Surviving salt fluctuations: stress and recovery in Halobacterium salinarum, an extreme halophilic Archaeon.

Halophilic proteins subjected to below about 15% salt in vitro denature through misfolding, aggregation and/or precipitation. Halobacteria, however, have been detected in environments of fluctuating salinity such as coastal salterns and even around fresh water springs in the depths of the Dead Sea. In order to identify the underlying mechanisms of low salt survival, we explored the reactivation capacity of Halobacterium (Hbt) salinarum sub-populations after incubation in low salt media and recovery in physiological salt. Respiratory oxygen consumption was assessed in stressed cells and cell viability was estimated by Live/Dead staining and flow cytometry. In vivo neutron scattering experiments showed that the recovery of Hbt salinarum sub-populations exposed to severe low salt conditions is related to a rapid retrieval of functional molecular dynamics in the proteome. In the hypothesis that the observations on Hbt salinarum have wider relevance, they could be of key ecological significance for the dispersion of extremophiles when environmental fluctuations become severe.

High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes.

Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

Stress regulation of the PAN-proteasome system in the extreme halophilic archaeon Halobacterium.

In Archaea, the importance of the proteasome system for basic biological processes is only poorly understood. Proteasomes were partially purified from Halobacterium by native gradient density ultracentrifugation. The peptidase activity profiles showed that the 20S proteasome accumulation is altered depending on the physiological state of the cells. The amount of active 20S particles increases in Halobacterium cells as a response to thermal and low salt stresses. In the same conditions, Northern experiments showed a positive transcriptional regulation of the alpha and beta proteasome subunits as well as of the two proteasome regulatory ATPases, PANA and PANB. Co-immunoprecipitation experiments demonstrated the existence of a physical interaction between the two Proteasome Activating Nucleotidase (PAN) proteins in cell extracts. Thus, a direct regulation occurs on the PAN-proteasome components to adjust the protein degradation activity to growth and environmental constraints. These results also indicate that, in extreme halophiles, proteasome mediated proteolysis is an important aspect of low salt stress response. The tri-peptide vinyl sulfone inhibitor NLVS was used in cell cultures to study the in vivo function of proteasome in Halobacterium. The chemical inhibition of proteasomes was measured in the cellular extracts. It has no effect on cell growth and mortality under normal growth conditions as well as under heat shock conditions. These results suggest that the PAN activators or other proteases compensate for loss of proteasome activity in stress conditions.

Tetrahedral aminopeptidase: a novel large protease complex from archaea.

A dodecameric protease complex with a tetrahedral shape (TET) was isolated from Haloarcula marismortui, a salt-loving archaeon. The 42 kDa monomers in the complex are homologous to metal-binding, bacterial aminopeptidases. TET has a broad aminopeptidase activity and can process peptides of up to 30-35 amino acids in length. TET has a central cavity that is accessible through four narrow channels (<17 A wide) and through four wider channels (21 A wide). This architecture is different from that of all the proteolytic complexes described to date that are made up by rings or barrels with a single central channel and only two openings.

Characterization of the proteasome from the extremely halophilic archaeon Haloarcula marismortui.

A 20S proteasome, comprising two subunits alpha and beta, was purified from the extreme halophilic archaeon Haloarcula marismortui, which grows only in saturated salt conditions. The three-dimensional reconstruction of the H. marismortui proteasome (Hm proteasome), obtained from negatively stained electron micrographs, is virtually identical to the structure of a thermophilic proteasome filtered to the same resolution. The stability of the Hm proteasome was found to be less salt-dependent than that of other halophilic enzymes previously described. The proteolytic activity of the Hm proteasome was investigated using the malate dehydrogenase from H. marismortui (HmMalDH) as a model substrate. The HmMalDH denatures when the salt concentration is decreased below 2 M. Under these conditions, the proteasome efficiently cleaves HmMalDH during its denaturation process, but the fully denatured HmMalDH is poorly degraded. These in vitro experiments show that, at low salt concentrations, the 20S proteasome from halophilic archaea eliminates a misfolded protein.

Characterization of a novel complex from halophilic archaebacteria, which displays chaperone-like activities in vitro.

We isolated a protein, P45, from the extreme halophilic archaeon Haloarcula marismortui, which displays molecular chaperone activities in vitro. P45 is a weak ATPase that assembles into a large ring-shaped oligomeric complex comprising about 10 subunits. The protein shows no significant homology to any known protein. P45 forms complexes with halophilic malate dehydrogenase during its salt-dependent denaturation/renaturation and decreases the rate of deactivation of the enzyme in an ATP-dependent manner. Compared with other halophilic proteins, the P45 complex appears to be much less dependent on salt for its various activities or stability. In vivo experiments showed that P45 accumulates when cells are exposed to a low salt environment. We suggest, therefore, that P45 could protect halophilic proteins against denaturation under conditions of cellular hyposaline stress.

Comparison of the PR mutant with the wild-type strain of proteus mirabilis brings insight into peroxide resistance factors and regulation of catalase expression.

The peroxide resistant mutant (PR) of Proteus mirabilis was characterized by an increased constitutive catalase activity concomitant with a large production of specific mRNA. Survival toward hydrogen peroxide during exponential phase was increased by H2O2 pretreatment in the wild type but not in the mutant, although the catalase of both strains was not inducible under these conditions. In the mutant, besides catalase, over-produced proteins comprised two different alkyl hydroperoxide reductase subunit C (AhpC) proteins and a protein homologous to the stationary phase transcription factor SspA of Escherichia coli. Conversely, the flagellin A (FlaA) of P. mirabilis was repressed in the PR mutant. Genomic DNA fragments of 2.9 kb carrying the catalase gene (katA) together with the 5' and 3' flanking regions were isolated from both strains and found to be identical. Upstream of katA, a Fur box-like sequence was found, but surprisingly, restricting iron in the culture medium caused a decrease in catalase production. The PR mutant presents similarities with other peroxide resistant mutants, but the regulation of catalase biosynthesis in P. mirabilis seems somewhat different from other close species such as E. coli.

The protein sequence of an archaeal catalase-peroxidase.

The gene encoding a catalase-peroxidase of archaeal origin, the halophilic catalase-peroxidase from Haloarcula marismortui, was sequenced. The primary structure proposed was confirmed by Edman degradation and mass spectrometry analyses of proteolytic fragments of the purified protein. The open reading frame in the gene corresponds to 731 amino acids and the calculated mass of the mature protein (deleted of the N-terminal methionine) is 81,253.65 Da, in reasonable agreement with the value of 81,292 +/- 9 Da previously measured by mass spectrometry. Southern and Northern blot analyses showed that the protein is encoded by a single gene as a monocistronic transcript. The protein sequence shows a high level of identity with bacterial catalase-peroxidases, with strongly conserved regions around the heme binding histidines. Similarly to other soluble halophilic proteins, it shows the excess of acidic residues that has been associated with solvation in halophilic adaptation.

Biochemical and serological evidence for an RNase E-like activity in halophilic Archaea.

Endoribonuclease RNase E appears to control the rate-limiting step that mediates the degradation of many mRNA species in bacteria. In this work, an RNase E-like activity in Archaea is described. An endoribonucleolytic activity from the extreme halophile Haloarcula marismortui showed the same RNA substrate specificity as the Escherichia coli RNase E and cross-reacted with a monoclonal antibody raised against E. coli RNase E. The archaeal RNase E activity was partially purified from the extreme halophilic cells and shown, contrary to the E. coli enzyme, to require a high salt concentration for cleavage specificity and stability. These data indicate that a halophilic RNA processing enzyme can specifically recognize and cleave mRNA from E. coli in an extremely salty environment (3 M KCI). Having recently been shown in mammalian cells (A. Wennborg, B. Sohlberg, D. Angerer, G. Klein, and A. von Gabain, Proc. Natl. Acad. Sci. USA 92:7322-7326, 1995), RNase E-like activity has now been identified in all three evolutionary domains: Archaea, Bacteria, and Eukarya. This strongly suggests that mRNA decay mechanisms are highly conserved despite quite different environmental conditions.

Halophilic protein stabilization by the mild solubilizing agents nondetergent sulfobetaines.

In this work, experiments performed on pig heart and halophilic malate dehydrogenase as well as halophilic elongation factor Tu demonstrate a protein stabilization property from the recently described mild solubilizing agents nondetergent sulfobetaines. A practical application is given by the separation of halophilic bacteria elongation factor Tu and halophilic malate dehydrogenase by high-performance ion-exchange chromatography achieved at reduced salt levels without significant loss of activity.

Structure and expression of the nuclear gene coding for the chloroplast ribosomal protein L21: developmental regulation of a housekeeping gene by alternative promoters.

We have cloned and sequenced the nuclear gene of the chloroplast ribosomal protein L21 (rpl21) of Spinacia oleracea. The gene consists of five exons and four introns. All introns are located in the sequence which corresponds to the Escherichia coli-like central core of the protein. L21 mRNA is present in photosynthetic (leaves) and nonphotosynthetic (roots and seeds) plant organs, although large quantitative differences exist. Primer extension and S1 nuclease mapping experiments revealed the existence of two types of transcripts in leaves. The two corresponding start sites were defined as P1 and P2. In roots and seeds, we found only the shorter of the two transcripts (initiated at P2). The nucleotide sequence surrounding P2 resembles promoters for housekeeping and vertebrate r-protein genes. Analysis of several promoter constructions by transient expression confirmed that both transcripts originate from transcription initiation. Results are interpreted to mean that the expression of the rpl21 gene is regulated by alternative promoters. One of the promoters (P2) is constitutive, and the other one (P1) is specifically induced in leaves, i.e., its activation should be related to the transformation of amyloplasts or proplastids to chloroplasts. The gene thus represents the first example of a housekeeping gene which is regulated by the organ-specific usage of alternative promoters. Primer extension analysis and S1 nuclease mapping of another nucleus-encoded chloroplast ribosomal protein gene (rps1) give evidence that the same type of regulation by two-promoter usage might be a more general phenomenon of plant chloroplast-related ribosomal protein genes. Preliminary results indicate that presence of conserved sequences within the rpl21 and rps1 promoter regions which compete for the same DNA binding activities.

Characterization and RNA-binding properties of a chloroplast S1-like ribosomal protein.

Control of translation is an important step in chloroplast gene expression. A first control can be exerted during the initiation complex formation which, in Escherichia coli, involves the ribosomal protein (r-protein) S1. A cDNA clone have been characterized which codes for the precursor of the chloroplast r-protein CS1. The mature protein consists of a central core which shows 31.5% amino acid homology to the E. coli protein S1. The CS1 is considerably shorter (40 kDa) than the protein S1 (61 kDa). The core fragment contains three degenerated repeats which show homology to both the ribosome- and the RNA-binding domain of S1. RNA-protein CS1 interactions were studied by UV cross-linking and toe-printing. CS1 has been over-expressed in E. coli, and after purification its RNA-binding properties were studied in vitro. We conclude that the CS1 exhibits an RNA-binding activity which is actively involved in the chloroplast initiation complex formation. It is shown that the CS1 binds to poly(A) in contrast with S1 which binds strongly to poly(U). These results are interpreted in relation to the presence of poly(A)-rich regions in chloroplast transcripts of higher plants.

Structure and expression of the nuclear gene coding for the plastid CS1 ribosomal protein from spinach.

The chloroplast ribosomal protein CS1 is an essential component of the plastids translational machinery involved in translation initiation. Southern analysis suggests that the corresponding nuclear gene is present in one copy in the spinach genome. We have isolated and sequenced the gene (rps1) to study its expression at the transcriptional level. The gene consists of 7 exons and 6 introns including an unusually large intron in the 5' coding region. No canonical TATA-box is found in the 5' upstream region of the gene. rps1 transcripts are detected early during germination and a significant accumulation is observed after the protrusion of the radicle. CS1 mRNAs are present in all organs of young seedlings although there are dramatic differences in the steady state level of the mRNAs between leaves and roots tissues. Transcripts accumulate independently of the presence or absence of light. Band shift analysis shows that the +1, -400 bp region of the gene can bind different sets of proteins isolated from roots and leaves nuclei. We suggest that the expression of the housekeeping plastid-related rps1 gene is regulated in a tissue-specific manner by transcriptional trans-acting factors.