RESUMEN
Biological nitrogen fixation catalyzed by nitrogenase contributes greatly to the global nitrogen cycle. Nitrogenase expression is subject to regulation in response to nitrogen availability. However, the mechanism through which the transcriptional activator NifA regulates nitrogenase expression by interacting with PII nitrogen regulatory proteins remains unclear in diazotrophic proteobacteria lacking NifL. Here, we demonstrate that in Rhodopseudomonas palustris grown with ammonium, NifA bound deuridylylated PII proteins to form an inactive NifA-PII complex, thereby inhibiting the expression of nitrogenase. Upon nitrogen limitation, the dissociation of uridylylated PII proteins from NifA resulted in the full restoration of NifA activity, and, simultaneously, uridylylation of the significantly up-regulated PII protein GlnK2 led to the increased expression of NifA in R. palustris. This insight into how NifA interacts with PII proteins and controls nitrogenase expression sets the stage for creating highly efficient diazotrophs, reducing the need for energy-intensive chemical fertilizers and helping to diminish carbon emissions.
Asunto(s)
Compuestos de Amonio , Proteínas Bacterianas , Fijación del Nitrógeno , Proteínas PII Reguladoras del Nitrógeno , Factores de Transcripción , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Compuestos de Amonio/metabolismo , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Proteínas PII Reguladoras del Nitrógeno/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Regulación Bacteriana de la Expresión Génica , Nitrogenasa/metabolismo , Rhodopseudomonas/metabolismo , Rhodopseudomonas/genéticaRESUMEN
Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4, have greatly contributed to our understanding of the nitrogen interaction network of cyanobacteria, the main hubs of which are the trimeric PII and the monomeric PipX regulators. The bacterial two-hybrid system, based on the reconstitution in the E. coli cytoplasm of the adenylate cyclase of Bordetella pertussis, should provide a relatively faster and presumably more physiological assay for cyanobacterial proteins than the yeast system. Here, we used the bacterial two-hybrid system to gain additional insights into the cyanobacterial PipX interaction network while simultaneously assessing the advantages and limitations of the two most popular two-hybrid systems. A comprehensive mutational analysis of PipX and bacterial two-hybrid assays were performed to compare the outcomes between yeast and bacterial systems. We detected interactions that were previously recorded in the yeast two-hybrid system as negative, as well as a "false positive", the self-interaction of PipX, which is rather an indirect interaction that is dependent on PII homologues from the E. coli host, a result confirmed by Western blot analysis with relevant PipX variants. This is, to our knowledge, the first report of the molecular basis of a false positive in the bacterial two-hybrid system.
Asunto(s)
Proteínas Bacterianas , Cianobacterias , Proteínas PII Reguladoras del Nitrógeno , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Cianobacterias/metabolismo , Cianobacterias/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Proteínas PII Reguladoras del Nitrógeno/genética , Unión Proteica , Técnicas del Sistema de Dos HíbridosRESUMEN
Members of the PII superfamily are versatile, multitasking signaling proteins ubiquitously found in all domains of life. They adeptly monitor and synchronize the cell's carbon, nitrogen, energy, redox, and diurnal states, primarily by binding interdependently to adenyl-nucleotides, including charged nucleotides (ATP, ADP, and AMP) and second messengers such as cyclic adenosine monophosphate (cAMP), cyclic di-adenosine monophosphate (c-di-AMP), and S-adenosylmethionine-AMP (SAM-AMP). These proteins also undergo a variety of posttranslational modifications, such as phosphorylation, adenylation, uridylation, carboxylation, and disulfide bond formation, which further provide cues on the metabolic state of the cell. Serving as precise metabolic sensors, PII superfamily proteins transmit this information to diverse cellular targets, establishing dynamic regulatory assemblies that fine-tune cellular homeostasis. Recently discovered, PII-like proteins are emerging families of signaling proteins that, while related to canonical PII proteins, have evolved to fulfill a diverse range of cellular functions, many of which remain elusive. In this review, we focus on the evolution of PII-like proteins and summarize the molecular mechanisms governing the assembly dynamics of PII complexes, with a special emphasis on the PII-like protein SbtB.
Asunto(s)
Homeostasis , Transducción de Señal , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Proteínas PII Reguladoras del Nitrógeno/genética , Procesamiento Proteico-Postraduccional , Bacterias/metabolismo , Bacterias/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genéticaRESUMEN
Coordinated carbon and nitrogen metabolism is crucial for bacteria living in the fluctuating environments. Intracellular carbon and nitrogen homeostasis is maintained by a sophisticated network, in which the widespread signaling protein PII acts as a major regulatory hub. In cyanobacteria, PII was proposed to regulate the nitrate uptake by an ABC (ATP-binding cassette)-type nitrate transporter NrtABCD, in which the nucleotide-binding domain of NrtC is fused with a C-terminal regulatory domain (CRD). Here, we solved three cryoelectron microscopy structures of NrtBCD, bound to nitrate, ATP, and PII, respectively. Structural and biochemical analyses enable us to identify the key residues that form a hydrophobic and a hydrophilic cavity along the substrate translocation channel. The core structure of PII, but not the canonical T-loop, binds to NrtC and stabilizes the CRD, making it visible in the complex structure, narrows the substrate translocation channel in NrtB, and ultimately locks NrtBCD at an inhibited inward-facing conformation. Based on these results and previous reports, we propose a putative transport cycle driven by NrtABCD, which is allosterically inhibited by PII in response to the cellular level of 2-oxoglutarate. Our findings provide a distinct regulatory mechanism of ABC transporter via asymmetrically binding to a signaling protein.
Asunto(s)
Cianobacterias , Transportadores de Nitrato , Nitratos/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Alostérica , Microscopía por Crioelectrón , Cianobacterias/metabolismo , Adenosina Trifosfato/metabolismo , Nitrógeno/metabolismo , Carbono/metabolismo , Proteínas PII Reguladoras del Nitrógeno/genética , Proteínas PII Reguladoras del Nitrógeno/metabolismoRESUMEN
A suite of molecular sensory systems enables Caulobacter to control growth, development, and reproduction in response to levels of essential elements. The bacterial enhancer-binding protein (bEBP) NtrC and its cognate sensor histidine kinase, NtrB, are key regulators of nitrogen assimilation in many bacteria, but their roles in Caulobacter metabolism and development are not well defined. Notably, Caulobacter NtrC is an unconventional bEBP that lacks the σ54-interacting loop commonly known as the GAFTGA motif. Here we show that deletion of Caulobacter crescentus ntrC slows cell growth in complex medium and that ntrB and ntrC are essential when ammonium is the sole nitrogen source due to their requirement for glutamine synthetase expression. Random transposition of a conserved IS3-family mobile genetic element frequently rescued the growth defect of ntrC mutant strains by restoring transcription of the glnBA operon, revealing a possible role for IS3 transposition in shaping the evolution of Caulobacter populations during nutrient limitation. We further identified dozens of direct NtrC-binding sites on the C. crescentus chromosome, with a large fraction located near genes involved in polysaccharide biosynthesis. The majority of binding sites align with those of the essential nucleoid-associated protein, GapR, or the cell cycle regulator, MucR1. NtrC is therefore predicted to directly impact the regulation of cell cycle and cell development. Indeed, loss of NtrC function led to elongated polar stalks and elevated synthesis of cell envelope polysaccharides. This study establishes regulatory connections between NtrC, nitrogen metabolism, polar morphogenesis, and envelope polysaccharide synthesis in Caulobacter. IMPORTANCE Bacteria balance cellular processes with the availability of nutrients in their environment. The NtrB-NtrC two-component signaling system is responsible for controlling nitrogen assimilation in many bacteria. We have characterized the effect of ntrB and ntrC deletion on Caulobacter growth and development and uncovered a role for spontaneous IS element transposition in the rescue of transcriptional and nutritional deficiencies caused by ntrC mutation. We further defined the regulon of Caulobacter NtrC, a bacterial enhancer-binding protein, and demonstrate that it shares specific binding sites with essential proteins involved in cell cycle regulation and chromosome organization. Our work provides a comprehensive view of transcriptional regulation mediated by a distinctive NtrC protein, establishing its connection to nitrogen assimilation and developmental processes in Caulobacter.
Asunto(s)
Caulobacter , Secuencia de Bases , Caulobacter/genética , Nitrógeno/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Polisacáridos , Regulación Bacteriana de la Expresión Génica , Proteínas PII Reguladoras del Nitrógeno/genética , Proteínas PII Reguladoras del Nitrógeno/metabolismoRESUMEN
The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is co-transcribed with the conserved PII-like protein, SbtB, to augment intracellular inorganic carbon levels for efficient CO2 fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cyclic AMP (cAMP) and c-di-AMP. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge of potential redox-regulatory function, which we call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of hydrolysis reactions from ATP over ADP to AMP in crystallographic snapshots and unravel the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin, suggesting that it is generally linked to cellular metabolism, which is supported by physiological alterations in site-specific mutants of the SbtB protein. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding but also from the day/night status reported by the C-terminal redox switch.
Asunto(s)
Apirasa , Cianobacterias , Apirasa/genética , Apirasa/metabolismo , Bicarbonatos/metabolismo , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Cianobacterias/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas PII Reguladoras del Nitrógeno/metabolismoRESUMEN
PII proteins are signal processor proteins that regulate the cellular metabolism of Bacteria, Archea and plant chloroplasts typically in response to the cellular nitrogen status. Here, we report the first biochemical characterization of a novel PII-like protein PotN from Lentilactobacillus hilgardii. PotN is encoded in an operon together with the potABCD genes, encoding the ABC transporter for spermidine/putrescine. Like canonical PII proteins, the native PotN has a trimeric structure and competitively binds ATP and ADP, but it does not bind 2-oxoglutarate. Immunoprecipitation and pull-down experiments revealed that PotN is associated in vivo with the transcriptional regulator GlnR and the beta-subunit of pyruvate/2-oxoglutarate/acetoin dehydrogenase AcoB. Moreover, in vitro assays revealed that the ATPase domain of PotA also is able to interact with PotN. Interaction analyses demonstrated that PotN preferentially associates with PotA in the ADP state, whereas it binds to GlnR at elevated ATP levels. This suggests that PotN regulates the transport of polyamines and GlnR-dependent gene expression in response to the energy availability for the cell.
Asunto(s)
Firmicutes , Ácidos Cetoglutáricos , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Firmicutes/metabolismo , Ácidos Cetoglutáricos/metabolismo , Nitrógeno/metabolismo , Proteínas PII Reguladoras del Nitrógeno/metabolismoRESUMEN
PII proteins are multitasking information-processing proteins occurring in bacteria, archaea, and plastids, decoding the metabolic state of the cells and providing this information to various regulatory targets. Research in recent years identified a wide range of novel PII targets mainly through ligand fishing assays, indicating that PII proteins evolved into major regulatory hubs of cellular metabolism. PII proteins orchestrate not only key steps of nitrogen and carbon metabolism but rather control a wide range of transporters and can also regulate the production of signaling molecules (c-di-GMP) and cofactors (NAD+). A recently identified class of PII-interacting proteins, which by themselves have no enzymatic activity, modulate cellular processes through protein interactions, further extending the regulatory range of PII proteins.
Asunto(s)
Nitrógeno , Transducción de Señal , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Nitrógeno/metabolismo , Proteínas PII Reguladoras del Nitrógeno/genética , Proteínas PII Reguladoras del Nitrógeno/metabolismoRESUMEN
The PII protein is an evolutionary, highly conserved regulatory protein found in both bacteria and higher plants. In bacteria, it modulates the activity of several enzymes, transporters, and regulatory factors by interacting with them and thereby regulating important metabolic hubs, such as carbon/nitrogen homeostasis. More than two decades ago, the PII protein was characterized for the first time in plants, but its physiological role is still not sufficiently resolved. To gain more insights into the function of this protein, we investigated the interaction behavior of AtPII with candidate proteins by BiFC and FRET/FLIM in planta and with GFP/RFP traps in vitro. In the course of these studies, we found that AtPII interacts in chloroplasts with itself as well as with known interactors such as N-acetyl-L-glutamate kinase (NAGK) in dot-like aggregates, which we named PII foci. In these novel protein aggregates, AtPII also interacts with yet unknown partners, which are known to be involved in plastidic protein degradation. Further studies revealed that the C-terminal component of AtPII is crucial for the formation of PII foci. Altogether, the discovery and description of PII foci indicate a novel mode of interaction between PII proteins and other proteins in plants. These findings may represent a new starting point for the elucidation of physiological functions of PII proteins in plants.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Proteínas PII Reguladoras del Nitrógeno/química , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Dominios Proteicos , Mapas de Interacción de Proteínas , ProteolisisRESUMEN
The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by its promoter-recognition sigma subunit. The model prokaryote E. coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. Using genomic SELEX (gSELEX) screening in vitro, we identified the whole set of 'constitutive' promoters recognized by the reconstituted RNAP holoenzyme alone, containing RpoD (σ70), RpoS (σ38), RpoH (σ32), RpoF (σ28) or RpoE (σ24), in the absence of other supporting regulatory factors. In contrast, RpoN sigma (σ54), involved in expression of nitrogen-related genes and also other cellular functions, requires an enhancer (or activator) protein, such as NtrC, for transcription initiation. In this study, a series of gSELEX screenings were performed to search for promoters recognized by the RpoN RNAP holoenzyme in the presence and absence of the major nitrogen response enhancer NtrC, the best-characterized enhancer. Based on the RpoN holoenzyme-binding sites, a total of 44 to 61 putative promoters were identified, which were recognized by the RpoN holoenzyme alone. In the presence of the enhancer NtrC, the recognition target increased to 61-81 promoters. Consensus sequences of promoters recognized by RpoN holoenzyme in the absence and presence of NtrC were determined. The promoter activity of a set of NtrC-dependent and -independent RpoN promoters was verified in vivo under nitrogen starvation, in the presence and absence of RpoN and/or NtrC. The promoter activity of some RpoN-recognized promoters increased in the absence of RpoN or NtrC, supporting the concept that the promoter-bound NtrC-enhanced RpoN holoenzyme functions as a repressor against RpoD holoenzyme. Based on our findings, we propose a model in which the RpoN holoenzyme fulfils the dual role of repressor and transcriptase for the same set of genes. We also propose that the promoter recognized by RpoN holoenzyme in the absence of enhancers is the 'repressive' promoter. The presence of high-level RpoN sigma in growing E. coli K-12 in rich medium may be related to the repression role of a set of genes needed for the utilization of ammonia as a nitrogen source in poor media. The list of newly identified regulatory targets of RpoN provides insight into E. coli survival under nitrogen-depleted conditions in nature.
Asunto(s)
Escherichia coli K12 , Factor sigma , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Elementos de Facilitación Genéticos , Escherichia coli , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa Sigma 54 , Factor sigma/genética , Factor sigma/metabolismo , Factores de Transcripción/genéticaRESUMEN
Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs). Successful urinary tract colonization requires appropriate expression of virulence factors in response to host environmental cues, such as limited oxygen and iron availability. Hemolysin is a pore-forming toxin, and its expression correlates with the severity of UPEC infection. Previously, we showed that hemolysin expression is enhanced under anaerobic conditions; however, the genetic basis and regulatory mechanisms involved remain undefined. Here, a transposon-based forward screen identified bis-molybdopterin guanine dinucleotide cofactor (bis-MGD) biosynthesis as an important factor for a full transcription of hemolysin under anaerobiosis but not under aerobiosis. bis-MGD positively influences hemolysin transcription via c3566-c3568, an operon immediately upstream of and cotranscribed with hlyCABD. Furthermore, suppressor mutation analysis identified the nitrogen regulator NtrC as a direct repressor of c3566-c3568-hlyCABD expression, and intact bis-MGD biosynthesis downregulated ntrC expression, thus at least partially explaining the positive role of bis-MGD in modulating hemolysin expression. Finally, bis-MGD is involved in hemolysin-mediated uroepithelial cell death and contributes to the competitive fitness of UPEC in a murine model of UTI. Collectively, our data establish that bis-MGD biosynthesis plays a crucial role in UPEC fitness in vivo, thus providing a potential target for combatting UTIs.
Asunto(s)
Infecciones por Escherichia coli/microbiología , Nucleótidos de Guanina/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Pterinas/metabolismo , Escherichia coli Uropatógena/genética , Escherichia coli Uropatógena/metabolismo , Anaerobiosis , Animales , Muerte Celular , Línea Celular , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos CBA , Mutagénesis Insercional , Operón , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Factores de Transcripción/metabolismo , Transcriptoma , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Protein inhibition is a natural regulatory process to control cellular metabolic fluxes. PII-family signal-transducing effectors are in this matter key regulators of the nitrogen metabolism. Their interaction with their various targets is governed by the cellular nitrogen level and the energy charge. Structural studies on GlnK, a PII-family inhibitor of the ammonium transporters (Amt), showed that the T-loops responsible for channel obstruction are displaced upon the binding of 2-oxoglutarate, magnesium and ATP in a conserved cleft. However, GlnK from Methanocaldococcus jannaschii was shown to bind 2-oxoglutarate on the tip of its T-loop, causing a moderate disruption to GlnK-Amt interaction, raising the question if methanogenic archaea use a singular adaptive strategy. Here we show that membrane fractions of Methanothermococcus thermolithotrophicus released GlnKs only in the presence of Mg-ATP and 2-oxoglutarate. This observation led us to structurally characterize the two GlnK isoforms apo or in complex with ligands. Together, our results show that the 2-oxoglutarate binding interface is conserved in GlnKs from Methanococcales, including Methanocaldococcus jannaschii, emphasizing the importance of a free carboxy-terminal group to facilitate ligand binding and to provoke the shift of the T-loop positions.
Asunto(s)
Compuestos de Amonio/metabolismo , Ácidos Cetoglutáricos/metabolismo , Methanococcales/metabolismo , Proteínas PII Reguladoras del Nitrógeno , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Transporte Iónico , Redes y Vías Metabólicas , Modelos Moleculares , Nitrógeno/metabolismo , Proteínas PII Reguladoras del Nitrógeno/química , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas/fisiología , Análisis de Secuencia de ProteínaRESUMEN
PII proteins are ubiquitous signaling proteins that are involved in the regulation of the nitrogen/carbon balance in bacteria, archaea, and some plants and algae. Signal transduction via PII proteins is modulated by effector molecules and post-translational modifications in the PII T-loop. Whereas the binding of ADP, ATP and the concomitant binding of ATP and 2-oxoglutarate (2OG) engender two distinct conformations of the T-loop that either favor or disfavor the interaction with partner proteins, the structural consequences of post-translational modifications such as phosphorylation, uridylylation and adenylylation are far less well understood. In the present study, crystal structures of the PII protein GlnK from Corynebacterium glutamicum have been determined, namely of adenylylated GlnK (adGlnK) and unmodified unadenylylated GlnK (unGlnK). AdGlnK has been proposed to act as an inducer of the transcription repressor AmtR, and the adenylylation of Tyr51 in GlnK has been proposed to be a prerequisite for this function. The structures of unGlnK and adGlnK allow the first atomic insights into the structural implications of the covalent attachment of an AMP moiety to the T-loop. The overall GlnK fold remains unaltered upon adenylylation, and T-loop adenylylation does not appear to interfere with the formation of the two major functionally important T-loop conformations, namely the extended T-loop in the canonical ADP-bound state and the compacted T-loop that is adopted upon the simultaneous binding of Mg-ATP and 2OG. Thus, the PII-typical conformational switching mechanism appears to be preserved in GlnK from C. glutamicum, while at the same time the functional repertoire becomes expanded through the accommodation of a peculiar post-translational modification.
Asunto(s)
Proteínas Bacterianas/química , Corynebacterium glutamicum , Proteínas PII Reguladoras del Nitrógeno/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Conformación Proteica , Procesamiento Proteico-Postraduccional , Estructura Cuaternaria de ProteínaRESUMEN
Among prokaryotes, cyanobacteria have an exclusive position as they perform oxygenic photosynthesis. Cyanobacteria substantially differ from other bacteria in further aspects, e.g., they evolved a plethora of unique regulatory mechanisms to control primary metabolism. This is exemplified by the regulation of glutamine synthetase (GS) via small proteins termed inactivating factors (IFs). Here, we reveal another small protein, encoded by the ssr0692 gene in the model strain Synechocystis sp. PCC 6803, that regulates flux into the ornithine-ammonia cycle (OAC), the key hub of cyanobacterial nitrogen stockpiling and remobilization. This regulation is achieved by the interaction with the central carbon/nitrogen control protein PII, which commonly controls entry into the OAC by activating the key enzyme of arginine synthesis, N-acetyl-l-glutamate kinase (NAGK). In particular, the Ssr0692 protein competes with NAGK for PII binding and thereby prevents NAGK activation, which in turn lowers arginine synthesis. Accordingly, we termed it PII-interacting regulator of arginine synthesis (PirA). Similar to the GS IFs, PirA accumulates in response to ammonium upshift due to relief from repression by the global nitrogen control transcription factor NtcA. Consistent with this, the deletion of pirA affects the balance of metabolite pools of the OAC in response to ammonium shocks. Moreover, the PirA-PII interaction requires ADP and is prevented by PII mutations affecting the T-loop conformation, the major protein interaction surface of this signal processing protein. Thus, we propose that PirA is an integrator determining flux into N storage compounds not only depending on the N availability but also the energy state of the cell.IMPORTANCE Cyanobacteria contribute a significant portion to the annual oxygen yield and play important roles in biogeochemical cycles, e.g., as major primary producers. Due to their photosynthetic lifestyle, cyanobacteria also arouse interest as hosts for the sustainable production of fuel components and high-value chemicals. However, their broad application as microbial cell factories is hampered by limited knowledge about the regulation of metabolic fluxes in these organisms. Our research identified a novel regulatory protein that controls nitrogen flux, in particular arginine synthesis. Besides its role as a proteinogenic amino acid, arginine is a precursor for the cyanobacterial storage compound cyanophycin, which is of potential interest to biotechnology. Therefore, the obtained results will not only enhance our understanding of flux control in these organisms but also help to provide a scientific basis for targeted metabolic engineering and, hence, the design of photosynthesis-driven biotechnological applications.
Asunto(s)
Amoníaco/metabolismo , Ornitina/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Arginina/biosíntesis , Arginina/metabolismo , Nitrógeno/metabolismo , Proteínas PII Reguladoras del Nitrógeno/genética , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Transducción de SeñalRESUMEN
In cyanobacteria, the PII protein (the glnB gene product) regulates a number of proteins involved in nitrogen assimilation including PipX, the coactivator of the global nitrogen regulator protein NtcA. In Synechococcus elongatus PCC 7942, construction of a PII-less mutant retaining the wild-type pipX gene is difficult because of the toxicity of uncontrolled action of PipX and the other defect(s) resulting from the loss of PIIper se, but the nature of the PipX toxicity and the PipX-independent defect(s) remains unclear. Characterization of a PipX-less glnB mutant (PD4) in this study showed that the loss of PII increases the sensitivity of PSII to ammonium. Ammonium was shown to stimulate the formation of reactive oxygen species in the mutant cells. The ammonium-sensitive growth phenotype of PD4 was rescued by the addition of an antioxidant α-tocopherol, confirming that photo-oxidative damage was the major cause of the growth defect. A targeted PII mutant retaining wild-type pipX was successfully constructed from the wild-type S. elongatus strain (SPc) in the presence of α-tocopherol. The resulting mutant (PD1X) showed an unusual chlorophyll fluorescence profile, indicating extremely slow reduction and re-oxidation of QA, which was not observed in mutants defective in both glnB and pipX. These results showed that the aberrant action of uncontrolled PipX resulted in an impairment of the electron transport reactions in both the reducing and oxidizing sides of QA.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Synechococcus/crecimiento & desarrollo , Synechococcus/metabolismo , Compuestos de Amonio/metabolismo , Compuestos de Amonio/farmacología , Proteínas Bacterianas/genética , Clorofila/química , Clorofila/metabolismo , Medios de Cultivo/química , Medios de Cultivo/farmacología , Fluorescencia , Mutación , Proteínas PII Reguladoras del Nitrógeno/genética , Paraquat/farmacología , Especies Reactivas de Oxígeno , Synechococcus/efectos de los fármacos , Synechococcus/genética , alfa-Tocoferol/farmacologíaRESUMEN
Nitrogen limitation imposes a major transition in the lifestyle of nondiazotrophic cyanobacteria that is controlled by a complex interplay of regulatory factors involving the pervasive signal processor PII Immediately upon nitrogen limitation, newly fixed carbon is redirected toward glycogen synthesis. How the metabolic switch for diverting fixed carbon toward the synthesis of glycogen or of cellular building blocks is operated was so far poorly understood. Here, using the nondiazotrophic cyanobacterium Synechocystis sp. PCC 6803 as model system, we identified a novel PII interactor, the product of the sll0944 gene, which we named PirC. We show that PirC binds to and inhibits the activity of 2,3-phosphoglycerate-independent phosphoglycerate mutase (PGAM), the enzyme that deviates newly fixed CO2 toward lower glycolysis. The binding of PirC to either PII or PGAM is tuned by the metabolite 2-oxoglutarate (2-OG), which accumulates upon nitrogen starvation. In these conditions, the high levels of 2-OG dissociate the PirC-PII complex to promote PirC binding to and inhibition of PGAM. Accordingly, a PirC-deficient mutant showed strongly reduced glycogen levels upon nitrogen deprivation, whereas polyhydroxybutyrate granules were overaccumulated compared to wild-type. Metabolome analysis revealed an imbalance in 3-phosphoglycerate to pyruvate levels in the pirC mutant, confirming that PirC controls the carbon flux in cyanobacteria via mutually exclusive interaction with either PII or PGAM.
Asunto(s)
Proteínas Bacterianas/genética , Cianobacterias/genética , Proteínas PII Reguladoras del Nitrógeno/genética , Fosfoglicerato Mutasa/genética , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Cianobacterias/metabolismo , Nitrógeno/metabolismo , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Fosfoglicerato Mutasa/metabolismo , Synechocystis/genética , Synechocystis/metabolismoRESUMEN
Escherichia coli uses the C4-dicarboxylate transporter DcuA for L-aspartate/fumarate antiport, which results in the exploitation of L-aspartate for fumarate respiration under anaerobic conditions and for nitrogen assimilation under aerobic and anaerobic conditions. L-Aspartate represents a high-quality nitrogen source for assimilation. Nitrogen assimilation from L-aspartate required DcuA, and aspartase AspA to release ammonia. Ammonia is able to provide by established pathways the complete set of intracellular precursors (ammonia, L-aspartate, L-glutamate, and L-glutamine) for synthesizing amino acids, nucleotides, and amino sugars. AspA was regulated by a central regulator of nitrogen metabolism, GlnB. GlnB interacted with AspA and stimulated its L-aspartate deaminase activity (NH3 -forming), but not the reverse amination reaction. GlnB stimulation required 2-oxoglutarate and ATP, or uridylylated GlnB-UMP, consistent with the activation of nitrogen assimilation under nitrogen limitation. Binding to AspA was lost in the GlnB(Y51F) mutant of the uridylylation site. AspA, therefore, represents a new type of GlnB target that binds GlnB (with ATP and 2-oxoglutarate), or GlnB-UMP (with or without effectors), and both situations stimulate AspA deamination activity. Thus, AspA represents the central enzyme for nitrogen assimilation from L-aspartate, and AspA is integrated into the nitrogen assimilation network by the regulator GlnB.
Asunto(s)
Aspartato Amoníaco-Liasa/metabolismo , Ácido Aspártico/metabolismo , Proteínas Bacterianas/metabolismo , Transportadores de Ácidos Dicarboxílicos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Escherichia coli/fisiología , Nitrógeno/metabolismo , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Amoníaco/metabolismo , Ácido Aspártico/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Redes y Vías Metabólicas , Mutación , Proteínas PII Reguladoras del Nitrógeno/química , Proteínas PII Reguladoras del Nitrógeno/genética , Dominios y Motivos de Interacción de ProteínasRESUMEN
Nitrogen is a most important nutrient resource for Escherichia coli and other bacteria that harbor the glnKamtB operon, a high-affinity ammonium uptake system highly interconnected with cellular metabolism. Although this system confers an advantage to bacteria when growing under nitrogen-limiting conditions, little is known about the impact of these genes on microbial fitness under nutrient-rich conditions. Here, the genetically tractable E. coli BW25113 strain and its glnKamtB-null mutant (JW0441) were used to analyze the impact of GlnK-AmtB on growth rates and oxidative stress tolerance. Strain JW0441 showed a shorter initial lag phase, higher growth rate, higher citrate synthase activity, higher oxidative stress tolerance and lower expression of serA than strain BW25113 under nutrient-rich conditions, suggesting a fitness cost to increase metabolic plasticity associated with serine metabolism. The overexpression of serA in strain JW0441 resulted in a decreased growth rate and stress tolerance in nutrient-rich conditions similar to that of strain BW25113, suggesting that the negative influence on bacterial fitness imposed by GlnK-AmtB can be traced to the control of serine biosynthesis. Finally, we discuss the potential applications of glnKamtB mutants in bioproduction processes.
Asunto(s)
Proteínas de Transporte de Catión/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Nucleotidiltransferasas/genética , Proteínas PII Reguladoras del Nitrógeno/genética , Serina/biosíntesis , Proteínas de Transporte de Catión/metabolismo , Proteínas de Escherichia coli/metabolismo , Microbiología Industrial , Mutación , Nucleotidiltransferasas/metabolismo , Operón/genética , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Serina/genéticaRESUMEN
Listeria monocytogenes is a fastidious bacterial pathogen that can utilize only a limited number of nitrogen sources for growth. Both glutamine and ammonium are common nitrogen sources used in listerial defined growth media, but little is known about the regulation of their uptake or utilization. The functional role of L. monocytogenes GlnR, the transcriptional regulator of nitrogen metabolism genes in low-G+C Gram-positive bacteria, was determined using transcriptome sequencing and real-time reverse transcription-PCR experiments. The GlnR regulon included transcriptional units involved in ammonium transport (amtB glnK) and biosynthesis of glutamine (glnRA) and glutamate (gdhA) from ammonium. As in other bacteria, GlnR proved to be an autoregulatory repressor of the glnRA operon. Unexpectedly, GlnR was most active during growth with ammonium as the nitrogen source and less active in the glutamine medium, apparently because listerial cells perceive growth with glutamine as a nitrogen-limiting condition. Therefore, paradoxically, expression of the glnA gene, encoding glutamine synthetase, was highest in the glutamine medium. For the amtB glnK operon, GlnR served as both a negative regulator in the presence of ammonium and a positive regulator in the glutamine medium. The gdhA gene was subject to a third mode of regulation that apparently required an elevated level of GlnR for repression. Finally, activity of glutamate dehydrogenase encoded by the gdhA gene appeared to correlate inversely with expression of gltAB, the operon that encodes the other major glutamate-synthesizing enzyme, glutamate synthase. Both gdhA and amtB were also regulated, in a negative manner, by the global transcriptional regulator CodY.IMPORTANCEL. monocytogenes is a widespread foodborne pathogen. Nitrogen-containing compounds, such as the glutamate-containing tripeptide, glutathione, and glutamine, have been shown to be important for expression of L. monocytogenes virulence genes. In this work, we showed that a transcriptional regulator, GlnR, controls expression of critical listerial genes of nitrogen metabolism that are involved in ammonium uptake and biosynthesis of glutamine and glutamate. A different mode of GlnR-mediated regulation was found for each of these three pathways.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Nitrógeno/metabolismo , Compuestos de Amonio/metabolismo , Glutamato-Amoníaco Ligasa/genética , Glutamato-Amoníaco Ligasa/metabolismo , Ácido Glutámico/biosíntesis , Ácido Glutámico/genética , Glutamina/biosíntesis , Glutamina/genética , Listeria monocytogenes/crecimiento & desarrollo , Mutación , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Operón , Proteínas PII Reguladoras del Nitrógeno/genética , Proteínas PII Reguladoras del Nitrógeno/metabolismo , Regiones Promotoras Genéticas , RNA-Seq , Regulón , Transactivadores/genética , Transactivadores/metabolismo , Transcriptoma , Virulencia/genéticaRESUMEN
All cyanobacteria produce a neurotoxic non-protein amino acid ß-N-methylamino-L-alanine (BMAA). However, the biological function of BMAA in the regulation of cyanobacteria metabolism still remains undetermined. It is known that BMAA suppresses the formation of heterocysts in diazotrophic cyanobacteria under nitrogen starvation conditions, and BMAA induces the formation of heterocyst-like cells under nitrogen excess conditions, by causing the expression of heterocyst-specific genes that are usually "silent" under nitrogen-replete conditions, as if these bacteria receive a nitrogen deficiency intracellular molecular signal. In order to find out the molecular mechanisms underlying this unexpected BMAA effect, we studied the proteome of cyanobacterium Nostoc sp. PCC 7120 grown under BMAA treatment in nitrogen-replete medium. Experiments were performed in two experimental settings: (1) in control samples consisted of cells grown without the BMAA treatment and (2) the treated samples consisted of cells grown with addition of an aqueous solution of BMAA (20 µM). In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by LC-MS/MS spectrometry. Among them, 80 proteins belonging to different functional categories were chosen for further functional analysis and interpretation of obtained proteomic data. Here, we provide the evidence that a pleiotropic regulatory effect of BMAA on the proteome of cyanobacterium was largely different under conditions of nitrogen-excess compared to its effect under nitrogen starvation conditions (that was studied in our previous work). The most significant difference in proteome expression between the BMAA-treated and untreated samples under different growth conditions was detected in key regulatory protein PII (GlnB). BMAA downregulates protein PII in nitrogen-starved cells and upregulates this protein in nitrogen-replete conditions. PII protein is a key signal transduction protein and the change in its regulation leads to the change of many other regulatory proteins, including different transcriptional factors, enzymes and transporters. Complex changes in key metabolic and regulatory proteins (RbcL, RbcS, Rca, CmpA, GltS, NodM, thioredoxin 1, RpbD, ClpP, MinD, RecA, etc.), detected in this experimental study, could be a reason for the appearance of the "starvation" state in nitrogen-replete conditions in the presence of BMAA. In addition, 15 proteins identified in this study are encoded by genes, which are under the control of NtcA-a global transcriptional regulator-one of the main protein partners and transcriptional regulators of PII protein. Thereby, this proteomic study gives a possible explanation of cyanobacterium starvation under nitrogen-replete conditions and BMAA treatment. It allows to take a closer look at the regulation of cyanobacteria metabolism affected by this cyanotoxin.