Application of coincidence index in the discovery of co-expressed metabolic pathways.

Analyzing transcription data requires intensive statistical analysis to obtain useful biological information and knowledge. A significant portion of this data is affected by random noise or even noise intrinsic to the modeling of the experiment. Without robust treatment, the data might not be explored thoroughly, and incorrect conclusions could be drawn. Examining the correlation between gene expression profiles is one way bioinformaticians extract information from transcriptomic experiments. However, the correlation measurements traditionally used have worrisome shortcomings that need to be addressed. This paper compares five already published and experimented-with correlation measurements to the newly developed coincidence index, a similarity measurement that combines Jaccard and interiority indexes and generalizes them to be applied to vectors containing real values. We used microarray and RNA-Seq data from the archaeonHalobacterium salinarumand the bacteriumEscherichia coli, respectively, to evaluate the capacity of each correlation/similarity measurement. The utilized method explores the co-expressed metabolic pathways by measuring the correlations between the expression levels of enzymes that share metabolites, represented in the form of a weighted graph. It then searches for local maxima in this graph using a simulated annealing algorithm. We demonstrate that the coincidence index extracts larger, more comprehensive, and more statistically significant pathways for microarray experiments. In RNA-Seq experiments, the results are more limited, but the coincidence index managed the largest percentage of significant components in the graph.

Halobacterium salinarum: Life with more than a grain of salt.

Halobacterium salinarum is a halophilic (salt-loving) archaeon that grows in salt concentrations near or at saturation. Although isolated from salted fish a century ago, it was the 1971 discovery of bacteriorhodopsin, the light-driven proton pump, that raised interest in Hbt. salinarum across a range of disciplines, including biophysics, chemistry, molecular evolution and biotechnology. Hbt. salinarum have since contributed to numerous discoveries, such as advances in membrane protein structure determination and the first example of a non-eukaryal glycoprotein. Work on Hbt. salinarum, one of the species used to define Archaea, has also elucidated molecular workings in the third domain. Finally, Hbt. salinarum presents creative solutions to the challenges of life in high salt.

Revisiting N-glycosylation in Halobacterium salinarum: Characterizing a dolichol phosphate- and glycoprotein-bound tetrasaccharide.

Although Halobacterium salinarum provided the first example of N-glycosylation outside the Eukarya, much regarding such post-translational modification in this halophilic archaea remains either unclear or unknown. The composition of an N-linked glycan decorating both the S-layer glycoprotein and archaellins offers one such example. Originally described some 40 years ago, reports from that time on have presented conflicted findings regarding the composition of this glycan, as well as differences between the protein-bound glycan and that version of the glycan attached to the lipid upon which it is assembled. To clarify these points, liquid chromatography-electrospray ionization mass spectrometry was employed here to revisit the composition of this glycan both when attached to selected asparagine residues of target proteins and when bound to the lipid dolichol phosphate upon which the glycan is assembled. Such efforts revealed the N-linked glycan as corresponding to a tetrasaccharide comprising a hexose, a sulfated hexuronic acid, a hexuronic acid and a second sulfated hexuronic acid. When attached to dolichol phosphate but not to proteins, the same tetrasaccharide is methylated on the final sugar. Moreover, in the absence of the oligosaccharyltransferase AglB, there is an accumulation of the dolichol phosphate-linked methylated and disulfated tetrasaccharide. Knowing the composition of this glycan at both the lipid- and protein-bound stages, together with the availability of gene deletion approaches for manipulating Hbt. salinarum, will allow delineation of the N-glycosylation pathway in this organism.

Structure and RNA-Binding Properties of Lsm Protein from Halobacterium salinarum.

The structure and the RNA-binding properties of the Lsm protein from Halobacterium salinarum have been determined. A distinctive feature of this protein is the presence of a short L4 loop connecting the ß3 and ß4 strands. Since bacterial Lsm proteins (also called Hfq proteins) have a short L4 loop and form hexamers, whereas archaeal Lsm proteins (SmAP) have a long L4 loop and form heptamers, it has been suggested that the length of the L4 loop may affect the quaternary structure of Lsm proteins. Moreover, the L4 loop covers the region of SmAP corresponding to one of the RNA-binding sites in Hfq, and thus can affect the RNA-binding properties of the protein. Our results show that the SmAP from H. salinarum forms heptamers and possesses the same RNA-binding properties as homologous proteins with the long L4 loop. Therefore, the length of the L4 does not govern the number of monomers in the protein particles and does not affect the RNA-binding properties of Lsm proteins.

Resistance of a Halobacterium salinarum isolate from a solar saltern to cadmium, lead, nickel, zinc, and copper.

The current study focuses on the tolerance of a strain of Halobacterium salinarum isolated from Sfax solar saltern (Tunisia) towards cadmium (Cd), lead (Pb), nickel (Ni), zinc (Zn), and copper (Cu) by using agar dilution methods in complex and minimal media. The results showed the least inhibitory metals based on Minimum Inhibitory Concentrations (MICs) were lead (MIC = 4.5 mM), cadmium (MIC = 4 mM), and nickel (MIC = 2.5 mM) in complex medium. The MICs of these metals were more inhibitory (MIC < 2 mM) in the other tested media. The archaeal strain revealed a high sensitivity for copper and zinc, with MICs below 0.5 mM for both metals. Growth kinetics in complex and minimal media showed the strain to be more sensitive to the metals in liquid media than in solid media. The growth kinetic assays indicated the presence of selected heavy metals resulted in a lower growth rate and lower total cell mass relative to the control. Despite that cadmium and lead are nonessential and have no nutrient value, they were the most tolerated metals by H. salinarum strain. In addition, pigment intensity in the strain was inhibited by the presence of the heavy metals relative to the control.

In silico and experimental improvement of bacteriorhodopsin production in Halobacterium salinarum R1 by increasing DNA-binding affinity of Bat through Q661R/Q665R substitutions in HTH motif.

DNA-binding motif of bacterioopsin activator (Bat) protein is a Helix-Turn-Helix motif, which binds to bop promoter and induces bacterioopsin (Bop) expression under light and low oxygen tension. Bacterioopsin is linked to retinal to produce bacteriorhodopsin (BR), which in turn supplies energy source in Halobacterium salinarum. In this study, effect of Bat HTH motif-promoter DNA interaction on bacterioopsin (Bop) expression was investigated using in silico and experimental approaches. Molecular docking showed that the most stable DNA-protein complex was generated by Q661R/Q665R mutant. Based on the in silico analysis, HTH motif was mutated using site-directed mutagenesis and Hbt. salinarum recombinant strains were developed by introduction of mutant bat genes. Double positively charged amino acid substitutions (Q661R/Q665R) in second helix of HTH motif increased whereas deletion of this region decreased BR production. However, other single substitutions (Q665R and Q661H) did not change BR production. These findings represent key role of HTH motif stability for DNA binding and regulation of bacterioopsin (Bop) expression and bacteriorhodopsin (BR) production independent of environmental condition.

Halobacterium salinarum storage and rehydration after spray drying and optimization of the processes for preservation of carotenoids.

Spray drying is appropriate for the preservation of halophilic microorganisms due to the nature of these microorganisms, as they survive in adverse environmental conditions by being encapsulated in salt crystals. Artificial neural networks were in this study used to optimize practically significant spray-drying regimes of the C50-carotenoids producer Halobacterium salinarum. Immediately after drying, the samples contained up to 54% halobacterial biomass and less than 5% moisture, and the level of preservation of carotenoids was 95-97%. The storage of biomass at 4 °C resulted in the gradual degradation of the carotenoids, which reached 58-64% in the best samples after 1 year. A comprehensive study of changes in halobacteria biomass after spray drying and the nature of the damage provided new data on the survival and preservation of cells and biologically active substances in the various spray-drying regimes and at different storage times.

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea.

Prokaryotic genomes show a high level of information compaction often with different molecules transcribed from the same locus. Although antisense RNAs have been relatively well studied, RNAs in the same strand, internal RNAs (intraRNAs), are still poorly understood. The question of how common is the translation of overlapping reading frames remains open. We address this question in the model archaeon Halobacterium salinarum. In the present work we used differential RNA-seq (dRNA-seq) in H. salinarum NRC-1 to locate intraRNA signals in subsets of internal transcription start sites (iTSS) and establish the open reading frames associated to them (intraORFs). Using C-terminally flagged proteins, we experimentally observed isoforms accurately predicted by intraRNA translation for kef1, acs3 and orc4 genes. We also recovered from the literature and mass spectrometry databases several instances of protein isoforms consistent with intraRNA translation such as the gas vesicle protein gene gvpC1. We found evidence for intraRNAs in horizontally transferred genes such as the chaperone dnaK and the aerobic respiration related cydA in both H. salinarum and Escherichia coli. Also, intraRNA translation evidence in H. salinarum, E. coli and yeast of a universal elongation factor (aEF-2, fusA and eEF-2) suggests that this is an ancient phenomenon present in all domains of life.

Cryo-electron microscopy of an extremely halophilic microbe: technical aspects.

Most halophilic Archaea of the class Halobacteriaceae depend on the presence of several molar sodium chloride for growth and cell integrity. This poses problems for structural studies, particularly for electron microscopy, where the high salt concentration results in diminished contrast. Since cryo-electron microscopy of intact cells provides new insights into the cellular and molecular organization under close-to-live conditions, we evaluated strategies and conditions to make halophilic microbes available for investigations in situ. Halobacterium salinarum, the test organism for this study, usually grows at 4.3 M NaCl. Adaptation to lower concentrations and subsequent NaCl reduction via dialysis led to still vital cells at 3 M salt. A comprehensive evaluation of vitrification parameters, thinning of frozen cells by focused-ion-beam micromachining, and cryo-electron microscopy revealed that structural studies under high salt conditions are possible in situ.

Impact of Protease on Ultraviolet Photodissociation Mass Spectrometry for Bottom-up Proteomics.

Recent mass spectrometric studies have reported enhanced proteome coverage by employing multiple proteases or by using multiple or alternative activation methods such as electron-transfer dissociation in combination with collisional-activated dissociation (CAD). In this study the use of 193 nm ultraviolet photodissociation for the analysis of thousands of Halobacterium salinarum peptides generated by four proteases (trypsin, LysC, GluC, and chymotrypsin) was evaluated in comparison with higher energy CAD (HCD). Proteins digested by trypsin resulted in greater sequence coverage for HCD over UVPD. LysC digestion resulted in similar sequence coverages for UVPD and HCD; however, for proteins digested by GluC and chymotrypsin 5-10% more sequence coverage on average was achieved by UVPD. HCD resulted in more peptide identifications (at 1% false discovery rate) for trypsin (4356 peptides by HCD versus 3907 peptides by UVPD), whereas UVPD identified greater numbers of peptides for LysC digests (1033 peptides by UVPD versus 844 HCD), chymotrypsin digests (3219 peptides for UVPD versus 2921 for HCD), and GluC digests (2834 peptides for UVPD and 2393 for HCD) and correspondingly greater numbers of proteins.

Solid-state fermentation as a potential technique for esterase/lipase production by halophilic archaea.

Halophilic archaea are extremophiles, adapted to high-salt environments, showing a big biotechnological potential as enzyme, lipids and pigments producers. Four inert supports (perlite, vermiculite, polyurethane foam and glass fiber) were employed for solid-state fermentation (SSF) of the halophilic archaeon Natronococcus sp. TC6 to investigate biomass and esterase production. A very low esterase activity and high water activity were observed when perlite, vermiculite and polyurethane were used as supports. When glass fiber was employed, an important moisture loss was observed (8.6%). Moreover, moisture retention was improved by mixing polyurethane and glass fiber, resulting in maximal biomass and esterase production. Three halophilic archaea: Natronococcus sp. TC6, Halobacterium sp. NRC-1 and Haloarcula marismortui were cultured by submerged fermentation (SmF) and by SSF; an improvement of 1.3- to 6.2-fold was observed in the biomass and esterase production when SSF was used. Growth was not homogeneous in the mixture, but was predominant in the glass fiber thus was probably because the glass fiber provides a holder to the cells, while the polyurethane acts as an impregnation medium reservoir. To the best of our knowledge, this work is the first report on haloarchaea cultivation by SSF aiming biomass and esterase/lipase activity production.

Similar mutation rates but different mutation spectra in moderate and extremely halophilic archaea.

Archaea are a major part of Earth's microbiota and extremely diverse. Yet, we know very little about the process of mutation that drives such diversification. To expand beyond previous work with the moderate halophilic archaeal species Haloferax volcanii, we performed a mutation-accumulation experiment followed by whole-genome sequencing in the extremely halophilic archaeon Halobacterium salinarum. Although Hfx. volcanii and Hbt. salinarum have different salt requirements, both species have highly polyploid genomes and similar GC content. We accumulated mutations for an average of 1250 generations in 67 mutation accumulation lines of Hbt. salinarum, and revealed 84 single-base substitutions and 10 insertion-deletion mutations. The estimated base-substitution mutation rate of 3.99 × 10-10 per site per generation or 1.0 × 10-3 per genome per generation in Hbt. salinarum is similar to that reported for Hfx. volcanii (1.2 × 10-3 per genome per generation), but the genome-wide insertion-deletion rate and spectrum of mutations are somewhat dissimilar in these archaeal species. The spectra of spontaneous mutations were AT biased in both archaea, but they differed in significant ways that may be related to differences in the fidelity of DNA replication/repair mechanisms or a simple result of the different salt concentrations.

Agl28 and Agl29 are key components of a Halobacterium salinarum N-glycosylation pathway.

Although Halobacterim salinarum provided the first example of N-glycosylation outside the Eukarya, only recently has attention focused on delineating the pathway responsible for the assembly of the N-linked tetrasaccharide decorating selected proteins in this haloarchaeon. In the present report, the roles of VNG1053G and VNG1054G, two proteins encoded by genes clustered together with a set of genes demonstrated to encode N-glycosylation pathway components, were considered. Relying on both bioinformatics and gene deletion and subsequent mass spectrometry analysis of known N-glycosylated proteins, VNG1053G was determined to be the glycosyltransferase responsible for addition of the linking glucose, while VNG1054G was deemed to be the flippase that translocates the lipid-bound tetrasaccharide across the plasma membrane to face the cell exterior, or to contribute to such activity. As observed with Hbt. salinarum lacking other components of the N-glycosylation machinery, both cell growth and motility were compromised in the absence of VNG1053G or VNG1054G. Thus, given their demonstrated roles in Hbt. salinarum N-glycosylation, VNG1053G and VNG1054G were re-annotated as Agl28 and Agl29, according to the nomenclature used to define archaeal N-glycosylation pathway components.

Comparative Genomics of Halobacterium salinarum Strains Isolated from Salted Foods Reveals Protechnological Genes for Food Applications.

Archaeal cell factories are becoming of great interest given their ability to produce a broad range of value-added compounds. Moreover, the Archaea domain often includes extremophilic microorganisms, facilitating their cultivation at the industrial level under nonsterile conditions. Halophilic archaea are studied for their ability to grow in environments with high NaCl concentrations. In this study, nine strains of Halobacterium salinarum were isolated from three different types of salted food, sausage casings, salted codfish, and bacon, and their genomes were sequenced along with the genome of the collection strain CECT 395. A comparative genomic analysis was performed on these newly sequenced genomes and the publicly available ones for a total of 19 H. salinarum strains. We elucidated the presence of unique gene clusters of the species in relation to the different ecological niches of isolation (salted foods, animal hides, and solar saltern sediments). Moreover, genome mining at the single-strain level highlighted the metabolic potential of H. salinarum UC4242, which revealed the presence of different protechnological genes (vitamins and myo-inositol biosynthetic pathways, aroma- and texture-related features, and antimicrobial compounds). Despite the presence of genes of potential concern (e.g., those involved in biogenic amine production), all the food isolates presented archaeocin-related genes (halocin-C8 and sactipeptides).

Direct Observation of Archaellar Motor Rotation by Single-Molecular Imaging Techniques.

Single-molecular techniques have characterized dynamics of molecular motors such as flagellum in bacteria and myosin, kinesin, and dynein in eukaryotes. We can apply these techniques to a motility machine of archaea, namely, the archaellum, composed of a thin helical filament and a rotary motor. Although the size of the motor hinders the characterization of its motor function under a conventional optical microscope, fluorescence-labeling techniques allow us to visualize the architecture and function of the archaellar filaments in real time. Furthermore, a tiny polystyrene bead attached to the filament enables the visualization of motor rotation through the bead rotation and quantification of biophysical properties such as speed and torque produced by the rotary motor imbedded in the cell membrane. In this chapter, I describe the details of the above biophysical method based on an optical microscope.

One Advantage of Being Polyploid: Prokaryotes of Various Phylogenetic Groups Can Grow in the Absence of an Environmental Phosphate Source at the Expense of Their High Genome Copy Numbers.

Genomic DNA has high phosphate content; therefore, monoploid prokaryotes need an external phosphate source or an internal phosphate storage polymer for replication and cell division. For two polyploid prokaryotic species, the halophilic archaeon Haloferax volcanii and the cyanobacterium Synechocystis PCC 6803, it has been reported that they can grow in the absence of an external phosphate source by reducing the genome copy number per cell. To unravel whether this feature might be widespread in and typical for polyploid prokaryotes, three additional polyploid prokaryotic species were analyzed in the present study, i.e., the alphaproteobacterium Zymomonas mobilis, the gammaproteobacterium Azotobacter vinelandii, and the haloarchaeon Halobacterium salinarum. Polyploid cultures were incubated in the presence and in the absence of external phosphate, growth was recorded, and genome copy numbers per cell were quantified. Limited growth in the absence of phosphate was observed for all three species. Phosphate was added to phosphate-starved cultures to verify that the cells were still viable and growth-competent. Remarkably, stationary-phase cells grown in the absence or presence of phosphate did not become monoploid but stayed oligoploid with about five genome copies per cell. As a negative control, it was shown that monoploid Escherichia coli cultures did not exhibit any growth in the absence of phosphate. Taken together, all five polyploid prokaryotic species that have been characterized until now can grow in the absence of environmental phosphate by reducing their genome copy numbers, indicating that cell proliferation outperforms other evolutionary advantages of polyploidy.

Recent Advances in the Study of Gas Vesicle Proteins and Application of Gas Vesicles in Biomedical Research.

The formation of gas vesicles has been investigated in bacteria and haloarchaea for more than 50 years. These air-filled nanostructures allow cells to stay at a certain height optimal for growth in their watery environment. Several gvp genes are involved and have been studied in Halobacterium salinarum, cyanobacteria, Bacillus megaterium, and Serratia sp. ATCC39006 in more detail. GvpA and GvpC form the gas vesicle shell, and additional Gvp are required as minor structural proteins, chaperones, an ATP-hydrolyzing enzyme, or as gene regulators. We analyzed the Gvp proteins of Hbt. salinarum with respect to their protein-protein interactions, and developed a model for the formation of these nanostructures. Gas vesicles are also used in biomedical research. Since they scatter waves and produce ultrasound contrast, they could serve as novel contrast agent for ultrasound or magnetic resonance imaging. Additionally, gas vesicles were engineered as acoustic biosensors to determine enzyme activities in cells. These applications are based on modifications of the surface protein GvpC that alter the mechanical properties of the gas vesicles. In addition, gas vesicles have been decorated with GvpC proteins fused to peptides of bacterial or viral pathogens and are used as tools for vaccine development.

Low Salt Influences Archaellum-Based Motility, Glycerol Metabolism, and Gas Vesicles Biogenesis in Halobacterium salinarum.

Halobacterium salinarum NRC-1 is an extremophile that grows optimally at 4.3 M NaCl concentration. In spite of being an established model microorganism for the archaea domain, direct comparisons between its proteome and transcriptome during osmotic stress are still not available. Through RNA-seq-based transcriptomics, we compared a low salt (2.6 M NaCl) stress condition with 4.3 M of NaCl and found 283 differentially expressed loci. The more commonly found classes of genes were: ABC-type transporters and transcription factors. Similarities, and most importantly, differences between our findings and previously published datasets in similar experimental conditions are discussed. We validated three important biological processes differentially expressed: gas vesicles production (due to down-regulation of gvpA1b, gvpC1b, gvpN1b, and gvpO1b); archaellum formation (due to down-regulation of arlI, arlB1, arlB2, and arlB3); and glycerol metabolism (due to up-regulation of glpA1, glpB, and glpC). Direct comparison between transcriptomics and proteomics showed 58% agreement between mRNA and protein level changes, pointing to post-transcriptional regulation candidates. From those genes, we highlight rpl15e, encoding for the 50S ribosomal protein L15e, for which we hypothesize an ionic strength-dependent conformational change that guides post-transcriptional processing of its mRNA and, thus, possible salt-dependent regulation of the translation machinery.

Identifying Components of a Halobacterium salinarum N-Glycosylation Pathway.

Whereas N-glycosylation is a seemingly universal process in Archaea, pathways of N-glycosylation have only been experimentally verified in a mere handful of species. Toward expanding the number of delineated archaeal N-glycosylation pathways, the involvement of the putative Halobacterium salinarum glycosyltransferases VNG1067G, VNG1066C, and VNG1062G in the assembly of an N-linked tetrasaccharide decorating glycoproteins in this species was addressed. Following deletion of each encoding gene, the impact on N-glycosylation of the S-layer glycoprotein and archaellins, major glycoproteins in this organism, was assessed by mass spectrometry. Likewise, the pool of dolichol phosphate, the lipid upon which this glycan is assembled, was also considered in each deletion strain. Finally, the impacts of such deletions were characterized in a series of biochemical, structural and physiological assays. The results revealed that VNG1067G, VNG1066C, and VNG1062G, renamed Agl25, Agl26, and Agl27 according to the nomenclature used for archaeal N-glycosylation pathway components, are responsible for adding the second, third and fourth sugars of the N-linked tetrasaccharide decorating Hbt. salinarum glycoproteins. Moreover, this study demonstrated how compromised N-glycosylation affects various facets of Hbt. salinarum cell behavior, including the transcription of archaellin-encoding genes.

Halobacterium salinarum and Haloferax volcanii Comparative Transcriptomics Reveals Conserved Transcriptional Processing Sites.

Post-transcriptional processing of messenger RNA is an important regulatory strategy that allows relatively fast responses to changes in environmental conditions. In halophile systems biology, the protein perspective of this problem (i.e., ribonucleases which implement the cleavages) is generally more studied than the RNA perspective (i.e., processing sites). In the present in silico work, we mapped genome-wide transcriptional processing sites (TPS) in two halophilic model organisms, Halobacterium salinarum NRC-1 and Haloferax volcanii DS2. TPS were established by reanalysis of publicly available differential RNA-seq (dRNA-seq) data, searching for non-primary (monophosphorylated RNAs) enrichment. We found 2093 TPS in 43% of H. salinarum genes and 3515 TPS in 49% of H. volcanii chromosomal genes. Of the 244 conserved TPS sites found, the majority were located around start and stop codons of orthologous genes. Specific genes are highlighted when discussing antisense, ribosome and insertion sequence associated TPS. Examples include the cell division gene ftsZ2, whose differential processing signal along growth was detected and correlated with post-transcriptional regulation, and biogenesis of sense overlapping transcripts associated with IS200/IS605. We hereby present the comparative, transcriptomics-based processing site maps with a companion browsing interface.