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1.
Nat Rev Microbiol ; 18(2): 67-83, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31857715

RESUMO

The number and diversity of known CRISPR-Cas systems have substantially increased in recent years. Here, we provide an updated evolutionary classification of CRISPR-Cas systems and cas genes, with an emphasis on the major developments that have occurred since the publication of the latest classification, in 2015. The new classification includes 2 classes, 6 types and 33 subtypes, compared with 5 types and 16 subtypes in 2015. A key development is the ongoing discovery of multiple, novel class 2 CRISPR-Cas systems, which now include 3 types and 17 subtypes. A second major novelty is the discovery of numerous derived CRISPR-Cas variants, often associated with mobile genetic elements that lack the nucleases required for interference. Some of these variants are involved in RNA-guided transposition, whereas others are predicted to perform functions distinct from adaptive immunity that remain to be characterized experimentally. The third highlight is the discovery of numerous families of ancillary CRISPR-linked genes, often implicated in signal transduction. Together, these findings substantially clarify the functional diversity and evolutionary history of CRISPR-Cas.


Assuntos
Archaea/genética , Bactérias/genética , Sistemas CRISPR-Cas/genética , Evolução Molecular , Regulação da Expressão Gênica em Archaea/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Sistemas CRISPR-Cas/fisiologia
2.
Int J Mol Sci ; 20(19)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561502

RESUMO

Haloferax volcanii, a well-developed model archaeon for genomic, transcriptomic, and proteomic analyses, can grow on a defined medium of abundant and intermediate levels of fixed nitrogen. Here we report a global profiling of gene expression of H. volcanii grown on ammonium as an abundant source of fixed nitrogen compared to l-alanine, the latter of which exemplifies an intermediate source of nitrogen that can be obtained from dead cells in natural habitats. By comparing the two growth conditions, 30 genes were found to be differentially expressed, including 16 genes associated with amino acid metabolism and transport. The gene expression profiles contributed to mapping ammonium and l-alanine usage with respect to transporters and metabolic pathways. In addition, conserved DNA motifs were identified in the putative promoter regions and transcription factors were found to be in synteny with the differentially expressed genes, leading us to propose regulons of transcriptionally co-regulated operons. This study provides insight to how H. volcanii responds to and utilizes intermediate vs. abundant sources of fixed nitrogen for growth, with implications for conserved functions in related halophilic archaea.


Assuntos
Regulação da Expressão Gênica em Archaea , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Fixação de Nitrogênio , Nitrogênio/metabolismo , Aminoácidos/metabolismo , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Redes e Vias Metabólicas , Transcriptoma
3.
Genes (Basel) ; 10(5)2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083437

RESUMO

Zinc finger domains are highly structured and can mediate interactions to DNA, RNA, proteins, lipids, and small molecules. Accordingly, zinc finger proteins are very versatile and involved in many biological functions. Eukaryotes contain a wealth of zinc finger proteins, but zinc finger proteins have also been found in archaea and bacteria. Large zinc finger proteins have been well studied, however, in stark contrast, single domain zinc finger µ-proteins of less than 70 amino acids have not been studied at all, with one single exception. Therefore, 16 zinc finger µ-proteins of the haloarchaeon Haloferax volcanii were chosen and in frame deletion mutants of the cognate genes were generated. The phenotypes of mutants and wild-type were compared under eight different conditions, which were chosen to represent various pathways and involve many genes. None of the mutants differed from the wild-type under optimal or near-optimal conditions. However, 12 of the 16 mutants exhibited a phenotypic difference under at least one of the four following conditions: Growth in synthetic medium with glycerol, growth in the presence of bile acids, biofilm formation, and swarming. In total, 16 loss of function and 11 gain of function phenotypes were observed. Five mutants indicated counter-regulation of a sessile versus a motile life style in H. volcanii. In conclusion, the generation and analysis of a set of deletion mutants demonstrated the high importance of zinc finger µ-proteins for various biological functions, and it will be the basis for future mechanistic insight.


Assuntos
Proteínas Arqueais/genética , Haloferax volcanii/genética , Dedos de Zinco/genética , Adaptação Fisiológica , Ácidos e Sais Biliares/farmacologia , Biofilmes , Deleção de Genes , Regulação da Expressão Gênica em Archaea , Haloferax volcanii/fisiologia , Estresse Fisiológico
4.
PLoS One ; 14(5): e0216089, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31048872

RESUMO

We theoretically study the effects of non-monotonic response curves in genetic auto-regulation by exploring the possible dynamical behaviors for such systems. Our motivation is twofold: we aim at conceiving the simplest genetic circuits for synthetic biology and at understanding the natural auto-regulation of the LrpB protein of the Sulfolobus solfataricus archaeon which exhibits non-monotonicity. We analyzed three toy models, based on mass-action kinetics, with increasing complexity and sought for oscillations and (fast) bistable switching. We performed large parameter scans and sensitivity analyses, and quantified the quality of the oscillators and switches by computing relative volumes in parameter space reproducing the sought dynamical behavior. All single gene systems need finely tuned parameters in order to oscillate, but bistable switches are more robust against parameter changes. We expected non-monotonic switches to be faster than monotonic ones, however solutions combining both auto-activation and repression in the physiological range to obtain fast switches are scarce. Our analysis shows that the Ss-LrpB system can not provide a bistable switch and that robust oscillations are unlikely. Gillespie simulations suggest that the function of the natural Ss-LrpB system is sensing via a spiking behavior, which is in line with the fact that this protein has a metabolic regulatory function and binds to a ligand.


Assuntos
Regulação da Expressão Gênica em Archaea/genética , Redes Reguladoras de Genes/genética , Sulfolobus solfataricus/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Regulação da Expressão Gênica/genética , Cinética , Modelos Teóricos , Regiões Promotoras Genéticas/genética , Biologia Sintética/métodos
5.
PLoS One ; 14(4): e0215986, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31039177

RESUMO

Haloferax volcanii is a well-established model species for haloarchaea. Small scale RNomics and bioinformatics predictions were used to identify small non-coding RNAs (sRNAs), and deletion mutants revealed that sRNAs have important regulatory functions. A recent dRNA-Seq study was used to characterize the primary transcriptome. Unexpectedly, it was revealed that, under optimal conditions, H. volcanii contains more non-coding sRNAs than protein-encoding mRNAs. However, the dRNA-Seq approach did not contain any length information. Therefore, a mixed RNA-Seq approach was used to determine transcript length and to identify additional transcripts, which are not present under optimal conditions. In total, 50 million paired end reads of 150 nt length were obtained. 1861 protein-coding RNAs (cdRNAs) were detected, which encoded 3092 proteins. This nearly doubled the coverage of cdRNAs, compared to the previous dRNA-Seq study. About 2/3 of the cdRNAs were monocistronic, and 1/3 covered more than one gene. In addition, 1635 non-coding sRNAs were identified. The highest fraction of non-coding RNAs were cis antisense RNAs (asRNAs). Analysis of the length distribution revealed that sRNAs have a median length of about 150 nt. Based on the RNA-Seq and dRNA-Seq results, genes were chosen to exemplify characteristics of the H. volcanii transcriptome by Northern blot analyses, e.g. 1) the transcript patterns of gene clusters can be straightforward, but also very complex, 2) many transcripts differ in expression level under the four analyzed conditions, 3) some genes are transcribed into RNA isoforms of different length, which can be differentially regulated, 4) transcripts with very long 5'-UTRs and with very long 3'-UTRs exist, and 5) about 30% of all cdRNAs have overlapping 3'-ends, which indicates, together with the asRNAs, that H. volcanii makes ample use of sense-antisense interactions. Taken together, this RNA-Seq study, together with a previous dRNA-Seq study, enabled an unprecedented view on the H. volcanii transcriptome.


Assuntos
Haloferax volcanii/crescimento & desenvolvimento , Haloferax volcanii/genética , Transcriptoma/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Regulação da Expressão Gênica em Archaea , Família Multigênica , Fases de Leitura Aberta/genética , Óperon/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo
6.
Genes (Basel) ; 10(4)2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30959844

RESUMO

Antisense RNAs (asRNAs) are present in diverse organisms and play important roles in gene regulation. In this work, we mapped the primary antisense transcriptome in the halophilic archaeon Halobacterium salinarum NRC-1. By reanalyzing publicly available data, we mapped antisense transcription start sites (aTSSs) and inferred the probable 3' ends of these transcripts. We analyzed the resulting asRNAs according to the size, location, function of genes on the opposite strand, expression levels and conservation. We show that at least 21% of the genes contain asRNAs in H. salinarum. Most of these asRNAs are expressed at low levels. They are located antisense to genes related to distinctive characteristics of H. salinarum, such as bacteriorhodopsin, gas vesicles, transposases and other important biological processes such as translation. We provide evidence to support asRNAs in type II toxin⁻antitoxin systems in archaea. We also analyzed public Ribosome profiling (Ribo-seq) data and found that ~10% of the asRNAs are ribosome-associated non-coding RNAs (rancRNAs), with asRNAs from transposases overrepresented. Using a comparative transcriptomics approach, we found that ~19% of the asRNAs annotated in H. salinarum belong to genes with an ortholog in Haloferax volcanii, in which an aTSS could be identified with positional equivalence. This shows that most asRNAs are not conserved between these halophilic archaea.


Assuntos
Perfilação da Expressão Gênica , Halobacterium salinarum/genética , RNA Antissenso/genética , Transcriptoma/genética , Regulação da Expressão Gênica em Archaea/genética , Genoma Arqueal/genética , RNA não Traduzido/genética , Ribossomos/genética , Sítio de Iniciação de Transcrição
7.
Biochimie ; 164: 3-16, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30995539

RESUMO

Prokaryotes encounter constant and often brutal modifications to their environment. In order to survive, they need to maintain fitness, which includes adapting their protein expression patterns. Many factors control gene expression but this review focuses on just one, namely antisense RNAs (asRNAs), a class of non-coding RNAs (ncRNAs) characterized by their location in cis and their perfect complementarity with their targets. asRNAs were considered for a long time to be trivial and only to be found on mobile genetic elements. However, recent advances in methodology have revealed that their abundance and potential activities have been underestimated. This review aims to illustrate the role of asRNA in various physiologically crucial functions in both archaea and bacteria, which can be regrouped in three categories: cell maintenance, horizontal gene transfer and virulence. A literature survey of asRNAs demonstrates the difficulties to characterize and assign a role to asRNAs. With the aim of facilitating this task, we describe recent technological advances that could be of interest to identify new asRNAs and to discover their function.


Assuntos
Archaea , Bactérias , Fenômenos Fisiológicos Bacterianos/genética , Transferência Genética Horizontal/genética , RNA Antissenso , Virulência/genética , Archaea/genética , Archaea/patogenicidade , Archaea/fisiologia , Bactérias/genética , Bactérias/patogenicidade , Regulação da Expressão Gênica em Archaea , Regulação Bacteriana da Expressão Gênica , RNA Antissenso/genética , RNA Antissenso/fisiologia , RNA Arqueal/genética , RNA Arqueal/fisiologia , RNA Bacteriano/genética , RNA Bacteriano/fisiologia
8.
J Biol Chem ; 294(19): 7821-7832, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30918025

RESUMO

Archaea are a distinct and deeply rooted lineage that harbor eukaryotic-like mechanisms, including several that manage chromosome function. In previous work, the thermoacidophilic crenarchaeon, Sulfolobus solfataricus, was subjected to adaptive laboratory evolution to produce three strains, called SARC, with a new heritable trait of super acid resistance. These strains acquired heritable conserved transcriptomes, yet one strain contained no mutations. Homologous recombination without allele replacement at SARC acid resistance genes caused changes in both phenotype and expression of the targeted gene. As recombination displaces chromatin proteins, their involvement was predicted in the SARC trait. Native chromatin proteins are basic and highly abundant and undergo post-translational modification through lysine monomethylation. In this work, their modification states were investigated. In all SARC lines, two chromatin proteins, Cren7 and Sso7d, were consistently undermethylated, whereas other chromatin proteins were unaltered. This pattern was heritable in the absence of selection and independent of transient exposure to acid stress. The bulk of Sso7d was undermethylated at three contiguous N-terminal lysine residues but not at central or C-terminal regions. The N-terminal region formed a solvent-exposed patch located on the opposite side of the binding domain associated with the DNA minor groove. By analogy to eukaryotic histones, this patch could interact with other chromosomal proteins and be modulated by differential post-translational modification. Previous work established an epigenetic-like mechanism of adaptation and inheritance in S. solfataricus The identification of heritable epigenetic marks in this work further supports the occurrence of an epigenetic process in archaea.


Assuntos
Proteínas Arqueais , Proteínas de Ligação a DNA , Evolução Molecular Direcionada , Epigênese Genética , Regulação da Expressão Gênica em Archaea , Locos de Características Quantitativas , Sulfolobus solfataricus , Adaptação Fisiológica , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Sulfolobus solfataricus/química , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismo , Transcriptoma
9.
mBio ; 10(2)2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837343

RESUMO

Control of electron flux is critical in both natural and bioengineered systems to maximize energy gains. Both small molecules and proteins shuttle high-energy, low-potential electrons liberated during catabolism through diverse metabolic landscapes. Ferredoxin (Fd) proteins-an abundant class of Fe-S-containing small proteins-are essential in many species for energy conservation and ATP production strategies. It remains difficult to model electron flow through complicated metabolisms and in systems in which multiple Fd proteins are present. The overlap of activity and/or limitations of electron flux through each Fd can limit physiology and metabolic engineering strategies. Here we establish the interplay, reactivity, and physiological role(s) of the three ferredoxin proteins in the model hyperthermophile Thermococcus kodakarensis We demonstrate that the three loci encoding known Fds are subject to distinct regulatory mechanisms and that specific Fds are utilized to shuttle electrons to separate respiratory and energy production complexes during different physiological states. The results obtained argue that unique physiological roles have been established for each Fd and that continued use of T. kodakarensis and related hydrogen-evolving species as bioengineering platforms must account for the distinct Fd partnerships that limit flux to desired electron acceptors. Extrapolating our results more broadly, the retention of multiple Fd isoforms in most species argues that specialized Fd partnerships are likely to influence electron flux throughout biology.IMPORTANCE High-energy electrons liberated during catabolic processes can be exploited for energy-conserving mechanisms. Maximal energy gains demand these valuable electrons be accurately shuttled from electron donor to appropriate electron acceptor. Proteinaceous electron carriers such as ferredoxins offer opportunities to exploit specific ferredoxin partnerships to ensure that electron flux to critical physiological pathways is aligned with maximal energy gains. Most species encode many ferredoxin isoforms, but very little is known about the role of individual ferredoxins in most systems. Our results detail that ferredoxin isoforms make largely unique and distinct protein interactions in vivo and that flux through one ferredoxin often cannot be recovered by flux through a different ferredoxin isoform. The results obtained more broadly suggest that ferredoxin isoforms throughout biological life have evolved not as generic electron shuttles, but rather serve as selective couriers of valuable low-potential electrons from select electron donors to desirable electron acceptors.


Assuntos
Transporte de Elétrons , Metabolismo Energético , Ferredoxinas/metabolismo , Thermococcus/enzimologia , Thermococcus/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação da Expressão Gênica em Archaea , Thermococcus/genética
10.
J Biol Chem ; 294(18): 7460-7471, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30902813

RESUMO

Phosphorylation-dependent interactions play crucial regulatory roles in all domains of life. Forkhead-associated (FHA) and von Willebrand type A (vWA) domains are involved in several phosphorylation-dependent processes of multiprotein complex assemblies. Although well-studied in eukaryotes and bacteria, the structural and functional contexts of these domains are not yet understood in Archaea. Here, we report the structural base for such an interacting pair of FHA and vWA domain-containing proteins, ArnA and ArnB, in the thermoacidophilic archaeon Sulfolobus acidocaldarius, where they act synergistically and negatively modulate motility. The structure of the FHA domain of ArnA at 1.75 Å resolution revealed that it belongs to the subclass of FHA domains, which recognizes double-pSer/pThr motifs. We also solved the 1.5 Å resolution crystal structure of the ArnB paralog vWA2, disclosing a complex topology comprising the vWA domain, a ß-sandwich fold, and a C-terminal helix bundle. We further show that ArnA binds to the C terminus of ArnB, which harbors all the phosphorylation sites identified to date and is important for the function of ArnB in archaellum regulation. We also observed that expression levels of the archaellum components in response to changes in nutrient conditions are independent of changes in ArnA and ArnB levels and that a strong interaction between ArnA and ArnB observed during growth on rich medium sequentially diminishes after nutrient limitation. In summary, our findings unravel the structural features in ArnA and ArnB important for their interaction and functional archaellum expression and reveal how nutrient conditions affect this interaction.


Assuntos
Proteínas Arqueais/metabolismo , Regulação da Expressão Gênica em Archaea , Genes Arqueais , Sulfolobus acidocaldarius/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Cristalografia por Raios X , Meios de Cultura , Fosforilação , Conformação Proteica , Sulfolobus acidocaldarius/metabolismo
11.
J Microbiol ; 57(2): 138-142, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30706342

RESUMO

Thermococcus onnurineus NA1, an obligate anaerobic hyperthermophilic archaeon, showed variable oxygen (O2) sensitivity depending on the types of substrate employed as an energy source. Unexpectedly, the culture with yeast extract as a sole energy source showed enhanced growth by 2-fold in the presence of O2. Genome-wide transcriptome analysis revealed the upregulation of several antioxidant-related genes encoding thioredoxin peroxidase (TON_0862), rubrerythrin (TON_0864), rubrerythrin-related protein (TON_0873), NAD(P)H rubredoxin oxidoreductase (TON_0865), or thioredoxin reductase (TON_1603), which can couple the detoxification of reactive oxygen species with the regeneration of NAD(P)+ from NAD(P)H. We present a plausible mechanism by which O2 serves to maintain the intracellular redox balance. This study demonstrates an unusual strategy of an obligate anaerobe underlying O2-mediated growth enhancement despite not having heme-based or cytochrome-type proteins.


Assuntos
Oxigênio/metabolismo , Thermococcus/enzimologia , Thermococcus/crescimento & desenvolvimento , Thermococcus/genética , Antioxidantes , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citocromos/genética , Citocromos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica em Archaea , Genes Arqueais/genética , Hemeproteínas/genética , Hemeproteínas/metabolismo , Hemeritrina/genética , Hemeritrina/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade , Rubredoxinas/genética , Rubredoxinas/metabolismo , Thermococcus/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Transcriptoma , Regulação para Cima
12.
Nucleic Acids Res ; 47(4): 1964-1976, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30605516

RESUMO

Random mutagenesis for the hyperthermophilic archaeon Thermococcus kodakarensis was established by the insertion of an artificial transposon designed to allow easy identification of the transposon-inserted locus. The phenotypic screening was applied for the isolation of thermosensitive mutants of T. kodakarensis, which resulted in the isolation of 16 mutants showing defective growth at the supraoptimal temperature 93°C. The high occurrence of the mutants suggested that the high thermotolerance of hyperthermophiles was achieved by a combination of diverse gene functions. The transposon insertion sites in two-thirds of the mutants were identified in a group of genes responsible for tRNA modifications including 7-formamidino-7-deaza-guanosine (archaeosine), N1-methyladenosine/N1-methylinosine, N4-acetylcytidine, and N2-dimethylguanosine/N2,N2-dimethylguanosine. LC-MS/MS analyses of tRNA nucleosides and fragments exhibited disappearance of the corresponding modifications in the mutants. The melting temperature of total tRNA fraction isolated from the mutants lacking archaeosine or N1-methyladenosine/N1-methylinosine decreased significantly, suggesting that the thermosensitive phenotype of these mutants was attributed to low stability of the hypomodified tRNAs. Genes for metabolism, transporters, and hypothetical proteins were also identified in the thermosensitive mutants. The present results demonstrated the usefulness of random mutagenesis for the studies on the hyperthermophile, as well as crucial roles of tRNA modifications in cellular thermotolerance.


Assuntos
Guanosina/análogos & derivados , Mutagênese/genética , RNA de Transferência/genética , Thermococcus/genética , Sequência de Bases , Cromatografia Líquida , Regulação da Expressão Gênica em Archaea , Guanosina/química , Guanosina/genética , Nucleosídeos/química , Nucleosídeos/genética , Espectrometria de Massas em Tandem , Temperatura
13.
Appl Environ Microbiol ; 85(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30504216

RESUMO

The discovery of hyperthermophiles has dramatically changed our understanding of the habitats in which life can thrive. However, the extreme high temperatures in which these organisms live have severely restricted the development of genetic tools. The archaeon Pyrococcus yayanosii A1 is a strictly anaerobic and piezophilic hyperthermophile that is an ideal model for studies of extreme environmental adaptation. In the present study, we identified a high hydrostatic pressure (HHP)-inducible promoter (P hhp ) that controls target gene expression under HHP. We developed an HHP-inducible toxin-antitoxin cassette (HHP-TAC) containing (i) a counterselectable marker in which a gene encoding a putative toxin (virulence-associated protein C [PF0776 {VapC}]) controlled by the HHP-inducible promoter was used in conjunction with the gene encoding antitoxin PF0775 (VapB), which was fused to a constitutive promoter (P hmtB ), and (ii) a positive marker with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-encoding gene from P. furiosus controlled by the constitutive promoter P gdh The HHP-TAC was constructed to realize markerless gene disruption directly in P. yayanosii A1 in rich medium. The pop-out recombination step was performed using an HHP-inducible method. As proof, the PYCH_13690 gene, which encodes a 4-α-glucanotransferase, was successfully deleted from the strain P. yayanosii A1. The results showed that the capacity for starch hydrolysis in the Δ1369 mutant decreased dramatically compared to that in the wild-type strain. The inducible toxin-antitoxin system developed in this study greatly increases the genetic tools available for use in hyperthermophiles.IMPORTANCE Genetic manipulations in hyperthermophiles have been studied for over 20 years. However, the extremely high temperatures under which these organisms grow have limited the development of genetic tools. In this study, an HHP-inducible promoter was used to control the expression of a toxin. Compared to sugar-inducible and cold-shock-inducible promoters, the HHP-inducible promoter rarely has negative effects on the overall physiology and central metabolism of microorganisms, especially piezophilic hyperthermophiles. Previous studies have used auxotrophic strains as hosts, which may interfere with studies of adaptation and metabolism. Using an inducible toxin-antitoxin (TA) system as a counterselectable marker enables the generation of a markerless gene disruption strain without the use of auxotrophic mutants and counterselection with 5-fluoroorotic acid. TA systems are widely distributed in bacteria and archaea and can be used to overcome the limitations of high growth temperatures and dramatically extend the selectivity of genetic tools in hyperthermophiles.


Assuntos
Adaptação Fisiológica/genética , Antitoxinas/genética , Archaea/genética , Proteínas Arqueais/metabolismo , Pressão Hidrostática , Pyrococcus/genética , Toxinas Biológicas/genética , Archaea/fisiologia , Proteínas Arqueais/genética , Proteínas de Bactérias , Sequência de Bases , DNA Arqueal , Proteínas de Ligação a DNA , Regulação da Expressão Gênica em Archaea , Genes Arqueais/genética , Temperatura Alta , Fontes Hidrotermais , Hidroximetilglutaril-CoA Redutases/genética , Glicoproteínas de Membrana , Ácido Orótico/análogos & derivados , Regiões Promotoras Genéticas , Pyrococcus/fisiologia , Toxinas Biológicas/metabolismo , Transformação Genética
14.
Plant Cell Physiol ; 60(2): 448-461, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30407601

RESUMO

During litchi (Litchi chinensis Sonn.) fruit ripening, two major physiological changes, degreening (Chl degradation) and pigmentation (anthocyanin biosynthesis), are visually apparent. However, the specific factor triggering this important transition is still unclear. In the present study, we found that endogenous ABA content increased sharply when Chl breakdown was initiated and the ABA level peaked just before the onset of anthocyanin accumulation, suggesting that ABA plays an important role during litchi fruit pigmentation. We characterized three ABSCISIC ACID RESPONSE ELEMENT-BINDING FACTORs (LcABF1/2/3) belonging to group A of the basic leucine zipper (bZIP) transcription factors previously shown to be involved in ABA signaling under abiotic stress. LcABF1 transcripts increased at the onset of Chl degradation, and the expression of LcABF3 accumulated in parallel with anthocyanin biosynthesis. In addition, dual luciferase and yeast one-hybrid assays indicated that LcABF1/2 recognized ABA-responsive elements in the promoter region of Chl degradation-related genes (PAO and SGR), while LcABF2/3 bound the promoter region of LcMYB1 and anthocyanin biosynthesis-related structural genes. Indeed, Nicotiana benthamiana leaves transiently expressing LcABF1/2 showed a senescence phenomenon with Chl degradation, and LcABF3 overexpression increased the accumulation of anthocyanin via activation of LcMYB1, which is the key determinant of anthocyanin biosynthesis. These data indicate that LcABF1/2/3 are important transcriptional regulators of ABA-dependent litchi fruit ripening involved in both Chl degradation and anthocyanin biosynthesis.


Assuntos
Antocianinas/biossíntese , Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Clorofila/metabolismo , Frutas/crescimento & desenvolvimento , Litchi/metabolismo , Proteínas de Plantas/fisiologia , Ácido Abscísico/metabolismo , Ácido Abscísico/fisiologia , Frutas/metabolismo , Regulação da Expressão Gênica em Archaea , Genes de Plantas/fisiologia , Litchi/genética , Litchi/crescimento & desenvolvimento , Filogenia , Reguladores de Crescimento de Planta/metabolismo , Reguladores de Crescimento de Planta/fisiologia , Plantas Geneticamente Modificadas , Alinhamento de Sequência , Tabaco
15.
J Bacteriol ; 201(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30373756

RESUMO

Halophilic Archaea are a distinctive pink color due to a carotenoid pigment called bacterioruberin. To sense or utilize light, many halophilic Archaea also produce rhodopsins, complexes of opsin proteins with a retinal prosthetic group. Both bacterioruberin and retinal are synthesized from isoprenoid precursors, with lycopene as the last shared intermediate. We previously described a regulatory mechanism by which Halobacterium salinarum bacterioopsin and Haloarcula vallismortis cruxopsin inhibit bacterioruberin synthesis catalyzed by lycopene elongase. In this work, we found that opsins in all three major Halobacteria clades inhibit bacterioruberin synthesis, suggesting that this regulatory mechanism existed in the common Halobacteria ancestor. Halophilic Archaea, which are generally heterotrophic and aerobic, likely evolved from an autotrophic, anaerobic methanogenic ancestor by acquiring many genes from Bacteria via lateral gene transfer. These bacterial "imports" include genes encoding opsins and lycopene elongases. To determine if opsins from Bacteria inhibit bacterioruberin synthesis, we tested bacterial opsins and found that an opsin from Curtobacterium, in the Actinobacteria phylum, inhibits bacterioruberin synthesis catalyzed by its own lycopene elongase, as well as that catalyzed by several archaeal enzymes. We also determined that the lycopene elongase from Halococcus salifodinae, a species from a family of Halobacteria lacking opsin homologs, retained the capacity to be inhibited by opsins. Together, our results indicate that opsin-mediated inhibition of bacterioruberin biosynthesis is a widely distributed mechanism found in both Archaea and Bacteria, possibly predating the divergence of the two domains. Further analysis may provide insight into the acquisition and evolution of the genes and their host species.IMPORTANCE All organisms use a variety of mechanisms to allocate limited resources to match their needs in their current environment. Here, we explore how halophilic microbes use a novel mechanism to allow efficient production of rhodopsin, a complex of an opsin protein and a retinal prosthetic group. We previously demonstrated that Halobacterium salinarum bacterioopsin directs available resources toward retinal by inhibiting synthesis of bacterioruberin, a molecule that shares precursors with retinal. In this work, we show that this mechanism can be carried out by proteins from halophilic Archaea that are not closely related to H. salinarum and those in at least one species of Bacteria Therefore, opsin-mediated inhibition of bacterioruberin synthesis may be a highly conserved, ancient regulatory mechanism.


Assuntos
Carotenoides/biossíntese , Halobacteriales/efeitos dos fármacos , Halobacteriales/metabolismo , Opsinas/metabolismo , Actinobacteria/química , Aerobiose , Anaerobiose , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/metabolismo , Regulação da Expressão Gênica em Archaea , Opsinas/isolamento & purificação
16.
Biosci Biotechnol Biochem ; 83(4): 695-704, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30582424

RESUMO

Replication protein A (RPA) is an essential component of DNA metabolic processes. RPA binds to single-stranded DNA (ssDNA) and interacts with multiple DNA-binding proteins. In this study, we showed that two DNA polymerases, PolB and PolD, from the hyperthermophilic archaeon Thermococcus kodakarensis interact directly with RPA in vitro. RPA was expected to play a role in resolving the secondary structure, which may stop the DNA synthesis reaction, in the template ssDNA. Our in vitro DNA synthesis assay showed that the pausing was resolved by RPA for both PolB and PolD. These results supported the fact that RPA interacts with DNA polymerases as a member of the replisome and is involved in the normal progression of DNA replication forks.


Assuntos
Proteínas Arqueais/genética , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , Regulação da Expressão Gênica em Archaea , Proteína de Replicação A/genética , Thermococcus/genética , Proteínas Arqueais/metabolismo , Clonagem Molecular , DNA Arqueal/genética , DNA Arqueal/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Replicação A/metabolismo , Thermococcus/metabolismo
17.
Appl Environ Microbiol ; 84(24)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30291120

RESUMO

"Candidatus Methanoperedens nitroreducens" is an archaeon that couples the anaerobic oxidation of methane to nitrate reduction. In natural and man-made ecosystems, this archaeon is often found at oxic-anoxic interfaces where nitrate, the product of aerobic nitrification, cooccurs with methane produced by methanogens. As such, populations of "Ca Methanoperedens nitroreducens" could be prone to regular oxygen exposure. Here, we investigated the effect of 5% (vol/vol) oxygen exposure in batch activity assays on a "Ca Methanoperedens nitroreducens" culture, enriched from an Italian paddy field. Metagenome sequencing of the DNA extracted from the enrichment culture revealed that 83% of 16S rRNA gene reads were assigned to a novel strain, "Candidatus Methanoperedens nitroreducens Verserenetto." RNA was extracted, and metatranscriptome sequencing upon oxygen exposure revealed that the active community changed, most notably in the appearance of aerobic methanotrophs. The gene expression of "Ca Methanoperedens nitroreducens" revealed that the key genes encoding enzymes of the methane oxidation and nitrate reduction pathways were downregulated. In contrast to this, we identified upregulation of glutaredoxin, thioredoxin family/like proteins, rubrerythrins, peroxiredoxins, peroxidase, alkyl hydroperoxidase, type A flavoproteins, FeS cluster assembly protein, and cysteine desulfurases, indicating the genomic potential of "Ca Methanoperedens nitroreducens Verserenetto" to counteract the oxidative damage and adapt in environments where they might be exposed to regular oxygen intrusion.IMPORTANCE "Candidatus Methanoperedens nitroreducens" is an anaerobic archaeon which couples the reduction of nitrate to the oxidation of methane. This microorganism is present in a wide range of aquatic environments and man-made ecosystems, such as paddy fields and wastewater treatment systems. In such environments, these archaea may experience regular oxygen exposure. However, "Ca Methanoperedens nitroreducens" is able to thrive under such conditions and could be applied for the simultaneous removal of dissolved methane and nitrogenous pollutants in oxygen-limited systems. To understand what machinery "Ca Methanoperedens nitroreducens" possesses to counteract the oxidative stress and survive, we characterized the response to oxygen exposure using a multi-omics approach.


Assuntos
Anaerobiose/fisiologia , Proteínas Arqueais/metabolismo , Regulação da Expressão Gênica em Archaea , Methanosarcinales/metabolismo , Estresse Oxidativo/fisiologia , Oxigênio/metabolismo , Anaerobiose/genética , Proteínas Arqueais/genética , Reatores Biológicos , Hidrolases de Éster Carboxílico/metabolismo , DNA Arqueal/isolamento & purificação , Ecossistema , Flavoproteínas/metabolismo , Glutarredoxinas/metabolismo , Hemeritrina/metabolismo , Metagenoma , Metano/metabolismo , Methanosarcinales/classificação , Methanosarcinales/genética , Nitratos/metabolismo , Oxirredução , Estresse Oxidativo/genética , Peroxidase/metabolismo , Peroxirredoxinas/metabolismo , Filogenia , RNA Ribossômico 16S/genética , Rubredoxinas/metabolismo , Análise de Sequência , Tiorredoxinas/metabolismo , Regulação para Cima , Águas Residuárias/microbiologia , Purificação da Água
18.
Annu Rev Genet ; 52: 465-487, 2018 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-30208289

RESUMO

Advances in genome-wide sequence technologies allow for detailed insights into the complexity of RNA landscapes of organisms from all three domains of life. Recent analyses of archaeal transcriptomes identified interaction and regulation networks of noncoding RNAs in this understudied domain. Here, we review current knowledge of small, noncoding RNAs with important functions for the archaeal lifestyle, which often requires adaptation to extreme environments. One focus is RNA metabolism at elevated temperatures in hyperthermophilic archaea, which reveals elevated amounts of RNA-guided RNA modification and virus defense strategies. Genome rearrangement events result in unique fragmentation patterns of noncoding RNA genes that require elaborate maturation pathways to yield functional transcripts. RNA-binding proteins, e.g., L7Ae and LSm, are important for many posttranscriptional control functions of RNA molecules in archaeal cells. We also discuss recent insights into the regulatory potential of their noncoding RNA partners.


Assuntos
Archaea/genética , Interação Gene-Ambiente , RNA Arqueal/genética , Pequeno RNA não Traduzido/genética , Archaea/metabolismo , Regulação da Expressão Gênica em Archaea , RNA Arqueal/metabolismo , Transdução de Sinais/genética
19.
PLoS Genet ; 14(9): e1007582, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30212449

RESUMO

The genomes of all organisms throughout the tree of life are compacted and organized in chromatin by association of chromatin proteins. Eukaryotic genomes encode histones, which are assembled on the genome into octamers, yielding nucleosomes. Post-translational modifications of the histones, which occur mostly on their N-terminal tails, define the functional state of chromatin. Like eukaryotes, most archaeal genomes encode histones, which are believed to be involved in the compaction and organization of their genomes. Instead of discrete multimers, in vivo data suggest assembly of "nucleosomes" of variable size, consisting of multiples of dimers, which are able to induce repression of transcription. Based on these data and a model derived from X-ray crystallography, it was recently proposed that archaeal histones assemble on DNA into "endless" hypernucleosomes. In this review, we discuss the amino acid determinants of hypernucleosome formation and highlight differences with the canonical eukaryotic octamer. We identify archaeal histones differing from the consensus, which are expected to be unable to assemble into hypernucleosomes. Finally, we identify atypical archaeal histones with short N- or C-terminal extensions and C-terminal tails similar to the tails of eukaryotic histones, which are subject to post-translational modification. Based on the expected characteristics of these archaeal histones, we discuss possibilities of involvement of histones in archaeal transcription regulation.


Assuntos
Archaea/fisiologia , DNA/metabolismo , Regulação da Expressão Gênica em Archaea/fisiologia , Histonas/fisiologia , Cristalografia por Raios X , Genoma Arqueal/fisiologia , Histonas/química , Nucleossomos/metabolismo , Transcrição Genética/fisiologia
20.
Metallomics ; 10(10): 1476-1500, 2018 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-30191942

RESUMO

The ArsR-SmtB family of proteins displays the greatest diversity among the bacterial metal-binding transcriptional regulators with regard to the variety of metal ions that they can sense. In the presence of increased levels of toxic heavy metals, these proteins dissociate from their cognate DNA upon the direct binding of metal ions to the appropriate sites, designated motifs on the proteins, either at the interface of the dimers or at the intra-subunit locations. In addition to the metal-mediated regulation, some proteins were also found to control transcription via redox reactions. In the present work, we have identified several new sequence motifs and expanded the knowledge base of metal binding sites in the ArsR-SmtB family of transcriptional repressors, and characterized them in terms of the ligands to the metal, distribution among different phyla of bacteria and archaea, amino acid propensities, protein length distributions and evolutionary interrelationships. We built structural models of the motifs to show the importance of specific residues in an individual motif. The wide abundance of these motifs in sequences of bacteria and archaea indicates the importance of these regulators in combating metal-toxicity within and outside of the hosts. We also show that by using residue composition, one can distinguish the ArsR-SmtB proteins from other metalloregulatory families. In addition, we show the importance of horizontal gene transfer in microorganisms, residing in similar habitats, on the evolution of the structural motifs in the family. Knowledge of the diverse metalloregulatory systems in microorganisms could enable us to manipulate specific genes that may result in a toxic metal-free environment.


Assuntos
Motivos de Aminoácidos , Archaea/metabolismo , Proteínas Arqueais/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Simulação por Computador , Metais/metabolismo , Transcrição Genética , Sequência de Aminoácidos , Archaea/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Regulação da Expressão Gênica em Archaea , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Modelos Moleculares , Família Multigênica , Ligação Proteica , Conformação Proteica
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