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Mycobacterium smegmatis Growth Using Nitrate as a Nitrogen Source LC-MSMS

ABSTRACT: PII proteins are signal transduction proteins that belong to a widely distributed family involved in modulating various bacterial metabolisms. These homotrimeric proteins carry a flexible loop, known as the T-loop, whose conformation changes upon recognition of key metabolites such as ADP, ATP, and 2-oxoglutarate. PII proteins interact with various partners to primarily regulate nitrogen pathways, and in some organisms, they can also control carbon metabolism by interacting with the biotin carboxyl carrier protein (BCCP), a critical component of the acetyl-CoA carboxylase (ACC) enzyme complex, inhibiting its activity and consequently reducing fatty acid biosynthesis. In mycobacteria, only one PII protein has been identified; however, its physiological role remains unknown. In this study, we constructed a M. smegmatis deletion mutant, ∆MsPII, which exhibited growth deficiency when nitrate and nitrite were the sole nitrogen sources, leading to nitrite accumulation in the culture supernatant.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mycobacterium Smegmatis (strain Atcc 700084 / Mc(2)155) Bacteria

SUBMITTER: Delfina Ensinck

LAB HEAD: Hugo Cesar Gramajo

PROVIDER: PXD049128 | Pride | 2024-03-15

REPOSITORIES: Pride

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Project description:Unlike all other bacteria, the physiology of cyanobacteria is based on the assimilation of organic matter from inorganic carbon driven by oxygenic photosynthesis. This setting poses special regulatory challenges, particularly in integrating carbon and nitrogen metabolism. Several regulatory factors control the primary nitrogen metabolism, such as the PII protein and proteins interacting with it (PirA, PirC or PipX), or the inactivating factors IF7 and IF17 impacting glutamine synthetase activity. However, regulatory proteins of other enzymes in the nitrogen assimilation pathway have not been described thus far. Here, we show that the 81 amino acids regulatory protein NirP1 in the cyanobacterium Synechocystis sp. PCC 6803 functions as an inhibitor of nitrite reductase, a central enzyme in the assimilation of ammonia from nitrate/nitrite. Ectopic overexpression of the nirP1 gene under standard growth conditions led to a pigmentation phenotype and delayed growth, which was correlated by the excretion of nitrite and dramatic changes in metabolite pools. We show that nitrite excretion occurs in Synechocystis wild type cells upon the shift from high CO2 (HC) to low CO2 (LC) conditions when NirP1 expression becomes activated. The expression of nirP1 is controlled by two transcription factors, NtcA, which binds to a recognition site in a repressive position above the transcription start site (TSS), and an activator, which probably binds to an element 60 to 43 nt upstream of the TSS. Therefore, the joint activity of NtcA and this activator leads to the observed induction of NirP1 under LC conditions, whereas it is repressed by shifts to nitrogen starvation. The data demonstrate that NirP1 plays a crucial role in the coordination of C and N primary metabolism in cyanobacteria by targeting the activity of one of the central enzymes. In natural environments, the excreted nitrite will be utilized by other microorganisms; therefore, NirP1 will ultimately impact the activities and composition of the surrounding microbiome.

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Mycobacterium smegmatis Growth Using Nitrate as a Nitrogen Source LC-MSMS

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