Characterization of Flagellum Gene Families of Methanogenic Archaea and Localization of Novel...

JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.00�0 DOI: 10.1128/JB.183.24.7154–7164.2001

Dec. 2001, p. 7154–7164 Vol. 183, No. 24

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Characterization of Flagellum Gene Families of MethanogenicArchaea and Localization of Novel Flagellum

Accessory ProteinsNIKHIL A. THOMAS AND KEN F. JARRELL*

Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario, Canada K7L 3N6

Received 18 April 2001/Accepted 21 September 2001

Archaeal flagella are unique motility structures, and the absence of bacterial structural motility genes in thecomplete genome sequences of flagellated archaeal species suggests that archaeal flagellar biogenesis is likelymediated by novel components. In this study, a conserved flagellar gene family from each of Methanococcusvoltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii has beencharacterized. These species possess multiple flagellin genes followed immediately by eight known and sup-posed flagellar accessory genes, flaCDEFGHIJ. Sequence analyses identified a conserved Walker box A motifin the putative nucleotide binding proteins FlaH and FlaI that may be involved in energy production forflagellin secretion or assembly. Northern blotting studies demonstrated that all the species have abundantpolycistronic mRNAs corresponding to some of the structural flagellin genes, and in some cases severalflagellar accessory genes were shown to be cotranscribed with the flagellin genes. Cloned flagellar accessorygenes of M. voltae were successfully overexpressed as His-tagged proteins in Escherichia coli. These recombinantflagellar accessory proteins were affinity purified and used as antigens to raise polyclonal antibodies forlocalization 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 isolatedflagellar filaments. Interestingly, flaD was found to encode two proteins, each translated from a separateribosome binding site. These protein expression data indicate for the first time that the putative flagellaraccessory genes of M. voltae, and likely those of other archaeal species, do encode proteins that can be detectedin the cell.

The Archaea represent a unique group of organisms thathave features in common with both the Bacteria and the Eu-carya while also possessing numerous traits that appear to bespecific to the archaeal lineage (22, 40). Interestingly, it ap-pears that gene products unique to the Archaea are responsiblefor the structure and assembly of the flagellar filament. Indeed,no homologues of genes for bacterial flagellar structure andassembly have been found in archaeal genomes (13).

Archaeal flagellins are initially made as preproteins that areprocessed prior to assembly into the flagellar filament (10, 45).It has been demonstrated for some methanogens that leaderpeptides are cleaved from preflagellins upon export (2, 14, 24).This is likely true of all archaeal flagellins, based on sequencealignments (45). In addition, many archaeal species haveflagellins that are glycosylated (5, 12, 33), although the exactbiological significance of this posttranslational modification isnot known. The gene products responsible for these specificposttranslational events have not been identified.

To date, two gene families that are involved in archaealflagellar biosynthesis and function have been identified. A setof bacterial-gene-like chemotaxis genes, including those en-coding CheA, CheW, and CheY, has been identified in Ar-chaeoglobus fulgidus and Halobacterium salinarum (29, 42). Inaddition, a gene family encoding at least some structural fea-

tures of the flagellum, as well as other products required forflagellation, has been identified (3, 24, 47). The compositionand order of the genes in this gene family can vary amongarchaea (45). Mutations of this gene family indicate that it is amotility gene locus (45, 47). Within the gene family, there aregenerally multiple flagellin genes that are immediately fol-lowed by a number of downstream genes of unknown function.Although the multiple flagellin genes within a particular familyshare considerable sequence similarity, mutant studies withMethanococcus voltae and H. salinarum suggest that the pro-teins encoded by the multiple flagellin genes are not simplyinterchangeable and likely have specific roles within the fila-ment (23, 44). This is supported by the fact that archaealflagellar filaments tend to be composed of a mixture of differ-ent flagellins, present in varying amounts (2, 16).

Transcriptional studies on the motility locus mentionedabove have been reported for some archaea. In M. voltae, twotranscriptional units have been identified (24). One unit con-tains flaA, a single flagellin gene that is weakly transcribed. Anadjacent unit encodes three flagellin genes (flaB1, flaB2, andflaB3) along with a number of immediate downstream genes.In H. salinarum, two transcriptional units encoding a total offive flagellin genes have been identified (17). One unit (the Alocus) contains two flagellin genes, while the other (the Blocus) contains three. The two loci are not physically linked onthe chromosome, although all of the flagellin genes are ex-pressed and all the flagellins are found in H. salinarum flagellarfilaments (16). The flagellar accessory genes of this halophileare not transcribed with any of the flagellin genes (17). Lastly,

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, Queen’s University, Kingston, Ontario, Can-ada K7L 3N6. Phone: (613) 533-2456. Fax: (613) 533-6796. E-mail:[email protected].

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the flagellin genes and a number of downstream open readingframes (ORFs) appear to be transcribed from a single pro-moter in Pyrococcus kodakaraensis KOD1 (36).

The majority of the conserved genes immediately down-stream of archaeal flagellins appear to be unique to archaealspecies. Moreover, no biochemical functions have been exper-imentally determined for any of these genes. One protein,called FlaI, is homologous to PilT (3), a protein involved intype II secretion and type IV pilus-mediated twitching motilityin bacteria (50, 52). The presence of a type II secretion homo-logue, along with the amino-terminal sequence similarity ofarchaeal flagellins and type IV pilins (14), suggests that ar-chaeal flagella may be assembled via a mechanism similar tothat for type IV pili (45).

Within the genus Methanococcus, a rare situation is found:species grow at very different temperature optima. Specifically,this genus contains closely related mesophiles, thermophiles,and hyperthermophiles. To further the understanding of ar-chaeal flagellation, the flagellar gene families and flagella ofselected methanogenic archaea were isolated and studied with

respect to gene composition, expression, and filament thermo-stability. This study presents novel protein expression and lo-calization data on specific conserved proteins that at presenthave no specific known functions but are known to be involvedin flagellar biogenesis in the mesophilic methanogen M. voltae.

MATERIALS AND METHODS

Microbial strains and growth conditions. M. voltae (obtained from G. D.Sprott, National Research Council of Canada, Ottawa, Ontario, Canada) andMethanococcus maripaludis (obtained from W. B. Whitman, University of Geor-gia, Athens, Ga.) were grown in Balch medium 3 at 37°C as previously described(26), while Methanococcus thermolithotrophicus (obtained from R. M. Sparling,University of Manitoba, Winnipeg, Manitoba, Canada) was grown in 10 ml ofBalch medium 3 in serum bottles at 60°C. Methanococcus jannaschii (obtainedfrom G. D. Sprott) and Methanococcus igneus (DSM 5666) cultures were grownas previously described (15) in modified 1-liter bottles at 80°C. Escherichia coliDH5� was used for all in vitro cloning and was grown in Luria-Bertani (LB)medium at 37°C. E. coli BL21(DE3) and E. coli BL21(DE3)/pLysS were used asexpression strains. Antibiotics were added for selection of plasmids whenneeded. Strains and plasmids used in this study are listed in Table 1.

DNA manipulations. Flagellin genes from various methanococci were initiallyidentified by Southern hybridization experiments. A short DNA fragment com-

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Description Source

StrainsM. voltae Wild type G. D. SprottM. maripaludis Wild type W. B. WhitmanM.thermolithotrophicus

Wild type R. M. Sparling

M. jannaschii Wild type G. D. SprottM. igneus DSM 5666 Wild type Deutsche Sammulung von

MikroorganismenE. coli

DH5� Laboratory K-12 strain for in vitro cloningBL21(DE3) Expression host for T7 promoter-based plasmids NovagenBL21(DE3)/pLysS Expression host for T7 promoter-based plasmids with pLysS for tight repression Novagen

PlasmidspKJ60 pUC18 derivative carrying a 3.3-kb PstI flaC-flaF M. voltae fragmentpKJ184 pUC18 derivative carrying a 0.7-kb EcoRI flaB1 M. maripaludis fragment This studypKJ188 pUC18 derivative carrying a 0.6-kb EcoRI flaB3 M. thermolithotrophicus fragment This studypKJ191 pUC18 derivative carrying a 1.8-kb EcoRI flaB2-flaC M. maripaludis fragment This studypKJ228 pUC21 derivative carrying a 1.2-kb PstI flaB2 M. thermolithotrophicus fragment This studypKJ229 pUC21 derivative carrying a 0.8-kb PstI flaB1 M. thermolithotrophicus fragment This studypKJ262 pUC18 derivative carrying a 1.2-kb EcoRI flaC-flaE M. maripaludis fragment This studypKJ263 pUC18 derivative carrying a 3.8-kb PvuII M. thermolithotrophicus fragment corresponding

to sequence upstream of flaB1; cloned into the SmaI site of the plasmid polylinkerThis study

pKJ267 pUC18 derivative carrying a 2.1-kb inverse-PCR product amplified from M. maripaludisgenomic DNA; cloned into the SmaI site of the plasmid polylinker

This study

pKJ307 pUC21 derivative carrying a 4.0-kb SmaI/HindIII flaF-flaJ M. maripaludis fragment This studypKJ312 pUC21 derivative carrying a 6.7-kb NcoI flaB3-flaJ M. thermolithotrophicus fragment This studypKJ315 pUC18 derivative carrying a 0.9-kb inverse-PCR product amplified from M. thermolitho-

trophicus genomic DNA into the SmaI site of the plasmid polylinkerThis study

pET23a� Expression vector with T7 promoter and sequence for a C-terminal polyhistidine tag onthe target protein

Novagen

pKJ194 pET23a� derivative carrying flaEMv PCR amplified without a stop codon as an NdeI/XhoI fragment

This study

pKJ198 pET23a� derivative carrying flaDMv PCR amplified without a stop codon as an NdeI/XhoI fragment

This study

pKJ199 pET23a� derivative carrying flaCMv PCR amplified without a stop codon as an NdeI/XhoI fragment

This study

pKJ200 pET23a� derivative carrying flaHMv PCR amplified without a stop codon as an NdeI/XhoI fragment

This study

pKJ210 pET23a� derivative carrying flaFMv PCR amplified without a stop codon as an NdeI/XhoI fragment

This study

pKJ266 pET23a� derivative carrying flaIMv PCR amplified without a stop codon as an NdeI/XhoIfragment

This study

pSJS1240 pACYC184 derivative containing ileX (encoding tRNAAUA) and argU (encodingtRNAAGA/AGG)

S. Sandler

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plementary to the first 135 bp of flaB1 of M. voltae was labeled with digoxigenin(DIG) and used as a probe against restriction enzyme digests of chromosomalDNA (isolated as previously described [18]) from different species of methano-cocci. Restriction enzyme-digested genomic DNA fragments approximately thesizes of those identified by Southern hybridization were gel extracted and puri-fied using a PREP-A-GENE matrix (Bio-Rad, Hercules, Calif.) and then clonedinto the pUC18 or pUC21 vector digested with the appropriate enzyme togenerate size-selected libraries.

In M. maripaludis, all three flagellin genes are located on two linked EcoRIfragments that were isolated from an M. maripaludis size-selected EcoRI library.An EcoRI fragment of approximately 1.2 kb linked to the flagellin genes wasidentified by Southern hybridization with an M. voltae flaC gene probe. Aninverse-PCR strategy was employed to obtain downstream sequence using DNAdigested with EcoRV and ligated under low DNA concentrations to promotemonomeric, intramolecular ligation (9). An aliquot of the ligation reaction prod-uct was used as a template in a PCR with the divergent primers 5�-ATACTTATCAGAAAGAGTGG and 5�-AATGCATCTTCTGGAACAGC. The resulting2.1-kb product was cloned into SmaI Ready to Go/BAP pUC18 (AmershamPharmacia Biotech, Baie D’Urfe, Quebec, Canada). The inverse-PCR productwas then DIG labeled and used as a probe in Southern blotting to identify a finaloverlapping DNA fragment that was cloned into pUC21 as a 4.7-kbSmaI/HindIII fragment to complete the flagellar gene family.

For M. thermolithotrophicus, a 0.6-kb EcoRI flagellin gene fragment identifiedby Southern hybridization was cloned and isolated from a pUC18/EcoRI library.In addition, Southern hybridization identified two PstI flagellin gene fragmentsof 0.8 and 1.2 kb that were cloned and isolated from a pUC21/PstI genomiclibrary. It was hypothesized that the flagellin genes were located close together,so a PCR-based strategy was used to link the fragments. Two divergent primerslocated on different DNA strands were designed from each isolated flagellingene fragment and were used in a PCR with genomic DNA to close the gapsbetween the fragments and to determine the physical arrangement of the geneson the chromosome. Primers 5�-AATGGAACTACAGTATGG and 5�-AATGCAGATGGTGCATC gave a PCR product of 778 bp, while primers 5�-AAGGTAACAGTATTATCC and 5�-CTGATTCAAATACTGATCC gave a PCRproduct of 865 bp. These PCR products linked the previously identified flagellingene fragments. Southern hybridization using the 0.6-kb EcoRI flagellin genefragment as a probe resulted in the identification and cloning of an overlapping6-kb NcoI fragment containing a fourth flagellin gene and additional flagellaraccessory genes. An inverse-PCR strategy was used to obtain a final DNAfragment for the complete M. thermolithotrophicus flagellar gene family. Thisinvolved DraI-digested DNA ligated as described above and used as a templatein PCR with the divergent primers 5�-CGCTTCATACGGAGTCGC and 5�-GGATTCAGTCATACCTCC. A 0.9-kb PCR product was amplified and thencloned as a blunt-end DNA fragment into SmaI/BAP Ready to Go pUC18.

RNA isolation. Total cellular RNA was isolated using a RNeasy Mini Kit(Qiagen) with minor modifications as previously described (47).

Southern and Northern hybridizations. Southern and Northern hybridiza-tions were performed as previously described (47). Probes were generated byusing a DIG-Labeling Kit (Roche Molecular Biochemicals). Specific DNA frag-ments were gel purified and then random prime labeled according to the man-ufacturer’s directions. Alternatively, DIG-labeled dUTP was directly incorpo-rated into probes using PCR, followed by purification of the PCR products witha Qiaquick PCR purification kit (Qiagen).

Construction of flagellar accessory protein overexpression clones. M. voltaeflagellar accessory genes flaC, flaD, flaE, and flaF (GenBank accession numberU97040) were PCR amplified using pKJ60, which contains a 3.3-kb PstI M. voltaegenomic fragment, as a template. For flaI and flaJ, genomic DNA served as atemplate in the PCR amplification. For each gene, primers introduced an NdeIsite at the 5� end of the gene and an XhoI site at the 3� end. Each PCR productwas cloned into the corresponding restriction sites in the pET23a� plasmid(Novagen, Inc., Madison, Wis.). The genes were amplified without their respec-tive stop codons to create an in-frame fusion with the sequence coding for thesix-histidine tag upon ligation in the pET23a� plasmid. All constructs wereverified by DNA sequencing.

Overexpression and purification of His-tagged flagellar accessory proteins. T7RNA polymerase-directed expression (43) of the cloned flagellar accessory geneswas performed in E. coli BL21(DE3). Plasmids pKJ199 and pKJ194 were trans-formed into E. coli BL21(DE3) cells and plated onto LB agar plates containing100 �g of ampicillin/ml. Similarly, plasmids pKJ198, pKJ200, and pKJ210 weretransformed into E. coli BL21(DE3)/pLysS cells and plated onto LB agar platescontaining 100 �g of ampicillin/ml and 30 �g of chloramphenicol/ml. For plasmidpKJ266, E. coli BL21(DE3) containing pSJS1240 was used as the expression host.pSJS1240 is a pACYC derivative (compatible with pET23a�) carrying the argU

and ileX genes encoding tRNAs (tRNAAGA/AGG and tRNAAUA) (28). The lattertransformants were plated onto plates containing 100 �g of ampicillin/ml and 50�g of streptomycin/ml. Cell cultures, induced for 2 to 4 h with 0.4 mM isopropyl-�-D-thiogalactopyranoside (IPTG; Life Technologies), were harvested at 5,000 �g for 15 min, and the resulting cell pellets were frozen overnight at �20°C.Overexpression of recombinant FlaD in E. coli was problematic but eventuallywas achieved with meticulous attention to plasmid stability. It was not possible totransform E. coli BL21(DE3) with pKJ198. The construct, however, could betransformed into E. coli BL21(DE3)/pLysS, although a small- and a large-colonytype were observed. Analysis of late-log-phase cultures initially inoculated withthe small-colony type revealed that most of the cells produced large colonieswhen grown on LB agar plates. Amelioration of an induction procedure forrecombinant FlaD expression demonstrated that the small-colony type possessedthe ability to express the target protein, whereas the large-colony type did notproduce an induction product.

All of the overexpressed target proteins were purified under denaturing con-ditions with the exception of FlaF and FlaH (see below). Nickel affinity purifi-cation was carried out by using a His-bind kit (Novagen) under denaturingconditions, as directed by the manufacturer. The purified proteins were thenconcentrated and desalted (using sterile distilled water) to a volume of approx-imately 2 ml using spin filters (Centricon YM-10 or YM-30 [Millipore, Bedford,Mass.], depending on the protein being isolated). Protein concentrations weremeasured spectrophotometrically by the Bradford binding assay using bovineserum albumin as the standard (Pierce, Rockford, Ill.). Depending on the flagel-lar accessory gene expressed in E. coli, approximately 2 to 4 mg of target proteinwas obtained using this protocol. In the case of FlaF overexpression, most of theprotein was located in inclusion bodies that were difficult to solubilize and couldnot be used for affinity chromatography. For FlaH, the soluble fraction of the cellculture lysate was used in a nondenaturing purification procedure using theHis-bind kit according to the manufacturer’s instructions.

Production of polyclonal antibodies to flagellar accessory proteins. Polyclonalantibodies against purified histidine-tagged M. voltae proteins FlaC, FlaD, FlaE,FlaH, and FlaI were raised in chickens and isolated from eggs. Antibodies wereproduced by RCH Antibodies, Sydenham, Ontario, Canada.

Immunoblotting. Membrane and cytoplasmic fractions of M. voltae (4) andflagellar preparations (see below) were subjected to SDS-PAGE as described byLaemmli (32) and then transferred to Immobilon-P transfer membranes (Milli-pore) as previously described (48). Primary antibodies were diluted to workingconcentrations. The secondary antibody was a horseradish peroxidase-conju-gated rabbit anti-chicken immunoglobulin Y (Jackson Immunoresearch Labora-tories, West Grove, Pa.) used at a 1:50,000 dilution. For immunodetection ofoverexpressed His-tagged proteins, approximately 1 �g (total protein) was sep-arated by SDS-PAGE and transferred to Immobilon-P membranes. Penta-Hisantibodies (0.2 mg/ml) (Qiagen) were used at a 1:2,000 dilution. The secondaryantibody was a peroxidase-linked sheep anti-mouse immunoglobulin G (JacksonImmunoresearch Laboratories) used at a 1:10,000 dilution. Blots were developedusing a chemiluminescent detection kit (Roche Molecular Biochemicals) accord-ing to the manufacturer’s directions.

N-terminal sequencing. Samples for N-terminal sequencing were preparedand analyzed as previously described (4).

Isolation of flagellar filaments. Crude preparations of flagellar filaments wereisolated by shearing, using a previously described protocol (26). For the isolationof flagella containing some attached basal structure, six liters of cells was pelletedby centrifugation at 5,900 � g for 15 min. The pellet was resuspended in 40 mlof suspension buffer (10 mM Tris [pH 7.0], 2% [wt/vol] NaCl, 0.28% [wt/vol]MgCl2, 0.35% [wt/vol] MgSO4) and treated with DNase and RNase (Life Tech-nologies). OP-10 detergent (a gift from S.-I. Aizawa) was added to a finalconcentration of 1% (vol/vol), and the suspension was incubated at room tem-perature for 30 min with occasional inverting. After centrifugation at 5,900 � gfor 15 min, the supernatant was collected and then mixed with 1 ml of precipi-tation buffer (1 M NaCl, 20% [wt/vol] polyethylene glycol 8000) (53), followed byincubation on ice for 1 h with gentle shaking. The mixture was then centrifugedat 7,800 � g for 10 min, and the pellet was resuspended in 10 mM Tris-HCl (pH7.0) prior to being loaded onto a KBr gradient, as previously described (21).

Thermostability of flagellar filaments and electron microscopy. Crude flagel-lar filament preparations (50-�l aliquots in Eppendorf microtubes) were incu-bated at 90, 80, 70, 60, 50, or 40°C for 30 min. After incubation, the flagellarfilament preparations were absorbed onto copper grids and negatively stainedwith 2% (wt/vol) uranyl acetate for 2 min. Grids were viewed on a HitachiH-7000 electron microscope operating at 75 kV.

Nucleotide sequence accession numbers. The nucleotide sequences of the M.maripaludis and M. thermolithotrophicus flagellar gene families have been depos-

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ited in GenBank under accession numbers AF333233 and AF333250, respec-tively.

RESULTS

Identification of methanogen flagellin genes. Previous stud-ies have identified a motility gene locus in M. voltae (23). Inaddition, flagellar gene families have been identified by com-plete genome sequencing of other archaeal species. Initialevaluation of these gene families suggested that the contentand number of genes within this motility locus are variable. Toobtain a better understanding of methanogen flagellar genet-ics, the flagellar gene families of closely related methanococcithat grow at very different temperature optima were charac-terized.

Initially, methanogen flagellin gene fragments were identi-fied using a short DNA probe corresponding to the 5� end offlaB1 of M. voltae. Chromosomal DNA restriction enzyme di-gests of M. maripaludis, M. thermolithotrophicus, and M. jann-aschii gave strong hybridization signals in Southern blots (Fig.1), consistent with the known conservation of the 5� ends ofmethanogen flagellin genes (25). The presence of more thanone signal (depending on the restriction digest) indicated thepresence of multiple flagellin genes in the genome. Surpris-ingly, no hybridization signals were obtained for the hyperther-mophile M. igneus, although it is believed that all members ofthe Methanococcales are flagellated (51). However, it is unclearwhether a few filamentous structures observed on the surfaceof M. igneus are, in fact, flagella (7). Using various libraries,overlapping DNA fragments were isolated, completing theidentification of the flagellin genes for each methanogen.

For M. maripaludis, three linked flagellin genes with shortintergenic regions (35 and 37 bp) were identified on two EcoRIrestriction fragments (Fig. 2). The flagellin genes, arranged intandem, are each about 650 bp in length, a size similar to thoseof flagellin genes of other mesophilic methanococci (2, 24),

and each one is preceded by a nucleotide sequence resemblinga methanogen ribosome binding site (41).

For M. thermolithotrophicus, four flagellin genes of differentsizes, arranged in tandem, were identified (Fig. 2). Each genehas its own putative ribosome binding site. Unexpectedly, M.thermolithotrophicus flaB1 and flaB2 (flaB1Mt and flaB2Mt) are986 and 1,305 bp, respectively, much longer than other meth-anogen flagellin genes. The remaining two flagellin genes aresimilar in size to those of other methanococci (at 650 bp). Thelong flagellin genes (flaB1Mt and flaB2Mt) share high sequenceidentity; however, flaB2Mt appears to encode an internal 220-amino-acid (220-aa) sequence that is unique to this flagellin. Aprevious study reported three major proteins of 62, 44, and26 kDa in M. thermolithotrophicus flagellar filaments (30). Ouranalysis of KBr gradient-purified M. thermolithotrophicusflagellar filaments resulted in the identification of proteins withapparent molecular masses of 62, 44, 37, and 26 kDa by SDS-PAGE (data not shown), while the flagellin genes are pre-dicted to encode proteins with molecular masses of 46.2, 34.9,22.9, and 22.7 kDa. Glycosylation does not appear to accountfor the apparent discrepancy in molecular masses, since noneof these flagellins stained as glycoproteins with the periodicacid-Schiff stain (data not shown).

From the complete genome sequence of M. jannaschii (6),three adjacent flagellin genes of approximately 650 bp eachwere identified (Fig. 2). The intergenic regions are larger (85and 102 bp) than those seen in the other methanococci. Inter-estingly, 183 nucleotides downstream from a putative pro-moter and 49 nucleotides upstream from the presumed trans-lational start site for the first flagellin gene, there is a 10-basedirect-repeat sequence (capital letters) separated by threebases (lowercase letters), GTGTGGGGGAattGTGTGGGGGA, that may serve as an element for regulating flagellargene family expression.

Identification of preflagellin leader sequences. Short leadersequences have been determined for some methanococcalflagellins (2, 14, 24). All of the methanogen flagellins identifiedin this study are predicted to possess leader sequences similarto those reported for other methanococci. Each leader se-quence has an overall net positive charge, an invariant glycineat position �1 (relative to the predicted cleavage site), and apositively charged amino acid at position �2 (either arginineor lysine).

Identification and characterization of methanogen flagellaraccessory genes. Immediately downstream of the flagellingenes in each methanogen there are 8 ORFs that are homol-ogous to flaC, flaD, flaE, flaF, flaG, flaH, flaI, and flaJ of M.voltae (3) (Fig. 2). A summary of identifiable properties of therespective gene products is presented in Table 2. The genes inthe cluster are arranged contiguously, either overlapping orseparated by short intergenic regions. Larger regions (40 to 60nucleotides) generally separate the flaF and flaG homologues.Alignment of the various methanococcal flaD genes suggestedthat the sequence of M. voltae flaD (flaDMv) as deposited inGenBank (1,026 bp; accession number U97040) did not rep-resent the entire gene due to a sequencing error. This wasconfirmed by resequencing. The correct flaDMv sequence is1,089 bp, with the new sequence extending the 5� end of thegene by 63 nucleotides. This new sequence overlaps with the 3�end of flaCMv, as found in the other methanococci.

FIG. 1. Identification of methanococcal flagellin gene fragmentsby Southern hybridization. EcoRI restriction enzyme-digested chromo-somal DNAs from various methanococci were probed with a DIG-labeled DNA fragment corresponding to the first 135 bp of M voltaeflaB1. Lane 1, DIG-labeled � HindIII molecular size standards; lane 2,M. voltae DNA; lane 3, M. maripaludis DNA; lane 4, M. thermolitho-trophicus DNA; lane 5, M. jannaschii DNA; lane 6, M. igneus DNA.



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Biochemical functions have yet to be determined for any ofthe respective gene products, although mutant studies with M.voltae have demonstrated that flaH, flaI, and flaJ are involvedin flagellar biogenesis (47; also unpublished data). It has beenreported that FlaI is homologous to PilB and PilT (3), twoproteins involved in type II secretion and type IV pilus bio-genesis in gram-negative bacteria (38, 39, 50). These putativenucleotide binding proteins have a Walker box A motif (49).Furthermore, FlaH (from each methanogen) has a putativeWalker box A motif at the N-terminal region of the protein.Interestingly, sequence alignments of the FlaD and FlaEhomologues from each methanogen reveal that the 135 C-terminal-most amino acids of FlaD (entire length, 360 aa)share approximately 65% sequence similarity with the entireFlaE protein (135 aa). No flaF homologues have been foundin other flagellated archaeal species, suggesting that the genemight be specific to methanogens. FlaF and FlaG are twoproteins with hydrophobic N-terminal regions, while FlaI ispredicted to possess a hydrophobic C-terminal domain. FlaJ ispredicted by dense alignment surface analysis to be an integralmembrane protein with 7 to 9 transmembrane domains (11).

Transcriptional analysis of flagellar gene families. The tran-scriptional profiles of the flagellar gene families of the threemethanogens were determined by Northern blotting.

The 0.7-kb EcoRI fragment containing flaB1 of M. maripalu-dis (flaB1Mm) was DIG labeled and used as a probe. This probeproduced three signals of different intensities corresponding tothree mRNA species of approximately 1.3, 2.0, and 4.0 kb (Fig.3A). Although no data are available for sequences upstream offlaB1Mm, presumably all the messages detected are expressedfrom a promoter upstream of this gene, as is the case for M.voltae. Furthermore, no nucleotide sequences resembling themethanogenic archaeal consensus box A promoter sequence[TTTA(T/A)ATA] (41) are evident 5� to any of the down-stream genes. The 1.3-kb message therefore likely encodes thefirst two flagellin genes, flaB1Mm and flaB2Mm. Moreover, apoly(T) stretch immediately after flaB2 may serve as a termi-nator, reducing transcription levels of downstream genes (24,41). Transcriptional read-through presumably produces the2.0-kb message, which would include all three flagellin genes.Additional read-through likely produces the 4-kb mRNA spe-cies. A flaDMm probe detected only a 4-kb mRNA species (Fig.

FIG. 2. Maps of flagellar gene families of M. maripaludis (A), M. thermolithotrophicus (B), and M. jannaschii (C). The M. jannaschii flagellargene family sequence and its annotation have been determined previously by complete genome sequencing (6). Gene designations are given in thecorresponding open arrows. Each arrow points in the direction of transcription. Bent arrows indicate putative promoters of the respective genefamilies. Lines above the genetic maps represent cloned restriction fragments that were used to obtain sequence data. Restriction site designations:D, DraI; E, EcoRI; H, HindIII; N, NcoI; P, PstI; PV, PvuII; RV, EcoRV; S, SmaI. Intergenic PCR (denoted by “PCR”) and inverse PCR(INV-PCR) were used to close gaps and to obtain flanking sequences, respectively. Probes used in Northern blotting experiments are shown asshaded rectangles below the respective flagellar gene families. The sequence of a 10-base direct repeat between the putative upstream promoterand the translational start site of flaB1 from M. jannaschii is shown.



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3A), whereas a flaEMm probe did not detect any mRNAs. Thus,the 4-kb message likely extends from flaB1Mm to flaDMm.

For M. thermolithotrophicus, only two mRNA species per-taining to the flagellar gene family could be detected. A nu-cleotide sequence with similarity to the methanogen consensuspromoter sequence is located 94 bp upstream of flaB1Mt. A0.8-kb PstI fragment corresponding to flaB1Mt was used as aprobe and detected a transcript of approximately 3 kb (Fig.3B). If this message originates upstream of flaB1Mt, the tran-script would include flaB1Mt, flaB2Mt, and flaB3Mt. Analysis ofthe intergenic region between flaB3Mt and flaB4Mt revealed thepresence of a short poly(T) stretch that may act as a termina-tor. Interestingly, a probe corresponding to flaB3Mt detectedtwo mRNA species of 3 and 0.6 kb (Fig. 3B). To determinewhether the 0.6-kb mRNA species corresponded to flaB3Mt orto flaB4Mt, which is immediately downstream, an internal andunique sequence for flaB4Mt was generated by PCR and usedas a probe. No mRNA species were detected using this probe,indicating that the 0.6-kb message does not correspond toflaB4Mt. One possible explanation for the 0.6-kb mRNA spe-

cies is mRNA processing, a feature that has been reported forsome polycistronic mRNAs in methanogens (1, 27, 34). mRNAprocessing may generate the stable 0.6-kb message carryingflaB3Mt from the larger 3-kb mRNA, since no obvious meth-anogen promoter sequences are present in the intergenic re-gion between flaB2Mt and flaB3Mt. Although no mRNAs couldbe detected for the downstream genes, presumably they aretranscribed, but at low levels that are not readily detectable.Since no promoter sequences are apparent 5� to any of theaccessory genes, transcription may be driven by the promoterupstream of flaB1Mt and followed by read-through of putativeterminator elements immediately after flaB3Mt, resulting inlimited expression of flaB4Mt and downstream accessory genes.Presumably, the genes transcribed at lower levels encode prod-ucts needed in smaller amounts than the major structuralflagellins.

For the hyperthermophile M. jannaschii, a 1.5-kb RNAspecies was detected by use of a probe for M. jannaschii flaB1(flaB1Mj) (Fig. 3C). A nucleotide sequence exactly matchingthe consensus box A methanogen promoter sequence (TTTA

FIG. 3. Northern blot analyses of methanococcal flagellar gene families. The DIG-labeled DNA probe used is given above each blot. (A) M.maripaludis RNA; (B) M. thermolithotrophicus RNA; (C) M. jannaschii RNA.

TABLE 2. Properties of the gene products encoded by methanococcal flagellar gene families

Gene product Characteristics Subcellular locationa

FlaB1/FlaB2b Highly conserved, mainly hydrophobic N-terminal region with sequence similarity to type IV pilins;synthesized as preproteins with a leader peptide that is removed by a preflagellin peptidase be-fore assembly into flagellar filaments

Flagellar filament

FlaC High charged-amino-acid content (34%) MembraneFlaD Putative polyproteinc with C-terminal region exhibiting sequence similarity to FlaE MembraneFlaE Sequence similarity to the C-terminal region and short version of FlaD MembraneFlaF Limited to methanococcal flagellar gene familiesd; N-terminal hydrophobic region with hydropathy

profile similar to that of flagellinsNot determined

FlaG N-terminal hydrophobic region with weak sequence similarity to flagellins Not determinedFlaH Walker box A motif, putative nucleotide binding protein; found in all flagellated archaea where

sequence data are availableMembrane

FlaI Bacterial type II secretion homologue with sequence similarity to PilB/PilT and other supposedtype II secretion archaeal homologues; Walker box A motif, putative nucleotide-binding protein;found in all flagellated archaea where sequence data are available

Membrane

FlaJ Presumed integral membrane protein with 7 to 9 predicted transmembrane domains; found in allflagellated archaea where sequence data are available

Not determined

a Determined by immunoblot analyses of M. voltae cell fractions and purified flagellar filaments.b The major flagellins found in Methanococcus sp. flagellar filaments are listed, although other flagellins possess the same features and are likely present as minor

components of flagella.c As demonstrated by overexpression studies with M. voltae and suggested by the presence of a second ribosome binding site within the ORF.d Not present in flagellar gene families outside of Methanococcus spp. that are currently available in public databases.



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TATA) was found 255 bp upstream of the putative transla-tional start site of flaB1Mj. This 1.5-kb message likely includesflaB1Mj and flaB2Mj. As with M. maripaludis, a poly(T) se-quence is located immediately after flaB2Mj. Although aflaB3Mj-specific probe did not detect any mRNAs, a sequenceclose to the methanogen consensus promoter sequence can beidentified 5� to flaB3Mj (ATTATATA); this sequence mayserve to drive transcription of this last flagellin gene and thedownstream genes. However, this may be a fortuitous resultdue to the high AT content of intergenic regions common tomethanogens (41).

Overexpression of recombinant flagellar accessory proteinsin E. coli. Since much structural information pertaining toflagella has been amassed for M. voltae (26, 31) and mutantswith changes in some of its flagellar accessory genes havepreviously been isolated, the flagellar accessory gene productsfrom this methanogen were overexpressed in E. coli. In thisstudy, six of the eight flagellar accessory genes of M. voltaewere successfully overexpressed as His-tagged proteins. Thecharacteristics of each of the expression clones are describedbelow.

Successful overexpression of recombinant FlaC, FlaD, FlaE,FlaF, FlaH, and FlaI was achieved in E. coli (Fig. 4A). In mostcases, the apparent molecular weights of the recombinant pro-teins are higher than the predicted molecular weights (Table3). Interestingly, upon IPTG induction (for FlaD expression)of E. coli BL21(DE3)/pLysS harboring pKJ198, two stableproducts with apparent molecular masses of approximately 49and 16 kDa were produced (Fig. 4A, lane 5). The flaD ORFthat was cloned is predicted to encode a protein with a molec-ular mass of 39.5 kDa. Purification of the induction productsrevealed that the 16-kDa species is a C-terminal His-taggedpolypeptide of FlaD. Furthermore, immunoblotting with ananti-His antibody revealed that both polypeptides were pro-duced with the polyhistidine tag (Fig. 4B, lane 5), and similar

amounts of the two induction products were obtained afternickel affinity chromatography (data not shown). N-terminalsequencing of the 16-kDa induction product revealed theamino acid sequence (MESEIAKINE) that corresponds toamino acid positions 232 to 242 in the FlaD sequence. Asequence with similarity to a methanogen ribosome bindingsite is located 7 bp upstream of the initiating methionine of the16-kDa induction product. Indeed, translation from this puta-tive ribosome binding site in E. coli would produce a 131-aapolypeptide with a predicted molecular mass of 15.5 kDa.These data suggest that the 49-kDa protein represents theentire protein, whereas the 16-kDa polypeptide is an inductionproduct that is translated from a second in-frame putativedistal start site.

No obvious recombinant FlaF production was apparent after2 h postinduction; however, immunoblotting with anti-His an-tibodies revealed that a His-tagged protein was being pro-duced, albeit at a minimal level (Fig. 4). The codon usage and

FIG. 4. Detection of overexpressed His-tagged M. voltae flagellar accessory proteins FlaC, FlaD, FlaE, FlaF, FlaH, and FlaI derived from a T7expression system. E. coli cultures harboring the respective flagellar accessory gene cloned into pET23a� were grown to an optical density at 600nm of 0.6 and then induced with 0.4 mM IPTG. Background vector controls were included in the analyses. Two hours postinduction, bacteriallysates (20 �g of total protein per lane) were analyzed by SDS-PAGE (12.5% gel) and the proteins were visualized by Coomassie blue staining.Arrows point to the gene-specific expressed proteins. (A) Lane M, molecular mass standards (in kilodaltons); lane 1, E. coli BL21(DE3)/pET23a�;lane 2, E. coli BL21(DE3)/pLysS/pET23a�; lane 3, E. coli BL21(DE3)/pKJ265/pET23a�; lane 4, E. coli BL21(DE3)/pKJ199 (FlaC); lane 5, E. coliBL21(DE3)/pLysS/pKJ198 (FlaD); lane 6, E. coli BL21(DE3)/pKJ194 (FlaE); lane 7, E. coli BL21(DE3)/pLysS/pKJ210 (FlaF); lane 8, E. coliBL21(DE3)/pLysS/pKJ200 (FlaH); lane 9, E. coli BL21(DE3)/pKJ265/pKJ266 (FlaI). (B) Immunoblot, developed with an anti-His monoclonalantibody, of a gel similar to that shown in panel A but containing 1/10 the amount of protein per lane.

TABLE 3. Summary of M. voltae flagellar accessoryprotein characteristics

Protein Size (aa) Predicted pIMolecular mass (kDa)

Predicted Apparenta

FlaC 189 4.22 21.5 31FlaD 363 4.53 41.9 52, 15b

FlaE 136 5.40 15.4 15FlaF 133 5.21 14.6 14FlaG 151 5.88 16.2 NDFlaH 231 8.97 25.8 30FlaI 553 5.80 63.1 73FlaJ 599 8.31 63.0 ND

a Determined by immunoblotting of SDS-PAGE-separated proteins. ND, notdetermined.

b flaD encodes two proteins, as determined by immunoblotting of cell frac-tions.



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hydrophobic character of FlaF may be factors that limit itsexpression in E. coli. Attempts to increase the level of expres-sion with a plasmid carrying specific tRNA genes (28) or byinducing at lower temperatures were not successful. Further-more, most of the target protein accumulated in inclusionbodies that, when solubilized, released only small amounts ofrecombinant FlaF. From these expression experiments, and inour hands, it was not possible to affinity purify FlaF.

Interestingly, overexpression of recombinant FlaI was aidedby the presence of a plasmid (pSJS1240) that possesses thegenes for tRNAAUA and tRNAAGA/AGG, which are rarely usedin E. coli (28) but are favored in Methanococcus species, whosegenomes are AT rich (41). A strong induction product ofapproximately 73 kDa was observed in SDS-PAGE of celllysates (Fig. 4A, lane 9), although flaI is predicted to encode aprotein with a molecular mass of 63.1 kDa.

Overexpression of recombinant FlaG and FlaJ was notachieved, even after thorough efforts. The use of plasmidscarrying tRNA genes or lower induction temperatures wasineffective in both cases, as were attempts to clone and expressa truncated version of flaJ.

Localization of flagellar accessory proteins in M. voltae. Cellfractions (membrane or particulate [insoluble] and cytoplasmic[soluble]) and purified flagella were generated and used inimmunoblots developed with polyclonal antibodies raisedagainst the recombinant flagellar accessory proteins. A non-flagellated M. voltae mutant, P2, with an insertion in flaB2 thatdoes not express any of the downstream genes (23) was used asa negative control. A summary of the localization data is pre-sented in Table 2. An antibody raised against recombinantFlaC detected a protein with an apparent molecular mass of 31kDa exclusively in the membrane fractions of cells (Fig. 5).This molecular mass compares closely with that of the His-tagged FlaC that was overexpressed in E. coli. Two proteinswith apparent molecular masses of approximately 52 and 15kDa were detected solely in the membrane fractions of cellswith an antibody raised against recombinant FlaD. In agree-ment with the corrected flaD gene sequence, the apparentmolecular mass of the native FlaD (52 kDa) of M. voltae isgreater than that of the protein that is expressed in E. coli (49kDa), since the latter is missing the first 21 aa of the protein.Moreover, the detection of a protein with an apparent molec-ular mass of 15 kDa in M. voltae with the polyclonal anti-FlaDantibody suggests that translation may also initiate at a down-stream location within the ORF. As observed from flaD ex-pression in E. coli, a putative second ribosome binding site mayallow for translation of the 15-kDa protein.

Polyclonal antibodies raised against recombinant FlaE de-tected a 15-kDa protein in the membrane fraction of M. voltae.Since FlaD and FlaE share sequence similarity, there was apossibility that the 15-kDa protein identified was a polypeptidederived from FlaD. An experiment to determine if the anti-FlaE antibodies could detect recombinant FlaD was per-formed. The reciprocal experiment to determine if polyclonalanti-FlaD could detect recombinant FlaE was also carried out.In these experiments the respective antibody preparationsdemonstrated specificity for the proteins they were raisedagainst and did not exhibit any significant cross-reaction withother proteins (data not shown).

Immunoblots developed with anti-FlaH antibodies detected

a 30-kDa protein in the membrane fractions of cells (Fig.5). Interestingly, recombinant-FlaH expressed in E. coli wasmostly soluble and remained in the cytoplasm. Immunoblot-ting with anti-FlaI antibodies detected three protein species(with approximate molecular masses of 85, 70, and 55 kDa) inthe membrane fractions of M. voltae cells (Fig. 5). However,when an induced culture of E. coli BL21(DE3) harboringpKJ266 was analyzed, a single protein of approximately 73 kDa(corresponding to recombinant FlaI) was detected by the anti-FlaI antibodies. It is possible that the polyclonal antibody prep-aration recognizes different forms of the protein in M. voltae orthat homologues with epitopes similar to FlaI exist.

Flagella with some attached basal structure were isolated byusing OP-10 detergent and were used in immunoblot analyseswith anti-FlaC (�-FlaC), �-FlaD, �-FlaE, �-FlaH, or �-FlaIantibodies. No cross-reacting proteins were revealed by any ofthe antibody preparations, indicating that none of the putativeflagellar accessory proteins are major components of flagellarfilaments.

FIG. 5. Subcellular localization of flagellar accessory proteins in M.voltae. Fractionated M. voltae cells and purified flagellar filaments weresubjected to immunoblotting with polyclonal antibodies raised againstpurified recombinant flagellar accessory proteins. A nonflagellated M.voltae mutant, P2, which does not express any of the flagellar accessorygenes due to insertional inactivation of the flagellar gene family (23),was included as a negative control. Approximately 20 �g of protein wasloaded per lane. For FlaC and FlaH, 40 �g of protein was required forimmunodetection. The antibodies used in the detection are indicatedto the right of each panel. Lane designations apply to all the panels.Lane 1, M. voltae membrane fraction; lane 2, M. voltae cytoplasmicfraction; lane 3, P2 membrane fraction; lane 4, P2 cytoplasmic fraction;lane 5, purified M. voltae flagellar filaments. The �-FlaD antibodypreparation cross-reacts with a soluble protein present in both the wildtype and the P2 mutant; however FlaD localization is confirmed by theabsence of a signal in the membrane fraction of the P2 mutant. Threeprotein species are recognized by the �-FlaI antibodies.



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Thermostability of methanogen flagellar filaments. To eval-uate flagellar filament thermostability, crude flagellar filamentpreparations were isolated from each of M. maripaludis, M.thermolithotrophicus, and M. jannaschii and were then incu-bated at 40, 50, 60, 70, 80, or 90°C. M. maripaludis flagellarfilaments retained structure up to 70°C, as viewed by electronmicroscopy of negatively stained, heat-treated preparations,whereas samples incubated at 80 or 90°C showed aggregatedproteins and no evidence of intact filament structures (data notshown). Flagellar filaments isolated from M. thermolithotrophi-cus and M. jannaschii retained a filament structure up to 90°C,as viewed by electron microscopy.

DISCUSSION

The data presented here characterize flagellar gene familiesof closely related methanococci and demonstrate for the firsttime the expression and subcellular localization of specificknown and putative flagellar accessory proteins in M. voltae.

The deduced flagellin sequences from each of the methano-cocci studied exhibit considerable sequence conservation overthe first 35 aa of the mature protein. Multiple flagellin geneswith high sequence similarity are common in the genomes offlagellated archaeal species, although mutant studies withsome archaea demonstrate that the different flagellins are notsimply interchangeable in vivo and likely have defined roles(23, 44). In Methanococcus spp., it appears that not all of theflagellins are present in large amounts within the filament.Furthermore, differential transcription patterns of specificflagellin genes suggest that some may be expressed at lowerlevels in the cell (24). Interestingly, the flagellin genes tran-scribed at the lowest levels in their respective gene families(flaB3Mm, flaB4Mt, and flaB3Mj) possess shorter leader peptides(11 aa, compared to the typical 12), as first observed in flaB3Mv

(24). Although the overall leader sequence is not highly con-served, a net positive charge is present, and the three aminoacids preceding the cleavage site tend to be KKG, KRG, orRRG. It has been demonstrated that a positively chargedamino acid at position �2 and a small amino acid (glycine oralanine) at position �1 are important for preflagellin process-ing in M. voltae (46). Furthermore, that study identified otheramino acids involved in processing that are also found in theflagellins identified in this study.

The cotranscription of at least some of the flagellar acces-sory genes with the flagellin genes may allow for their con-certed regulation by transcriptional activators or repressorsand DNA sequence elements. In M. jannaschii, a 10-nucleotidedirect-repeat sequence is located upstream of the first flagellingene. This sequence could potentially serve as a regulatoryelement for the flagellar gene family’s expression. Examples ofDNA elements involved in regulating gene expression in meth-anogens include heptameric direct-repeat sequences that reg-ulate selenium-free hydrogenase gene expression in M. voltae(37) and an inverted repeat that acts as a repressor binding siteto regulate nif (nitrogen fixation) gene expression in M. mari-paludis (8). Interestingly, a hydrogen partial-pressure controlon the expression of flagella in M. jannaschii has been reported(35), although the mechanism of the regulation was not deter-mined.

The flagellar gene families identified in this study are com-

posed of multiple flagellin genes followed immediately by anumber of conserved genes, some of which have been shown tobe involved in flagellation (23, 47). It is likely that additionalgenes exist, but sequencing downstream of flaJ in M. voltae, M.maripaludis, and M. thermolithotrophicus has identified genesthat are not predicted to be involved in flagellar biogenesis(unpublished data). In M. jannaschii, there are some genesdownstream of flaJ that could be cotranscribed and possiblyinvolved in flagellation, but as yet no data are available tosupport their role in flagellation. Although the functions ofFlaCDEFGHIJ have not been determined, valuable informa-tion on their gene products is presented here. FlaC, FlaD,FlaE, FlaH, and FlaI were all detected in the membrane frac-tions of cells, although transmembrane segment models do notpredict these proteins to have membrane-spanning domains,except for FlaI, which is predicted to have a C-terminal mem-brane-spanning domain. Moreover, the flagellar accessory pro-teins are not major components of flagella, although there stillremains the possibility that these proteins interact with flagellabut are not strongly associated and are therefore lost in flagel-lum isolation procedures. Alternatively, these proteins mayhave a role in the secretion and translocation of flagellins orpossibly in the assembly of flagella.

FlaD and FlaE of M. voltae were readily detected, in agree-ment with the findings of a study describing two-dimensionalprotein gel analysis of M. jannaschii (grown under low H2

partial pressure), where mass spectroscopy analysis of selectedpolypeptide spots identified FlaD, FlaE, and other proteins(35). Strangely, only a 16-kDa C-terminal portion of FlaDwas detected in that study, and as a result it was considered tobe a cleavage product of the entire FlaD protein (calculatedmolecular mass, 39.95 kDa). The N-terminal sequencing datapresented here for the 16-kDa overexpressed protein derivedfrom pKJ198 strongly suggest that an internal ribosome bind-ing site within the ORF results in a shorter version of FlaD.Our ability to readily detect this shorter version of FlaD, aswell as the full-length 52-kDa FlaD protein, in M. voltae dem-onstrates their presence in vivo. No mRNA for flaD can bedetected in a flaB2 insertional mutant (unpublished data) thatexhibits transcriptional polar effects on downstream genes(23); therefore, the 15-kDa FlaD species is likely translatedfrom a downstream ribosome binding site and not derivedfrom a different transcript. Putative downstream ribosomebinding sites followed by in-frame translational start codonsare evident within the flaD homologues of M. maripaludis, M.thermolithotrophicus, and M. jannaschii. The short versions ofFlaD (predicted to be approximately 135 aa long) are similar insequence, size, and pI to FlaE of the respective methanogen.Thus, flaD might encode two proteins, one of which is verysimilar to FlaE. Further studies characterizing FlaD and FlaEare required to assess the functions of these proteins.

Since the flagellins identified for the methanogens in thisstudy share considerable sequence similarity, it was hypothe-sized that minor differences in flagellin amino acid composi-tion are responsible for the observed increases in thermosta-bility for the flagellar filaments of M. thermolithotrophicus andM. jannaschii. Indeed, a study evaluating large amounts ofsequence data from closely related mesophilic and thermo-philic Methanococcus species has identified recurring themesfor thermal adaptation (20). The protein properties that were



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found to correlate most closely with thermophiles includehigher residue volume, higher residue hydrophobicity, morecharged amino acids, and fewer uncharged polar residues. Byuse of the amino acid category assignments of Haney et al.(20), the flagellins of M. thermolithotrophicus and M. jannaschiido indeed have slight increases in hydrophobic amino acidcontent (42 to 45%) compared with the mesophilic methano-coccal flagellins (40%) and also have a lower polar unchargedresidue content. However, these flagellins do not have a highercharged-amino-acid content than their mesophilic counter-parts. Nonetheless, hydrophobic interactions have previouslybeen associated with increased thermostability of proteins inmethanogens (19). Since the flagella of methanococcal hyper-thermophiles and thermophiles are present in high-tempera-ture environments, slight increases in hydrophobic interactions(along with other stabilizing forces) may serve to achieveflagellar filament thermostability, as could posttranslationalmodifications.

A common trend for this flagellar gene family is multipleflagellin genes followed by at least the flaHIJ genes, which arefound in all flagellated archaea for which data are currentlyavailable. In all available flaI genes from diverse archaeal spe-cies, an invariant Walker box A sequence is present (Fig. 6),indicating an essential role of this motif in flagellar biogenesis.Based on the homology of FlaI to PilB (3), a demonstratedtype II secretion protein, and the finding that FlaH and FlaIlocalize to the membrane, it is presumed that FlaH and FlaIare involved in flagellin secretion. Indeed, an M. voltae strainwith an insertional mutation in flaH (affecting flaI as well, dueto transcriptional polar effects) makes abundant levels offlagellin but does not secrete flagellins and does not formflagella on the cell surface (47). Furthermore, flaJ (immediate-ly downstream of flaH and flaI) is predicted to be a proteinwith 7 to 9 transmembrane domains that may form a secretorycomplex with FlaH and FlaI (45). Additional work will berequired to determine the biological functions of these geneproducts in order to elucidate their involvement in flagellarbiogenesis in the methanogenic archaea.

ACKNOWLEDGMENTS

We thank Sonia Bardy for isolating purified flagellar filaments andAleksandra Lalovic for cloning part of the M. maripaludis flagellar

gene family. pSJS1240 was kindly provided by Steven Sandler. OP-10detergent was a generous gift from S.-I. Aizawa.

This work was supported by an operating grant from the NaturalSciences and Engineering Research Council of Canada (NSERC) toK.F.J. N.A.T. was the recipient of an Ontario Graduate Scholarship.

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FIG. 6. Partial amino acid sequence alignment of all archaeal FlaI proteins currently available in public databases. The bordering amino acidpositions of each polypeptide are shown. The putative Walker box A motif is boxed. Asterisks indicate invariant amino acids; colons indicate similaramino acids. Abbreviations: Mv, M. voltae; Mm, M. maripaludis; Mt, M. thermolithotrophicus; Mj, M. jannaschii; Ta, Thermoplasma acidophilum;Pa, Pyrococcus abyssi; Ph, Pyrococcus horikoshii; Af, A. fulgidus; Ap, Aeropyrum pernix; Hs, H. salinarum.



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