Mutants in flaI and flaJ of the archaeon Methanococcus voltae are deficient in flagellum assembly

Thomas, Nikhil A.; Mueller, Steffen; Klein, Albrecht; Jarrell, Ken F.

doi:10.1046/j.1365-2958.2002.03220.x

Cited by 50 publications

(53 citation statements)

References 30 publications

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“…The genes Mv990, Mv991, and Mv891 were individually targeted for knockout by removing the genomic copy of the gene and replacing it with a selectable antibiotic marker (puromycin cassette) in M. voltae PS*. The puromycin cassette used in this study was designed with an additional promoter after the antibiotic resistance terminator sequence to allow the restarting of a multigene transcript (36). For Mv891 and Mv991, the puromycin cassette was inserted so that the additional promoter was facing downstream genes.…”

Section: Selection Of M Voltae Genes To Target For Studymentioning

confidence: 99%

AglC and AglK Are Involved in Biosynthesis and Attachment of Diacetylated Glucuronic Acid to the N-Glycan in Methanococcus voltae

et al. 2009

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Recent advances in the field of prokaryotic N-glycosylation have established a foundation for the pathways and proteins involved in this important posttranslational protein modification process. To continue the study of the Methanococcus voltae N-glycosylation pathway, characteristics of known eukaryotic, bacterial, and archaeal proteins involved in the N-glycosylation process were examined and used to select candidate M. voltae genes for investigation as potential glycosyl transferase and flippase components. The targeted genes were knocked out via linear gene replacement, and the resulting effects on N-glycan assembly were identified through flagellin and surface (S) layer protein glycosylation defects. This study reports the finding that deletion of two putative M. voltae glycosyl transferase genes, designated aglC (for archaeal glycosylation) and aglK, interfered with proper N-glycosylation. This resulted in flagellin and S-layer proteins with significantly reduced apparent molecular masses, loss of flagellar assembly, and absence of glycan attachment. Given previous knowledge of both the N-glycosylation pathway in M. voltae and the general characteristics of N-glycosylation components, it appears that AglC and AglK are involved in the biosynthesis or transfer of diacetylated glucuronic acid within the glycan structure. In addition, a knockout of the putative flippase candidate gene (Mv891) had no effect on N-glycosylation but did result in the production of giant cells with diameters three to four times that of wild-type cells.It has become widely accepted that glycosylation is an important posttranslational protein modification within all three domains of life. Long recognized and studied in eukaryotes, glycosylation pathways in prokaryotes have received more attention in recent years. Several reviews summarizing the current state of knowledge of protein glycosylation in Archaea (2, 11, 42) and flagellar glycosylation in Bacteria and Archaea (25) attest to the progress that has been made in understanding this important process from a prokaryotic perspective. Specifically, significant advances in comprehending the process of N-linked glycosylation, which is the attachment of polysaccharide structures to specific Asn residues within a conserved Asn-Xaa-Ser/ Thr motif (where Xaa is any amino acid except Pro) have occurred. Of note is the N-glycosylation system in Campylobacter jejuni, where the gene products from the pgl locus assemble a branched heptasaccharide on a membrane-bound lipid carrier and then translocate the glycan across the cytoplasmic membrane to facilitate transfer to the appropriate Asn residue of the target protein (24, 34).The study of archaeal glycosylation in recent years has begun to yield an understanding of how these organisms modify proteins. The first requirement for assembling a glycan of any linkage type is a pool of nucleotide-activated monosaccharide precursors. Several enzymes have been identified from Methanococcus maripaludis that are required for UDP-acetamido sugar synthesis...

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Section: Selection Of M Voltae Genes To Target For Studymentioning

confidence: 99%

AglC and AglK Are Involved in Biosynthesis and Attachment of Diacetylated Glucuronic Acid to the N-Glycan in Methanococcus voltae

et al. 2009

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“…In this approach, recombination between a truncated version of the gene and its chromosomal copy leads to an insertion/disruption mutation ( Figure 2B). A targeted version of insertional mutagenesis has been used in M. voltae, to characterise genes encoding flagellins and hydrogenases [95][96][97] . For random mutagenesis, a genomic library of small fragments (less than a full-length gene) is used to target recombination.…”

Section: Transformationmentioning

confidence: 99%

Archaeal genetics — the third way

2005

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| For decades, archaea were misclassified as bacteria on account of their prokaryotic morphology. Molecular phylogeny eventually revealed that archaea, like bacteria and eukaryotes, are a fundamentally distinct domain of life. Genome analyses have confirmed that archaea share many features with eukaryotes, particularly in information processing, and therefore can serve as streamlined models for understanding eukaryotic biology. Biochemists and structural biologists have embraced the study of archaea but geneticists have been more wary, despite the fact that genetic techniques for archaea are quite sophisticated. It is high time for geneticists to start asking fundamental questions about our distant relatives.

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“…Similarly, archaeal flagellin precursors contain a class III signal peptide that is processed by a prepilin-specific peptidase homolog (FlaK/ PibD) (3,8,10,11). Moreover, flagellar assembly involves homologs of components involved in the biosynthesis of bacterial type IV pili, including FlaI, an ATPase homologous to PilB, and FlaJ, a multispanning membrane protein that may provide a platform for flagellar assembly, similar to the proposed role for PilC in pilus assembly (38,44,53,54). These genes, as well as a number of others that encode proteins often required for either flagellar assembly or function (flaCDEFG and flaH), are frequently coregulated with the flg genes (11,26,44,54).…”

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Haloferax volcanii Flagella Are Required for Motility but Are Not Involved in PibD-Dependent Surface Adhesion

2010

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Although the genome of Haloferax volcanii contains genes (flgA1-flgA2) that encode flagellins and others that encode proteins involved in flagellar assembly, previous reports have concluded that H. volcanii is nonmotile. Contrary to these reports, we have now identified conditions under which H. volcanii is motile. Moreover, we have determined that an H. volcanii deletion mutant lacking flagellin genes is not motile. However, unlike flagella characterized in other prokaryotes, including other archaea, the H. volcanii flagella do not appear to play a significant role in surface adhesion. While flagella often play similar functional roles in bacteria and archaea, the processes involved in the biosynthesis of archaeal flagella do not resemble those involved in assembling bacterial flagella but, instead, are similar to those involved in producing bacterial type IV pili. Consistent with this observation, we have determined that, in addition to disrupting preflagellin processing, deleting pibD, which encodes the preflagellin peptidase, prevents the maturation of other H. volcanii type IV pilin-like proteins. Moreover, in addition to abolishing swimming motility, and unlike the flgA1-flgA2 deletion, deleting pibD eliminates the ability of H. volcanii to adhere to a glass surface, indicating that a nonflagellar type IV pilus-like structure plays a critical role in H. volcanii surface adhesion.To escape toxic conditions or to acquire new sources of nutrients, prokaryotes often depend on some form of motility. Swimming motility, a common means by which many bacteria move from one place to another, usually depends on flagellar rotation to propel cells through liquid medium (24,26,34). These motility structures are also critical for the effective attachment of bacteria to surfaces.As in bacteria, rotating flagella are responsible for swimming motility in archaea, and recent studies suggest that archaea, like bacteria, also require flagella for efficient surface attachment (37, 58). However, in contrast to bacterial flagellar subunits, which are translocated via a specialized type III secretion apparatus, archaeal flagellin secretion and flagellum assembly resemble the processes used to translocate and assemble the subunits of bacterial type IV pili (34,38,54).

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Mutants in flaI and flaJ of the archaeon Methanococcus voltae are deficient in flagellum assembly

Cited by 50 publications

References 30 publications

AglC and AglK Are Involved in Biosynthesis and Attachment of Diacetylated Glucuronic Acid to the N-Glycan in Methanococcus voltae

AglC and AglK Are Involved in Biosynthesis and Attachment of Diacetylated Glucuronic Acid to the N-Glycan in Methanococcus voltae

Archaeal genetics — the third way

Haloferax volcanii Flagella Are Required for Motility but Are Not Involved in PibD-Dependent Surface Adhesion

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