Identification of Haloferax volcanii Pilin N-Glycans with Diverse Roles in Pilus Biosynthesis, Adhesion, and Microcolony Formation

Esquivel, Rianne N.; Schulze, Stefan; Xu, Rachel; Hippler, Michael; Pohlschröder, Mechthild

doi:10.1074/jbc.m115.693556

Cited by 55 publications

(77 citation statements)

References 45 publications

(142 reference statements)

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“…In agreement with earlier efforts addressing the impact of compromised N-glycosylation on the functions of other H. volcanii glycoproteins (18,41), perturbed S-layer glycoprotein N-glycosylation was shown to have physiological implications. Still, heterogeneity in S-layer glycoprotein N-glycosylation may be responsible for more-subtle effects than suggested here (38).…”

Section: Discussionsupporting

confidence: 86%

N-Glycosylation Is Important for Proper Haloferax volcanii S-Layer Stability and Function

Tamir

Eichler

2017

Appl Environ Microbiol

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N-Glycosylation, the covalent linkage of glycans to select Asn residues of target proteins, is an almost universal posttranslational modification in archaea. However, whereas roles for N-glycosylation have been defined in eukarya and bacteria, the function of archaeal N-glycosylation remains unclear. Here, the impact of perturbed N-glycosylation on the structure and physiology of the haloarchaeon Haloferax volcanii was considered. Cryo-electron microscopy was used to examine right-side-out membrane vesicles prepared from cells of a parent strain and from strains lacking genes encoding glycosyltransferases involved in assembling the N-linked pentasaccharide decorating the surface layer (S-layer) glycoprotein, the sole component of the S-layer surrounding H. volcanii cells. Whereas a regularly repeating S-layer covered the entire surface of vesicles prepared from parent strain cells, vesicles from the mutant cells were only partially covered. To determine whether such N-glycosylation-related effects on S-layer assembly also affected cell function, the secretion of a reporter protein was addressed in the parent and N-glycosylation mutant strains. Compromised S-layer glycoprotein N-glycosylation resulted in impaired transfer of the reporter past the S-layer and into the growth medium. Finally, an assessment of S-layer glycoprotein susceptibility to added proteases in the mutants revealed that in cells lacking AglD, which is involved in adding the final pentasaccharide sugar, a distinct S-layer glycoprotein conformation was assumed in which the N-terminal region was readily degraded. Perturbed N-glycosylation thus affects S-layer glycoprotein folding. These findings suggest that H. volcanii could adapt to changes in its surroundings by modulating N-glycosylation so as to affect S-layer architecture and function.IMPORTANCE Long held to be a process unique to eukaryotes, it is now accepted that bacteria and archaea also perform N-glycosylation, namely, the covalent attachment of sugars to select asparagine residues of target proteins. Yet, while information on the importance of N-glycosylation in eukaryotes and bacteria is available, the role of this posttranslational modification in archaea remains unclear. Here, insight into the purpose of archaeal N-glycosylation was gained by addressing the surface layer (S-layer) surrounding cells of the halophilic species Haloferax volcanii. Relying on mutant strains defective in N-glycosylation, such efforts revealed that compromised N-glycosylation affected S-layer integrity and the transfer of a secreted reporter protein across the S-layer into the growth medium, as well as the conformation of the S-layer glycoprotein, the sole component of the S-layer. Thus, by modifying N-glycosylation, H. volcanii cells can change how they interact with their surroundings.

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Section: Discussionsupporting

confidence: 86%

N-Glycosylation Is Important for Proper Haloferax volcanii S-Layer Stability and Function

Tamir

Eichler

2017

Appl Environ Microbiol

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“…While surface filaments could be observed in both strains, fewer cells had filaments compared to the wild-type strain. Likewise, while wild-type cells generally have 1–12 long filaments [19], none of the hvo_2876::tn or Δ hvo_2876 cells examined had more than one long filament. Intriguingly, these cells had a significant number of short filamentous surface structures.…”

Section: Resultsmentioning

confidence: 99%

“…When these pilins are incorporated into pili, this sequestered factor is released and inhibits motility. N -glycosylation also appears to play roles in regulating both motility and the early steps of biofilm formation [18,19]. Both the pilins and the flagellins can be modified by N -glycosylation, but interestingly, while flagella-dependent motility is inhibited in cells that are defective for N -glycosylation, certain pilins switch their functions depending on their glycosylation state [19].…”

Section: Introductionmentioning

confidence: 99%

“…N -glycosylation also appears to play roles in regulating both motility and the early steps of biofilm formation [18,19]. Both the pilins and the flagellins can be modified by N -glycosylation, but interestingly, while flagella-dependent motility is inhibited in cells that are defective for N -glycosylation, certain pilins switch their functions depending on their glycosylation state [19]. However, not all components of this archaeal glycosylation pathway, or of the other recently characterized glycosylation pathways, have been identified [20].…”

Section: Introductionmentioning

confidence: 99%

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Screening of a Haloferax volcanii Transposon Library Reveals Novel Motility and Adhesion Mutants

Legerme

Yang

Esquivel

et al. 2016

Life

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Archaea, like bacteria, use type IV pili to facilitate surface adhesion. Moreover, archaeal flagella—structures required for motility—share a common ancestry with type IV pili. While the characterization of archaeal homologs of bacterial type IV pilus biosynthesis components has revealed important aspects of flagellum and pilus biosynthesis and the mechanisms regulating motility and adhesion in archaea, many questions remain. Therefore, we screened a Haloferax volcanii transposon insertion library for motility mutants using motility plates and adhesion mutants, using an adapted air–liquid interface assay. Here, we identify 20 genes, previously unknown to affect motility or adhesion. These genes include potential novel regulatory genes that will help to unravel the mechanisms underpinning these processes. Both screens also identified distinct insertions within the genomic region lying between two chemotaxis genes, suggesting that chemotaxis not only plays a role in archaeal motility, but also in adhesion. Studying these genes, as well as hypothetical genes hvo_2512 and hvo_2876—also critical for both motility and adhesion—will likely elucidate how these two systems interact. Furthermore, this study underscores the usefulness of the transposon library to screen other archaeal cellular processes for specific phenotypic defects.

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“…Glycan structures alone are published for several other varied members of this domain [1,9,11]. The most commonly studied proteins modified by N -linked glycans in archaea are S-layer proteins [12–14], archaellins [15–19] and pilins [20–22]. The current model of the archaeal N -glycosylation pathway involves the sequential addition of sugar monomers onto a dolichol-type lipid carrier embedded in the cytoplasmic membrane, followed by a flipping of the lipid-linked glycan across the membrane.…”

Section: Introductionmentioning

confidence: 99%

Complementation of an aglB Mutant of Methanococcus maripaludis with Heterologous Oligosaccharyltransferases

et al. 2016

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The oligosaccharyltransferase is the signature enzyme for N-linked glycosylation in all domains of life. In Archaea, this enzyme termed AglB, is responsible for transferring lipid carrier-linked glycans to select asparagine residues in a variety of target proteins including archaellins, S-layer proteins and pilins. This study investigated the ability of a variety of AglBs to compensate for the oligosaccharyltransferase activity in Methanococcus maripaludis deleted for aglB, using archaellin FlaB2 as the reporter protein since all archaellins in Mc. maripaludis are modified at multiple sites by an N-linked tetrasaccharide and this modification is required for archaellation. In the Mc. maripaludis ΔaglB strain FlaB2 runs as at a smaller apparent molecular weight in western blots and is nonarchaellated. We demonstrate that AglBs from Methanococcus voltae and Methanothermococcus thermolithotrophicus functionally replaced the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain, both returning the apparent molecular weight of FlaB2 to wildtype size and restoring archaellation. This demonstrates that AglB from Mc. voltae has a relaxed specificity for the linking sugar of the transferred glycan since while the N-linked glycan present in Mc. voltae is similar to that of Mc. maripaludis, the Mc. voltae glycan uses N-acetylglucosamine as the linking sugar. In Mc. maripaludis that role is held by N-acetylgalactosamine. This study also identifies aglB from Mtc. thermolithotrophicus for the first time by its activity. Attempts to use AglB from Methanocaldococcus jannaschii, Haloferax volcanii or Sulfolobus acidocaldarius to functionally replace the oligosaccharyltransferase activity missing in the Mc. maripaludis ΔaglB strain were unsuccessful.

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Identification of Haloferax volcanii Pilin N-Glycans with Diverse Roles in Pilus Biosynthesis, Adhesion, and Microcolony Formation

Cited by 55 publications

References 45 publications

N-Glycosylation Is Important for Proper Haloferax volcanii S-Layer Stability and Function

N-Glycosylation Is Important for Proper Haloferax volcanii S-Layer Stability and Function

Screening of a Haloferax volcanii Transposon Library Reveals Novel Motility and Adhesion Mutants

Complementation of an aglB Mutant of Methanococcus maripaludis with Heterologous Oligosaccharyltransferases

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