Deletion of the archaeal histone in Methanosarcina mazei Gö1 results in reduced growth and genomic transcription.
ABSTRACT HMm is the only archaeal histone in Methanosarcina mazei Göl and recombinant HMm, synthesized by expression of MM1825 in Escherichia coli, has been purified and confirmed to have the DNA binding and compaction properties characteristic of an archaeal histone. Insertion of a puromycin resistance conferring cassette (pac) into MM1825 was not lethal but resulted in mutants (M. mazei MM1825::pac) that have impaired ability to grow on methanol and trimethylamine. Loss of HMm also resulted in increased sensitivity to UV light and decreased transcript levels for approximately 25% of all M. mazei genes. For most genes, the transcript decrease was 3- to 10-fold, but transcripts of MM483 (small heat-shock protein), MM1688 (trimethylamine:corrinoid methyl transferase) and MM3195 (transcription regulator), were reduced 100-, 100- and 25-fold, respectively, in M. mazei MM1825::pac cells. Transcripts of only five adjacent genes that appear to constitute an aromatic amino acid biosynthetic operon were elevated in M. mazei MM1825::pac cells. Complementary synthesis of HMm from a plasmid transformed into M. mazei MM1825::pac restored wild-type growth and transcript levels.
- SourceAvailable from: ncbi.nlm.nih.gov[Show abstract] [Hide abstract]
ABSTRACT: Archaeal histones wrap DNA into complexes, designated archaeal nucleosomes that resemble the tetrasome core of a eukaryotic nucleosome. Therefore, all DNA interactions in vivo in Thermococcus kodakarensis, the most genetically versatile model species for archaeal research, must occur in the context of a histone-bound chromosome. Here we report the construction and properties of T. kodakarensis strains that have TK1413 or TK2289 deleted, the genes that encode HTkA and HTkB, respectively, the two archaeal histones present in this Archaeon. All attempts to generate a strain with both TK1413 and TK2289 deleted were unsuccessful arguing that histone-mediated event(s) in T. kodakarensis are essential. The HTkA and HTkB amino acid sequences are 84% identical (56/67) and 94% (63/67) similar but despite this homology, and their apparent redundancy in terms of supporting viability, the absence of HTkA and HTkB resulted in differences in growth and in quantitative and qualitative differences in genome transcription. A most surprising result was that deletion of TK1413 (ΔhtkA) resulted in a T. kodakarensis strain that is no longer amenable to transformation whereas deletion of TK2289 (ΔhtkB) had no detrimental effects on transformation. Potential roles for the archaeal histones in regulating gene expression, and for HTkA in DNA uptake and recombination are discussed.Journal of bacteriology 10/2012; · 3.94 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The clustered regularly interspaced short palindromic repeats (CRISPR) system represents a highly adaptive and heritable defense system against foreign nucleic acids in bacteria and archaea. We analyzed the two CRISPR-Cas systems in Methanosarcina mazei strain Gö1. Although belonging to different subtypes (I-B and III-B), the leaders and repeats of both loci are nearly identical. Also, despite many point mutations in each array, a common hairpin motif was identified in the repeats by a bioinformatics analysis and in vitro structural probing. The expression and maturation of CRISPR-derived RNAs (crRNAs) were studied in vitro and in vivo. Both respective potential Cas6b-type endonucleases were purified and their activity tested in vitro. Each protein showed significant activity and could cleave both repeats at the same processing site. Cas6b of subtype III-B, however, was significantly more efficient in its cleavage activity compared with Cas6b of subtype I-B. Northern blot and differential RNaseq analyses were performed to investigate in vivo transcription and maturation of crRNAs, revealing generally very low expression of both systems, whereas significant induction at high NaCl concentrations was observed. crRNAs derived proximal to the leader were generally more abundant than distal ones and in vivo processing sites were clarified for both loci, confirming the previously well-established 8 nt 5' repeat tags. The 3'-ends were more diverse, but generally ended in a prefix of the following repeat sequence (3'-tag). The analysis further revealed a 5'-hydroxy and 3'-phosphate termini architecture of small crRNAs specific for cleavage products of Cas6 endonucleases from type I-E and I-F and type III-B.RNA biology 04/2013; 10(5). · 5.56 Impact Factor
Article: Genetic Techniques for the Archaea.[Show abstract] [Hide abstract]
ABSTRACT: Genetic techniques for the Archaea have undergone a rapid expansion in complexity, resulting in increased exploration of the role of Archaea in the environment and detailed analyses of the molecular physiology and information-processing systems in the third domain of life. Complementary gains in describing the ever-increasing diversity of archaeal organisms have allowed these techniques to be leveraged in new and imaginative ways to elucidate shared and unique aspects of archaeal diversity and metabolism. In this review, we introduce the four archaeal clades for which advanced genetic techniques are available-the methanogens, halophiles, Sulfolobales, and Thermococcales-with the aim of providing an overall profile of the advantages and disadvantages of working within each clade, as essentially all of the genetically accessible archaeal organisms require unique culturing techniques that present real challenges. We discuss the full repertoire of techniques possible within these clades while highlighting the recent advances that have been made by taking advantage of the most prominent techniques and approaches. Expected final online publication date for the Annual Review of Genetics Volume 47 is November 23, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.Annual Review of Genetics 09/2013; · 17.44 Impact Factor