Electron-ion equilibration in ultrafast heated graphite.

We have employed fast electrons produced by intense laser illumination to isochorically heat thermal electrons in solid density carbon to temperatures of ∼10,000 K. Using time-resolved x-ray diffraction, the temperature evolution of the lattice ions is obtained through the Debye-Waller effect, and this directly relates to the electron-ion equilibration rate. This is shown to be considerably lower than predicted from ideal plasma models. We attribute this to strong ion coupling screening the electron-ion interaction.

Purification and analysis of an extremely halophilic beta-galactosidase from Haloferax alicantei.

As a first step in the development of a reporter system for gene expression in halophilic archaea, a beta-galactosidase was purified 140-fold from Haloferax alicantei (previously phenon K, strain Aa2.2). An overproducing mutant was first isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contained 25-fold higher enzyme levels than the parent. Purification of the active enzyme was greatly facilitated by the ability of sorbitol to stabilise enzyme activity in the absence of salt, which allowed conventional purification methods (e.g., ion-exchange chromatography) to be utilised. The enzyme was optimally active at 4 M NaCl and was estimated to be 180 +/- 20 kDa in size, consisting of two monomers (each 78 +/- 3 kDa). It cleaves several different beta-galactoside substrates such as ONP-Gal, X-Gal and lactulose, but not lactose, and also has beta-D-fucosidase activity. No beta-glucosidase, beta-arabinosidase or beta-xylosidase activity could be detected. The amino-acid sequence at the N-terminus and of four proteolytic products has been determined.

Three different but related gene clusters encoding gas vesicles in halophilic archaea.

We present an analysis of the chromosomal region comprising the gene cluster involved in gas vesicle (Vac) synthesis in Haloferax mediterranei (mc-vac-region) and Halobacterium salinarium (c-vac-region) and compare both of them to the plasmid located p-vac-region of H. salinarium. The p-vac-region of 9000 base-pairs (9 kb) is more related to mc-vac (9.4 kb) of Hf. mediterranei than it is to the c-vac-region (8.3 kb) present in the same cell. The Vac- species Hf. volcanii becomes Vac+ following transformation with a fragment containing the entire mc-vac-region. Also the p-vac-region transforms Hf. volcanii to a Vac+ phenotype, indicating that this gene cluster is sufficient for gas vesicle synthesis and does not depend on products of the c-vac-region. Each of these vac-regions contains, in addition to gvpA encoding the major gas vesicle protein, 13 open reading frames named gvpC through gvpO. Ten of these, gvpD through gvpM, are located upstream from gvpA in opposite orientation, while gvpC, gvpN and gvpO are found 3' to gvpA. The absolute requirement of gvpO for gas vesicle synthesis was demonstrated by transformation experiments. Northern analyses with RNA samples isolated during the growth cycle of Hf. mediterranei or of H. salinarium PHH4 revealed that the mc-gvpD or c-gvpD mRNAs occur similar to the respective gvpA mRNA in stationary growth phase, while gvpF-gvpM are transcribed mainly during logarithmic growth. S1-nuclease mapping was performed to determine the transcriptional start site of the gvpD mRNA. The distance between the two divergent start sites of gvpA and gvpD mRNA is 109 base-pairs in mc-vac and p-vac, while in the case of c-vac this distance is 22 base-pairs larger. The conservation of the various gvp products, characteristic features and their possible functions in gas vesicle synthesis are discussed.

The transcriptional activator GvpE for the halobacterial gas vesicle genes resembles a basic region leucine-zipper regulatory protein.

The GvpE protein involved in the regulation of gas vesicles synthesis in halophilic archaea has been identified as the transcriptional activator for the promoter located upstream of the gvpA gene encoding the major gas vesicle structural protein GvpA. A closer inspection of the GvpE protein sequence revealed that GvpE resembles basic leucine-zipper proteins typically involved in the gene regulation of eukarya. A molecular modelling study of the C-terminal part implied a cluster of basic amino acid residues constituting the DNA-binding site (DNAB) followed by an amphiphilic helix, suitable for the formation of a leucine-zipper structure within a GvpE dimer. The model of a GvpE dimer docked onto DNA indicated that the side-chains of the basic residues could perfectly interact with the negatively charged phosphate groups of the DNA backbone. Substitution of three basic amino acid residues of this putative DNAB by alanine and/or glutamate generated mutated GvpE proteins. None of these was able to activate the c-gvpA promoter in vivo, indicating that these basic residues are required for GvpE activity. This identification of an archaeal gene regulator displaying similarity to eukaryal regulatory proteins implies that the basic transcription machinery of eukarya and archaea are closely related, and that the regulatory proteins have evolved according to common principles.

Improved shuttle vectors for Haloferax volcanii including a dual-resistance plasmid.

Two new Haloferax-Escherichia shuttle vectors are described, pMDS20 and pMLH3. These vectors contain the E. coli ColE1 plasmid ori region and ampicillin-resistance(ApR)-conferring bla gene, and the Haloferax pHK2 replicon region and novobiocin-resistance(NbR)-encoding gyrB gene, enabling maintenance and selection in both hosts. Plasmid pMLH3 has, in addition, a H. volcanii mevinolin-resistance (MvR) determinant and restriction sites allowing insertional inactivation of either marker, to facilitate the identification of Haloferax transformants harbouring cloned sequences. Sequencing of gyrA, within the NbR determinant, and the pHK2 ori region has been completed so the complete sequence of both pMDS20 and pMLH3 is now known.

Analysis of the halobacterial plasmid pHK2 minimal replicon.

The pMDS series of cloning vectors developed for use in halophilic archaea have utilized a 10.5-kb plasmid, pHK2, from Haloferax sp. Aa2.2. The minimal replicon of pHK2 has now been determined (3359 bp) and completely sequenced. No significant sequence similarity was found between the pHK2 subfragment and plasmid pHV2 from the closely related H. volcanii. However, a long open reading frame (ORF), named rep, was identified which encodes a putative protein with approx. 30% sequence identity to ORFs within plasmids pGRB1, pHGN1 and pHSB1 from Halobacterium sp. All these putative Rep proteins contain sequence motifs conserved in bacterial plasmids and phage genomes known to replicate via a rolling-circle mechanism.

[Helminth fauna of red foxes (Vulpes vulpes LINNE 1758) in southern Saxony--2: Nematodes]. / Zur Helminthenfauna des Rotfuchses (Vulpes vulpes LINNE 1758) im Süden Sachsen-Anhalts--Teil 2: Nematoden.

Between January 1993 and November 1994 a total of 1300 red foxes from the administrative districts Halle and Dessau were examined for the presence of nematodes in the stomach and the small intestine. The following nematodes were found: Toxocara canis (26.5%), Toxascaris leonina (10.5%), Uncinaria stenocephala (15.9%) Ancylostoma caninum (1.7%). The search for Trichinella spp. larvae was negative in all 780 examined foxes.

Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region, the gas vesicle gene cluster and insertion elements.

The DNA sequence of the 5.7 kb plasmid pHH9 containing the replicon region of the 150 kb plasmid pHH1 from Halobacterium salinarium was determined. The minimal region necessary for stable plasmid maintenance lies within a 2.9 kb fragment, as defined by transformation experiments. The DNA sequence contained two open reading frames arranged in opposite orientations, separated by an unusually high AT-rich (60-70% A+T) sequence of 350 bp. All H. salinarium strains (H. halobium, H. cutirubrum) investigated harbour endogenous plasmids containing the pHH1 replicon; however, these pHH1-type plasmids differ by insertions and deletions. Adjacent to the replicon, and separated by a copy of each of the insertion elements ISH27 and ISH26, is the 9 kb p-vac region required for gas vesicle synthesis. Analysis of these and other ISH element copies in pHH1 revealed that most of them lack the target DNA duplication usually found with recently transposed ISH elements. These results underline the plasticity of plasmid pHH1.

Complementation studies with the gas vesicle-encoding p-vac region of Halobacterium salinarium PHH1 reveal a regulatory role for the p-gvpDE genes.

Gas-vesicle (Vac) synthesis in Halobacterium salinarium PHH1 involves the expression of the p-vac region consisting of 14 different gvp genes that are arranged in two clusters: p-gvpACNO and, oppositely oriented, p-gvpDEFGHIJKLM. The latter cluster of genes is transcribed as two units: p-gvpDE and p-gvpF-M. The 5'-terminus of the p-gvpF-M mRNA was located 169 nucleotides upstream of p-gvpF within p-gvpE. The p-gvpG and p-gvpK gene was expressed in Escherichia coli and antibodies to proteins obtained were raised in rabbits. Both proteins could be detected in halobacterial cell lysates; in gas-vesicle preparations, however, neither GvpG nor GvpK could be found. The requirement for single p-gvp gene expression for gas-vesicle synthesis was determined by transformation experiments using the Vac- species Haloferax volcanii as recipient. Construct delta A containing all p-gvp genes except for p-gvpA, encoding the major gas-vesicle structural protein, produced Vac- transformants, but the addition of p-gvpA on a second vector restored gas-vesicle synthesis to wild-type level (Vac++). Similarly, double transformants containing p-gvpD-M plus p-gvpACNO, or p-gvpG-M (fused to the promoter of the halobacterial ferredoxin gene for expression) plus p-gvpFED-ACNO were Vac++. Transformants containing the p-vac region either lacking gvpA, gvpF, or gvpGHI were Vac-, indicating the absolute requirement of these gvp genes (or at least one in the case of gvpGHI) for gas-vesicle formation. Double transformants containing the constructs p-gvpF-M plus p-gvpACNO (delta DE) accumulated gas vesicles (Vac+) but synthesized fewer than the wild type, showing that the p-gvpDE genes are not necessary for gas-vesicle assembly. A repressor function affecting the synthesis of the p-gvpF-M mRNA could be suggested for p-gvpD and the 5'-region of its mRNA.

Expression of two gas vacuole protein genes in Halobacterium halobium and other related species.

The archaebacterium Halobacterium halobium contains two genes encoding gas vacuole proteins (vac). One resides on a large naturally occurring plasmid and encodes a protein of 76 amino acids (p-vac), while the other is a chromosomal gene that encodes a highly similar protein of 79 amino acids (c-vac). Northern analysis determined the c-vac and p-vac mRNA to be approximately 340 nucleotides in length, and S1 mapping of both transcripts indicated that the 5' terminus for each starts at the same relative nucleotide. Three other Halobacterium species producing gas vacuoles were investigated, H. spec. GN101, YC819-9, and SB3. All three contain only a chromosomal c-vac gene, and the 5' terminus of the 340 nucleotide mRNA starts at the same nucleotide as found for H. halobium. The c-vac gene region of H. spec. GN101 contains nine nucleotide exchanges, three of which occur in the coding region with no effect on the amino acid sequence. In contrast, the c-vac gene of H. spec. SB3 has an identical nucleotide sequence to the H. halobium c-vac gene. Gas vacuole production in each of these species was monitored during culture growth by phase contrast microscopy, and the vac mRNA level was determined for each time point. H. halobium p-vac deletion mutants, as well as the halobacterial species GN101 and YC819-9, start to synthesize gas vacuoles in early stationary growth phase with a maximal mRNA content in stationary phase. In contrast, H. halobium wild-type synthesizes gas vacuoles exclusively due to p-vac gene expression with a maximal mRNA level during logarithmic growth, and transcripts of the c-vac gene were not detectable.(ABSTRACT TRUNCATED AT 250 WORDS)

Insertion elements and deletion formation in a halophilic archaebacterium.

Deletion events that occur spontaneously in 36-kilobase-pair (kbp) plasmid pHH4 from the archaebacterium Halobacterium halobium were investigated. Four different deletion derivatives with sizes ranging from 5.7 to 17 kbp were isolated. Three of these deletion variants derived from pHH4 (pHH6 [17 kbp], pHH7 [16 kbp], and pHH8 [6.3 kbp]), whereas the 5.7-kbp plasmid pHH9 derived from pHH6. Strains containing pHH6, pHH7, or pHH9 each lacked the parental plasmid pHH4, while pHH8 occurred at a 1:1 ratio together with pHH4. Common to all of these plasmids was the 5.7-kbp region of pHH9 DNA. The regions containing the fusion site in the deletion derivatives were investigated and compared with the corresponding area of the parental plasmid. Each deletion occurred exactly at the terminus of an insertion element. In pHH6 and pHH7, a halobacterial insertion element (ISH2) was located at the deletion site. The DNA fused to ISH2 displayed a 7-base-pair (bp) (pHH7) or 10-bp (pHH6) sequence homology to the inverted repeat of ISH2. In the two smaller plasmids, pHH8 and pHH9, an ISH27 element was located at the deletion site. Most likely, all of these smaller plasmids resulted from an intramolecular transposition event. The ISH27 insertion sequence contains a 16-bp terminal inverted repeat and duplicates 5 bp of target DNA during the transposition with the specificity 5'ANNNT3'. Four ISH27 copies were analyzed, and two ISH27 element types were identified that have approximately 85% sequence similarity. The ISH27 insertion elements constitute a family which is related to the ISH51 family characterized for H. volcanii, another halophilic archaebacterium.

Function and biosynthesis of gas vesicles in halophilic Archaea.

The proteinaceous gas vesicles produced by various microorganisms including halophilic Archaea are hollow, gas-filled structures with a hydrophobic inner and a hydrophilic outer surface. The structural components of gas vesicles and their biosynthesis are still under investigation; an 8-kDa polypeptide appears to be the major constituent of the gas-vesicle envelope. Genetic analysis of the halobacterial gas-vesicle synthesis revealed an unexpected complexity: about 14 genes organized in three transcription units are involved in gas-vesicle structure, assembly, and gene regulation. Here we describe the comparison of three different genomic regions encoding gas vesicles in Halobacterium salinarium (p-vac and c-vac regions) and Haloferax mediterranei (mc-vac region) and speculate on the function of the gene products involved in gas-vesicle synthesis.

Analysis of gas vesicle gene expression in Haloferax mediterranei reveals that GvpA and GvpC are both gas vesicle structural proteins.

Gas vesicle synthesis in Haloferax mediterranei involves several gene products encoded by a 9.4-kilobase pair DNA region (mc-vac region) that contains 13 genes in addition to gvpA encoding the major structural gas vesicle protein. The expression of part of this region, encompassing the genes gvpA, gvpC, gvpN, and gvpO was investigated. These genes are transcribed from a common promoter located upstream of gvpA. Transcripts of 0.34 (gvpA only), 1.8 (gvpA/C), 2.4 (gvpA/C/N) and 3 kilobases (gvpA/C/N/O) were observed, with the gvpA transcript being the predominant mRNA species. The majority of the mRNA formed terminates 64 base pairs downstream of gvpA at the cytosine of the sequence 5' TTTTTC 3'. The synthesis of the GvpA and GvpC proteins was investigated by Western analyses. An antiserum raised against isolated gas vesicles of Hf. mediterranei detects, in addition to gas vesicle fragments, the GvpA protein of the M(r) of approximately 8,000 in lysates derived from different halobacteria or from Escherichia coli expressing gvpA. In samples containing isolated gas vesicles, mainly partially disaggregated gas vesicle fragments hybridize, but a minor amount of monomeric GvpA is also seen. For the detection of the GvpC protein, two versions of the gvpC gene (full length and gvp delta C lacking the 3' part encoding the acidic C terminus) were expressed in E. coli, and the resulting proteins were purified. The two antisera raised against these GvpC versions indicate the expression of gvpC in different halobacteria. By Western analysis, GvpC is also detectable in samples containing isolated gas vesicles demonstrating that GvpC is a second, but minor, gas vesicle structural protein.

The characterization of the nv-gvpACNOFGH gene cluster involved in gas vesicle formation in Natronobacterium vacuolatum.

The haloalkaliphilic archaeon Natronobacterium vacuolatum forms cylinder-shaped gas vesicles throughout the growth cycle when grown in media containing 15-25% NaCl. Cells cultivated in media containing 13% NaCl are, however, gas-vesicle-free. The major gas vesicle structural protein, nv-GvpA, was detected by an antiserum raised against the gas vesicles of Haloferax mediterranei; the antiserum reacted with an 8.3-kDa protein in samples containing cell extracts or purified gas vesicles of N. vacuolatum. The gene encoding nv-GvpA was isolated together with six additional gvp genes; these genes are arranged consecutively in a cluster as nv-gvpACNOFGH and are cotranscribed. Transcript analysis by primer extension revealed only one start site three nucleotides upstream of the nv-gvpA reading frame. This arrangement of gvp genes differs from that of the gas-vesicle-encoding genes in Halobacterium salinarium and Hf. mediterranei. The comparison of the deduced Gvp protein sequences indicated similarities with the respective halobacterial Gvp proteins, with GvpA exhibiting the highest degree of conservation (97-100%). The second gas vesicle structural protein, nv-GvpC, was 150-250 amino acids longer than all other halobacterial GvpC proteins and was much less conserved (48-73%). The expression of the nv-gvp genes was monitored in N. vacuolatum cells cultivated in 20 or 13% salt media. Northern and Western analyses showed that despite the lack of gas vesicles in cells grown in 13% salt medium, the gvpACNOFGH gene cluster was transcribed and GvpA protein was synthesized, suggesting that the absence of gas vesicles is not due to a lack of transcription.

Transcript analysis of the c-vac region and differential synthesis of the two regulatory gas vesicle proteins GvpD and GvpE in Halobacterium salinarium PHH4.

Halobacterium salinarium PHH4 synthesizes gas vesicles in the stationary growth phase by the expression of 14 gyp genes arranged in two clusters. The chromosomal gvpACNO (c-gvpACNO) gene cluster (encoding the major structural gas vesicle protein GvpA and the minor structural protein GvpC was transcribed as three mRNA species starting at one promoter during the stationary phase of growth. The second gene cluster, c-gvpDEFGHIKLM), was transcribed during all stages of growth as a relatively unstable, single mRNA with a maximal length of 6 kb. In addition, a 1.7-kb c-gvpD transcript was synthesized during stationary growth starting at the same promotor as that of the cgvpDEFGHIJKLM mRNA. The expression of the first two genes located in this unit (c-gvpD and c-gvpE) was also monitored by Western blot (immunoblot) analyses using antisera raised against these proteins synthesized in Escherichia coli. While the cGvpD protein was present only during early exponential growth and disappeared during gas vesicle formation, the cGvpE protein was present during cGvpA and gas vesicle synthesis in the early stationary phase of growth. Previous data indicated that cGvpD is involved in repression of gas vesicle formation, whereas cGvpE is a transcriptional activator for the c-gvpA promoter. The appearance of both proteins during the growth cycle is in line with the functions of these proteins in gas vesicle synthesis. The mechanism of the differential translation of cGvpD and cGvpE from the c-gvpDEFGHIJKLM rnRNA still has to be elucidated, but antisense RNAs complementary to the 5' terminus as well as the 3' portion of the c-gvpD mRNA might be involved in this regulation. Such RNAs occurred during early stationary growth when the cGvpD protein level decreased and may possibly inhibit the translation of the c-gvpD mRNA.

Genome organization in Halobacterium halobium: a 70 kb island of more (AT) rich DNA in the chromosome.

The more A + T rich fractionated component (FII DNA) of the Halobacterium halobium genome constitutes one third of the total DNA and upon isolation consists of covalently closed circular DNA (pHH1 and minor cccDNA) and nonsupercoiled sequences. We have investigated the physical organization of the non cccDNA in FII by a chromosome walk using one copy of the halobacterial insertion element ISH1 as a start point. This chromosome walk led to the isolation of 160 kb of chromosomal DNA containing 70 kb of FII DNA covalently linked to more G + C rich sequences (FI DNA). Copies of three previously characterized insertion elements (ISH1, ISH2, and ISH26) as well as at least 10 other repeated sequences are clustered within this chromosomal FII DNA "island". Unique sequences are found in the FI DNA flanking the FII DNA island as well as in 40 kb of FI DNA surrounding the bacterio-opsin gene. The presence of pHH1 in H. halobium and closely related species correlates with the occurrence of the characterized chromosomal FII DNA island. Halophilic purple membrane producing isolates YC81819-9, GN101, SB3 and GRA lack pHH1 and the 70 kb FII DNA, but contain all of the FI DNA sequences tested. We propose that pHH1 and this chromosomal FII DNA are characteristic genomic components of H. halobium and closely related species, and, that the 70 kb FII DNA might represent a large insertion in the chromosome of H. halobium and closely related species. The conservation of both FI and FII DNA sequences can be used for strain classification and determination of evolutionary relationships among halo-bacteria.

Influence of salt on the transcription of the gas-vesicle genes of Haloferax mediterranei and identification of the endogenous transcriptional activator gene.

The transcription of the 14 gvp genes of the gas-vesicle-encoding mc-vac region was investigated, using RNA from 25% and 15% (w/v) salt cultures of the moderately halophilic archaeon Haloferax mediterranei. Transcription occurred only from two promoters, located in front of the mc-gvpA and mc-gvpD genes. In both cultures transcripts spanning the entire mc-gvpDEFGHIJKLM transcription unit were formed only during the exponential growth phase. Amounts of these transcripts were larger in the 25% salt culture, in which the 2.0 kb mc-gvpD transcripts were also synthesized during the stationary phase. The levels of the mc-gvpD transcripts and of the 324 nt mc-gvpA mRNA increased in parallel during the stationary phase of the 25% salt culture. Only under these conditions were mRNAs spanning the entire mc-gvpACNO transcription unit observed, and gas-vesicles were formed. Investigation of the influence of the mc-gvpDE genes on both mc-vac promoters in transformants revealed that by themselves they were nearly inactive. The addition of mc-gvpE, however, resulted in a high level of constitutively produced mc-gvpA and mc-gvpD mRNA, indicating a transcriptional activator function for the mc-gvpE product.

Transformation of Halobacterium halobium: development of vectors and investigation of gas vesicle synthesis.

We developed vector plasmids for the transformation of Halobacterium halobium, using the replicon region from the halobacterial phage H or from the plasmid pHH1 together with a DNA fragment conferring resistance to mevinolin. H. halobium P03, a strain lacking pHH1 as well as the restriction endonuclease activity found in wild-type H. halobium, was used as the recipient strain. All H. halobium fragments tested for autonomous replication as well as the Haloferax volcanii vector pWL102 enabled stable plasmid maintenance in this strain. A frequent loss of all vectors (including pWL102) was observed in Hf. volcanii, where >90% of the mevinolin-resistant colonies obtained after transformation had lost the vector, presumably because of restriction endonuclease activity and concomitant recombination of the mevinolin resistance marker with the chromosome. The expression of gas vesicle-encoding genes (vac) was analyzed by using a 4.5-kilobase-pair (kbp) fragment containing the plasmid-encoded p-vac gene from H. halobium or an 11-kbp fragment containing the mc-vac chromosomal gene from Haloferax mediterranei for transformation experiments with H. halobium and Hf. volcanii. These experiments indicated that the mc-vac fragment contains all information necessary to synthesize gas vesicles, whereas in the case of the smaller p-vac fragment, complementation by other genes was required for a Vac+ phenotype.

Transposition burst of the ISH27 insertion element family in Halobacterium halobium.

Investigation of the plasmid pHH4 in single colonies of Halobacterium halobium PHH4 indicated transposition of insertion elements in 20% of the colonies. Seven ISH27 insertions were observed as well as one ISH23 insertion. The various copies of ISH27 were compared to the two ISH27 elements already present in pHH4, and to the ISH27 element that was identified in the bacteriopsin (bop) gene of a Bop mutant. These ten copies of ISH27 constitute three types on the basis of DNA sequence identity: ISH27-1 (1398 bp), ISH27-2, and ISH27-3 (1389 bp each). The DNA sequence comparison between the three types indicates a region of 1200 bp where the identity between ISH27-1 and ISH27-2 or ISH27-3 is 82-83%. ISH27-2 and ISH27-3 are 95% identical in this region. The remaining region exhibits a lower DNA similarity (64-74% identity) between the different copies. An open reading frame of 1167 nucleotides spans the more conserved region, and a corresponding transcript could be detected in H. halobium PHH4, but not in H. halobium wild-type. ISH27-1 is 91% identical to members of the insertion sequence-like elements ISH51 of Haloferax volcanii, whereas the other two ISH27 element types are 82-83% identical to ISH51. The transposition 'burst' of ISH27 was only seen after storage of the cells for more than two years at 4 degrees C. Upon continuous cultivation at 37 degrees C no transposition event could be observed, suggesting that stress factor(s) might have caused the high transposition rate.