Halobacterial Proteins Research Articles

Resurrection of Ancestral Malate Dehydrogenases Reveals the Evolutionary History of Halobacterial Proteins: Deciphering Gene Trajectories and Changes in Biochemical Properties.

Extreme halophilic Archaea thrive in high salt, where, through proteomic adaptation, they cope with the strong osmolarity and extreme ionic conditions of their environment. In spite of wide fundamental interest, however, studies providing insights into this adaptation are scarce, because of practical difficulties inherent to the purification and characterization of halophilic enzymes. In this work, we describe the evolutionary history of malate dehydrogenases (MalDH) within Halobacteria (a class of the Euryarchaeota phylum). We resurrected nine ancestors along the inferred halobacterial MalDH phylogeny, including the Last Common Ancestral MalDH of Halobacteria (LCAHa) and compared their biochemical properties with those of five modern halobacterial MalDHs. We monitored the stability of these various MalDHs, their oligomeric states and enzymatic properties, as a function of concentration for different salts in the solvent. We found that a variety of evolutionary processes, such as amino acid replacement, gene duplication, loss of MalDH gene and replacement owing to horizontal transfer resulted in significant differences in solubility, stability and catalytic properties between these enzymes in the three Halobacteriales, Haloferacales, and Natrialbales orders since the LCAHa MalDH. We also showed how a stability trade-off might favor the emergence of new properties during adaptation to diverse environmental conditions. Altogether, our results suggest a new view of halophilic protein adaptation in Archaea.

Exploiting lipid interactions to control membrane protein insertion, folding and stability

Efficient systems need to be developed to unfold and re-fold alpha helical membrane proteins. We have been developing in vitro lipid-bilayer folding systems for membrane proteins. The stored curvature elastic stress of model bilayers can be used to optimise the rate and yield of folded protein. We have shown that events such as transmembrane helix insertion, as well as tertiary and quaternary structure formation are altered by the stored curvature stress of the bilayer. The curvature stress of a phosphatoidylcholine lipid bilayer can be altered by changing either the lipid chains or introducing a different headgroup such as phosphoethanolamine. We have developed the approach on a halobacterial protein and since applied it to other membrane proteins.

Genes for DNA cytosine methyltransferases and structural proteins, expressed during lytic growth by the phage phi H of the archaebacterium Halobacterium salinarium.

Lytic genes and transcription from the Halobacterium salinarium phage phi H were studied. Genes for three structural proteins were located to the left arm of the linear phage genome. The right arm was shown to encode three DNA cytosine methyltransferases, the first such sequences reported from an archaebacterium. One cytosine methyltransferase is of the N(4)-methyltransferase type. The other two open reading frames (ORFs) seem to be parts of the same gene, which has been split by a recombination event. This gene product is of the C5-methyltransferase type. The methyltransferase genes are the first phi H genes detected showing high homology to eubacterial proteins. Five of the six described gene products have a higher proportion acidic over basic amino acid residues, a common characteristic of halobacterial proteins. Lytic phi H transcription was shown to produce three RNA species, two shorter species encoding the methyltransferase genes and one large species transcribed from both the right and the left phage arm and subsequently being processed upstream of the region encoding the structural proteins.

Compilation of Halobacterial Protein Coding Genes, the Halobacterial Codon Usage Table and its Use

Before 1988 only four sequences of halobacterial protein coding genes were known, but in recent years the number has increased dramatically. Today 130 protein coding genes have been sequenced, belonging to a limited set of protein families. Here a compilation of these genes is given. Most of the genes have the same codon preference. A halobacterial codon usage table is presented and its characteristic features are discussed. The genes of the gas vesicle gene clusters exhibit a different codon usage, which is also shown. The use of the halobacterial codon usage table for the identification of protein coding genes as well as its application as a guide in sequencing halobacterial DNA and construct oligonucleotides are exemplified.

Biochemical, Structural, and Molecular Genetic Aspects of Halophilism

The study of halobacteria relates to a better understanding of evolutionary relationships. Halophilic enzymes are very unstable in low salt concentrations. Because some of the important fractionation methods in protein chemistry, such as electrophoresis or ion-exchange chromatography, cannot be applied at high salt concentrations, the available fractionation methods are limited. The existing purification procedures fall into two groups: the nonhalophilic approach and the halophilic approach. This chapter reviews the developments in the molecular characterization of halobacterial proteins, starting with the methodology of their purification. The biochemical and biophysical structural analyses of some enzymatic systems for which extensive knowledge is accumulated is described.. Macromolecular structures from halophilic bacteria are discussed to improve the understanding of the molecular mechanisms of adaptation to high salt concentration environments by considering genome organization, genetic tools, isolation of genes, or transcript organization, and structure. The structural aspects of halophilism can be determined by ribosomal subunits, surface layers, purple membrane, or halophilic malate dehydrogenase.

An unusual class I (Schiff base) fructose‐1,6‐bisphosphate aldolase from the halophilic archaebacterium Haloarcula vallismortis

An electrophoretically homogeneous class I (Schiff base) alsolase has been isolated for the first time from the archaebacterial halophile Haloarcula (Halobacterium) vallismortis. The aldolase was characterized with respect to its molecular mass, amino acid composition, salt dependency, immunological cross-reactivity and kinetic properties. The subunit mass of aldolase is 27 kDa, which is much smaller than other class I aldolases. By the gel filtration method, the molecular mass of the halobacterial enzyme was estimated as 280 +/- 10 kDa, suggesting a decameric nature. In contrast to many halobacterial proteins, the H. vallismortis aldolase, though a halophilic enzyme, did not show an excess of acidic residues. Unlike the eukaryotic aldolases, the activity of the halobacterial enzyme was not affected by carboxypeptidase digestion. The general catalytic features of the enzyme were similar to its counterparts from other sources. No antigenic similarity could be detected between the H. vallismortis aldolase and class I aldolase from eubacteria and eukaryotes or class II halobacterial aldolases.

A 13-amino acid peptide in three yeast glycosyltransferases may be involved in dolichol recognition.

A 13-amino acid peptide was identified in three glycosyltransferases of the yeast endoplasmic reticulum. These enzymes, the products of the ALG1, ALG7, and DPM1 genes, catalyze the transfer of sugars from nucleotide sugars to dolichol phosphate derivatives. The consensus sequence for the conserved peptide was Leu-Phe-Val-Xaa-Phe-Xaa-Xaa-Ile-Pro-Phe-Xaa-Phe-Tyr. A sequence resembling the conserved peptide was also found in the predicted SEC59 protein, which is suspected to participate in assembly of the lipid-linked precursor oligosaccharide, although its specific function is unknown. All of the identified sequences contain an isoleucine at position 8 and phenylalanine or tyrosine at positions 2, 5, and 12. We believe this peptide may be involved in dolichol recognition for the following reasons. (i) The conserved sequence occurs in potential membrane-spanning regions. (ii) The ALG7 and DPM1 proteins are known to recognize the isoprenoid region of dolichol phosphate specifically; this recognition presumably occurs in the membrane since dolichol is very hydrophobic. (iii) The consensus sequence is similar to a region of two halobacterial proteins implicated in binding of the isoprenoid region of retinal. (iv) If the consensus sequence is represented as an alpha-helix, the conserved residues lie on one face of the helix. An alpha-helical structure is likely since the conserved regions are in potential membrane-spanning domains.

Extended N-terminal sequencing of proteins of archaebacterial ribosomes blotted from two-dimensional gels onto glass fiber and poly(vinylidene difluoride) membrane.

Previously uncharacterized proteins from intact ribosomes and ribosomal subunits of the extreme halophile Halobacterium marismortui (Haloarcula marismortui) were isolated and separated by high-resolution two-dimensional electrophoresis (2DE). N-Terminal amino acid sequences of 14 of these acidic large-subunit proteins were obtained by direct blotting of the separated proteins from two-dimensional electrophoresis gels to sequencer-stable supports followed by excision of the protein spots and sequencing. Furthermore, long internal sequences were obtained by in situ enzymatic cleavage of halobacterial proteins in gel pieces obtained from two-dimensional gels followed by electrophoretic separation of the fragments, blotting, and sequencing. Precautions are outlined for avoidance of N-terminal blockage of proteins, and the preparation and selection of suitable supports for obtaining extended N-terminal sequences are described. The results suggest that when prior fractionation is carried out to enrich for cell organelles, subcellular components of cells, or cell membranes, it is routinely possible to obtain numerous N-terminal sequences from one or a few 2DE gels of such fractions. Our results also indicate that, with appropriate precautions, proteins are routinely obtainable from 2DE gels in a form suitable for both N-terminal and internal sequence determination and show no detectable evidence for N-terminal blockage or destruction or modification of labile amino acid residues.

Primary structures of three highly acidic ribosomal proteins S6, S12 and S15 from the archaebacterium Halobacterium marismortui

The amino acid sequences of three extremely acidic ribosomal proteins, S6, S12, and S15, from Halobacterium marismortui have been determined. The sequences were obtained by the sequence analysis of peptides derived by enzymatic digestion with trypsin. Stapylococcus aureus protease and chymotrypsin, as well as by cleavage with dilute HCl. The proteins, S6, S12 and S15, consist of 116, 147 and 102 amino acid residues, and have molecular masses of 12251, 16440 and 11747 Da, respectively. Comparison of the amino acid sequences of these proteins with ribosomal protein sequences of other organisms revealed that halobacterial protein S12 has homology with the eukaryotic protein S16A from Saccharomyces cerevisiae, while S15 is significantly related to the Xenopus laevis S19 protein. No homology was found between these halobacterial proteins and any eubacterial ribosomal proteins.

Physical chemistry and evolution of salt tolerance in halobacteria

The cellular constituents of extremely halophilic bacteria not only tolerate high salt concentration, but in many cases require it for optical functioning. The characteristics affected by salt include enzyme activity, stability, allosteric regulation, conformation and subunit association. The salt effects are of two major kinds: electrostatic shielding of negative charges by cations at low salt concentration, and hydrophobic stabilization by salting-out type salts at high salt concentration. The composition of halobacterial proteins shows an excess of acidic amino acids and a deficiency of nonpolar amino acids, which accounts for these effects. Since the cohesive forces are weaker and the repulsing forces are stronger in these proteins, preventing aggregation in salt, these structures are no longer suited for functioning in the absence of high salt concentrations. Unlike these nonspecific effects, ribosomes in halobacteria show marked preference for potassium over sodium ions. To ensure the proper intracellular ionic composition, powerful ion transport systems have evolved in the halobacteria, resulting in the extrusion of sodium ions and their replacement by potassium. It is likely that such membrane transport system for ionic movements is a necessary requisite for salt tolerance.