Transformation of the archaebacterium Halobacterium volcanii with genomic DNA.

We describe optimization of a transformation system for the halophilic archaebacterium Halobacterium volcanii. Transformation of spheroplasts in the presence of polyethylene glycol permits the uptake and expression of high-molecular-weight linear fragments of genomic DNA as well as plasmid or bacteriophage DNA. Transformations can be performed with either fresh or frozen cell preparations. Auxotrophic mutants were transformed to prototrophy with genomic DNA from wild-type cells with efficiencies of 5 x 10(4)/micrograms of DNA and frequencies of 8 x 10(-5) per regenerated spheroplast. The overall efficiency of transformation with genomic DNA implies that genetic recombination is an efficient process in H. volcanii.     

Sequence analysis of the insertion element ISH1.8 and of associated structural changes in the genome of phage PhiH of the archaebacterium Halobacterium halobium

We have sequenced the insertion element ISH1.8 which can be present in one or two copies in the genome of phage ΦH of Halobacterium halobium. ISH1.8 is 1895 bp long, has no inverted repeat at its ends, and one only of the two copies is flanked by two 5-bp duplications. An 8-bp sequence composed of 4 bp from each end of ISH1.8 is present in both sites lacking the element. This 8-bp sequence could either be a specific insertion sequence or a part of the element that is left behind upon deletion. The plasmid pΦHL, consisting of the invertible L segment of the phage genome which is, in ΦH2 and ΦH5, flanked by two copies of ISH1.8, contains 112 bp of ISH1.8 and is released from the phage genome by recombination within a direct repeat of 9 bp. This 9-bp sequence (TCCCGCCCT) exists as an inverted repeat in ISH1.8 and therefore as two distinct repeats in phage genomes containing two copies of ISH1.8 in inverted orientation.     

Dihydrofolate reductase of the extremely halophilic archaebacterium Halobacterium volcanii. The enzyme and its coding gene

Halobacterium volcanii mutants that are resistant to the dihydrofolate reductase inhibitor trimethoprim contain DNA sequence amplifications. This paper describes the cloning and nucleic acid sequencing of the amplified DNA sequence of the H. volcanii mutant WR215. This sequence contains an open reading frame that codes for an amino acid sequence that is homologous to the amino acid sequences of dihydrofolate reductases from different sources. As a result of the gene amplification, the trimethoprim-resistant mutant overproduces dihydrofolate reductase. This enzyme was purified to homogeneity using ammonium sulfate-mediated chromatographies. It is shown that the enzyme comprises 5% of the cell protein. The amino acid sequence of the first 15 amino acids of the enzyme fits the coding sequence of the gene. Preliminary biochemical characterization shows that the enzyme is unstable at salt concentrations lower than 2 M and that its activity increases with increase in the KCl or NaCl concentrations.     

Efficient transfection of the archaebacterium Halobacterium halobium.

We developed an efficient polyethylene glycol-mediated spheroplast transfection method for the extremely halophilic archaebacterium Halobacterium halobium. The 59-kilobase-pair linear phage phi H DNA molecule routinely produced between 5 X 10(6) and 2 X 10(7) transfectants per microgram of DNA. Between 0.5 and 1% of spheroplasts were transfected per microgram of luminal diameter H DNA. Under our conditions, survival and regeneration of H. halobium spheroplasts were also quite efficient, suggesting that this method will be useful for introducing other DNAs into these bacteria.     

The alpha-operon equivalent genome region in the extreme halophilic archaebacterium Haloarcula (Halobacterium) marismortui.

The genome region of the extreme halophilic archaebacterium Haloarcula marismortui equivalent to the alpha-operon of Escherichia coli has been characterized. In H. marismortui, the alpha-operon was found to be located immediately upstream from the S9 gene cluster. The gene order in the halobacterial alpha-operon, given according to the gene products, is tRNA(Ser), HmaS13, HmaS4, HmaS11, and HmaRp alpha. Compared to the corresponding operon from E. coli, the halobacterial gene organization differs in (i) the presence of a gene for tRNA(Ser) (GCU), (ii) the reversed order of the genes for the ribosomal proteins HmaS11 and HmaS4, and (iii) the absence of the gene coding for the ribosomal protein L17. The primary structure of HmaRp alpha shows high similarity to a subunit of eukaryotic RNA polymerase II (YeaRpB3, HsaRpB33), whereas the similarity to the eubacterial alpha-subunit of RNA polymerase is only weak.     

Novel insertion sequence-like elements in phytoplasma strains of the aster yellows group are putative new members of the IS3 family

Novel insertion sequence (IS)-like elements were isolated and characterized from phytoplasma strains in the aster yellows (AY) group (16SrI). The IS-like elements were cloned from phytoplasma strains AY1 and NJAY or PCR-amplified from 15 additional strains representing nine subgroups in the AY group using primers based on sequences of the putative transposases (Tpases). All IS-like elements contained sequences encoding similar Tpases of 321 amino acids (320 for strain CPh). Substantial amino acid sequence variability suggested multiple species of Tpases or IS-like elements exist in the AY phytoplasma group. These Tpases have an identical DDE motif that is most similar to the DDE consensus of Tpases in the IS3 family.     

Chromosomal structure of the halophilic archaebacterium Halobacterium salinarium.

The chromosomal structure of the extremely halophilic archaebacterium Halobacterium salinarium was examined. Sheared chromosomes prepared from the bacteria in the late exponential phase were separated into two peaks (peaks I and II) by sucrose gradient centrifugation, suggesting that the chromosomes consist of two parts differing in quality. The UV spectra of peaks I and II resembled those of DNA and eukaryotic chromatin, respectively. Electron microscopic observations revealed that the major component of peak I was protein-free DNA, while the major components of peak II were rugged thick fibers with a diameter of 17 to 20 nm. The rugged fibers basically consisted of bacterial nucleosome-like structures composed of DNA and protein, as demonstrated in experiments with proteinase and nuclease digestion. Whole-mount electron microscopic observations of the chromosomes directly spread onto a water surface revealed a configuration in which the above-described regions were localized on a continuous DNA fiber. From these results it is concluded that the H. salinarium chromosome is composed of regions of protein-free DNA and DNA associated with nucleosome-like structures. Peaks I and II were predominant in the early exponential phase and stationary phase, respectively; therefore, the transition of the chromosome structure between non-protein-associated and protein-associated forms seems to be related to the bacterial growth phase.     

Variability in the distribution and composition of insertion sequence-like elements in strains of Mycoplasma fermentans

Mycoplasma fermentans has been reported to be pathogenic for man. All fourteen strains tested contain an insertion sequence-like element (ISLE) which may be present in multiple copies. To determine whether ISLE copies are similarly distributed in different strains of M. fermentans, restriction enzyme digest fragments of genomic DNA from 14 isolates, from a variety of sources, were separated by electrophoresis, blotted and hybridized to a biotin labelled polymerase chain reaction (PCR) amplified fragment of ISLE. A range of patterns was observed suggesting that the element has a tendency to undergo rearrangement within the genome. Analysis of ISLE sequences revealed inter- and intra-strain polymorphisms.     

Detailed physical map and set of overlapping clones covering the genome of the archaebacterium Haloferax volcanii DS2.

An integrated approach of "bottom up" and "top down" mapping has produced a minimal set of overlapping cosmid clones covering 96% of the 4140 kilobase-pairs (kbp) Haloferax volcanii DS2 genome and a completely closed physical map. This genome is partitioned into five replicons: a 2920 kbp chromosome and four plasmids, of 690 kbp (pHV4), 442 kbp (pHV3), 86 kbp(pHV1) and 6.4 kbp (pHV2). A restriction map for six infrequently-cutting restriction enzymes was constructed, representing a total of 903 sites in the cloned DNA. We have placed the two ribosomal RNA operons, the genes for 7 S RNA and for RNaseP RNA and 22 protein-coding genes on the map. Restriction site frequencies show significant variation in different portions of the genome. The regions of high site density correspond to halobacterial satellite or FII DNA which includes two small regions of the chromosome, the plasmids pHV1 and pHV2, and half of pHV4, but not pHV3.     

Genes for tryptophan biosynthesis in the halophilic archaebacterium Haloferax volcanii: the trpDFEG cluster.

Tryptophan auxotrophs of the archaebacterium Haloferax volcanii define a cluster of overlapping genes homologous to eubacterial-eukaryotic trpD, -F, -E, and -G, linked in that order and each preceded by a possible ribosome binding site. Residues involved in feedback inhibition of eubacterial anthranilate synthetases are conserved.