We screened for microorganisms that can be utilized as new host cells for mosquito larvicides. As... more We screened for microorganisms that can be utilized as new host cells for mosquito larvicides. As long persistence in the environment is required of host cells, we examined the bacterial populations in the guts of mosquito larvae collected from natural breeding habitats. Larvae of Aedes aegypti and Culex quinquefasciatus were examined, and Bacillus species, particularly Bacillus cereus, were found to be the dominant bacterial species in their guts. To investigate the relationship between these Bacillus strains and the mosquito larvae, we re-introduced the bacteria into larvae of Aedes aegypti, C. quinquefasciatus and another common mosquito strain, Anopheles dirus. The cell numbers of Bacillus cereus strains Ae10 and Cx5 in the guts were consistent throughout a 7-d period without food supplementation, suggesting that these strains were able to colonize in the guts of the larvae. To confirm this, we introduced a plasmid containing a kanamycin resistance marker into Ae10 and Cx5 and fed these recombinant strains to C. quinquefasciatus larvae. Even when food was supplemented for 7 d, the recombinant strains, particularly Ae10, were still found in the guts. Under similar conditions, B. thuringiensis serovar israelensis c4Q2-72 was hardly detectable after 2 d, while Escherichia coli could not be detected at all. Their stable retention in mosquito larvae guts and the feasibility of genetic manipulation indicates these strains possess high potential as novel host cells for application in mosquito control.
The use of Lactobacillus plantarum BCC 9546 (LpBCC9546) as a starter culture for Nham, a traditio... more The use of Lactobacillus plantarum BCC 9546 (LpBCC9546) as a starter culture for Nham, a traditional Thai fermented pork sausage ensures product quality and consistency. However, no direct evidence has confirmed the growth of this starter during Nham fermentation. In order to investigate its role during Nham fermentation, LpBCC9546 was genetically modified to distinguish it from the natural microflora in Nham. LpBCC9546 was transformed with a recombinant plasmid pRV85 to produce the recombinant strain LpG11, which is resistant to erythromycin and emits green fluorescence. LpG11 was used as a starter culture for Nham fermentation, and its growth was monitored by plating on a selective medium and assay of fluorescent activity. During Nham fermentation the numbers of LpG11 increased ten fold during the first 12 h of fermentation, reaching maximum numbers of between 10 7 and 10 8 cfu g − 1 after 24 h, and then declining after 60 h to 10 5 cfu g − 1 at 168 h. The growth of LpG11 starter culture during Nham fermentation was very similar to that of the untransformed LpBCC9546, although after a prolonged period of fermentation the recombinant LpG11 bacteria appeared to lose the plasmid, or were outgrown by naturally present L. plantarum. The acidity, texture and color of fermented Nham inoculated with recombinant LpG11 or untransformed LpBCC9546 were similar. These results indicated that the recombinant L. plantarum strain LpG11 is a suitable starter culture for Nham fermentation, and that the ability to monitor its growth directly during Nham fermentation could be exploited to further improve Nham production.
... 3, subiiiis ANA-! (£/rg-/j" listiR hstiM JacrAj Amy-Npr ") was a derivative strain ... more ... 3, subiiiis ANA-! (£/rg-/j" listiR hstiM JacrAj Amy-Npr ") was a derivative strain of 3. subiiiis N'AI i 13). ... ì;le mosquito larvicidal activity ,md no n-li aza niou:- properties, aloni; with their orinimi features, their broad ,iost ran^e specificity and stable habitation in li'.c environ-in e ¡it. ...
The mosquito-larvicidal binary toxin of Bacillus sphaericus 2297 was expressed in Enterobacter am... more The mosquito-larvicidal binary toxin of Bacillus sphaericus 2297 was expressed in Enterobacter amnigenus, a Gram-negative bacterium isolated from Anopheles dirus larvae gut. The toxin was placed under the regulation of various promoters in order to improve the expression level of the toxin. Amongst the recombinants obtained, E. amnigenus harboring pBS373, a plasmid which contains the toxin genes under the control of the native B. sphaericus promoter, expressed a significant amount of protein, comparable to that found in B. sphaericus 2297. In addition, this recombinant provided approximately twenty times higher toxicity against second-instar Anopheles dirus larvae when compared to B. sphaericus 2297. The procedure of obtaining this environmentally isolated bacterium from larvae gut and introducing the system for mosquito-larvicidal toxin synthesis is noteworthy. The promising result presented here provides a substantial degree of confidence for further field studies.
The gram-negative bacterium, An11/2 G1, isolated from the guts of Anopheles dirus mosquito larvae... more The gram-negative bacterium, An11/2 G1, isolated from the guts of Anopheles dirus mosquito larvae, was identi®ed as Enterobacter amnigenus. The E. amnigenus was able to recolonize in the gut of An. dirus larva but not in those of Aedes aegypti and Culex quinquefasciatus larvae. It was able to¯oat in water for a longer period than Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus. These are desirable characteristics for a delivery vehicle of mosquito-larvicidal toxins for the control of mosquito larvae, and E. amnigenus was therefore used as a host to express the cryIVB gene of B. thuringiensis subsp. israelensis and the binary toxin genes of B. sphaericus. The recombinant E. amnigenus produced a high level of CryIVB protein, which was toxic to larvae of Ae. aegypti and An. dirus. Another E. amnigenus producing the 51-kDa protein of B. sphaericus was toxic to larvae of An. dirus and Cx. quinquefasciatus. The recombinant plasmids were stable in E. amnigenus without the presence of selective pressure for at least 23 generations. The recombinant E. amnigenus should represent a desirable biological agent for controlling mosquito larvae.
A Gram-positive, endospore-forming, rod-shaped bacterium, strain PD-A10 T , was isolated from sal... more A Gram-positive, endospore-forming, rod-shaped bacterium, strain PD-A10 T , was isolated from salted crab (poo-khem) in Thailand and subjected to a taxonomic study. Phenotypic and chemotaxonomic characteristics, including phylogenetic analyses, showed that the novel strain was a member of the genus Bacillus. The novel strain grew in medium with 0-14 % (w/v) NaCl, at 4-55 6C and at pH 4.5-9. The predominant quinone was a menaquinone with seven isoprene units (MK-7). The major fatty acids were anteiso-C 15 : 0 and anteiso-C 17 : 0 . Polar lipid analysis revealed the presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, lysylphosphatidylglycerol, glycolipid and unknown lipids. The DNA G+C content was 41.4 mol%. The 16S rRNA gene sequence similarities between strain PD-A10 T and Bacillus amyloliquefaciens NBRC 15535 T , Bacillus subtilis DSM 10 T , Bacillus vallismortis DSM 11031 T and Bacillus mojavensis IFO 15718 T were 99.5, 99.4, 99.4 and 99.2 %, respectively. Strain PD-A10 T showed a low degree similarity of rep-PCR fingerprints and low DNA-DNA relatedness with the above-mentioned species. On the basis of the data gathered in this study, strain PD-A10 T should be classified as representing a novel species of the genus Bacillus, for which the name Bacillus siamensis sp. nov. is proposed. The type strain is PD-A10 T (5BCC 22614 T 5KCTC 13613 T ).
We screened for microorganisms that can be utilized as new host cells for mosquito larvicides. As... more We screened for microorganisms that can be utilized as new host cells for mosquito larvicides. As long persistence in the environment is required of host cells, we examined the bacterial populations in the guts of mosquito larvae collected from natural breeding habitats. Larvae of Aedes aegypti and Culex quinquefasciatus were examined, and Bacillus species, particularly Bacillus cereus, were found to be the dominant bacterial species in their guts. To investigate the relationship between these Bacillus strains and the mosquito larvae, we re-introduced the bacteria into larvae of Aedes aegypti, C. quinquefasciatus and another common mosquito strain, Anopheles dirus. The cell numbers of Bacillus cereus strains Ae10 and Cx5 in the guts were consistent throughout a 7-d period without food supplementation, suggesting that these strains were able to colonize in the guts of the larvae. To confirm this, we introduced a plasmid containing a kanamycin resistance marker into Ae10 and Cx5 and fed these recombinant strains to C. quinquefasciatus larvae. Even when food was supplemented for 7 d, the recombinant strains, particularly Ae10, were still found in the guts. Under similar conditions, B. thuringiensis serovar israelensis c4Q2-72 was hardly detectable after 2 d, while Escherichia coli could not be detected at all. Their stable retention in mosquito larvae guts and the feasibility of genetic manipulation indicates these strains possess high potential as novel host cells for application in mosquito control.
The use of Lactobacillus plantarum BCC 9546 (LpBCC9546) as a starter culture for Nham, a traditio... more The use of Lactobacillus plantarum BCC 9546 (LpBCC9546) as a starter culture for Nham, a traditional Thai fermented pork sausage ensures product quality and consistency. However, no direct evidence has confirmed the growth of this starter during Nham fermentation. In order to investigate its role during Nham fermentation, LpBCC9546 was genetically modified to distinguish it from the natural microflora in Nham. LpBCC9546 was transformed with a recombinant plasmid pRV85 to produce the recombinant strain LpG11, which is resistant to erythromycin and emits green fluorescence. LpG11 was used as a starter culture for Nham fermentation, and its growth was monitored by plating on a selective medium and assay of fluorescent activity. During Nham fermentation the numbers of LpG11 increased ten fold during the first 12 h of fermentation, reaching maximum numbers of between 10 7 and 10 8 cfu g − 1 after 24 h, and then declining after 60 h to 10 5 cfu g − 1 at 168 h. The growth of LpG11 starter culture during Nham fermentation was very similar to that of the untransformed LpBCC9546, although after a prolonged period of fermentation the recombinant LpG11 bacteria appeared to lose the plasmid, or were outgrown by naturally present L. plantarum. The acidity, texture and color of fermented Nham inoculated with recombinant LpG11 or untransformed LpBCC9546 were similar. These results indicated that the recombinant L. plantarum strain LpG11 is a suitable starter culture for Nham fermentation, and that the ability to monitor its growth directly during Nham fermentation could be exploited to further improve Nham production.
... 3, subiiiis ANA-! (£/rg-/j" listiR hstiM JacrAj Amy-Npr ") was a derivative strain ... more ... 3, subiiiis ANA-! (£/rg-/j" listiR hstiM JacrAj Amy-Npr ") was a derivative strain of 3. subiiiis N'AI i 13). ... ì;le mosquito larvicidal activity ,md no n-li aza niou:- properties, aloni; with their orinimi features, their broad ,iost ran^e specificity and stable habitation in li'.c environ-in e ¡it. ...
The mosquito-larvicidal binary toxin of Bacillus sphaericus 2297 was expressed in Enterobacter am... more The mosquito-larvicidal binary toxin of Bacillus sphaericus 2297 was expressed in Enterobacter amnigenus, a Gram-negative bacterium isolated from Anopheles dirus larvae gut. The toxin was placed under the regulation of various promoters in order to improve the expression level of the toxin. Amongst the recombinants obtained, E. amnigenus harboring pBS373, a plasmid which contains the toxin genes under the control of the native B. sphaericus promoter, expressed a significant amount of protein, comparable to that found in B. sphaericus 2297. In addition, this recombinant provided approximately twenty times higher toxicity against second-instar Anopheles dirus larvae when compared to B. sphaericus 2297. The procedure of obtaining this environmentally isolated bacterium from larvae gut and introducing the system for mosquito-larvicidal toxin synthesis is noteworthy. The promising result presented here provides a substantial degree of confidence for further field studies.
The gram-negative bacterium, An11/2 G1, isolated from the guts of Anopheles dirus mosquito larvae... more The gram-negative bacterium, An11/2 G1, isolated from the guts of Anopheles dirus mosquito larvae, was identi®ed as Enterobacter amnigenus. The E. amnigenus was able to recolonize in the gut of An. dirus larva but not in those of Aedes aegypti and Culex quinquefasciatus larvae. It was able to¯oat in water for a longer period than Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus. These are desirable characteristics for a delivery vehicle of mosquito-larvicidal toxins for the control of mosquito larvae, and E. amnigenus was therefore used as a host to express the cryIVB gene of B. thuringiensis subsp. israelensis and the binary toxin genes of B. sphaericus. The recombinant E. amnigenus produced a high level of CryIVB protein, which was toxic to larvae of Ae. aegypti and An. dirus. Another E. amnigenus producing the 51-kDa protein of B. sphaericus was toxic to larvae of An. dirus and Cx. quinquefasciatus. The recombinant plasmids were stable in E. amnigenus without the presence of selective pressure for at least 23 generations. The recombinant E. amnigenus should represent a desirable biological agent for controlling mosquito larvae.
A Gram-positive, endospore-forming, rod-shaped bacterium, strain PD-A10 T , was isolated from sal... more A Gram-positive, endospore-forming, rod-shaped bacterium, strain PD-A10 T , was isolated from salted crab (poo-khem) in Thailand and subjected to a taxonomic study. Phenotypic and chemotaxonomic characteristics, including phylogenetic analyses, showed that the novel strain was a member of the genus Bacillus. The novel strain grew in medium with 0-14 % (w/v) NaCl, at 4-55 6C and at pH 4.5-9. The predominant quinone was a menaquinone with seven isoprene units (MK-7). The major fatty acids were anteiso-C 15 : 0 and anteiso-C 17 : 0 . Polar lipid analysis revealed the presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, lysylphosphatidylglycerol, glycolipid and unknown lipids. The DNA G+C content was 41.4 mol%. The 16S rRNA gene sequence similarities between strain PD-A10 T and Bacillus amyloliquefaciens NBRC 15535 T , Bacillus subtilis DSM 10 T , Bacillus vallismortis DSM 11031 T and Bacillus mojavensis IFO 15718 T were 99.5, 99.4, 99.4 and 99.2 %, respectively. Strain PD-A10 T showed a low degree similarity of rep-PCR fingerprints and low DNA-DNA relatedness with the above-mentioned species. On the basis of the data gathered in this study, strain PD-A10 T should be classified as representing a novel species of the genus Bacillus, for which the name Bacillus siamensis sp. nov. is proposed. The type strain is PD-A10 T (5BCC 22614 T 5KCTC 13613 T ).
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