Characterization of mosquito larvicidal parasporal inclusions of a Bacillus thuringiensis serovar higo strain

The parasporal inclusion proteins of the type strain of Bacillus thuringiensis serovar higo (H44), that have moderate mosquitocidal activity, were characterized. The purified parasporal inclusions, spherical in shape, were examined for activity against the two mosquito species, Culex pipiens molestus and Anopheles stephensi and the moth-fly, Telmatoscopus albipunctatus . The LC₅₀ values of the inclusion for the two mosquitoes were 3·41 and 0·15 μg ml⁻¹, respectively. No mortality was shown for T. albipunctatus larvae by the inclusions at concentrations up to 1 mg ml⁻¹. Solubilized parasporal inclusions exhibited no haemolytic activity against sheep erythrocytes. Parasporal inclusions consisted of eight proteins with molecular masses of 98, 91, 71, 63, 59, 50, 44 and 27 kDa. Of these, the 50 and 44 kDa proteins were the major components. Analysis with immunoblotting revealed that, among several inclusion proteins of B. thuringiensis serovar israelensis, only two proteins of 130 kDa and 110 kDa reacted weakly with antibodies against higo proteins. N-terminal amino acid sequences of the 98, 91, and 71 kDa proteins showed 85–100% identity to those of the two established Cry protein classes, Cry4A and Cry10A.     

Naturally occurring Bacillus thuringiensis in Indonesia

S. HASTOWO, B.W. LAY AND M. OHBA. 1992. A total of 135 strains of Bacillus thuringiensis were isolated from soils of sericultural and natural environments of various regions in Indonesia: Sumatra, Java, Bali, Timor, Sulawesi and Kalimantan. Serologically, 63 strains were assigned to flagella (H) serotypes 3abc, 3ade, 4ac, 5ac, 6ab, 8ab, 9, 11, 17, 20ac, and 24, indicating a varied flora of B. thuringiensis in Indonesia. Of these, the serotype 3ade predominated, followed by the serotypes 3abc and 6ab. The other 72 strains (53·3%) were not sero-reactive against any of the H antisera to B. thuringiensis H serotypes 1–24. In toxicity tests, 34 strains belonging to serotypes 3abc, 3ade, 4ac, and 8ab showed larvicidal activity to the silkworm, Bombyx mori, while 74·8% did not exhibit insecticidal activity against larvae of B. mori and the mosquitoes, Aedes aegypti and Culex quinquefasciatus. Morphotypes of parasporal inclusions were not correlated with insecticidal activities.     

Characterization of Bacillus thuringiensis serovar bolivia (serotype H63), a novel serovar isolated from the Bolivian high valleys

The type strain Bacillus thuringiensis var. bolivia (serotype H63), isolated from the Bolivian high valleys, has been characterized at different levels. Its parasporal crystal has an unusual shape and it is composed of a protein of 155 kDa which shows two bands of 75 and 80 kDa after activation. Analysis by PCR shows the presence of cry1 genes, and amplification with specific primers gave products for cry1 E, cry1 D, cry4 A and cry4 B with sizes different to those expected. Immunoblotting tests showed positive reaction for Cry1 E, Cry3 A, Cry4 A and Cry11 A crystal proteins. The plasmid pattern revealed two large and two small plasmids. Toxicity tests were performed against 14 insects and a slight toxicity was found against Plutella xylotella and Trichoplusia ni.     

Anti-Helicobacter pylori activity of Lactobacillus delbrueckii subsp. bulgaricus strains: preliminary report

Aims:  To evaluate the activities of six Lactobacillus delbrueckii subsp. bulgaricus (LB) strains against 30 Helicobacter pylori strains by agar-well diffusion method.
Methods and Results:  LB cultures [4 × 10⁸–4 × 10⁹ CFU ml⁻¹) either were prepared in milk at their native pH, 3·8–5·0, or were adjusted to pH 6·4–7·7. At low and neutralized pH, LB strains inhibited the growth by 40–86·7% and 16·7–66·7% of H. pylori strains, respectively. LB activity was strain-dependent. At low and neutralized pH, one and five H. pylori strains, respectively, were not inhibited by any LB strain. LB2 and LB3, taken together, were active against most metronidazole and clarithromycin resistant strains.
Conclusions:  All LB strains inhibited a number of H. pylori strains, including also antibiotic resistant strains. LB activity was strain-dependent and better at low pH. At low pH values, the most active LB strains were LB1, LB2 and LB3, inhibiting 86·7% of H. pylori strains, while at neutralized pH values, the most active LB strains were LB2 and LB3, inhibiting 53·3 and 66·7% of H. pylori strains, respectively.
Significance and Impact of the Study:  LB could be utilized in the treatment or prophylaxis of H. pylori infection and warrants clinical investigations.     

Characterization of Bacillus thuringiensis ser. jordanica (serotype H71), a novel serovariety isolated in Jordan

The novel strain of Bacillus thuringiensis J112 isolated from a soil sample in Jordan was classified and characterized in terms of toxicity against dipteran and nematode larvae, crystal protein pattern, plasmid profile, and cry gene content. A new name, Bacillus thuringiensis serovariety jordanica (H serotype 71), is proposed for the reference strain J112. The parasporal crystal proteins were toxic to 3(rd) instar larvae of Drosophila melanogaster and to 2(nd) stage juveniles of root knot nematodes Meloidogyne javanica and M. incognita, but showed poor mosquitocidal activity towards Culex pipiens molestus and Culiseta longiareolata larvae. Solubilized and trypsin-digested crystal proteins possessed moderate hemolytic activity against sheep erythrocytes. SDS-polyacrylamide gel electrophoresis revealed that crystals are composed of several polypeptides ranging from 24 to 170 kDa, of which the 20-, 42-, 140-, and 170-kDa proteins were the major components. Analysis of the plasmid pattern of J112 revealed the presence of two large plasmidic bands of about 160 and 205 kbp. PCR with total DNA from strain J112 and specific primers for cry1, cry2, cry3, cry4, and cyt2A genes revealed that cry1, cry3A, cry4, cry5 and cyt2a genes are present.     

Crystal proteins from Bacillus thuringiensis serovar. medellin

Colombian strains 163-131 and 24-726 of the Bacillus thuringiensis serovar. medellin (Btmed), serotype H-30, are very toxic to mosquito larvae. Strain 24-726 was serologically and biochemically characterized. It is almost identical to the reference-strain 163-131. The parasporal inclusion of Btmed strain 163-131 was analysed by electron microscopy. The crystal protein matrix was very similar to that observed in B. thuringiensis serovar. israelensis (Bti). Aedes aegypti, Anopheles albimanus and Culex quinquefasciatus larvae were exposed to 500× the half-lethal concentration (LC₅₀) of Btmed strains, Bti strain 1884 and B. thuringiensis serovar. morrisoni (Btm) strain PG-14. Mortality of Aedes aegypti occurred within approx. 60 min with the four strains, whereas C. quinquefasciatus mortality was three times slower with Btmed than with strains 1884 and PG14. The onset mortality of Anopheles albimanus starts when other species are already dead. The thermolabilities of the mosquitocidal activities of the crystal proteins were tested by incubation of cultures of 20 min at various temperatures. Btmed lost all mosquitocidal activity at 73°C, and 1884 and PG-14 at 79°C. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of the crystals purified from strain 163-131 shows polypeptides at 100 kDa, multiple bands at 80, 75, 70, 67, and 65 kDa, and two doubles at 40–41 and 28–30 kDa. Immunodetection with antibodies raised against Bti toxins shows cross-reaction between the 30-kDa and to a lesser extent the 28-kDa polypeptides of Btmed crystals and Cyt A of Bti. A slight response is observed with the 65-kDa Btmed to serum raised against Cry IV D of Bti. Correspondence to: I. Thiéry     

Delta-endotoxin proteins associated with spherical parasporal inclusions of the four Lepidoptera-specific Bacillus thuringiensis strains

Four Lepidoptera-specific reference strains of Bacillus thuringiensis , belonging to serovars sumiyoshiensis (H3a:3d), fukuokaensis (H3a:3d:3e), darmstadiensis (H10a:10b) and japonensis (H23), which produce spherical parasporal inclusions, were examined for comparative characterization of δ-endotoxins. SDS-PAGE profiles of the alkali-solubilized parasporal inclusions revealed the presence of single major protein bands of 130 kDa in the four strains. Chymotrypsin and trypsin treatment of the proteins gave profiles different from those of the strains HD-1 (serovar kurstaki , H3a:3b:3c) and T84 A1 (serovar sotto , H4a:4b). Also, minor variations were observed in proteolysis profiles among the four strains. The LC₅₀ values of purified parasporal inclusions for the silkworm ( Bombyx mori ) larvae were 7·35, 6·45, 3·08 and 2·63 μg g⁻¹ diet, respectively, showing that their toxicity levels were 5–15 times lower than that of the strain HD-1 (0·49 μg g⁻¹ diet). Analysis by immunodiffusion and immunoblotting with polyclonal antisera revealed that parasporal inclusion proteins of the four strains are highly related, whereas they shared few or no common antigens with those of the strains HD-1, T84 A1 and Buibui (serovar japonensis ).     

A comparative study of the extracellular glucosyl- and fructosyltransferases from cariogenic and non-cariogenic Streptococcus mutans strains of two different serotypes

Extracellular proteins from continuous cultures of serotype c and g Streptococcus mutans strains were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Gels stained with raffinose after electrophoresis revealed that although serotype c strains secrete two fructosyltransferases of molecular mass 68 kDa and 79 kDa, no fructosyltransferase was secreted by the serotype g strain K1. A sucrose activity stain was used to detect two glucosyltransferases (GTF) of molecular mass 162 kDa (bifunctional 1,6-alpha-D-glucan 3-alpha- and 6-alpha GTF or 'dextransucrase') and 153 kDa (a 1,3-alpha-D-glucan 3-alpha-GTF) in samples from cariogenic serotype c strains. Neither the 153 kDa protein nor the corresponding GTF activity was secreted by the non-cariogenic mutant C 67-25. The molecular masses of the corresponding 1,3-alpha and 1,6-alpha-GTF proteins from the serotype g strain K1 were 164 kDa and 158 kDa, respectively. All of the GTF proteins were degraded to discrete bands of lower molecular mass on storage at 4 degrees C even after extensive purification. The results provide an explanation for several outstanding controversies in the GTF literature.     

Laboratory growth of larval lampreys (Lampetra (Entosphenus) tridentata Richardson) at different food concentrations and animal densities

Lampreys are important research animals. This study investigates some of the Parameters important for culturing the Suspension feeding larvae: food concentration, temperature and crowding. Large larvae ( Lampetra ( Entosphenus ) tridentata Richardson) were used, weigh-ing from l·5 to 3·0 g (wet). Two food types were employed: suspended yeast cells ( Saccharo-myces cerevisiae , 0–20 mg1 – (dry), or, in a few tests, a fine particulate fish food, Liquifry® (Interpet LTD, 0–13 mg l^-1). At both 14 and 4°C, yeast could sustain weight increases comparable to those in nature: >6% month^-1 for up to 6 months, the duration of the study. In a single lest, a vitamin Supplement failed to improve growth on yeast. Growth-was fastest at 14°C (+41% month^-1, max. weight increase), although also substantial at 4°C (+11% month^-1, max). Growth could not be sustained at 20°C, due perhaps to difficulty in removing products of food decay from the aquaria. Food level being constant, growth rate varied inversely with animal density. It is suggested that larval lampreys release a growth-inhibiting substance into the sand which they inhabit. Overall, the best growth was obtained at 14° C, with <0·05g of animal (wet weight) – aquarium water and average daily yeast concentrations between 4 and 13 mg –. Liquifry was associated with lowered growth rates when present continually above 4 mg (dry weight) – (14° C), although growth did occur at lower concentrations.     

Larval development of the Patagonian brotula Cataetyx messieri (Pisces, Bythitidae) from fjords of southern Chile

Larvae of Cataetyx messieri (Bythitidae) are described from 14 individuals (12·3–27·9 mm body length) collected in deep fjords and channels of the inland sea of southern Chile (41–55° S), between 137 and 604 m depth. The larvae are elongate, with a large, flattened mouth and a coiled gut. They are lightly pigmented and show three distinctive features: a band of melanophores from the snout to the opercle, melanophores spread over the dorsolateral surface of the stomach and the intestine and a band of melanophores on both dorsal and ventral margins of the tip of the tail and adjacent finfold.     

The Isolation and Some Characteristics of Photosynthetic Bacteria (Chromatiaceae and Chlorobiaceae) from Antarctic Marine Sediments

Sediment samples taken at depths from 5 to 50 m in Borge Bay, Signy Island, Antarctica yielded purple ( Chromatiaceae ) and green ( Chlorobiaceae ) phototrophic bacteria when inoculated into Winogradsky columns or directly into Pfennig's medium. Pure cultures of Thiocapsa roseopersicina (2 strains), Thiocapsa pfennigii, Chromatium gracile, Chromatium vinosum, Thiocystis violaceae, Chlorobium limicola, Chlorobium vibrioforme and Prostaecochloris aestuarii were subsequently isolated from these enrichments. Thiocapsa roseopersicina and Chromatium vinosum were the species isolated most commonly. None of the isolates showed evidence of temperature adaptation; optimum growth was achieved between 25–30°C but all grew readily at low white light levels (spectral irradiance 160 μW/cm²) with mean generation times ranging from 8·9 to 14·2 h at 23°C depending upon the isolate. The green phototrophs grew readily in blue light (440–510 nm) of low irradiance (126 μW/cm²) which penetrates to a depth of 40–50 m in coastal waters surrounding Signy Island. Under these conditions Chlorobium limicola and Chlorobium vibrioforme had mean generation times of 22 and 12·2 h respectively compared with 10·3 and 8·9 when grown in white light of similar total irradiance. None of the purple phototrophs grew under blue light but they grew under yellow light (peak transmission 590 nm, irradiance 137 μW/cm²). Chromatium gracile and Thiocapsa roseopersicina had mean generation times of 22·3 and 18·3 h respectively compared with 14·2 and 12·3 h in white light of similar total irradiance.     

Ontogeny of the gastrointestinal tract and selected digestive enzymes in cobia Rachycentron canadum (L.)

The ontogeny of the digestive system of cobia Rachycentron canadum from hatching to 22 days post-hatch (dph) (20·1 mm standard length) was examined with light microscopy. The activities of selected pancreatic enzymes were also determined during this period in order to optimize current rearing methods for this species. At hatching (3·6 mm), the digestive tract consisted of a relatively undifferentiated, straight tube positioned dorsally to the yolk sac. The major morphological changes in the digestive tract primarily occurred over the first 1–4 dph (3·6–4·4 mm). During this time, larvae began exogenous feeding (3 dph) and the digestive tract differentiated into five histologically distinct regions: buccopharynx, oesophagus, stomach anlage, anterior intestine and posterior intestine. Yolk reserves were exhausted by 5 dph (4·5 mm) and the oil globule began rapidly decreasing in size disappearing entirely by 9–10 dph (6·3–6·8 mm). Gastric glands differentiated at this time, and by 12 dph (8·1 mm) surface mucous cells of the stomach anlage stained positive for neutral mucosubstances. By 16 dph (11·6 mm), the blind sac (fundic region) of the stomach formed as did the pyloric caecae which initially appeared as a single protrusion of the anterior intestine just ventral to the pyloric sphincter. Generally, enzyme activities (U larva⁻¹) for amylase (0·0–1·8), chymotrypsin (0·0–7902·4), trypsin (0·2–16·6) and lipase (9·3–1319·0) were measurable at or soon after hatching and increased steadily from c. 8–22 dph (5·7–20·1 mm). The results of this study are discussed in terms of current and future weaning practices of this species.     

Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish

Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, α-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 °C for 15, 30 and 60 min, or 121 °C for 15 min. Lactococcin R remained active after storage at −20 and −70 °C for 3 months and after exposure to a pH of 2–9. The molecular weight of lactococcin R was about 2·5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml⁻¹) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0·5% glucose, at 6·5–7·0 initial pH values, 30 °C temperature and 18–24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6–7 (95%). Crude lactococcin R at 1280 AU ml⁻¹ was bactericidal, reducing colony counts of Listeria monocytogenes by 99·98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2·5 kDa vs 3·4 kDa, and in being sensitive to pepsin and α-chymotrypsin to which nisin is resistant.     

Plasmids coding for heat-labile enterotoxin production isolated from Escherichia coli O78: comparison of properties.

Nineteen enterotoxigenic Escherichia coli strains of serogroup O78, isolated in different geographical areas from humans with diarrheal diseases, were tested for their ability to transfer enterotoxin production. All of the strains originally produced heat-labile enterotoxin, and 16 also produced heat-stable enterotoxin and colonization factor antigen I. Plasmids coding for the production of heat-labile enterotoxin only were transferred from 13 strains. Some properties of these plasmids were compared. All were fi+, but they could be divided into three groups on the basis of their incompatibility reactions, ability to restrict E. coli K-12 phages, and size. The three heat-labile enterotoxin plasmids isolated from African strains all belonged to one enterotoxin plasmid group. The heat-labile enterotoxin plasmids from the Asian strains were divided into two groups, those from serotype O78.H11 differing from those from serotype O78.H12.     

Bacillus thuringiensis populations naturally occurring on mulberry leaves: a possible source of the populations associated with silkworm-rearing insectaries

Mulberry leaves were examined for the occurrence of Bacillus thuringiensis. This organism was recovered from both abaxial and adaxial surfaces: a total of 186 B. thuringiensis colonies were isolated from 24 (96·0%) out of 25 mulberry trees, and from 112 (11·2%) out of 1004 leaves from 25 trees. The frequency of B. thuringiensis colonies was 3·2% among 5900 colonies belonging to the Bacillus cereus/B. thuringiensis group. Single colonies were associated with 75·9% of the B. thuringiensis -positive leaves and 2–16 colonies were occasionally found on a single phylloplane. Flagellar (H) serotypying of the isolates revealed that, among the 19 H serotypes (serovars) detected, the H serotype 13 (serovar pakistani ) was the predominant, followed by the H serotypes 3abc ( kurstaki ), 6ac ( oyamensis ), 16 ( indiana ), 24 ( neoleonesis ), 4ac ( kenyae ), 7 ( aizawai ) and 10 ( darmstadiensis ). Larvicidal activity, against the silkworm ( Bombyx mori ) and/or the mosquito ( Aedes aegypti ), was exhibited by 18 isolates (9·7%) belonging to H serovars kurstaki, kenyae, canadensis and aizawai , and an unidentified H serogroup.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Characteristics of Yersinia spp. isolated from wild and zoo animals

Thirteen strains of Yersinia spp. were isolated at the Rome zoo and at Castelporziano, a game preserve near Rome. The strains were tested for calcium dependency, autoagglutination, heat-stable toxin production, 50% minimum lethal dose in mice (LD50), pyrazinamidase activity and content of plasmids by electrophoresis in agarose gel. The former three tests were negative for all strains, the LD50 was always greater than or equal to 1 X 10(7.6) CFU/ml and pyrazinamidase activity was positive for all strains. Electrophoresis revealed the presence of two plasmids of 27 and 66 megadaltons (MDa) in the two strains of Y. enterocolitica of serotype 027 isolated from animals in the zoo. The two strains of the same species and serotype, isolated from wild animals harboured a 42-MDa plasmid. A small plasmid of 2 MDa was found in two strains of Y. enterocolitica of serotype 07.8 from two subsequent samples of a zoo animal.     

Studies on Bacillus thuringiensis H-14 strains isolated in Egypt—V. Composition and toxicity of the mosquitocidal parasporal inclusions

Parasporal inclusions of Bacillus thuringlensis H-14 strains M1 and S128 were characterized by solubilization, electron microscopy, polyacrylamide gel electrophoresis, amino acid analysis and insecticidal activity. Inclusions of both strains are composed largely of protein with 8 to 9% carbohydrate. Amino acid analysis of the purlfied inclusions revealed that the two strains produce inclusions that are closely related to each other but significantly different from lepidopteran-toxic B. thuringiensis parasporal crystals. The LC₅₀ values of the purlfied inclusions of strains M1 and S128 were 3.4 and 2.9 ng/ml, respectively, for fourth instar larvae of Aedes aegypti. Inclusions from strain M1 were resolved into two inclusion bands on the basis of their densities possibly formed as a result of disruption of some envelopes during sonication. Both inclusion types contained proteins of approximately 27, 38 and 66 kDa. The heavlest and more predominant type had an envelope and was either spherical or irregular being composed of several subunits which varied in shape, size and staining densities. The LC₅₀ value was 2.2 ng/ml and the major protein was of approximately 27 kDa. The lightest inclusions type did not have an envelope and showed clear crystal lattices. They were 10 times less toxic to A. aegypti larvae, as compared to the heavy-type inclusions and contained major protein of approximately 66 kDa.     

Purification and characterization of thermophilin T, a novel bacteriocin produced by Streptococcus thermophilus ACA-DC 0040

ACA-DC 0040 produced an antimicrobial agent, which was named thermophilin T, active against several lactic acid bacteria strains of different species and food spoilage bacteria, such as Clostridium sporogenes C22/10 and Cl. tyrobutyricum NCDO-1754. The crude antimicrobial compound is sensitive to proteolytic enzymes and α-amylase, heat-stable (100 °C for 30 min), resistant to pH exposure at pH 1–12 and demonstrates a bactericidal mode of action against the sensitive strain Lactococcus cremoris CNRZ-117. The production of bacteriocin was optimized approximately 10-fold in an aerobic fermenter held at constant pH 5·8 and 6·2. Ultrafiltration experiments with culture supernatant fluids containing the bacteriocin, and further estimation of molecular weight with gel filtration chromatography, revealed that bacteriocin in the native form has a molecular weight in excess of 300 kDa. SDS-gel electrophoresis of partially purified thermophilin T showed that bacteriocin activity was associated with a protein band of approximately 2·5 kDa molecular mass.