Genetic analysis of a type IV pili-like locus in the archaeon Methanococcus maripaludis.

Methanococcus maripaludis is a stringently anaerobic archaeon with two studied surface structures, archaella and type IV pili. Previously, it was shown that three pilin genes (mmp0233 [epdA], mmp0236 [epdB] and mmp0237 [epdC]) located within an 11 gene cluster in the genome were necessary for normal piliation. This study focused on analysis of the remaining genes to determine their potential involvement in piliation. Reverse transcriptase PCR experiments demonstrated the 11 genes formed a single transcriptional unit. Deletions were made in all the non-pilin genes except mmp0231. Electron microscopy revealed that all the genes in the locus except mmp0235 and mmp0238 were essential for piliation. Complementation with a plasmid-borne wild-type copy of the deleted gene restored at least some piliation. We identified genes for an assembly ATPase and two versions of the conserved pilin platform forming protein necessary for pili assembly at a separate genetic locus.

The structure of an archaeal pilus.

Bacterial pili are involved in a host of activities, including motility, adhesion, transformation, and immune escape. Structural studies of these pili have shown that several distinctly different classes exist, with no common origin. Remarkably, it is now known that the archaeal flagellar filament appears to have a common origin with the bacterial type IV pilus, and assembly in both systems involves hydrophobic N-terminal alpha-helices that form three-stranded coils in the center of these filaments. Recent work has identified further genes in archaea as being similar to bacterial type IV pilins, but the function or structures formed by such gene products was unknown. Using electron cryo-microscopy, we show that an archaeal pilus from Methanococcus maripaludis has a structure entirely different from that of any of the known bacterial pili. Two subunit packing arrangements were identified: one has rings of four subunits spaced by approximately 44 A and the other has a one-start helical symmetry with approximately 2.6 subunits per turn of a approximately 30 A pitch helix. Remarkably, these schemes appear to coexist within the same filaments. For the segments composed of rings, the twist between adjacent rings is quite variable, while for the segments having a one-start helix there is a large variability in both the axial rise and the twist per subunit. Since this pilus appears to be assembled from a type IV pilin-like protein with a hydrophobic N-terminal helix, it provides yet another example of how different quaternary structures can be formed from similar building blocks. This result has many implications for understanding the evolutionary divergence of bacteria and archaea.

Systematic deletion analyses of the fla genes in the flagella operon identify several genes essential for proper assembly and function of flagella in the archaeon, Methanococcus maripaludis.

The archaeal flagellum is a unique motility apparatus in the prokaryotic domain, distinct from the bacterial flagellum. Most of the currently recognized archaeal flagella-associated genes fall into a single fla operon that contains the genes for the flagellin proteins (two or more genes designated as flaA or flaB), some variation of a set of conserved proteins of unknown function (flaC, flaD, flaE, flaF, flaG and flaH), an ATPase (flaI) and a membrane protein (flaJ). In addition, the flaD gene has been demonstrated to encode two proteins: a full-length gene product and a truncated product derived from an alternate, internal start site. A systematic deletion approach was taken using the methanogen Methanococcus maripaludis to investigate the requirement and a possible role for these proposed flagella-associated genes. Markerless in-frame deletion strains were created for most of the genes in the M. maripaludis fla operon. In addition, a strain lacking the truncated FlaD protein [FlaD M(191)I] was also created. DNA sequencing and Southern blot analysis confirmed each mutant strain, and the integrity of the remaining operon was confirmed by immunoblot. With the exception of the DeltaFlaB3 and FlaD M(191)I strains, all mutants were non-motile by light microscopy and non-flagellated by electron microscopy. A detailed examination of the DeltaFlaB3 mutant flagella revealed that these structures had no hook region, while the FlaD M(191)I strain appeared identical to wild type. Each deletion strain was complemented, and motility and flagellation was restored. Collectively, these results demonstrate for first time that these fla operon genes are directly involved and critically required for proper archaeal flagella assembly and function.

Identification of genes involved in the biosynthesis and attachment of Methanococcus voltae N-linked glycans: insight into N-linked glycosylation pathways in Archaea.

N-linked glycosylation is recognized as an important post-translational modification across all three domains of life. However, the understanding of the genetic pathways for the assembly and attachment of N-linked glycans in eukaryotic and bacterial systems far outweighs the knowledge of comparable processes in Archaea. The recent characterization of a novel trisaccharide [beta-ManpNAcA6Thr-(1-4)-beta-GlcpNAc3NAcA-(1-3)-beta-GlcpNAc]N-linked to asparagine residues in Methanococcus voltae flagellin and S-layer proteins affords new opportunities to investigate N-linked glycosylation pathways in Archaea. In this contribution, the insertional inactivation of several candidate genes within the M. voltae genome and their resulting effects on flagellin and S-layer glycosylation are reported. Two of the candidate genes were shown to have effects on flagellin and S-layer protein molecular mass and N-linked glycan structure. Further examination revealed inactivation of either of these two genes also had effects on flagella assembly. These genes, designated agl (archaeal glycosylation) genes, include a glycosyl transferase (aglA) involved in the attachment of the terminal sugar to the glycan and an STT3 oligosaccharyl transferase homologue (aglB) involved in the transfer of the complete glycan to the flagellin and S-layer proteins. These findings document the first experimental evidence for genes involved in any glycosylation process within the domain Archaea.

Recent advances in the structure and assembly of the archaeal flagellum.

Archaeal motility occurs through the rotation of flagella that are distinct from the flagella found on bacteria. The differences between the two structures include the multi-flagellin nature of the archaeal filament, the widespread posttranslational modification of the flagellins and the presence of a short signal peptide on each flagellin that is cleaved by a specific signal peptidase prior to the incorporation of the mature flagellin into the flagellar filament. Research has revealed similarities between the archaeal flagellum and the type IV pilus, including the presence of similar unusual signal peptides on the flagellins and pilins, similarities in the amino acid sequences of the major structural proteins themselves, as well as similarities between potential assembly and processing components. The recent suggestion that type IV pili are part of a family of cell surface complexes, coupled with the similarities between type IV pili and archaeal flagella, raise questions about the evolution of these systems and possible inclusion of archaeal flagella into this surface complex family.

Structure and subunit arrangement of the A-type ATP synthase complex from the archaeon Methanococcus jannaschii visualized by electron microscopy.

In Archaea, bacteria, and eukarya, ATP provides metabolic energy for energy-dependent processes. It is synthesized by enzymes known as A-type or F-type ATP synthase, which are the smallest rotatory engines in nature (Yoshida, M., Muneyuki, E., and Hisabori, T. (2001) Nat. Rev. Mol. Cell. Biol. 2, 669-677; Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315). Here, we report the first projected structure of an intact A(1)A(0) ATP synthase from Methanococcus jannaschii as determined by electron microscopy and single particle analysis at a resolution of 1.8 nm. The enzyme with an overall length of 25.9 nm is organized in an A(1) headpiece (9.4 x 11.5 nm) and a membrane domain, A(0) (6.4 x 10.6 nm), which are linked by a central stalk with a length of approximately 8 nm. A part of the central stalk is surrounded by a horizontal-situated rodlike structure ("collar"), which interacts with a peripheral stalk extending from the A(0) domain up to the top of the A(1) portion, and a second structure connecting the collar structure with A(1). Superposition of the three-dimensional reconstruction and the solution structure of the A(1) complex from Methanosarcina mazei Gö1 have allowed the projections to be interpreted as the A(1) headpiece, a central and the peripheral stalk, and the integral A(0) domain. Finally, the structural organization of the A(1)A(0) complex is discussed in terms of the structural relationship to the related motors, F(1)F(0) ATP synthase and V(1)V(0) ATPases.

Methanocaldococcus indicus sp. nov., a novel hyperthermophilic methanogen isolated from the Central Indian Ridge.

An autotrophic, hyperthermophilic methanogen, strain SL43(T), was isolated from a deep-sea hydrothermal chimney sample collected on the Central Indian Ridge at a depth of 2420 m. The coccoid, surface-layer-carrying, Gram-negative-staining cells were heavily flagellated and exhibited a slight tumbling motility. The temperature range for growth at pH 6.5 was 50-86 degrees C, with optimum growth at 85 degrees C. The optimum pH for growth was 6.6 and the optimum NaCl concentration for growth was 30 g l(-1). The novel isolate used H(2) and CO(2) as the only substrates for growth and produced methane. Selenium and yeast extract stimulated growth significantly. In the presence of CO(2) and H(2), the organism reduced elemental sulfur to hydrogen sulfide. Growth was inhibited by chloramphenicol and rifampicin, but not by ampicillin, kanamycin, penicillin or streptomycin. The G+C content of the genomic DNA was 30.7 mol%. On the basis of 16S rRNA gene sequence analysis, this organism was most closely related to Methanocaldococcus infernus ME(T) (3.2 % distance). Its phylogenetic distinctiveness was confirmed by RFLP analysis of the 16S rDNA, a reliable tool for differentiating hyperthermophilic methanococci. On the basis of phylogenetic and physiological characteristics, it is proposed that strain SL43(T) (=DSM 15027(T)=JCM 11886(T)) be designated as the type strain of a novel species, Methanocaldococcus indicus sp. nov.

Cleavage of preflagellins by an aspartic acid signal peptidase is essential for flagellation in the archaeon Methanococcus voltae.

The differences between archaeal and bacterial flagella are becoming more apparent as research on the archaeal structure progresses. One crucial difference is the presence of a leader peptide on archaeal preflagellins, which is removed from the flagellin prior to its incorporation into the flagellar filament. The enzyme responsible for the removal of the flagellin leader peptide was identified as FlaK. FlaK of Methanococcus voltae retains its preflagellin peptidase activity when expressed in Escherichia coli and used in an in vitro assay. Homologous recombination of an integration vector into the chromosomal copy of flaK resulted in a non-motile, non-flagellated phenotype. The flagellins of the mutant had larger molecular weights than their wild-type counterparts, as expected if they retained their 11- to 12-amino-acid leader peptide. Membranes of the flaK mutant were unable to process preflagellin in the in vitro assay. Site-directed mutagenesis demonstrated that two aspartic acid residues conserved with ones in type IV prepilin peptidases were necessary for proper recognition or processing of the preflagellin. As bacterial flagellins lack a leader peptide and a peptidase is not required for export and assembly, the requirement for FlaK further emphasizes the similarity archaeal flagella have with type IV pili, rather than with bacterial flagella.

Identification and localization of flagellins FlaA and FlaB3 within flagella of Methanococcus voltae.

Methanococcus voltae possesses four flagellin genes, two of which (flaB1 and flaB2) have previously been reported to encode major components of the flagellar filament. The remaining two flagellin genes, flaA and flaB3, are transcribed at lower levels, and the corresponding proteins remained undetected prior to this work. Electron microscopy examination of flagella isolated by detergent extraction of whole cells revealed a curved, hook-like region of varying length at the end of a long filament. Enrichment of the curved region of the flagella resulted in the identification of FlaB3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal sequencing, and the localization of this flagellin to the cell-proximal portion of the flagellum was confirmed through immunoblotting and immunoelectron microscopy with FlaB3-specific antibodies, indicating that FlaB3 likely composes the curved portion of the flagella. This could represent a unique case of a flagellin performing the role of the bacterial hook protein. FlaA-specific antibodies were used in immunoblotting to determine that FlaA is found throughout the flagellar filament. M. voltae cells were transformed with a modified flaA gene containing a hemagglutinin (HA) tag introduced into the variable region. Transformants that had replaced the wild-type copy of the flaA gene with the HA-tagged version incorporated the HA-tagged version of FlaA into flagella which appeared normal by electron microscopy.

Insertional inactivation of the flaH gene in the archaeon Methanococcus voltae results in non-flagellated cells.

The marine methanogen Methanococcus voltae possesses two transcriptional units that encode a total of four flagellins. Immediately downstream of the flagellin genes are a number of ORFs, some of which are cotranscribed with the flagellin genes. These putative genes have been named flaCDEFGHIJ, although no biochemical data has implicated them in flagellar morphogenesis. None of the flaC-J genes has homology to any bacterial gene, with the exception of flaI, which shows homology to pilT, a gene that encodes a nucleotide binding protein of the type IV pilus family. In this study, insertional mutations in flaH of M. voltae were identified. The mutants were non-motile and non-flagellated as determined by electron microscopy. Southern hybridization experiments confirmed the insertion of a mutagenic vector into flaH and indicated that two, tandem, copies of the vector were present. It is believed that insertion of the vector into flaH should disrupt the transcription of flaIJ due to polar effects. The flaH mutant displayed the same pattern of multiple mRNA transcripts, all originating upstream of flaB1, as the wild-type cells. Northern hybridization experiments failed to detect a flaHIJ transcript in either wild-type or mutant cells. Immunoblotting experiments indicated, however, that the mutants produced similar amounts of flagellin, FlaD and FlaE to wild-type cells. Flagellin localization experiments suggest that the flaH mutant is deficient in flagellin secretion and/or assembly. The mutant also displayed similar preflagellin peptidase activity to the wild-type cells, indicating that none of the genes flaHIJ is likely to be the gene that encodes this enzyme, which is required for cleaving the leader peptide from the preflagellins prior to their incorporation into the flagellar filament. This is the first data indicating that the flaHIJ gene cluster is essential for flagellation in methanogens.

Characterization of Methanococcus voltaei strain P2F9701a: a new methanogen isolated from estuarine environment.

A new methanogen, designated as strain P2F9701a (= OCM 745), was isolated from a water sample of estuarine environment in Elrin Shi, Taiwan. Cells of strain P2F9701a were motile coccus (0.7 approximately 1.1 micron) with tufts of flagella. Gas vacuoles were observed, and the protein cell wall was composed of S-layer protein subunit with Mr of 74,700. Cells catabolized formate and H2+CO2 to produce methane, but not acetate, methanol, and trimethylamine. Strain P2F9701a grew in the range of 30-42 degrees C, with optimal growth temperature at 37 degrees C, but did not grow below 28 degrees C or above 42 degrees C. This estuarine isolate P2F9701a tolerated well the NaCl concentration between 0.02 and 1.03 m, and the optimal salt for growth was 0.17 m. Although phylogenetic analytic results indicated that P2F9701a belongs to the mesophilic, hydrogenotrophic marine methanogen of Methanococcus voltaei, the occurrence of gas vacuoles, tufts of flagella, eury-halotolerant and steno-thermotolerant characters of strain P2F9701a are different from mesophilic Methanococcus spp. that had been reported.

Helical tubes of FtsZ from Methanococcus jannaschii.

Bacterial cell division depends on the formation of a cytokinetic ring structure, the Z-ring. The bacterial tubulin homologue FtsZ is required for Z-ring formation. FtsZ assembles into various polymeric forms in vitro, indicating a structural role in the septum of bacteria. We have used recombinant FtsZ1 protein from M. jannaschii to produce helical tubes and sheets with high yield using the GTP analogue GMPCPP [guanylyl-(alpha,beta)-methylene-diphosphate]. The sheets appear identical to the previously reported Ca++-induced sheets of FtsZ from M. jannaschii that were shown to consist of 'thick'-filaments in which two protofilaments run in parallel. Tubes assembled either in Ca++ or in GMPCPP contain filaments whose dimensions indicate that they could be equivalent to the 'thick'-filaments in sheets. Some tubes are hollow but others are filled by additional protein density. Helical FtsZ tubes differ from eukaryotic microtubules in that the filaments curve around the filament axis with a pitch of approximately 430 A for Ca++-induced tubes or 590 - 620 A for GMPCPP. However, their assembly in vitro as well-ordered polymers over distances comparable to the inner circumference of a bacterium may indicate a role in vivo. Their size and stability make them suitable for use in motility assays.

A novel pH2 control on the expression of flagella in the hyperthermophilic strictly hydrogenotrophic methanarchaeaon Methanococcus jannaschii.

The methanarchaeon, Methanococcus jannaschii, a hyperthermophilic, autotrophic, and strictly hydrogenotrophic inhabitant of submarine hydrothermal vents, was cultivated in a reactor at two hydrogen partial pressure (p(H(2))) values, 178 kPa (high) and 650 Pa (ultralow), and the cells were subjected to a comparative proteome analysis. From these studies, it was discovered that, when p(H(2)) was high and the cell density was low (a combination representing a hydrogen-excess condition), the cells possessed very low or undetectable levels of four flagella-related polypeptides (FlaB2, FlaB3, FlaD, and FlaE); electron microscopic examination showed that most of these cells were devoid of flagella. Flagella synthesis occurred when hydrogen became limiting either at high cell density under high p(H(2)) or at low cell density under low p(H(2)). The results from a p(H(2))-shift experiment corroborated the above observations. The p(H(2))-dependent changes in the levels of two methanogenic enzymes (MTD and HMDX) were as expected, and thus they served as internal controls. To our knowledge, this is the first example for the regulation of expression of flagella by hydrogen in any domain of life and for a control of any kind on flagella synthesis in the archaea. Our work also provides the only known example for each of the following: (i) the pure culture cultivation of a methanogen at an ultralow, near ecologically relevant p(H(2)); (ii) experimental functional genomics for M. jannaschii; and (iii) the use of proteomics with M. jannaschii.

Bacteriophage-like particles associated with the gene transfer agent of methanococcus voltae PS.

The methanogenic archaeobacterium Methanococcus voltae (strain PS) is known to produce a filterable, DNase-resistant agent (called VTA, for voltae transfer agent), which carries very small fragments (4400 bp) of bacterial DNA and is able to transduce bacterial genes between derivatives of the strain. Examination by electron microscopy of two preparations of VTA that were concentrated and partially purified by different methods showed virus-like particles with isometric heads, about 40 nm in diameter, and with 61 nm long tails. These particles co-sedimented with the minute bacteriophage φX174 in a sucrose density gradient.

Nucleoid structure and partition in Methanococcus jannaschii: an archaeon with multiple copies of the chromosome.

We measured different cellular parameters in the methanogenic archaeon Methanococcus jannaschii. In exponential growth phase, the cells contained multiple chromosomes and displayed a broad variation in size and DNA content. In most cells, the nucleoids were organized into a thread-like network, although less complex structures also were observed. During entry into stationary phase, chromosome replication continued to termination while no new rounds were initiated: the cells ended up with one to five chromosomes per cell with no apparent preference for any given DNA content. Most cells in stationary phase contained more than one genome equivalent. Asymmetric divisions were detected in stationary phase, and the nucleoids were found to be significantly more compact than in exponential phase.

Immunoelectron microscopic studies indicate the existence of a cell shape preserving cytoskeleton in prokaryotes.

Immunoelectron microscopic studies of prokaryotes were performed with anti-actin antibodies directed against the C terminus of actin. Studies on ultrathin sections revealed high proportions of the overall label close to the cell periphery in Escherichia coli, Ralstonia eutropha, Thermoanaerobacterium thermosulfurigenes, T. thermosaccharolyticum, and Methanococcus jannaschii. Substantial label also in the cytoplasm was observed in Bacillus sp., Methanococcus voltae, and Methanobacterium thermoautotrophicum. Only very minor amounts of label were found in the nucleoid region of the cells. Whole-mount immunogold studies, combined with negative staining, revealed the existence of an intracellular network of fibrils which could be labeled by anti-actin antibodies. This network is assumed to be located below the cytoplasmic membrane all around the cytoplasm. It appears to have properties that would allow its function as a cytoskeleton-like structure preserving cell shape.

Characterization of a membrane-associated ATPase from Methanococcus voltae, a methanogenic member of the Archaea.

A membrane-associated ATPase with an M(r) of approximately 510,000 and containing subunits with M(r)s of 80,000 (alpha), 55,000 (beta), and 25,000 (gamma) was isolated from the methanogen Methanococcus voltae. Enzymatic activity was not affected by vanadate or azide, inhibitors of P- and F1-ATPase, respectively, but was inhibited by nitrate and bafilomycin A1, inhibitors of V1-type ATPases. Since dicyclohexylcarbodiimide inhibited the enzyme when it was present in membranes but not after the ATPase was solubilized, we suggest the presence of membrane-associated component analogous to the F0 and V0 components of both F-type and V-type ATPases. N-terminal amino acid sequence analysis of the alpha subunit showed a higher similarity to ATPases of the V-type family than to those of the F-type family.