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1.
Annu Rev Microbiol ; 76: 91-111, 2022 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-35417196

RESUMO

Since Jacques Monod's foundational work in the 1940s, investigators studying bacterial physiology have largely (but not exclusively) focused on the exponential phase of bacterial cultures, which is characterized by rapid growth and high biosynthesis activity in the presence of excess nutrients. However, this is not the predominant state of bacterial life. In nature, most bacteria experience nutrient limitation most of the time. In fact, investigators even prior to Monod had identified other aspects of bacterial growth, including what is now known as the stationary phase, when nutrients become limiting. This review will discuss how bacteria transition to growth arrest in response to nutrient limitation through changes in transcription, translation, and metabolism. We will then examine how these changes facilitate survival during potentially extended periods of nutrient limitation, with particular attention to the metabolic strategies that underpin bacterial longevity in this state.


Assuntos
Bactérias , Longevidade , Bactérias/genética , Fenômenos Fisiológicos Bacterianos , Divisão Celular , Regulação Bacteriana da Expressão Gênica
2.
Annu Rev Microbiol ; 74: 247-266, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32660386

RESUMO

The enzyme molybdenum nitrogenase converts atmospheric nitrogen gas to ammonia and is of critical importance for the cycling of nitrogen in the biosphere and for the sustainability of life. Alternative vanadium and iron-only nitrogenases that are homologous to molybdenum nitrogenases are also found in archaea and bacteria, but they have a different transition metal, either vanadium or iron, at their active sites. So far alternative nitrogenases have only been found in microbes that also have molybdenum nitrogenase. They are less widespread than molybdenum nitrogenase in bacteria and archaea, and they are less efficient. The presumption has been that alternative nitrogenases are fail-safe enzymes that are used in situations where molybdenum is limiting. Recent work indicates that vanadium nitrogenase may play a role in the global biological nitrogen cycle and iron-only nitrogenase may contribute products that shape microbial community interactions in nature.


Assuntos
Bactérias/metabolismo , Ferro/metabolismo , Nitrogênio/metabolismo , Nitrogenase/metabolismo , Archaea/enzimologia , Archaea/metabolismo , Bactérias/enzimologia , Molibdênio/metabolismo , Fixação de Nitrogênio
3.
Proc Natl Acad Sci U S A ; 119(18): e2117633119, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35476526

RESUMO

Surface sensing is a critical process that promotes the transition to a biofilm lifestyle. Several surface-sensing mechanisms have been described for a range of species, most involving surface appendages, such as flagella and pili. Pseudomonas aeruginosa uses the Wsp chemosensory-like signal transduction pathway to sense surfaces and promote biofilm formation. The methyl-accepting chemotaxis protein WspA recognizes an unknown surface-associated signal and initiates a phosphorylation cascade that activates the diguanylate cyclase WspR. We conducted a screen for Wsp-activating compounds and found that chemicals that impact the cell envelope induce Wsp signaling, increase intracellular c-di-GMP levels, and can promote surface attachment. To isolate the Wsp system from other P. aeruginosa surface-sensing systems, we heterologously expressed it in Escherichia coli and found it sufficient for sensing surfaces and the chemicals identified in our screen. Using well-characterized reporters for different E. coli cell envelope stress responses, we then determined that Wsp sensitivity overlapped with multiple E. coli cell envelope stress-response systems. Using mutational and CRISPRi analysis, we found that misfolded proteins in the periplasm appear to be a major stimulus of the Wsp system. Finally, we show that surface attachment appears to have an immediate, observable effect on cell envelope integrity. Collectively, our results provide experimental evidence that cell envelope stress represents an important feature of surface sensing in P. aeruginosa.


Assuntos
Parede Celular , Pseudomonas aeruginosa , Biofilmes , Membrana Celular/metabolismo , Periplasma , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo
4.
Appl Environ Microbiol ; 90(1): e0174123, 2024 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-38078768

RESUMO

Denitrification is a form of anaerobic respiration wherein nitrate (NO3-) is sequentially reduced via nitrite (NO2-), nitric oxide, and nitrous oxide (N2O) to dinitrogen gas (N2) by four reductase enzymes. Partial denitrifying bacteria possess only one or some of these four reductases and use them as independent respiratory modules. However, it is unclear if partial denitrifiers sense and respond to denitrification intermediates outside of their reductase repertoire. Here, we tested the denitrifying capabilities of two purple nonsulfur bacteria, Rhodopseudomonas palustris CGA0092 and Rhodobacter capsulatus SB1003. Each had denitrifying capabilities that matched their genome annotation; CGA0092 reduced NO2- to N2, and SB1003 reduced N2O to N2. For each bacterium, N2O reduction could be used both for electron balance during growth on electron-rich organic compounds in light and for energy transformation via respiration in darkness. However, N2O reduction required supplementation with a denitrification intermediate, including those for which there was no associated denitrification enzyme. For CGA0092, NO3- served as a stable, non-catalyzable molecule that was sufficient to activate N2O reduction. Using a ß-galactosidase reporter, we found that NO3- acted, at least in part, by stimulating N2O reductase gene expression. In SB1003, NO2- but not NO3- activated N2O reduction, but NO2- was slowly removed, likely by a promiscuous enzyme activity. Our findings reveal that partial denitrifiers can still be subject to regulation by denitrification intermediates that they cannot use.IMPORTANCEDenitrification is a form of microbial respiration wherein nitrate is converted via several nitrogen oxide intermediates into harmless dinitrogen gas. Partial denitrifying bacteria, which individually have some but not all denitrifying enzymes, can achieve complete denitrification as a community by cross-feeding nitrogen oxide intermediates. However, the last intermediate, nitrous oxide (N2O), is a potent greenhouse gas that often escapes, motivating efforts to understand and improve the efficiency of denitrification. Here, we found that at least some partial denitrifying N2O reducers can sense and respond to nitrogen oxide intermediates that they cannot otherwise use. The regulatory effects of nitrogen oxides on partial denitrifiers are thus an important consideration in understanding and applying denitrifying bacterial communities to combat greenhouse gas emissions.


Assuntos
Gases de Efeito Estufa , Óxido Nitroso , Óxido Nitroso/metabolismo , Desnitrificação , Nitratos/metabolismo , Gases de Efeito Estufa/metabolismo , Dióxido de Nitrogênio/metabolismo , Dióxido de Nitrogênio/farmacologia , Bactérias/genética , Óxido Nítrico/metabolismo , Oxirredutases/metabolismo
5.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33649224

RESUMO

A number of plant-associated proteobacteria have LuxR family transcription factors that we refer to as PipR subfamily members. PipR proteins play roles in interactions between bacteria and their plant hosts, and some are important for bacterial virulence of plants. We identified an ethanolamine derivative, N-(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), as a potent effector of PipR-mediated gene regulation in the plant endophyte Pseudomonas GM79. HEHEAA-dependent PipR activity requires an ATP-binding cassette-type active transport system, and the periplasmic substrate-binding protein (SBP) of that system binds HEHEAA. To begin to understand the molecular basis of PipR system responses to plant factors we crystallized a HEHEAA-responsive SBP in the free- and HEHEAA-bound forms. The SBP, which is similar to peptide-binding SBPs, was in a closed conformation. A narrow cavity at the interface of its two lobes is wide enough to bind HEHEAA, but it cannot accommodate peptides with side chains. The polar atoms of HEHEAA are recognized by hydrogen-bonding interactions, and additional SBP residues contribute to the binding site. This binding mode was confirmed by a structure-based mutational analysis. We also show that a closely related SBP from the plant pathogen Pseudomonas syringae pv tomato DC3000 does not recognize HEHEAA. However, a single amino acid substitution in the presumed effector-binding pocket of the P. syringae SBP converted it to a weak HEHEAA-binding protein. The P. syringae PipR depends on a plant effector for activity, and our findings imply that different PipR-associated SBPs bind different effectors.


Assuntos
Acetamidas/química , Proteínas de Bactérias/química , Pseudomonas syringae/química , Acetamidas/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Pseudomonas syringae/metabolismo
6.
J Bacteriol ; 204(4): e0047921, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35285725

RESUMO

Hospital environments are excellent reservoirs for the opportunistic pathogen Acinetobacter baumannii in part because it is exceptionally tolerant to desiccation. We found that relative to other A. baumannii strains, the virulent strain AB5075 was strikingly desiccation resistant at 2% relative humidity (RH), suggesting that it is a good model for studies of the functional basis of this trait. Consistent with results from other A. baumannii strains at 40% RH, we found the global posttranscriptional regulator CsrA to be critically important for desiccation tolerance of AB5075 at 2% RH. Proteomics experiments identified proteins that were differentially present in wild-type and csrA mutant cells. Subsequent analysis of mutants in genes encoding some of these proteins revealed six genes that were required for wild-type levels of desiccation tolerance. These include genes for catalase, a universal stress protein, a hypothetical protein, and a biofilm-associated protein. Two genes of unknown function had very strong desiccation phenotypes, with one of the two genes predicting an intrinsically disordered protein (IDP) that binds to DNA. Intrinsically disordered proteins are widespread in eukaryotes but less so in prokaryotes. Our results suggest there are new mechanisms underlying desiccation tolerance in bacteria and identify several key functions involved. IMPORTANCE Acinetobacter baumannii is found in terrestrial environments but can cause nosocomial infections in very sick patients. A factor that contributes to the prevalence of A. baumannii in hospital settings is that it is intrinsically resistant to dry conditions. Here, we established the virulent strain A. baumannii AB5075 as a model for studies of desiccation tolerance at very low relative humidity. Our results show that this trait depends on two proteins of unknown function, one of which is predicted to be an intrinsically disordered protein. This category of protein is critical for the small animals named tardigrades to survive desiccation. Our results suggest that A. baumannii may have novel strategies to survive desiccation that have not previously been seen in bacteria.


Assuntos
Acinetobacter baumannii , Proteínas Intrinsicamente Desordenadas , Acinetobacter baumannii/metabolismo , Animais , Biofilmes , Dessecação , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteômica
7.
Proc Natl Acad Sci U S A ; 115(39): 9785-9790, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30190434

RESUMO

Certain plant-associated Proteobacteria sense their host environment by detecting an unknown plant signal recognized by a member of a LuxR subfamily of transcription factors. This interkingdom communication is important for both mutualistic and pathogenic interactions. The Populus root endophyte Pseudomonas sp. GM79 possesses such a regulator, named PipR. In a previous study we reported that PipR activates an adjacent gene (pipA) coding for a proline iminopeptidase in response to Populus leaf macerates and peptides and that this activation is dependent on a putative ABC-type transporter [Schaefer AL, et al. (2016) mBio 7:e01101-16]. In this study we identify a chemical derived from ethanolamine that induces PipR activity at picomolar concentrations, and we present evidence that this is the active inducer present in plant leaf macerates. First, a screen of more than 750 compounds indicated ethanolamine was a potent inducer for the PipR-sensing system; however, ethanolamine failed to bind to the periplasmic-binding protein (PBP) required for the signal response. This led us to discover that a specific ethanolamine derivative, N-(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), binds to the PBP and serves as a potent PipR-dependent inducer. We also show that a compound, which coelutes with HEHEAA in HPLC and induces pipA gene expression in a PipR-dependent manner, can be found in Populus leaf macerates. This work sheds light on how plant-associated bacteria can sense their environment and on the nature of inducers for a family of plant-responsive LuxR-like transcription factors found in plant-associated bacteria.


Assuntos
Acetamidas/metabolismo , Endófitos/fisiologia , Etanolamina/metabolismo , Reguladores de Crescimento de Plantas/fisiologia , Populus/microbiologia , Pseudomonas/fisiologia , Acetamidas/farmacologia , Endófitos/metabolismo , Regulação Bacteriana da Expressão Gênica , Espectrometria de Massas , Proteínas Periplásmicas de Ligação/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/metabolismo , Raízes de Plantas/microbiologia , Populus/metabolismo , Pseudomonas/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Transativadores/metabolismo , Transativadores/fisiologia
8.
J Biol Chem ; 294(8): 2854-2861, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30578298

RESUMO

Spontaneous formation of isoaspartates (isoDs) often causes protein damage. l-Isoaspartate O-methyltransferase (PIMT) repairs isoD residues by catalyzing the formation of an unstable l-isoaspartyl methyl ester that spontaneously converts to an l-aspartyl residue. PIMTs are widely distributed in all three domains of life and have been studied most intensively in connection with their role in protein repair and aging in plants and animals. Studies of bacterial PIMTs have been limited to Escherichia coli, which has one PIMT. The α-proteobacterium Rhodopseudomonas palustris has three annotated PIMT genes, one of which (rpa2580) has been found to be important for cellular longevity in a growth-arrested state. However, the biochemical activities of these three R. palustris PIMTs are unknown. Here, we expressed and characterized all three annotated PIMT proteins, finding that two of them, RPA0376 and RPA2838, had PIMT activity, whereas RPA2580 did not. RPA0376 and RPA2838 single- and double-deletion mutants did not differ in longevity from WT R. palustris and did not exhibit elevated levels of isoD residues in aged cells. Comparative sequence analyses revealed that RPA2580 belongs to a separate phylogenetic group of annotated PIMT proteins present in the α-proteobacteria. Our results suggest that this group of proteins is not involved in repair of protein isoD residues. In addition, the bona fide bacterial PIMT enzymes may play a different or subtler role in bacterial physiology than previously suggested.


Assuntos
Ácido Isoaspártico/metabolismo , Filogenia , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/química , Proteína D-Aspartato-L-Isoaspartato Metiltransferase/metabolismo , Rodopseudomonas/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Homologia de Sequência , Especificidade por Substrato
9.
Appl Environ Microbiol ; 86(11)2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32220835

RESUMO

The purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris strain CGA009 uses the three-carbon dicarboxylic acid malonate as the sole carbon source under phototrophic conditions. However, this bacterium grows extremely slowly on this compound and does not have operons for the two pathways for malonate degradation that have been detected in other bacteria. Many bacteria grow on a spectrum of carbon sources, some of which are classified as poor growth substrates because they support low growth rates. This trait is rarely addressed in the literature, but slow growth is potentially useful in biotechnological applications where it is imperative for bacteria to divert cellular resources to value-added products rather than to growth. This prompted us to explore the genetic and physiological basis for the slow growth of R. palustris with malonate as a carbon source. There are two unlinked genes annotated as encoding a malonyl coenzyme A (malonyl-CoA) synthetase (MatB) and a malonyl-CoA decarboxylase (MatA) in the genome of R. palustris, which we verified as having the predicted functions. Additionally, two tripartite ATP-independent periplasmic transporters (TRAP systems) encoded by rpa2047 to rpa2049 and rpa2541 to rpa2543 were needed for optimal growth on malonate. Most of these genes were expressed constitutively during growth on several carbon sources, including malonate. Our data indicate that R. palustris uses a piecemeal approach to growing on malonate. The data also raise the possibility that this bacterium will evolve to use malonate efficiently if confronted with an appropriate selection pressure.IMPORTANCE There is interest in understanding how bacteria metabolize malonate because this three-carbon dicarboxylic acid can serve as a building block in bioengineering applications to generate useful compounds that have an odd number of carbons. We found that the phototrophic bacterium Rhodopseudomonas palustris grows extremely slowly on malonate. We identified two enzymes and two TRAP transporters involved in the uptake and metabolism of malonate, but some of these elements are apparently not very efficient. R. palustris cells growing with malonate have the potential to be excellent biocatalysts, because cells would be able to divert cellular resources to the production of value-added compounds instead of using them to support rapid growth. In addition, our results suggest that R. palustris is a candidate for directed evolution studies to improve growth on malonate and to observe the kinds of genetic adaptations that occur to make a metabolic pathway operate more efficiently.


Assuntos
Malonatos/metabolismo , Redes e Vias Metabólicas , Rodopseudomonas/genética , Biodegradação Ambiental , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Rodopseudomonas/crescimento & desenvolvimento , Rodopseudomonas/metabolismo
10.
Proc Natl Acad Sci U S A ; 114(48): 12809-12814, 2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-29133402

RESUMO

In contrast to Escherichia coli, a model organism for chemotaxis that has 5 chemoreceptors and a single chemosensory pathway, Pseudomonas aeruginosa PAO1 has a much more complex chemosensory network, which consists of 26 chemoreceptors feeding into four chemosensory pathways. While several chemoreceptors were rigorously linked to specific pathways in a series of experimental studies, for most of them this information is not available. Thus, we addressed the problem computationally. Protein-protein interaction network prediction, coexpression data mining, and phylogenetic profiling all produced incomplete and uncertain assignments of chemoreceptors to pathways. However, comparative sequence analysis specifically targeting chemoreceptor regions involved in pathway interactions revealed conserved sequence patterns that enabled us to unambiguously link all 26 chemoreceptors to four pathways. Placing computational evidence in the context of experimental data allowed us to conclude that three chemosensory pathways in P. aeruginosa utilize one chemoreceptor per pathway, whereas the fourth pathway, which is the main system controlling chemotaxis, utilizes the other 23 chemoreceptors. Our results show that while only a very few amino acid positions in receptors, kinases, and adaptors determine their pathway specificity, assigning receptors to pathways computationally is possible. This requires substantial knowledge about interacting partners on a molecular level and focusing comparative sequence analysis on the pathway-specific regions. This general principle should be applicable to resolving many other receptor-pathway interactions.


Assuntos
Proteínas de Bactérias/genética , Quimiotaxia/genética , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/genética , Receptores de Superfície Celular/genética , Transdução de Sinais , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Fatores Quimiotáticos/química , Fatores Quimiotáticos/metabolismo , Biologia Computacional/métodos , Mineração de Dados/estatística & dados numéricos , Redes Reguladoras de Genes , Ligantes , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Pseudomonas aeruginosa/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/classificação , Receptores de Superfície Celular/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
11.
Proc Natl Acad Sci U S A ; 114(49): 13006-13011, 2017 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-29158389

RESUMO

Quorum sensing (QS) signals are used by bacteria to regulate biological functions in response to cell population densities. Cyclic diguanosine monophosphate (c-di-GMP) regulates cell functions in response to diverse environmental chemical and physical signals that bacteria perceive. In Burkholderia cenocepacia, the QS signal receptor RpfR degrades intracellular c-di-GMP when it senses the QS signal cis-2-dodecenoic acid, also called Burkholderia diffusible signal factor (BDSF), as a proxy for high cell density. However, it was unclear how this resulted in control of BDSF-regulated phenotypes. Here, we found that RpfR forms a complex with a regulator named GtrR (BCAL1536) to enhance its binding to target gene promoters under circumstances where the BDSF signal binds to RpfR to stimulate its c-di-GMP phosphodiesterase activity. In the absence of BDSF, c-di-GMP binds to the RpfR-GtrR complex and inhibits its ability to control gene expression. Mutations in rpfR and gtrR had overlapping effects on both the B. cenocepacia transcriptome and BDSF-regulated phenotypes, including motility, biofilm formation, and virulence. These results show that RpfR is a QS signal receptor that also functions as a c-di-GMP sensor. This protein thus allows B. cenocepacia to integrate information about its physical and chemical surroundings as well as its population density to control diverse biological functions including virulence. This type of QS system appears to be widely distributed in beta and gamma proteobacteria.


Assuntos
Proteínas de Bactérias/genética , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/patogenicidade , GMP Cíclico/análogos & derivados , Ácidos Graxos Monoinsaturados/metabolismo , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum/genética , Animais , Carga Bacteriana , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Infecções por Burkholderia/microbiologia , Infecções por Burkholderia/patologia , Burkholderia cenocepacia/crescimento & desenvolvimento , GMP Cíclico/metabolismo , Camundongos , Mutação , Fenótipo , Transdução de Sinais , Virulência
12.
J Biol Chem ; 293(30): 11727-11735, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-29794028

RESUMO

The MarR family transcriptional regulator CouR, from the soil bacterium Rhodopseudomonas palustris CGA009, has recently been shown to negatively regulate a p-coumarate catabolic operon. Unlike most characterized MarR repressors that respond to small metabolites at concentrations in the millimolar range, repression by CouR is alleviated by the 800-Da ligand p-coumaroyl-CoA with high affinity and specificity. Here we report the crystal structures of ligand-free CouR as well as the complex with p-coumaroyl-CoA, each to 2.1-Å resolution, and the 2.85-Å resolution cocrystal structure of CouR bound to an oligonucleotide bearing the cognate DNA operator sequence. In combination with binding experiments that uncover specific residues important for ligand and DNA recognition, these structures provide glimpses of a MarR family repressor in all possible states, providing an understanding of the molecular basis of DNA binding and the conformation alterations that accompany ligand-induced dissociation for activation of the operon.


Assuntos
Acil Coenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon , Proteínas Repressoras/metabolismo , Rodopseudomonas/genética , Acil Coenzima A/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Ácidos Cumáricos/metabolismo , Cristalografia por Raios X , Conformação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Rodopseudomonas/química , Rodopseudomonas/metabolismo , Ativação Transcricional
13.
Appl Environ Microbiol ; 85(9)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30824440

RESUMO

The anoxygenic phototrophic bacterium Rhodopseudomonas palustris produces methane (CH4) from carbon dioxide (CO2) and hydrogen (H2) from protons (H+) when it expresses a variant form of molybdenum (Mo) nitrogenase that has two amino acid substitutions near its active site. We examined the influence of light energy and electron availability on in vivo production of these biofuels. Nitrogenase activity requires large amounts of ATP, and cells exposed to increasing light intensities produced increasing amounts of CH4 and H2 As expected for a phototroph, intracellular ATP increased with increasing light intensity, but there was only a loose correlation between ATP content and CH4 and H2 production. There was a much stronger correlation between decreased intracellular ADP and increased gas production with increased light intensity, suggesting that the rate-limiting step for CH4 and H2 production by R. palustris is inhibition of nitrogenase by ADP. Increasing the amounts of electrons available to nitrogenase by providing cells with organic alcohols, using nongrowing cells, blocking electrons from entering the Calvin cycle, or blocking H2 uptake resulted in higher yields of H2 and, in some cases, CH4 Our results provide a more complete understanding of the constraints on nitrogenase-based production of biofuels.IMPORTANCE A variant form of Mo nitrogenase catalyzes the conversion of CO2 and protons to the biofuels CH4 and H2 A constant supply of electrons and ATP is needed to drive these reduction reactions. The bacterium R. palustris generates ATP from light and has a versatile metabolism that makes it ideal for manipulating electron availability intracellularly. We therefore explored its potential as a biocatalyst for CH4 and H2 production. We found that intracellular ADP had a major effect on biofuel production, more pronounced than the effect caused by ATP. This is probably due to inhibition of nitrogenase activity by ADP. In general, the amount of CH4 produced by the variant nitrogenase in vivo was affected by electron availability much less than was the amount of H2 produced. This study shows the nature of constraints on in vivo biofuel production by variant Mo nitrogenase.


Assuntos
Proteínas de Bactérias/genética , Elétrons , Metabolismo Energético , Hidrogênio/metabolismo , Metano/metabolismo , Nitrogenase/genética , Rodopseudomonas/metabolismo , Proteínas de Bactérias/metabolismo , Molibdênio/metabolismo , Nitrogenase/metabolismo , Rodopseudomonas/enzimologia , Rodopseudomonas/genética
14.
Proc Natl Acad Sci U S A ; 113(46): 13174-13179, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27799551

RESUMO

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls diverse functions in bacteria, including transitions from planktonic to biofilm lifestyles, virulence, motility, and cell cycle. Here we describe TolR, a hybrid two-component system (HTCS), from the ß-proteobacterium Azoarcus sp. strain CIB that degrades c-di-GMP in response to aromatic hydrocarbons, including toluene. This response protects cells from toluene toxicity during anaerobic growth. Whereas wild-type cells tolerated a sudden exposure to a toxic concentration of toluene, a tolR mutant strain or a strain overexpressing a diguanylate cyclase gene lost viability upon toluene shock. TolR comprises an N-terminal aromatic hydrocarbon-sensing Per-Arnt-Sim (PAS) domain, followed by an autokinase domain, a response regulator domain, and a C-terminal c-di-GMP phosphodiesterase (PDE) domain. Autophosphorylation of TolR in response to toluene exposure initiated an intramolecular phosphotransfer to the response regulator domain that resulted in c-di-GMP degradation. The TolR protein was engineered as a functional sensor histidine kinase (TolRSK) and an independent response regulator (TolRRR). This classic two-component system (CTCS) operated less efficiently than TolR, suggesting that TolR was evolved as a HTCS to optimize signal transduction. Our results suggest that TolR enables Azoarcus sp. CIB to adapt to toxic aromatic hydrocarbons under anaerobic conditions by modulating cellular levels of c-di-GMP. This is an additional role for c-di-GMP in bacterial physiology.


Assuntos
Azoarcus/metabolismo , Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Membrana/metabolismo , Tolueno/toxicidade , Azoarcus/efeitos dos fármacos , Proteínas de Bactérias/genética , GMP Cíclico/metabolismo , Proteínas de Membrana/genética
15.
Proc Natl Acad Sci U S A ; 113(2): E209-18, 2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26712005

RESUMO

Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ(54)-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ's AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP-complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , GMP Cíclico/análogos & derivados , Pseudomonas aeruginosa/fisiologia , Transativadores/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Sítios de Ligação , Calorimetria , Sequência Conservada , Reagentes de Ligações Cruzadas , Cristalografia por Raios X , GMP Cíclico/farmacologia , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Regiões Promotoras Genéticas/genética , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Alinhamento de Sequência , Soluções , Temperatura , Transativadores/química , Transcrição Gênica
16.
Proc Natl Acad Sci U S A ; 113(36): 10163-7, 2016 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-27551090

RESUMO

Nitrogenase is an ATP-requiring enzyme capable of carrying out multielectron reductions of inert molecules. A purified remodeled nitrogenase containing two amino acid substitutions near the site of its FeMo cofactor was recently described as having the capacity to reduce carbon dioxide (CO2) to methane (CH4). Here, we developed the anoxygenic phototroph, Rhodopseudomonas palustris, as a biocatalyst capable of light-driven CO2 reduction to CH4 in vivo using this remodeled nitrogenase. Conversion of CO2 to CH4 by R. palustris required constitutive expression of nitrogenase, which was achieved by using a variant of the transcription factor NifA that is able to activate expression of nitrogenase under all growth conditions. Also, light was required for generation of ATP by cyclic photophosphorylation. CH4 production by R. palustris could be controlled by manipulating the distribution of electrons and energy available to nitrogenase. This work shows the feasibility of using microbes to generate hydrocarbons from CO2 in one enzymatic step using light energy.


Assuntos
Proteínas de Bactérias/genética , Dióxido de Carbono/metabolismo , Metano/biossíntese , Nitrogenase/genética , Fotossíntese/genética , Rodopseudomonas/genética , Trifosfato de Adenosina/biossíntese , Substituição de Aminoácidos , Proteínas de Bactérias/metabolismo , Expressão Gênica , Engenharia Genética/métodos , Cinética , Luz , Molibdoferredoxina/metabolismo , Nitrogenase/metabolismo , Oxirredução , Fotofosforilação , Rodopseudomonas/enzimologia , Rodopseudomonas/efeitos da radiação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
J Bacteriol ; 200(10)2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29483165

RESUMO

Nitrogenase catalyzes the reduction of dinitrogen (N2) using low-potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP-dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O2)-sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds, including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd-/Fld-reducing enzymes in 359 genomes of putative N2 fixers (diazotrophs). Six distinct lineages of nitrogenase were identified, and their distributions largely corresponded to differences in the host cells' ability to integrate O2 or light into energy metabolism. The predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the levels of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. The energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N2 reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation.IMPORTANCE Nitrogen fixation supplies fixed nitrogen to cells from a variety of genomic and metabolic backgrounds, including those of aerobes, facultative anaerobes, chemotrophs, and phototrophs. Here, using informatics approaches applied to genomic data, we show that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O2 or light into their energy metabolism. The acquisition of two key enzyme complexes enabled aerobic and facultatively anaerobic phototrophic taxa to generate electrons of sufficiently low potential to reduce nitrogenase: the bifurcation of electrons via the Fix complex or the coupling of Fd reduction to reverse ion translocation via the Rhodobacter nitrogen fixation (Rnf) complex.


Assuntos
Bactérias/genética , Ferredoxinas/química , Flavodoxina/química , Fungos/genética , Nitrogenase/metabolismo , Aerobiose , Anaerobiose , Bactérias/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte de Elétrons , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Genoma Bacteriano , Genoma Fúngico , Nitrogenase/genética , Filogenia , Transdução de Sinais
18.
Environ Microbiol ; 20(7): 2500-2508, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29708646

RESUMO

The phototrophic alpha-proteobacterium, Rhodopseudomonas palustris, is a model for studies of regulatory and physiological parameters that control the activity of nitrogenase. This enzyme produces the energy-rich compound H2 , in addition to converting N2 gas to NH3 . Nitrogenase is an ATP-requiring enzyme that uses large amounts of reducing power, but the electron transfer pathway to nitrogenase in R. palustris was incompletely known. Here, we show that the ferredoxin, Fer1, is the primary but not sole electron carrier protein encoded by R. palustris that serves as an electron donor to nitrogenase. A flavodoxin, FldA, is also an important electron donor, especially under iron limitation. We present a model where the electron bifurcating complex, FixABCX, can reduce both ferredoxin and flavodoxin to transfer electrons to nitrogenase, and we present bioinformatic evidence that FixABCX and Fer1 form a conserved electron transfer pathway to nitrogenase in nitrogen-fixing proteobacteria. These results may be useful in the design of strategies to reroute electrons generated during metabolism of organic compounds to nitrogenase to achieve maximal activity.


Assuntos
Nitrogenase/metabolismo , Rodopseudomonas/metabolismo , Transporte de Elétrons , Ferredoxinas/metabolismo , Nitrogênio/metabolismo
19.
J Bacteriol ; 199(21)2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28808132

RESUMO

Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes.IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.


Assuntos
Archaea/enzimologia , Bactérias/enzimologia , Flavoproteínas Transferidoras de Elétrons/classificação , Flavoproteínas Transferidoras de Elétrons/metabolismo , Motivos de Aminoácidos , Archaea/genética , Bactérias/genética , Biologia Computacional , Flavoproteínas Transferidoras de Elétrons/química , Flavoproteínas Transferidoras de Elétrons/genética , Modelos Moleculares , Oxirredução , Conformação Proteica
20.
Environ Microbiol ; 19(9): 3567-3578, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28677146

RESUMO

Tn-seq was used to identify genes essential for phototrophic growth by the purple bacterium Rhodopseudomonas palustris. About 167 genes required for anaerobic growth on acetate in light were identified, 35 of which are annotated as photosynthesis genes. The essentiality of many of these genes by analysing the phenotypes of independently generated mutants that had altered pigmentation was verified. Three genes were identified, two possibly involved in biogenesis of the membrane-bound photosynthetic apparatus and one for phosphatidylcholine biosynthesis, that were not known to be essential for phototrophic growth. Site-directed mutagenesis was used to show that the NADH:quinone oxidoreductase complex IE was essential for phototrophic growth under strictly anaerobic conditions and appeared to play a role in reverse electron transport to generate NADH. A homologous NADH:quinone oxidoreductase complex IA likely operates in the opposite direction to oxidize NADH. The operation of the two enzymes in opposition would allow R. palustris to maintain redox balance. As a complement to the genetic data, proteomics experiments were carried out in which it was found that 408 proteins were present in significantly higher amounts in cells grown anaerobically in light compared with aerobically. Among these were proteins encoded by subset of the phototrophic growth-essential genes.


Assuntos
Acetatos/metabolismo , Transporte de Elétrons/genética , Processos Fototróficos/genética , Rodopseudomonas/crescimento & desenvolvimento , Rodopseudomonas/genética , Anaerobiose/fisiologia , Complexo I de Transporte de Elétrons/genética , Luz , Oxirredução , Fosfatidilcolinas/biossíntese , Fotossíntese , Processos Fototróficos/fisiologia
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