Chaperone patterns in vernal keratoconjunctivitis are distinctive of cell and Hsp type and are modified by inflammatory stimuli.

BACKGROUND: Vernal keratoconjunctivitis (VKC) is a severe ocular allergy with pathogenic mechanism poorly understood and no efficacious treatment. The aims of the study were to determine quantities and distribution of Hsp chaperones in the conjunctiva of VKC patients and assess their levels in conjunctival epithelial and fibroblast cultures exposed to inflammatory stimuli. METHODS: Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, Hsp105, and Hsp110 were determined in conjunctiva biopsies from nine patients and nine healthy age-matched normal subjects, using immunomorphology and qPCR. Conjunctival epithelial cells and fibroblasts were cultured and stimulated with IL-1ß, histamine, IL-4, TNF-α, or UV-B irradiation, and changes in Hsp levels were determined by Western blotting. RESULTS: Hsp27, Hsp40, Hsp70, and Hsp90 levels increased in the patients' conjunctiva, whereas Hsp10, Hsp60, Hsp100, and Hsp105 did not. Double immunofluorescence demonstrated colocalization of Hsp27, Hsp40, Hsp70, and Hsp90 with CD68 and tryptase. Testing of cultured conjunctival cells revealed an increase in the levels of Hsp27 in fibroblasts stimulated with IL-4; Hsp40 in epithelial cells stimulated with IL-4 and TNF-α and in fibroblasts stimulated with IL-4, TNF-α, and IL-1ß; Hsp70 in epithelial cells stimulated with histamine and IL-4; and Hsp90 in fibroblasts stimulated with IL-1ß, TNF-α, and IL-4. UV-B did not induce changes. CONCLUSIONS: VKC conjunctiva displays distinctive quantitative patterns of Hsps as compared with healthy controls. Cultured conjunctival cells respond to cytokines and inflammatory stimuli with changes in the Hsps quantitative patterns. The data suggest that interaction between the chaperoning and the immune systems drives disease progression.

Hsp10, Hsp70, and Hsp90 immunohistochemical levels change in ulcerative colitis after therapy.

Ulcerative colitis (UC) is a form of inflammatory bowel disease (IBD) characterized by damage of large bowel mucosa and frequent extra-intestinal autoimmune comorbidities. The role played in IBD pathogenesis by molecular chaperones known to interact with components of the immune system involved in inflammation is unclear. We previously demonstrated that mucosal Hsp60 decreases in UC patients treated with conventional therapies (mesalazine, probiotics), suggesting that this chaperonin could be a reliable biomarker useful for monitoring response to treatment, and that it might play a role in pathogenesis. In the present work we investigated three other heat shock protein/molecular chaperones: Hsp10, Hsp70, and Hsp90. We found that the levels of these proteins are increased in UC patients at the time of diagnosis and decrease after therapy, supporting the notion that these proteins deserve attention in the study of the mechanisms that promote the development and maintenance of IBD, and as biomarkers of this disease (e.g., to monitor response to treatment at the histological level).

Upon oxidative stress, the antiapoptotic Hsp60/procaspase-3 complex persists in mucoepidermoid carcinoma cells.

Hsp60, a mitochondrial chaperonin highly conserved during evolution, has been found elevated in the cytosol of cancer cells, both in vivo and in vitro, but its role in determining apoptosis during oxidative stress (OS) has not yet been fully elucidated. The aim of the present work was to study the effects of OS on Hsp60 levels and its interactions with procaspase- 3 (p-C3) and p53 in tumor cells. NCI-H292 (mucoepidermoid carcinoma) cells were exposed to various concentrations of hydrogen peroxide (H2O2) for 24 hours. Cell viability was determined by Trypan blue and MTT assays. DNA damage was assessed by the Comet assay, and apoptosis was measured by the AnnexinV cytofluorimetric test. Exposure to increasing concentrations of H2O2 resulted in a reduction of cell viability, DNA damage, and early apoptotic phenomena. Hsp60, p-C3, p53, and p21 were assessed by Western blotting and immunocytochemistry before and after OS. Hsp60 and p-C3 were present before and after OS induction. Immunoprecipitation experiments showed an Hsp60/p-C3 complex before OS that persisted after it, while an Hsp60/p53 complex was not detected in either condition. The presence of wild type (wt) p53 was confirmed by RT-PCR, and p21 detection suggested p53 activation after OS. We postulate that, although OS may induce early apoptosis in NCI-H292 cells, Hsp60 exerts an anti-apoptotic effect in these cells and, by extension, it may do so in other cancer cells.

The basal transcription factors TBP and TFB from the mesophilic archaeon Methanosarcina mazeii: structure and conformational changes upon interaction with stress-gene promoters.

Transcription of archaeal non-stress genes involves the basal factors TBP and TFB, homologs of the eucaryal TATA-binding protein and transcription factor IIB, respectively. No comparable information exists for the archaeal molecular-chaperone, stress genes hsp70(dnaK), hsp40(dnaJ), and grpE. These do not occur in some archaeal species, but are present in others possibly due to lateral transfer from bacteria, which provides a unique opportunity to study regulation of stress-inducible bacterial genes in organisms with eukaryotic-like transcription machinery. Among the Archaea with the genes, those from the mesophilic methanogen Methanosarcina mazeii are the only ones whose basal (constitutive) and stress-induced transcription patterns have been determined. To continue this work, tbp and tfb were cloned from M. mazeii, sequenced, and the encoded recombinant proteins characterized in solution, separately and in complex with each other and with DNA. M. mazeii TBP ranks among the shortest within Archaea and, contrary to other archaeal TBPs, it lacks tryptophan or an acidic tail at the C terminus and has a basic N-terminal third. M. mazeii TFB is similar in length to archaeal and eucaryal homologs and all have a zinc finger and HTH motifs. Phylogenetically, the archaeal and eucaryal proteins form separate clusters and the M. mazeii molecules are closer to the homologs from Archaeoglobus fulgidus than to any other. Antigenically, M. mazeii TBP and TFB are close to archaeal homologs within each factor family, but the two families are unrelated. The purified recombinant factors were functionally active in a cell-free in vitro transcription system, and were interchangeable with the homologs from Methanococcus thermolithotrophicus. The M. mazeii factors have a similar secondary structure by circular dichroism (CD). The CD spectra changed upon binding to the promoters of the stress genes grpE, dnaK, and dnaJ, with the changes being distinctive for each promoter; in contrast, no effect was produced by the promoter of a non-stress-gene. Factor(s)-DNA modeling predicted that modifications of H bonds are caused by TBP binding, and that these modifications are distinctive for each promoter. It also showed which amino acid residues would contact an extended TATA box with a B recognition element, and evolutionary conservation of the TBP-TFB-DNA complex orientation between two archaeal organisms with widely different optimal temperature for growth (37 and 100 degrees C).

The molecular chaperone system and other anti-stress mechanisms in archaea.

This article presents a brief review of stressors, their cellular and intracellular targets, stress proteins, molecular chaperones, and other anti-stress mechanisms. New data are reported on cochaperones and multicellular structures in archaea. The molecular chaperoning systems of bacteria and eukaryotes have been studied for many years and are relatively well known in terms of their components and mechanisms of action, although many details remain to be elucidated and almost certainly other components will be discovered in the future. By comparison, the molecular chaperoning system of archaea is still unexplored. Since archaea have some molecular genetic and physiologic features similar to those of bacteria and some resembling those of eukaryotes, extrapolation from what is known of organisms from these two phylogenetic domains to archaeal species is unwarranted. For example, the components of the molecular chaperone machine, Hsp70(DnaK), Hsp40(DnaJ), and GrpE, in the archaeal species that have it, are closely related to bacterial counterparts, whereas the archaeal chaperonins are like the eukaryotic equivalents. Furthermore, many archaeal species lack the chaperone machine, in contrast to bacteria and eukaryotes that have it without any known exception. A search for the cochaperones trigger factor, Hop, Hip, BAG-1, and NAC in archaeal genomes demonstrated no conserved equivalents, but two families of archaeal molecules were identified that might be related to NAC and Hop, respectively. Multicellular structures with a single species such as packet and lamina are formed by Methanosarcina species, among which the best studied is M. mazeii. Multispecies multicellular structures are formed by a variety of archaeal organisms, which are either flat (biofilm) or globular (granule) and constitute a functional association or consortium. Details of morphology, formation, and internal organization are described for representative examples of multicellular structures. These may be seen as the result of primitive histogenesis reflecting primeval mechanisms of differentiation-development that might have evolved driven by environmental stressors. Cells in these complex threedimensional arrangements are not only positioned so they can interact with each other for more efficient functioning as in a tissue or organ, but are also protected from stressors. Single cells lacking the protective shield of other cells packed together with intercellular connective material, which is typical of multicellular structures, are directly exposed to environmental stressors and, thus, are at a disadvantage from the evolutionary standpoint. It seems reasonable to argue that differentiation-development leading to histogenesis might have arisen in primeval times as a consequence of the harsh conditions that primitive life forms had to endure, and that the ability to form tissue-like structures was a primary characteristic that ensured positive selection.

Stress and molecular chaperones in disease.

Stress, a common phenomenon in today's society, is suspected of playing a role in the development of disease. Stressors of various types, psychological, physical, and biological, abound. They occur in the working and social environments, in air, soil, water, food, and medicines. Stressors impact on cells directly or indirectly, cause protein denaturation, and elicit a stress response. This is mediated by stress (heat-shock) genes and proteins, among which are those named molecular chaperones because they assist other proteins to achieve and maintain a functional shape (the native configuration), and to recover it when partially lost due to stress. Denatured proteins tend to aggregate and precipitate. The same occurs with abnormal proteins due to mutations, or to failure of post-transcriptional or post-translational mechanisms. These abnormal proteins need the help of molecular chaperones as much as denatured molecules do, especially during stress. A cell with normal antistress mechanisms, including a complete and functional set of chaperones, may be able to withstand stress if its intensity is not beyond that which will cause irreversible protein damage. There is a certain threshold that normal cells have above which they cannot cope with stress. A cell with an abnormal protein that has an intrinsic tendency to misfold and aggregate is more vulnerable to stress than normal counterparts. Furthermore, these abnormal proteins may precipitate even in the absence of stress and cause diseases named proteinopathies. It is possible that stress contributes to the pathogenesis of proteinopathies by promoting protein aggregation, even in cells that possess a normal chaperoning system. Examples of proteinopathies are age-related degenerative disorders with protein deposits in various tissues, most importantly in the brain where the deposits are associated with neuronal degeneration. It is conceivable that stress enhances the progression of these diseases by facilitating protein unfolding and misfolding, which lead to aggregation and deposition. A number of reports in the last few years have described research aimed at elucidating the role of heatshock proteins, molecular chaperones in particular, in the pathogenesis of neurodegenerative disorders. The findings begin to shed light on the molecular mechanism of protein aggregation and deposition, and of the ensuing cell death. The results also begin to elucidate the role of molecular chaperones in pathogenesis. This is a fascinating area of research with great clinical implications. Although there are already several experimental models for the study of proteinopathies, others should be developed using organisms that are better known now than only a few years ago and that offer unique advantages. Use of these systems and of information available in databases from genome sequencing efforts should boost research in this field. It should be possible in the not-too-distant future to develop therapeutic and preventive means for proteinopathies based on the use of heat-shock protein and molecular chaperone genes and proteins.

Stressors, stress and survival: overview.

This overview introduces the contributions in this Special Issue with the aim of presenting an integrated picture of it. The contributions cover several important areas: protein stability and function under extreme conditions, osmotic stress and osmoadaptation, the structural features of the cell membrane and their possible significance with regard to heat stress, the molecular chaperone machine and multicellular structures as anti-stress mechanisms, peptidyl-prolyl cis-trans isomerases, proteases and the proteasome, and oxidative stress and the role of superoxide dismutase. These topics are briefly discussed to explain the basic concepts underpinning them, quoting for the most part introductory articles or reviews that might help the non-specialist to become familiar with the central themes of the Special Issue. As mentioned in the Preface every effort has been made to discuss the archaeal features within the context of other disciplines and biology in general, against the background of what is known for bacteria and eucarya. Hopefully, this approach will help the reader in understanding what is unique to the archaea, what is shared between them and the members of the other two phylogenetic domains, and how studies in archaea impact on other fields of science.

Identification of genes in the genome of the archaeon Methanosarcina mazeii that code for homologs of nuclear eukaryotic molecules involved in RNA processing.

A 2.6kb fragment of chromosomal DNA from the archaeon Methanosarcina mazeii was sequenced and analyzed, and it was found to contain coding regions for three proteins that were 321, 234, and 193 amino acids (aa) in length. Homologs of the 321-aa protein were found in all archaeal genomes examined, but not in eukaryotic or bacterial genomes, with one exception in the latter. The protein with 234aa (named PrpM) was most similar to the putative protein Prp31p from Methanobacterium thermoautotrophicum, while the 193-aa protein (named FibM) was identified as an archaeal fibrillarin homolog. Prp and fibrillarin proteins are involved in RNA processing in eukaryotes, but their functions in archaea are not yet understood. The M. mazeii PrpM was also similar to three proteins from Saccharomyces cerevisiae: Prp31p, Nop56p, and Nop58p. Prp31p is a pre-mRNA processing protein, while Nop56p and Nop58p are involved in rRNA processing and interact with fibrillarin. No homologs of either protein were found in bacteria. The archaeal fibrillarin was shorter than its eukaryotic counterpart because it lacked the N-terminal glycine-arginine-rich (GAR) domain, present in most eukaryal homologs. The archaeal prp and fibrillarin gene homologs were found adjacent to each other, whereas in eukarya these genes are on separate chromosomes. Sequence signatures typical of the eukaryal molecules were identified in the M. mazeii and the other archaeal molecules studied. The close proximity of the prp and fib genes raises the possibility of a Prp-fibrillarin interaction in archaea.

Stress genes and proteins in the archaea.

The field covered in this review is new; the first sequence of a gene encoding the molecular chaperone Hsp70 and the first description of a chaperonin in the archaea were reported in 1991. These findings boosted research in other areas beyond the archaea that were directly relevant to bacteria and eukaryotes, for example, stress gene regulation, the structure-function relationship of the chaperonin complex, protein-based molecular phylogeny of organisms and eukaryotic-cell organelles, molecular biology and biochemistry of life in extreme environments, and stress tolerance at the cellular and molecular levels. In the last 8 years, archaeal stress genes and proteins belonging to the families Hsp70, Hsp60 (chaperonins), Hsp40(DnaJ), and small heat-shock proteins (sHsp) have been studied. The hsp70(dnaK), hsp40(dnaJ), and grpE genes (the chaperone machine) have been sequenced in seven, four, and two species, respectively, but their expression has been examined in detail only in the mesophilic methanogen Methanosarcina mazei S-6. The proteins possess markers typical of bacterial homologs but none of the signatures distinctive of eukaryotes. In contrast, gene expression and transcription initiation signals and factors are of the eucaryal type, which suggests a hybrid archaeal-bacterial complexion for the Hsp70 system. Another remarkable feature is that several archaeal species in different phylogenetic branches do not have the gene hsp70(dnaK), an evolutionary puzzle that raises the important question of what replaces the product of this gene, Hsp70(DnaK), in protein biogenesis and refolding and for stress resistance. Although archaea are prokaryotes like bacteria, their Hsp60 (chaperonin) family is of type (group) II, similar to that of the eukaryotic cytosol; however, unlike the latter, which has several different members, the archaeal chaperonin system usually includes only two (in some species one and in others possibly three) related subunits of approximately 60 kDa. These form, in various combinations depending on the species, a large structure or chaperonin complex sometimes called the thermosome. This multimolecular assembly is similar to the bacterial chaperonin complex GroEL/S, but it is made of only the large, double-ring oligomers each with eight (or nine) subunits instead of seven as in the bacterial complex. Like Hsp70(DnaK), the archaeal chaperonin subunits are remarkable for their evolution, but for a different reason. Ubiquitous among archaea, the chaperonins show a pattern of recurrent gene duplication-hetero-oligomeric chaperonin complexes appear to have evolved several times independently. The stress response and stress tolerance in the archaea involve chaperones, chaperonins, other heat shock (stress) proteins including sHsp, thermoprotectants, the proteasome, as yet incompletely understood thermoresistant features of many molecules, and formation of multicellular structures. The latter structures include single- and mixed-species (bacterial-archaeal) types. Many questions remain unanswered, and the field offers extraordinary opportunities owing to the diversity, genetic makeup, and phylogenetic position of archaea and the variety of ecosystems they inhabit. Specific aspects that deserve investigation are elucidation of the mechanism of action of the chaperonin complex at different temperatures, identification of the partners and substitutes for the Hsp70 chaperone machine, analysis of protein folding and refolding in hyperthermophiles, and determination of the molecular mechanisms involved in stress gene regulation in archaeal species that thrive under widely different conditions (temperature, pH, osmolarity, and barometric pressure). These studies are now possible with uni- and multicellular archaeal models and are relevant to various areas of basic and applied research, including exploration and conquest of ecosystems inhospitable to humans and many mammals and plants.

The genes coding for the hsp70 (dnaK) molecular chaperone machine occur in the moderate thermophilic archaeon Methanosarcina thermophila TM-1.

The hsp70(dnaK) locus of the moderate thermophilic archaeon Methanosarcina thermophila TM-1 was cloned, sequenced, and tested in vitro to measure gene induction by heat and ammonia, i.e., stressors pertinent to the biotechnological ecosystem of this methanogen that plays a key role in anaerobic bioconversions. The locus' genes and organization, 5'-grpE-hsp70(dnaK)-hsp40 (dnaJ)-trkA-3', are the same as those of the closely related mesophile Methanosarcina mazei S-6, but different from those of the only other archaeon for which comparable sequence data exist, the thermophile Methanobacterium thermoautotrophicum deltaH, from another genus, in which trkA is not part of the locus. The proteins encoded in the TM-1 genes are very similar to the S-6 homologs, but considerably less similar to the deltaH proteins. The TM-1 Hsp70(DnaK) protein has the 23-amino acid deletion--by comparison with homologs from gram-negative bacteria first described in the S-6 molecule and later found to be present in all homologs from archaea and gram positives. The genes responded to a temperature elevation in a manner that demonstrated that they are heat-shock genes, functionally active in vivo. Ammonia also induced a heat-shock type of response by hsp70(dnaK), and a similar response by trkA. The data suggest that the moderate thermophile TM-1 has an active Hsp70(DnaK)-chaperone machine in contrast to hyperthermophilic archaea, and that trkA is a stress gene, inasmuch as it responds like classic heat-shock genes to stressors that induce a typical heat-shock response.

The archaeal molecular chaperone machine: peculiarities and paradoxes.

A major finding within the field of archaea and molecular chaperones has been the demonstration that, while some species have the stress (heat-shock) gene hsp70(dnaK), others do not. This gene encodes Hsp70(DnaK), an essential molecular chaperone in bacteria and eukaryotes. Due to the physiological importance and the high degree of conservation of this protein, its absence in archaeal organisms has raised intriguing questions pertaining to the evolution of the chaperone machine as a whole and that of its components in particular, namely, Hsp70(DnaK), Hsp40(DnaJ), and GrpE. Another archaeal paradox is that the proteins coded by these genes are very similar to bacterial homologs, as if the genes had been received via lateral transfer from bacteria, whereas the upstream flanking regions have no bacterial markers, but instead have typical archaeal promoters, which are like those of eukaryotes. Furthermore, the chaperonin system in all archaea studied to the present, including those that possess a bacterial-like chaperone machine, is similar to that of the eukaryotic-cell cytosol. Thus, two chaperoning systems that are designed to interact with a compatible partner, e.g., the bacterial chaperone machine physiologically interacts with the bacterial but not with the eucaryal chaperonins, coexist in archaeal cells in spite of their apparent functional incompatibility. It is difficult to understand how these hybrid characteristics of the archaeal chaperoning system became established and work, if one bears in mind the classical ideas learned from studying bacteria and eukaryotes. No doubt, archaea are intriguing organisms that offer an opportunity to find novel molecules and mechanisms that will, most likely, enhance our understanding of the stress response and the protein folding and refolding processes in the three phylogenetic domains.

Methanobacterium subterraneum sp. nov., a new alkaliphilic, eurythermic and halotolerant methanogen isolated from deep granitic groundwater.

Deep subterranean granitic aquifers have not been explored regarding methanogens until now. Three autotrophic methane-producing Archaea were isolated from deep granitic groundwater at depths of 68, 409 and 420 m. These organisms were non-motile, small, thin rods, 0.1-0.15 micron in diameter, and they could use hydrogen and carbon dioxide or formate as substrates for growth and methanogenesis. One of the isolates, denoted A8p, was studied in detail. It grew with a doubling time of 2.5 h under optimal conditions (20-40 degrees C, pH 7.8-8.8 and 0.2-1.2 M NaCl). Strain A8p is eurythermic as it grew between 3.6 and 45 degrees C. It was resistant to up to 20 mg bacitracin l-1. The G + C content was 54.5 mol%, as determined by thermal denaturation. Phylogenetic studies based upon 16S rRNA gene sequence comparisons placed the isolate A8p in the genus Methanobacterium. Phenotypic and phylogenetic characters indicate that the alkaliphilic, halotolerant strain A8p represents a new species. We propose the name Methanobacterium subterraneum for this species, and strain A8p (= DSM 11074T) is the type strain.

Structure, organization, and expression of genes coding for envelope components in the archaeon Methanosarcina mazei S-6.

The antigenic mosaics of archaeal species are complex and lead to the distinction of different immunotypes. We began the dissection of the antigenic mosaic of the methanogen Methanosarcina mazei S-6 by gene cloning and sequencing. The analysis of the sequence, organization, and in vitro (heterologous) and in vivo expression of two three-gene clusters that encode proteins localized to the cell envelope and that are recognized by antibodies for surface structures is presented in this report. The amino acid sequences and compositions share characteristics with S-layer proteins and, most notably, have repeats of conserved sequences and secondary structures. Expressed genes produced proteins with a tendency to oligomerize, and one of these proteins was susceptible to breakdown at regular intervals. Altogether, the data reveal a modular system (clusters of homologous genes, proteins of similar sequences with conserved repeats) seemingly suitable for assembling an enormous variety of final molecular structures by rearranging and combining genes, proteins, and repeats, and thus generate the observed wide spectrum of antigenic diversity.

Heat-shock response in Methanosarcina mazei S-6.

The dnaK locus of Methanosarcina mazei S-6, a mesophilic organism of the phylogenetic domain Archaea, contains the heat-shock genes 5'-grpE-dnaK-dnaJ-3'. Parameters known to affect the response of these genes in organisms of the other two domains, Bacteria and Eucarya, were tested to determine their effects on the archaeal homologs. The mRNA from the three genes increased after heat shock more in lamina than in single cells (these S-6 morphologic stages can be grown in the same substrate). Single cells in early stationary phase showed the highest levels of dnaK mRNA after heat shock, as compared with cells in exponential, or in late stationary, phase. The dnaK mRNA always had the size of a monocistronic transcript. dnaK was also found in the thermophileMethanosarcina thermophila TM-1, and its response to heat shock showed distinctive characteristics. However, dnaK was not revealed in other archaea: three hyperthermophiles (Methanothermus fervidus,Methanococcus jannaschii, and Sulfolobus sp.), and one mesophilic methanogen (Methanospirillum hungateii).

Molecular biology of S-layers.

In this chapter we report on the molecular biology of crystalline surface layers of different bacterial groups. The limited information indicates that there are many variations on a common theme. Sequence variety, antigenic diversity, gene expression, rearrangements, influence of environmental factors and applied aspects are addressed. There is considerable variety in the S-layer composition, which was elucidated by sequence analysis of the corresponding genes. In Corynebacterium glutamicum one major cell wall protein is responsible for the formation of a highly ordered, hexagonal array. In contrast, two abundant surface proteins from the S-layer of Bacillus anthracis. Each protein possesses three S-layer homology motifs and one protein could be a virulence factor. The antigenic diversity and ABC transporters are important features, which have been studied in methanogenic archaea. The expression of the S-layer components is controlled by three genes in the case of Thermus thermophilus. One has repressor activity on the S-layer gene promoter, the second codes for the S-layer protein. The rearrangement by reciprocal recombination was investigated in Campylobacter fetus. 7-8 S-layer proteins with a high degree of homology at the 5' and 3' ends were found. Environmental changes influence the surface properties of Bacillus stearothermophilus. Depending on oxygen supply, this species produces different S-layer proteins. Finally, the molecular bases for some applications are discussed. Recombinant S-layer fusion proteins have been designed for biotechnology.

An ABC-transporter system homolog in an organism of the phylogenetic domain Archaea.

A cluster of genes was identified in an archaeal organism, the methanogen Methanosarcina mazei S-6, that was a homolog of the ABC-transporter system loci of several organisms belonging to the phylogenetic domain Bacteria. The gene number, size, and organization were also similar. The proteins encoded by these genes were similar in structure, hydrophilicity-hydrophobicity profiles, and motifs to the equivalent components of homolog systems in bacteria and eucarya.

ABC transporters in Archaea: two genes encoding homologs of the nucleotide-binding components in the methanogen Methanosarcina mazei S-6.

Two genes, 5'-orfD-orfF-3', were found in the genome of the archaeon Methanosarcina mazei S-6 that encode the deduced proteins, OrfD and OrfF, with structural motifs typical of the nucleotide-binding components of the ABC-transporter systems of Bacteria and Eukarya. These motifs, and other similarities of OrfD and OrfF with bacterial and eukaryal counterparts, indicate that the two archaeal molecules belong to the ATP-binding cassette (ABC)-transporter family.

Integration of foreign DNA in an intergenic region of the archaeon Methanosarcina mazei without effect on transcription of adjacent genes.

Transformation systems for methanogenic archaea are scarce, none has been reported for the genus Methanosarcina, and plasmids useful as vectors for cloning foreign DNA into methanogens that stably replicate as extrachromosomal elements are not available. We developed an integration vector for transformation of a member of the genus Methanosarcina, i.e. Methanosarcina mazei, using a segment (Int alpha; 1015 bp) which encompasses the intergenic region (431 bp) between the stress (heat-shock) genes grpE and dnaK. This segment also includes the 3' end (270 bp) of the grpE protein-coding region and the 5' end (314 bp) of the dnaK protein-coding region. Int alpha has an EcoRI site, useful for cloning, situated in the 3' direction beyond the grpE transcription termination region, and far upstream from the dnaK promoter. This location of the site, and the monocistronic mode of transcription of grpE and dnaK in M. mazei, suggested to us that a foreign insert in the site would not affect transcription of either flanking gene. A puromycin-resistance cassette (pac cassette) was inserted in the EcoRI site of Int alpha already inserted in pUC18, to obtain a vector which integrated the pac cassette in the chromosome between grpE and dnaK. The pac gene was transcribed and the transformants acquired puromycin resistance. Constitutive and heat-shock-induced transcription of grpE and dnaK in the transformants was the same as in wild-type cells. The two vectors found with transforming ability differed in the orientation of the pac cassette but both had M. mazei's DNA on each flank of the cassette, with the same orientation as that of the homologous segments in the chromosome.

An archaeal gene upstream of grpE different from eubacterial counterparts.

In some eubacteria with a dnaK locus in which grpE is close upstream of dnaK, grpE is preceded by an open reading frame (orf) believed to be a heat-shock gene. We also found an orf, orf16, upstream of grpE in the archaeon Methanosarcina mazei S-6, but this gene differs from the eubacterial counterpart: it is shorter, does not respond to a temperature upshift as heat-shock genes do, and the deduced protein Orf16, does not resemble the proteins coded by the eubacterial equivalents. orf16 is expressed monocistronically, with a transcription initiation site 24 bases upstream of the translation start codon, 22 bases downstream of a putative promoter identical to the consensus promoter for genes in methanogens. This initiation site is used by heat-shocked and non-heat-shocked cells in the two morphologic stages of M. mazei S-6 tested, i.e., packets and single cells. Three transcription termination sites were identified, one of which is detectable only in non-heat-shocked cells. Data from comparative analyses of the Orf16 deduced amino acid sequence and those of other known proteins, as well as the apparent biochemical characteristics of Orf16, suggest that the latter is a membrane molecule.