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Photosynthesis Research Nov 2019The influence of temperature on photosynthetic reactions was investigated by a combination of time-resolved bacteriochlorophyll fluorescence, steady-state and...
The influence of temperature on photosynthetic reactions was investigated by a combination of time-resolved bacteriochlorophyll fluorescence, steady-state and differential absorption spectroscopy, and polarographic respiration measurements in intact cells of purple non-sulphur bacterium Rhodospirillum rubrum. Using variable bacteriochlorophyll fluorescence, it was found that the electron-transport activity increased with the increasing temperature up to 41 °C. The fast and medium components of the fluorescence decay kinetics followed the ideal Arrhenius equation. The calculated activation energy for the fast component was E = 16 kJ mol, while that of the medium component was more than double, with E = 38 kJ mol. At temperatures between 41 and 59 °C, the electron transport was gradually, irreversibly inhibited. Interestingly, the primary charge separation remained fully competent from 20 to 59 °C as documented by both BChl fluorescence and differential absorption spectroscopy of the P signal. At temperatures above 60 °C, the primary photochemistry became reversibly inhibited, which was manifested by an increase in minimal fluorescence, F, whereas maximal fluorescence, F, slowly declined. Finally, above 71 °C, the photosynthetic complexes began to disassemble as seen in the decline of all fluorometric parameters and the disappearance of the LH1 absorption band at 880 nm. The extended optimal temperature of photosynthetic reaction centre in a model species of Rhodospirillales adds on the evidence that the good thermostability of the photosynthetic reaction centres is present across all Alphaproteobacteria.
Topics: Cell Respiration; Fluorescence; Kinetics; Light-Harvesting Protein Complexes; Photosynthesis; Rhodospirillum rubrum; Temperature
PubMed: 31267356
DOI: 10.1007/s11120-019-00652-7 -
MBio Jul 2018In bacteria and eukaryotes alike, proper cellular physiology relies on robust subcellular organization. For the phototrophic purple nonsulfur bacteria (PNSB), this...
In bacteria and eukaryotes alike, proper cellular physiology relies on robust subcellular organization. For the phototrophic purple nonsulfur bacteria (PNSB), this organization entails the use of a light-harvesting, membrane-bound compartment known as the intracytoplasmic membrane (ICM). Here we show that ICMs are spatially and temporally localized in diverse patterns among PNSB. We visualized ICMs in live cells of 14 PNSB species across nine genera by exploiting the natural autofluorescence of the photosynthetic pigment bacteriochlorophyll (BChl). We then quantitatively characterized ICM localization using automated computational analysis of BChl fluorescence patterns within single cells across the population. We revealed that while many PNSB elaborate ICMs along the entirety of the cell, species across as least two genera restrict ICMs to discrete, nonrandom sites near cell poles in a manner coordinated with cell growth and division. Phylogenetic and phenotypic comparisons established that ICM localization and ICM architecture are not strictly interdependent and that neither trait fully correlates with the evolutionary relatedness of the species. The natural diversity of ICM localization revealed herein has implications for both the evolution of phototrophic organisms and their light-harvesting compartments and the mechanisms underpinning spatial organization of bacterial compartments. Many bacteria organize their cellular space by constructing subcellular compartments that are arranged in specific, physiologically relevant patterns. The purple nonsulfur bacteria (PNSB) utilize a membrane-bound compartment known as the intracytoplasmic membrane (ICM) to harvest light for photosynthesis. It was previously unknown whether ICM localization within cells is systematic or irregular and if ICM localization is conserved among PNSB. Here we surveyed ICM localization in diverse PNSB and show that ICMs are spatially organized in species-specific patterns. Most strikingly, several PNSB resolutely restrict ICMs to regions near the cell poles, leaving much of the cell devoid of light-harvesting machinery. Our results demonstrate that bacteria of a common lifestyle utilize unequal portions of their intracellular space to harvest light, despite light harvesting being a process that is intuitively influenced by surface area. Our findings therefore raise fundamental questions about ICM biology and evolution.
Topics: Bacteriochlorophylls; Cell Membrane; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Organelle Biogenesis; Rhodospirillaceae; Spatial Analysis
PubMed: 29970460
DOI: 10.1128/mBio.00780-18 -
Parasites & Vectors Mar 2016Malaria still remains a serious health burden in developing countries, causing more than 1 million deaths annually. Given the lack of an effective vaccine against its...
BACKGROUND
Malaria still remains a serious health burden in developing countries, causing more than 1 million deaths annually. Given the lack of an effective vaccine against its major etiological agent, Plasmodium falciparum, and the growing resistance of this parasite to the currently available drugs repertoire and of Anopheles mosquitoes to insecticides, the development of innovative control measures is an imperative to reduce malaria transmission. Paratransgenesis, the modification of symbiotic organisms to deliver anti-pathogen effector molecules, represents a novel strategy against Plasmodium development in mosquito vectors, showing the potential to reduce parasite development. However, the field application of laboratory-based evidence of paratransgenesis imposes the use of more realistic confined semi-field environments.
METHODS
Large cages were used to evaluate the ability of bacteria of the genus Asaia expressing green fluorescent protein (Asaia (gfp)), to diffuse in Anopheles stephensi and Anopheles gambiae target mosquito populations. Asaia (gfp) was introduced in large cages through the release of paratransgenic males or by sugar feeding stations. Recombinant bacteria transmission was directly detected by fluorescent microscopy, and further assessed by molecular analysis.
RESULTS
Here we show the first known trial in semi-field condition on paratransgenic anophelines. Modified bacteria were able to spread at high rate in different populations of An. stephensi and An. gambiae, dominant malaria vectors, exploring horizontal ways and successfully colonising mosquito midguts. Moreover, in An. gambiae, vertical and trans-stadial diffusion mechanisms were demonstrated.
CONCLUSIONS
Our results demonstrate the considerable ability of modified Asaia to colonise different populations of malaria vectors, including pecies where its association is not primary, in large environments. The data support the potential to employ transgenic Asaia as a tool for malaria control, disclosing promising perspective for its field application with suitable effector molecules.
Topics: Acetobacteraceae; Animals; Anopheles; Gene Transfer Techniques; Green Fluorescent Proteins; Insect Vectors; Microscopy, Fluorescence; Molecular Biology; Pilot Projects; Recombinant Proteins; Staining and Labeling
PubMed: 26965746
DOI: 10.1186/s13071-016-1427-3 -
Nature Communications Oct 2019Gluconacetobacter xylinus (G. xylinus) metabolism is activated by oxygen, which makes the formation of an air-medium interface critical. Here we report solid...
Gluconacetobacter xylinus (G. xylinus) metabolism is activated by oxygen, which makes the formation of an air-medium interface critical. Here we report solid matrix-assisted 3D printing (SMAP) of an incubation medium surface and the 3D fabrication of bacterial cellulose (BC) hydrogels by in situ biosynthesis of G. xylinus. A printing matrix of polytetrafluoroethylene (PTFE) microparticles and a hydrogel ink containing an incubation medium, bacteria, and cellulose nanofibers (CNFs) are used in the SMAP process. The hydrogel ink can be printed in the solid matrix with control over the topology and dimensional stability. Furthermore, bioactive bacteria produce BC hydrogels at the surface of the medium due to the permeability of oxygen through the PTFE microparticle layer. The flexibility of the design is verified by fabricating complex 3D structures that were not reported previously. The resulting tubular BC structures suggest future biomedical applications, such as artificial blood vessels and engineered vascular tissue scaffolding. The fabrication of a versatile free-form structure of BC has been challenged due to restricted oxygen supplies at the medium and the dimensional instability of hydrogel printing. SMAP is a solution to the problem of fabricating free-form biopolymer structures, providing both printability and design diversity.
Topics: Cell Culture Techniques; Cellulose; Culture Media; Gluconacetobacter xylinus; Hydrogels; Nanofibers; Oxygen; Printing, Three-Dimensional; Tissue Engineering; Tissue Scaffolds
PubMed: 31604956
DOI: 10.1038/s41467-019-12585-9 -
Current Biology : CB Jan 2024Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix...
Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. Although bacterial cellulose (BC) was first described in the 19 century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold-the "cortical belt"-which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing β- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers.
Topics: Cellulose; Proteobacteria; Gluconacetobacter; Bacteria; Biofilms
PubMed: 38141614
DOI: 10.1016/j.cub.2023.11.057 -
Applied and Environmental Microbiology May 2016Magnetotactic bacteria biosynthesize specific organelles, the magnetosomes, which are membrane-enclosed crystals of a magnetic iron mineral that are aligned in a linear...
UNLABELLED
Magnetotactic bacteria biosynthesize specific organelles, the magnetosomes, which are membrane-enclosed crystals of a magnetic iron mineral that are aligned in a linear chain. The number and size of magnetosome particles have to be critically controlled to build a sensor sufficiently strong to ensure the efficient alignment of cells within Earth's weak magnetic field while at the same time minimizing the metabolic costs imposed by excessive magnetosome biosynthesis. Apart from their biological function, bacterial magnetosomes have gained considerable interest since they provide a highly useful model for prokaryotic organelle formation and represent biogenic magnetic nanoparticles with exceptional properties. However, potential applications have been hampered by the difficult cultivation of these fastidious bacteria and their poor yields of magnetosomes. In this study, we found that the size and number of magnetosomes within the cell are controlled by many different Mam and Mms proteins. We present a strategy for the overexpression of magnetosome biosynthesis genes in the alphaproteobacterium Magnetospirillum gryphiswaldense by chromosomal multiplication of individual and multiple magnetosome gene clusters via transposition. While stepwise amplification of the mms6 operon resulted in the formation of increasingly larger crystals (increase of ∼35%), the duplication of all major magnetosome operons (mamGFDC, mamAB, mms6, and mamXY, comprising 29 genes in total) yielded an overproducing strain in which magnetosome numbers were 2.2-fold increased. We demonstrate that the tuned expression of the mam and mms clusters provides a powerful strategy for the control of magnetosome size and number, thereby setting the stage for high-yield production of tailored magnetic nanoparticles by synthetic biology approaches.
IMPORTANCE
Before our study, it had remained unknown how the upper sizes and numbers of magnetosomes are genetically regulated, and overproduction of magnetosome biosynthesis had not been achieved, owing to the difficulties of large-scale genome engineering in the recalcitrant magnetotactic bacteria. In this study, we established and systematically explored a strategy for the overexpression of magnetosome biosynthesis genes by genomic amplification of single and multiple magnetosome gene clusters via sequential chromosomal insertion by transposition. Our findings also indicate that the expression levels of magnetosome proteins together limit the upper size and number of magnetosomes within the cell. We demonstrate that tuned overexpression of magnetosome gene clusters provides a powerful strategy for the precise control of magnetosome size and number.
Topics: Gene Dosage; Genes, Bacterial; Magnetosomes; Magnetospirillum; Multigene Family; Organelle Biogenesis
PubMed: 26969709
DOI: 10.1128/AEM.03860-15 -
Scientific Reports Dec 2019Protein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the...
Protein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy Förster resonance energy transfer (FLIM-FRET) and stimulated emission depletion (STED) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.
Topics: Bacterial Proteins; Fluorescence Resonance Energy Transfer; Magnetosomes; Magnetospirillum; Microscopy, Fluorescence
PubMed: 31873083
DOI: 10.1038/s41598-019-55804-5 -
Applied and Environmental Microbiology Oct 2016The purple nonsulfur alphaproteobacterium Rhodospirillum rubrum S1 was genetically engineered to synthesize a heteropolymer of mainly 3-hydroxydecanoic acid and...
UNLABELLED
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 CO-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 P 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 (PHA), enhanced gene expression through the P promoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the P 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 PHA 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.
Topics: Gases; Metabolic Engineering; Polyhydroxyalkanoates; Rhodospirillum rubrum
PubMed: 27520812
DOI: 10.1128/AEM.01744-16 -
Microbiological Research 2003Magnetotactic bacteria are microorganisms that respond to magnetic fields. We have studied the surface ultrastructure of Magnetospirillum magnetotacticum and uncultured...
Magnetotactic bacteria are microorganisms that respond to magnetic fields. We have studied the surface ultrastructure of Magnetospirillum magnetotacticum and uncultured magnetotactic bacteria from a marine environment using transmission electron microscopy and freeze-etching. Numerous membrane vesicles were observed on the surface of Magnetospirillum magnetotacticum bacteria. All uncultured magnetotactic bacteria presented membrane vesicles on their surface in addition to an extensive capsular material and an S-layer formed by particles arranged in a hexagonal symmetry. We did not observe any indication of electron-dense precipitation on the surface of these microorganisms. Our results indicate that membrane vesicles are a common characteristic of magnetotactic bacteria in natural sediments.
Topics: Bacteria; Cell Membrane; Culture Media; Ferric Compounds; Freeze Etching; Magnetics; Magnetospirillum; Microscopy, Electron; Water Microbiology
PubMed: 14717452
DOI: 10.1078/0944-5013-00210 -
Journal of Bacteriology Aug 2017Hydroxyprolines, such as -4-hydroxy-l-proline (T4LHyp), -3-hydroxy-l-proline (T3LHyp), and -3-hydroxy-l-proline (C3LHyp), are present in some proteins including...
Hydroxyprolines, such as -4-hydroxy-l-proline (T4LHyp), -3-hydroxy-l-proline (T3LHyp), and -3-hydroxy-l-proline (C3LHyp), are present in some proteins including collagen, plant cell wall, and several peptide antibiotics. In bacteria, genes involved in the degradation of hydroxyproline are often clustered on the genome (l-Hyp gene cluster). We recently reported that an aconitase X (AcnX)-like gene from an l-Hyp gene cluster functions as a monomeric C3LHyp dehydratase (AcnX). However, the physiological role of C3LHyp dehydratase remained unclear. We here demonstrate that NBRC 102289, an aerobic nitrogen-fixing bacterium, robustly grows using not only T4LHyp and T3LHyp but also C3LHyp as the sole carbon source. The small and large subunits of the gene ( and , respectively) from NBRC 102289 are located separately from the l-Hyp gene cluster and encode a C3LHyp dehydratase with a novel heterodimeric structure (AcnX). A strain disrupted in the gene did not grow on C3LHyp, suggesting its involvement in C3LHyp metabolism. Furthermore, C3LHyp induced transcription of not only the genes but also the gene encoding Δ-pyrroline-2-carboxylate reductase, which is involved in T3LHyp, d-proline, and d-lysine metabolism. On the other hand, the l-Hyp gene cluster of some other bacteria contained not only the AcnX gene but also two putative proline racemase-like genes ( and ). Despite having the same active sites (a pair of Cys/Cys) as hydroxyproline 2-epimerase, which is involved in the metabolism of T4LHyp, the dominant reaction by HypA2 was clearly the dehydration of T3LHyp, a novel type of T3LHyp dehydratase that differed from the known enzyme (Cys/Thr). More than 50 years after the discovery of -4-hydroxy-l-proline (generally called l-hydroxyproline) degradation in aerobic bacteria, its genetic and molecular information has only recently been elucidated. l-Hydroxyproline metabolic genes are often clustered on bacterial genomes. These loci frequently contain a hypothetical gene(s), whose novel enzyme functions are related to the metabolism of -3-hydroxyl-proline and/or -3-hydroxyl-proline, a relatively rare l-hydroxyproline in nature. Several l-hydroxyproline metabolic enzymes show no sequential similarities, suggesting their emergence by convergent evolution. Furthermore, transcriptional regulation by -4-hydroxy-l-proline, -3-hydroxy-l-proline, and/or -3-hydroxy-l-proline significantly differs between bacteria. The results of the present study show that several l-hydroxyprolines are available for bacteria as carbon and energy sources and may contribute to the discovery of potential metabolic pathways of another hydroxyproline(s).
Topics: Azospirillum brasilense; Carbon; Gene Knockout Techniques; Hydro-Lyases; Hydroxyproline; Multigene Family; Protein Subunits; Transcription, Genetic
PubMed: 28559297
DOI: 10.1128/JB.00255-17