Papers by Georgios Tsiotis
Proceedings of the National Academy of Sciences
Light energy absorption and transfer are very important processes in photosynthesis. In green sul... more Light energy absorption and transfer are very important processes in photosynthesis. In green sulfur bacteria light is absorbed primarily by the chlorosomes and its energy is transferred via the Fenna–Matthews–Olson (FMO) proteins to a homodimeric reaction center (RC). Here, we report the cryogenic electron microscopic structure of the intact FMO-RC apparatus from Chlorobaculum tepidum at 2.5 Å resolution. The FMO-RC apparatus presents an asymmetric architecture and contains two FMO trimers that show different interaction patterns with the RC core. Furthermore, the two permanently bound transmembrane subunits PscC, which donate electrons to the special pair, interact only with the two large PscA subunits. This structure fills an important gap in our understanding of the transfer of energy from antenna to the electron transport chain of this RC and the transfer of electrons from reduced sulfur compounds to the special pair.
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Microorganisms
Pseudomonas sp. phDV1 is a polyhydroxyalkanoate (PHA) producer. The presence of the endogenous PH... more Pseudomonas sp. phDV1 is a polyhydroxyalkanoate (PHA) producer. The presence of the endogenous PHA depolymerase (phaZ) responsible for the degradation of the intracellular PHA is one of the main shortages in the bacterial production of PHA. Further, the production of PHA can be affected by the regulatory protein phaR, which is important in accumulating different PHA-associated proteins. PHA depolymerase phaZ and phaR knockout mutants of Pseudomonas sp. phDV1 were successfully constructed. We investigate the PHA production from 4.25 mM phenol and grape pomace of the mutants and the wild type. The production was screened by fluorescence microscopy, and the PHA production was quantified by HPLC chromatography. The PHA is composed of Polydroxybutyrate (PHB), as confirmed by 1H-nuclear magnetic resonance analysis. The wildtype strain produces approximately 280 μg PHB after 48 h in grape pomace, while the phaZ knockout mutant produces 310 μg PHB after 72 h in the presence of phenol per gr...
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PROTEOMICS
Chlorobaculum tepidum is an anaerobic green sulfur bacterium which oxidizes sulfide, elemental su... more Chlorobaculum tepidum is an anaerobic green sulfur bacterium which oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. It can also oxidize sulfide to produce extracellular S0 globules, which can be further oxidized to sulfate and used as an electron donor. Here, we performed label‐free quantitative proteomics on total cell lysates prepared from different metabolic states, including a sulfur production state (10 h post‐incubation [PI]), the beginning of sulfur consumption (20 h PI), and the end of sulfur consumption (40 h PI), respectively. We observed an increased abundance of the sulfide:quinone oxidoreductase (Sqr) proteins in 10 h PI indicating a sulfur production state. The periplasmic thiosulfate‐oxidizing Sox enzymes and the dissimilatory sulfite reductase (Dsr) subunits showed an increased abundance in 20 h PI, corresponding to the sulfur‐consuming state. In addition, we found that the abundance of the heterodisulfide‐reductase and the sulfhydrogena...
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Current Research in Biotechnology
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Photosynthetic RCs are classified according to the nature of their terminal electron acceptors. Q... more Photosynthetic RCs are classified according to the nature of their terminal electron acceptors. Q-type RCs (type II) reduce a mobile quinone. Examples are found in purple bacteria and in the photosystem II (PSII) of cyanobacteria and higher plants [1]. The RC of PSII consists of the PsbA, PsbD, PsbE, PsbF and PsbI subunits. The PsbA/PsbD heterodimer binds a variety of cofactors. These include the chlorophylls of the primary donor P680, the primary acceptor pheophytin, QA, the non-heme iron, QB, and the radicals TyrZ+ and TyrD+. Four additional chlorophylls, the “non-photochemical” pheophytin, and one or two s-carotene are also bound. A 9 kDa and a 5 kDa membrane protein, the psbE and psbF gene products respectively, form the cyt b559 which is required for the PSII assembly. Cyt b559 is thought to be involved in cyclic electron flow around PSII since it has been shown to be both, photooxydized by P680+ and photoreduced by plastoquinol [2]. The function of the small 4 kDa membrane sub...
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Chemical Communications, 2021
The rationally designed variant V37A of L. syltensis (R)-selective transaminase exhibits improved... more The rationally designed variant V37A of L. syltensis (R)-selective transaminase exhibits improved activity towards bulkier substrates compared to the wild-type.
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Protein Expression and Purification, 2021
Coxiella burnetii, the causative agent of Q fever, is an intracellular bacterial pathogen. Studie... more Coxiella burnetii, the causative agent of Q fever, is an intracellular bacterial pathogen. Studies on Coxiella have shown that a type IVB secretion system (T4BSS) contributes to the establishment of the infection by transferring protein molecules. In this report, we focus on two core proteins of the Coxiella T4BSS, namely the IcmG/DotF protein (CBU_1626) and the IcmK/DotH protein (CBU_1628). Here we present a method for the recombinant expression of IcmG and IcmK in E. coli. IcmG was purified by Strep-Tactin affinity chromatography and size exclusion chromatography, while for the purification of IcmK an additional anion exchange chromatography step was introduced. The yields of the purified IcmG and IcmK proteins were 1.2 mg/L and 3 mg/L, respectively. The purified proteins showed predominant band on SDS-PAGE gel of 37 kDa for the IcmG and 40 kDa for the IcmK. Protein folding is confirmed by circular dichroism spectroscopy. The dynamic light scattering experiment indicated that IcmG and IcmK existed in a homogenous form. Further Blue native PAGE indicates the presences of a monomeric form for the IcmK and IcmG. Our work lays the basis for functional exploration and structural determination of IcmG and IcmK proteins of Coxiella's secretion system.
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PROTEOMICS, 2020
The degradation of aromatic compounds comprises an important step in the removal of pollutants an... more The degradation of aromatic compounds comprises an important step in the removal of pollutants and re‐utilization of plastics and other non‐biological polymers. Here, Pseudomonas sp. strain phDV1, a gram‐negative bacterium that is selected for its ability to degrade aromatic compounds is studied. In order to understand how the aromatic compounds and their degradation products are reintroduced in the metabolism of the bacteria and the systematic/metabolic response of the bacterium to the new carbon source, the proteome of this strain is analyzed in the presence of succinate, phenol, and o‐, m‐, and p‐cresol as the sole carbon source. As a reference proteome, the bacteria are grown in succinate and then compared with the respective proteomes of bacteria grown on phenol and different cresols. In total, 2295 proteins are identified; 1908 proteins are used for quantification between different growth conditions. The carbon source affects the synthesis of enzymes related to aromatic compou...
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Frontiers in Cellular and Infection Microbiology, 2020
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PROTEOMICS, 2019
Cyanobacteria are oxygenic photosynthetic prokaryotes and play a crucial role in the Earth's ... more Cyanobacteria are oxygenic photosynthetic prokaryotes and play a crucial role in the Earth's carbon and nitrogen cycles. The photoautotrophic cyanobacterium Anabaena sp. PCC 7120 has the ability to fix atmospheric nitrogen in heterocysts and produce hydrogen as a byproduct through a nitrogenase. In order to improve hydrogen production, mutants from Anabaena sp. PCC 7120 are constructed by inactivation of the uptake hydrogenase (ΔhupL) and the bidirectional hydrogenase (ΔhoxH) in previous studies. Here the proteomic differences of enriched heterocysts between these mutants cultured in N2‐fixing conditions are investigated. Using a label‐free quantitative proteomics approach, a total of 2728 proteins are identified and it is found that 79 proteins are differentially expressed in the ΔhupL and 117 proteins in the ΔhoxH variant. The results provide for the first time comprehensive information on proteome regulation of the uptake hydrogenase and the bidirectional hydrogenase, as well...
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ChemInform, 1989
The dicarbonitriles (I) react with ammonium azide, generated in situ from NaN3 (II), to give the ... more The dicarbonitriles (I) react with ammonium azide, generated in situ from NaN3 (II), to give the bis(tetrazolyl)pyrazines (III) which are alkylated with α,ω‐dibromoalkanes (IV), forming the azacyclophanes (V) in low yields.
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ChemInform, 1989
The chlorine atoms of the easily accessible pyrazine (I) can be readily exchanged by amines such ... more The chlorine atoms of the easily accessible pyrazine (I) can be readily exchanged by amines such as (II) or (VI) yielding the new compounds (III) or (VII) which form the nitrogen‐rich heterocycles (IV) or (VIII) upon treatment with hydrazine hydrate (V).
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European Journal of Biochemistry, 1995
A monoclonal antibody was derived from mice immunized with the native trimeric, photosystem I (PS... more A monoclonal antibody was derived from mice immunized with the native trimeric, photosystem I (PSI) complex from the cyanobacterium Synechococcus PCC 7002 which reacts with a conformational epitope of the PSI complex. As seen by immunoelectron microscopy, the mAb bound to the stromal side of the thylakoid membranes. The DNA sequence encoding variable regions of the mAb was cloned into recombinant plasmids, sequenced and expressed in Escherichia coli. ELISA, Western blots and immunoelectron microscopy provided evidence that the expressed paired variable domain (Fv) fragments bind to the antigen in the same way as the parent mAb.A one‐step purification was applied to purify the trimeric PSI complex using an affinity tag attached to the Fv fragment. Analysis by gel electrophoresis and N‐terminal sequencing revealed the presence of the psaA, psaB, psaC, psaD, psaE, psaF and psaL gene products. The antenna size of the isolated PSI/Fv was 139±9 chlorophyll a/primary electron donor. Flash‐induced absorption‐change measurements showed that the complex exhibited electron transfer from the primary electron donor, P700, to the Fe‐S center, FA/FB. The position of the bound Fv fragment on the trimeric PSI surface was determined by high‐resolution electron microscopy and digital image processing.
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Concepts in Photobiology, 1999
... of photoautotrophic growth, oxygen evolution, and marked declines in the amounts of other rea... more ... of photoautotrophic growth, oxygen evolution, and marked declines in the amounts of other reaction center proteins (Dl, D2 and CP 47)(Kuhn and Vermaas ... D Crystallization of Photosystem II There have been two reports of the 3-D crystallization of a PS II core complex (Adir et al ...
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Photosynthesis: Mechanisms and Effects, 1998
Functional Photosystem I (PSI) from the thermophilic cyanobacterium Synechococcus sp. OD24 was pu... more Functional Photosystem I (PSI) from the thermophilic cyanobacterium Synechococcus sp. OD24 was purified and crystallized into two-dimensional (2D) crystals in the presence of the lipid dimyristoyl phosphatidylcholine [1,2]. This cyanobacterial PSI consists of 11 protein subunits, assembled into a 340 kDa complex [3]. The subunits PsaA and -B are each 83 kDa while the other 9 subunits (PsaC, -D, -E, -F, -I, -J, -K, -L, and -M) are smaller having masses below 20 kDa. All subunits are intrinsic except PsaC, -D and -E, which are extrinsic and located on the stromal side of the thylakoid membrane. A wealth of information about the PSI complex is available from different structure determination methods. Nevertheless, detailed information on the surface topography of this fascinating enzyme is lacking.
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Photosynthesis: from Light to Biosphere, 1995
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Membrane Protein Purification and Crystallization, 2003
The net charge of an amphoteric molecule is determined by the pH of its local environment. In the... more The net charge of an amphoteric molecule is determined by the pH of its local environment. In the case of protein molecules this is the sum of all positive and negative charges of the ionizable amino acid side chains and prosthetic groups. For every protein there is a specific pH at which the net charge is zero. This isoelectric pH value (p I ) is a characteristic physicochemical property of every protein. The amphoteric nature of proteins can be used as the basis for isolectric focusing (lEF). In this method proteins are separated according to their p I in a pH gradient gel. Stable, linear pH gradients are the keys to successful lEF. lEF is a high-resolution technique that can resolve proteins differing in p I by less that 0.05 pH units. The technique is rapid and nondenaturating and can be run preparatively as well as analytically. lEF is well suited for use at any stage of a preparative scheme, and it is particularly effective in the early stages of purification. Electrofocusing differs from conventional electrophoresis by using pH gradient gels. Either the pH gradient within the gel preexists if ampholytes covalently bound to acrylamide are used for casting gels, or the pH gradient is formed during electrofocusing if mobile carrier ampholytes are used. This chapter describes isoelectric focusing using mobile ampholytes. During the experiment the carrier ampholytes arrange according to their isoelectric points, and a pH gradient develops.
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European Journal of Biochemistry, 1999
The photosystem II (PSII) reaction center (RC) complex was isolated from spinach and characterize... more The photosystem II (PSII) reaction center (RC) complex was isolated from spinach and characterized by gel electrophoresis, gel filtration and analytical ultracentrifugation. The purified complex contained the PsbA, PsbD, PsbE, PsbF and PsbI subunits. Gel filtration and analytical ultracentrifugation indicated the presence of a homogeneous complex. The mass of the RC complexes was found to be 107 kDa by analytical ultracentrifugation and 132 kDa by scanning transmission electron microscopy (STEM). The mass obtained showed the isolated complex to exist as a monomer and only one cytochrome b559 (cyt b559) to be associated with the RC complex. Digital images of negatively stained RC complexes were recorded by STEM and analyzed by single‐particle averaging. The complex was 9 nm long and 5 nm wide, and exhibited a pronounced quasi‐twofold symmetry. This supports the symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged Psb...
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Photosynthesis Research, 1996
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Journal of Proteomics, 2014
Ever since antibiotics were used to help humanity battle infectious diseases, microorganisms stra... more Ever since antibiotics were used to help humanity battle infectious diseases, microorganisms straight away fought back. Antibiotic resistance mechanisms indeed provide microbes with possibilities to by-pass and survive the action of antibiotic drugs. Several methods have been employed to identify these microbial resistance mechanisms in an ongoing effort to reduce the steadily increasing number of treatment failures due to multi-drug-resistant microbes. Proteomics has evolved to an important tool for this area of research. Following rapid advances in whole genome sequencing, proteomic technologies have been widely used to investigate microbial gene expression. This review highlights the contribution of proteomics in identifying microbial drug resistance mechanisms. It summarizes different proteomic studies on bacteria resistant to different antibiotic drugs. The review further includes an overview of the methodologies used, as well as lists key proteins identified, thus providing the reader not only a summary of research already done, but also directions for future research. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.
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Papers by Georgios Tsiotis