Long stretches of short tandem repeats are present in the largest replicons of the Archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning

We report the presence of long stretches of tandem repeats in the genome of the halophilic Archaea Haloferax mediterranei and Haloferax volcanii A 30 bp sequence with dyad symmetry (including 5 bp inverted repeats) was repeated in tandem, interspersed with 33–39 bp unique sequences. This structure extends for long stretches — 1.4kb at one location in H. mediterranei chromosome and about 3kb in the H. volcanii chromosome. The tandem repeats (designated TREPs) show a similar distribution in both organisms, appearing once or twice in the H. volcanii and H. mediterranei chromosomes, and once in the largest, probably essential megaplasmid of each organism but not in the smaller replicons. Sequencing of the structures in both H. volcanii replicons revealed an extremely high sequence conservation in both replicons within the species, as well as in the different organisms. Homologous sequences have also been found in other more distantly related halophilic members of the Archaea. Transformation of H. volcanii with a recombinant plasmid containing a 1.1 kb fragment of the TREPs produced significant alterations in the host cells, particularly in terms of cell viability. The introduction of extra copies of TREPs within the vector significantly alters the distribution of the genome among the daughter cells, as observed by DAPI staining. Although the precise biological role cannot be completely ascertained, all the data conform with the tandem repeats being involved in replicon partitioning in halobacteria.     

Alignment of genes and SwaI restriction sites to the BamHI genomic map of Haloferax mediterranei

Abstract Eleven Swa I restriction sites and ten genes have been aligned to the Bam HI physical map of the main chromosome of the halobacterium Haloferax mediterranei ATCC33500, using two-dimensional pulsed field gel electrophoresis and hybridization experiments.     

Regulatory RNAs in Haloferax volcanii

In organisms of all three domains of life, a plethora of sRNAs (small regulatory RNAs) exists in addition to the well-known RNAs such as rRNAs, tRNAs and mRNAs. Although sRNAs have been well studied in eukaryotes and in bacteria, the sRNA population in archaea has just recently been identified and only in a few archaeal species. In the present paper, we summarize our current knowledge about sRNAs and their function in the halophilic archaeon Haloferax volcanii. Using two different experimental approaches, 111 intergenic and 38 antisense sRNAs were identified, as well as 42 tRFs (tRNA-derived fragments). Observation of differential expression under various conditions suggests that these sRNAs might be active as regulators in gene expression like their bacterial and eukaryotic counterparts. The severe phenotypes observed upon deletion and overexpression of sRNA genes revealed that sRNAs are involved in, and important for, a variety of biological functions in H. volcanii and possibly other archaea. Investigation of the Haloferax Lsm protein suggests that this protein is involved in the archaeal sRNA pathway.     

地中海富盐菌中phaB基因的鉴定及PHBV前体供应途径的分析

【目的】进一步揭示地中海富盐菌(Haloferax mediterranei)中聚羟基丁酸羟基戊酸酯[poly(3-hydroxybutyrate-co-3-hydroxyvalerate),PHBV]前体供应的途径并鉴定其中的关键基因。【方法】将细菌及其他古菌中已鉴定的与聚羟基脂肪酸酯前体(3-羟基脂酰-CoA)供应相关的酶与地中海富盐菌预测的全蛋白质组进行同源性比对,得到相似性比较高的5个基因,分别命名为:phaB1,phaB2,phaJ1,phaJ2和phaJ3。首先利用RT-PCR检测了5个基因在产PHBV的条件下的转录情况。然后利用同源重组双交换的方法,将5个基因分别或组合敲除,得到突变株:ΔphaB1,ΔphaB2,ΔphaJ1,ΔphaJ2,ΔphaJ3,ΔphaB1phaB2,ΔphaJ1phaJ2和ΔphaJ1phaJ2phaJ3。并在突变株ΔphaB1phaB2中分别互补phaB1和phaB2基因。【结果】无论是将3个phaJ基因单独敲除,还是组合敲除,对地中海富盐菌PHBV的积累都没有明显影响。单独敲除phaB1基因对PHBV的积累没有明显影响,单独敲除phaB2基因导致突变株PHBV产量明显下降,而且3-HV单体组分所占的摩尔比例也有所下降。将phaB1和phaB2基因同时敲除后,得到的突变株不再产生PHBV。【结论】在地中海富盐菌中可能主要存在由乙酰-CoA和丙酰-CoA提供PHBV前体的途径,其中编码乙酰乙酰-CoA还原酶的两个基因即为phaB1和phaB2。     

The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases

The gene encoding dihydrofolate reductase, hdrA, from the extremely halophilic archaeon Haloferax volcanii was previously isolated from a spontaneous trimethoprim-resistant mutant in a DNA sequence that had undergone amplification. Here, we show that deletion of hdrA did not affect growth in minimal medium and that the strain carrying the deletion remained sensitive to trimethoprim. A spontaneous trimethoprim-resistant colony was isolated in the hdrA deletion strain and found to possess a new DNA amplification. Sequencing of the amplification revealed a second, substantially different, dihydrofolate reductase gene, hdrB, which was found to be located immediately downstream of the thymidylate synthase gene, hts. The physiological role of hDHFR-1 and hDHFR-2 was determined by generating Haloferax volcanii strains in which each gene, hdrA or hdrB, or both genes were deleted. It was found that hdrB alone can support growth of Haloferax volcanii in minimal medium, whereas hdrA alone can support growth of Haloferax volcanii in minimal medium only when the medium is supplemented with thymidine. It was also shown that, in contrast to Escherichia coli, the DeltahdrA, DeltahdrB double deletion mutant is viable in the presence of a functional thymidylate synthase gene. The hdrB gene was overexpressed in Escherichia coli and the enzyme purified to homogeneity. The biochemical properties of the new enzyme (hDHFR-2) are markedly different from those of hDHFR-1. The use of the dihydrofolate reductase and thymidylate synthase genes as stable selectable markers is described.     

Characterization of a gene involved in histidine biosynthesis in Halobacterium (Haloferax) volcanii: isolation and rapid mapping by transformation of an auxotroph with cosmid DNA.

Techniques for the transformation of halophilic archaebacteria have been developed recently and hold much promise for the characterization of these organisms at the molecular level. In order to understand genome organization and gene regulation in halobacteria, we have begun the characterization of genes involved in amino acid biosynthesis in Halobacterium (Haloferax) volcanii. These studies are facilitated by the many auxotrophic mutants of H. volcanii that have been isolated. In this project we demonstrate that cosmid DNA prepared from Escherichia coli can be used to transform an H. volcanii histidine auxotroph to prototrophy. A set of cosmid clones covering most of the genome of H. volcanii was used to isolate the gene which is defective in H. volcanii WR256. Subcloning identified a 1.6-kilobase region responsible for transformation. DNA sequence analysis of this region revealed an open reading frame encoding a putative protein 361 amino acids in length. A search of the DNA and protein data bases revealed that this open reading frame encodes histidinol-phosphate aminotransferase (EC 2.6.1.9), the sequence of which is also known for E. coli, Bacillus subtilis, and Saccharomyces cerevisiae.     

Genomic organization of the halophilic archaeon Haloferax mediterranei: physical map of the chromosome.

Pulsed field gel electrophoresis (PFG) has been used to study the genomic organization of the halophilic archaeon Haloferax mediterranei. Analysis of the different genomic elements as well as the restriction patterns obtained with several endonucleases revealed that this microorganism has a circular chromosome of 2.9 Mb and, at least, three extrachromosomal elements of 490, 320 and 130 kb respectively. The complete physical map of the chromosome for the endonucleases PacI and BamHI has been constructed, and several BcII, BgIII and DraI restriction fragments have been aligned on these maps. The localization of heterologous and homologous genes on the physical map, including those for rRNA, lay the ground work for the construction of a genetic map.     

Complete Genome Sequence of the Metabolically Versatile Halophilic Archaeon Haloferax mediterranei, a Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Producer

Haloferax mediterranei, an extremely halophilic archaeon, has shown promise for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated cheap carbon sources. Here we report the complete genome (3,904,707 bp) of H. mediterranei CGMCC 1.2087, consisting of one chromosome and three megaplasmids.     

Characterization of a tightly controlled promoter of the halophilic archaeon Haloferax volcanii and its use in the analysis of the essential cct1 gene

A system where archaeal gene expression could be controlled by simple manipulation of growth conditions would enable the construction of conditional lethal mutants in essential genes, and permit the controlled overproduction of proteins in their native host. As tools for the genetic manipulation of Haloferax volcanii are well developed, we set out to identify promoters with a wide dynamic range of expression in this organism. Tryptophan is the most costly amino acid for the cell to make, so we reasoned that tryptophan-regulated promoters might be good candidates. Microarray analysis of H. volcanii gene expression in the presence and absence of tryptophan identified a tryptophanase gene (tna) that showed strong induction in the presence of tryptophan. qRT-PCR revealed a very fast response and an up to 100-fold induction after tryptophan addition. This result has been confirmed using three independent reporter genes (cct1, pyrE2 and bgaH). Vectors containing this promoter will be very useful for investigating gene function in H. volcanii and potentially in other halophilic archaea. To demonstrate this, we used the promoter to follow the consequences of depletion of the essential chaperonin protein CCT1, and to determine the ability of heterologous CCT proteins to function in H. volcanii.     

Functional role for a 2-oxo acid dehydrogenase in the halophilic archaeon Haloferax volcanii

The archaeon Haloferax volcanii was previously shown to contain and transcribe the genes for a 2-oxo acid dehydrogenase (OADH) complex, but their presence remained a mystery because no enzymatic activity with any of the known OADH substrates could be found, and an inactivation of one of the genes did not lead to any phenotype. Here we report the identification of an additional oadh gene cluster in the genome of H. volcanii. In contrast to previously known oadh loci, it contains three genes, oadh2A1, oadh2A2, and oadh2ld, with coding capacity for the E1alpha and E1beta subunits and an unattached lipoyl domain, but it is devoid of the genes for a complete E2 and an E3. The genes were isolated by complementation of a nitrate respiration-deficient mutant of H. volcanii and therefore were shown to be functional in vivo. Phylogenetic analyses revealed that the deduced E1alpha and E1beta subunits of OADH2 group with bacterial acetoin dehydrogenases but not with the OADH1 subunits, and thus, H. volcanii has obtained the two gene groups independently. Comparison of the wild type and the mutant allowed us to exclude a function of OADH2 in the aerobic or anaerobic degradation of acetoin or glucose. Instead, it could be shown that OADH2 is important during nitrate-respirative growth on Casamino Acids. Many physiological and biochemical experiments failed to indicate that OADH2 uses any of the previously known OADH substrates. Growth potentials of the mutant were markedly different in media with a single carbon source versus media with mixed carbon sources.     

Glucose inhibits the formation of gas vesicles in Haloferax volcanii transformants

The effect of glucose on the formation of gas vesicles was investigated in Haloferax mediterranei and Hfx.volcanii transformants containing the mc- gvp gene cluster of Hfx. mediterranei (mc-vac transformants). Increasing amounts of glucose in the medium resulted in a successive decrease in the amount of gas vesicles in both species, with a complete inhibition of their formation at glucose concentrations of > 70 mM in mc-vac transformants, and 100 mM in Hfx. mediterranei . Maltose and sucrose imposed a similar inhibitory effect, whereas xylose, arabinose, lactose, pyruvate and 2-deoxy-glucose had no influence on the gas vesicle formation in mc-vac transformants. The activities of the two mc-vac promoters were strongly reduced in mc-vac transformants grown in the presence of > 50 mM glucose. The gas vesicle overproducing ΔD transformant (lacking the repressing protein GvpD) also showed a glucose-induced lack of gas vesicles, indicating that GvpD is not involved in the repression. The addition of glucose was useful to block gas vesicle formation at a certain stage during growth, and vice versa , gas vesicle synthesis could be induced when a glucose-grown culture was shifted to medium lacking glucose. Both procedures will enable the investigation of defined stages during gas vesicle formation.     

Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes

Since most archaea are extremophilic and difficult to cultivate, our current knowledge of their biology is confined largely to comparative genomics and biochemistry. Haloferax volcanii offers great promise as a model organism for archaeal genetics, but until now there has been a lack of a wide variety of selectable markers for this organism. We describe here isolation of H. volcanii leuB and trpA genes encoding 3-isopropylmalate dehydrogenase and tryptophan synthase, respectively, and development of these genes as a positive selection system. DeltaleuB and DeltatrpA mutants were constructed in a variety of genetic backgrounds and were shown to be auxotrophic for leucine and tryptophan, respectively. We constructed both integrative and replicative plasmids carrying the leuB or trpA gene under control of a constitutive promoter. The use of these selectable markers in deletion of the lhr gene of H. volcanii is described.     

Pseudouridine formation in archaeal RNAs: The case of Haloferax volcanii

Pseudouridine (Ψ), the isomer of uridine, is commonly found at various positions of noncoding RNAs of all organisms. Ψ residues are formed by a number of single- or multisite specific Ψ synthases, which generally act as stand-alone proteins. In addition, in Eukarya and Archaea, specific ribonucleoprotein complexes, each containing a distinct box H/ACA guide RNA and four core proteins, can produce Ψ at many sites of different cellular RNAs. Cbf5 is the core Ψ synthase in these complexes. Using Haloferax volcanii as an archaeal model organism, we show that, contrary to eukaryotes, the Cbf5 homolog (HVO_2493) is not essential in this archaeon. The Cbf5-deleted strain of H. volcanii completely lacks Ψ at positions 1940, 1942, 2605, and 2591 (Escherichia coli positions 1915, 1917, 2572, and 2586) of its 23S rRNA, and contains reduced steady-state levels of some box H/ACA RNAs. Archaeal Cbf5 is known to have tRNA Ψ55 synthase activity in vitro but we could not confirm this activity in vivo in H. volcanii. Conversely, the Pus10 (previously PsuX) homolog (HVO_1979), which can produce tRNA Ψ55, as well as Ψ54 in vitro, is shown here to be essential in H. volcanii, whereas the corresponding tRNA Ψ55 synthases, Pus4 and TruB, are not essential in yeast and E. coli, respectively. Finally, we demonstrate that HVO_1852, the TruA/Pus3 homolog, is responsible for the pseudouridylation of position 39 in H. volcanii tRNAs and that the corresponding gene is not essential.     

Purification and characterisation of a possible assimilatory nitrite reductase from the halophile archaeon Haloferax mediterranei

The nitrite reductase from the extreme halophilic archaeon, Haloferax mediterranei, has been purified and characterised. H. mediterranei is capable of growing in a minimal medium (inorganic salts and glucose as a carbon source) with nitrate as the only nitrogen source. The overall purification was 46-fold with about 4% recovery of activity. The enzyme is a monomeric protein of approximately 66 kDa. A pH of 7.5 and high temperatures up to 60 degrees C are necessary for optimum activity. Reduced methyl viologen has been found to be an electron donor as effective as ferredoxin. NADPH and NADH, which are electron donors in nitrite reductases from different non-photosynthetic bacteria, were not effective with nitrite reductase from H. mediterranei.     

Cloning and sequencing of ftsZ homolog from extremely halophilic archaeon Haloarcula japonica strain TR-1.

The gene encoding FtZ was cloned from triangular disc-shaped extremely halophilic archaeon Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the possible ftsZ gene revealed that the structural gene consisted of an open reading frame of 1,182 nucleotides encoding 394 amino acids. The deduced amino acid sequence of the Ha. japonica FtsZ showed high identities with those Halobacterium salinarom, Haloferax volcanii and Haloferax mediterranei FtsZs.     

A bioluminescent reporter for the halophilic archaeon Haloferax volcanii

Haloarchaea have evolved to thrive in hypersaline environments. Haloferax volcanii is of particular interest due to its genetic tractability; however, few in vivo reporters exist for halophiles. Haloarchaeal proteins evolved characteristics that promote proper folding and function at high salt concentrations, but many mesophilic reporter proteins lack these characteristics. Mesophilic proteins that acquire salt-stabilizing mutations, however, can lead to proper function in haloarchaea. Using laboratory-directed evolution, we developed and demonstrated an in vivo luciferase that functions in the hypersaline cytosol of H. volcanii.