{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/256731"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/256731","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Molecular and phylogenetic analysis of a Bacillus thuringiensis genetic locus","abstract":"The diptericidal $\\textit{Bacillus thuringiensis}$ (Bt) ssp. $\\textit{fukuokaensis}$ strains 84-I and 17A were investigated for the presence of novel Cry proteins. N-terminal amino acid, immunological and PCR analysis indicated that both strains contain a novel set of $\\delta$-endotoxins. N-terminal amino acid sequence analysis indicated that the larger proteins from each strain (90 and 72-kDa of 84-I and 70 and 65-kDa of 17A) were related to the Cry proteins of Bt ssp. $\\textit{israelensis}$(Bti). Immunoblotting experiments confirmed that Cry10A-type proteins were present in both strains although subsequent PCR did not give a positive reaction for either strain using $\\textit{cry10A}$ specific primers indicating that the Cry10-types were indeed novel. To further investigate the 65-kDa protein of 17A, the gene encoding it was cloned from a size-enriched plasmid DNA library. Unsuccessful attempts were also made to clone the 90-kDa protein of 84-I. Sequence alignments of the deduced protein product of the 17A gene ($\\textit{am1}$) showed it to represent the second identification of a natural C-terminal truncate of a Cry4-type protein, the first being Cry10A. The missing C-terminal region of AMl appears to be encoded as a complete Orf ($\\textit{am2}$) immediately downstream of the first protein gene. When DNA containing both the $\\textit{am1}$ and $\\textit{am2}$ genes was subcloned into the pSVP27A expression vector high levels of expression of both proteins were observed in acrystalliferous Bt. The protein was deposited in inclusion bodies which were found to be toxic to $\\textit{Dacus oleae}$. Extensive phylogenetic analysis was carried out to determine the relationship between, and possible evolutionary origins of, AMl, the Cry proteins of Bti and two further Cry10A-type $\\delta$-endotoxins (Cry19A from Bt ssp. $\\textit{jegathesan}$ and Cry20A from 84-I) identified in other laboratories during the course of this project. Based on the amino acid sequence alignment, all seven proteins appear to have evolved from a common ancestor to form three distinct groups which mirror the structural organisation of the genes. Based on these groupings and a previous hypothesis of Dervyn $\\textit{et al.}$ (1995), a hypothesis was proposed as to the evolution of the 130-kDa Cry4-type proteins from a 70-kDa Cry2-type ancestor. The above hypothesis is based on the assumption that transfer of $\\delta$-endotoxin genes between subspecies has occurred at some point in evolutionary history. Evidence for this transfer was found when the genetic context of the $\\textit{am1}$ gene was investigated. Two novel insertion sequences (Tl) and (T2) were identified with sequence similarity to IS$\\textit{240A}$ from Bti and an insertion sequence associated with the $\\textit{Orf1}$ gene of 84-I. The identification of a further incomplete reading frame with similarity to integrase/recombinase proteins involved in Class II transposition raises the possibility that T1 and T2 form part of a novel Class II transposon. A novel $\\alpha$/$\\beta$-type small, acid soluble protein (SASP) gene was also discovered. This gene, which may be plasmid encoded, showed considerable sequence similarity to $\\alpha$/$\\beta$-type SASP from $\\textit{Bacillus megaterium}$. The discovery of this gene raises new questions about taxonomic relations between the $\\textit{Bacilli}$.","abstract_html":"The diptericidal <span class=\"etd-inline-math\"><em>Bacillus thuringiensis</em></span> (Bt) ssp. <span class=\"etd-inline-math\"><em>fukuokaensis</em></span> strains 84-I and 17A were investigated for the presence of novel Cry proteins. N-terminal amino acid, immunological and PCR analysis indicated that both strains contain a novel set of <span class=\"etd-inline-math\">&delta;</span>-endotoxins. N-terminal amino acid sequence analysis indicated that the larger proteins from each strain (90 and 72-kDa of 84-I and 70 and 65-kDa of 17A) were related to the Cry proteins of Bt ssp. <span class=\"etd-inline-math\"><em>israelensis</em></span>(Bti). Immunoblotting experiments confirmed that Cry10A-type proteins were present in both strains although subsequent PCR did not give a positive reaction for either strain using <span class=\"etd-inline-math\"><em>cry10A</em></span> specific primers indicating that the Cry10-types were indeed novel. To further investigate the 65-kDa protein of 17A, the gene encoding it was cloned from a size-enriched plasmid DNA library. Unsuccessful attempts were also made to clone the 90-kDa protein of 84-I. Sequence alignments of the deduced protein product of the 17A gene (<span class=\"etd-inline-math\"><em>am1</em></span>) showed it to represent the second identification of a natural C-terminal truncate of a Cry4-type protein, the first being Cry10A. The missing C-terminal region of AMl appears to be encoded as a complete Orf (<span class=\"etd-inline-math\"><em>am2</em></span>) immediately downstream of the first protein gene. When DNA containing both the <span class=\"etd-inline-math\"><em>am1</em></span> and <span class=\"etd-inline-math\"><em>am2</em></span> genes was subcloned into the pSVP27A expression vector high levels of expression of both proteins were observed in acrystalliferous Bt. The protein was deposited in inclusion bodies which were found to be toxic to <span class=\"etd-inline-math\"><em>Dacus oleae</em></span>. Extensive phylogenetic analysis was carried out to determine the relationship between, and possible evolutionary origins of, AMl, the Cry proteins of Bti and two further Cry10A-type <span class=\"etd-inline-math\">&delta;</span>-endotoxins (Cry19A from Bt ssp. <span class=\"etd-inline-math\"><em>jegathesan</em></span> and Cry20A from 84-I) identified in other laboratories during the course of this project. Based on the amino acid sequence alignment, all seven proteins appear to have evolved from a common ancestor to form three distinct groups which mirror the structural organisation of the genes. Based on these groupings and a previous hypothesis of Dervyn <span class=\"etd-inline-math\"><em>et al.</em></span> (1995), a hypothesis was proposed as to the evolution of the 130-kDa Cry4-type proteins from a 70-kDa Cry2-type ancestor. The above hypothesis is based on the assumption that transfer of <span class=\"etd-inline-math\">&delta;</span>-endotoxin genes between subspecies has occurred at some point in evolutionary history. Evidence for this transfer was found when the genetic context of the <span class=\"etd-inline-math\"><em>am1</em></span> gene was investigated. Two novel insertion sequences (Tl) and (T2) were identified with sequence similarity to IS<span class=\"etd-inline-math\"><em>240A</em></span> from Bti and an insertion sequence associated with the <span class=\"etd-inline-math\"><em>Orf1</em></span> gene of 84-I. The identification of a further incomplete reading frame with similarity to integrase/recombinase proteins involved in Class II transposition raises the possibility that T1 and T2 form part of a novel Class II transposon. A novel <span class=\"etd-inline-math\">&alpha;</span>/<span class=\"etd-inline-math\">&beta;</span>-type small, acid soluble protein (SASP) gene was also discovered. This gene, which may be plasmid encoded, showed considerable sequence similarity to <span class=\"etd-inline-math\">&alpha;</span>/<span class=\"etd-inline-math\">&beta;</span>-type SASP from <span class=\"etd-inline-math\"><em>Bacillus megaterium</em></span>. The discovery of this gene raises new questions about taxonomic relations between the <span class=\"etd-inline-math\"><em>Bacilli</em></span>.","abstract_has_math":true,"creators":["Mowbray, Alison"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Ellar, David"],"committee_chairs":[],"committee_members":[],"year":1999,"date_issued":"1999-05-04","date_published":"1999-05-04","updated_at":"2026-07-22T22:24:06Z","subjects":["Research Subject Categories::NATURAL SCIENCES::Chemistry::Biochemistry","molecular analysis","phylogenetic analysis","Bacillus thuringiensis","Cry proteins","genetics"],"languages":["en"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/750796a7-b709-4eb4-b6f1-7fbc2d3043d5/download","https://www.rioxx.net/licenses/all-rights-reserved/","https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/fa26eb48-286e-426d-a0b3-e62ccc202d6b/download"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.667","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Ellar, David"]},{"key":"dc:creator","label":"Author","values":["Mowbray, Alison"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["1999-05-04"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/256731"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Research Subject Categories::NATURAL SCIENCES::Chemistry::Biochemistry","molecular analysis","phylogenetic analysis","Bacillus thuringiensis","Cry proteins","genetics"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/750796a7-b709-4eb4-b6f1-7fbc2d3043d5/download","https://www.rioxx.net/licenses/all-rights-reserved/","https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/fa26eb48-286e-426d-a0b3-e62ccc202d6b/download"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.667"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1fec3d59-2cb0-4a32-8a4c-328be04bb4e0/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The diptericidal $\\textit{Bacillus thuringiensis}$ (Bt) ssp. $\\textit{fukuokaensis}$ strains 84-I and 17A were investigated for the presence of novel Cry proteins. N-terminal amino acid, immunological and PCR analysis indicated that both strains contain a novel set of $\\delta$-endotoxins. N-terminal amino acid sequence analysis indicated that the larger proteins from each strain (90 and 72-kDa of 84-I and 70 and 65-kDa of 17A) were related to the Cry proteins of Bt ssp. $\\textit{israelensis}$(Bti). Immunoblotting experiments confirmed that Cry10A-type proteins were present in both strains although subsequent PCR did not give a positive reaction for either strain using $\\textit{cry10A}$ specific primers indicating that the Cry10-types were indeed novel. To further investigate the 65-kDa protein of 17A, the gene encoding it was cloned from a size-enriched plasmid DNA library. Unsuccessful attempts were also made to clone the 90-kDa protein of 84-I. Sequence alignments of the deduced protein product of the 17A gene ($\\textit{am1}$) showed it to represent the second identification of a natural C-terminal truncate of a Cry4-type protein, the first being Cry10A. The missing C-terminal region of AMl appears to be encoded as a complete Orf ($\\textit{am2}$) immediately downstream of the first protein gene. When DNA containing both the $\\textit{am1}$ and $\\textit{am2}$ genes was subcloned into the pSVP27A expression vector high levels of expression of both proteins were observed in acrystalliferous Bt. The protein was deposited in inclusion bodies which were found to be toxic to $\\textit{Dacus oleae}$. Extensive phylogenetic analysis was carried out to determine the relationship between, and possible evolutionary origins of, AMl, the Cry proteins of Bti and two further Cry10A-type $\\delta$-endotoxins (Cry19A from Bt ssp. $\\textit{jegathesan}$ and Cry20A from 84-I) identified in other laboratories during the course of this project. Based on the amino acid sequence alignment, all seven proteins appear to have evolved from a common ancestor to form three distinct groups which mirror the structural organisation of the genes. Based on these groupings and a previous hypothesis of Dervyn $\\textit{et al.}$ (1995), a hypothesis was proposed as to the evolution of the 130-kDa Cry4-type proteins from a 70-kDa Cry2-type ancestor. The above hypothesis is based on the assumption that transfer of $\\delta$-endotoxin genes between subspecies has occurred at some point in evolutionary history. Evidence for this transfer was found when the genetic context of the $\\textit{am1}$ gene was investigated. Two novel insertion sequences (Tl) and (T2) were identified with sequence similarity to IS$\\textit{240A}$ from Bti and an insertion sequence associated with the $\\textit{Orf1}$ gene of 84-I. The identification of a further incomplete reading frame with similarity to integrase/recombinase proteins involved in Class II transposition raises the possibility that T1 and T2 form part of a novel Class II transposon. A novel $\\alpha$/$\\beta$-type small, acid soluble protein (SASP) gene was also discovered. This gene, which may be plasmid encoded, showed considerable sequence similarity to $\\alpha$/$\\beta$-type SASP from $\\textit{Bacillus megaterium}$. The discovery of this gene raises new questions about taxonomic relations between the $\\textit{Bacilli}$."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["330516bfae78b0d909aae1a8965b7e5c","87eda9de84448d1f82354d60eee3eb5f","94489d24e8f5d87896c69e02a6d0af21"]},{"key":"dc:title","label":"Title","values":["Molecular and phylogenetic analysis of a Bacillus thuringiensis genetic locus"]}]}],"canonical_facts":{"dc:contributor.advisor":["Ellar, David"],"dc:creator":["Mowbray, Alison"],"dc:date.issued":["1999-05-04"],"dc:description.abstract":["The diptericidal $\\textit{Bacillus thuringiensis}$ (Bt) ssp. $\\textit{fukuokaensis}$ strains 84-I and 17A were investigated for the presence of novel Cry proteins. N-terminal amino acid, immunological and PCR analysis indicated that both strains contain a novel set of $\\delta$-endotoxins. N-terminal amino acid sequence analysis indicated that the larger proteins from each strain (90 and 72-kDa of 84-I and 70 and 65-kDa of 17A) were related to the Cry proteins of Bt ssp. $\\textit{israelensis}$(Bti). Immunoblotting experiments confirmed that Cry10A-type proteins were present in both strains although subsequent PCR did not give a positive reaction for either strain using $\\textit{cry10A}$ specific primers indicating that the Cry10-types were indeed novel. To further investigate the 65-kDa protein of 17A, the gene encoding it was cloned from a size-enriched plasmid DNA library. Unsuccessful attempts were also made to clone the 90-kDa protein of 84-I. Sequence alignments of the deduced protein product of the 17A gene ($\\textit{am1}$) showed it to represent the second identification of a natural C-terminal truncate of a Cry4-type protein, the first being Cry10A. The missing C-terminal region of AMl appears to be encoded as a complete Orf ($\\textit{am2}$) immediately downstream of the first protein gene. When DNA containing both the $\\textit{am1}$ and $\\textit{am2}$ genes was subcloned into the pSVP27A expression vector high levels of expression of both proteins were observed in acrystalliferous Bt. The protein was deposited in inclusion bodies which were found to be toxic to $\\textit{Dacus oleae}$. Extensive phylogenetic analysis was carried out to determine the relationship between, and possible evolutionary origins of, AMl, the Cry proteins of Bti and two further Cry10A-type $\\delta$-endotoxins (Cry19A from Bt ssp. $\\textit{jegathesan}$ and Cry20A from 84-I) identified in other laboratories during the course of this project. Based on the amino acid sequence alignment, all seven proteins appear to have evolved from a common ancestor to form three distinct groups which mirror the structural organisation of the genes. Based on these groupings and a previous hypothesis of Dervyn $\\textit{et al.}$ (1995), a hypothesis was proposed as to the evolution of the 130-kDa Cry4-type proteins from a 70-kDa Cry2-type ancestor. The above hypothesis is based on the assumption that transfer of $\\delta$-endotoxin genes between subspecies has occurred at some point in evolutionary history. Evidence for this transfer was found when the genetic context of the $\\textit{am1}$ gene was investigated. Two novel insertion sequences (Tl) and (T2) were identified with sequence similarity to IS$\\textit{240A}$ from Bti and an insertion sequence associated with the $\\textit{Orf1}$ gene of 84-I. The identification of a further incomplete reading frame with similarity to integrase/recombinase proteins involved in Class II transposition raises the possibility that T1 and T2 form part of a novel Class II transposon. A novel $\\alpha$/$\\beta$-type small, acid soluble protein (SASP) gene was also discovered. This gene, which may be plasmid encoded, showed considerable sequence similarity to $\\alpha$/$\\beta$-type SASP from $\\textit{Bacillus megaterium}$. 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