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

Abstract

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.

FIG. 1

Identification of methanococcal flagellin gene fragments by Southern hybridization. EcoRI restriction enzyme-digested chromosomal DNAs from various methanococci were probed with a DIG-labeled DNA fragment corresponding to the first 135 bp of M voltae flaB1. Lane 1, DIG-labeled λ HindIII molecular size standards; lane 2, M. voltae DNA; lane 3, M. maripaludis DNA; lane 4, M. thermolithotrophicus DNA; lane 5, M. jannaschii DNA; lane 6, M. igneus DNA.

FIG. 2

Maps of flagellar gene families of M. maripaludis (A), M. thermolithotrophicus (B), and M. jannaschii (C). The M. jannaschii flagellar gene family sequence and its annotation have been determined previously by complete genome sequencing (6). Gene designations are given in the corresponding open arrows. Each arrow points in the direction of transcription. Bent arrows indicate putative promoters of the respective gene families. 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 as shaded rectangles below the respective flagellar gene families. The sequence of a 10-base direct repeat between the putative upstream promoter and the translational start site of flaB1 from M. jannaschii is shown.

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.

FIG. 4

Detection of overexpressed His-tagged M. voltae flagellar accessory proteins FlaC, FlaD, FlaE, FlaF, FlaH, and FlaI derived from a T7 expression system. E. coli cultures harboring the respective flagellar accessory gene cloned into pET23a+ were grown to an optical density at 600 nm of 0.6 and then induced with 0.4 mM IPTG. Background vector controls were included in the analyses. Two hours postinduction, bacterial lysates (20 μg 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. coli BL21(DE3)/pLysS/pKJ198 (FlaD); lane 6, E. coli BL21(DE3)/pKJ194 (FlaE); lane 7, E. coli BL21(DE3)/pLysS/pKJ210 (FlaF); lane 8, E. coli BL21(DE3)/pLysS/pKJ200 (FlaH); lane 9, E. coli BL21(DE3)/pKJ265/pKJ266 (FlaI). (B) Immunoblot, developed with an anti-His monoclonal antibody, of a gel similar to that shown in panel A but containing 1/10 the amount of protein per lane.

FIG. 5

Subcellular localization of flagellar accessory proteins in M. voltae. Fractionated M. voltae cells and purified flagellar filaments were subjected to immunoblotting with polyclonal antibodies raised against purified recombinant flagellar accessory proteins. A nonflagellated M. voltae mutant, P2, which does not express any of the flagellar accessory genes due to insertional inactivation of the flagellar gene family (23), was included as a negative control. Approximately 20 μg of protein was loaded per lane. For FlaC and FlaH, 40 μg of protein was required for immunodetection. The antibodies used in the detection are indicated to the right of each panel. Lane designations apply to all the panels. Lane 1, M. voltae membrane fraction; lane 2, M. voltae cytoplasmic fraction; lane 3, P2 membrane fraction; lane 4, P2 cytoplasmic fraction; lane 5, purified M. voltae flagellar filaments. The α-FlaD antibody preparation cross-reacts with a soluble protein present in both the wild type and the P2 mutant; however FlaD localization is confirmed by the absence of a signal in the membrane fraction of the P2 mutant. Three protein species are recognized by the α-FlaI antibodies.

FIG. 6

Partial amino acid sequence alignment of all archaeal FlaI proteins currently available in public databases. The bordering amino acid positions of each polypeptide are shown. The putative Walker box A motif is boxed. Asterisks indicate invariant amino acids; colons indicate similar amino 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.