Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense / Caracterização da glutamina sintetase da estipe excretora de amônio HM053 de Azospirillum brasilense

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense

Abstract Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

Resumo A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense / Caracterização da glutamina sintetase da estipe excretora de amônio HM053 de Azospirillum brasilense

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.(AU)

A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.(AU)

Draft genome sequence of Paraburkholderia tropica Ppe8 strain, a sugarcane endophytic diazotrophic bacterium

Abstract Paraburkholderia tropica (syn Burkholderia tropica) are nitrogen-fixing bacteria commonly found in sugarcane. The Paraburkholderia tropica strain Ppe8 is part of the sugarcane inoculant consortium that has a beneficial effect on yield. Here, we report a draft genome sequence of this strain elucidating the mechanisms involved in its interaction mainly with Poaceae. A genome size of approximately 8.75 Mb containing 7844 protein coding genes distributed in 526 subsystems was de novo assembled with ABySS and annotated by RAST. Genes related to the nitrogen fixation process, the secretion systems (I, II, III, IV, and VI), and related to a variety of metabolic traits, such as metabolism of carbohydrates, amino acids, vitamins, and proteins, were detected, suggesting a broad metabolic capacity and possible adaptation to plant association.

Draft genome sequence of Paraburkholderia tropica Ppe8 strain, a sugarcane endophytic diazotrophic bacterium

Paraburkholderia tropica (syn Burkholderia tropica) are nitrogen-fixing bacteria commonly found in sugarcane. The Paraburkholderia tropica strain Ppe8 is part of the sugarcane inoculant consortium that has a beneficial effect on yield. Here, we report a draft genome sequence of this strain elucidating the mechanisms involved in its interaction mainly with Poaceae. A genome size of approximately 8.75 Mb containing 7844 protein coding genes distributed in 526 subsystems was de novo assembled with ABySS and annotated by RAST. Genes related to the nitrogen fixation process, the secretion systems (I, II, III, IV, and VI), and related to a variety of metabolic traits, such as metabolism of carbohydrates, amino acids, vitamins, and proteins, were detected, suggesting a broad metabolic capacity and possible adaptation to plant association.(AU)