Genetic techniques for the archaea.

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.

Construction of a stable replicating shuttle vector for Caldicellulosiruptor species: use for extending genetic methodologies to other members of this genus.

The recalcitrance of plant biomass is the most important barrier to its economic conversion by microbes to products of interest. Thermophiles have special advantages for biomass conversion and members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic microbes known. In this study, we report the construction of a replicating shuttle vector for Caldicellulosiruptor species based on pBAS2, the smaller of two native C. bescii plasmids. The entire plasmid was cloned into an E. coli cloning vector containing a pSC101 origin of replication and an apramycin resistance cassette for selection in E. coli. The wild-type C. bescii pyrF locus was cloned under the transcriptional control of the regulatory region of the ribosomal protein S30EA (Cbes2105), and the resulting vector was transformed into a new spontaneous deletion mutant in the pyrFA locus of C. bescii that allowed complementation with the pyrF gene alone. Plasmid DNA was methylated in vitro with a recently described cognate methyltransferase, M.CbeI, and transformants were selected for uracil prototrophy. The plasmid was stably maintained in low copy with selection but rapidly lost without selection. There was no evidence of DNA rearrangement during transformation and replication in C. bescii. A similar approach was used to screen for transformability of other members of this genus using M.CbeI to overcome restriction as a barrier and was successful for transformation of C. hydrothermalis, an attractive species for many applications. Plasmids containing a carbohydrate binding domain (CBM) and linker region from the C. bescii celA gene were maintained with selection and were structurally stable through transformation and replication in C. bescii and E. coli.

Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement.

BACKGROUND: Thermophilic microorganisms have special advantages for the conversion of plant biomass to fuels and chemicals. Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria known. They have the ability to grow on a variety of non-pretreated biomass substrates at or near ~80°C and hold promise for converting biomass to bioproducts in a single step. As for all such relatively uncharacterized organisms with desirable traits, the ability to genetically manipulate them is a prerequisite for making them useful. Metabolic engineering of pathways for product synthesis is relatively simple compared to engineering the ability to utilize non-pretreated biomass. RESULTS: Here we report the construction of a deletion of cbeI (Cbes2438), which encodes a restriction endonuclease that is as a major barrier to DNA transformation of C. bescii. This is the first example of a targeted chromosomal deletion generated by homologous recombination in this genus and the resulting mutant, JWCB018 (ΔpyrFA ΔcbeI), is readily transformed by DNA isolated from E. coli without in vitro methylation. PCR amplification and sequencing suggested that this deletion left the adjacent methyltransferase (Cbes2437) intact. This was confirmed by the fact that DNA isolated from JWCB018 was protected from digestion by CbeI and HaeIII. Plasmid DNA isolated from C. hydrothermalis transformants were readily transformed into C. bescii. Digestion analysis of chromosomal DNA isolated from seven Caldicellulosiruptor species by using nine different restriction endonucleases was also performed to identify the functional restriction-modification activities in this genus. CONCLUSION: Deletion of the cbeI gene removes a substantial barrier to routine DNA transformation and chromosomal modification of C. bescii. This will facilitate the functional analyses of genes as well as metabolic engineering for the production of biofuels and bioproducts from biomass. An analysis of restriction-modification activities in members of this genus suggests a way forward to eliminating restriction as a barrier to DNA transformation and efficient genetic manipulation of this important group of hyperthermophiles.

Detection of a novel active transposable element in Caldicellulosiruptor hydrothermalis and a new search for elements in this genus.

We show that a previously annotated hypothetical protein is the transposase of a new and active IS element, ISCahy1, widespread in Caldicellulosiruptor species. Transposition generated an 11-bp direct repeat at the insertion site in Caldicellulosiruptor hydrothermalis, suggesting a cut-and-paste mechanism. The discovery of an active insertion sequence in Caldicellulosiruptor species led to a survey of potential IS elements in the genome sequences of eight Caldicellulosiruptor species that identified several new elements, including one novel to this genus.

Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii.

Methods for efficient growth and manipulation of relatively uncharacterized bacteria facilitate their study and are essential for genetic manipulation. We report new growth media and culture techniques for Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium known. A low osmolarity defined growth medium (LOD) was developed that avoids problems associated with precipitates that form in previously reported media allowing the monitoring of culture density by optical density at 680 nm (OD(680)) and more efficient DNA transformation by electroporation. This is a defined minimal medium and does not support growth when a carbon source is omitted, making it suitable for selection of nutritional markers as well as the study of biomass utilization by C. bescii. A low osmolarity complex growth medium (LOC) was developed that dramatically improves growth and culture viability during storage, making it a better medium for routine growth and passaging of C. bescii. Both media contain significantly lower solute concentration than previously published media, allowing for flexibility in developing more specialized media types while avoiding the issues of growth inhibition and cell lysis due to osmotic stress. Plating on LOD medium solidified by agar results in ~1,000-fold greater plating efficiency than previously reported and allows the isolation of discrete colonies. These new media represent a significant advance for both genetic manipulation and the study of biomass utilization in C. bescii, and may be applied broadly across the Caldicellulosiruptor genus.

Methylation by a unique α-class N4-cytosine methyltransferase is required for DNA transformation of Caldicellulosiruptor bescii DSM6725.

Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute) and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.

Recombinogenic properties of Pyrococcus furiosus strain COM1 enable rapid selection of targeted mutants.

We recently reported the isolation of a mutant of Pyrococcus furiosus, COM1, that is naturally and efficiently competent for DNA uptake. While we do not know the exact nature of this mutation, the combined transformation and recombination frequencies of this strain allow marker replacement by direct selection using linear DNA. In testing the limits of its recombination efficiency, we discovered that marker replacement was possible with as few as 40 nucleotides of flanking homology to the target region. We utilized this ability to design a strategy for selection of constructed deletions using PCR products with subsequent excision, or "pop-out," of the selected marker. We used this method to construct a "markerless" deletion of the trpAB locus in the GLW101 (COM1 ΔpyrF) background to generate a strain (JFW02) that is a tight tryptophan auxotroph, providing a genetic background with two auxotrophic markers for further strain construction. The utility of trpAB as a selectable marker was demonstrated using prototrophic selection of plasmids and genomic DNA containing the wild-type trpAB alleles. A deletion of radB was also constructed that, surprisingly, had no obvious effect on either recombination or transformation, suggesting that its gene product is not involved in the COM1 phenotype. Attempts to construct a radA deletion mutation were unsuccessful, suggesting that this may be an essential gene. The ease and speed of this procedure will facilitate the construction of strains with multiple genetic changes and allow the construction of mutants with deletions of virtually any nonessential gene.

Defining components of the chromosomal origin of replication of the hyperthermophilic archaeon Pyrococcus furiosus needed for construction of a stable replicating shuttle vector.

We report the construction of a series of replicating shuttle vectors that consist of a low-copy-number cloning vector for Escherichia coli and functional components of the origin of replication (oriC) of the chromosome of the hyperthermophilic archaeon Pyrococcus furiosus. In the process of identifying the minimum replication origin sequence required for autonomous plasmid replication in P. furiosus, we discovered that several features of the origin predicted by bioinformatic analysis and in vitro binding studies were not essential for stable autonomous plasmid replication. A minimum region required to promote plasmid DNA replication was identified, and plasmids based on this sequence readily transformed P. furiosus. The plasmids replicated autonomously and existed in a single copy. In contrast to shuttle vectors based on a plasmid from the closely related hyperthermophile Pyrococcus abyssi for use in P. furiosus, plasmids based on the P. furiosus chromosomal origin were structurally unchanged after transformation and were stable without selection for more than 100 generations.

Identification and characterization of CbeI, a novel thermostable restriction enzyme from Caldicellulosiruptor bescii DSM 6725 and a member of a new subfamily of HaeIII-like enzymes.

Potent HaeIII-like DNA restriction activity was detected in cell-free extracts of Caldicellulosiruptor bescii DSM 6725 using plasmid DNA isolated from Escherichia coli as substrate. Incubation of the plasmid DNA in vitro with HaeIII methyltransferase protected it from cleavage by HaeIII nuclease as well as cell-free extracts of C. bescii. The gene encoding the putative restriction enzyme was cloned and expressed in E. coli with a His-tag at the C-terminus. The purified protein was 38 kDa as predicted by the 981-bp nucleic acid sequence, was optimally active at temperatures between 75°C and 85°C, and was stable for more than 1 week when stored at 35°C. The cleavage sequence was determined to be 5'-GG/CC-3', indicating that CbeI is an isoschizomer of HaeIII. A search of the C. bescii genome sequence revealed the presence of both a HaeIII-like restriction endonuclease (Athe 2438) and DNA methyltransferase (Athe 2437). Preliminary analysis of other Caldicellulosiruptor species suggested that this restriction/modification activity is widespread in this genus. A phylogenetic analysis based on sequence alignment and conserved motif searches identified features of CbeI distinct from other members of this group and classified CbeI as a member of a novel subfamily of HaeIII-like enzymes.

Which Sry locus is the hypertensive Y chromosome locus?

The Y chromosome of the spontaneously hypertensive rat (SHR) contains a genetic component that raises blood pressure compared with the Wistar-Kyoto (WKY) Y chromosome. This research tests the Sry gene complex as the hypertensive component of the SHR Y chromosome. The Sry loci were sequenced in 1 strain with a hypertensive Y chromosome (SHR/Akr) and 2 strains with a normotensive Y chromosome (SHR/Crl and WKY/Akr). Both SHR strains have 7 Sry loci, whereas the WKY strain has 6. The 6 loci in common between SHR and WKY strains were identical in the sequence compared (coding region, 392-bp 5' prime flanking, 1200-bp 3' flanking). Both SHR strains have a locus (Sry3) not found in WKY rats, but this locus is different between SHR/Akr and SHR/Crl rats. Six mutations have accumulated in Sry3 between the SHR strains, whereas the other 6 Sry loci are identical. This pattern of an SHR-specific locus and mutation in this locus in SHR/Crl coinciding with the loss of Y chromosome hypertension is an expected pattern if Sry3 is the Y chromosome-hypertensive component. The SHR/y strain showed a significant increase in total Sry expression in the kidney between 4 and 15 weeks of age. There are significant differences in Sry expression between adrenal glands and the kidney (15 to 30 times higher in kidneys) but no significant differences between strains. These results, along with previous studies demonstrating an interaction of Sry with the tyrosine hydroxylase promoter and increased blood pressure with exogenous Sry expression, suggest the Sry loci as the hypertensive component of the SHR Y chromosome.

Genomic and expression analysis of multiple Sry loci from a single Rattus norvegicus Y chromosome.

BACKGROUND: Sry is a gene known to be essential for testis determination but is also transcribed in adult male tissues. The laboratory rat, Rattus norvegicus, has multiple Y chromosome copies of Sry while most mammals have only a single copy. DNA sequence comparisons with other rodents with multiple Sry copies are inconsistent in divergence patterns and functionality of the multiple copies. To address hypotheses of divergence, gene conversion and functional constraints, we sequenced Sry loci from a single R. norvegicus Y chromosome from the Spontaneously Hypertensive Rat strain (SHR) and analyzed DNA sequences for homology among copies. Next, to determine whether all copies of Sry are expressed, we developed a modification of the fluorescent marked capillary electrophoresis method to generate three different sized amplification products to identify Sry copies. We applied this fragment analysis method to both genomic DNA and cDNA prepared from mRNA from testis and adrenal gland of adult male rats. RESULTS: Y chromosome fragments were amplified and sequenced using primers that included the entire Sry coding region and flanking sequences. The analysis of these sequences identified six Sry loci on the Y chromosome. These are paralogous copies consistent with a single phylogeny and the divergence between any two copies is less than 2%. All copies have a conserved reading frame and amino acid sequence consistent with function. Fragment analysis of genomic DNA showed close approximations of experimental with predicted values, validating the use of this method to identify proportions of each copy. Using the fragment analysis procedure with cDNA samples showed the Sry copies expressed were significantly different from the genomic distribution (testis p < 0.001, adrenal gland p < 0.001), and the testis and adrenal copy distribution in the transcripts were also significantly different from each other (p < 0.001). Total Sry transcript expression, analyzed by real-time PCR, showed significantly higher levels of Sry in testis than adrenal gland (p, 0.001). CONCLUSION: The SHR Y chromosome contains at least 6 full length copies of the Sry gene. These copies have a conserved coding region and conserved amino acid sequence. The pattern of divergence is not consistent with gene conversion as the mechanism for this conservation. Expression studies show multiple copies expressed in the adult male testis and adrenal glands, with tissue specific differences in expression patterns. Both the DNA sequence analysis and RNA transcript expression analysis are consistent with more than one copy having function and selection preventing divergence although we have no functional evidence.