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1.
Biochim Biophys Acta Bioenerg ; 1865(3): 149050, 2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-38806091

RESUMEN

Purple phototrophic bacteria possess light-harvesting 1 and reaction center (LH1-RC) core complexes that play a key role in converting solar energy to chemical energy. High-resolution structures of LH1-RC and RC complexes have been intensively studied and have yielded critical insight into the architecture and interactions of their proteins, pigments, and cofactors. Nevertheless, a detailed picture of the structure and assembly of LH1-only complexes is lacking due to the intimate association between LH1 and the RC. To study the intrinsic properties and structure of an LH1-only complex, a genetic system was constructed to express the Thermochromatium (Tch.) tepidum LH1 complex heterologously in a modified Rhodospirillum rubrum mutant strain. The heterologously expressed Tch. tepidum LH1 complex was isolated in a pure form free of the RC and exhibited the characteristic absorption properties of Tch. tepidum. Cryo-EM structures of the LH1-only complexes revealed a closed circular ring consisting of either 14 or 15 αß-subunits, making it the smallest completely closed LH1 complex discovered thus far. Surprisingly, the Tch. tepidum LH1-only complex displayed even higher thermostability than that of the native LH1-RC complex. These results reveal previously unsuspected plasticity of the LH1 complex, provide new insights into the structure and assembly of the LH1-RC complex, and show how molecular genetics can be exploited to study membrane proteins from phototrophic organisms whose genetic manipulation is not yet possible.


Asunto(s)
Chromatiaceae , Complejos de Proteína Captadores de Luz , Complejos de Proteína Captadores de Luz/metabolismo , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/genética , Chromatiaceae/metabolismo , Chromatiaceae/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismo
2.
Protein Sci ; 33(4): e4920, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38501449

RESUMEN

L-Asparaginases (ASNases) catalyze the hydrolysis of L-Asn to L-Asp and ammonia. Members of the ASNase family are used as drugs in the treatment of leukemia, as well as in the food industry. The protomers of bacterial ASNases typically contain 300-400 amino acids (typical class 1 ASNases). In contrast, the chain of ASNase from Rhodospirillum rubrum, reported here and referred to as RrA, consists of only 172 amino acid residues. RrA is homologous to the N-terminal domain of typical bacterial class 1 ASNases and exhibits millimolar affinity for L-Asn. In this study, we demonstrate that RrA belongs to a unique family of cytoplasmic, short-chain ASNases (scASNases). These proteins occupy a distinct region in the sequence space, separate from the regions typically assigned to class 1 ASNases. The scASNases are present in approximately 7% of eubacterial species, spanning diverse bacterial lineages. They seem to be significantly enriched in species that encode for more than one class 1 ASNase. Here, we report biochemical, biophysical, and structural properties of RrA, a member of scASNases family. Crystal structures of the wild-type RrA, both with and without bound L-Asp, as well as structures of several RrA mutants, reveal topologically unique tetramers. Moreover, the active site of one protomer is complemented by two residues (Tyr21 and Asn26) from another protomer. Upon closer inspection, these findings clearly outline scASNases as a stand-alone subfamily of ASNases that can catalyze the hydrolysis of L-Asn to L-Asp despite the lack of the C-terminal domain that is present in all ASNases described structurally to date.


Asunto(s)
Asparaginasa , Rhodospirillum rubrum , Asparaginasa/química , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismo , Subunidades de Proteína , Ácido Aspártico , Dominio Catalítico
3.
Appl Microbiol Biotechnol ; 108(1): 258, 2024 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-38466440

RESUMEN

Environmental concerns about residues and the traditional disposal methods are driving the search for more environmentally conscious processes, such as pyrolysis and gasification. Their main final product is synthesis gas (syngas) composed of CO, CO2, H2, and methane. Syngas can be converted into various products using CO-tolerant microorganisms. Among them, Rhodospirillum rubrum is highlighted for its biotechnological potential. However, the extent to which high doses of CO affect its physiology is still opaque. For this reason, we have studied R. rubrum behavior under high levels of this gas (up to 2.5 bar), revealing a profound dependence on the presence or absence of light. In darkness, the key variable affected was the lag phase, where the highest levels of CO retarded growth to more than 20 days. Under light, R. rubrum ability to convert CO into CO2 and H2 depended on the presence of an additional carbon source, such as acetate. In those conditions where CO was completely exhausted, CO2 fixation was unblocked, leading to a diauxic growth. To enhance R. rubrum tolerance to CO in darkness, a UV-accelerated adaptive laboratory evolution (UVa-ALE) trial was conducted to isolate clones with shorter lag phases, resulting in the isolation of clones 1.4-2B and 1.7-2A. The adaptation of 1.4-2B was mainly based on mutated enzymes with a metabolic function, while 1.7-3A was mostly affected at regulatory genes, including the anti-repressor PpaA/AerR. Despite these mutations having slight effects on biomass and pigment levels, they successfully provoked a significant reduction in the lag phase (-50%). KEYPOINTS: • CO affects principally R. rubrum lag phase (darkness) and growth rate (light) • CO is converted to CO2/H2 during acetate uptake and inhibits CO2 fixation (light) • UVa-ALE clones showed a 50% reduction in the lag phase (darkness).


Asunto(s)
Monóxido de Carbono , Rhodospirillum rubrum , Monóxido de Carbono/metabolismo , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismo , Dióxido de Carbono/metabolismo , Acetatos/metabolismo
4.
mSystems ; 8(6): e0070223, 2023 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-38054698

RESUMEN

IMPORTANCE: Rhodospirillum rubrum vast metabolic versatility places it as a remarkable model bacterium and an excellent biotechnological chassis. The key component of photosynthesis (PS) studied in this work (HP1) stands out among the other members of PpaA/AerR anti-repressor family since it lacks the motif they all share: the cobalamin B-12 binding motif. Despite being reduced and poorly conserved, HP1 stills controls PS as the other members of the family, allowing a fast response to changes in the redox state of the cell. This work also shows that HP1 absence affects genes from relevant biological processes other than PS, including nitrogen fixation and stress response. From a biotechnological perspective, HP1 could be manipulated in approaches where PS is not necessary, such as hydrogen or polyhydroxyalkanoates production, to save energy.


Asunto(s)
Rhodospirillum rubrum , Rhodospirillum rubrum/genética , Fotosíntesis , Oxidación-Reducción , Bacterias/metabolismo , Proteínas Cromosómicas no Histona/metabolismo
5.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-34445230

RESUMEN

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is one of the best studied enzymes. It is crucial for photosynthesis, and thus for all of biosphere's productivity. There are four isoforms of this enzyme, differing by amino acid sequence composition and quaternary structure. However, there is still a group of organisms, dinoflagellates, single-cell eukaryotes, that are confirmed to possess Rubisco, but no successful purification of the enzyme of such origin, and hence a generation of a crystal structure was reported to date. Here, we are using in silico tools to generate the possible structure of Rubisco from a dinoflagellate representative, Symbiodinium sp. We selected two templates: Rubisco from Rhodospirillum rubrum and Rhodopseudomonas palustris. Both enzymes are the so-called form II Rubiscos, but the first is exclusively a homodimer, while the second one forms homo-hexamers. Obtained models show no differences in amino acids crucial for Rubisco activity. The variation was found at two closely located inserts in the C-terminal domain, of which one extends a helix and the other forms a loop. These inserts most probably do not play a direct role in the enzyme's activity, but may be responsible for interaction with an unknown protein partner, possibly a regulator or a chaperone. Analysis of the possible oligomerization interface indicated that Symbiodinium sp. Rubisco most likely forms a trimer of homodimers, not just a homodimer. This hypothesis was empowered by calculation of binding energies. Additionally, we found that the protein of study is significantly richer in cysteine residues, which may be the cause for its activity loss shortly after cell lysis. Furthermore, we evaluated the influence of the loop insert, identified exclusively in the Symbiodinium sp. protein, on the functionality of the recombinantly expressed R. rubrum Rubisco. All these findings shed new light onto dinoflagellate Rubisco and may help in future obtainment of a native, active enzyme.


Asunto(s)
Multimerización de Proteína , Rhodospirillum rubrum/enzimología , Ribulosa-Bifosfato Carboxilasa/química , Dominios Proteicos , Rhodospirillum rubrum/genética , Ribulosa-Bifosfato Carboxilasa/genética
6.
Adv Biol (Weinh) ; 5(9): e2101017, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34296829

RESUMEN

Recently, the photosynthetic Rhodospirillum rubrum has been endowed with the ability of magnetosome biosynthesis by transfer and expression of biosynthetic gene clusters from the magnetotactic bacterium Magnetospirillum gryphiswaldense. However, the growth conditions for efficient magnetite biomineralization in the synthetic R. rubrum "magneticum", as well as the particles themselves (i.e., structure and composition), have so far not been fully characterized. In this study, different cultivation strategies, particularly the influence of temperature and light intensity, are systematically investigated to achieve optimal magnetosome biosynthesis. Reduced temperatures ≤16 °C and gradual increase in light intensities favor magnetite biomineralization at high rates, suggesting that magnetosome formation might utilize cellular processes, cofactors, and/or pathways that are linked to photosynthetic growth. Magnetosome yields of up to 13.6 mg magnetite per liter cell culture are obtained upon photoheterotrophic large-scale cultivation. Furthermore, it is shown that even more complex, i.e., oligomeric, catalytically active functional moieties like enzyme proteins can be efficiently expressed on the magnetosome surface, thereby enabling the in vivo functionalization by genetic engineering. In summary, it is demonstrated that the synthetic R. rubrum "magneticum" is a suitable host for high-yield magnetosome biosynthesis and the sustainable production of genetically engineered, bioconjugated magnetosomes.


Asunto(s)
Magnetosomas , Magnetospirillum , Rhodospirillum rubrum , Óxido Ferrosoférrico , Magnetospirillum/genética , Rhodospirillum rubrum/genética
7.
Plant Cell ; 32(9): 2898-2916, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32647068

RESUMEN

Engineering improved Rubisco for the enhancement of photosynthesis is challenged by the alternate locations of the chloroplast rbcL gene and nuclear RbcS genes. Here we develop an RNAi-RbcS tobacco (Nicotiana tabacum) master-line, tobRrΔS, for producing homogenous plant Rubisco by rbcL-rbcS operon chloroplast transformation. Four genotypes encoding alternative rbcS genes and adjoining 5'-intergenic sequences revealed that Rubisco production was highest (50% of the wild type) in the lines incorporating a rbcS gene whose codon use and 5' untranslated-region matched rbcL Additional tobacco genotypes produced here incorporated differing potato (Solanum tuberosum) rbcL-rbcS operons that either encoded one of three mesophyll small subunits (pS1, pS2, and pS3) or the potato trichome pST-subunit. The pS3-subunit caused impairment of potato Rubisco production by ∼15% relative to the lines producing pS1, pS2, or pST However, the ßA-ßB loop Asn-55-His and Lys-57-Ser substitutions in the pS3-subunit improved carboxylation rates by 13% and carboxylation efficiency (CE) by 17%, relative to potato Rubisco incorporating pS1 or pS2-subunits. Tobacco photosynthesis and growth were most impaired in lines producing potato Rubisco incorporating the pST-subunit, which reduced CE and CO2/O2 specificity 40% and 15%, respectively. Returning the rbcS gene to the plant plastome provides an effective bioengineering chassis for introduction and evaluation of novel homogeneous Rubisco complexes in a whole plant context.


Asunto(s)
Cloroplastos/genética , Nicotiana/fisiología , Ribulosa-Bifosfato Carboxilasa/metabolismo , Solanum tuberosum/fisiología , Proteínas Bacterianas/genética , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas , Operón , Iniciación de la Cadena Peptídica Traduccional , Fotosíntesis/fisiología , Hojas de la Planta/fisiología , Plantas Modificadas Genéticamente , Subunidades de Proteína , Interferencia de ARN , Rhodospirillum rubrum/genética , Ribulosa-Bifosfato Carboxilasa/genética , Solanum tuberosum/genética , Nicotiana/genética , Nicotiana/crecimiento & desarrollo
8.
BMC Microbiol ; 20(1): 126, 2020 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-32434546

RESUMEN

BACKGROUND: The great metabolic versatility of the purple non-sulfur bacteria is of particular interest in green technology. Rhodospirillum rubrum S1H is an α-proteobacterium that is capable of photoheterotrophic assimilation of volatile fatty acids (VFAs). Butyrate is one of the most abundant VFAs produced during fermentative biodegradation of crude organic wastes in various applications. While there is a growing understanding of the photoassimilation of acetate, another abundantly produced VFA, the mechanisms involved in the photoheterotrophic metabolism of butyrate remain poorly studied. RESULTS: In this work, we used proteomic and functional genomic analyses to determine potential metabolic pathways involved in the photoassimilation of butyrate. We propose that a fraction of butyrate is converted to acetyl-CoA, a reaction shared with polyhydroxybutyrate metabolism, while the other fraction supplies the ethylmalonyl-CoA (EMC) pathway used as an anaplerotic pathway to replenish the TCA cycle. Surprisingly, we also highlighted a potential assimilation pathway, through isoleucine synthesis and degradation, allowing the conversion of acetyl-CoA to propionyl-CoA. We tentatively named this pathway the methylbutanoyl-CoA pathway (MBC). An increase in isoleucine abundance was observed during the early growth phase under butyrate condition. Nevertheless, while the EMC and MBC pathways appeared to be concomitantly used, a genome-wide mutant fitness assay highlighted the EMC pathway as the only pathway strictly required for the assimilation of butyrate. CONCLUSION: Photoheterotrophic growth of Rs. rubrum with butyrate as sole carbon source requires a functional EMC pathway. In addition, a new assimilation pathway involving isoleucine synthesis and degradation, named the methylbutanoyl-CoA (MBC) pathway, could also be involved in the assimilation of this volatile fatty acid by Rs. rubrum.


Asunto(s)
Proteínas Bacterianas/metabolismo , Butiratos/metabolismo , Proteómica/métodos , Rhodospirillum rubrum/crecimiento & desarrollo , Acilcoenzima A/metabolismo , Proteínas Bacterianas/genética , Fermentación , Aptitud Genética , Isoleucina/metabolismo , Redes y Vías Metabólicas , Mutación , Pentanoles/metabolismo , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismo
9.
Mol Microbiol ; 113(5): 923-937, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-31950558

RESUMEN

S-adenosyl-l-methionine (SAM) is a necessary cosubstrate for numerous essential enzymatic reactions including protein and nucleotide methylations, secondary metabolite synthesis and radical-mediated processes. Radical SAM enzymes produce 5'-deoxyadenosine, and SAM-dependent enzymes for polyamine, neurotransmitter and quorum sensing compound synthesis produce 5'-methylthioadenosine as by-products. Both are inhibitory and must be addressed by all cells. This work establishes a bifunctional oxygen-independent salvage pathway for 5'-deoxyadenosine and 5'-methylthioadenosine in both Rhodospirillum rubrum and Extraintestinal Pathogenic Escherichia coli. Homologous genes for this pathway are widespread in bacteria, notably pathogenic strains within several families. A phosphorylase (Rhodospirillum rubrum) or separate nucleoside and kinase (Escherichia coli) followed by an isomerase and aldolase sequentially function to salvage these two wasteful and inhibitory compounds into adenine, dihydroxyacetone phosphate and acetaldehyde or (2-methylthio)acetaldehyde during both aerobic and anaerobic growth. Both SAM by-products are metabolized with equal affinity during aerobic and anaerobic growth conditions, suggesting that the dual-purpose salvage pathway plays a central role in numerous environments, notably the human body during infection. Our newly discovered bifunctional oxygen-independent pathway, widespread in bacteria, salvages at least two by-products of SAM-dependent enzymes for carbon and sulfur salvage, contributing to cell growth.


Asunto(s)
Proteínas Bacterianas/metabolismo , Desoxiadenosinas/metabolismo , Escherichia coli/metabolismo , Rhodospirillum rubrum/metabolismo , S-Adenosilmetionina/metabolismo , Tionucleósidos/metabolismo , Proteínas Bacterianas/genética , Carbono/metabolismo , Dihidroxiacetona Fosfato/metabolismo , Escherichia coli/genética , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/metabolismo , Isomerasas/genética , Isomerasas/metabolismo , Redes y Vías Metabólicas/genética , Metionina/metabolismo , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , Oxígeno/metabolismo , Fosforilasas/genética , Fosforilasas/metabolismo , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Rhodospirillum rubrum/genética
10.
Microbiology (Reading) ; 166(2): 199-211, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31774392

RESUMEN

Purple non-sulfur bacteria (PNSB) use light for energy and organic substrates for carbon and electrons when growing photoheterotrophically. This lifestyle generates more reduced electron carriers than are required for biosynthesis, even during consumption of some of the most oxidized organic substrates like malate and fumarate. Reduced electron carriers not used in biosynthesis must still be oxidized for photoheterotrophic growth to occur. Diverse PNSB commonly rely on the CO2-fixing Calvin cycle to oxidize reduced electron carriers. Some PNSB also produce H2 or reduce terminal electron acceptors as alternatives to the Calvin cycle. Rhodospirillum rubrum Calvin-cycle mutants defy this trend by growing phototrophically on malate or fumarate without H2 production or access to terminal electron acceptors. We used 13C-tracer experiments to examine how a Rs. rubrum Calvin-cycle mutant maintains electron balance under such conditions. We detected the reversal of some tricarboxylic acid cycle enzymes, carrying reductive flux from malate or fumarate to αKG. This pathway and the reductive synthesis of αKG-derived amino acids are likely important for electron balance, as supplementing the growth medium with αKG-derived amino acids prevented Rs. rubrum Calvin-cycle-mutant growth unless a terminal electron acceptor was provided. Flux estimates also suggested that the Calvin-cycle mutant preferentially synthesized isoleucine using the reductive threonine-dependent pathway instead of the less-reductive citramalate-dependent pathway. Collectively, our results suggest that alternative biosynthetic pathways can contribute to electron balance within the constraints of a relatively constant biomass composition.


Asunto(s)
Aminoácidos/biosíntesis , Ciclo del Ácido Cítrico/fisiología , Electrones , Fotosíntesis/genética , Rhodospirillum rubrum/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biomasa , Vías Biosintéticas , Fumaratos/metabolismo , Isoleucina/biosíntesis , Ácidos Cetoglutáricos/metabolismo , Malatos/metabolismo , Mutación , Oxidación-Reducción , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/crecimiento & desarrollo
11.
PLoS One ; 14(5): e0217281, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31112563

RESUMEN

The lipophilic electron-transport cofactor rhodoquinone (RQ) facilitates anaerobic metabolism in a variety of bacteria and selected eukaryotic organisms in hypoxic environments. We have shown that an intact rquA gene in Rhodospirillum rubrum is required for RQ production and efficient growth of the bacterium under anoxic conditions. While the explicit details of RQ biosynthesis have yet to be fully delineated, ubiquinone (Q) is a required precursor to RQ in R. rubrum, and the RquA gene product is homologous to a class I methyltransferase. In order to identify any additional requirements for RQ biosynthesis or factors influencing RQ production in R. rubrum, we performed transcriptome analysis to identify differentially expressed genes in anoxic, illuminated R. rubrum cultures, compared with those aerobically grown in the dark. To further select target genes, we employed a bioinformatics approach to assess the likelihood that a given differentially expressed gene under anoxic conditions may also have a direct role in RQ production or regulation of its levels in vivo. Having thus compiled a list of candidate genes, nine were chosen for further study by generation of knockout strains. RQ and Q levels were quantified using liquid chromatography-mass spectrometry, and rquA gene expression was measured using the real-time quantitative polymerase chain reaction. In one case, Q and RQ levels were decreased relative to wild type; in another case, the opposite effect was observed. These results comport with the crucial roles of rquA and Q in RQ biosynthesis, and reveal the existence of potential modulators of RQ levels in R. rubrum.


Asunto(s)
Genes Bacterianos , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismo , Ubiquinona/análogos & derivados , Secuencia de Bases , Vías Biosintéticas/genética , Cromatografía Liquida , ADN Bacteriano/genética , Expresión Génica , Técnicas de Inactivación de Genes , Espectrometría de Masa por Ionización de Electrospray , Ubiquinona/biosíntesis , Ubiquinona/genética
12.
J Biol Chem ; 294(19): 7601-7614, 2019 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-30858174

RESUMEN

Activation of nickel enzymes requires specific accessory proteins organized in multiprotein complexes controlling metal transfer to the active site. Histidine-rich clusters are generally present in at least one of the metallochaperones involved in nickel delivery. The maturation of carbon monoxide dehydrogenase in the proteobacterium Rhodospirillum rubrum requires three accessory proteins, CooC, CooT, and CooJ, dedicated to nickel insertion into the active site, a distorted [NiFe3S4] cluster coordinated to an iron site. Previously, CooJ from R. rubrum (RrCooJ) has been described as a nickel chaperone with 16 histidines and 2 cysteines at its C terminus. Here, the X-ray structure of a truncated version of RrCooJ, combined with small-angle X-ray scattering data and a modeling study of the full-length protein, revealed a homodimer comprising a coiled coil with two independent and highly flexible His tails. Using isothermal calorimetry, we characterized several metal-binding sites (four per dimer) involving the His-rich motifs and having similar metal affinity (KD = 1.6 µm). Remarkably, biophysical approaches, site-directed mutagenesis, and X-ray crystallography uncovered an additional nickel-binding site at the dimer interface, which binds Ni(II) with an affinity of 380 nm Although RrCooJ was initially thought to be a unique protein, a proteome database search identified at least 46 bacterial CooJ homologs. These homologs all possess two spatially separated nickel-binding motifs: a variable C-terminal histidine tail and a strictly conserved H(W/F)X2HX3H motif, identified in this study, suggesting a dual function for CooJ both as a nickel chaperone and as a nickel storage protein.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Níquel/química , Multimerización de Proteína , Rhodospirillum rubrum/química , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Proteínas Portadoras/genética , Mutagénesis Sitio-Dirigida , Rhodospirillum rubrum/genética
13.
World J Microbiol Biotechnol ; 34(12): 184, 2018 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-30488133

RESUMEN

Nitrogen fixation is one of the major biogeochemical contributions carried out by diazotrophic microorganisms. The goal of this research is study of posttranslational modification of dinitrogenase reductase (Fe protein), the involvement of malate and pyruvate in generation of reductant in Rhodospirillum rubrum. A procedure for the isolation of the Fe protein from cell extracts was developed and used to monitor the modification of the Fe protein in vivo. The subunit pattern of the isolated the Fe protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was assayed by Western blot analysis. Whole-cell nitrogenase activity was also monitored during the Fe protein modification by gas chromatograpy, using the acetylene reduction assay. It has been shown, that the addition of fluoroacetate, ammonia and darkness resulted in the loss of whole-cell nitrogenase activity and the in vivo modification of the Fe protein. For fluoroacetate, ammonia and darkness, the rate of loss of nitrogenase activity was similar to that for the Fe protein modification. The addition of NADH and reillumination of a culture incubated in the dark resulted in the rapid restoration of nitrogenase activity and the demodification of the Fe protein. Fluoroacetate inhibited the nitrogenase activity of R. rubrum and resulted in the modification of the Fe protein in cells, grown on pyruvate or malate as the endogeneous electron source. The nitrogenase activity in draTG mutant (lacking DRAT/DRAG system) decreased after the addition of fluoroacetate, but the Fe protein remained completely unmodified. The results showed that the reduced state of cell, posttranslational modifications of the Fe protein and the DRAT/DRAG system are important for nitrogenase activity and the regulation of nitrogen fixation.


Asunto(s)
Proteínas Bacterianas/metabolismo , Dinitrogenasa Reductasa/metabolismo , Fluoroacetatos/metabolismo , Rhodospirillum rubrum/enzimología , Proteínas Bacterianas/genética , Dinitrogenasa Reductasa/genética , Regulación Bacteriana de la Expresión Génica , Fijación del Nitrógeno , Procesamiento Proteico-Postraduccional , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismo
14.
Microbiology (Reading) ; 164(11): 1416-1431, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30222098

RESUMEN

The in vivo physiological role of the gene cobZ, which encodes precorrin-3B synthase, which catalyzes the initial porphyrin ring contraction step of cobalamin biosynthesis via the cob pathway, has been demonstrated here for the first time. Cobalamin is known to be essential for an early step of bacteriochlorophyll biosynthesis in anoxygenic purple bacteria. The cobZ (cobZRR) gene of the purple bacterium Rhodospirillum rubrum was localized to a 23.5 kb insert of chromosomal DNA contained on the cosmid pSC4. pSC4 complemented several mutants of bacteriochlorophyll and carotenoid biosynthesis, due to the presence of the bchCX and crtCDEF genes at one end of the cosmid insert, flanking cobZRR. A second gene, citB/tcuB, immediately downstream of cobZRR, shows homologies to both a tricarballylate oxidoreductase (tcuB) and a gene (citB) involved in signal transduction during citrate uptake. CobZRR shows extensive homology to the N-terminal domain of the bifunctional CobZ from Rhodobacter capsulatus, and the R. rubrum citB/tcuB gene is homologous to the CobZ C-terminal domain. A mutant, SERGK25, containing a terminatorless kanamycin interposon inserted into cobZRR, could not grow by anaerobic photosynthesis, but grew normally under dark, aerobic and microaerophilic conditions with succinate and fructose as carbon sources. The anaerobic in vivo activity of CobZ indicates that it does not require oxygen as a substrate. The mutant excreted large amounts of protoporphyrin IX-monomethylester, a brown precursor of bacteriochlorophyll biosynthesis. The mutant was complemented either by the cobZRR gene in trans, or when exogenous cobalamin was added to the medium. A deletion mutant of tcuB/citB did not exhibit the cob phenotype. Thus, a role for tcuB/citB in cobalamin biosynthesis could not be confirmed.


Asunto(s)
Fotosíntesis/fisiología , Rhodospirillum rubrum , Vitamina B 12/biosíntesis , Secuencia de Aminoácidos , Bacterioclorofilas/biosíntesis , Carotenoides/biosíntesis , Cósmidos/genética , ADN Bacteriano/genética , Eliminación de Gen , Metiltransferasas/genética , Oxidorreductasas/genética , Oxígeno/metabolismo , Porfirinas/metabolismo , Rhodospirillum rubrum/enzimología , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismo
15.
FEMS Microbiol Lett ; 365(16)2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-30010831

RESUMEN

Metabolic regulation of Rhodospirillum rubrum nitrogenase is mediated at the post-translational level by the enzymes DraT and DraG when subjected to changes in nitrogen or energy status. DraT is activated during switch-off, while DraG is inactivated by reversible membrane association. We confirm here that the ammonium transporter, AmtB1, rather than its paralog AmtB2, is required for ammonium induced switch-off. Amongst several substitutions at the N100 position in DraG, only N100K failed to locate to the membrane following ammonium shock, suggesting loss of interaction through charge repulsion. When switch-off was induced by lowering energy levels, either by darkness during photosynthetic growth or oxygen depletion under respiratory conditions, reversible membrane sequestration of DraG was independent of AmtB proteins and occurred even under non-diazotrophic conditions. We propose that under these conditions, changes in redox status or possibly membrane potential induce interactions between DraG and another membrane protein in response to the energy status.


Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Proteínas de Transporte de Membrana/metabolismo , N-Glicosil Hidrolasas/metabolismo , Rhodospirillum rubrum/enzimología , Secuencias de Aminoácidos , Compuestos de Amonio/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Metabolismo Energético , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/genética , N-Glicosil Hidrolasas/química , N-Glicosil Hidrolasas/genética , Fijación del Nitrógeno , Unión Proteica , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismo
16.
Microbiology (Reading) ; 164(4): 625-634, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29493489

RESUMEN

Polyhydroxybutyrate (PHB), a biodegradable polymer accumulated by bacteria is deposited intracellularly in the form of inclusion bodies often called granules. The granules are supramolecular complexes harbouring a varied number of proteins on their surface, which have specific but incompletely characterised functions. By comparison with other organisms that produce biodegradable polymers, only two phasins have been described to date for Rhodosprillum rubrum, raising the possibility that more await discovery. Using a comparative proteomics strategy to compare the granules of wild-type R. rubrum with a PHB-negative mutant housing artificial PHB granules, we identified four potential PHB granules' associated proteins. These were: Q2RSI4, an uncharacterised protein; Q2RWU9, annotated as an extracellular solute-binding protein; Q2RQL4, annotated as basic membrane lipoprotein; and Q2RQ51, annotated as glucose-6-phosphate isomerase. In silico analysis revealed that Q2RSI4 harbours a Phasin_2 family domain and shares low identity with a single-strand DNA-binding protein from Sphaerochaeta coccoides. Fluorescence microscopy found that three proteins Q2RSI4, Q2EWU9 and Q2RQL4 co-localised with PHB granules. This work adds three potential new granule associated proteins to the repertoire of factors involved in bacterial storage granule formation, and confirms that proteomics screens are an effective strategy for discovery of novel granule associated proteins.


Asunto(s)
Proteínas Bacterianas/análisis , Biopolímeros/metabolismo , Gránulos Citoplasmáticos/química , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Rhodospirillum rubrum/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , Proteínas de Unión al ADN/química , Microscopía Fluorescente , Anotación de Secuencia Molecular , Mutación , Dominios Proteicos , Proteómica , Rhodospirillum rubrum/citología , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismo
17.
Appl Environ Microbiol ; 84(3)2018 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-29180364

RESUMEN

Purple nonsulfur bacteria represent a promising resource for biotechnology because of their great metabolic versatility. Rhodospirillum rubrum has been widely studied regarding its metabolism of volatile fatty acid, mainly acetate. As the glyoxylate shunt is unavailable in Rs. rubrum, the citramalate cycle pathway and the ethylmalonyl-coenzyme A (CoA) pathway are proposed as alternative anaplerotic pathways for acetate assimilation. However, despite years of debate, neither has been confirmed to be essential. Here, using functional genomics, we demonstrate that the ethylmalonyl-CoA pathway is required for acetate photoassimilation. Moreover, an unexpected reversible long-term adaptation is observed, leading to a drastic decrease in the lag phase characterizing the growth of Rs. rubrum in the presence of acetate. Using proteomic and genomic analyses, we present evidence that the adaptation phenomenon is associated with reversible amplification and overexpression of a 60-kb genome fragment containing key enzymes of the ethylmalonyl-CoA pathway. Our observations suggest that a genome duplication and amplification phenomenon is not only involved in adaptation to acute stress but can also be important for basic carbon metabolism and the redox balance.IMPORTANCE Purple nonsulfur bacteria represent a major group of anoxygenic photosynthetic bacteria that emerged as a promising resource for biotechnology because of their great metabolic versatility and ability to grow under various conditions. Rhodospirillum rubrum S1H has notably been selected by the European Space Agency to colonize its life support system, called MELiSSA, due to its capacity to perform photoheterotrophic assimilation of volatile fatty acids (VFAs), mainly acetate. VFAs are valuable carbon sources for many applications, combining bioremediation of contaminated environments with the generation of added-value products. Acetate is one of the major volatile fatty acids generated as a by-product of fermentation processes. In Rs. rubrum, purple nonsulfur bacteria, the assimilation of acetate is still under debate since two different pathways have been proposed. Here, we clearly demonstrate that the ethylmalonyl-CoA pathway is the major anaplerotic pathway for acetate assimilation in this strain. Interestingly, we further observed that gene duplication and amplification, which represent a well-known phenomenon in antibiotic resistance, also play a regulatory function in carbon metabolism and redox homeostasis.


Asunto(s)
Acetatos/metabolismo , Adaptación Fisiológica/genética , Redes y Vías Metabólicas/genética , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/metabolismo , Acilcoenzima A , Carbono/metabolismo , Duplicación de Gen , Genoma Bacteriano , Genómica , Homeostasis , Oxidación-Reducción , Proteómica , Rhodospirillum rubrum/crecimiento & desarrollo
18.
Microb Biotechnol ; 10(6): 1300-1301, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28840979

RESUMEN

Biodegradable polymers such as polyhydroxybutyrate (PHB) are part of the emerging portfolio of renewable materials, which are addressing the issue of plastic waste. Syngas, as a cheap, renewable and sustainable resource that can be obtained from biomass or waste, is viewed as an excellent feedstock for different bioprocesses, including syngas to PHB bioconversion. However, due to the hazardous nature of syngas, it is of utmost importance to consider safety aspects of the process. This recently developed tailor-made platform for safe syngas fermentation and PHB production addresses safety aspects and demonstrates the importance of robust online and in-line analytical tools allowing for monitoring and controlling of this bioprocess.


Asunto(s)
Gases/metabolismo , Hidroxibutiratos/metabolismo , Rhodospirillum rubrum/metabolismo , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , Reactores Biológicos , Fermentación , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Plásticos/metabolismo , Rhodospirillum rubrum/enzimología , Rhodospirillum rubrum/genética , Rhodospirillum rubrum/crecimiento & desarrollo , Residuos Sólidos/análisis
19.
Biomed Khim ; 63(1): 62-74, 2017 Jan.
Artículo en Ruso | MEDLINE | ID: mdl-28251953

RESUMEN

The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrАE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.


Asunto(s)
Antineoplásicos/farmacología , Asparaginasa/farmacología , Proteínas Bacterianas/farmacología , Mutación Puntual , Rhodospirillum rubrum/enzimología , Telomerasa/antagonistas & inhibidores , Telómero/efectos de los fármacos , Secuencia de Aminoácidos , Antineoplásicos/química , Antineoplásicos/metabolismo , Asparaginasa/química , Asparaginasa/genética , Asparaginasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Células HL-60 , Humanos , Células Jurkat , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Pectobacterium carotovorum/química , Pectobacterium carotovorum/enzimología , Pectobacterium carotovorum/genética , Plásmidos/química , Plásmidos/metabolismo , Estructura Secundaria de Proteína , Rhodospirillum rubrum/química , Rhodospirillum rubrum/genética , Especificidad de la Especie , Relación Estructura-Actividad , Telomerasa/genética , Telomerasa/metabolismo , Telómero/química , Wolinella/química , Wolinella/enzimología , Wolinella/genética
20.
Appl Environ Microbiol ; 82(20): 6132-6140, 2016 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-27520812

RESUMEN

The purple nonsulfur alphaproteobacterium Rhodospirillum rubrum S1 was genetically engineered to synthesize a heteropolymer of mainly 3-hydroxydecanoic acid and 3-hydroxyoctanoic acid [P(3HD-co-3HO)] from CO- and CO2-containing artificial synthesis gas (syngas). For this, genes from Pseudomonas putida KT2440 coding for a 3-hydroxyacyl acyl carrier protein (ACP) thioesterase (phaG), a medium-chain-length (MCL) fatty acid coenzyme A (CoA) ligase (PP_0763), and an MCL polyhydroxyalkanoate (PHA) synthase (phaC1) were cloned and expressed under the control of the CO-inducible promoter PcooF from R. rubrum S1 in a PHA-negative mutant of R. rubrum P(3HD-co-3HO) was accumulated to up to 7.1% (wt/wt) of the cell dry weight by a recombinant mutant strain utilizing exclusively the provided gaseous feedstock syngas. In addition to an increased synthesis of these medium-chain-length PHAs (PHAMCL), enhanced gene expression through the PcooF promoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the Plac promoter, which regulated expression on the original vector. The recombinant strains were able to partially degrade the polymer, and the deletion of phaZ2, which codes for a PHA depolymerase most likely involved in intracellular PHA degradation, did not reduce mobilization of the accumulated polymer significantly. However, an amino acid exchange in the active site of PhaZ2 led to a slight increase in PHAMCL accumulation. The accumulated polymer was isolated; it exhibited a molecular mass of 124.3 kDa and a melting point of 49.6°C. With the metabolically engineered strains presented in this proof-of-principle study, we demonstrated the synthesis of elastomeric second-generation biopolymers from renewable feedstocks not competing with human nutrition. IMPORTANCE: Polyhydroxyalkanoates (PHAs) are natural biodegradable polymers (biopolymers) showing properties similar to those of commonly produced petroleum-based nondegradable polymers. The utilization of cheap substrates for the microbial production of PHAs is crucial to lower production costs. Feedstock not competing with human nutrition is highly favorable. Syngas, a mixture of carbon monoxide, carbon dioxide, and hydrogen, can be obtained by pyrolysis of organic waste and can be utilized for PHA synthesis by several kinds of bacteria. Up to now, the biosynthesis of PHAs from syngas has been limited to short-chain-length PHAs, which results in a stiff and brittle material. In this study, the syngas-utilizing bacterium Rhodospirillum rubrum was genetically modified to synthesize a polymer which consisted of medium-chain-length constituents, resulting in a rubber-like material. This study reports the establishment of a microbial synthesis of these so-called medium-chain-length PHAs from syngas and therefore potentially extends the applications of syngas-derived PHAs.


Asunto(s)
Gases/metabolismo , Ingeniería Metabólica , Polihidroxialcanoatos/biosíntesis , Rhodospirillum rubrum/genética , Gases/síntesis química , Polihidroxialcanoatos/química , Rhodospirillum rubrum/química , Rhodospirillum rubrum/metabolismo
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