{"id":{"repo_id":"aachen","oai_identifier":"oai:publications.rwth-aachen.de:61380"},"canonical_url":"https://search.dev.ndltd.org/etd/aachen/oai:publications.rwth-aachen.de:61380","repository":{"repo_id":"aachen","name":"RWTH Aachen University","base_url":"https://publications.rwth-aachen.de/oai2d"},"display":{"title":"Physiologische und genetische Charakterisierung der Gamma-Aminobutyrat-(GABA)-Aminotransferase in Rhizobium leguminosarum bv. viciae VF39","abstract":"In the frame of this work the gabRTD region of Rhizobium leguminosarum bv. viciae VF39 was identified and systematically analysed. Starting from a Tn5-B20 mutant PH10 which was found to show increasing induction of the lacZ-reporter on low pH media, the transposon was cloned and 2.5 kb of flanking DNA were sequenced. The sequence contained the full gabT open reading frame, coding for a GABA aminotransferase. Downstream of gabT the 5'-coding region of a succinate semialdehyde dehydrogenase (SSDH) was found and designated gabD. Both enzymes are part of the so called GABA shunt, which is the major pathway for GABA metabolism and in combination with glutamate decarboxylase a theoretical bypass of the 2-oxoglutarat dehydrogenase complex of the TCA cycle. Upstream of gabT a third ORF oriented in the opposite direction and carefully designated gabR shows homology to a MerR type regulator. Starting the analysis of the PH10 mutant phenotype, it was shown, that growth on GABA as a sole carbon and nitrogen source was not different from wild-type. Enzyme assays from protein crude extracts showed moderate 2-oxoglutarat dependent GABA aminotransferase activities in wild-type and the absence of activity in the mutant. In contrast pyruvate dependent GABA aminotransferase activities where very high in both strains, which easily explained the mutant growth phenotype on GABA. For the further analysis of gabT expression, a promoter-probe vector pJP2 was constructed. This plasmid contains a promoterless uidA gen, which is able to express GUS as a reporterenzyme. Further features of the vector are the par genes from RK2 maintaining stability during symbiosis. Stable plasmids are interesting tools in Rhizobium-research. Therefore pJP2 is an important result of this work. After cloning the gabT promoter-region into pJP2 and introducing the plasmid into wild-type VF39, pH dependent expression was measured. The results were compa-rable to the lacZ induction of PH10. Then the involvement of gabT in the GABA metabolism was investigated during growth on related carbon and nitrogen sources. GABA was shown to induce the fusion. During symbiosis with Pisum sativum, gabT was induced during bacteroid differentiation and strongly expressed in the symbiotic zone. However, the PH10 mutant formed normal nitrogen fixing nodules. The above mentioned gabR open reading frame was analysed for being involved in gabT regulation, because of its homology to regulatory genes. In a gabR mutant, gabT expression was strongly increased, what led to the conclusion, that gabR must act as a repressor or repressor/activator of gabT. The role of gabD could be only poorly described. In gabT mutants NAD+ dependent, SSDH activities were unaffected and there was no measurable promotor activity expressed by the gabT and gabD intergenic region. Furthermore it was not possible to identify other GABA metabolising enzymes with classical transposon mutagenesis.","abstract_html":"In the frame of this work the gabRTD region of Rhizobium leguminosarum bv. viciae VF39 was identified and systematically analysed. Starting from a Tn5-B20 mutant PH10 which was found to show increasing induction of the lacZ-reporter on low pH media, the transposon was cloned and 2.5 kb of flanking DNA were sequenced. The sequence contained the full gabT open reading frame, coding for a GABA aminotransferase. Downstream of gabT the 5'-coding region of a succinate semialdehyde dehydrogenase (SSDH) was found and designated gabD. Both enzymes are part of the so called GABA shunt, which is the major pathway for GABA metabolism and in combination with glutamate decarboxylase a theoretical bypass of the 2-oxoglutarat dehydrogenase complex of the TCA cycle. Upstream of gabT a third ORF oriented in the opposite direction and carefully designated gabR shows homology to a MerR type regulator. Starting the analysis of the PH10 mutant phenotype, it was shown, that growth on GABA as a sole carbon and nitrogen source was not different from wild-type. Enzyme assays from protein crude extracts showed moderate 2-oxoglutarat dependent GABA aminotransferase activities in wild-type and the absence of activity in the mutant. In contrast pyruvate dependent GABA aminotransferase activities where very high in both strains, which easily explained the mutant growth phenotype on GABA. For the further analysis of gabT expression, a promoter-probe vector pJP2 was constructed. This plasmid contains a promoterless uidA gen, which is able to express GUS as a reporterenzyme. Further features of the vector are the par genes from RK2 maintaining stability during symbiosis. Stable plasmids are interesting tools in Rhizobium-research. Therefore pJP2 is an important result of this work. After cloning the gabT promoter-region into pJP2 and introducing the plasmid into wild-type VF39, pH dependent expression was measured. The results were compa-rable to the lacZ induction of PH10. Then the involvement of gabT in the GABA metabolism was investigated during growth on related carbon and nitrogen sources. GABA was shown to induce the fusion. During symbiosis with Pisum sativum, gabT was induced during bacteroid differentiation and strongly expressed in the symbiotic zone. However, the PH10 mutant formed normal nitrogen fixing nodules. The above mentioned gabR open reading frame was analysed for being involved in gabT regulation, because of its homology to regulatory genes. In a gabR mutant, gabT expression was strongly increased, what led to the conclusion, that gabR must act as a repressor or repressor/activator of gabT. The role of gabD could be only poorly described. In gabT mutants NAD+ dependent, SSDH activities were unaffected and there was no measurable promotor activity expressed by the gabT and gabD intergenic region. Furthermore it was not possible to identify other GABA metabolising enzymes with classical transposon mutagenesis.","abstract_has_math":false,"creators":["Prell, Jürgen"],"institution":"Publikationsserver der RWTH Aachen University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Priefer, Ursula B."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2003,"date_issued":"2003","date_published":"2003","updated_at":"2026-07-30T19:43:10Z","subjects":["info:eu-repo/classification/ddc/570","Biowissenschaften, Biologie"],"languages":["ger"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123049%22"],"render_values":[{"text":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123049%22","href":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123049%22","code":true}]}]},"links":{"outbound_url":"https://publications.rwth-aachen.de/record/61380","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Priefer, Ursula B."]},{"key":"dc:creator","label":"Author","values":["Prell, Jürgen"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:coverage","label":"Dc Coverage","values":["DE"]},{"key":"dc:date","label":"Dc Date","values":["2003"]},{"key":"dc:publisher","label":"Institution","values":["Publikationsserver der RWTH Aachen University"]},{"key":"dc:relation","label":"Dc Relation","values":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-5260"]},{"key":"dc:type","label":"Dc Type","values":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["info:eu-repo/classification/ddc/570","Biowissenschaften, Biologie"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["ger"]},{"key":"dc:rights","label":"Dc Rights","values":["info:eu-repo/semantics/openAccess"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/record/61380","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123049%22"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["In the frame of this work the gabRTD region of Rhizobium leguminosarum bv. viciae VF39 was identified and systematically analysed. Starting from a Tn5-B20 mutant PH10 which was found to show increasing induction of the lacZ-reporter on low pH media, the transposon was cloned and 2.5 kb of flanking DNA were sequenced. The sequence contained the full gabT open reading frame, coding for a GABA aminotransferase. Downstream of gabT the 5'-coding region of a succinate semialdehyde dehydrogenase (SSDH) was found and designated gabD. Both enzymes are part of the so called GABA shunt, which is the major pathway for GABA metabolism and in combination with glutamate decarboxylase a theoretical bypass of the 2-oxoglutarat dehydrogenase complex of the TCA cycle. Upstream of gabT a third ORF oriented in the opposite direction and carefully designated gabR shows homology to a MerR type regulator. Starting the analysis of the PH10 mutant phenotype, it was shown, that growth on GABA as a sole carbon and nitrogen source was not different from wild-type. Enzyme assays from protein crude extracts showed moderate 2-oxoglutarat dependent GABA aminotransferase activities in wild-type and the absence of activity in the mutant. In contrast pyruvate dependent GABA aminotransferase activities where very high in both strains, which easily explained the mutant growth phenotype on GABA. For the further analysis of gabT expression, a promoter-probe vector pJP2 was constructed. This plasmid contains a promoterless uidA gen, which is able to express GUS as a reporterenzyme. Further features of the vector are the par genes from RK2 maintaining stability during symbiosis. Stable plasmids are interesting tools in Rhizobium-research. Therefore pJP2 is an important result of this work. After cloning the gabT promoter-region into pJP2 and introducing the plasmid into wild-type VF39, pH dependent expression was measured. The results were compa-rable to the lacZ induction of PH10. Then the involvement of gabT in the GABA metabolism was investigated during growth on related carbon and nitrogen sources. GABA was shown to induce the fusion. During symbiosis with Pisum sativum, gabT was induced during bacteroid differentiation and strongly expressed in the symbiotic zone. However, the PH10 mutant formed normal nitrogen fixing nodules. The above mentioned gabR open reading frame was analysed for being involved in gabT regulation, because of its homology to regulatory genes. In a gabR mutant, gabT expression was strongly increased, what led to the conclusion, that gabR must act as a repressor or repressor/activator of gabT. The role of gabD could be only poorly described. In gabT mutants NAD+ dependent, SSDH activities were unaffected and there was no measurable promotor activity expressed by the gabT and gabD intergenic region. Furthermore it was not possible to identify other GABA metabolising enzymes with classical transposon mutagenesis."]},{"key":"dc:source","label":"Dc Source","values":["Aachen : Publikationsserver der RWTH Aachen University 127 S. : Ill., graph. Darst. (2003). = Aachen, Techn. Hochsch., Diss., 2003"]},{"key":"dc:title","label":"Title","values":["Physiologische und genetische Charakterisierung der Gamma-Aminobutyrat-(GABA)-Aminotransferase in Rhizobium leguminosarum bv. viciae VF39"]}]}],"canonical_facts":{"dc:contributor":["Priefer, Ursula B."],"dc:coverage":["DE"],"dc:creator":["Prell, Jürgen"],"dc:date":["2003"],"dc:description":["In the frame of this work the gabRTD region of Rhizobium leguminosarum bv. viciae VF39 was identified and systematically analysed. Starting from a Tn5-B20 mutant PH10 which was found to show increasing induction of the lacZ-reporter on low pH media, the transposon was cloned and 2.5 kb of flanking DNA were sequenced. The sequence contained the full gabT open reading frame, coding for a GABA aminotransferase. Downstream of gabT the 5'-coding region of a succinate semialdehyde dehydrogenase (SSDH) was found and designated gabD. Both enzymes are part of the so called GABA shunt, which is the major pathway for GABA metabolism and in combination with glutamate decarboxylase a theoretical bypass of the 2-oxoglutarat dehydrogenase complex of the TCA cycle. Upstream of gabT a third ORF oriented in the opposite direction and carefully designated gabR shows homology to a MerR type regulator. Starting the analysis of the PH10 mutant phenotype, it was shown, that growth on GABA as a sole carbon and nitrogen source was not different from wild-type. Enzyme assays from protein crude extracts showed moderate 2-oxoglutarat dependent GABA aminotransferase activities in wild-type and the absence of activity in the mutant. In contrast pyruvate dependent GABA aminotransferase activities where very high in both strains, which easily explained the mutant growth phenotype on GABA. For the further analysis of gabT expression, a promoter-probe vector pJP2 was constructed. This plasmid contains a promoterless uidA gen, which is able to express GUS as a reporterenzyme. Further features of the vector are the par genes from RK2 maintaining stability during symbiosis. Stable plasmids are interesting tools in Rhizobium-research. Therefore pJP2 is an important result of this work. After cloning the gabT promoter-region into pJP2 and introducing the plasmid into wild-type VF39, pH dependent expression was measured. The results were compa-rable to the lacZ induction of PH10. Then the involvement of gabT in the GABA metabolism was investigated during growth on related carbon and nitrogen sources. GABA was shown to induce the fusion. During symbiosis with Pisum sativum, gabT was induced during bacteroid differentiation and strongly expressed in the symbiotic zone. However, the PH10 mutant formed normal nitrogen fixing nodules. The above mentioned gabR open reading frame was analysed for being involved in gabT regulation, because of its homology to regulatory genes. In a gabR mutant, gabT expression was strongly increased, what led to the conclusion, that gabR must act as a repressor or repressor/activator of gabT. The role of gabD could be only poorly described. In gabT mutants NAD+ dependent, SSDH activities were unaffected and there was no measurable promotor activity expressed by the gabT and gabD intergenic region. Furthermore it was not possible to identify other GABA metabolising enzymes with classical transposon mutagenesis."],"dc:identifier":["https://publications.rwth-aachen.de/record/61380","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123049%22"],"dc:language":["ger"],"dc:publisher":["Publikationsserver der RWTH Aachen University"],"dc:relation":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-5260"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Aachen : Publikationsserver der RWTH Aachen University 127 S. : Ill., graph. Darst. (2003). = Aachen, Techn. Hochsch., Diss., 2003"],"dc:subject":["info:eu-repo/classification/ddc/570","Biowissenschaften, Biologie"],"dc:title":["Physiologische und genetische Charakterisierung der Gamma-Aminobutyrat-(GABA)-Aminotransferase in Rhizobium leguminosarum bv. viciae VF39"],"dc:type":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]},"updated_at":"2026-07-30T19:43:10Z"}