Spherical particles of halophilic archaea correlate with exposure to low water activity--implications for microbial survival in fluid inclusions of ancient halite.

Viable extremely halophilic archaea (haloarchaea) have been isolated from million-year-old salt deposits around the world; however, an explanation of their supposed longevity remains a fundamental challenge. Recently small roundish particles in fluid inclusions of 22 000- to 34 000-year-old halite were identified as haloarchaea capable of proliferation (Schubert BA, Lowenstein TK, Timofeeff MN, Parker MA, 2010, Environmental Microbiology, 12, 440-454). Searching for a method to produce such particles in the laboratory, we exposed rod-shaped cells of Halobacterium species to reduced external water activity (a(w)). Gradual formation of spheres of about 0.4 µm diameter occurred in 4 M NaCl buffer of a(w) ≤ 0.75, but exposure to buffered 4 M LiCl (a(w) ≤ 0.73) split cells into spheres within seconds, with concomitant release of several proteins. From one rod, three or four spheres emerged, which re-grew to normal rods in nutrient media. Biochemical properties of rods and spheres were similar, except for a markedly reduced ATP content (about 50-fold) and an increased lag phase of spheres, as is known from dormant bacteria. The presence of viable particles of similar sizes in ancient fluid inclusions suggested that spheres might represent dormant states of haloarchaea. The easy production of spheres by lowering a(w) should facilitate their investigation and could help to understand the mechanisms for microbial survival over geological times.

Radiation sensitivity of N. pharaonis in comparison with E. coli K12 strains.

To investigate the radiation sensitivity of the natronobacterium Natronomonas pharaonis in comparison with Escherichia coli strains (N. pharaonis DSM 2160T, E. coli strains AB1157 and K12 lambda s) were exposed to gamma-radiation (60Co-gamma-source, 100 Gy min-1) in the presence of oxygen (air) and under strongly reduced oxygen conditions (argon-saturated medium). After irradiation, the colony-forming ability (dose-survival curves) and the D37 dose were determined. The oxygen content of the solutions containing high NaCl concentrations was measured with an oxygen electrode (Clark electrode). It was found that N. pharaonis can tolerate a remarkably higher irradiation dose than the two E. coli strains (approx. 1.5-fold of K12 lambda s and approx. 4-fold of AB1157). The oxygen enhancement ratio (OER) is 2.8 for N. pharaonis and 2.6 for both E. coli strains. Therefore the higher radiation resistance of the N. pharaonis is not due to the low oxygen content of the cell solution (high salt concentration) but is probably related to the higher DNA repair ability of this archaebacteria strain.

Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt.

Prokaryotic diversity in Alpine salt sediments was investigated by polymerase chain reaction (PCR) amplification of 16S rRNA genes, sequencing of cloned products, and comparisons with culturable strains. DNA was extracted from the residue following filtration of dissolved Permo-Triassic rock salt. Fifty-four haloarchaeal sequences were obtained, which could be grouped into at least five distinct clusters. Similarity values of three clusters to known 16S rRNA genes were less than 90%-95%, suggesting the presence of uncultured novel taxa; two clusters were 98% and 99% similar to isolates from Permo-Triassic or Miocene salt from England and Poland, and to Halobacterium salinarum, respectively. Some rock salt samples, including drilling cores, yielded no amplifiable DNA and no cells or only a few culturable cells. This result suggested a variable distribution of haloarchaea within different strata, probably consistent with the known geologic heterogeneity of Alpine salt deposits. We recently reported identical culturable Halococcus salifodinae strains in Permo-Triassic salt sediments from England, Germany, and Austria; together with the data presented here, those results suggest one plausible scenario to be an ancient continuous hypersaline ocean (Zechstein sea) populated by haloarchaea, whose descendants are found today in the salt sediments. The novelty of the sequences also suggested avoidance of haloarchaeal contaminants during our isolation of strains, preparation of DNA, and PCR reactions.

Isolation of a chymotrypsinogen B-like enzyme from the archaeon Natronomonas pharaonis and other halobacteria.

A protease of a molecular mass of approximately 30kDa was isolated and purified from the haloalkaliphilic archaeon Natronomonas (formerly Natronobacterium) pharaonis. The enzyme hydrolyzed synthetic peptides, preferentially at the carboxyl terminus of phenylalanine or leucine, as well as large proteins. Hydrolysis occurred over the range of pH from 6 to 12, with an optimum at pH 10. The temperature optimum was 61 degrees C. The enzyme was nearly equally active over the range of salt concentration from 0.5 to 4M (NaCl or KCl). A strong cross-reaction with a polyclonal antiserum against human chymotrypsin was observed. Enzymatic activity was inhibited by typical serine protease inhibitors. There was significant homology between N-terminal and internal sequences from autolytic fragments and the sequence of bovine chymotrypsinogen B; the overall amino acid composition was similar to that of vertebrate chymotrypsinogens. Evidence for a zymogen-like processing of the protease was obtained. Cell extracts from other halobacteria exhibited similar proteolytic activity and immunoreactivity. The data suggested a widespread distribution of a chymotrypsinogen B-like protease among halo- and haloalkaliphilic Archaea.

Marinobacter aquaeolei sp. nov., a halophilic bacterium isolated from a Vietnamese oil-producing well.

Several strains of moderately halophilic and mesophilic bacteria were isolated at the head of an oil-producing well on an offshore platform in southern Vietnam. Cells were Gram-negative, non-spore-forming, rod-shaped and motile by means of a polar flagellum. Growth occurred at NaCl concentrations between 0 and 20%; the optimum was 5% NaCl. One strain, which was designated VT8T, could degrade n-hexadecane, pristane and some crude oil components. It grew anaerobically in the presence of nitrate on succinate, citrate or acetate, but not on glucose. Several organic acids and amino acids were utilized as sole carbon and energy sources. The major components of its cellular fatty acids were C12:0 3-OH, C16:1, omega 9c, C16:0 and C18:1 omega 9c. The DNA G + C content was 55.7 mol%. 16S rDNA sequence analysis indicated that strain VT8T was closely related to Marinobacter sp. strain CAB (99.8% similarity) and Marinobaster hydrocarbonoclasticus (99.4% similarity). Its antibiotic resistance, isoprenoid quinones and fatty acids were similar to those of Marinobacter hydrocarbonoclasticus and Pseudomonas nautica. However, the whole-cell protein pattern of VT8T differed from that of other halophilic marine isolates, including P. nautica. DNA-DNA hybridization indicated that the level of relatedness to Marinobacter hydrocarbonoclasticus was 65% and that to P. nautica was 75%. Further differences were apparent in Fourier-transformed IR spectra of cells and lipopolysaccharide composition. It is proposed that VT8T should be the type strain of a new species and should be named Marinobacter aquaeolei. P. nautica may have been misclassified, as suggested previously, and may also belong to the genus Marinobacter.

F-and V-ATPases in the genus Thermus and related species.

The discovery of a V-type ATPase in the gram-negative bacterium Thermus thermophilus HB8 (YOKOYAMA et al., J. Biol. Chem. 265, 21946, 1990) was unexpected, since only eukaryotic endomembranes and archaea were thought to contain this enzyme complex, and horizontal gene transfer was suggested to explain the finding. We examined membrane-associated ATPases from representatives of several groups of the genus Thermus. The enzymes were extracted with chloroform and purified by ion exchange chromatography or native gel electrophoresis. One novel Islandic isolate, T. scotoductus SE-1, as well as strain T. filiformis from New Zealand, possessed F-ATPases, as judged by the typical five subunit composition of the F1-moiety, sensitivity to azide, insensitivity to nitrate and a strong crossreaction with antibodies against the F1-ATPase from E. coli. In addition, N-terminal amino acid sequencing of the beta subunit from T. scotoductus SE-1 confirmed its homology with beta subunits from known F-ATPases. In contrast, the same extraction procedure released a V-ATPase from the membranes of T. thermophilus HB27 and T. aquaticus YT-1. The related species Meiothermus (formerly Thermus) chliarophilus ALT-8 also possessed a V-ATPase. All V-ATPases examined in this study contained larger major subunits than F-ATPases, crossreacted with antiserum against subunit A of the V-ATPase from the archaeon Halobacterium saccharovorum, and the N-terminal sequences of their major subunits were homologous to those of other V-ATPases. Sequences of the 16S rRNA gene clearly placed T. scotoductus SE-1, along with other non-pigmented Thermus strains, as a distinct species close to T. aquaticus. Our results suggested that at least two members of the genus, T. scotoductus SE-1 and T. filiformis, contain an F-ATPase, whereas several others possess a V-ATPase. These data could indicate a greater diversity of the genus Thermus than was previously thought. Alternatively, the genus may consist of species where horizontal gene transfer has occurred and others, where it has not.

Comparison of membrane ATPases from extreme halophiles isolated from ancient salt deposits.

Halophilic microorganisms were isolated from Triassic and Permian salt deposits. Two were rods and grew as red colonies; another was a coccus and produced pink colonies. The rods lysed in solutions that lacked added sodium chloride. Growth of all isolates was inhibited by aphidicolin and their bulk proteins were acidic as judged from isoelectric focusing. Therefore, these organisms were tentatively identified as extreme halophiles. Whole cell proteins patterns of the isolates following gel electrophoresis were distinct and differed from those of representative type strains of halophilic bacteria. The membrane ATPases from the rods were similar to the enzyme from Halobacterium saccharovorum with respect to subunit composition, enzymatic properties and immunological cross-reaction, but differed slightly in amino acid composition. If the age of the microbial isolated is similar to that of the salt deposits, they can be considered repositories of molecular information of great evolutionary interest.

Presence of methyl sterol and bacteriohopanepolyol in an outer-membrane preparation from Methylococcus capsulatus (Bath).

Cytoplasmic/intracytoplasmic and outer membrane preparations of Methylococcus capsulatus (Bath) were isolated by sucrose density gradient centrifugation of a total membrane fraction prepared by disruption using a French pressure cell. The cytoplasmic and/or intracytoplasmic membrane fraction consisted of two distinct bands, Ia and Ib (buoyant densities 1.16 and 1.8 g ml-1, respectively) that together contained 57% of the protein, 68% of the phospholipid, 73% of the ubiquinone and 89% of the CN-sensitive NADH oxidase activity. The only apparent difference between these two cytoplasmic bands was a much higher phospholipid content for Ia. The outer membrane fraction (buoyant density 1.23 - 1.24 g ml-1) contained 60% of the lipopolysaccharide-associated, beta-hydroxypalmitic acid, 74% of the methylsterol, and 66% of the bacteriohopanepolyol (BHP); phospholipid to methyl sterol or BHP ratios were 6:1. Methanol dehydrogenase activity and a c-type cytochrome were also present in this outer membrane fraction. Phospholipase A activity was present in both the cytoplasmic membrane and outer membrane fractions. The unique distribution of cyclic triterpenes may reflect a specific role in conferring outer membrane stability in this methanotrophic bacterium.

Nucleotide-protectable labeling of sulfhydryl groups in subunit I of the ATPase from Halobacterium saccharovorum.

A membrane-bound ATPase from the archaebacterium Halobacterium saccharovorum is inhibited by N-ethylmaleimide in a nucleotide-protectable manner (Stan-Lotter et al., 1991, Arch. Biochem. Biophys. 284, 116-119). When the enzyme was incubated with N-[14C]ethylmaleimide, the bulk of radioactivity was associated with the 87,000-Da subunit (subunit I). ATP, ADP, or AMP reduced incorporation of the inhibitor. No charge shift of subunit I was detected following labeling with N-ethylmaleimide, indicating an electroneutral reaction. The results are consistent with the selective modification of sulfhydryl groups in subunit I at or near the catalytic site and are further evidence of a resemblance between this archaebacterial ATPase and the vacuolar-type ATPases.

Purification and properties of an ATPase from Sulfolobus solfataricus.

A sulfite-activated ATPase isolated from Sulfolobus solfataricus had a relative molecular mass of 370,000. It was composed of three subunits whose relative molecular masses were 63,000, 48,000, and 24,000. The enzyme was inhibited by the vacuolar ATPase inhibitors nitrate and N-ethylmaleimide; 4-chloro-7-nitrobenzofurazan (NBD-Cl) was also inhibitory. N-Ethylmaleimide was predominately bound to the largest subunit while NBD-Cl was bound to both subunits. ATPase activity was inhibited by low concentrations of p-chloromercuriphenyl sulfonate and the inhibition was reversed by cysteine which suggested that thiol groups were essential for activity. While the ATPase from S. solfataricus shared several properties with the ATPase from S. acidocaldarius there were significant differences. The latter enzyme was activated by sulfate and chloride and was unaffected by N-ethylmaleimide, whereas the S. solfataricus ATPase was inhibited by these anions as well as N-ethylmaleimide. These differences as well as differences that occur in other vacuolar-like ATPases isolated from the methanogenic and the extremely halophilic bacteria suggest the existence of several types of archaeal ATPases, none of which have been demonstrated to synthesize ATP.

Relationship of the membrane ATPase from Halobacterium saccharovorum to vacuolar ATPases.

Polyclonal antiserum against subunit A (67 kDa) of the vacuolar ATPase from Neurospora crassa reacted with subunit I (87 kDa) from a membrane ATPase of the extremely halophilic archaebacterium Halobacterium saccharovorum. The halobacterial ATPase was inhibited by nitrate and N-ethylmaleimide; the extent of the latter inhibition was diminished in the presence of adenosine di- or triphosphates. 4-Chloro-7-nitrobenzofurazan inhibited the halobacterial ATPase also in a nucleotide-protectable manner; the bulk of inhibitor was associated with subunit II (60 kDa). The data suggested that this halobacterial ATPase may have conserved structural features from both the vacuolar and the F-type ATPases.

A comparison of an ATPase from the archaebacterium Halobacterium saccharovorum with the F1 moiety from the Escherichia coli ATP synthase.

A purified ATPase associated with membranes from Halobacterium saccharovorum was compared with the F1 moiety from the Escherichia coli ATP synthase. The halobacterial enzyme was composed of two major (I and II) and two minor subunits (III and IV), whose molecular masses were 87 kDa, 60 kDa, 29 kDa and 20 kDa, respectively. The isoelectric points of these subunits ranged from 4.1 to 4.8, which in the case of the subunits I and II was consistent with the presence of an excess of acidic amino acids (20-22 mol/100 mol). Peptide mapping of subunits I and II denatured with sodium dodecyl sulfate showed no relationship between the primary structures of the individual halobacterial subunits or similarities to the subunits of the F1 ATPase from E. coli. Trypsin inactivation of the halobacterial ATPase was accompanied by the partial degradation of the major subunits. This observation, taken in conjunction with molecular masses of the subunits and the native enzyme, was consistent with the previously proposed stoichiometry of 2:2:1:1. These results suggest that H. saccharovorum, and possibly, halobacteria in general, possess an ATPase which is unlike the ubiquitous F0F1 ATP synthase.

Electrophoresis and isoelectric focusing of whole cell and membrane proteins from the extremely halophilic archaebacteria.

The isoelectric points of most proteins from the extremely halophilic archaebacteria are between 4.0 and 4.65 which agrees with the generally high content of glutamic and aspartic acid in proteins from halobacteria. The subunits from two purified halobacterial membrane enzymes (ATPase and nitrate reductase) behaved differently with respect to isoelectric focusing, silver staining and interaction with ampholytes. Differential behavior was also observed in whole cell proteins from Halobacterium saccharovorum regarding resolution in two-dimensional gels and silver staining. We propose that these differences reflect the existence of two classes of halobacterial proteins, one resembling non-halophilic proteins, and the other possessing unique properties that may be related to salt dependence.

Conformational interactions between alpha and beta subunits in the F1 ATPase of Escherichia coli as shown by chemical modification of uncA401 and uncD412 mutant enzymes.

In contrast to wild-type F1 adenosine triphosphatase, the beta subunits of soluble ATPase from Escherichia coli mutant strains AN120 (uncA401) and AN939 (uncD412) were not labeled by the fluorescent thiol-specific reagents 5-iodoacetamidofluorescein, 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid or 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-diazole. The mutation in the alpha subunit (uncA401) of F1 ATPase thus influences the accessibility of the single cysteinyl residue in the beta subunit. Following reaction of ATPase with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or N,N'-dicyclohexylcarbodiimide, the alpha and beta subunits of the uncA401, but not of the uncD412 mutant F1 ATPase were intensely labeled by a fluorescent thiol reagent. The mutation in the beta subunit (uncD412) thus influences the accessibility of the cysteinyl residues in the alpha subunit. In other work [Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 248] we have shown that 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid react with a different beta subunit from that labeled by N,N'-dicyclohexylcarbodiimide. This asymmetry with respect to modification by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and N,N'-dicyclohexylcarbodiimide was seen in both mutant enzymes. In addition, the modification of one beta subunit of the uncA401 F1 ATPase induced the previously unreactive sulfhydryl group of another beta subunit to react with 2-(4'-iodoacetamidoanilino-naphthalene-6-sulfonic acid. These results provide evidence for at least three types of conformational interactions of the major subunits of F1 ATPase: from alpha to beta, from beta to alpha, and from beta to beta. As in wild-type ATPase, labeling of membrane-bound unc mutant ATPase by a fluorescent thiol reagent modified the alpha subunits. This suggests that a conformational change of yet a different type occurs when the enzyme binds to the membrane.

N,N'-dicyclohexylcarbodiimide and 4-chloro-7-nitrobenzofurazan bind to different beta subunits of the F1 ATPase of Escherichia coli.

The fluorescent thiol reagent 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid (IAANS) labels the gamma, delta, and one of the three beta subunits of the F1 ATPase from Escherichia coli (ECF1). This is the same beta subunit which incorporates 4-chloro-7-nitrobenzofurazan (Nbf) [H. Stan-Lotter and P. D. Bragg (1986) Eur. J. Biochem. 154, 321-327]. After inactivation of ECF1 with N,N'-dicyclohexylcarbodiimide (DCCD), IAANS labels in addition to the beta, gamma, and delta subunits also the alpha subunit. This suggests a conformational change of ECF1 upon binding of DCCD. The beta subunit which incorporates DCCD does does not bind IAANS. Likewise, IAANS-modified ECF1 does not incorporate DCCD into the same beta subunit. It is concluded that DCCD and Nbf bind to different beta subunits. Since neither of these reagents binds to that beta subunit which can be crosslinked to to the epsilon subunit by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, these data show that there is a difference in the chemical reactivity of each of the three beta subunits of ECF1, despite their identical primary structures. This suggests that there is an asymmetry in the F1 molecule.

Isolation of a fourth cysteinyl-containing peptide of the alpha-subunit of the F1 ATPase from Escherichia coli necessitates revision of the DNA sequence.

The rapid determination of cysteinyl residues by Creighton's method [(1980) Nature 284, 487-489] led to the discovery of a discrepancy between protein and DNA sequence data in the alpha-subunit of the F1 ATPase from Escherichia coli [(1984) Arch. Biochem. Biophys. 229, 320-328]. We have isolated a cysteinyl-containing decapeptide from the alpha-subunit with a protein sequence (AGCAMGEYFR) which is only partially recognizable from DNA data. Re-sequencing of DNA in the region coding for the peptide has resulted in two corrections: insertion of a cytosine before position 715 and deletion of a thymine at position 731 of the uncA gene.

Electrophoretic determination of sulfhydryl groups and its application to complex protein samples, in vitro protein synthesis mixtures, and cross-linked proteins.

Without prior fractionation, the number of sulfhydryl groups of individual polypeptides in a protein mixture can be determined, provided their molecular weights and approximate isoelectric points are known. Urea-denatured protein samples are reacted with iodoacetamide and iodoacetate in a modified version of Creighton's procedure. After separation by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and isoelectric focusing, the number of sulfhydryl groups is determined by counting the protein bands which have additional negative charges. This method requires little material and provides an additional parameter, besides the molecular weight and isoelectric point, for the identification and characterization of a protein. The sensitivity may be enhanced for nonradioactive proteins by using 14C-labeled iodoacetamide and iodoacetate. The procedure has been applied to prokaryotic in vitro protein synthesis mixtures, bacterial membrane protein, and trypsin-cleaved or chemically cross-linked subunits of the F1 ATPase from Escherichia coli.

Thiol modification as a probe of conformational forms of the F1 ATPase of Escherichia coli and of the structural asymmetry of its beta subunits.

The sulfhydryl groups of soluble and membrane-bound F1 adenosine triphosphatase of Escherichia coli were modified by reaction with the fluorescent thiol reagents 5-iodoacetamidofluorescein, 2-[(4'-iodoacetamido)anilino]naphthalene-6-sulfonic acid 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-d iaz ole and 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid. Whereas gamma and delta subunits were always labeled by these reagents, the beta subunit reacted preferentially in the soluble enzyme, and the alpha subunit in the membrane-bound enzyme. This suggests that the soluble enzyme undergoes a conformational change on binding to the membrane. The three beta subunits of the soluble ATPase did not react with chemical reagents in a similar manner. One beta subunit was cross-linked to the epsilon subunit on treatment of the ATPase with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide, as observed previously by Lötscher et al. [Biochemistry (1984) 23, 4134-4140]. A second beta subunit, which did not cross-link to the epsilon subunit, was modified preferentially by the fluorescent thiol reagents and by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The third beta subunit was less chemically reactive than the others. Both alpha and beta subunits of the soluble 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole-modified enzyme were labeled by the fluorescent thiol reagents. Thus, the modified enzyme, which is inactive, probably has a different conformation from the native soluble ATPase.