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Indole and its derivatives are widespread across different life forms, functioning as signalling molecules in prokaryotes and with more diverse roles in eukaryotes. A majority of indoles found in the environment are attributed to bacterial enzymes converting tryptophan into indole and its derivatives. The involvement of indoles among lower organisms as an interspecies and intraspecies signal is well known, with many reports showing that inter-kingdom interactions involving microbial indole compounds are equally important as they influence defence systems and even the behaviour of higher organisms. This review summarizes recent advances in our understanding of the functional properties of indole and indole derivatives in diverse eukaryotes. Furthermore, we discuss current perspectives on the role of microbial indoles in human diseases such as diabetes, obesity, atherosclerosis, and cancers. Deciphering the function of indoles as biomarkers of metabolic state will facilitate the formulation of diet-based treatments and open unique therapeutic opportunities.

Summary Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P. acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6‐DMP, and non‐conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well‐studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties., Prokaryotic laccases are emergent biocatalysts. However, they have not been broadly found and characterized in bacterial organisms, especially in lactic acid bacteria. Recently, a prokaryotic laccase from the lactic acid bacterium Pediococcus acidilactici 5930, which can degrade biogenic amines, was discovered. Thus, our study aimed to shed light on laccases from lactic acid bacteria focusing on two Pediococcus laccases, P . acidilactici 5930 and Pediococcus pentosaceus 4816, which have provided valuable information on their biochemical activities on redox mediators and biogenic amines. Both laccases are able to oxidize canonical substrates as ABTS, ferrocyanide and 2,6‐DMP, and non‐conventional substrates as biogenic amines. With ABTS as a substrate, they prefer an acidic environment and show sigmoidal kinetic activity, and are rather thermostable. Moreover, this study has provided the first structural view of two lactic acid bacteria laccases, revealing new structural features not seen before in other well‐studied laccases, but which seem characteristic for this group of bacteria. We believe that understanding the role of laccases in lactic acid bacteria will have an impact on their biotechnological applications and provide a framework for the development of engineered lactic acid bacteria with enhanced properties.

Lateral gene transfer (LGT) occurs in a broad range of prokaryotes and eukaryotes, occasionally promoting adaptation. LGT of functional nuclear genes has been reported among some plants, but systematic studies are needed to assess the frequency and facilitators of LGT. We scanned the genomes of a diverse set of 17 grass species that span more than 50 Ma of divergence and include major crops to identify grass-to-grass protein-coding LGT. We identified LGTs in 13 species, with significant variation in the amount each received. Rhizomatous species acquired statistically more genes, probably because this growth habit boosts opportunities for transfer into the germline. In addition, the amount of LGT increases with phylogenetic relatedness, which might reflect genomic compatibility among close relatives facilitating successful transfers. However, genetic exchanges among highly divergent species indicates that transfers can occur across almost the entire family. Overall, we showed that LGT is a widespread phenomenon in grasses that has moved functional genes across the grass family into domesticated and wild species alike. Successful LGTs appear to increase with both opportunity and compatibility.

Liquid-liquid phase separation (LLPS) forms biomolecular condensates or coacervates in cells. Metabolic enzymes can form phase-separated subcellular compartments that enrich enzymes, cofactors, and substrates. Herein, we report the construction of synthetic multienzyme condensates that catalyze the biosynthesis of a terpene, α-farnesene, in the prokaryote E. coli. RGGRGG derived from LAF-1 was used as the scaffold protein to form the condensates by LLPS. Multienzyme condensates were then formed by assembling two enzymes Idi and IspA through an RIAD/RIDD interaction. Multienzyme condensates constructed inside E. coli cells compartmentalized the cytosolic space into regions of high and low enzyme density and led to a significant enhancement of α-farnesene production. This work demonstrates LLPS-driven compartmentalization of the cytosolic space of prokaryotic cells, condensation of a biosynthetic pathway, and enhancement of the biosynthesis of α-farnesene.

S‐layers are paracrystalline proteinaceous lattices that surround prokaryotic cells, forming a critical interface between the cells and their extracellular environment. Here, we report the discovery of a novel S‐layer protein present in the Gram‐negative marine organism, Pseudoalteromonas tunicata D2. An uncharacterized protein (EAR28894) was identified as the most abundant protein in planktonic cultures and biofilms. Bioinformatic methods predicted a beta‐helical structure for EAR28894 similar to the Caulobacter S‐layer protein, RsaA, despite sharing less than 20% sequence identity. Transmission electron microscopy revealed that purified EAR28894 protein assembled into paracrystalline sheets with a unique square lattice symmetry and a unit cell spacing of ~9.1 nm. An S‐layer was found surrounding the outer membrane in wild‐type cells and completely removed from cells in an EAR28894 deletion mutant. S‐layer material also appeared to be “shed” from wild‐type cells and was highly abundant in the extracellular matrix where it is associated with outer membrane vesicles and other matrix components. EAR28894 and its homologs form a new family of S‐layer proteins that are widely distributed in Gammaproteobacteria including species of Pseudoalteromonas and Vibrio, and found exclusively in marine metagenomes. We propose the name Slr4 for this novel protein family., The S‐layer is the outermost protein layer surrounding bacterial cells. We discovered a new family of S‐layer proteins in marine bacteria that forms a paracrystalline square lattice surrounding cells. In addition, the S‐layer was found surrounding extracellular components in biofilms, suggesting that it may provide physical protection not only to cells but also to extracellular structures.

Phosphorylation at aspartic acid residues represents an abundant and critical post-translational modification (PTM) in prokaryotes. In contrast to most characterized PTMs, such as phosphorylation at serine or threonine, the phosphoaspartate moiety is intrinsically labile, and therefore incompatible with common proteomic profiling methods. Herein, we report a nucleophilic, desthiobiotin-containing hydroxylamine (DBHA) chemical probe that covalently labels modified aspartic acid residues in native proteomes. DBHA treatment coupled with LC-MS/MS analysis enabled detection of known phosphoaspartate modifications, as well as novel aspartic acid sites in the E. coli proteome. Coupled with isotopic labelling, DBHA-dependent proteomic profiling also permitted global quantification of changes in endogenous protein modification status, as demonstrated with the detection of increased E. coli OmpR phosphorylation, but not abundance, in response to changes in osmolarity.

Phycobiliproteins (PBPs) are colored fluorescent proteins present in cyanobacteria, red alga, and cryptophyta. These proteins have many potential uses in biotechnology going from food colorants to medical applications. Allophycocyanin, the simplest PBP, is a heterodimer of αβ subunits that oligomerizes as a trimer (αβ)3. Each subunit contains a phycocyanobilin, bound to a cysteine residue, which is responsible for its spectroscopic properties. In this article, we are reporting the expression of recombinant allophycocyanin (rAPC) from the eukaryotic red algae Agarophyton chilensis in Escherichia coli, using prokaryotic accessory enzymes to obtain a fully functional rAPC. Three duet vectors were used to include coding sequences of α and β subunits from A. chilensis and accessorial enzymes (heterodimeric lyase cpc S/U, heme oxygenase 1, phycocyanobilin oxidoreductase) from cyanobacteria Arthrospira maxima. rAPC was purified using several chromatographic steps. The characterization of the pure rAPC indicates very similar spectroscopic properties, λmax Abs, λmax Em, fluorescence lifetime, and chromophorylation degree, with native allophycocyanin (nAPC) from A. chilensis. This method, to produce high‐quality recombinant allophycocyanin, can be used to express and characterize other macroalga phycobiliproteins, to be used for biotechnological or biomedical purposes., A heterologous expression of Allophycocyanin from A.chilensis in E.coli was successfully accomplished. The enzymes for the synthesis of the holo‐APC were obtained from Arthrospira maxima, and they were active to introduce phycocyanobilin to Allophycocyanin. The spectroscopic and biochemical characterization of the protein confirmed a trimeric recombinant Allophycocyanin. The trimeric recombinant APC produced was completely functional, with high quality and it showed similar properties with native APC.

Fermentation, also known as aging, is vital for enhancing the quality of flue‐cured tobacco leaves (FTLs). Aged FTLs demonstrate high‐quality sensory characteristics, while unaged FTLs do not. Microbes play important roles in the FTL fermentation process. However, the eukaryotic microbial community diversity is poorly understood, as are microbial associations within FTLs. We aimed to characterize and compare the microbiota associated with two important categories, fresh and strong flavor style FTLs, and to reveal correlations between the microbial taxa within them. Based on 16S and 18S rRNA Illumina MiSeq sequencing, the community richness and diversity of prokaryotes were almost as high as that of eukaryotes. The dominant microbes of FTLs belonged to seven genera, including Pseudomonas, Bacillus, Methylobacterium, Acinetobacter, Sphingomonas, Neophaeosphaeria, and Cladosporium, of the Proteobacteria, Firmicutes, and Ascomycota phyla. According to partial least square discriminant analysis (PLS‐DA), Xanthomonas, Franconibacter, Massilia, Quadrisphaera, Staphylococcus, Cladosporium, Lodderomyces, Symmetrospora, Golovinomyces, and Dioszegia were significantly positively correlated with fresh flavor style FTLs, while Xenophilus, Fusarium, unclassified Ustilaginaceae, Tilletiopsis, Cryphonectria, Colletotrichum, and Cyanodermella were significantly positively correlated with strong flavor style FTLs. Network analysis identified seven hubs, Aureimonas, Kocuria, Massilia, Brachybacterium, Clostridium, Dietzia, and Vishniacozyma, that may play important roles in FTL ecosystem stability, which may be destroyed by Myrmecridium. FTL microbiota was found to be correlated with flavor style. Species present in lower numbers than the dominant microbes might be used as microbial markers to discriminate different flavor style samples and to stabilize FTL microbial communities. This research advances our understanding of FTL microbiota and describes a means of discriminating between fresh and strong flavor FTLs based on their respective stable microbiota., In this study, we use 16S and 18S rRNA Illumina MiSeq sequencing data to characterize and compare the microbiota associated with fresh and strong flavor style flue‐cured tobacco leaves and reveal correlations between the microbial taxa within them. Flue‐cured tobacco leaf microbiota are correlated with flavor style, and species present at low numbers within the microbial communities can be used as microbial markers to discriminate between samples of different flavor styles and to stabilize flue‐cured tobacco leaf microbial communities.

Metazoans evolved from a single protist lineage. While all eukaryotes share a conserved actin and tubulin-based cytoskeleton, it is commonly perceived that intermediate filaments (IFs), including lamin, vimentin or keratin among many others, are restricted to metazoans. Actin and tubulin proteins are conserved enough to be detectable across all eukaryotic genomes using standard phylogenetic methods, but IF proteins, in contrast, are notoriously difficult to identify by such means. Since the 1950s, dozens of cytoskeletal proteins in protists have been identified that seemingly do not belong to any of the IF families described for metazoans, yet, from a structural and functional perspective fit criteria that define metazoan IF proteins. Here, we briefly review IF protein discovery in metazoans and the implications this had for the definition of this protein family. We argue that the many cytoskeletal and filament-forming proteins of protists should be incorporated into a more comprehensive picture of IF evolution by aligning it with the recent identification of lamins across the phylogenetic diversity of eukaryotic supergroups. This then brings forth the question of how the diversity of IF proteins has unfolded. The evolution of IF proteins likely represents an example of convergent evolution, which, in combination with the speed with which these cytoskeletal proteins are evolving, generated their current diversity. IF proteins did not first emerge in metazoa, but in protists. Only the emergence of cytosolic IF proteins that appear to stem from a nuclear lamin is unique to animals and coincided with the emergence of true animal multicellularity.

SELEX (Systematic Evolution of Ligands by EXponential enrichment) has been widely used for the generation of aptamers against target proteins. However, its requirement for pure target proteins remains a major problem in aptamer selection, as procedures for protein purification from crude bio-samples are not only complicated but also time and labor consuming. This is because native proteins can be found in a large number of diverse forms because of posttranslational modifications and their complicated molecular conformations. Moreover, several proteins are difficult to purify owing to their chemical fragility and/or rarity in native samples. An alternative route is the use of recombinant proteins for aptamer selection, because they are homogenous and easily purified. However, aptamers generated against recombinant proteins produced in prokaryotic cells may not interact with the same proteins expressed in eukaryotic cells because of posttranslational modifications. Moreover, to date recombinant proteins have been constructed for only a fraction of proteins expressed in the human body. Therefore, the demand for advanced SELEX methods not relying on complicated purification processes from native samples or recombinant proteins is growing. This review article describes several such techniques that allow researchers to directly develop an aptamer from various unpurified samples, such as whole cells, tissues, serum, and cell lysates. The key advantages of advanced SELEX are that it does not require a purification process from a crude bio-sample, maintains the functional states of target proteins, and facilitates the development of aptamers against unidentified and uncharacterized proteins in unpurified biological samples.

Ferritin is considered the major iron storage protein which maintains a large iron core in its cavity and has ferroxidase activity. There are many types of ferritin particularly in prokaryotes that include the canonical 24-mer FTN molecules, the heme-containing BFR, the smaller 12-mer DPS and the newly recognized EncFtn of encapsulin that forms a very large iron storage compartment. Recent studies show that ferritin function is more dynamic than previous depicted and new mechanisms of ferritin iron recycling are emerging. They participate to the regulation of cellular iron homeostasis as those of ferritin biosynthesis, cooperating also with the iron-dependent mechanism of cellular iron secretion. Some of these basic processes are in common between unicellular and animal cells, and this review aims at discussing the findings on the connections between iron storage, cellular iron regulation and ferritin iron recycling that have been explored in unicellular organisms and in animals. © 2017 IUBMB Life, 69(6):414-422, 2017.

Since their discovery, small regulatory RNAs (sRNAs) were thought to be regulated exclusively at the transcriptional level. However, accumulating data from recent reports indicate that posttranscriptional signals can also modulate the function and stability of sRNAs. One of these posttranscriptional signals are competing endogenous RNAs (ceRNAs). Commonly called RNA sponges, ceRNAs can effectively sequester sRNAs and prevent them from binding their cognate target messenger RNAs (mRNAs). Subsequently, they prevent sRNA-dependent regulation of translation and stability of mRNA targets. While some ceRNAs seem to be expressed constitutively, others are intricately regulated according to environmental conditions. The outcome of ceRNA binding to a sRNA reaches beyond simple sequestration. Various effects observed on sRNA functions extend from reducing transcriptional noise to promote RNA turnover. Here, we present a historical perspective of the discovery of ceRNAs in eukaryotic organisms and mainly focus on the synthesis and function of select, well-described, ceRNAs in bacterial cells. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions Translation > Translation Regulation RNA Turnover and Surveillance > Regulation of RNA Stability.

M24B peptidases cleaving Xaa-Pro bond in dipeptides are prolidases whereas those cleaving this bond in longer peptides are aminopeptidases-P. Bacteria have small aminopeptidases-P (36-39 kDa), which are diverged from canonical aminopeptidase-P of Escherichia coli (50 kDa). Structure-function studies of small aminopeptidases-P are lacking. We report crystal structures of small aminopeptidases-P from E. coli and Deinococcus radiodurans, and report substrate-specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are aminopeptidases-P, structurally close to small prolidases except for absence of dipeptide-selectivity loop. We noticed absence of this loop and conserved arginine in canonical archaeal prolidase (Maher et al., Biochemistry. 43, 2004, 2771-2783) and questioned its classification. Our enzymatic assays show that this enzyme is an aminopeptidase-P. Further, our mutagenesis studies illuminate importance of DXRY sequence motif in bacterial small aminopeptidases-P and suggest common evolutionary origin with human XPNPEP1/XPNPEP2. Our analyses reveal sequence/structural features distinguishing small aminopeptidases-P from other M24B peptidases.

The identification of individual eukaryotic and prokaryotic cells is the backbone of clinical pathology and provides crucial information about the genesis and progression of a disease. While most commonly fluorescent-label based methods are applied, label-free methods, such as Raman spectroscopy, are elegant alternatives. A major disadvantage of Raman spectroscopy is the low signal yield resulting in long acquisition times, making it impractical for high-throughput clinical analysis. As a rule, Raman-based cell identification relies on high-resolution Raman spectra. This comes at a cost of detected Raman photons. In this letter we show that while the proper biochemical characterization of cells requires high-resolution Raman spectra, the proper classification of cells does not. By varying the slit-width between 50 µm and 500 µm it is possible to show that detected Raman signal from eukaryotic cells increased up to seven-fold. Raman-based cell classification was performed on three cancer cell lines: Jurkat, MiaPaca2, and Capan1, at three different resolutions 8 cm-1 , 24 cm-1 , and 48 cm-1 . Moreover, we have simulated the resolution decrease due to low-diffraction gratings by binning neighboring pixels together. In both cases the cells were well classifiable using support vectors machine (SVM). For anyone working in the field of Raman spectroscopy this picture of Sir C.V. Raman is recognizable, even with reduced spatial resolution. Raman spectra of eukaryotic cells can also be recognized even with six fold reduced spectral resolution.

CRISPR-Cas are self-/nonself-discriminating systems found in prokaryotic cells. They represent a remarkable example of molecular memory that is hereditarily transmitted. Their discovery can be considered as one of the first fruits of the systematic exploration of prokaryotic genomes. Although this genomic feature was serendipitously discovered in molecular biology studies, it was the availability of multiple complete genomes that shed light about their role as a genetic immune system. Here we tell the story of how this discovery originated and was slowly and painstakingly advanced to the point of understating the biological role of what initially was just an odd genomic feature. FJMM is funded by the Spanish Ministerio de Economía y Competitividad (BIO2014-53029P) and the European Commission/Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (291815 Era-Net ANIHWA). FR-V is funded by projects MEDIMAX BFPU2013-48007-P from the Spanish Ministerio de Economía y Competitividad, MaCuMBA Project 311975 of the European Commission FP7 and PROMETEO II/2014/012 project AQUAMET from the Generalitat Valenciana.

From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell-free systems are of enormous interest. The modification of the genetic code to incorporate non-canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell-free projects. Many sophisticated cell-free systems for manifold applications have been established. This review describes the recent advances in cell-free protein synthesis and details the expanding applications in this field.

Classical zinc finger (ZF) domains were thought to be confined to the eukaryotic kingdom until the transcriptional regulator Ros protein was identified in Agrobacterium tumefaciens. The Ros Cys2His2 ZF binds DNA in a peculiar mode and folds in a domain significantly larger than its eukaryotic counterpart consisting of 58 amino acids (the 9-66 region) arranged in a βββαα topology, and stabilized by a conserved, extensive, 15-residue hydrophobic core. The prokaryotic ZF domain, then, shows some intriguing new features that make it interestingly different from its eukaryotic counterpart. This review will focus on the prokaryotic ZFs, summarizing and discussing differences and analogies with the eukaryotic domains and providing important insights into their structure/function relationships. The prokaryotic domain, found for the first time in the protein Ros from A. tumefaciens, shows some interesting structural and functional features that differentiate it from its eukaryotic counterpart. The review summarizes and discusses differences and analogies with the eukaryotic domains providing important insights into their structure/function relationships.

Summary SecReT6 (http://db-mml.sjtu.edu.cn/SecReT6/) is an integrated database providing comprehensive information on type VI secretion systems (T6SSs) in bacteria. T6SSs are a class of sophisticated cell contact-dependent apparatuses involved in mediating antagonistic or synergistic communications between bacteria and/or bacteria and eukaryotes. These apparatuses have recently been found to be widely distributed among Gram-negative bacterial species. SecReT6 offers a unique, readily explorable archive of known and putative T6SSs, and cognate effectors found in bacteria. It currently contains data on 11 167 core T6SS components mapping to 906 T6SSs found in 498 bacterial strains representing 240 species, as well as a collection of over 600 directly relevant references. Also collated and archived were 1340 diverse candidate secreted effectors which were experimentally shown and/or predicted to be delivered by T6SSs into target eukaryotic and/or prokaryotic cells as well as 196 immunity proteins. A broad range of T6SS gene cluster detection and comparative analysis tools are readily accessible via SecReT6, which may aid identification of effectors and immunity proteins around the T6SS core components. This database will be regularly updated to ensure its ongoing maximal utility and relevance to the scientific research community.

This work was supported by a college studentship of Queen Mary University of London, and the College Central Research Fund. Work in Seville was supported by Grant No. BFU2011-22762 from Plan Nacional de Investigacion, Spain, co-financed by FEDER.

DNA sequencing of ancient permafrost samples can be used to reconstruct past plant, animal and bacterial communities. In this study, we assess the small-scale reproducibility of taxonomic composition obtained from sequencing four molecular markers (mitochondrial 12S ribosomal DNA (rDNA), prokaryote 16S rDNA, mitochondrial cox1 and chloroplast trnL intron) from two soil cores sampled 10 cm apart. In addition, sequenced control reactions were used to produce a contaminant library that was used to filter similar sequences from sample libraries. Contaminant filtering resulted in the removal of 1% of reads or 0.3% of operational taxonomic units. We found similar richness, overlap, abundance and taxonomic diversity from the 12S, 16S and trnL markers from each soil core. Jaccard dissimilarity across the two soil cores was highest for metazoan taxa detected by the 12S and cox1 markers. Taxonomic community distances were similar for each marker across the two soil cores when the chi-squared metric was used; however, the 12S and cox1 markers did not cluster well when the Goodall similarity metric was used. A comparison of plant macrofossil vs. read abundance corroborates previous work that suggests eastern Beringia was dominated by grasses and forbs during cold stages of the Pleistocene, a habitat that is restricted to isolated sites in the present-day Yukon.

During cellular stress response, a widespread inhibition of transcription and blockade of splicing and other post-transcriptional processing is detected, while certain specific genes are induced. In particular, free-living cells constantly monitor temperature. When the thermal condition changes, they activate a set of genes coding for proteins that participate in the response. Non-coding RNAs, ncRNAs, and conformational changes in specific regions of mRNAs seem also to be crucial regulators that enable the cell to adjust its physiology to environmental changes. They exert their effects following the same principles in all organisms and may affect all steps of gene expression. These ncRNAs and structural elements as related to thermal stress response in bacteria are reviewed. The resemblances to eukaryotic ncRNAs are highlighted.

The detection of recombinant human growth hormone (rhGH) doping using the World Anti-Doping Agency (WADA) approved kits is reported in this research. Twenty-five young male students were selected and divided randomly into two groups with six belonging to the placebo and nineteen to the administration group. Thirteen volunteers in one group were administered with a Chinese preparation of rhGH while six volunteers included in the other group were given rhGH made in Switzerland. Both preparations were administered at a dose of 0.1 IU/kg body weight, one injection per day for 14 consecutive days. Blood samples were collected using WADA guidelines and all blood samples were analyzed with WADA-approved Kits 1 and 2. The time window for detection of rhGH doping using WADA-approved kits and criteria are discussed. Based on the comparison of the data obtained from this excretion study and from our routine (Chinese population as reference), consideration of the recent WADA criteria for rhGH AAF (Analytical Adverse Findings) is reported statistically. A comparison of data obtained from the two sample groups administered with pharmaceutical preparations, one Chinese rhGH (GenHeal®, S19990019, 1.6 mg (4 IU), Shanghai, China) obtained from prokaryotic cells and the other (Saizen®, S20080036, 1.33 mg (4 IU), Laboratoires Serone S.A., Switzerland) from eukaryotic cells is reported and did not show any significant difference for the detection of doping with rhGH. Copyright © 2011 John Wiley & Sons, Ltd.

The evolutionary success of an organism is a testament to its inherent capacity to keep pace with environmental conditions that change over short and long periods. Mechanisms underlying adaptive processes are being investigated with renewed interest and excitement. This revival is partly fueled by powerful technologies that can probe molecular phenomena at a systems scale. Such studies provide spectacular insight into the mechanisms of adaptation, including rewiring of regulatory networks via natural selection of horizontal gene transfers, gene duplication, deletion, readjustment of kinetic parameters, and myriad other genetic reorganizational events. Here, we will discuss advances in prokaryotic systems biology from the perspective of evolutionary principles that have shaped regulatory networks for dynamic adaptation to environmental change.

Cyclization via head-to-tail linkage of the termini of a peptide chain occurs in only a small percentage of proteins, but engenders the resultant cyclic proteins with exceptional stability. The mechanisms involved are poorly understood and this review attempts to summarize what is known of the events that lead to cyclization. Cyclic proteins are found in both prokaryotic and eukaryotic species. The prokaryotic circular proteins include the bacteriocins and pilins. The eukaryotic circular proteins in mammals include the theta defensins, found in rhesus macaques, and the retrocyclins. Two types of cyclic proteins have been found in plants, the sunflower trypsin inhibitor and the larger, more prolific, group known as cyclotides. The cyclotides from Oldenlandia affinis, the plant in which these cyclotides were first discovered, are processed by an asparaginyl endopeptidase which is a cysteine protease. Cysteine proteases are commonly associated with transpeptidation reactions, which, for suitable substrates can lead to cyclization events. These proteases cleave an amide bond and form an acyl enzyme intermediate before nucleophilic attack by the amine group of the N-terminal residue to form a peptide bond, resulting in a cyclic peptide. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 573–583, 2010.

Prokaryotic genomes of endosymbionts and parasites are examples of naturally evolved minimal cells, the study of which can shed light on life in its minimum form. Their diverse biology, their lack of a large set of orthologous genes and the existence of essential linage (and environmentally) specific genes all illustrate the diversity of genes building up naturally evolved minimal cells. This conclusion is reinforced by the fact that sometimes the same essential function is performed by genes from different evolutionary origins. Nevertheless, all cells perform a set of life-essential functions however different their cell machinery and environment in which they thrive. An upcoming challenge for biologists will be to discern, by studying differences and similarities in current biodiversity, how cells with reduced genomes have adapted while retaining all basic life-supporting functions.

Because over expression of Hsp70 molecular chaperones suppresses the toxicity of aberrantly folded proteins that occur in Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis, and various polyQ-diseases (Huntington's disease and ataxias), Hsp70 is garnering attention as a possible therapeutic agent for these various diseases. Here, I review progress in this fascinating field of molecular chaperones and neurodegeneration and describe our current understanding of the mechanisms by which Hsp70 protects cells from the PD-related protein called alpha-synuclein (alpha-syn).

We report the expression profile of acyl-lipid Delta12-desaturase (desA) gene from Synechocystis sp. PCC6803 and its effect on cell membrane lipid composition and cold tolerance in prokaryotic (Escherichia coli) and eukaryotic (Solanum tuberosum) cells. For this purpose, a hybrid of desA and reporter gene encoding thermostable lichenase (licBM3) was constructed and used to transform these cells. The expression of this hybrid gene was measured using qualitative (Petri dish test, electrophoregram and zymogram) and quantitative methods (spectrometry and gas liquid chromatography assays). The maximum level of linoleic acid in the bacterial cells containing hybrid gene was 1.9% of total fatty acids. Cold stress tolerance assays using plant damage index and growth parameters showed that cold tolerance was enhanced in primary transgenic lines because of increased unsaturated fatty acid concentration in their lipids. The greatest content of 18:2 and 18:3 fatty acids in primary transgenic plants was observed for lines 2 (73%) and 3 (41%). Finally, our results showed that desaturase could enhance tolerance to cold stress in potato, and desaturase and lichenase retain their functionality in the structure of the hybrid protein where the enzymatic activity of target gene product was higher than in the case of reporter lichenase gene absence in the construction.

Between prokaryotic cells and eukaryotic cells there is dramatic difference in complexity which represents a problem for the current version of the cell theory, as well as for the current version of evolution theory. In the past few decades, the serial endosymbiotic theory of Lynn Margulis has been confirmed. This results in a radical departure from our understanding of living systems: the eukaryotic cell represents de facto"cells-within-cell." Higher order "cells-within-cell" situations are obvious at the eukaryotic cell level in the form of secondary and tertiary endosymbiosis, or in the male and female gametophytes of higher plants. The next challenge of the current version of the cell theory is represented by the fact that the multicellular fungi and plants are, in fact, supracellular assemblies as their cells are not physically separated from each other. Moreover, there are also examples of alliances and mergings between multicellular organisms. Infection, especially the viral one, but also bacterial and fungal infections, followed by symbiosis, is proposed to act as the major force that drives the biological evolution toward higher complexity.

Understanding how the gulf between prokaryotic and eukaryotic cellular design arose is a major challenge. The viral eukaryogenesis (VE) hypothesis addresses the challenge of eukaryotic origins by suggesting the first eukaryotic cell was a multimember consortium consisting of a viral ancestor of the nucleus, an archaeal ancestor of the eukaryotic cytoplasm, and a bacterial ancestor of the mitochondria. Using only prokaryotes and their viruses, and invoking selective pressures observed in modern organisms, the VE hypothesis can explain the origins of the eukaryotic cell, sex, and meiosis. In the VE hypothesis, a cell wall-less archaeon and an alpha-proteobacterium established a syntrophic relationship, and then a complex DNA virus permanently lysogenized the archaeal syntroph to produce a consortium of three organisms that evolved into the eukaryotic cell. The mechanisms by which the virus replicated, controlled its copy number, and segregated to daughter cells led to the evolution of the asexual mitotic replication cycle and the sexual meiotic replication cycle. The VE hypothesis conceptually unifies prokaryotic and eukaryotic sex into variants of a single process.

Mutation and lateral transfer are two categories of processes generating genetic diversity in prokaryotic genomes. Their relative importance varies between lineages, yet both are complementary rather than independent, separable evolutionary forces. The replication process inevitably merges together their effects on the genome. We develop the concept of "open lineages" to characterize evolutionary lineages that over time accumulate more changes in their genomes by lateral transfer than by mutation. They contrast with "closed lineages," in which most of the changes are caused by mutation. Open and closed lineages are interspersed along the branches of any tree of prokaryotes. This patchy distribution conflicts with the basic assumptions of traditional phylogenetic approaches. As a result, a tree representation including both open and closed lineages is a misrepresentation. The evolution of all prokaryotic lineages cannot be studied under a single model unless new phylogenetic approaches that are more pluralistic about lineage evolution are designed.

Three different classes of thiol-oxidoreductases that facilitate the formation of protein disulfide bonds have been identified. They are the Ero1 and SOX/ALR family members in eukaryotic cells, and the DsbB family members in prokaryotic cells. These enzymes transfer oxidizing potential to the proteins PDI or DsbA, which are responsible for directly introducing disulfide bonds into substrate proteins during oxidative protein folding in eukaryotes and prokaryotes, respectively. A comparison of the recent X-ray crystal structure of Ero1 with the previously solved structure of the SOX/ALR family member Erv2 reveals that, despite a lack of primary sequence homology between Ero1 and Erv2, the core catalytic domains of these two proteins share a remarkable structural similarity. Our search of the DsbB protein sequence for features found in the Ero1 and Erv2 structures leads us to propose that, in a fascinating example of structural convergence, the catalytic core of this integral membrane protein may resemble the soluble catalytic domain of Ero1 and Erv2. Our analysis of DsbB also identified two new groups of DsbB proteins that, based on sequence homology, may also possess a catalytic core similar in structure to the catalytic domains of Ero1 and Erv2.

In this article, we present the cloning of Hscm1, a gene for chorismate mutase (CM) from the beet cyst nematode Heterodera schachtii. CM is a key branch-point enzyme of the shikimate pathway, and secondary metabolites that arise from this pathway control developmental programmes and defence responses of the plant. By manipulating the plant's endogenous shikimate pathway, the nematode can influence the plant physiology for its own benefit. Hscm1 is a member of the CM gene family and is expressed during the pre-parasitic and parasitic stages of the nematode's life cycle. In situ mRNA hybridization reveals an expression pattern specific to the subventral and dorsal pharyngeal glands. The predicted protein has a signal peptide for secretion in addition to two domains. The N-terminal domain of the mature protein, which is only found in cyst nematodes, contains six conserved cysteine residues, which may reflect the importance of disulphide bond formation for protein stabilization. The C-terminal domain holds a single catalytic site and has similarity to secreted CMs of pathogenic bacteria, classifying HsCM1 as an AroQ(gamma) enzyme. The presumed catalytic residues are discussed in detail, and genetic complementation experiments indicate that the C-terminal domain is essential for enzyme activity. Finally, we show how the modular design of the protein is mirrored in the genomic sequence by the intron/exon organization, suggesting exon shuffling as a mechanism for the evolutionary assembly of this protein.

Suppression of viral infection by RNA in a nucleotide sequence homology-dependent manner was first reported in plants in early 1990s. Studies in the past 15 years have established a completely new RNA-based immune system against viruses that is mechanistically Riverside, CA, USA. related to RNA silencing or RNA interference (RNAi). This viral immunity begins with recognition of viral double-stranded or structured RNA by the Dicer nuclease family of host immune receptors. In fungi, plants and invertebrates, the viral RNA trigger is processed into small interfering RNAs (siRNAs) to direct specific silencing of the homologous viral genomic and/or messenger RNAs by an RNaseH-like Argonaute protein. Deep sequencing of virus-derived siRNAs indicates that the immunity against viruses with a positive-strand RNA genome is induced by Dicer recognition of dsRNA formed during the initiation of viral progeny (+)RNA synthesis. The RNA-based immune pathway in these organisms overlaps the canonical dsRNA-siRNA pathway of RNAi and may require amplification of viral siRNAs by host RNA-dependent RNA polymerase in plants and nematodes. Production of virus-derived small RNAs is undetectable in mammalian cells infected with RNA viruses. However, infection of mammals with several nucleus-replicating DNA viruses induces production of virus-derived microRNAs capable of silencing host and viral mRNAs as found for viral siRNAs. Remarkably, recent studies indicate that prokaryotes also produce virus-derived small RNAs known as CRISPR RNAs to guide antiviral defense in a manner that has yet to be defined. In this article, we review the recent progress on the identification and mechanism of the key components including viral sensors, viral triggers, effectors, and amplifiers, of the small RNA-directed viral immunity. We also highlight some of the many unresolved questions.

In spite of numerous publications on potential diagnostic application of circulating DNA and transrenal nucleic acid (Tr-NA) analysis, few, if any, tests based on this technology are available in clinical labs. This delay in test development and implementation is caused, at least in part, by the deficit in robust methods for isolation of short nucleic acid fragments from bodily fluids, as well as in techniques for analyzing these fragments. We have developed a new anion exchanger-based method for the isolation of cell-free nucleic acid fragments from large volumes of bodily fluids, and analyzed these fragments by PCR techniques specially designed to amplify "ultrashort" templates. The combination of these two techniques not only revealed the presence in urine of 10-150 bases or bp DNA and RNA fragments in addition to previously observed 150-200-bp DNA fragments and high molecular weight DNA, but also significantly increased the sensitivity of Tr-DNA detection. Additionally, we detected in urine a variety of miRNAs, including those excreted transrenally, thereby opening new diagnostic possibilities for Tr-NA analysis.

Summary Experimental and computational studies show that tran-scriptional regulatory networks in both eukaryotic andprokaryotic microbes have a large amount of variationeven between closely related species. While the architec-ture of bacterial genomes is unexpectedly diverse, evenwithin short evolutionary scales, the variations in eithergene content or gene order must be tolerated by andintegrated into the regulatory networks. These are intrin-sically unstable because mutation and lateral gene trans-fer introduce modifications in the set of potential bindingsites of the regulatory proteins within each genome, andbecause regulatory mutations are tolerated better thanmutations in structural genes. The intrinsically lower sta-bility of the regulatory networks, together with the extraor-dinary ability of genetic programs to adapt to changes ingenome contents and gene organization, provide a fasttrack to evolutionary divergence and the generation ofbiodiversity in the microbial world. Acknowledgements

Selenium (Se) can provide unique biochemical and biological functions, and properties to macromolecules, including protein and RNA. Although Se has not yet been found in DNA, identification of the presence of Se in natural tRNAs has led to discovery of the naturally occurring 2-selenouridine and 5-[(methylamino)methyl]-2-selenouridine (mnm(5)se(2)U). The Se-atoms at C(2) of the modified uridines are introduced by 2-selenouridine synthase via displacement of the S-atoms in the corresponding 2-thiouridine nucleotides of the tRNAs, and selenophosphate is used as the Se donor. The research indicated that mnm(5)se(2)U is located at the first or wobble position of the anticodons in several bacterial tRNAs, including tRNA(Lys), tRNA(Glu), and tRNA(Gln). The 2-seleno functionality on this modified nucleotide probably improves the translation accuracy and/or efficiency. These observations in vivo suggest that the presence of Se can provide natural RNAs with useful properties to better function and survival. To further investigate the biochemical and structural properties of Se-derivatized nucleic acids (SeNA), we have pioneered chemical and enzymatic synthesis of Se-derivatized nucleic acids, and introduced Se into both RNA and DNA at a variety of positions by atom-specific replacement of oxygen. This review outlines the recent advancements in chemical and biochemical syntheses, and studies of SeNAs, and their potential applications in structural and functional investigation of nucleic acids and their protein complexes.

Synonymous codon usage analysis between thermophilic and mesophilic prokaryotes has gained wide attention in recent years. Although it is known that thermophilic and mesophilic prokaryotes use different subset of synonymous codons, no reason for this difference is known so far. In the present communication, by analyzing a large number of thermophilic and mesophilic prokaryotes, we provide evidence that bias in the selection of synonymous codons between thermophilic and mesophilic prokaryotes is related to differential folding pattern of mRNA secondary structures. Moreover, we observe that error-minimizing property has significant influence in differentiating the synonymous codon usage between thermophilic and mesophilic prokaryotes. Biological implications of these results are discussed.

MS combined with database searching has become the preferred method for identifying proteins present in cell or tissue samples. The technique enables us to execute large-scale proteome analyses of species whose genomes have already been sequenced. Searching mass spectrometric data against protein databases composed of annotated genes has been widely conducted. However, there are some issues with this technique; wrong annotations in protein databases cause deterioration in the accuracy of protein identification, and only proteins that have already been annotated can be identified. We propose a new framework that can detect correct ORFs by integrating an MS/MS proteomic data mapping and a knowledge-based system regarding the translation initiation sites. This technique can provide correction of predicted coding sequences, together with the possibility of identifying novel genes. We have developed a computational system; it should first conduct the probabilistic peptide-matching against all possible translational frames using MS/MS data, then search for discriminative DNA patterns around the detected peptides, and lastly integrate the facts using empirical knowledge stored in knowledge bases to obtain correct ORFs. We used photosynthetic bacteria Synechocystis sp. PCC6803 as a sample prokaryote, resulting in the finding of 14 N-terminus annotation errors and several new candidate genes.

Background At present, identification of antibodies against high-frequency antigens is limited to reference laboratories having panels of rare red blood cell (RBC) specimens in stock. Antibodies against Lu(b) are among the most frequent clinically relevant antibody specificities directed against high-frequency antigens. Study design and methods Soluble recombinant Lu(b) fusion proteins consisting of the first three N-terminal immunoglobulin superfamily domains and a V5-His tag were generated. Eukaryotic recombinant Lu(b) proteins were isolated from cell culture supernatant of stably transfected HEK293 cells with anti-V5 Sepharose. Prokaryotic Lu(b) fusion proteins were expressed in Escherichia coli, purified by Ni-NTA, and refolded by chromatographic procedures. Ten anti-Lu(b) serum samples, 6 anti-Lu(a) serum samples, 30 serum samples directed against other blood group antigens, 10 serum samples from patients with RBC autoantibodies, and 100 serum samples from randomly selected donors were used for antibody screening. Results Eukaryotic and prokaryotic recombinant Lu(b) proteins proved to be equally suited for identification of anti-Lu(b). Recombinant Lu(b) protein-based enzyme-linked immunosorbent assay correctly identified samples containing anti-Lu(b) sera, and the titers were at least two times higher than those measured by the gel agglutination-based indirect antiglobulin test. In hemagglutination inhibition assays, recombinant Lu(b) protein neutralized all anti-Lu(b), but none of the other alloantibodies decreased in reactivity. Conclusion Antibody detection systems based on soluble eukaryotic or prokaryotic recombinant blood group proteins have the potential to replace current systems with rare RBCs for identification of alloantibodies against high- or low-frequency antigens. This innovation could bring routine laboratories one step closer to specialized antibody diagnostics.

The use of multidimensional capillary HPLC combined with tandem mass spectrometry has allowed high qualitative and quantitative proteome coverage of prokaryotic organisms. The determination of protein abundance change between two or more conditions has matured to the point that false discovery rates can be very low and for smaller proteomes coverage is sufficiently high to explicitly consider false negative error. Selected aspects of using these methods for global protein abundance assessments are reviewed. These include instrumental issues that influence the reliability of abundance ratios; a comparison of sources of non-linearity, errors, and data compression in proteomics and spotted cDNA arrays; strengths and weaknesses of spectral counting versus stable isotope metabolic labeling; and a survey of microbiological applications of global abundance analysis at the protein level. Proteomic results for two organisms that have been studied extensively using these methods are reviewed in greater detail. Spectral counting and metabolic labeling data are compared and the utility of proteomics for global gene regulation studies are discussed for the methanogenic Archaeon Methanococcus maripaludis. The oral pathogen Porphyromonas gingivalis is discussed as an example of an organism where a large percentage of the proteome differs in relative abundance between the intracellular and extracellular phenotype.

Motifs that are evolutionarily conserved in proteins are crucial to their structure and function. In one of our earlier studies, we demonstrated that the conserved motifs occurring invariantly across several organisms could act as structural determinants of the proteins. We observed the abundance of glycyl residues in these invariantly conserved motifs. The role of glycyl residues in highly conserved motifs has not been studied extensively. Thus, it would be interesting to examine the structural perturbations induced by mutation in these conserved glycyl sites. In this work, we selected a representative set of invariant signature (IS) peptides for which both the PDB structure and mutation information was available. We thoroughly analyzed the conformational features of the glycyl sites and their local interactions with the surrounding residues. Using Ramachandran angles, we showed that the glycyl residues occurring in these IS peptides, which have undergone mutation, occurred more often in the L-disallowed as compared with the L-allowed region of the Ramachandran plot. Short range contacts around the mutation site were analyzed to study the steric effects. With the results obtained from our analysis, we hypothesize that any change of activity arising because of such mutations must be attributed to the long-range interaction(s) of the new residue if the glycyl residue in the IS peptide occurred in the L-allowed region of the Ramachandran plot. However, the mutation of those conserved glycyl residues that occurred in the L-disallowed region of the Ramachandran plot might lead to an altered activity of the protein as a result of an altered conformation of the backbone in the immediate vicinity of the glycyl residue, in addition to long range effects arising from the long side chains of the new residue. Thus, the loss of activity because of mutation in the conserved glycyl site might either relate to long range interactions or to local perturbations around the site depending upon the conformational preference of the glycyl residue.

The activity of transcription factors is usually governed by allosteric physicochemical signals or metabolites, which are in turn produced in the cell or obtained from the environment by the activity of the products of effector genes. Previously, we identified a collection of more than 110 transcription factors and their corresponding effector genes in Escherichia coli K-12. Here, we introduce the notion of “triferog”, which relates to the identification of orthologous transcription factors and effector genes across genomes and show that transcriptional sensing systems known in E. coli are poorly conserved beyond Salmonella. We also find that enzymes that act as effector genes for the production of endogenous effector metabolites are more conserved than their corresponding effector genes encoding for transport and two-component systems for sensing exogenous signals. Finally, we observe that on an evolutionary scale enzymes are more conserved than their respective TFs, suggesting a homogenous cellular metabolism across genomes and the conservation of transcriptional control of critical cellular processes like DNA replication by a common endogenous signal. We hypothesize that extensive variation in the domain architecture of TFs and changes in endogenous conditions at large phylogenetic distances could be the major contributing factors for the observed differential conservation of TFs and their corresponding effector genes encoding for enzymes, causing variations in transcriptional responses across organisms.

Intraflagellar transport (IFT) is essential machinery for biogenesis and maintenance of cilia in many eukaryotic and prokaryotic cells. A large number of polypeptides are known to be involved in IFT, but the physiological role of each component is not fully elucidated. Here, we identified a C. elegans orthologue of a Chlamydomonas reinhardtii IFT component, IFT-81, and found that its loss-of-function mutants show an unusual behavioral property and small body size. IFT-81 is expressed in sensory neurons, and localized at the base of cilia. The similar phenotypes with ift-81 mutants were also observed in several IFT mutants, suggesting these defects are caused by inability of IFT. We also demonstrated that IFT-81 interacts and co-localizes with IFT-74, which is another putative component of IFT. The ift-74 loss-of-function mutants showed phenocopies with ift-81 mutants, suggesting IFT-81 and IFT-74 play comparable functions. Moreover, ift-81 and ift-74 mutants similarly exhibited weak anomalies in cilia formation and obvious disruptions of transport in mature cilia. Thus, we conclude that IFT-81 and IFT-74 coordinately act in IFT in C. elegans sensory cilia.

The review focuses attention on the different kinds of equipment which are used to grow large quantities of the two types of animal cells; the monolayer or surface-dependent cell and the suspension or surface-independent cell. The methodologies used to obtain reliable performance are described as are some examples of the application of microprocessors to monitor and control operational parameters. The kinds of products which are currently obtained from animal cells are listed and the future potential of genetically engineered animal and prokaryotic cells is discussed.

Summary Bdellovibrio -and-like organisms (BALOs) are peculiar, ubiquitous, small-sized, highly motile Gram-negative bacteria that are obligatory predators of other bacte- ria. Typically, these predators invade the periplasm of their prey where they grow and replicate. To date, BALOs constitute two highly diverse families affili- ated with the d - proteobacteria class. In this study, Micavibrio spp., a BALO lineage of epibiotic preda- tors, were isolated from soil. These bacteria attach to digest and grow at the expense of other prokaryotes, much like other BALOs. Multiple phylogenetic analy- ses based on six genes revealed that they formed a deep branch within the a - proteobacteria , not affiliated with any of the a -proteobacterial orders. The presence of BALOs deep among the a - proteobacteria suggests that their peculiar mode of parasitism maybe an ancestral character in this proteobacterial class. The origin of the mitochondrion from an a - proteobacte- rium endosymbiont is strongly supported by molecu- lar phylogenies. Accumulating data suggest that the endosymbiont's host was also a prokaryote. As prokaryotes are unable to phagocytose, the means by which the endosymbiont gained access into its host remains mysterious. We here propose a scenario based on the BALO feeding-mode to hypothesize a mechanism at play at the origin of the mitochondrial endosymbiosis.

Almost all aspects of protein traffic in bacteria were covered at the ASM-FEMS meeting on the topic in Iraklio, Crete in May 2006. The studies presented ranged from mechanistic analysis of specific events leading proteins to their final destinations to the physiological roles of the targeted proteins. Among the highlights from the meeting that are reviewed here are the molecular dynamics of SecA protein, membrane protein insertion, type III secretion needles and chaperones, type IV secretion, the two partner and autosecretion systems, the 'secretion competent state', and the recently discovered type VI secretion system.