquitocidal and/or cytolytic from serovars of B.t. israelensis, morrisoni, darmstadiensis, medellin, kyushuensis, and fukuokaensis. The electrophoretic protein profiles of purified crystals and immunological relationships with B.t.i. polypeptides were studied. Five out of the six new strains showed the same larvicidal and hemolytic activities and the same crystal proteins and toxin genes as B.t.i. One strain, B.t. jegathesan 367, presented a novel pattern of larvicidal activity and a protein profile different from those of other strains.     

Phenol degradation by immobilized cold-adapted yeast strains of Cryptococcus terreus and Rhodotorula creatinivora

Three strains were isolated from hydrocarbon-polluted alpine habitats and were representatives of Cryptococcus terreus (strain PB4) and Rhodotorula creatinivora (strains PB7, PB12). All three strains synthesized and accumulated glycogen (both acid- and alkali-soluble) and trehalose during growth in complex medium containing glucose as carbon source and in minimal salt medium (MSM) with phenol as sole carbon and energy source. C. terreus strain PB4 showed a lower total accumulation level of storage compounds and a lower extracellular polysaccharides (EPS) production than the two R. creatinivora strains, PB7 and PB12. Biofilm formation and phenol degradation by yeast strains attached to solid carriers of zeolite or filter sand were studied at 10°C. Phenol degradation by immobilized yeast strains was always higher on zeolite compared with filter sand under normal osmotic growth conditions. The transfer of cells immobilized on both solid supports to a high osmotic environment decreased phenol degradation activity by all strains. However, both R. creatinivora PB7 and PB12 strains maintained higher ability to degrade phenol compared with C. terreus strain PB4, which almost completely lost its phenol degradation activity. Moreover, R. creatinivora strain PB7 showed the highest ability to form biofilm on both carriers under high osmotic conditions of cultivation.     

Various levels of cross-resistance to Bacillus sphaericus strains in Culex pipiens (Diptera: Culicidae) colonies resistant to B. sphaericus strain 2362.

We studied the cross-resistance to three highly toxic Bacillus sphaericus strains, IAB-59 (serotype H6), IAB-881 (serotype H3), and IAB-872 (serotype H48), of four colonies of the Culex pipiens complex resistant to B. sphaericus 2362 and 1593, both of which are serotype H5a5b strains. Two field-selected highly resistant colonies originating from India (KOCHI, 17,000-fold resistance) and France (SPHAE, 23,000-fold resistance) and a highly resistant laboratory-selected colony from California (GeoR, 36,000-fold resistance) showed strong cross-resistance to strains IAB-881 and IAB-872 but significantly weaker cross-resistance to IAB-59 (3- to 43-fold resistance). In contrast, a laboratory-selected California colony with low-level resistance (JRMM-R, 5-fold resistance) displayed similar levels of resistance (5- to 10-fold) to all of the B. sphaericus strains tested. Thus, among the mosquitocidal strains of B. sphaericus we identified a strain, IAB-59, which was toxic to several Culex colonies that were highly resistant to commercial strains 2362 and 1593. Our analysis also indicated that strain IAB-59 may possess other larvicidal factors. These results could have important implications for the development of resistance management strategies for area-wide mosquito control programs based on the use of B. sphaericus preparations.     

The Introduction into Bacillus sphaericus of the Bacillus thuringiensis subsp. medellin cyt1Ab1 Gene Results in Higher Susceptibility of Resistant Mosquito Larva Populations to B. sphaericus

The fragment containing the gene encoding the cytolytic Cyt1Ab1 protein from Bacillus thuringiensis subsp. medellin and its flanking sequences (I. Thiery, A. Delécluse, M. C. Tamayo, and S. Orduz, Appl. Environ. Microbiol. 63:468–473, 1997) was introduced into Bacillus sphaericus toxic strains 2362, 2297, and Iab872 by electroporation with the shuttle vector pMK3. Only small amounts of the protein were produced in recombinant strains 2362 and Iab872. The protein was detected in these strains only by Western blotting and immunodetection with antibody raised against Cyt1Ab1 protein. Large amounts of Cyt1Ab1 protein were produced in B. sphaericus recombinant strain 2297, and there was an additional crystal, other than that of the binary toxin, within the exosporium. The production of the Cyt1Ab1 protein in addition to the binary toxin did not increase the larvicidal activity of the B. sphaericus recombinant strain against susceptible mosquito populations of Culex pipiens or Aedes aegypti. However, it partially restored (10 to 20 times) susceptibility of the resistant mosquito populations of C. pipiens (SPHAE) and Culex quinquefasciatus (GeoR) to the binary toxin. The Cyt1Ab1 protein produced in recombinant B. thuringiensis SPL407(pcyt1Ab1) was synthesized in two types of crystal—one round and with various dense areas, surrounded by an envelope, and the other a regular cuboid crystal, very similar to that found in the B. sphaericus recombinant strain.     

Screening of highly cellulolytic fungi and the action of their cellulase enzyme systems

Over 100 strains of wood-rotting fungi were compared for their ability to degrade wood blocks. Some of these strains were then assayed for extracellular cellulase [1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] activity using a variety of different solid media containing carboxymethyl cellulose or acid swollen cellulose. The diameter of clearing on these plates gave an approximate indication of the order of cellulase activities obtained from culture filtrates of these strains. Trichoderma strains grown on Vogels medium gave the highest cellulase yields. The cellulase enzyme production of T. reesei C30 and QM9414 was compared with that of eight other Trichoderma strains. Trichoderma strain E58 had comparable endoglucanase and filter paper activities with the mutant strains while the β- -glucosidase [β- -glucoside glucohydrolase, EC 3.2.1.21] activity was approximately six to nine times greater.