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Applied and Environmental Microbiology Mar 2022Poly(hydroxybutyrate--hydroxyhexanoate) [P(HB--HHx)] and poly(hydroxybutyrate--hydroxyvalerate-hydroxyhexanoate) [P(HB--HV--HHx)] demonstrate interesting mechanical and...
Poly(hydroxybutyrate--hydroxyhexanoate) [P(HB--HHx)] and poly(hydroxybutyrate--hydroxyvalerate-hydroxyhexanoate) [P(HB--HV--HHx)] demonstrate interesting mechanical and thermal properties as well as excellent biocompatibility, making them suitable for multiple applications and notably biomedical purposes. The production of such polymers was described in Rhodospirillum rubrum, a purple nonsulfur bacteria in a nutrient-lacking environment where the HHx synthesis is triggered by the presence of hexanoate in the medium. However, the production of P(HB--HHx) under nutrient-balanced growth conditions in has not been described so far, and the assimilation of hexanoate is poorly documented. In this study, we used proteomic analysis and a mutant fitness assay to demonstrate that hexanoate assimilation involve β-oxidation and the ethylmalonyl-coenzyme A (CoA) (EMC) and methylbutanoyl-CoA (MBC) pathways, both being anaplerotic pathways already described in . Polyhydroxyalkanoate (PHA) production is likely to involve the fatty acid synthesis pathway. Concerning the polymer composition, HB is the main component of the polymer, probably as acetyl-CoA and butyryl-CoA are intermediates of hexanoate assimilation pathways. When no essential nutrient is lacking in the medium, the synthesis of PHA seems to help maintain the redox balance of the cell. In this framework, we showed that the fixation of CO is required to sustain the growth. An increase in the proportion of HHx in the polymer was observed when redox stress was engendered in the cell under bicarbonate-limiting growth conditions. The addition of isoleucine or valerate in the medium also increased the HHx content of the polymer and allowed the production of a terpolymer of P(HB--HV--HHx). The use of purple bacteria, which can assimilate volatile fatty acids, for biotechnological applications has increased, since they reduce the production costs of added-value compounds such as PHA. P(HB--HHx) and P(HB--HV--HHx) have demonstrated interesting properties, notably for biomedical applications. In a nutrient-lacking environment, is known to synthesize such polymers when hexanoate is used as the carbon source. However, their production in in non-nutrient-lacking growth conditions has not been described so far, and the assimilation of hexanoate is poorly documented. As the carbon source and its assimilation directly impact the polymer composition, we studied under non-nutrient-lacking growth conditions the assimilation pathway of hexanoate and PHA production in Proteomic analysis and mutant fitness assays allowed us to explain PHA production and composition. An increase in HHx content of the polymer and production of P(HB--HV--HHx) was possible using the knowledge gained on metabolism under hexanoate growth conditions.
Topics: Biotechnology; Hydroxybutyrates; Polyhydroxyalkanoates; Proteomics; Rhodospirillum rubrum
PubMed: 35080906
DOI: 10.1128/AEM.01586-21 -
International Journal of Molecular... Aug 2021Ribulose-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...
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, sp. We selected two templates: Rubisco from and . 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 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 sp. protein, on the functionality of the recombinantly expressed Rubisco. All these findings shed new light onto dinoflagellate Rubisco and may help in future obtainment of a native, active enzyme.
Topics: Protein Domains; Protein Multimerization; Rhodospirillum rubrum; Ribulose-Bisphosphate Carboxylase
PubMed: 34445230
DOI: 10.3390/ijms22168524 -
Applied and Environmental Microbiology Sep 2020Purple nonsulfur bacteria are increasingly recognized for industrial applications in bioplastics, pigment, and biomass production. In order to optimize the yield of...
Purple nonsulfur bacteria are increasingly recognized for industrial applications in bioplastics, pigment, and biomass production. In order to optimize the yield of future biotechnological processes, the assimilation of different carbon sources by has to be understood. As they are released from several fermentation processes, volatile fatty acids (VFAs) represent a promising carbon source in the development of circular industrial applications. To obtain an exhaustive characterization of the photoheterotrophic metabolism of in the presence of valerate, we combined phenotypic, proteomic, and genomic approaches. We obtained evidence that valerate is cleaved into acetyl coenzyme A (acetyl-CoA) and propionyl-CoA and depends on the presence of bicarbonate ions. Genomic and enzyme inhibition data showed that a functional methylmalonyl-CoA pathway is essential. Our proteomic data showed that the photoheterotrophic assimilation of valerate induces an intracellular redox stress which is accompanied by an increased abundance of phasins (the main proteins present in polyhydroxyalkanoate [PHA] granules). Finally, we observed a significant increase in the production of the copolymer P(HB--HV), accounting for a very high (>80%) percentage of HV monomer. Moreover, an increase in the PHA content was obtained when bicarbonate ions were progressively added to the medium. The experimental conditions used in this study suggest that the redox imbalance is responsible for PHA production. These findings also reinforce the idea that purple nonsulfur bacteria are suitable for PHA production through a strategy other than the well-known feast-and-famine process. The use and the littering of plastics represent major issues that humanity has to face. Polyhydroxyalkanoates (PHAs) are good candidates for the replacement of oil-based plastics, as they exhibit comparable physicochemical properties but are biobased and biodegradable. However, the current industrial production of PHAs is curbed by the production costs, which are mainly linked to the carbon source. Volatile fatty acids issued from the fermentation processes constitute interesting carbon sources, since they are inexpensive and readily available. Among them, valerate is gaining interest regarding the ability of many bacteria to produce a copolymer of PHAs. Here, we describe the photoheterotrophic assimilation of valerate by , a purple nonsulfur bacterium mainly known for its metabolic versatility. Using a knowledge-based optimization process, we present a new strategy for the improvement of PHA production, paving the way for the use of in industrial processes.
Topics: Heterotrophic Processes; Phototrophic Processes; Polyhydroxyalkanoates; Rhodospirillum rubrum; Valerates
PubMed: 32651203
DOI: 10.1128/AEM.00901-20 -
MBio Mar 2021is a Gram-negative alphaproteobacterium that is capable of differentiating into dormant cysts that are metabolically inactive and desiccation resistant. Like spores...
is a Gram-negative alphaproteobacterium that is capable of differentiating into dormant cysts that are metabolically inactive and desiccation resistant. Like spores synthesized by many Gram-positive species, dormant cysts germinate in response to an environmental signal, indicating that conditions favor survival and proliferation. Factors that induce germination are called germinants and are often both niche and species specific. In this study, we have identified photosynthesis as a niche-specific germinant for cyst germination. Specifically, excitation of wild-type cysts suspended in a nutrient-free buffer with far-red light at >750 nm results in rapid germination. This is in stark contrast to mutant strains deficient in photosynthesis that fail to germinate upon exposure to far-red light under all assayed conditions. We also show that photosynthesis-induced germination occurs in a carbon- and nitrogen-free buffer even in strains that are deficient in carbon or nitrogen fixation. These results demonstrate that photosynthesis not only is necessary for germination but is itself sufficient for the germination of cysts. Environmental cues that signal Gram-positive spores to germinate (termed germinants) have been identified for several and species. These studies showed that germinants are niche and species specific. For example, spores sense bile salts as a germinant as their presence informs these cells of an intestinal environment. spores use uric acid as a germinant that is present in soil and poultry litter as this species inhabits poultry litter. It is evident from these studies that dormant cells sample their environment to assess whether conditions are advantageous for the propagation and survival of vegetative cells. To date, a limited number of germinants have been defined for only a few Gram-positive spore-forming species. Beyond that group, there is scant information on what cues signal dormant cells to exit dormancy. In our study, we show that the versatile Gram-negative photosynthetic bacterium uses light-driven photosynthesis, and not the availability of nutrients, to trigger the germination of dormant cysts. This use of light-driven photosynthesis as a germinant is surprising as this species is also capable of growing under dark conditions using exogenous carbon sources for energy. Consequently, photosynthetic growth appears to be the preferred growth mechanism by this species.
Topics: Bacterial Proteins; Biofilms; Photosynthesis; Rhodospirillum centenum; Signal Transduction; Spores, Bacterial
PubMed: 33727361
DOI: 10.1128/mBio.03619-20 -
Quarterly Reviews of Biophysics Feb 2002
Review
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Biophysical Phenomena; Biophysics; Chlorophyll; Crystallography, X-Ray; Electrons; Energy Transfer; Light-Harvesting Protein Complexes; Models, Genetic; Models, Molecular; Models, Statistical; Photons; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Rhodopseudomonas; Rhodospirillum; Time Factors
PubMed: 11997980
DOI: 10.1017/s0033583501003754 -
Applied Microbiology Jan 1964The interaction between photosynthetic microorganisms and an inert electrode material was examined. Cathodic polarization values of platinum-bearing marine algae were...
The interaction between photosynthetic microorganisms and an inert electrode material was examined. Cathodic polarization values of platinum-bearing marine algae were obtained over a wide current-density range under both illumination and dark conditions. A potential shift of 0.6 v in the cathodic direction occurred upon illumination at a current density of 4.3 mua/cm(2). Similar photo-induced results, involving anodic polarization, were obtained by use of resting cells of Rhodospirillum rubrum supplemented with malate. Appropriate combinations of such bioelectrodes were used to assemble an electrochemical cell capable of light-dependent production of electrical energy.
Topics: Electrophysiology; Eukaryota; Light; Malates; Metabolism; Photosynthesis; Platinum; Research; Rhodospirillum; Rhodospirillum rubrum
PubMed: 14106931
DOI: 10.1128/am.12.1.10-12.1964 -
Journal of Bacteriology Aug 1999The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni(2+) transport assay was used to monitor...
The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni(2+) transport assay was used to monitor uptake and accumulation of (63)Ni(2+) into R. rubrum and to observe the effect of mutations in the cooC, cooT, and cooJ genes on (63)Ni(2+) transport and accumulation. Cells grown either in the presence or absence of CO transported Ni(2+) with a K(m) of 19 +/- 4 microM and a V(max) of 310 +/- 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the cooCTJ genes, either individually or all three genes simultaneously, transported Ni(2+) the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn(2+), Mg(2+), Ca(2+), and Zn(2+) showed no inhibition of (63)Ni(2+) transport but Co(2+), Cd(2+), and Cu(2+) inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4 degrees C), by protonophores (carbonyl cyanide m-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (N, N'-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni(2+) transport. (63)Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated (63)Ni(2+) accumulation coincided with the appearance of CODH activity in the culture, suggesting that the (63)Ni(2+) was accumulating in CODH. The cooC, cooT, and cooJ genes are required for the increased (63)Ni(2+) accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated (63)Ni(2+) like cells unexposed to CO.
Topics: 2,4-Dinitrophenol; Aldehyde Oxidoreductases; Biological Transport; Carbon Monoxide; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cations, Divalent; Dicyclohexylcarbodiimide; Hydroxylamine; Ionomycin; Kinetics; Metals; Multienzyme Complexes; Nickel; Proton-Translocating ATPases; Rhodospirillum rubrum; Sodium Azide
PubMed: 10419953
DOI: 10.1128/JB.181.15.4554-4560.1999 -
The Journal of Biological Chemistry Jun 1988In Rhodospirillum rubrum, the genes coding for the alpha and beta polypeptides of the B880 antenna (pufA,B) and the L and M polypeptides of the photoreaction center...
In Rhodospirillum rubrum, the genes coding for the alpha and beta polypeptides of the B880 antenna (pufA,B) and the L and M polypeptides of the photoreaction center (pufL,M) are clustered on operon puf. In oxygen-limited cells, the puf mRNA is present as species of 2561, 640, and 617 nucleotides. Aerated cells contain only traces of these mRNAs. The large mRNA encodes the alpha,beta, L, and M polypeptides, whereas the small mRNAs encode only alpha and beta. S1 nuclease protection mapping showed these transcripts to have a common 5' end, immediately downstream of a region of dyad symmetry and at 166 nucleotides upstream of the initiation codon of pufB. The 3' termini of the small transcripts are located in the intercistronic region between pufA and pufL, downstream of another region of dyad symmetry. This region is highly conserved in Rhodospirillum rubrum, Rhodobacter capsulatus, and Rhodobacter sphaeroides and shares 61% sequence similarity with the repetitive extragenic palindromic sequences of Escherichia coli. The slightly heterogeneous 3' termini of the large transcript are downstream of a region of dyad symmetry characteristic of rho-independent transcription termination. Following a shift from oxygen-limited to aerated conditions, the pufL,M and the pufA,B mRNAs decayed with respective half-lives of 9 and 20 min. These high relative stabilities, attributed to secondary structure, are in accord with the mole ratio (2:1) of the pufA,B/pufL,M messages. While the differential expression of alpha,beta/L,M congruent to 15 is thought to be due, in part, to this relative stability, the main factor may be a more efficient translation initiation for pufA,B than for pufL,M.
Topics: Bacterial Proteins; Base Sequence; Chromosome Mapping; Gene Expression Regulation; Nucleic Acid Conformation; Operon; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; RNA, Messenger; Rhodospirillum rubrum
PubMed: 3131324
DOI: No ID Found -
The ISME Journal Dec 2009In view of long-haul space exploration missions, the European Space Agency initiated the Micro-Ecological Life Support System Alternative (MELiSSA) project targeting the...
In view of long-haul space exploration missions, the European Space Agency initiated the Micro-Ecological Life Support System Alternative (MELiSSA) project targeting the total recycling of organic waste produced by the astronauts into oxygen, water and food using a loop of bacterial and higher plant bioreactors. In that purpose, the alpha-proteobacterium, Rhodospirillum rubrum S1H, was sent twice to the International Space Station and was analyzed post-flight using a newly developed R. rubrum whole genome oligonucleotide microarray and high throughput gel-free proteomics with Isotope-Coded Protein Label technology. Moreover, in an effort to identify a specific response of R. rubrum S1H to space flight, simulation of microgravity and space-ionizing radiation were performed on Earth under identical culture set-up and growth conditions as encountered during the actual space journeys. Transcriptomic and proteomic data were integrated and permitted to put forward the importance of medium composition and culture set-up on the response of the bacterium to space flight-related environmental conditions. In addition, we showed for the first time that a low dose of ionizing radiation (2 mGy) can induce a significant response at the transcriptomic level, although no change in cell viability and only a few significant differentially expressed proteins were observed. From the MELiSSA perspective, we could argue the effect of microgravity to be minimized, whereas R. rubrum S1H could be more sensitive to ionizing radiation during long-term space exploration mission.
Topics: Bacterial Proteins; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Oligonucleotide Array Sequence Analysis; Proteome; Radiation, Ionizing; Rhodospirillum rubrum; Space Flight; Stress, Physiological; Weightlessness
PubMed: 19571896
DOI: 10.1038/ismej.2009.74 -
Journal of Bacteriology Oct 2007The nitrogen regulatory protein P(II) and the ammonia gas channel AmtB are both found in most prokaryotes. Interaction between these two proteins has been observed in...
The nitrogen regulatory protein P(II) and the ammonia gas channel AmtB are both found in most prokaryotes. Interaction between these two proteins has been observed in several organisms and may regulate the activities of both proteins. The regulation of their interaction is only partially understood, and we show that in Rhodospirillum rubrum one P(II) homolog, GlnJ, has higher affinity for an AmtB(1)-containing membrane than the other two P(II) homologs, GlnB and GlnK. This interaction strongly favors the nonuridylylated form of GlnJ and is disrupted by high levels of 2-ketoglutarate (2-KG) in the absence of ATP or low levels of 2-KG in the presence of ATP. ADP inhibits the destabilization of the GlnJ-AmtB(1) complex in the presence of ATP and 2-KG, supporting a role for P(II) as an energy sensor measuring the ratio of ATP to ADP. In the presence of saturating levels of ATP, the estimated K(d) of 2-KG for GlnJ bound to AmtB(1) is 340 microM, which is higher than that required for uridylylation of GlnJ in vitro, about 5 microM. This supports a model where multiple 2-KG and ATP molecules must bind a P(II) trimer to stimulate release of P(II) from AmtB(1), in contrast to the lower 2-KG requirement for productive uridylylation of P(II) by GlnD.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Bacterial Proteins; Blotting, Western; Cation Transport Proteins; Ketoglutaric Acids; PII Nitrogen Regulatory Proteins; Protein Binding; Quaternary Ammonium Compounds; Rhodospirillum rubrum
PubMed: 17644595
DOI: 10.1128/JB.00759-07