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.

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.

NAD+ biosynthesis in bacteria is controlled by global carbon/nitrogen levels via PII signaling.

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+ Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+ This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

Regulation of Herbaspirillum seropedicae NifA by the GlnK PII signal transduction protein is mediated by effectors binding to allosteric sites.

Herbaspirillum seropedicae is a plant growth promoting bacterium that is able to fix nitrogen and to colonize the surface and internal tissues of important crops. Nitrogen fixation in H. seropedicae is regulated at the transcriptional level by the prokaryotic enhancer binding protein NifA. The activity of NifA is negatively affected by oxygen and positively stimulated by interaction with GlnK, a PII signaling protein that monitors intracellular levels of the key metabolite 2-oxoglutarate (2-OG) and functions as an indirect sensor of the intracellular nitrogen status. GlnK is also subjected to a cycle of reversible uridylylation in response to intracellular levels of glutamine. Previous studies have established the role of the N-terminal GAF domain of NifA in intramolecular repression of NifA activity and the role of GlnK in relieving this inhibition under nitrogen-limiting conditions. However, the mechanism of this control of NifA activity is not fully understood. Here, we constructed a series of GlnK variants to elucidate the role of uridylylation and effector binding during the process of NifA activation. Our data support a model whereby GlnK uridylylation is not necessary to activate NifA. On the other hand, binding of 2-OG and MgATP to GlnK are very important for NifA activation and constitute the most important signal of cellular nitrogen status to NifA.

Fatty acid biosynthesis is enhanced in Escherichia coli strains with deletion in genes encoding the PII signaling proteins.

The committed and rate-limiting step in fatty acid biosynthesis is catalyzed by acetyl-CoA carboxylase (ACC). In previous studies we showed that ACC activity is inhibited through interactions with the PII signaling proteins in vitro. Here we provide in vivo support for that model; we noted that PII proteins are able to reduce malonyl-CoA levels in vivo in Escherichia coli. Furthermore, we show that fatty acid biosynthesis is strongly enhanced in E. coli strains carrying deletions in PII coding genes. Given that PII proteins act as conserved negative regulators of ACC in Bacteria, our findings may be explored to engineer other prokaryotes to improve fatty acid yields, thereby turning microbial biofuel production economically competitive in the future.

Tailoring recombinant lipases: keeping the His-tag favors esterification reactions, removing it favors hydrolysis reactions.

We determined the effect of the His-tag on the structure, activity, stability and immobilization of LipC12, a highly active lipase from a metagenomic library. We purified LipC12 with a N-terminal His-tag and then removed the tag using tobacco etch virus (TEV) protease. Circular dichroism analysis showed that the overall structure of LipC12 was largely unaffected by His-tag removal. The specific hydrolytic activities against natural and artificial substrates were significantly increased by the removal of the His-tag. On the other hand, His-tagged LipC12 was significantly more active and stable in the presence of polar organic solvents than untagged LipC12. The immobilization efficiency on Immobead 150 was 100% for both forms of LipC12 and protein desorption studies confirmed that the His-tag does not participate in the covalent binding of the enzyme. In the case of immobilized LipC12, the His-tag negatively influenced the hydrolytic activity, as it had for the free lipase, however, it positively influenced the esterification activity. These results raise the possibility of tailoring recombinant lipases for different applications, where the His-tag may be retained or removed, as appropriate for the desired activity.

Kinetics and structural features of dimeric glutamine-dependent bacterial NAD+ synthetases suggest evolutionary adaptation to available metabolites.

NADH (NAD+) and its reduced form NADH serve as cofactors for a variety of oxidoreductases that participate in many metabolic pathways. NAD+ also is used as substrate by ADP-ribosyl transferases and by sirtuins. NAD+ biosynthesis is one of the most fundamental biochemical pathways in nature, and the ubiquitous NAD+ synthetase (NadE) catalyzes the final step in this biosynthetic route. Two different classes of NadE have been described to date: dimeric single-domain ammonium-dependent NadENH3 and octameric glutamine-dependent NadEGln, and the presence of multiple NadE isoforms is relatively common in prokaryotes. Here, we identified a novel dimeric group of NadEGln in bacteria. Substrate preferences and structural analyses suggested that dimeric NadEGln enzymes may constitute evolutionary intermediates between dimeric NadENH3 and octameric NadEGln The characterization of additional NadE isoforms in the diazotrophic bacterium Azospirillum brasilense along with the determination of intracellular glutamine levels in response to an ammonium shock led us to propose a model in which these different NadE isoforms became active accordingly to the availability of nitrogen. These data may explain the selective pressures that support the coexistence of multiple isoforms of NadE in some prokaryotes.

Genome Sequence of Type Strain Fonsecaea multimorphosa CBS 980.96T, a Causal Agent of Feline Cerebral Phaeohyphomycosis.

A draft genome sequence of type strain Fonsecaea multimorphosa CBS 980.96T was obtained. This species was first isolated from a cat with cerebral phaeohyphomycosis in Queensland, Australia.

Genome Sequence of Bacillus mycoides B38V, a Growth-Promoting Bacterium of Sunflower.

Bacillus mycoides B38V is a bacterium isolated from the sunflower rhizosphere that is able to promote plant growth and N uptake. The genome of the isolate has approximately 5.80 Mb and presents sequence codifiers for plant growth-promoting characteristics, such as nitrate reduction and ammonification and iron-siderophore uptake.

Whole-Genome Shotgun Sequence of the Keratinolytic Bacterium Lysobacter sp. A03, Isolated from the Antarctic Environment.

Lysobacter sp. strain A03 is a protease-producing bacterium isolated from decomposing-penguin feathers collected in the Antarctic environment. This strain has the ability to degrade keratin at low temperatures. The A03 genome sequence provides the possibility of finding new genes with biotechnological potential to better understand its cold-adaptation mechanism and survival in cold environments.

Genome of Pseudomonas sp. FeS53a, a Putative Plant Growth-Promoting Bacterium Associated with Rice Grown in Iron-Stressed Soils.

Pseudomonas sp. FeS53a was isolated from the roots of rice plants cultivated in one area with a well-established history of iron toxicity. The FeS53a genome sequence provides the genetic basis for understanding its lifestyle and survival in association with rice in conditions of iron toxicity.

Genome of Rhizobium sp. UR51a, Isolated from Rice Cropped in Southern Brazilian Fields.

Rhizobium sp. UR51a is a Gram-negative bacterium isolated from roots of rice plants, and it presents plant growth-promoting abilities. The nutrient uptake in rice plants inoculated with UR51a was satisfactory. The genome of strain UR51a is composed of 5,233,443-bp and harbors 5,079 coding sequences.

First co-expression of a lipase and its specific foldase obtained by metagenomics.

BACKGROUND: Metagenomics is a useful tool in the search for new lipases that might have characteristics that make them suitable for application in biocatalysis. This paper reports the cloning, co-expression, purification and characterization of a new lipase, denominated LipG9, and its specific foldase, LifG9, from a metagenomic library derived from a fat-contaminated soil. RESULTS: Within the metagenomic library, the gene lipg9 was cloned jointly with the gene of the foldase, lifg9. LipG9 and LifG9 have 96% and 84% identity, respectively, with the corresponding proteins of Aeromonas veronii B565. LipG9 and LifG9 were co-expressed, both in N-truncated form, in Escherichia coli BL21(DE3), using the vectors pET28a(+) and pT7-7, respectively, and then purified by affinity chromatography using a Ni(2+) column (HiTrap Chelating HP). The purified enzyme eluted from the column complexed with its foldase. The molecular masses of the N-truncated proteins were 32 kDa for LipG9, including the N-terminal His-tag with 6 residues, and 23 kDa for LifG9, which did not have a His-tag. The biochemical and kinetic characteristics of the purified lipase-foldase preparation were investigated. This preparation was active and stable over a wide range of pH values (6.5-9.5) and temperatures (10-40°C), with the highest specific activity, of 1500 U mg(-1), being obtained at pH 7.5 at 30°C. It also had high specific activities against tributyrin, tricaprylin and triolein, with values of 1852, 1566 and 817 U mg(-1), respectively. A phylogenetic analysis placed LipG9 in the lipase subfamily I.1. A comparison of the sequence of LipG9 with those of other bacterial lipases in the Protein Data Bank showed that LipG9 contains not only the classic catalytic triad (Ser(103), Asp(250), His(272)), with the catalytic Ser occurring within a conserved pentapeptide, Gly-His-Ser-His-Gly, but also a conserved disulfide bridge and a conserved calcium binding site. The homology-modeled structure presents a canonical α/ß hydrolase folding type I. CONCLUSIONS: This paper is the first to report the successful co-expression of a lipase and its associated foldase from a metagenomic library. The high activity and stability of Lip-LifG9 suggest that it has a good potential for use in biocatalysis.