Abstract
Halobacterium salinarum are halophilic archaea that display directional swimming in response to various environmental signals, including light, chemicals and oxygen. In Hbt. salinarum, the building blocks (archaellins) of the archaeal swimming apparatus (the archaellum) are N-glycosylated. However, the physiological importance of archaellin N-glycosylation remains unclear. Here, a tetrasaccharide comprising a hexose and three hexuronic acids decorating the five archaellins was characterized by mass spectrometry. Such analysis failed to detect sulfation of the hexuronic acids, in contrast to earlier reports. To better understand the physiological significance of Hbt. salinarum archaellin N-glycosylation, a strain deleted of aglB, encoding the archaeal oligosaccharyltransferase, was generated. In this ΔaglB strain, archaella were not detected and only low levels of archaellins were released into the medium, in contrast to what occurs with the parent strain. Mass spectrometry analysis of the archaellins in ΔaglB cultures did not detect N-glycosylation. ΔaglB cells also showed a slight growth defect and were impaired for motility. Quantitative real-time PCR analysis revealed dramatically reduced transcript levels of archaellin-encoding genes in the mutant strain, suggesting that N-glycosylation is important for archaellin transcription, with downstream effects on archaellum assembly and function. Control of AglB-dependent post-translational modification of archaellins could thus reflect a previously unrecognized route for regulating Hbt. salinarum motility.
Highlights
In 1976, the surface (S)-layer glycoprotein from the hypersaline-adapted archaeon Halobacterium salinarum provided the first example of a glycosylated protein outside the Eukarya (Mescher and Strominger, 1976)
The present study provided the first direct demonstration that Hbt. salinarum AglB is necessary for N-glycosylation
Previous efforts had shown that Hbt. salinarum AglB could functionally replace its Hfx. volcanii counterpart, where the N-linked glycan that decorates cell surface glycoproteins is assembled on a dolichol phosphate carrier (Guan et al, 2010; Cohen-Rosenzweig et al, 2014)
Summary
In 1976, the surface (S)-layer glycoprotein from the hypersaline-adapted (halophilic) archaeon Halobacterium salinarum provided the first example of a glycosylated protein outside the Eukarya (Mescher and Strominger, 1976). Hbt. salinarum archaellins comprising the archaellum [the archaeal counterparts of bacterial flagellins and the flagellum, respectively (Jarrell and Albers, 2012)] were shown to be modified (Wieland et al, 1985). Both the S-layer glycoprotein and archaellins were reported to be N-glycosylated by a tetrasaccharide comprising a glucose and three sulfated glucuronic acids initially assembled on a dolichol phosphate carrier. N-Glycosylation of Hbt. salinarum Archaellins (Lechner et al, 1985a; Wieland et al, 1985). Despite the central role played by archaella in the directional taxis Hbt. salinarum displays in response to appropriate light, chemical, oxygen and other signals (Marwan et al, 1991), the importance of archaellin N-glycosylation in such directional swimming remains unclear
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