RESUMO
Organisms that use ammonium as the sole nitrogen source discriminate between [(15)N] and [(14)N] ammonium. This selectivity leaves an isotopic signature in their biomass that depends on the external concentration of ammonium. To dissect how differences in discrimination arise molecularly, we examined a wild-type (WT) strain of Escherichia coli K12 and mutant strains with lesions affecting ammonium-assimilatory proteins. We used isotope ratio mass spectrometry (MS) to assess the nitrogen isotopic composition of cell material when the strains were grown in batch culture at either high or low external concentrations of NH3 (achieved by controlling total NH4Cl and pH of the medium). At high NH3 (≥ 0.89 µM), discrimination against the heavy isotope by the WT strain (-19.2) can be accounted for by the equilibrium isotope effect for dissociation of NH4(+) to NH3 + H(+). NH3 equilibrates across the cytoplasmic membrane, and glutamine synthetase does not manifest an isotope effect in vivo. At low NH3 (≤ 0.18 µM), discrimination reflects an isotope effect for the NH4(+) channel AmtB (-14.1). By making E. coli dependent on the low-affinity ammonium-assimilatory pathway, we determined that biosynthetic glutamate dehydrogenase has an inverse isotope effect in vivo (+8.8). Likewise, by making unmediated diffusion of NH3 across the cytoplasmic membrane rate-limiting for cell growth in a mutant strain lacking AmtB, we could deduce an in vivo isotope effect for transport of NH3 across the membrane (-10.9). The paper presents the raw data from which our conclusions were drawn and discusses the assumptions underlying them.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Nitrogênio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Transporte de Íons/fisiologia , Isótopos de Nitrogênio/metabolismoRESUMO
In Escherichia coli, each subunit of the trimeric channel protein AmtB carries a hydrophobic pore for transport of NH(4)(+) across the cytoplasmic membrane. Positioned along this substrate conduction pathway are two conserved elements--a pair of hydrogen-bonded histidines (H168/H318) located within the pore itself and a set of aromatic residues (F107/W148/F215) at its periplasmic entrance--thought to be critical to AmtB function. Using site-directed mutagenesis and suppressor genetics, we examined the requirement for these elements in NH(4)(+) transport. This analysis shows that AmtB can accommodate, by either direct substitution or suppressor generation, acidic residues at one or both positions of the H168/H318 twin-histidine site while retaining near wild-type activity. Similarly, study of the F107/W148/F215 triad indicates that good-to-excellent AmtB function is preserved upon individual and simultaneous replacement of these aromatic amino acids with aliphatic residues. Our findings lead us to conclude that these elements and their component parts are not required for AmtB function, but instead serve to optimize its performance.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Histidina/metabolismo , Compostos de Amônio Quaternário/metabolismo , Aminoácidos/metabolismo , Proteínas de Transporte de Cátions/química , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Proteínas Mutantes/metabolismo , Transporte Proteico/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologiaRESUMO
The Rut pathway is composed of seven proteins, all of which are required by Escherichia coli K-12 to grow on uracil as the sole nitrogen source. The RutA and RutB proteins are central: no spontaneous suppressors arise in strains lacking them. RutA works in conjunction with a flavin reductase (RutF or a substitute) to catalyze a novel reaction. It directly cleaves the uracil ring between N-3 and C-4 to yield ureidoacrylate, as established by both nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Although ureidoacrylate appears to arise by hydrolysis, the requirements for the reaction and the incorporation of (18)O at C-4 from molecular oxygen indicate otherwise. Mass spectrometry revealed the presence of a small amount of product with the mass of ureidoacrylate peracid in reaction mixtures, and we infer that this is the direct product of RutA. In vitro RutB cleaves ureidoacrylate hydrolytically to release 2 mol of ammonium, malonic semialdehyde, and carbon dioxide. Presumably the direct products are aminoacrylate and carbamate, both of which hydrolyze spontaneously. Together with bioinformatic predictions and published crystal structures, genetic and physiological studies allow us to predict functions for RutC, -D, and -E. In vivo we postulate that RutB hydrolyzes the peracid of ureidoacrylate to yield the peracid of aminoacrylate. We speculate that RutC reduces aminoacrylate peracid to aminoacrylate and RutD increases the rate of spontaneous hydrolysis of aminoacrylate. The function of RutE appears to be the same as that of YdfG, which reduces malonic semialdehyde to 3-hydroxypropionic acid. RutG appears to be a uracil transporter.
Assuntos
Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Redes e Vias Metabólicas , Nitrogênio/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Uracila/metabolismo , Dióxido de Carbono/metabolismo , Escherichia coli K12/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Deleção de Genes , Espectroscopia de Ressonância Magnética , Malondialdeído/análogos & derivados , Malondialdeído/metabolismo , Espectrometria de Massas , Modelos Biológicos , Oxirredutases/química , Compostos de Amônio Quaternário/metabolismoRESUMO
The Amt/Mep ammonium channels are trimers in which each monomer contains a long, narrow, hydrophobic pore. Whether the substrate conducted by these pores is NH(3) or NH(4)(+) remains controversial. Substitution of leucine for the highly conserved tryptophan 148 residue at the external opening to Escherichia coli AmtB pores allowed us to address this issue. A strain carrying AmtB(W148L) accumulates much larger amounts of both [(14)C]methylammonium and [(14)C]methylglutamine in a washed cell assay than a strain carrying wild-type AmtB. Accumulation of methylammonium occurs within seconds and appears to reflect channel conductance, whereas accumulation of methylglutamine, which depends on the ATP-dependent activity of glutamine synthetase, increases for many minutes. Concentration of methylammonium was most easily studied in strains that lack glutamine synthetase. It is eliminated by the protonophore carbonyl cyanide m-chlorophenyl hydrazone and is approximately 10-fold higher in the strain carrying AmtB(W148L) than wild-type AmtB. The results indicate that AmtB allows accumulation of CH(3)NH(3)(+) ion in response to the electrical potential across the membrane and that the rate of flux through AmtB(W148L) is approximately 10 times faster than through wild-type AmtB. We infer that both mutant and wild-type proteins also carry NH(4)(+). Contrary to our previous views, we assess that E. coli AmtB does not differ from plant Amt proteins in this regard; both carry ions. We address the role of W148 in decreasing the activity and increasing the selectivity of AmtB and the implications of our findings with respect to the function of Rh proteins, the only known homologues of Amt/Mep proteins.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Transporte de Cátions/genética , Cromatografia em Camada Fina , Escherichia coli/citologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Íons/química , Íons/metabolismo , Lisina/genética , Lisina/metabolismo , Metilaminas/metabolismo , Viabilidade Microbiana , Mutação/genética , Triptofano/genética , Triptofano/metabolismoRESUMO
The AMT4 locus of the green alga Chlamydomonas reinhardtii, which we mapped to the long arm of chromosome 8, provides a good experimental system for the study of transposition. Most mutations that confer resistance to the toxic ammonium analog methylammonium are in AMT4 and a high proportion of spontaneous mutations are caused by transposon-related events. Among the 15 such events that we have characterized at the molecular level, 9 were associated with insertions of the retrotransposon TOC1, 2 with a small Gulliver-related transposon, and 1 with the Tcr1 transposon. We found that Tcr1 is apparently a foldback transposon with terminal inverted repeats that are much longer and more complex than previously realized. A duplication of Tcr1 yielded a configuration thought to be important for chromosomal evolution. Other mutations in AMT4 were caused by two mobile elements that have not been described before. The sequence of one, which we propose to call the Bill element, indicates that it probably transposes by way of a DNA intermediate and requires functions that it does not encode. The sequence of the other and bioinformatic analysis indicates that it derives from a miniature retrotransposon or TRIM, which we propose to call MRC1 (miniature retrotransposon of Chlamydomonas).
Assuntos
Chlamydomonas reinhardtii/genética , DNA de Algas/genética , Resistência a Medicamentos/genética , Genes de Protozoários/genética , Locos de Características Quantitativas/genética , Retroelementos/genética , Animais , Sequência de Bases , Chlamydomonas reinhardtii/metabolismo , Cromossomos/genética , Resistência a Medicamentos/efeitos dos fármacos , Evolução Molecular , Metilaminas/farmacologia , Dados de Sequência Molecular , MutaçãoRESUMO
Evidence in several microorganisms indicates that Amt proteins are gas channels for NH(3) and CH(3)NH(2), and this has been confirmed structurally. Chlamydomonas reinhardtii has at least four AMT genes, the most reported for a microorganism. Under nitrogen-limiting conditions all AMT genes are transcribed and Chlamydomonas is sensitive to methylammonium toxicity. All 16 spontaneous methylammonium-resistant mutants that we analyzed had defects in accumulation of [(14)C]methylammonium. Genetic crosses indicated that 12 had lesions in a single locus, whereas two each had lesions in other loci. Lesions in different loci were correlated with different degrees of defect in [(14)C]methylammonium uptake. One mutant in the largest class had an insert in the AMT4 gene, and the insert cosegregated with methylammonium resistance in genetic crosses. The other 11 strains in this class also had amt4 lesions, which we characterized at the molecular level. Properties of the amt4 mutants were clearly different from those of rh1 RNAi lines. They indicated that the physiological substrates for Amt and Rh proteins, the only two members of their protein superfamily, are NH(3) and CO(2), respectively.
Assuntos
Proteínas de Transporte de Cátions/genética , Mutação , Compostos de Amônio Quaternário/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Northern Blotting , Dióxido de Carbono/metabolismo , Proteínas de Transporte de Cátions/fisiologia , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cruzamentos Genéticos , DNA/química , Técnicas Genéticas , Genótipo , Metilaminas/farmacologia , Modelos Genéticos , Dados de Sequência Molecular , Nitrogênio/metabolismo , Peptídeos/química , Fenótipo , Reação em Cadeia da Polimerase , RNA/química , RNA/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Fatores de Tempo , Transcrição GênicaRESUMO
We have developed programs to facilitate analysis of microarray data in Escherichia coli. They fall into two categories: manipulation of microarray images and identification of known biological relationships among lists of genes. A program in the first category arranges spots from glass-slide DNA microarrays according to their position in the E. coli genome and displays them compactly in genome order. The resulting genome image is presented in a web browser with an image map that allows the user to identify genes in the reordered image. Another program in the first category aligns genome images from two or more experiments. These images assist in visualizing regions of the genome with common transcriptional control. Such regions include multigene operons and clusters of operons, which are easily identified as strings of adjacent, similarly colored spots. The images are also useful for assessing the overall quality of experiments. The second category of programs includes a database and a number of tools for displaying biological information about many E. coli genes simultaneously rather than one gene at a time, which facilitates identifying relationships among them. These programs have accelerated and enhanced our interpretation of results from E. coli DNA microarray experiments. Examples are given.
Assuntos
Escherichia coli/genética , Genoma Bacteriano , Análise de Sequência com Séries de Oligonucleotídeos , Mapeamento Cromossômico , Cromossomos Bacterianos/genética , Simulação por Computador , DNA Bacteriano/genética , SoftwareRESUMO
We here develop computational methods to facilitate use of 454 whole genome shotgun sequencing to identify mutations in Escherichia coli K12. We had Roche sequence eight related strains derived as spontaneous mutants in a background without a whole genome sequence. They provided difference tables based on assembling each genome to reference strain E. coli MG1655 (NC_000913). Due to the evolutionary distance to MG1655, these contained a large number of both false negatives and positives. By manual analysis of the dataset, we detected all the known mutations (24 at nine locations) and identified and genetically confirmed new mutations necessary and sufficient for the phenotypes we had selected in four strains. We then had Roche assemble contigs de novo, which we further assembled to full-length pseudomolecules based on synteny with MG1655. This hybrid method facilitated detection of insertion mutations and allowed annotation from MG1655. After removing one genome with less than the optimal 20- to 30-fold sequence coverage, we identified 544 putative polymorphisms that included all of the known and selected mutations apart from insertions. Finally, we detected seven new mutations in a total of only 41 candidates by comparing single genomes to composite data for the remaining six and using a ranking system to penalize homopolymer sequencing and misassembly errors. An additional benefit of the analysis is a table of differences between MG1655 and a physiologically robust E. coli wild-type strain NCM3722. Both projects were greatly facilitated by use of comparative genomics tools in the CoGe software package (http://genomevolution.org/).
Assuntos
Escherichia coli K12/genética , Genética Microbiana/métodos , Genoma Bacteriano , Análise de Sequência de DNA/métodos , Algoritmos , Biologia Computacional/métodos , Mapeamento de Sequências Contíguas/métodos , Escherichia coli/classificação , Escherichia coli/genética , Estudos de Associação Genética , Genômica/métodos , Modelos Biológicos , Anotação de Sequência Molecular/métodos , Polimorfismo Genético , Especificidade da EspécieRESUMO
The Escherichia coli ammonium channel AmtB is a trimer in which each monomer carries a pore for substrate conduction and a cytoplasmic C-terminal extension of approximately 25 residues. Deletion of the entire extension leaves the protein with intermediate activity, but some smaller lesions in this region completely inactivate AmtB, as do some lesions in its cytoplasmic loops. We here provide genetic evidence that inactivation depends on the essential protease HflB, which appears to cause inactivation not as a protease but as a chaperone. Selection for restored function of AmtB is a positive selection for loss of the ATPase/chaperone activity of HflB and reveals that the conditional lethal phenotype for hflB is cold sensitivity. Deletion of only a few residues from the C terminus of damaged AmtB proteins seems to prevent HflB from acting on them. Either yields the intermediate activity of a complete C-terminal deletion. HflB apparently "tacks" damaged AmtB tails to the adjacent monomers. Knowing that HflB has intervened is prerequisite to determining the functional basis for AmtB inactivation.
Assuntos
Proteases Dependentes de ATP/metabolismo , Proteínas de Transporte de Cátions/genética , Epistasia Genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/genética , Proteases Dependentes de ATP/química , Sequência de Aminoácidos , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Chaperonas Moleculares/química , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Conformação Proteica , Supressão GenéticaRESUMO
Ammonium channels, called Amt or Mep, concentrate NH(4)(+) against a gradient. Each monomer of the trimer has a pore through which substrate passes and a C-terminal cytoplasmic extension. The importance of the C-terminal extension to AmtB activity remains unclear. We have described lesions in conserved C-terminal residues that inactivate AmtB and here characterize 38 intragenic suppressors upstream of the C terminus ( approximately 1/3 of total suppressors). Three that occurred repeatedly, including the previously characterized W148L at the pore entry, restored growth at low NH(3) to nearly wild-type levels and hence restored high activity. V116L completely restored function to two of the mutant proteins and, when separated from other lesions, did not damage wild-type AmtB. A179E notably altered folding of AmtB, compensated for all inactivating C-terminal lesions, and damaged wild-type AmtB. V116L and A179E lie at the cytoplasmic end of transmembrane-spanning segments (TM) 3 and 5, respectively, and the proximal part of the C-terminal tail makes intimate contacts with the loops following them before crossing to the adjacent monomer. Collectively, the properties of intragenic suppressor strains lead us to postulate that the C-terminal tail facilitates an oscillation of TM 5 that is required for coordinated pore function and high AmtB activity. Movement of TM 5 appears to control the opening of both the periplasmic entry and the cytoplasmic exit to the pore.
Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Proteases Dependentes de ATP/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte de Cátions/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Marcação por Isótopo , Modelos Moleculares , Dados de Sequência Molecular , Nucleotidiltransferases/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , Conformação Proteica , Deleção de Sequência , Supressão GenéticaRESUMO
Chlamydomonas reinhardtii strains lacking phytoene synthase, the first enzyme of carotenoid biosynthesis, are white. They lack carotenoid pigments, have very low levels of chlorophyll, and can grow only heterotrophically in the dark. Our electron and fluorescence microscopic studies showed that such a mutant strain (lts1-204) had a proliferated plastid envelope membrane but no stacks of thylakoid membranes within the plastid. It accumulated cytoplasmic compartments that appeared to be autophagous vacuoles filled with membranous material. The lts1 mutants apparently lacked pyrenoid bodies, which normally house ribulose bisphosphate carboxylase-oxygenase (Rubisco), and accumulated many starch granules. Although these mutant strains cannot synthesize the carotenoid and carotenoid-derived pigments present in the phototactic organelle (eyespot), the mutant we examined made a vestigial eyespot that was disorganized and often mislocalized to the posterior end of the cell. The absence of a pyrenoid body, the accumulation of starch, and the disorganization of the eyespot may all result from the absence of thylakoids. The ultrastructure of lts1 mutant strains is similar to but distinct from that of previously described white and yellow mutant strains of C. reinhardtii and is similar to that of naturally colorless algae of the Polytoma group.
Assuntos
Alquil e Aril Transferases/deficiência , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/ultraestrutura , Pigmentação/fisiologia , Acetatos/farmacologia , Proteínas de Algas/metabolismo , Autofagia/efeitos dos fármacos , Western Blotting , Membrana Celular/ultraestrutura , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Flagelos/efeitos dos fármacos , Flagelos/ultraestrutura , Geranil-Geranildifosfato Geranil-Geraniltransferase , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/ultraestrutura , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Pigmentação/efeitos dos fármacos , Reprodutibilidade dos Testes , Amido/metabolismo , Tilacoides/efeitos dos fármacos , Tilacoides/metabolismo , Tilacoides/ultraestrutura , Vacúolos/efeitos dos fármacos , Vacúolos/ultraestruturaRESUMO
The major Rhesus (Rh) protein of the green alga Chlamydomonas reinhardtii, Rh1, is homologous to Rh proteins of humans. It is an integral membrane protein involved in transport of carbon dioxide. To localize a fusion of intact Rh1 to the green fluorescent protein (GFP), we used as host a white (lts1) mutant strain of C. reinhardtii, which is blocked at the first step of carotenoid biosynthesis. The lts1 mutant strain accumulated normal amounts of Rh1 heterotrophically in the dark and Rh1-GFP was at the periphery of the cell co-localized with the cytoplasmic membrane dye FM4-64. Although Rh1 carries a potential chloroplast targeting sequence at its N-terminus, Rh1-GFP was clearly not associated with the chloroplast envelope membrane. Moreover, the N-terminal half of the protein was not imported into chloroplasts in vitro and N-terminal regions of Rh1 did not direct import of the small subunit of ribulose bisphosphate carboxylase (SSU). Despite caveats to this interpretation, which we discuss, current evidence indicates that Rh1 is a cytoplasmic membrane protein and that Rh1-GFP is among the first cytoplasmic membrane protein fusions to be obtained in C. reinhardtii. Although lts1 (white) mutant strains cannot be used to localize proteins within sub-compartments of the chloroplast because they lack thylakoid membranes, they should nonetheless be valuable for localizing many GFP fusions in Chlamydomonas.
Assuntos
Proteínas de Algas/química , Chlamydomonas reinhardtii/metabolismo , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Membranas Intracelulares/metabolismo , Mutação/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Algas/metabolismo , Sequência de Aminoácidos , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/genética , Cloroplastos/metabolismo , DNA Complementar/genética , Fluorescência , Imageamento Tridimensional , Microscopia Confocal , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Transporte Proteico , Alinhamento de Sequência , Transformação GenéticaRESUMO
The b1012 operon of Escherichia coli K-12, which is composed of seven unidentified ORFs, is one of the most highly expressed operons under control of nitrogen regulatory protein C. Examination of strains with lesions in this operon on Biolog Phenotype MicroArray (PM3) plates and subsequent growth tests indicated that they failed to use uridine or uracil as the sole nitrogen source and that the parental strain could use them at room temperature but not at 37 degrees C. A strain carrying an ntrB(Con) mutation, which elevates transcription of genes under nitrogen regulatory protein C control, could also grow on thymidine as the sole nitrogen source, whereas strains with lesions in the b1012 operon could not. Growth-yield experiments indicated that both nitrogens of uridine and thymidine were available. Studies with [(14)C]uridine indicated that a three-carbon waste product from the pyrimidine ring was excreted. After trimethylsilylation and gas chromatography, the waste product was identified by mass spectrometry as 3-hydroxypropionic acid. In agreement with this finding, 2-methyl-3-hydroxypropionic acid was released from thymidine. Both the number of available nitrogens and the waste products distinguished the pathway encoded by the b1012 operon from pyrimidine catabolic pathways described previously. We propose that the genes of this operon be named rutA-G for pyrimidine utilization. The product of the divergently transcribed gene, b1013, is a tetracycline repressor family regulator that controls transcription of the b1012 operon negatively.
Assuntos
Escherichia coli/metabolismo , Pirimidinas/metabolismo , Carbono/metabolismo , Proliferação de Células , Biologia Computacional , Escherichia coli/química , Escherichia coli/citologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Estrutura Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Óperon/genética , Fenótipo , Pirimidinas/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
We previously characterized nutrient-specific transcriptional changes in Escherichia coli upon limitation of nitrogen (N) or sulfur (S). These global homeostatic responses presumably minimize the slowing of growth under a particular condition. Here, we characterize responses to slow growth per se that are not nutrient-specific. The latter help to coordinate the slowing of growth, and in the case of down-regulated genes, to conserve scarce N or S for other purposes. Three effects were particularly striking. First, although many genes under control of the stationary phase sigma factor RpoS were induced and were apparently required under S-limiting conditions, one or more was inhibitory under N-limiting conditions, or RpoS itself was inhibitory. RpoS was, however, universally required during nutrient downshifts. Second, limitation for N and S greatly decreased expression of genes required for synthesis of flagella and chemotaxis, and the motility of E. coli was decreased. Finally, unlike the response of all other met genes, transcription of metE was decreased under S- and N-limiting conditions. The metE product, a methionine synthase, is one of the most abundant proteins in E. coli grown aerobically in minimal medium. Responses of metE to S and N limitation pointed to an interesting physiological rationale for the regulatory subcircuit controlled by the methionine activator MetR.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Nitrogênio/metabolismo , Enxofre/metabolismo , Proteínas de Bactérias/fisiologia , Flagelos/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Fator sigma/fisiologia , Transcrição Gênica/fisiologiaRESUMO
We determined global transcriptional responses of Escherichia coli K-12 to sulfur (S)- or nitrogen (N)-limited growth in adapted batch cultures and cultures subjected to nutrient shifts. Using two limitations helped to distinguish between nutrient-specific changes in mRNA levels and common changes related to the growth rate. Both homeostatic and slow growth responses were amplified upon shifts. This made detection of these responses more reliable and increased the number of genes that were differentially expressed. We analyzed microarray data in several ways: by determining expression changes after use of a statistical normalization algorithm, by hierarchical and k-means clustering, and by visual inspection of aligned genome images. Using these tools, we confirmed known homeostatic responses to global S limitation, which are controlled by the activators CysB and Cbl, and found that S limitation propagated into methionine metabolism, synthesis of FeS clusters, and oxidative stress. In addition, we identified several open reading frames likely to respond specifically to S availability. As predicted from the fact that the ddp operon is activated by NtrC, synthesis of cross-links between diaminopimelate residues in the murein layer was increased under N-limiting conditions, as was the proportion of tripeptides. Both of these effects may allow increased scavenging of N from the dipeptide D-alanine-D-alanine, the substrate of the Ddp system.
Assuntos
Escherichia coli K12/metabolismo , Nitrogênio/metabolismo , Enxofre/metabolismo , Análise por Conglomerados , DNA Bacteriano/genética , DNA Complementar/genética , Escherichia coli K12/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Homeostase , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Peptidoglicano/genética , Transcrição GênicaRESUMO
The ammonium/methylammonium transport (Amt) proteins of enteric bacteria and their homologues, the methylammonium/ammonium permeases of Saccharomyces cerevisiae, are required for fast growth at very low concentrations of the uncharged species NH(3). For example, they are essential at low ammonium (NH(4)+ + NH(3)) concentrations under acidic conditions. Based on growth studies in batch culture, the Amt protein of Salmonella typhimurium (AmtB) cannot concentrate either NH(3) or NH(4)+ and this organism appears to have no means of doing so. We now show that S. typhimurium releases ammonium into the medium when grown on the alternative nitrogen source arginine and that outward diffusion of ammonium is enhanced by the activity of AmtB. The latter result indicates that AmtB acts bidirectionally. We also confirm a prediction that the AmtB protein would be required at pH 7.0 in ammonium-limited continuous culture, i.e., when the concentration of NH(3) is < or =50 nM. Together with our previous studies, current results are in accord with the view that Amt and methylammonium/ammonium permease proteins increase the rate of diffusion of the uncharged species NH(3) across the cytoplasmic membrane. These proteins are examples of protein facilitators for a gas.
Assuntos
Amônia/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Escherichia coli , Metilaminas/metabolismo , Compostos de Amônio Quaternário/metabolismo , Salmonella typhimurium/metabolismo , Amônia/farmacologia , Arginina/metabolismo , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte de Cátions/genética , Técnicas de Cultura de Células , Difusão/efeitos dos fármacos , Glicerol/metabolismo , Concentração de Íons de Hidrogênio , Nitrogênio/metabolismo , Compostos de Amônio Quaternário/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , TemperaturaRESUMO
Although Rhesus (Rh) proteins are best known as antigens on human red blood cells, they are not restricted to red cells or to mammals, and hence their primary biochemical functions can be studied in more tractable organisms. We previously established that the Rh1 protein of the green alga Chlamydomonas reinhardtii is highly expressed in cultures bubbled with air containing high CO(2) (3%), conditions under which Chlamydomonas grows rapidly. By RNA interference, we have now obtained Chlamydomonas rh mutants (epigenetic), which are among the first in nonhuman cells. These mutants have essentially no mRNA or protein for RH1 and grow slowly at high CO(2), apparently because they fail to equilibrate this gas rapidly. They grow as well as their parental strain in air and on acetate plus air. However, during growth on acetate, rh1 mutants fail to express three proteins that are known to be down-regulated by high CO(2): periplasmic and mitochondrial carbonic anhydrases and a chloroplast envelope protein. This effect is parsimoniously rationalized if the small amounts of Rh1 protein present in acetate-grown cells of the parental strain facilitate leakage of CO(2) generated internally. Together, these results support our hypothesis that the Rh1 protein is a bidirectional channel for the gas CO(2). Our previous studies in a variety of organisms indicate that the only other members of the Rh superfamily, the ammonium/methylammonium transport proteins, are bidirectional channels for the gas NH(3). Physiologically, both types of gas channels can apparently function in acquisition of nutrients and/or waste disposal.
Assuntos
Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Sistema do Grupo Sanguíneo Rh-Hr/metabolismo , Animais , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Expressão Gênica , Fenótipo , Compostos de Amônio Quaternário/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Sistema do Grupo Sanguíneo Rh-Hr/genéticaRESUMO
Transcription of an aqpZ-lac fusion in a single copy on the Escherichia coli chromosome increased as cells entered the stationary growth phase. This was true in a variety of media, and increased transcription in enriched medium required the RpoS sigma factor. Expression of the aqpZ-lac fusion was not affected by up- or downshifts in osmolality. Disruption of aqpZ had no detectable adverse effects.
Assuntos
Aquaporinas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Membrana , Aquaporinas/genética , Escherichia coli/crescimento & desenvolvimento , Deleção de Genes , Concentração OsmolarRESUMO
Ammonium transport (Amt) proteins appear to be bidirectional channels for NH(3). The amt genes of the hyperthermophiles Aquifex aeolicus and Methanococcus jannaschii complement enteric amtB mutants for growth at 25 nM NH(3) at 37 degrees C. To our knowledge, Amt proteins are the first hyperthermophilic membrane transport proteins shown to be active in a mesophilic bacterium. Despite low expression levels, His-tagged Aquifex Amt could be purified by heating and nickel chelate affinity chromatography. It could be studied genetically in Escherichia coli.
Assuntos
Amônia/metabolismo , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Mutação , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Teste de Complementação Genética , Mathanococcus/genética , Mathanococcus/metabolismo , TemperaturaRESUMO
The function of the Rhesus (Rh) complex in the human red cell membrane has been unknown for six decades. Based on the organismal, organ, and tissue distribution of Rh proteins, and on our evidence that their only known paralogues, the ammonium and methylammonium transport proteins (also called methylammonium permeases), are gas channels for NH(3), we recently speculated that Rh proteins are biological gas channels for CO(2). Like NH(3), CO(2) differs from other gases in being readily hydrated. We have now tested our speculation by studying expression of the RH1 gene in the photosynthetic microbe Chlamydomonas reinhardtii. Expression of RH1 was high for cells grown in air supplemented with 3% CO(2) or shifted from air to high CO(2) (3%) for 3 h. Conversely, RH1 expression was low for cells grown in air (0.035% CO(2)) or shifted from high CO(2) to air for 3 h. These results make viable the hypothesis that Rh1 and Rh proteins generally are gas channels for CO(2).