Structural features that stabilize halophilic malate dehydrogenase from an archaebacterium.

The high-resolution structure of halophilic malate dehydrogenase (hMDH) from the archaebacterium Haloarcula marismortui was determined by x-ray crystallography. Comparison of the three-dimensional structures of hMDH and its nonhalophilic congeners reveals structural features that may promote the stability of hMDH at high salt concentrations. These features include an excess of acidic over basic residues distributed on the enzyme surface and more salt bridges present in hMDH compared with its nonhalophilic counterparts. Other features that contribute to the stabilization of thermophilic lactate dehydrogenase and thermophilic MDH-the incorporation of alanine into alpha helices and the introduction of negatively charged amino acids near their amino termini, both of which stabilize the alpha helix as a result of interaction with the positive part of the alpha-helix dipole-also were observed in hMDH.

The mechanism of DNA transfer in the mating system of an archaebacterium.

The genetic transfer system in the extremely halophilic archaebacterium Halobacterium volcanii is the only archaebacterial mating system known. The mechanism of genetic transfer of this archaebacterium was studied by using the immobile plasmids pHV2 and pHV11 as cytoplasmic markers. It was found that the cytoplasms of the parental types do not mix during the mating process, that each parental type can serve both as a donor and as a recipient, and that cytoplasmic bridges, with dimensions of up to 2 micrometers long and 0.1 micrometer in diameter, were formed between the parental types. These bridges appear to be used for the transfer of DNA from one cell to another. If so, this archaebacterial mating system is different from both eubacterial conjugation and eukaryotic sexual cell fusion.

RNA polymerase activity in virions from Ustilago maydis.

RNA polymerase activity is associated with the double-stranded RNA virions of Ustilago maydis. The reaction products of the polymerase activity are single-stranded RNA molecules. The RNA molecules synthesized are homologous to the three classes of double-stranded RNA molecules that typify the viral genome. The single-stranded RNA synthesized is released from the virions. The molecular weight of the single-stranded RNA transcripts is about half the size of the double-stranded RNA segments, and thus, it appears that in the in vitro reaction, full-length transcripts can be obtained.

Structural features of halophilicity derived from the crystal structure of dihydrofolate reductase from the Dead Sea halophilic archaeon, Haloferax volcanii.

BACKGROUND: The proteins of halophilic archaea require high salt concentrations both for stability and for activity, whereas they denature at low ionic strength. The structural basis for this phenomenon is not yet well understood. The crystal structure of dihydrofolate reductase (DHFR) from Haloferax volcanii (hv-DHFR) reported here provides the third example of a structure of a protein from a halophilic organism. The enzyme is considered moderately halophilic, as it retains activity and secondary structure at monovalent salt concentrations as low as 0.5 M. RESULTS: The crystal structure of hv-DHFR has been determined at 2.6 A resolution and reveals the same overall fold as that of other DHFRs. The structure is in the apo state, with an open conformation of the active-site gully different from the open conformation seen in other DHFR structures. The unique feature of hv-DHFR is a shift of the alpha helix encompassing residues 46-51 and an accompanied altered conformation of the ensuing loop relative to other DHFRs. Analysis of the charge distribution, amino acid composition, packing and hydrogen-bonding pattern in hv-DHFR and its non-halophilic homologs has been performed. CONCLUSIONS: The moderately halophilic behavior of hv-DHFR is consistent with the lack of striking structural features expected to occur in extremely halophilic proteins. The most notable feature of halophilicity is the presence of clusters of non-interacting negatively charged residues. Such clusters are associated with unfavorable electrostatic energy at low salt concentrations, and may account for the instability of hv-DHFR at salt concentrations lower than 0.5 M. With respect to catalysis, the open conformation seen here is indicative of a conformational transition not reported previously. The impact of this conformation on function and/or halophilicity is unknown.

Amino acid sequence of 2Fe-2S ferredoxin from an extreme halophile, Halobacterium of the Dead Sea.

The primary structure of the 2Fe-2S ferredoxin from Halobacterium of the Dead Sea was determined and it consisted of 128 amino acid residues including an N epsilon-acetyllysyl residue. Due to a high degree of sequence homology between this ferredoxin and the one from Halobacterium halobium, all tryptic peptides could be aligned in order. Only 20 amino acid differences were observed between these two halobacterial ferredoxins. The distribution of cysteinyl residues involved in the iron chelation was similar to that of chloroplast-type ferredoxins.

Crystallization of halophilic malate dehydrogenase from Halobacterium marismortui.

Malate dehydrogenase from the extreme halophile Halobacterium marismortui crystallizes in highly concentrated phosphate solution in space group 12 with cell dimensions a = 113.8 A, b = 122.8 A, c = 126.7 A, beta = 98.1 degrees. The halophilic enzyme was found to be unstable at lower concentrations of phosphate. It associates with unusually large amounts of water and salt, and the combined particle volume shows a tight fit in the unit cell.

Microbial isopenicillin N synthase genes: structure, function, diversity and evolution.

Clinically and economically, penicillins and cephalosporins are the most important class of the beta-lactam antibiotics. They are produced by a wide variety of microorganisms including numerous species of Streptomyces, some unicellular bacteria and several filamentous fungi. A key step common to their biosynthetic pathways is the conversion of a linear, cysteine-containing tripeptide to a bicyclic beta-lactam antibiotic by isopenicillin N synthase. Recent successes in the cloning and expression of isopenicillin N synthase genes now permit production of a plentiful supply of this enzyme, which may be used for structural and mechanistic studies, or for biotechnological applications in the creation of novel beta-lactam compounds from peptide analogues. New ideas concerning the evolution and prevalence of the penicillin and cephalosporin biosynthetic genes have emerged from studies of isopenicillin N synthase genes.

Cloning and expression in Escherichia coli of an esterase-coding gene from the oil-degrading bacterium Acinetobacter calcoaceticus RAG-1.

A putative esterase gene (est) from Acinetobacter calcoaceticus RAG-1 has been cloned into Escherichia coli. Esterase-positive clones exhibited high levels of esterase activity even in intact cells. In addition, expression of the est gene conferred on E. coli the ability to grow on simple triglycerides such as triacetin (TAC). The original esterase-positive plasmid pRA17 carried a 2.2-kb insert from a partial MboI digest of RAG-1 DNA, which gave a single band with RAG-1 DNA following Southern hybridization. By subcloning and sequencing the est gene was found to contain a sequence of 870 bp which could be translated to yield a protein of Mr 32,700. In support of the sequencing results was the finding that when pRA17 was expressed in minicells, a unique peptide of Mr 32,500 was identified. This peptide was not found in minicells transformed with esterase-negative plasmids, such as pRA176, which contained a Tn5 insertion in the est gene. The fact that the production of active esterase depended on the orientation of the est gene within the vector suggested that transcription proceeded from the tet promoter in pBR322.

Cloning and nucleotide sequence determination of the isopenicillin N synthetase gene from Streptomyces clavuligerus.

The isopenicillin N synthetase (IPNS) gene from Streptomyces clavuligerus was isolated from an Escherichia coli plasmid library of S. clavuligerus genomic DNA fragments using a 44-mer mixed oligodeoxynucleotide probe. The nucleotide sequence of a 3-kb region of the cloned fragment from the plasmid, pBL1, was determined and analysis of the sequence showed an open reading frame that could encode a protein of 329 amino acids with an Mr of 36,917. When the S. clavuligerus DNA from pBL1 was introduced into an IPNS-deficient mutant of S. clavuligerus on the Streptomyces vector pIJ941, the recombinant plasmid was able to complement the mutation and restore IPNS activity. The protein coding region of the S. clavuligerus IPNS gene shows about 63% and 62% similarity to the Cephalosporium acremonium and Penicillium chrysogenum IPNS nucleotide sequences, respectively, and the predicted amino acid sequence of the encoded protein showed about 56% similarity to both fungal sequences.

Halophilic enzymes: proteins with a grain of salt.

Halophilic enzymes, while performing identical enzymatic functions as their non-halophilic counterparts, have been shown to exhibit substantially different properties, among them the requirement for high salt concentrations, in the 1-4 M range, for activity and stability, and a high excess of acidic over basic amino residues. The following communication reviews the functional and structural properties of two proteins isolated from the extremely halophilic archaeon Haloarcula marismortui: the enzyme malate-dehydrogenase (hMDH) and the 2Fe-2S protein ferredoxin. It is argued that the high negative surface charge of halophilic proteins makes them more soluble and renders them more flexible at high salt concentrations, conditions under which non-halophilic proteins tend to aggregate and become rigid. This high surface charge is neutralized mainly by tightly bound water dipoles. The requirement of high salt concentration for the stabilization of halophilic enzymes, on the other hand, is due to a low affinity binding of the salt to specific sites on the surface of the folded polypeptide, thus stabilizing the active conformation of the protein.

Isolation and partial characterization of plasmids found in three Halobacterium volcanii isolates.

Three new isolates of Halobacterium volcanii were screened for the presence of plasmids. Each of the different isolates was found to contain one plasmid. These plasmids do not show any homology to each other, nor to the previously isolated plasmid pHV2. Partial restriction maps of these plasmids were determined. One of the plasmids contains chromosomal repetitive sequences as judged by the existence of homologous sequences in the chromosomal DNA of the three isolates. Using the protoplast fusion technique, we showed that at least one of the newly isolated plasmids is compatible with pHV2.

Evidence for post-translational membrane insertion of the integral membrane protein bacterioopsin expressed in the heterologous halophilic archaeon Haloferax volcanii.

The gene coding for the integral membrane protein bacterioopsin (Bop), that is composed of seven transmembrane helices, was expressed in the halophilic archaeon Haloferax volcanii as a fusion protein with the halobacterial enzyme dihydrofolate reductase and with the cellulose binding domain of Clostridium thermocellum cellulosome. In each case, bacterioopsin was present both in the membrane and in the cytoplasmic fractions. Pulse-chase labeling experiments showed that the fusion protein in the cytoplasmic fraction is the precursor of the membrane-bound species. Bacterioopsin mutants that lack the seventh helix (BopDelta7) were found to accumulate only in the cytoplasmic fraction, whereas bacterioopsin mutants that lack either helices four and five (BopDelta4-5), or helices one and two (BopDelta1-2), were found in the cytoplasmic as well as in the membrane fractions. The seventh helix, when expressed alone, could target in trans the insertion of a separately expressed bacterioopsin mutant protein that has only the first six helices. These results support a model in which bacterioopsin is produced in H. volcanii as a soluble protein and in which its insertion into the membrane occurs post-translationally. According to this model, membrane insertion is directed by the seventh helix.

Genetic transfer in Halobacterium volcanii.

Auxotrophic mutants of Halobacterium volcanii generated by chemical mutagenesis were used to demonstrate a native genetic transfer system in this extremely halophilic member of the class Archaeobacteria.

High expression in Escherichia coli of the gene coding for dihydrofolate reductase of the extremely halophilic archaebacterium Haloferax volcanii. Reconstitution of the active enzyme and mutation studies.

The gene coding for the enzyme dihydrofolate reductase of the extremely halophilic archaebacterium Haloferax volcanii was recombined into the Escherichia coli expression vector pET11d. Following induction, the enzyme was produced in large quantities and accumulated in the cells in an insoluble form. The enzymic activity could be efficiently reconstituted by dissolving the aggregate in 6 M guanidine hydrochloride followed by dilution into salt solutions. Mutants were produced in which Lys30 was converted to Leu (K30L), Lys31 was converted to Ala (K31A) and a double mutant in which both lysines were converted (K30L, K31A). The mutated enzymes were produced in E. coli, activated and purified to homogeneity. The effect of the salt concentration on the steady-state kinetic parameters was determined. It was found that the salt concentration affects the Km but not kcat of the various mutants.

Nucleotide sequence of a cyanobacterial nifH gene coding for nitrogenase reductase.

The nucleotide sequence of nifH, the structural gene for nitrogenase reductase (component II or Fe protein of nitrogenase) from the cyanobacterium Anabaena 7120 has been determined. Also reported are 194 bases of the 5'-flanking sequence and 170 bases of the 3'-flanking sequence. The predicted amino acid sequence was compared with that determined for the complete nitrogenase reductase of Clostridium pasteurianum and the cysteine-containing peptides of the protein from Azotobacter vinelandii. Amino acid sequences around five cysteines, located in the NH(2)-terminal two-thirds of the protein, are highly conserved in all three species. Codon usage in the first gene from a cyanobacterium to be sequenced shows striking asymmetries for eight amino acids.

Malate dehydrogenase isolated from extremely halophilic bacteria of the Dead Sea. 1. Purification and molecular characterization.

The complete purification of malate dehydrogenase (EC 1.1.1.37) from extremely halophilic bacteria of the Dead Sea is described. The purification procedure includes (a) precipitation by ammonium sulfate, (b) fractionation on Sepharose 4B using a decreasing concentration gradient of ammonium sulfate, (c) gel permeation chromatography on Sephadex G-100, (d) chromatography on hydroxylapatite, and (e) affinity chromatography on 8-(6-aminohexyl)amino-NAD+-Sepharose at 4.26 M NaCl. The absorption and fluorescence spectra of the native and denatured enzyme were measured, and the extinction coefficient at 280 nm in 4.26 M NaCl was found to be 0.803 cm2mg-1. The amino acid composition analysis showed an excess of 10.4 mol % of acidic amino acids. The apparent specific "volume" phi' of the active enzyme at 4.26 M NaCl was found to be 0.680 +/- 0.015 mL/g. The molecular weight of the native enzyme was found to be 84 000 +/- 4000 determined in 4.26 M NaCl from equilibrium sedimentation data. The molecular weight of the subunits is 39 000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, the native enzyme is composed of two subunits.

Regulatory region of the metA gene of Escherichia coli K-12.

Transcription of the metA gene of Escherichia coli K-12 is from a promoter which is under methionine control and is located next to a region which has an extensive sequence homology with the operator regions of the metBL and metF genes. However, in the metA gene there is a second transcription start point which is located 74 base pairs upstream and which is independent of the intracellular methionine concentration.

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.

Detection of gastrin-specific mRNA using oligodeoxynucleotide probes of defined sequence.

Three oligodeoxynucleotides have been chemically synthesized and used as hybridization probes to detect gastrin-specific mRNA within an heterogeneous population of RNA molecules. Using these oligonucleotides whose nucleotide sequences were deduced from the amino acid sequence of the hormone, 0.2 fmol of gastrin mRNA can be detected/microgram of poly(A)-RNA from hog antrums. The 32P-labeled oligonucleotides hybridize specifically to RNA covalently coupled to DBM-paper (Alwine, J.C., Kemp, D.J., and Stark, G.R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5350-5354). Labeled oligonucleotides also hybridize specifically to RNA separated by gel electrophoresis in the presence of CH3HgOH and covalently coupled to DBM-paper. Using this procedure, we have found that the mRNA coding for gastrin contains about 620 nucleotides, which is in agreement with results obtained using gastrin cDNA to determine mRNA size.