The archaeal flagellum: a different kind of prokaryotic motility structure.

The archaeal flagellum is a unique motility apparatus distinct in composition and likely in assembly from the bacterial flagellum. Gene families comprised of multiple flagellin genes co-transcribed with a number of conserved, archaeal-specific accessory genes have been identified in several archaea. However, no homologues of any bacterial genes involved in flagella structure have yet been identified in any archaeon, including those archaea in which the complete genome sequence has been published. Archaeal flagellins possess a highly conserved hydrophobic N-terminal sequence that is similar to that of type IV pilins and clearly unlike that of bacterial flagellins. Also unlike bacterial flagellins but similar to type IV pilins, archaeal flagellins are initially synthesized with a short leader peptide that is cleaved by a membrane-located peptidase. With recent advances in genetic transfer systems in archaea, knockouts have been reported in several genes involved in flagellation in different archaea. In addition, techniques to isolate flagella with attached hook and anchoring structures have been developed. Analysis of these preparations is under way to identify minor structural components of archaeal flagella. This and the continued isolation and characterization of flagella mutants should lead to significant advances in our knowledge of the composition and assembly of archaeal flagella.

Correlation between glycosylation of flagellin proteins and sensitivity of flagellar filaments to Triton X-100 in methanogens.

The flagellins of Methanospirillum hungatei strains JF1 and GP1, Methanococcus deltae, and Methanothermus fervidus are glycosylated. Isolated flagellar filaments from these organisms are dissociated by low concentrations (0.5% (v/v)) of Triton X-100. Flagellar filaments from other methanogens (Methanococcus voltae, Methanococcus vannielii and Methanoculleus marisnigri) composed of non-glycosylated flagellins are resistant to Triton X-100 treatment. Consequently, the isolation techniques (employing Triton X-100) used to isolate basal body-hook-filament complexes in eubacteria may not be applicable to many methanogens.

Cloning and sequencing of a multigene family encoding the flagellins of Methanococcus voltae.

The flagellins of Methanococcus voltae are encoded by a multigene family of four related genes (flaA, flaB1, flaB2, and flaB3). All four genes map within the same region of the genome, with the last three arranged in a direct tandem. Northern (RNA) blot and primer extension analyses of total cellular RNA indicate that all four genes are transcribed. The flaB1, flaB2, and flaB3 flagellins are transcribed as part of a large polycistronic message which encodes at least one more protein which is not a flagellin. An intercistronic stem-loop followed by a poly(T) tract located between the flaB2 and flaB3 genes appears to increase the resistance of the flaB1/flaB2 portion of this polycistronic message to digestion by endogenous RNases. The flaA gene, located approximately 600 bp upstream from the tandem, is transcribed as a separate message at very low levels. The four open reading frames encode proteins of molecular weights 23,900, 22,400, 22,800, and 25,500, much less than the Mr values of 33,000 and 31,000 for the flagellins calculated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of isolated flagellar filaments. Each flagellin contains multiple eukaryotic glycosylation signals (Arg-X-Ser/Thr), although they do not appear to be glycoproteins, and each has an 11- or 12-amino-acid leader peptide. The N termini of all four flagellins (amino acids 1 through 47 of the mature protein) are very hydrophobic, and this region shows a high degree of homology with the flagellins from Halobacterium halobium.

Ultrastructure and biochemistry of the cell wall of Methanococcus voltae.

The ultrastructure and chemical composition of the cell wall of the marine archaebacterium Methanococcus voltae were studied by negative-staining and freeze-etch electron microscopy and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. M. voltae possesses a single regularly structured (RS) protein layer external to the plasma membrane. Freeze-etch preparations of cells indicated that the protein subunits are hexagonally arranged with a center-to-center spacing of approximately 10 nm. The extracted RS protein had a molecular weight of 76,000. It was present on envelopes prepared by shearing in a French press, osmotic lysis, or sonication, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NaCl was not required for attachment of the RS protein to the underlying plasma membrane. The hexagonal array could be demonstrated by platinum shadowing and freeze-etching of envelopes, but negative staining in the abscence of NaCl failed to stabilize the array. The RS protein could be solubilized by urea, guanidine hydrochloride, dithiothreitol, and several detergents, including Nonidet P-40, Triton X-100, and Tween 20. However, the most specific release of the wall protein from envelopes occurred after a heat treatment in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at 50 to 60 degrees C.

The effects of ionophores and metabolic inhibitors on methanogenesis and energy-related properties of Methanobacterium bryantii.

The effects of numerous ionophores and inhibitors were tested on methane synthesis, intracellular ATP and potassium concentrations, and the proton motive force of the methanogenic archaebacterium Methanobacterium bryantii. M. bryantii had an internal pH near 6.8 (and hence little delta pH during growth) with an electrical potential of --127 mV in growth medium and --105 mV in a pH 6.5 buffer. The study has identified agents which, in M. bryantii, can effectively cause a decline of intracellular ATP (gramicidin, acetylene) and potassium concentrations (gramicidin, nigericin), inhibit methane synthesis (acetylene, gramicidin, nigericin, triphenylmethylphosphonium bromide), eliminate the electrical potential (high extracellular potassium ion concentrations), and dissipate artificially imposed, inside alkaline, pH gradients (monensin, nigericin, carbonyl cyanide m-chlorophenylhydrazone). Carbonyl cyanide m-chlorophenylhydrazone was generally ineffective in media or buffers reduced with cysteine-sulfide but could be effective in cysteine-free solutions reduced with hydrogen sulfide.

Posttranslational processing of Methanococcus voltae preflagellin by preflagellin peptidases of M. voltae and other methanogens.

Methanococcus voltae is a mesophilic archaeon with flagella composed of flagellins that are initially made with 11- or 12-amino-acid leader peptides that are cleaved prior to incorporation of the flagellin into the growing filament. Preflagellin peptidase activity was demonstrated in immunoblotting experiments with flagellin antibody to detect unprocessed and processed flagellin subunits. Escherichia coli membranes containing the expressed M. voltae preflagellin (as the substrate) were combined in vitro with methanogen membranes (as the enzyme source). Correct processing of the preflagellin to the mature flagellin was also shown directly by comparison of the N-terminal sequences of the two flagellin species. M. voltae preflagellin peptidase activity was optimal at 37 degrees C and pH 8.5 and in the presence of 0.4 M KCl with 0.25% (vol/vol) Triton X-100.

Overexpression of Methanococcus voltae flagellin subunits in Escherichia coli and Pseudomonas aeruginosa: a source of archaeal preflagellin.

Methanococcus voltae is a flagellated member of the Archaea. Four highly similar flagellin genes have previously been cloned and sequenced, and the presence of leader peptides has been demonstrated. While the flagellins of M. voltae are predicted from their gene sequences to be approximately 22 to 25 kDa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified flagella revealed flagellin subunits with apparent molecular masses of 31 and 33 kDa. Here we describe the expression of a M. voltae flagellin in the bacteria Escherichia coli and Pseudomonas aeruginosa. Both of these systems successfully generated a specific expression product with an apparently uncleaved leader peptide migrating at approximately 26.5 kDa. This source of preflagellin was used to detect the presence of preflagellin peptidase activity in the membranes of M. voltae. In addition to the native flagellin, a hybrid flagellin gene containing the sequence encoding the M. voltae FlaB2 mature protein fused to the P. aeruginosa pilin (PilA) leader peptide was constructed and transformed into both wild-type P. aeruginosa and a prepilin peptidase (pilD) mutant of P. aeruginosa. Based on migration in SDS-PAGE, the leader peptide appeared to be cleaved in the wild-type cells. However, the archaeal flagellin could not be detected by immunoblotting when expressed in the pilD mutant, indicating a role of the peptidase in the ultimate stability of the fusion product. When the +5 position of the mature flagellin portion of the pilin-flagellin fusion was changed from glycine to glutamic acid (as in the P. aeruginosa pilin) and expressed in both wild-type and pilD mutant P. aeruginosa, the product detected by immunoblotting migrated slightly more slowly in the pilD mutant, indicating that the fusion was likely processed by the prepilin peptidase present in the wild type. Potential assembly of the cleaved fusion product by the type IV pilin assembly system in a P. aeruginosa PilA-deficient strain was tested, but no filaments were noted on the cell surface by electron microscopy.

Nickel transport in Methanobacterium bryantii.

Methanobacterium bryantii, grown autotrophically on H2-CO2, transported nickel against a concentration gradient by a high-affinity system (Km = 3.1 microM). The system had a pH optimum of 4.9 and a temperature optimum of 49 degrees C with an energy of activation of 7.8 kcal/mol (ca. 32.6 kJ/mol). A headspace of H2-CO2 (4:1, vol/vol) was required for maximum rate of transport. The system was highly specific for nickel and was unaffected by high levels of all monovalent and divalent ions tested (including Mg2+) with the sole exception of Co2+. Kinetic experiments indicated that accumulated nickel became increasingly incorporated into cofactor F430 and protein. Nickel transport was inhibited by nigericin, monensin, and gramicidin but not by carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone, carbonyl cyanide-m-chlorophenyl hydrazone, N,N'-dicyclohexylcarbodiimide, valinomycin plus potassium, or acetylene. The ineffectiveness of carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone, carbonyl cyanide-m-chlorophenyl hydrazone, and N,N'-dicyclohexylcarbodiimide may be related to difficulties in the penetration of these compounds through the outer cell barriers. Nickel uptake was greatly stimulated by an artificially imposed pH gradient (inside alkaline). The data suggest that nickel transport is not dependent on the membrane potential or on intracellular ATP, but is coupled to proton movement.

Sensitivity of methanogenic bacteria to dicyclohexylcarbodiimide.

Methanospirillum hungatei strains GP1 and JF1 when exposed to the adenosinetriphosphatase inhibitor N,N'-dicyclohexylcarbodiimide experienced a marked decline in growth rates, methane synthesis activities, and intracellular ATP concentrations. Although growth was inhibited, the intracellular ATP concentrations of all other methanogens tested were little affected by high concentrations of dicyclohexylcarbodiimide (500 microM). Thus, for studies of ATP synthesis, or for ATP depletion in whole cell suspensions of methanogens, the use of dicyclohexylcarbodiimide appears limited to M. hungatei.

The transmembrane electrical potential and intracellular pH in methanogenic bacteria.

The magnitudes of the electrical potential and proton gradient in Methanospirillum hungatei GP1 and Methanobacterium thermoautotrophicum were determined. No delta pH (inside alkaline) could be demonstrated in either organism suspended in growth media at normal growth pH values by the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO), butyrate, propionate, or methylamine. The internal pH, estimated to be approximately 6.7 under our growth conditions, was not constant, but varied as the external pH was adjusted. However, the internal pH was always more neutral than the external pH (except at pH 6.7 where the two were equal). The distribution of triphenylmethylphosphonium cation, in the presence of tetraphenylboron anion, gave estimates of 119 and 79 mV (interior negative) for the electrical potentials of the thermophile and mesophile, respectively, for cells suspended in a phosphate buffer (pH 7.0). The uptake of 86Rb+, in the presence of valinomycin, gave similar results for M. thermoautotrophicum, ranging from 143 mV (at pH 5.8) to 120 mV (at pH 8.0). Electrical potentials compared to the size of the respective K+ gradients, maintained between the cytoplasm and growth medium. The results are interpreted in terms of proton efflux and monovalent cation antiport activities at the cytoplasmic membrane, with possible proton pumping at the site of internal vesicles.

Isolation and characterization of a bacteriophage specific for the lipopolysaccharide of rough derivatives of Pseudomonas aeruginosa strain PAO.

A lipopolysaccharide (LPS)-defective (rough) mutant of Pseudomonas aeruginosa PAO was isolated by selection for resistance to the LPS-specific phage E79. The LPS of this mutant, AK-1012, lacked the O-antigenic side chain-specific amino sugar fucosamine as well as the core-specific sugars glucose and rhamnose. Using this strain, we isolated and characterized a phage, phi PLS27, which is specifically inactivated upon incubation with LPS extracted from rough mutants of P. aeruginosa PAO. phi PLS27 was found to be a Bradley type C phage and was very similar to coliphage T7 in a number of properties, including size, buoyant density, mass, and the number of structural proteins.

Pseudomonas aeruginosa bacteriophage phi PLS27-lipopolysaccharide interactions.

We investigated the phi PLS27 receptor in Pseudomonas aeruginosa strain PAO lipopolysaccharide (LPS) by analyzing a resistant mutant. This mutant, which was designated AK1282, had the most defective LPS yet reported for a P. aeruginosa rough mutant; this LPS contained only lipid A, 2-keto-3-deoxyoctonate, heptose, and alanine as major components. In addition, this LPS lacked galactosamine, which is present in the inner core of the LPS of other rough mutants. The loss of galactosamine but only a small decrease in the alanine content indicated that the core of strain PAO LPS differed from the core structure which has been suggested for the LPS of other well-characterized P. aeruginosa strains. Our analysis also indicated that galactosamine residues may be crucial for phi PLS27 receptor activity of the LPS. Electrodialysis of LPS and conversion to salt forms (sodium or triethylamine) influenced the phage-inactivating capacity of the LPS, as did the medium in which the inactivation occurred; experiments performed in 1/10-strength broth resulted in much lower PhI50 (concentration of LPS causing a 50% decrease in the titer of phage during 1 h of incubation at 37 degrees C) values than experiments performed in regular-strength broth. Sonication of the LPS also increased the phage-inactivating capacities of the LPS preparations.

K+, Na+, and Mg2+ content and permeability of Methanospirillum hungatei and Methanobacterium thermoautotrophicum.

The K+, Na+, and Mg2+ contents of Methanospirillum hungatei and of the thermophile Methanobacterium thermoautotrophicum were determined at various phases of growth. The intracellular K+ content of exponential phase cells of M. thermoautotrophicum (approximately 780 mM) was 5.4-fold higher than in M. hungatei, and decreased gradually as the culture entered the stationary phase. Both methanogens concentrated Mg2+, exhibiting an increased content as the cultures aged. Comparisons among extraction methods showed that most of the internal K+ was readily released, but a minimum of half of the Mg2+ in M. hungatei, and most of the M2+ in M. thermoautotrophicum, was in a bound form. Exponential phase of cells of M. hungatei established an intracellular level of Na+ lower than the outside medium, but the thermophile concentrated Na+. Dextran, inulin, sucrose, and glucose penetrated cell pellets to varying degrees and could be used to measure the space corresponding to cytoplasm and to cell wall permeability barriers. L-Phenylalanine penetrated fully and acetate accumulated in both methanogens. Acetate uptake in cell suspensions of M. hungatei was fully inhibited by oxygen. N-ethylmaleimide, or N,N'-dicyclohexylcarbodiimide, but was not affected by the proton conductor carbonylcyanide p-trifluoromethoxyphenyl-hydrazone. L-Malate, which penetrated M. hungatei cells poorly, was metabolized to glutamate, indicating the presence of an incomplete reductive carboxylic acid cycle.

Characterization of flagellum gene families of methanogenic archaea and localization of novel flagellum accessory proteins.

Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in the complete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likely mediated by novel components. In this study, a conserved flagellar gene family from each of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii has been characterized. These species possess multiple flagellin genes followed immediately by eight known and supposed flagellar accessory genes, flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motif in the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production for flagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundant polycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases several flagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessory genes of M. voltae were successfully overexpressed as His-tagged proteins in Escherichia coli. These recombinant flagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies for localization studies. Immunoblotting of fractionated M. voltae cells demonstrated that FlaC, FlaD, FlaE, FlaH, and FlaI are all present in the cell as membrane-associated proteins but are not major components of isolated flagellar filaments. Interestingly, flaD was found to encode two proteins, each translated from a separate ribosome binding site. These protein expression data indicate for the first time that the putative flagellar accessory genes of M. voltae, and likely those of other archaeal species, do encode proteins that can be detected in the cell.

Effect of gramicidin on methanogenesis by various methanogenic bacteria.

Methanogenesis by Methanobacterium thermoautotrophicum strains was extremely sensitive to gramicidin, total inhibition being observed at 0.2 mug/ml. In contrast, methane synthesis by Methanococcus voltae, Methanogenium marisnigri, Methanosarcina mazei, and Methanospirillum hungatei were resistant to the highest concentrations of gramicidin tested (40 mug/ml), although spheroplasts of Methanospirillum hungatei were extremely sensitive. Other species tested showed intermediate sensitivity to gramicidin, methanogenesis inhibition occurring at 4 to 20 mug/ml.

Physical characterization of the flagella and flagellins from Methanospirillum hungatei.

Flagellar filaments from Methanospirillum hungatei GP1 and JF1 were isolated and subjected to a variety of physical and chemical treatments. The filaments were stable to temperatures up to 80 degrees C and over the pH range of 4 to 10. The flagellar filaments were dissociated in the detergents (final concentration of 0.5%) Triton X-100, Tween 20, Tween 80, Brij 58, N-octylglucoside, cetyltrimethylammonium bromide, and Zwittergent 3-14, remaining intact in only two of the detergents tested, sodium deoxycholate and 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS). Spheroplasting techniques were used to separate the internal cells from the complex sheath, S-layer (cell wall), and end plugs of M. hungatei. The flagellar basal structure was visualized after solubilization of membranes by CHAPS or deoxycholate. The basal structure appeared to be a simple knob with no apparent ring or hook structures. The multiple, glycosylated flagellins constituting the flagellar filaments were cleaved by proteases and cyanogen bromide. The cyanogen bromide-generated fragments of M. hungatei GP1 flagellins were partially sequenced to provide internal sequence information. In addition, the amino acid composition of each flagellin was determined and indicated that the flagellins are distinct gene products, rather than differentially glycosylated forms of the same gene product.

Heat shock response of the archaebacterium Methanococcus voltae.

The general properties of the heat shock response of the archaebacterium Methanococcus voltae were characterized. The induction of 11 heat shock proteins, with apparent molecular weights ranging from 18,000 to 90,000, occurred optimally at 40 to 50 degrees C. Some of the heat shock proteins were preferentially enriched in either the soluble (cytoplasm) or particulate (membrane) fraction. Alternative stresses (ethanol, hydrogen peroxide, NaCl) stimulated the synthesis of subsets of the heat shock proteins as well as unique proteins. Western blot (immunoblot) analysis, in which antisera to Escherichia coli heat shock proteins (DnaK and GroEL) were used, did not detect any immunologically cross-reactive proteins. In addition, Southern blot analysis did not reveal any homology between M. voltae and four highly conserved heat shock genes, mopB and dnaK from E. coli and hsp70 genes from Drosophila species and Saccharomyces cerevisiae.

Inhibition of methanogenesis in pure cultures by ammonia, fatty acids, and heavy metals, and protection against heavy metal toxicity by sewage sludge.

The effect of ammonium chloride, sodium butyrate, sodium propionate, and the heavy metals nickel, zinc, and copper on methanogenesis by pure cultures of Methanospirillum hungatei, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, and Methanobacterium formicicum at pH 6.5 was studied. The latter three strains were resistant to greater than 60 g/L of the volatile fatty acids and to greater than 10 g/L of NH3 N. Methanospirillum hungatei was somewhat more sensitive with 50% inhibition of methanogenesis occurring at 4.2 g/L NH3 N, 27 g/L butyrate, and 41 g/L propionate. All strains were very sensitive to both copper (1-5 mg/L) and zinc (1-10 mg/L), but much more resistant to nickel. Zinc and copper concentrations 30 to 270 times higher were required to cause inhibition of Msp. hungatei incubated in sewage sludge compared with buffer, indicating a strong protective environment was afforded the methanogens against heavy metal toxicity in the sludge.

Relatedness of the flagellins from methanogens.

Purified flagellar filaments isolated from six methanogens were composed of multiple flagellins. Two flagellins were present in Methanococcus deltae (Mr = 34,000 and 32,000), Methanoculleus marisnigri (Mr = 31,000 and 25,500) and Methanococcus jannaschii (Mr = 31,000 and 27,500), three in Methanothermus fervidus (Mr = 34,000, 25,000 and 24,000) and four or more in both Methanococcus vannielii and Methanococcus maripaludis (Mr ranging from 27,500 to 32,000). The flagellins of M. fervidus and M. deltae reacted positively with glycoprotein-specific stains. The flagellins of M. deltae, M. maripaludis and M. vannielii were closely related to those of M. voltae based on cross-reactivity with antisera raised against M. voltae flagellins and homology with flagellin-specific oligonucleotide probes to the N-terminus and leader peptide of M. voltae flagellins. Similarities appear to exist among the flagellins of M. fervidus, M. marisnigri and Halobacterium halobium based on cross-reactivity with antisera produced against the flagella of Methanospirillum hungatei JF1. The N-termini of the flagellins from the mesophilic Methanococcus spp. and M. marisnigri show homology with the N-termini of other archaebacterial flagellins. These N-termini may undergo a modification involving removal of a leader peptide.

Isolation of flagella from the archaebacterium Methanococcus voltae by phase separation with Triton X-114.

The flagella of Methanococcus voltae were isolated by using three procedures. Initially, cells were sheared to release the filaments, which were purified by differential centrifugation and banding in KBr gradients. Flagella were also prepared by solubilization of cells with 1% (vol/vol) Triton X-100 and purified as described above. Both of these techniques resulted in variable recovery and poor yield of flagellar filaments. Purification of intact flagella (filament, hook, and basal body) was achieved by using phase transition separation with Triton X-114. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified flagella revealed two major proteins, with molecular weights of 33,000 and 31,000. This result indicates the likely presence of two flagellins. The filament had a diameter of 13 nm. The basal structure consisted of a small knob, while a slight thickening of the filament immediately adjacent to this area was the only evidence of a hook region. Flagella from three other Methanococcus species were isolated by this technique and found to have the same ultrastructure as flagella from M. voltae. Isolation of flagella from three eubacteria and another methanogen (Methanospirillum hungatei [M. hungatii]) by the phase separation technique indicated that the detergent treatment did not affect the structure of basal bodies. Intact ring structures and well-differentiated hook regions were apparent in each of these flagellar preparations.