The synergy between the PscC subunits for electron transfer to the P840 special pair in Chlorobaculum tepidum.

The primary photochemical reaction of photosynthesis in green sulfur bacteria occurs in the homodimer PscA core proteins by a special chlorophyll pair. The light induced excited state of the special pair producing P840+ is rapidly reduced by electron transfer from one of the two PscC subunits. Molecular dynamics (MD) simulations are combined with bioinformatic tools herein to provide structural and dynamic insight into the complex between the two PscA core proteins and the two PscC subunits. The microscopic dynamic model involves extensive sampling at atomic resolution and at a cumulative time-scale of 22µs and reveals well defined protein-protein interactions. The membrane complex is composed of the two PscA and the two PscC subunits and macroscopic connections are revealed within a putative electron transfer pathway from the PscC subunit to the special pair P840 located within the PscA subunits. Our results provide a structural basis for understanding the electron transport to the homodimer RC of the green sulfur bacteria. The MD based approach can provide the basis to further probe the PscA-PscC complex dynamics and observe electron transfer therein at the quantum level. Furthermore, the transmembrane helices of the different PscC subunits exert distinct dynamics in the complex.

Production of Polyhydroxybutyrate by Genetically Modified Pseudomonas sp. phDV1: A Comparative Study of Utilizing Wine Industry Waste as a Carbon Source.

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 gram of cells, respectively. The ability of the phaZ mutant to synthesize high levels of PHB in the presence of monocyclic aromatic compounds may open the possibility of reducing the costs of industrial PHB production.

Insights into the sulfur metabolism of Chlorobaculum tepidum by label-free quantitative proteomics.

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 sulfhydrogenase operons was influenced by electron donor availability and may be associated with sulfur metabolism. Further, we isolated and analyzed the extracellular sulfur globules in the different metabolic states to study their morphology and the sulfur cluster composition, yielding 58 previously uncharacterized proteins in purified globules. Our results show that C. tepidum regulates the cellular levels of enzymes involved in sulfur metabolism in response to the availability of reduced sulfur compounds.

Cryo-EM structure of the whole photosynthetic reaction center apparatus from the green sulfur bacterium Chlorobaculum tepidum.

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.

Rational engineering of Luminiphilus syltensis (R)-selective amine transaminase for the acceptance of bulky substrates.

Despite the plethora of information on (S)-selective amine transaminases, the (R)-selective ones are still not well-studied; only a few structures are known to date, and their substrate scope is limited, apart from a few stellar works in the field. Herein, the structure of Luminiphilus syltensis (R)-selective amine transaminase is elucidated to facilitate engineering towards variants active on bulkier substrates. The V37A variant exhibited increased activity towards 1-phenylpropylamine and to activity against 1-butylamine. In contrast, the S248 and T249 positions, located on the ß-turn in the P-pocket, seem crucial for maintaining the activity of the enzyme.

Polyhydroxyalkanoate (PHA) Production in Pseudomonas sp. phDV1 Strain Grown on Phenol as Carbon Sources.

Pseudomonas strains have a variety of potential uses in bioremediation and biosynthesis of biodegradable plastics. Pseudomonas sp. strain phDV1, a Gram-negative phenol degrading bacterium, has been found to utilize monocyclic aromatic compounds as sole carbon source via the meta-cleavage pathway. The degradation of aromatic compounds comprises an important step in the removal of pollutants. The present study aimed to investigate the ability of the Pseudomonas sp. strain phDV1 to produce polyhydroxyalkanoates (PHAs) and examining the effect of phenol concentration on PHA production. The bacterium was cultivated in minimal medium supplemented with different concentrations of phenol ranging from 200-600 mg/L. The activity of the PHA synthase, the key enzyme which produces PHA, was monitored spectroscopically in cells extracts. Furthermore, the PHA synthase was identified by mass spectrometry in cell extracts analyzed by SDS-PAGE. Transmission electron micrographs revealed abundant electron-transparent intracellular granules. The isolated biopolymer was confirmed to be polyhydroxybutyrate (PHB) by FTIR, NMR and MALDI-TOF/TOF analyses. The ability of strain Pseudomonas sp. phDV1 to remove phenol and to produce PHB makes the strain a promising biocatalyst in bioremediation and biosynthesis of biodegradable plastics.

Expression, purification and characterization of the IcmG and IcmK proteins of the type IVB secretion system from Coxiella burnetii.

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.

Proteomic Characterization of the Pseudomonas sp. Strain phDV1 Response to Monocyclic Aromatic Compounds.

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 compound degradation and in particular the enzyme involved in the meta-pathway of monocyclic aromatic compounds degradation. In addition, proteins involved in the production of polyhydroxyalkanoate (PHA), an attractive biomaterial, show higher abundance in the presence of monocyclic aromatic compounds. The results provide, for the first time, comprehensive information on the proteome response of this strain to monocyclic aromatic compounds.

In the Search of Potential Serodiagnostic Proteins to Discriminate Between Acute and Chronic Q Fever in Humans. Some Promising Outcomes.

Coxiella burnetii is the agent that causes acute and chronic Q fever infections in humans. Although the isolates studied so far have shown that the two forms of the disease differ in virulence potential thus, implying a variance in their proteomic profile, the methods used do not deliver enough discriminatory capability and often, human infections may be mis-diagnosed. The current study adds further knowledge to the results that we have already published on the Coxiella outer membrane protein 1 (Com1). Herein we identified the proteins GroEL, Ybgf, OmpH, and UPF0422 as candidates for serodiagnostics of Q fever; following cloning, expression and purification they were further used as antigens in ELISA for the screening of patients' sera associated with chronic Q fever endocarditis, sera negative for phase I IgG, sera with at least one sample positive for phase I IgG and sera from patients who suffered from various rheumatic diseases. Blood donors were used as the controls. Sensitivity, specificity, positive predictive value, negative predictive value, and Cohen's kappa coefficient (κ) were calculated and we also performed binary logistic regression analysis to identify combinations of proteins with increased diagnostic yield. We found that proteins GroEL and Ybgf, together with Com1, play the most significant role in the correct diagnosis of chronic Q fever. Of these three proteins, it was shown that Com1 and GroEL present the highest sensitivity and specificity altogether. The results add to the existing knowledge that an antigen-based serodiagnostic test that will be able to correctly diagnose chronic Q fever may not be far from reality.

Proteome Analysis of Enriched Heterocysts from Two Hydrogenase Mutants from Anabaena sp. PCC 7120.

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 as systematic data on the hydrogen related metabolism in Anabaena sp. PCC 7120.

Complete Genome Sequence of Pseudomonas sp. Strain phDV1, an Isolate Capable of Efficient Degradation of Aromatic Hydrocarbons.

Pseudomonas sp. strain phDV1 is a Gram-negative bacterium capable of degrading aromatic hydrocarbons. Here, we present the complete genome sequence of this strain, which consists of 4,727,682 bp, with a 62.3% G+C content and 4,574 genes. Multiple genes responsible for the degradation of aromatics are present in this strain.

The ultrastructure of Chlorobaculum tepidum revealed by cryo-electron tomography.

Chlorobaculum (Cba) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. As other anoxygenic green photosynthetic bacteria, Cba tepidum synthesizes bacteriochlorophylls for the assembly of a large light-harvesting antenna structure, the chlorosome. Chlorosomes are sac-like structures that are connected to the reaction centers in the cytoplasmic membrane through the BChl α-containing Fenna-Matthews-Olson protein. Most components of the photosynthetic machinery are known on a biophysical level, however, the structural integration of light harvesting with charge separation is still not fully understood. Despite over two decades of research, gaps in our understanding of cellular architecture exist. Here we present an in-depth analysis of the cellular architecture of the thermophilic photosynthetic green sulfur bacterium of Cba tepidum by cryo-electron tomography. We examined whole hydrated cells grown under different electron donor conditions. Our results reveal the distribution of chlorosomes in 3D in an unperturbed cell, connecting elements between chlorosomes and the cytoplasmic membrane and the distribution of reaction centers in the cytoplasmic membrane.

Proteome Changes Induced by Imatinib and Novel Imatinib Derivatives in K562 Human Chronic Myeloid Leukemia Cells.

Imatinib mesylate is the leading compound to treat chronic myeloid leukemia (CML) and other cancers, through its inhibition of Bcr-Abl tyrosine kinases. However, resistance to imatinib develops frequently, particularly in late-stage disease and has necessitated the development of new Bcr-Abl inhibitors. The synthesis of a new series of phenylaminopyrimidines, structurally related to imatinib, showed large interest since the introduction of nilotinib. Here, we compare the protein levels in K562 cells treated with either imatinib or with novel imatinib derivates. Our results revealed that among the 986 quantified proteins, 35 had significantly altered levels of expression by imatinib or its derivates. In a second series of experiments, we directly compared the proteomes of imatinib treated K562 cells with those K562 cells treated with any of the four imatinib derivates. More than 1029 protein were quantified, 80 of which had altered levels of expression. Both experiments pointed to changes in the expression of the ATP-dependent RNA helicase DDX3X and of two mitochondrial coiled-coil-helix-coiled-coil-helix domain-containing proteins.

Proteome studies of bacterial antibiotic resistance mechanisms.

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.

The contribution of proteomics towards deciphering the enigma of Coxiella burnetii.

Coxiella burnetii, the causative agent of Q fever, is an intracellular bacterium and a potential weapon for bioterrorism. The widespread throughout the world, zoonosis is manifested clinically as a self-limited febrile illness, as pneumonia (acute Q fever) or as a chronic illness with endocarditis being its major complication. The recent Netherlands Q fever outbreak has driven the bacterium from a relatively cryptic, underappreciated, "niche" microorganism on the sideline of bacteriology, to one of possibly great impact on public health. Advances in the study of this microorganism proceeded slowly, primarily due to the, until recently, obligatory intracellular nature of the pathogen that in its virulent phase I must be manipulated under biosafety level-3 conditions. Proteomic studies, in particular, have generated a vast amount of information concerning several aspects of the bacterium such as virulence factors, detection/diagnostic and immunogenic biomarkers, inter-/intraspecies variation, resistance to antibiotics, and secreted effector proteins with significant clinical impact. The phenomenon observed following the genomics era, that of generation and accumulation of huge amount of data that ultimately end up unexploited on several databases, begins to emerge in the proteomics field as well. This review will focus on the advances in the field of C. burnetii proteomics through MS, attempting in parallel to utilize some of the proteomics findings by suggesting future directions for the improvement of Q fever diagnosis and therapy.

Study of the whole cell lysate of two Coxiella burnetii strains using N-terminomics.

The etiological agent of Q fever is Coxiella burnetii , an obligate intracellular Gram-negative bacterium and the only bacterium known to date that survives and replicates within a vacuole of phagolysosomal characteristics. In humans, Q fever is characterized by a wide spectrum of clinical manifestations. Of note is that genetic diversity among C. burnetii strains has been reported. To further investigate C. burnetii's diversity, but now at the proteome level, we compared the proteomes of whole cell lysates from two reference strains, Nine Mile and Q212. Proteomes were isolated from each strain and subjected MS-driven combined fractional diagonal chromatography (COFRADIC), a peptide-centered proteomics technique, with a total of 322 proteins that were unambiguously identified. On the basis of their identified neo-N-terminal peptides that are highly likely generated upon in vivo processing by proteases, the most proteolytical sensitive proteins in these strains were identified, and a consensus cleavage pattern was obtained. Further, with the use of differential proteomics based on the here-identified N-terminal peptides, 44 proteins were found to be differentially expressed between the two C. burnetii strains, representing 13.6% of the here-identified C. burnetii proteome. Among these proteins, 10 proteins were found uniquely expressed in the NM strain including proteins with unknown functions as well as housekeeping enzymes, suggesting that strain-related proteins might be present among such uncharacterized proteins.

Quantitative proteome profiling of C. burnetii under tetracycline stress conditions.

The recommended antibiotic regimen against Coxiella burnetii, the etiological agent of Q fever, is based on a semi-synthetic, second-generation tetracycline, doxycycline. Here, we report on the comparison of the proteomes of a C. burnetii reference strain either cultured under control conditions or under tetracycline stress conditions. Using the MS-driven combined fractional diagonal chromatography proteomics technique, out of the 531 proteins identified, 5 and 19 proteins were found significantly up- and down-regulated respectively, under tetracycline stress. Although the predicted cellular functions of these regulated proteins did not point to known tetracycline resistance mechanisms, our data clearly reveal the plasticity of the proteome of C. burnetii to battle tetracycline stress. Finally, we raise several plausible hypotheses that could further lead to more focused experiments on studying tetracycline resistance in C. burnetii and thus reduced treatment failures of Q fever.

Investigation of rifampicin resistance mechanisms in Brucella abortus using MS-driven comparative proteomics.

Mutations in the rpoB gene have already been shown to contribute to rifampicin resistance in many bacterial strains including Brucella species. Resistance against this antibiotic easily occurs and resistant strains have already been detected in human samples. We here present the first research project that combines proteomic, genomic, and microbiological analysis to investigate rifampicin resistance in an in vitro developed rifampicin resistant strain of Brucella abortus 2308. In silico analysis of the rpoB gene was performed and several antibiotics used in the therapy of Brucellosis were used for cross resistance testing. The proteomic profiles were examined and compared using MS-driven comparative proteomics. The resistant strain contained an already described mutation in the rpoB gene, V154F. A correlation between rifampicin resistance and reduced susceptibility on trimethoprim/sulfamethoxazole was detected by E-test and supported by the proteomics results. Using 12 836 MS/MS spectra we identified 6753 peptides corresponding to 456 proteins. The resistant strain presented 39 differentially regulated proteins most of which are involved in various metabolic pathways. Results from our research suggest that rifampicin resistance in Brucella mostly involves mutations in the rpoB gene, excitation of several metabolic processes, and perhaps the use of the already existing secretion mechanisms at a more efficient level.

Proteome profiling of the green sulfur bacterium Chlorobaculum tepidum by N-terminal proteomics.

In this study, we performed the first large-scale identification of N-terminal peptides from the green sulfur bacterium Chlorobaculum tepidum. Combined fractional diagonal chromatography (COFRADIC) was used to isolate protein N-terminal peptides from three different proteome preparations, and following LC-MS/MS analysis, over 621 different proteins were identified by their N-terminal peptides. Our data constitute the largest data set currently available for protein N-termini of prokaryotic photosynthetic organisms.

Unraveling persistent host cell infection with Coxiella burnetii by quantitative proteomics.

The interaction between the immune system and invading bacteria is sufficient to eradicate microorganisms for the majority of bacterial infections, but suppression of the microbicidal response leads to reactivation or chronic evolution of infections and to bacterial persistence. To identify the cellular pathways affected by bacterial persistence, we applied the MS-driven combined fractional diagonal chromatography (COFRADIC) proteomics technique for a comparative study of protein expression in the C. burnetii strains Nine Mile (NM) and its respective strain (NMper) isolated from 18 months persistently infected cell cultures. In total, three different proteome comparisons were performed with the total bacterial proteome, potentially secreted bacterial proteins, and the eukaryotic infected proteome being assessed. Our results revealed that among the 547 identified bacterial proteins, 53 had significantly altered levels of expression and indicated potential metabolic differences between the two strains. Regarding differences in the secreted proteins between both strains and different modulation of the host cell, machineries reflect at least large rearrangements of both bacterial and eukaryotic proteomes during the persistent model of infection when compared to the acute one, which emphasizes that C. burnetii orchestrates a vast number of different bacterial and eukaryotic host cell processes to persist within its host.