Antenna modification leads to enhanced nitrogenase activity in a high light tolerant cyanobacterium

Biological nitrogen fixation is an energy intensive process that contributes significantly towards supporting life on this planet. Among nitrogen-fixing organisms, cyanobacteria remain unrivaled in their ability to fuel the energetically expensive nitrogenase reaction with photosynthetically harnessed solar energy. In heterocystous cyanobacteria light-driven, photosystem I (PSI)-mediated ATP synthesis plays a key role in propelling the nitrogenase reaction. Efficient light transfer to the photosystems rely on phycobilisomes (PBS), the major antenna protein complexes. PBS undergo degradation as a natural response to nitrogen starvation. Upon nitrogen availability, these proteins are resynthesized back to normal levels in vegetative cells, but their occurrence and function in heterocysts remains inconclusive. Anabaena 33047 is a heterocystous cyanobacterium that thrives under high light, harbors higher amounts of PBS in its heterocysts and fixes nitrogen at higher rates compared to other heterocystous cyanobacteria. To assess the relationship between PBS in heterocysts and nitrogenase function, we engineered a strain that retains high amounts of the antenna proteins in its heterocysts. Intriguingly, under high light intensities the engineered strain exhibited unusually high rates of nitrogenase activity compared to the wild type. Spectroscopic analysis revealed altered PSI kinetics in the mutant, with increased cyclic electron flow around PSI, a route that contributes to ATP generation and nitrogenase activity in heterocysts. Retaining higher levels of PBS in heterocysts appears to be an effective strategy to enhance nitrogenase function in cyanobacteria that are equipped with the machinery to operate under high light intensities.

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Antenna Modification Leads to Enhanced Nitrogenase Activity in a High Light-Tolerant Cyanobacterium

mBio ◽

10.1128/mbio.03408-21 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Anindita Bandyopadhyay ◽

Zi Ye ◽

Zuzana Benedikty ◽

Martin Trtilek ◽

Himadri B. Pakrasi

Keyword(s):

Direct Evidence ◽

Protein Complexes ◽

Nitrogenase Activity ◽

High Light ◽

Heterocystous Cyanobacterium ◽

Light Intensities

The function of phycobilisomes, the large antenna protein complexes in heterocysts has long been debated. This study provides direct evidence of the involvement of these proteins in supporting nitrogenase activity in Anabaena 33047, a heterocystous cyanobacterium that has an affinity for high light intensities.

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The desert green algae Chlorella ohadii thrives at excessive high light intensities by exceptionally enhancing the mechanisms that protect photosynthesis from photoinhibition

10.1101/2021.02.08.430232 ◽

2021 ◽

Author(s):

Guy Levin ◽

Sharon Kulikovsky ◽

Varda Liveanu ◽

Benjamin Eichenbaum ◽

Ayala Meir ◽

...

Keyword(s):

Molecular Mechanisms ◽

Electron Flow ◽

High Light ◽

Low Light ◽

Photosynthetic Organisms ◽

Light Intensities ◽

Protection Mechanisms ◽

Desert Algae ◽

Photo Damage ◽

Fast Repair

AbstractAlthough light is the driving force of photosynthesis, excessive light can be harmful. One of the main processes that limits photosynthesis is photoinhibition (PI), the process of light-induced photo-damage. When the absorbed light exceeds the amount that is dissipated by photosynthetic electron flow and other processes, damaging radicals are formed that mostly inactivate photosystem II (PSII). Damaged PSII must be replaced by a newly repaired complex in order to preserve full photosynthetic activity. Chlorella ohadii is a green micro-alga, isolated from biological soil crusts in the desert that thrive under extreme high light and is highly resistant to PI. Therefore, C. ohadii is an ideal candidate for study the molecular protection mechanisms from PI. To charac-terize these protection mechanisms in C. ohadii, we compared thylakoids of cells that were grown under low light versus extreme high light intensities. C. ohadii were found to employ all three known PI protection mechanisms: i) performance of massive reduction of the PSII antenna size; ii) accumulate protective carotenoids; and iii) possess a very fast repair cycle of photo-damaged reaction center proteins. This work elucidated the molecular mechanisms of photoinhibition resistance in one of the most light-tolerant photosynthetic organisms and shows how photoinhibition protection mechanisms evolved to marginal conditions enabling photosynthesis-dependent life in severe habitats.One Sentence HighlightAnalysis of the photosynthetic properties of a desert algae that thrives at extreme high light in-tensities reveals how protection from photoinhibition is achieved by a remarkable enhancement of three protection mechanisms.

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Structure of contact sites between the outer and inner mitochondrial membranes investigated by HVEM tomography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100167299 ◽

1996 ◽

Vol 54 ◽

pp. 966-967

Author(s):

C.A. Mannella ◽

K.F. Buttle ◽

K.A. O‘Farrell ◽

A. Leith ◽

M. Marko

Keyword(s):

Liver Mitochondrion ◽

Adenine Nucleotide ◽

Protein Complexes ◽

Mitochondrial Membranes ◽

Electron Microscopic ◽

Contact Sites ◽

Transmission Electron ◽

Precursor Proteins ◽

Membrane Contacts ◽

And Function

Early transmission electron microscopy of plastic-embedded, thin-sectioned mitochondria indicated that there are numerous junctions between the outer and inner membranes of this organelle. More recent studies have suggested that the mitochondrial membrane contacts may be the site of protein complexes engaged in specialized functions, e.g., import of mitochondrial precursor proteins, adenine nucleotide channeling, and even intermembrane signalling. It has been suggested that the intermembrane contacts may be sites of membrane fusion involving non-bilayer lipid domains in the two membranes. However, despite growing interest in the nature and function of intramitochondrial contact sites, little is known about their structure.We are using electron microscopic tomography with the Albany HVEM to determine the internal organization of mitochondria. We have reconstructed a 0.6-μm section through an isolated, plasticembedded rat-liver mitochondrion by combining 123 projections collected by tilting (+/- 70°) around two perpendicular tilt axes. The resulting 3-D image has confirmed the basic inner-membrane organization inferred from lower-resolution reconstructions obtained from single-axis tomography.

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Chaperonin as the normal controls and pathological anti-stress response in the human reproductive system

HEALTH OF WOMAN ◽

10.15574/hw.2016.111.126 ◽

2016 ◽

pp. 126-129

Author(s):

M. Makarenko ◽

◽

D. Hovsyeyev ◽

L. Sydoryk ◽

◽

...

Keyword(s):

Reproductive System ◽

Stress Proteins ◽

Physiological Stress ◽

Protein Complexes ◽

Reproductive Function ◽

Intercellular Signaling ◽

Toll Like Receptors ◽

Wide Range ◽

Protein Functions ◽

And Function

Different kinds of physiological stress cause mass changes in the cells, including the changes in the structure and function of the protein complexes and in separate molecules. The protein functions is determined by its folding (the spatial conclusion), which depends on the functioning of proteins of thermal shock- molecular chaperons (HSPs) or depends on the stress proteins, that are high-conservative; specialized proteins that are responsible for the correct proteinaceous folding. The family of the molecular chaperones/ chaperonins/ Hsp60 has a special place due to the its unique properties of activating the signaling cascades through the system of Toll-like receptors; it also stimulates the cells to produce anti- inflammatory cytokines, defensins, molecules of cell adhesion and the molecules of MHC; it functions as the intercellular signaling molecule. The pathological role of Hsp60 is established in a wide range of illnesses, from diabetes to atherosclerosis, where Hsp60 takes part in the regulation of both apoptosis and the autoimmune processes. The presence of the HSPs was found in different tissues that are related to the reproductive system. Key words: molecular chaperons (HSPs), Toll-like receptors, reproductive function, natural auto antibody.

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Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein

Methods and Protocols ◽

10.3390/mps4030051 ◽

2021 ◽

Vol 4 (3) ◽

pp. 51

Author(s):

Satish Kantipudi ◽

Daniel Harder ◽

Sara Bonetti ◽

Dimitrios Fotiadis ◽

Jean-Marc Jeckelmann

Keyword(s):

Amino Acid ◽

Pichia Pastoris ◽

Protein Complexes ◽

Amino Acid Transporter ◽

Amino Acid Transporters ◽

Expression Host ◽

Amino Acids And Derivatives ◽

Heterodimeric Complex ◽

Acid Transporter ◽

And Function

Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations.

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Mitochondrial microRNAs: A Putative Role in Tissue Regeneration

Biology ◽

10.3390/biology9120486 ◽

2020 ◽

Vol 9 (12) ◽

pp. 486

Author(s):

Sílvia C. Rodrigues ◽

Renato M. S. Cardoso ◽

Filipe V. Duarte

Keyword(s):

Tissue Regeneration ◽

Protein Complexes ◽

Mitochondrial Protein ◽

Noncoding Rnas ◽

Atp Synthesis ◽

Mitochondrial Proteins ◽

Cellular Mechanisms ◽

Small Noncoding Rnas ◽

Mitochondrial Ribosome

The most famous role of mitochondria is to generate ATP through oxidative phosphorylation, a metabolic pathway that involves a chain of four protein complexes (the electron transport chain, ETC) that generates a proton-motive force that in turn drives the ATP synthesis by the Complex V (ATP synthase). An impressive number of more than 1000 mitochondrial proteins have been discovered. Since mitochondrial proteins have a dual genetic origin, it is predicted that ~99% of these proteins are nuclear-encoded and are synthesized in the cytoplasmatic compartment, being further imported through mitochondrial membrane transporters. The lasting 1% of mitochondrial proteins are encoded by the mitochondrial genome and synthesized by the mitochondrial ribosome (mitoribosome). As a result, an appropriate regulation of mitochondrial protein synthesis is absolutely required to achieve and maintain normal mitochondrial function. Regarding miRNAs in mitochondria, it is well-recognized nowadays that several cellular mechanisms involving mitochondria are regulated by many genetic players that originate from either nuclear- or mitochondrial-encoded small noncoding RNAs (sncRNAs). Growing evidence collected from whole genome and transcriptome sequencing highlight the role of distinct members of this class, from short interfering RNAs (siRNAs) to miRNAs and long noncoding RNAs (lncRNAs). Some of the mechanisms that have been shown to be modulated are the expression of mitochondrial proteins itself, as well as the more complex coordination of mitochondrial structure and dynamics with its function. We devote particular attention to the role of mitochondrial miRNAs and to their role in the modulation of several molecular processes that could ultimately contribute to tissue regeneration accomplishment.

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Estimating Nitrogenase Activity of Faba Bean (Vicia faba) by Acetylene Reduction (Ar) Assay

Functional Plant Biology ◽

10.1071/pp9900489 ◽

1990 ◽

Vol 17 (5) ◽

pp. 489 ◽

Cited By ~ 20

Author(s):

Herdina ◽

JH Silsbury

Keyword(s):

Diurnal Variation ◽

Vicia Faba ◽

Faba Bean ◽

Closed System ◽

N2 Fixation ◽

Acetylene Reduction ◽

Nitrogenase Activity ◽

Electron Flow ◽

Specific Rate ◽

Rooting Medium

Methods of conducting acetylene reduction (AR) assay were appraised for estimating the nitrogenase activity of nodules of faba bean (Vicia faba L.). Factors considered were: (i) disturbance of plants when removing the rooting medium; (ii) assay temperature; (iii) the use of whole plants rather than detached, nodulated roots; (iv) diurnal variation in nodule activity; and (v) a decline in C2H4 production after exposure to C2H2. Plants growing in jars of 'oil dry' (calcined clay) had the same AR activity when assayed in situ in a closed system as when assayed after removal of the rooting medium. Assay temperatures of 12.5, 17.5 and 22.5°C influenced the specific rate of AR with the optimum at 17.5°C. Removal of the shoot resulted in a rapid decrease in AR activity in both vegetative and reproductive plants but the effect was much larger in the latter. AR and respiration by nodulated roots were closely linked and both varied markedly over a diurnal 12 h/12 h cycle. Since no fluctuation was found after nodules were detached, diurnal variation in the respiration of nodulated roots is attributed to change in nodule activity. Half of the dark respiration of nodulated roots was associated with respiration of the nodules and thus largely with N2 fixation. Since the AR assay provides no information on how electron flow in vivo is partitioned between reduction of N2 and reduction of protons, diurnal variation in hydrogen evolution (HE) in air and Ar/O2 in an open system was used to estimate this partitioning. Diurnal variation in apparent N2 fixation estimated in this manner was examined at a 'low' PPFD (300 μmol m-2 s-1) and at 'high' (1300 μmol m-2 s-1) to explore whether variation could be attributed to change in carbohydrate supply. Although HE in air and in Ar/O2 were both closely linked with the respiration of the nodulated root, apparent N2 fixation showed only a slight diurnal variation at 'low' light and almost none at 'high'. Vegetative plants showed no C2H2-induced decline in activity with exposure to C2H2 but reproductive plants did. This difference appears to be an age effect rather than attributable to flowering per se, since a decline occurred even when plants were kept vegetative by disbudding. A closed system for AR assay appears satisfactory for vegetative faba bean but such an assay over a 40-min period during the reproductive stage would underestimate nitrogenase activity by about 20%.

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Stoichiometry and Turnover of the Bacterial Flagellar Switch Protein FliN

mBio ◽

10.1128/mbio.01216-14 ◽

2014 ◽

Vol 5 (4) ◽

Cited By ~ 47

Author(s):

Nicolas J. Delalez ◽

Richard M. Berry ◽

Judith P. Armitage

Keyword(s):

Copy Number ◽

Motor Proteins ◽

Fluorescent Protein ◽

Protein Complexes ◽

Content Type ◽

Rotation Direction ◽

Cell Measurement ◽

Biological Complexes ◽

And Function

ABSTRACTSome proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, inEscherichia coli. Our results confirm that,in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments.IMPORTANCEThe flagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single-cell measurement, that both the copy number and the fraction of exchanging molecules vary with the rotational bias of the rotor. When the motor is locked in counterclockwise rotation, the copy number is similar to that determined by averaged, fixed methodologies, but when locked in a clockwise direction, the number is much lower, suggesting that that the switch complex ring is incomplete. Our results suggest that motor remodeling is an important component in tuning responses and adaptation at the motor.

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