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15 pages, 2496 KiB

Open AccessArticle

Divergent Roles of RPA Homologs of the Model Archaeon Halobacterium salinarum in Survival of DNA Damage

by Jessica J. Evans, Patrick E. Gygli, Julienne McCaskill and Linda C. DeVeaux

Genes 2018, 9(4), 223; https://doi.org/10.3390/genes9040223 - 20 Apr 2018

Cited by 5 | Viewed by 4912

Abstract

The haloarchaea are unusual in possessing genes for multiple homologs to the ubiquitous single-stranded DNA binding protein (SSB or replication protein A, RPA) found in all three domains of life. Halobacterium salinarum contains five homologs: two are eukaryotic in organization, two are prokaryotic [...] Read more.

The haloarchaea are unusual in possessing genes for multiple homologs to the ubiquitous single-stranded DNA binding protein (SSB or replication protein A, RPA) found in all three domains of life. Halobacterium salinarum contains five homologs: two are eukaryotic in organization, two are prokaryotic and are encoded on the minichromosomes, and one is uniquely euryarchaeal. Radiation-resistant mutants previously isolated show upregulation of one of the eukaryotic-type RPA genes. Here, we have created deletions in the five RPA operons. These deletion mutants were exposed to DNA-damaging conditions: ionizing radiation, UV radiation, and mitomycin C. Deletion of the euryarchaeal homolog, although not lethal as in Haloferax volcanii, causes severe sensitivity to all of these agents. Deletion of the other RPA/SSB homologs imparts a variable sensitivity to these DNA-damaging agents, suggesting that the different RPA homologs have specialized roles depending on the type of genomic insult encountered. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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16 pages, 57895 KiB

Open AccessArticle

Engineering the Salt-Inducible Ectoine Promoter Region of Halomonas elongata for Protein Expression in a Unique Stabilizing Environment

by Lisa M. Stiller, Erwin A. Galinski and Elisabeth M. H. J. Witt

Genes 2018, 9(4), 184; https://doi.org/10.3390/genes9040184 - 28 Mar 2018

Cited by 16 | Viewed by 7696

Abstract

It has been firmly established that organic osmolytes (compatible solutes) of halophilic Bacteria and Archaea have positive effects on conformation and activity of proteins, and may therefore improve their functional production. In particular, the amino acid derivative ectoine is known for its conformational [...] Read more.

It has been firmly established that organic osmolytes (compatible solutes) of halophilic Bacteria and Archaea have positive effects on conformation and activity of proteins, and may therefore improve their functional production. In particular, the amino acid derivative ectoine is known for its conformational stabilization, aggregation suppression, and radical protection properties. The natural producer and industrial production strain Halomonas elongata accumulates ectoine in the cytoplasm, and as a result offers a unique stabilizing environment for recombinant proteins. For the construction of broad hoast range vector systems with fluorescent reporter proteins, we chose the salt-inducible promoter region of the ectoine gene cluster (promA). A closer inspection of the genetic background revealed that its combination of sigma 38 (σ³⁸) and sigma 70 (σ⁷⁰) promoters was followed by a weak ribosomal binding site (RBS). This inspired a systematic approach for the construction of a promA-based vector series with a synthetic RBS region using the RBS Calculator v2.0, which resulted in a greatly improved salt-dependent expression—even in a deletion construct lacking the σ³⁸ promoter. To expand the application range of this expression system, we looked further into the possible export of recombinant proteins into the periplasm. Both sec and tat leader sequences from H. elongata proved to be suitable for directed periplasmic transport into an extreme environment of freely selectable ionic strength. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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17 pages, 2296 KiB

Open AccessArticle

Comparative Analysis of Surface Layer Glycoproteins and Genes Involved in Protein Glycosylation in the Genus Haloferax

by Yarden Shalev, Shannon M. Soucy, R. Thane Papke, J. Peter Gogarten, Jerry Eichler and Uri Gophna

Genes 2018, 9(3), 172; https://doi.org/10.3390/genes9030172 - 20 Mar 2018

Cited by 14 | Viewed by 6612

Abstract

Within the Haloferax genus, both the surface (S)-layer protein, and the glycans that can decorate it, vary between species, which can potentially result in many different surface types, analogous to bacterial serotypes. This variation may mediate phenotypes, such as sensitivity to different viruses [...] Read more.

Within the Haloferax genus, both the surface (S)-layer protein, and the glycans that can decorate it, vary between species, which can potentially result in many different surface types, analogous to bacterial serotypes. This variation may mediate phenotypes, such as sensitivity to different viruses and mating preferences. Here, we describe S-layer glycoproteins found in multiple Haloferax strains and perform comparative genomics analyses of major and alternative glycosylation clusters of isolates from two coastal sites. We analyze the phylogeny of individual glycosylation genes and demonstrate that while the major glycosylation cluster tends to be conserved among closely related strains, the alternative cluster is highly variable. Thus, geographically- and genetically-related strains may exhibit diverse surface structures to such an extent that no two isolates present an identical surface profile. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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22 pages, 3678 KiB

Open AccessArticle

Metagenomic Insights into the Phylogenetic and Metabolic Diversity of the Prokaryotic Community Dwelling in Hypersaline Soils from the Odiel Saltmarshes (SW Spain)

by Blanca Vera-Gargallo and Antonio Ventosa

Genes 2018, 9(3), 152; https://doi.org/10.3390/genes9030152 - 8 Mar 2018

Cited by 49 | Viewed by 7403

Abstract

Hypersaline environments encompass aquatic and terrestrial habitats. While only a limited number of studies on the microbial diversity of saline soils have been carried out, hypersaline lakes and marine salterns have been thoroughly investigated, resulting in an aquatic-biased knowledge about life in hypersaline [...] Read more.

Hypersaline environments encompass aquatic and terrestrial habitats. While only a limited number of studies on the microbial diversity of saline soils have been carried out, hypersaline lakes and marine salterns have been thoroughly investigated, resulting in an aquatic-biased knowledge about life in hypersaline environments. To improve our understanding of the assemblage of microbes thriving in saline soils, we assessed the phylogenetic diversity and metabolic potential of the prokaryotic community of two hypersaline soils (with electrical conductivities of ~24 and 55 dS/m) from the Odiel saltmarshes (Spain) by metagenomics. Comparative analysis of these soil databases with available datasets from salterns ponds allowed further identification of unique and shared traits of microbial communities dwelling in these habitats. Saline soils harbored a more diverse prokaryotic community and, in contrast to their aquatic counterparts, contained sequences related to both known halophiles and groups without known halophilic or halotolerant representatives, which reflects the physical heterogeneity of the soil matrix. Our results suggest that Haloquadratum and certain Balneolaeota members may preferentially thrive in aquatic or terrestrial habitats, respectively, while haloarchaea, nanohaloarchaea and Salinibacter may be similarly adapted to both environments. We reconstructed 4 draft genomes related to Bacteroidetes, Balneolaeota and Halobacteria and appraised their metabolism, osmoadaptation strategies and ecology. This study greatly improves the current understanding of saline soils microbiota. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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24 pages, 9334 KiB

Open AccessArticle

Novel Sequence Features of DNA Repair Genes/Proteins from Deinococcus Species Implicated in Protection from Oxidatively Generated Damage

by F. M. Nazmul Hassan and Radhey S. Gupta

Genes 2018, 9(3), 149; https://doi.org/10.3390/genes9030149 - 8 Mar 2018

Cited by 10 | Viewed by 5969

Abstract

Deinococcus species display a high degree of resistance to radiation and desiccation due to their ability to protect critical proteome from oxidatively generated damage; however, the underlying mechanisms are not understood. Comparative analysis of DNA repair proteins reported here has identified 22 conserved [...] Read more.

Deinococcus species display a high degree of resistance to radiation and desiccation due to their ability to protect critical proteome from oxidatively generated damage; however, the underlying mechanisms are not understood. Comparative analysis of DNA repair proteins reported here has identified 22 conserved signature indels (CSIs) in the proteins UvrA1, UvrC, UvrD, UvsE, MutY, MutM, Nth, RecA, RecD, RecG, RecQ, RecR, RuvC, RadA, PolA, DnaE, LigA, GyrA and GyrB, that are uniquely shared by all/most Deinococcus homologs. Of these CSIs, a 30 amino acid surface-exposed insert in the Deinococcus UvrA1, which distinguishes it from all other UvrA homologs, is of much interest. The uvrA1 gene in Deinococcus also exhibits specific genetic linkage (predicted operonic arrangement) to genes for three other proteins including a novel Deinococcus-specific transmembrane protein (designated dCSP-1) and the proteins DsbA and DsbB, playing central roles in protein disulfide bond formation by oxidation-reduction of CXXC (C represents cysteine, X any other amino acid) motifs. The CXXC motifs provide important targets for oxidation damage and they are present in many DNA repair proteins including five in UvrA, which are part of Zinc-finger elements. A conserved insert specific for Deinococcus is also present in the DsbA protein. Additionally, the uvsE gene in Deinococcus also shows specific linkage to the gene for a membrane-associated protein. To account for these novel observations, a model is proposed where specific interaction of the Deinococcus UvrA1 protein with membrane-bound dCSP-1 enables the UvrA1 to receive electrons from DsbA-DsbB oxido-reductase machinery to ameliorate oxidation damage in the UvrA1 protein. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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13 pages, 4604 KiB

Open AccessArticle

Overview of Oxidative Stress Response Genes in Selected Halophilic Fungi

by Cene Gostinčar and Nina Gunde-Cimerman

Genes 2018, 9(3), 143; https://doi.org/10.3390/genes9030143 - 6 Mar 2018

Cited by 30 | Viewed by 6071

Abstract

Exposure of microorganisms to stress, including to high concentrations of salt, can lead to increased production of reactive oxygen species in the cell. To limit the resulting damage, cells have evolved a variety of antioxidant defenses. The role of these defenses in halotolerance [...] Read more.

Exposure of microorganisms to stress, including to high concentrations of salt, can lead to increased production of reactive oxygen species in the cell. To limit the resulting damage, cells have evolved a variety of antioxidant defenses. The role of these defenses in halotolerance has been proposed before. Whole genome sequencing for some of the most halotolerant and halophilic fungal species has enabled us to investigate the possible links between oxidative and salt stress tolerance on the genomic level. We identified genes involved in oxidative stress response in the halophilic basidiomycete Wallemia ichthyophaga, and halotolerant ascomycetous black yeasts Hortaea werneckii and Aureobasidium pullulans, and compared them to genes from 16 other fungi, both asco- and basidiomycetes. According to our results, W. ichthyophaga can survive salinities detrimental to most other organisms with only a moderate number of oxidative stress response genes. In other investigated species, however, the maximum tolerated salinity correlated with the number of genes encoding three major enzymes of the cellular oxidative stress response: superoxide dismutases, catalases, and peroxiredoxins. This observation supports the hypothetical link between the antioxidant capacity of cells and their halotolerance. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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18 pages, 4254 KiB

Open AccessArticle

The Unexplored Diversity of Pleolipoviruses: The Surprising Case of Two Viruses with Identical Major Structural Modules

by Nina S. Atanasova, Camilla H. Heiniö, Tatiana A. Demina, Dennis H. Bamford and Hanna M. Oksanen

Genes 2018, 9(3), 131; https://doi.org/10.3390/genes9030131 - 28 Feb 2018

Cited by 9 | Viewed by 4632

Abstract

Extremely halophilic Archaea are the only known hosts for pleolipoviruses which are pleomorphic non-lytic viruses resembling cellular membrane vesicles. Recently, pleolipoviruses have been acknowledged by the International Committee on Taxonomy of Viruses (ICTV) as the first virus family that contains related viruses with [...] Read more.

Extremely halophilic Archaea are the only known hosts for pleolipoviruses which are pleomorphic non-lytic viruses resembling cellular membrane vesicles. Recently, pleolipoviruses have been acknowledged by the International Committee on Taxonomy of Viruses (ICTV) as the first virus family that contains related viruses with different DNA genomes. Genomic diversity of pleolipoviruses includes single-stranded and double-stranded DNA molecules and their combinations as linear or circular molecules. To date, only eight viruses belong to the family Pleolipoviridae. In order to obtain more information about the diversity of pleolipoviruses, further isolates are needed. Here we describe the characterization of a new halophilic virus isolate, Haloarcula hispanica pleomorphic virus 4 (HHPV4). All pleolipoviruses and related proviruses contain a conserved core of approximately five genes designating this virus family, but the sequence similarity among different isolates is low. We demonstrate that over half of HHPV4 genome is identical to the genome of pleomorphic virus HHPV3. The genomic regions encoding known virion components are identical between the two viruses, but HHPV4 includes unique genetic elements, e.g., a putative integrase gene. The co-evolution of these two viruses demonstrates the presence of high recombination frequency in halophilic microbiota and can provide new insights considering links between viruses, membrane vesicles, and plasmids. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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23 pages, 5184 KiB

Open AccessArticle

Characterizing the DNA Methyltransferases of Haloferax volcanii via Bioinformatics, Gene Deletion, and SMRT Sequencing

by Matthew Ouellette, J. Peter Gogarten, Jessica Lajoie, Andrea M. Makkay and R. Thane Papke

Genes 2018, 9(3), 129; https://doi.org/10.3390/genes9030129 - 27 Feb 2018

Cited by 18 | Viewed by 7825

Abstract

DNA methyltransferases (MTases), which catalyze the methylation of adenine and cytosine bases in DNA, can occur in bacteria and archaea alongside cognate restriction endonucleases (REases) in restriction-modification (RM) systems or independently as orphan MTases. Although DNA methylation and MTases have been well-characterized in [...] Read more.

DNA methyltransferases (MTases), which catalyze the methylation of adenine and cytosine bases in DNA, can occur in bacteria and archaea alongside cognate restriction endonucleases (REases) in restriction-modification (RM) systems or independently as orphan MTases. Although DNA methylation and MTases have been well-characterized in bacteria, research into archaeal MTases has been limited. A previous study examined the genomic DNA methylation patterns (methylome) of the halophilic archaeon Haloferax volcanii, a model archaeal system which can be easily manipulated in laboratory settings, via single-molecule real-time (SMRT) sequencing and deletion of a putative MTase gene (HVO_A0006). In this follow-up study, we deleted other putative MTase genes in H. volcanii and sequenced the methylomes of the resulting deletion mutants via SMRT sequencing to characterize the genes responsible for DNA methylation. The results indicate that deletion of putative RM genes HVO_0794, HVO_A0006, and HVO_A0237 in a single strain abolished methylation of the sole cytosine motif in the genome (C^m4TAG). Amino acid alignments demonstrated that HVO_0794 shares homology with characterized cytosine CTAG MTases in other organisms, indicating that this MTase is responsible for C^m4TAG methylation in H. volcanii. The CTAG motif has high density at only one of the origins of replication, and there is no relative increase in CTAG motif frequency in the genome of H. volcanii, indicating that CTAG methylation might not have effectively taken over the role of regulating DNA replication and mismatch repair in the organism as previously predicted. Deletion of the putative Type I RM operon rmeRMS (HVO_2269-2271) resulted in abolished methylation of the adenine motif in the genome (GCA^m6BN₆VTGC). Alignments of the MTase (HVO_2270) and site specificity subunit (HVO_2271) demonstrate homology with other characterized Type I MTases and site specificity subunits, indicating that the rmeRMS operon is responsible for adenine methylation in H. volcanii. Together with HVO_0794, these genes appear to be responsible for all detected methylation in H. volcanii, even though other putative MTases (HVO_C0040, HVO_A0079) share homology with characterized MTases in other organisms. We also report the construction of a multi-RM deletion mutant (ΔRM), with multiple RM genes deleted and with no methylation detected via SMRT sequencing, which we anticipate will be useful for future studies on DNA methylation in H. volcanii. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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15 pages, 1993 KiB

Open AccessArticle

Biofilm Formation by the Acidophile Bacterium Acidithiobacillus thiooxidans Involves c-di-GMP Pathway and Pel exopolysaccharide

by Mauricio Díaz, Matias Castro, Sylvia Copaja and Nicolas Guiliani

Genes 2018, 9(2), 113; https://doi.org/10.3390/genes9020113 - 21 Feb 2018

Cited by 41 | Viewed by 7399

Abstract

Acidophile bacteria belonging to the Acidithiobacillus genus are pivotal players for the bioleaching of metallic values such as copper. Cell adherence to ores and biofilm formation, mediated by the production of extracellular polymeric substances, strongly favors bioleaching activity. In recent years, the second [...] Read more.

Acidophile bacteria belonging to the Acidithiobacillus genus are pivotal players for the bioleaching of metallic values such as copper. Cell adherence to ores and biofilm formation, mediated by the production of extracellular polymeric substances, strongly favors bioleaching activity. In recent years, the second messenger cyclic diguanylate (c-di-GMP) has emerged as a central regulator for biofilm formation in bacteria. C-di-GMP pathways have been reported in different Acidithiobacillus species; however, c-di-GMP effectors and signal transduction networks are still largely uncharacterized in these extremophile species. Here we investigated Pel exopolysaccharide and its role in biofilm formation by sulfur-oxidizing species Acidithiobacillus thiooxidans. We identified 39 open reading frames (ORFs) encoding proteins involved in c-di-GMP metabolism and signal transduction, including the c-di-GMP effector protein PelD, a structural component of the biosynthesis apparatus for Pel exopolysaccharide production. We found that intracellular c-di-GMP concentrations and transcription levels of pel genes were higher in At. thiooxidans biofilm cells compared to planktonic ones. By developing an At. thiooxidans ΔpelD null-mutant strain we revealed that Pel exopolysaccharide is involved in biofilm structure and development. Further studies are still necessary to understand how Pel biosynthesis is regulated in Acidithiobacillus species, nevertheless these results represent the first characterization of a c-di-GMP effector protein involved in biofilm formation by acidophile species. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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19 pages, 1265 KiB

Open AccessArticle

Small RNAs of Haloferax mediterranei: Identification and Potential Involvement in Nitrogen Metabolism

by Gloria Payá, Vanesa Bautista, Mónica Camacho, Natalia Castejón-Fernández, Luís A. Alcaraz, María-José Bonete and Julia Esclapez

Genes 2018, 9(2), 83; https://doi.org/10.3390/genes9020083 - 10 Feb 2018

Cited by 15 | Viewed by 4839

Abstract

Small RNAs have been studied in detail in domains Bacteria and Eukarya but, in the case of the domain Archaea, the knowledge is scarce and the physiological function of these small RNAs (sRNAs) is still uncertain. To extend the knowledge of sRNAs in [...] Read more.

Small RNAs have been studied in detail in domains Bacteria and Eukarya but, in the case of the domain Archaea, the knowledge is scarce and the physiological function of these small RNAs (sRNAs) is still uncertain. To extend the knowledge of sRNAs in the domain Archaea and their possible role in the regulation of the nitrogen assimilation metabolism in haloarchaea, Haloferax mediterranei has been used as a model microorganism. The bioinformatic approach has allowed for the prediction of 295 putative sRNAs genes in the genome of H. mediterranei, 88 of which have been verified by means of RNA-Sequencing (RNA-Seq). The secondary structure of these sRNAs and their possible targets have been identified. Curiously, some of them present as possible target genes relating to nitrogen assimilation, such as glutamate dehydrogenase and the nitrogen regulatory PII protein. Analysis of RNA-Seq data has also revealed differences in the expression pattern of 16 sRNAs according to the nitrogen source. Consequently, RNomic and bioinformatic approaches used in this work have allowed for the identification of new sRNAs in H. mediterranei, some of which show different expression patterns depending on the nitrogen source. This suggests that these sRNAs could be involved in the regulation of nitrogen assimilation and can constitute an important gene regulatory network. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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13 pages, 2036 KiB

Open AccessArticle

Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus

by Tiphaine Birien, Axel Thiel, Ghislaine Henneke, Didier Flament, Yann Moalic and Mohamed Jebbar

Genes 2018, 9(2), 77; https://doi.org/10.3390/genes9020077 - 7 Feb 2018

Cited by 18 | Viewed by 4852

Abstract

A gene disruption system for Thermococcus barophilus was developed using simvastatin (HMG-CoA reductase encoding gene) for positive selection and 5-Fluoroorotic acid (5-FOA), a pyrF gene for negative selection. Multiple gene mutants were constructed with this system, which offers the possibility of complementation in [...] Read more.

A gene disruption system for Thermococcus barophilus was developed using simvastatin (HMG-CoA reductase encoding gene) for positive selection and 5-Fluoroorotic acid (5-FOA), a pyrF gene for negative selection. Multiple gene mutants were constructed with this system, which offers the possibility of complementation in trans, but produces many false positives (<80%). To significantly reduce the rate of false positives, we used another counterselective marker, 6-methylpurine (6-MP), a toxic analog of adenine developed in Thermococcus kodakarensis, consistently correlated with the TK0664 gene (encoding a hypoxanthine-guanine phosphoribosyl-transferase). We thus replaced pyrF by TK0664 on our suicide vector and tested T. barophilus strain sensitivity to 6-MP before and after transformation. Wild-Type (WT) T. barophilus is less sensitive to 6-MP than WT T. kodakarensis, and an increase of cell resistance was achieved after deletion of the T. barophilus TERMP_00517 gene homologous to T. kodakarensis TK0664. Results confirmed the natural resistance of T. barophilus to 6-MP and show that TK0664 can confer sensitivity. This new counterselection system vastly improves genetic manipulations in T. barophilus MP, with a strong decrease in false positives to <15%. Using this genetic tool, we have started to investigate the functions of several genes involved in genomic maintenance (e.g., polB and rnhB). Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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18 pages, 1577 KiB

Open AccessArticle

Proteomic Analysis of Methanonatronarchaeum thermophilum AMET1, a Representative of a Putative New Class of Euryarchaeota, “Methanonatronarchaeia”

by Manuel Ferrer, Dimitry Y. Sorokin, Yuri I. Wolf, Sergio Ciordia, María C. Mena, Rafael Bargiela, Eugene V. Koonin and Kira S. Makarova

Genes 2018, 9(2), 28; https://doi.org/10.3390/genes9020028 - 23 Jan 2018

Cited by 4 | Viewed by 5095

Abstract

The recently discovered Methanonatronarchaeia are extremely halophilic and moderately thermophilic methyl-reducing methanogens representing a novel class-level lineage in the phylum Euryarchaeota related to the class Halobacteria. Here we present a detailed analysis of 1D-nano liquid chromatography–electrospray ionization tandem mass spectrometry data obtained for [...] Read more.

The recently discovered Methanonatronarchaeia are extremely halophilic and moderately thermophilic methyl-reducing methanogens representing a novel class-level lineage in the phylum Euryarchaeota related to the class Halobacteria. Here we present a detailed analysis of 1D-nano liquid chromatography–electrospray ionization tandem mass spectrometry data obtained for “Methanonatronarchaeum thermophilum” AMET1 grown in different physiological conditions, including variation of the growth temperature and substrates. Analysis of these data allows us to refine the current understanding of the key biosynthetic pathways of this triple extremophilic methanogenic euryarchaeon and identify proteins that are likely to be involved in its response to growth condition changes. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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13 pages, 2187 KiB

Open AccessArticle

Metagenomics of Bacterial Diversity in Villa Luz Caves with Sulfur Water Springs

by Giuseppe D’Auria, Alejandro Artacho, Rafael A. Rojas, José S. Bautista, Roberto Méndez, María T. Gamboa, Jesús R. Gamboa and Rodolfo Gómez-Cruz

Genes 2018, 9(1), 55; https://doi.org/10.3390/genes9010055 - 22 Jan 2018

Cited by 25 | Viewed by 6471

Abstract

New biotechnology applications require in-depth preliminary studies of biodiversity. The methods of massive sequencing using metagenomics and bioinformatics tools offer us sufficient and reliable knowledge to understand environmental diversity, to know new microorganisms, and to take advantage of their functional genes. Villa Luz [...] Read more.

New biotechnology applications require in-depth preliminary studies of biodiversity. The methods of massive sequencing using metagenomics and bioinformatics tools offer us sufficient and reliable knowledge to understand environmental diversity, to know new microorganisms, and to take advantage of their functional genes. Villa Luz caves, in the southern Mexican state of Tabasco, are fed by at least 26 groundwater inlets, containing 300–500 mg L⁻¹ H₂S and <0.1 mg L⁻¹ O₂. We extracted environmental DNA for metagenomic analysis of collected samples in five selected Villa Luz caves sites, with pH values from 2.5 to 7. Foreign organisms found in this underground ecosystem can oxidize H₂S to H₂SO₄. These include: biovermiculites, a bacterial association that can grow on the rock walls; snottites, that are whitish, viscous biofilms hanging from the rock walls, and sacks or bags of phlegm, which live within the aquatic environment of the springs. Through the emergency food assistance program (TEFAP) pyrosequencing, a total of 20,901 readings of amplification products from hypervariable regions V1 and V3 of 16S rRNA bacterial gene in whole and pure metagenomic DNA samples were generated. Seven bacterial phyla were identified. As a result, Proteobacteria was more frequent than Acidobacteria. Finally, acidophilic Proteobacteria was detected in UJAT5 sample. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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18 pages, 1002 KiB

Open AccessArticle

Differential Gene Expression in Response to Salinity and Temperature in a Haloarcula Strain from Great Salt Lake, Utah

by Swati Almeida-Dalmet, Carol D. Litchfield, Patrick Gillevet and Bonnie K. Baxter

Genes 2018, 9(1), 52; https://doi.org/10.3390/genes9010052 - 22 Jan 2018

Cited by 12 | Viewed by 5779 | Correction

Abstract

Haloarchaea that inhabit Great Salt Lake (GSL), a thalassohaline terminal lake, must respond to the fluctuating climate conditions of the elevated desert of Utah. We investigated how shifting environmental factors, specifically salinity and temperature, affected gene expression in the GSL haloarchaea, NA6-27, which [...] Read more.

Haloarchaea that inhabit Great Salt Lake (GSL), a thalassohaline terminal lake, must respond to the fluctuating climate conditions of the elevated desert of Utah. We investigated how shifting environmental factors, specifically salinity and temperature, affected gene expression in the GSL haloarchaea, NA6-27, which we isolated from the hypersaline north arm of the lake. Combined data from cultivation, microscopy, lipid analysis, antibiotic sensitivity, and 16S rRNA gene alignment, suggest that NA6-27 is a member of the Haloarcula genus. Our prior study demonstrated that archaea in the Haloarcula genus were stable in the GSL microbial community over seasons and years. In this study, RNA arbitrarily primed PCR (RAP-PCR) was used to determine the transcriptional responses of NA6-27 grown under suboptimal salinity and temperature conditions. We observed alteration of the expression of genes related to general stress responses, such as transcription, translation, replication, signal transduction, and energy metabolism. Of the ten genes that were expressed differentially under stress, eight of these genes responded in both conditions, highlighting this general response. We also noted gene regulation specific to salinity and temperature conditions, such as osmoregulation and transport. Taken together, these data indicate that the GSL Haloarcula strain, NA6-27, demonstrates both general and specific responses to salinity and/or temperature stress, and suggest a mechanistic model for homeostasis that may explain the stable presence of this genus in the community as environmental conditions shift. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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Open AccessArticle

Genetic Analysis of the Major Capsid Protein of the Archaeal Fusellovirus SSV1: Mutational Flexibility and Conformational Change

by Eric A. Iverson, David A. Goodman, Madeline E. Gorchels and Kenneth M. Stedman

Genes 2017, 8(12), 373; https://doi.org/10.3390/genes8120373 - 8 Dec 2017

Cited by 16 | Viewed by 5049

Abstract

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is [...] Read more.

Viruses with spindle or lemon-shaped virions are rare in the world of viruses, but are common in viruses of archaeal extremophiles, possibly due to the extreme conditions in which they thrive. However, the structural and genetic basis for the unique spindle shape is unknown. The best-studied spindle-shaped virus, Sulfolobus Spindle-shaped Virus 1 (SSV1), is composed mostly of the major capsid protein VP1. Similar to many other viruses, proteolytic cleavage of VP1 is thought to be critical for virion formation. Unlike half of the genes in SSV1, including the minor capsid protein gene VP3, the VP1 gene does not tolerate deletion or transposon insertion. To determine the role of the VP1 gene and its proteolysis for virus function, we developed techniques for site-directed mutagenesis of the SSV1 genome and complemented deletion mutants with VP1 genes from other SSVs. By analyzing these mutants, we demonstrate that the N-terminus of the VP1 protein is required, but the N-terminus, or entire SSV1 VP1 protein, can be exchanged with VP1s from other SSVs. However, the conserved glutamate at the cleavage site is not essential for infectivity. Interestingly, viruses containing point mutations at this position generate mostly abnormal virions. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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6456 KiB

Open AccessArticle

Hierarchical Control of Nitrite Respiration by Transcription Factors Encoded within Mobile Gene Clusters of Thermus thermophilus

by Laura Alvarez, Nieves G. Quintáns, Alba Blesa, Ignacio Baquedano, Mario Mencía, Carlos Bricio and José Berenguer

Genes 2017, 8(12), 361; https://doi.org/10.3390/genes8120361 - 1 Dec 2017

Cited by 5 | Viewed by 4330

Abstract

Denitrification in Thermus thermophilus is encoded by the nitrate respiration conjugative element (NCE) and nitrite and nitric oxide respiration (nic) gene clusters. A tight coordination of each cluster’s expression is required to maximize anaerobic growth, and to avoid toxicity by intermediates, especially [...] Read more.

Denitrification in Thermus thermophilus is encoded by the nitrate respiration conjugative element (NCE) and nitrite and nitric oxide respiration (nic) gene clusters. A tight coordination of each cluster’s expression is required to maximize anaerobic growth, and to avoid toxicity by intermediates, especially nitric oxides (NO). Here, we study the control of the nitrite reductases (Nir) and NO reductases (Nor) upon horizontal acquisition of the NCE and nic clusters by a formerly aerobic host. Expression of the nic promoters PnirS, PnirJ, and PnorC, depends on the oxygen sensor DnrS and on the DnrT protein, both NCE-encoded. NsrR, a nic-encoded transcription factor with an iron–sulfur cluster, is also involved in Nir and Nor control. Deletion of nsrR decreased PnorC and PnirJ transcription, and activated PnirS under denitrification conditions, exhibiting a dual regulatory role never described before for members of the NsrR family. On the basis of these results, a regulatory hierarchy is proposed, in which under anoxia, there is a pre-activation of the nic promoters by DnrS and DnrT, and then NsrR leads to Nor induction and Nir repression, likely as a second stage of regulation that would require NO detection, thus avoiding accumulation of toxic levels of NO. The whole system appears to work in remarkable coordination to function only when the relevant nitrogen species are present inside the cell. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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Review

Jump to: Research, Other

58 pages, 11749 KiB

Open AccessReview

Role of the Extremolytes Ectoine and Hydroxyectoine as Stress Protectants and Nutrients: Genetics, Phylogenomics, Biochemistry, and Structural Analysis

by Laura Czech, Lucas Hermann, Nadine Stöveken, Alexandra A. Richter, Astrid Höppner, Sander H. J. Smits, Johann Heider and Erhard Bremer

Genes 2018, 9(4), 177; https://doi.org/10.3390/genes9040177 - 22 Mar 2018

Cited by 170 | Viewed by 16920

Abstract

Fluctuations in environmental osmolarity are ubiquitous stress factors in many natural habitats of microorganisms, as they inevitably trigger osmotically instigated fluxes of water across the semi-permeable cytoplasmic membrane. Under hyperosmotic conditions, many microorganisms fend off the detrimental effects of water efflux and the [...] Read more.

Fluctuations in environmental osmolarity are ubiquitous stress factors in many natural habitats of microorganisms, as they inevitably trigger osmotically instigated fluxes of water across the semi-permeable cytoplasmic membrane. Under hyperosmotic conditions, many microorganisms fend off the detrimental effects of water efflux and the ensuing dehydration of the cytoplasm and drop in turgor through the accumulation of a restricted class of organic osmolytes, the compatible solutes. Ectoine and its derivative 5-hydroxyectoine are prominent members of these compounds and are synthesized widely by members of the Bacteria and a few Archaea and Eukarya in response to high salinity/osmolarity and/or growth temperature extremes. Ectoines have excellent function-preserving properties, attributes that have led to their description as chemical chaperones and fostered the development of an industrial-scale biotechnological production process for their exploitation in biotechnology, skin care, and medicine. We review, here, the current knowledge on the biochemistry of the ectoine/hydroxyectoine biosynthetic enzymes and the available crystal structures of some of them, explore the genetics of the underlying biosynthetic genes and their transcriptional regulation, and present an extensive phylogenomic analysis of the ectoine/hydroxyectoine biosynthetic genes. In addition, we address the biochemistry, phylogenomics, and genetic regulation for the alternative use of ectoines as nutrients. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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13 pages, 1716 KiB

Open AccessReview

The Non-Coding Regulatory RNA Revolution in Archaea

by Diego Rivera Gelsinger and Jocelyne DiRuggiero

Genes 2018, 9(3), 141; https://doi.org/10.3390/genes9030141 - 5 Mar 2018

Cited by 29 | Viewed by 8315

Abstract

Small non-coding RNAs (sRNAs) are ubiquitously found in the three domains of life playing large-scale roles in gene regulation, transposable element silencing and defense against foreign elements. While a substantial body of experimental work has been done to uncover function of sRNAs in [...] Read more.

Small non-coding RNAs (sRNAs) are ubiquitously found in the three domains of life playing large-scale roles in gene regulation, transposable element silencing and defense against foreign elements. While a substantial body of experimental work has been done to uncover function of sRNAs in Bacteria and Eukarya, the functional roles of sRNAs in Archaea are still poorly understood. Recently, high throughput studies using RNA-sequencing revealed that sRNAs are broadly expressed in the Archaea, comprising thousands of transcripts within the transcriptome during non-challenged and stressed conditions. Antisense sRNAs, which overlap a portion of a gene on the opposite strand (cis-acting), are the most abundantly expressed non-coding RNAs and they can be classified based on their binding patterns to mRNAs (3′ untranslated region (UTR), 5′ UTR, CDS-binding). These antisense sRNAs target many genes and pathways, suggesting extensive roles in gene regulation. Intergenic sRNAs are less abundantly expressed and their targets are difficult to find because of a lack of complete overlap between sRNAs and target mRNAs (trans-acting). While many sRNAs have been validated experimentally, a regulatory role has only been reported for very few of them. Further work is needed to elucidate sRNA-RNA binding mechanisms, the molecular determinants of sRNA-mediated regulation, whether protein components are involved and how sRNAs integrate with complex regulatory networks. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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15 pages, 650 KiB

Open AccessReview

Archaeal Viruses from High-Temperature Environments

by Jacob H. Munson-McGee, Jamie C. Snyder and Mark J. Young

Genes 2018, 9(3), 128; https://doi.org/10.3390/genes9030128 - 27 Feb 2018

Cited by 51 | Viewed by 6283

Abstract

Archaeal viruses are some of the most enigmatic viruses known, due to the small number that have been characterized to date. The number of known archaeal viruses lags behind known bacteriophages by over an order of magnitude. Despite this, the high levels of [...] Read more.

Archaeal viruses are some of the most enigmatic viruses known, due to the small number that have been characterized to date. The number of known archaeal viruses lags behind known bacteriophages by over an order of magnitude. Despite this, the high levels of genetic and morphological diversity that archaeal viruses display has attracted researchers for over 45 years. Extreme natural environments, such as acidic hot springs, are almost exclusively populated by Archaea and their viruses, making these attractive environments for the discovery and characterization of new viruses. The archaeal viruses from these environments have provided insights into archaeal biology, gene function, and viral evolution. This review focuses on advances from over four decades of archaeal virology, with a particular focus on archaeal viruses from high temperature environments, the existing challenges in understanding archaeal virus gene function, and approaches being taken to overcome these limitations. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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28 pages, 6500 KiB

Open AccessReview

In a quest for engineering acidophiles for biomining applications: challenges and opportunities

by Yosephine Gumulya, Naomi J. Boxall, Himel N. Khaleque, Ville Santala, Ross P. Carlson and Anna H. Kaksonen

Genes 2018, 9(2), 116; https://doi.org/10.3390/genes9020116 - 21 Feb 2018

Cited by 82 | Viewed by 13274

Abstract

Biomining with acidophilic microorganisms has been used at commercial scale for the extraction of metals from various sulfide ores. With metal demand and energy prices on the rise and the concurrent decline in quality and availability of mineral resources, there is an increasing [...] Read more.

Biomining with acidophilic microorganisms has been used at commercial scale for the extraction of metals from various sulfide ores. With metal demand and energy prices on the rise and the concurrent decline in quality and availability of mineral resources, there is an increasing interest in applying biomining technology, in particular for leaching metals from low grade minerals and wastes. However, bioprocessing is often hampered by the presence of inhibitory compounds that originate from complex ores. Synthetic biology could provide tools to improve the tolerance of biomining microbes to various stress factors that are present in biomining environments, which would ultimately increase bioleaching efficiency. This paper reviews the state-of-the-art tools to genetically modify acidophilic biomining microorganisms and the limitations of these tools. The first part of this review discusses resilience pathways that can be engineered in acidophiles to enhance their robustness and tolerance in harsh environments that prevail in bioleaching. The second part of the paper reviews the efforts that have been carried out towards engineering robust microorganisms and developing metabolic modelling tools. Novel synthetic biology tools have the potential to transform the biomining industry and facilitate the extraction of value from ores and wastes that cannot be processed with existing biomining microorganisms. Full article

(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)

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