Dataset Information

Agl16, a thermophilic glycosyltransferase mediating the last step of N-Glycan biosynthesis in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

ABSTRACT: Recently, the S-layer protein of Sulfolobus acidocaldarius was shown to be N-linked with a tribranched hexasaccharide, composed of Man2Glc1GlcNAc2 and a sulfated sugar called sulfoquinovose. To identify genes involved in the biosynthesis and attachment of this glycan, markerless in-frame deletions of genes coding for predicted glycosyltransferases were created. The successful deletion of agl16, coding for a glycosyltransferase, resulted in the S-layer protein and archaellins having reduced molecular weights, as visualized by Coomassie staining or immunoblotting. This analysis indicated a change in the N-glycan composition. Nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses confirmed that the glycan of the S-layer protein from the agl16 deletion mutant was a pentasaccharide, which was missing a terminal hexose residue. High-performance liquid chromatography (HPLC) analyses of the hydrolyzed N-glycan indicated that the missing hexose is a glucose residue. A physiological characterization of the agl16 deletion mutant revealed a significant effect on the growth at elevated salt concentrations. At 300 mM NaCl, the doubling time of the ?agl16 mutant was increased 2-fold compared to that of the background strain. Furthermore, the incomplete glycan structure of the ?agl16 deletion strain affected the assembly and function of the archaellum, as exemplified by semisolid Gelrite plate analysis, in which the motility is decreased according to the N-glycan size.

SUBMITTER: Meyer BH

PROVIDER: S-EPMC3650533 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Agl16, a thermophilic glycosyltransferase mediating the last step of N-Glycan biosynthesis in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

Meyer Benjamin H BH Peyfoon Elham E Dietrich Carsten C Hitchen Paul P Panico Maria M Morris Howard R HR Dell Anne A Albers Sonja-Verena SV

Journal of bacteriology 20130308 10

Recently, the S-layer protein of Sulfolobus acidocaldarius was shown to be N-linked with a tribranched hexasaccharide, composed of Man2Glc1GlcNAc2 and a sulfated sugar called sulfoquinovose. To identify genes involved in the biosynthesis and attachment of this glycan, markerless in-frame deletions of genes coding for predicted glycosyltransferases were created. The successful deletion of agl16, coding for a glycosyltransferase, resulted in the S-layer protein and archaellins having reduced molec ...[more]

PMID: 23475978

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Project description:High-temperature stress is critical for all organisms and induces a profound cellular response. For Crenarchaeota, little information is available on how heat shock affects cellular processes and on how this response is regulated. We set out to study heat shock response in the thermoacidophilic model crenarchaeon Sulfolobus acidocaldarius, which thrives in volcanic hot springs and has an optimal growth temperature of 75°C. Pulse-labeling experiments demonstrated that a temperature shift to 86°C induces a drastic reduction of the transcriptional and translational activity, but that RNA and protein neosynthesis still occurs. By combining RNA sequencing and mass spectrometry, an integrated mapping of the transcriptome and proteome was performed. This revealed that heat shock causes an immediate change in the gene expression profile, with RNA levels of half of the genes being affected, followed by a more subtle reprogramming of the protein landscape. Functional enrichment analysis indicated that nearly all cellular processes are affected by heat shock. A limited correlation was observed in the differential expression on the RNA and protein level, suggesting a prevalence of post-transcriptional and post-translational regulation. Furthermore, promoter sequence analysis of heat shock regulon genes demonstrated the conservation of strong transcription initiation elements for highly induced genes, but an absence of a conserved protein-binding motif. It is, therefore, hypothesized that histone-lacking archaea such as Sulfolobales use an evolutionarily ancient regulatory mechanism that relies on temperature-responsive changes in DNA organization and compaction induced by the action of nucleoid-associated proteins, as well as on enhanced recruitment of initiation factors. IMPORTANCE Heat shock response is the ability to respond adequately to sudden temperature increases that could be harmful for cellular survival and fitness. It is crucial for microorganisms living in volcanic hot springs that are characterized by high temperatures and large temperature fluctuations. In this study, we investigated how S. acidocaldarius, which grows optimally at 75°C, responds to heat shock by altering its gene expression and protein production processes. We shed light on which cellular processes are affected by heat shock and propose a hypothesis on underlying regulatory mechanisms. This work is not only relevant for the organism's lifestyle, but also with regard to its evolutionary status. Indeed, S. acidocaldarius belongs to the archaea, an ancient group of microbes that is more closely related to eukaryotes than to bacteria. Our study thus also contributes to a better understanding of the early evolution of heat shock response.

Project description:DNA methylation is the most common epigenetic modification observed in the genomic DNA (gDNA) of prokaryotes and eukaryotes. Methylated nucleobases, N6-methyl-adenine (m6A), N4-methyl-cytosine (m4C), and 5-methyl-cytosine (m5C), detected on gDNA represent the discrimination mark between self and non-self DNA when they are part of restriction-modification systems in prokaryotes (Bacteria and Archaea). In addition, m5C in Eukaryotes and m6A in Bacteria play an important role in the regulation of key cellular processes. Although archaeal genomes present modified bases as in the two other domains of life, the significance of DNA methylations as regulatory mechanisms remains largely uncharacterized in Archaea. Here, we began by investigating the DNA methylome of Sulfolobus acidocaldarius. The strategy behind this initial study entailed the use of combined digestion assays, dot blots, and genome resequencing, which utilizes specific restriction enzymes, antibodies specifically raised against m6A and m5C and single-molecule real-time (SMRT) sequencing, respectively, to identify DNA methylations occurring in exponentially growing cells. The previously identified restriction-modification system, specific of S. acidocaldarius, was confirmed by digestion assay and SMRT sequencing while, the presence of m6A was revealed by dot blot and identified on the characteristic Dam motif by SMRT sequencing. No m5C was detected by dot blot under the conditions tested. Furthermore, by comparing the distribution of both detected methylations along the genome and, by analyzing DNA methylation profiles in synchronized cells, we investigated in which cellular pathways, in particular the cell cycle, this m6A methylation could be a key player. The analysis of sequencing data rejected a role for m6A methylation in another defense system and also raised new questions about a potential involvement of this modification in the regulation of other biological functions in S. acidocaldarius.

Project description:High-temperature stress is a critical environmental parameter for all organisms and induces a cellular heat shock response, characterized by an upregulation of heat shock proteins. For Crenarchaeota, it is unknown how this response is regulated, given the lack of conservation of known prokaryotic or eukaryotic heat shock-regulating transcription factors and how cellular processes are affected. To this end, we studied the heat shock response of the thermoacidophilic Sulfolobus acidocaldarius, thriving in volcanic hot springs at 75°C, upon shift to 86°C, at the transcriptome and proteome level. By pulse-labeling of neosynthesized RNA and proteins upon heat shock, we show a drastic reduction of the cell’s major transcriptional activity immediately after shift and neosynthesis of certain abundant (heat shock) proteins, including the thermosome. By combining RNA-sequencing and mass spectrometry, we show that RNA levels of half of the genes are affected immediately after temperature shift and that reprogramming of the protein landscape takes at least one hour and changes are more subtle. Correlation analysis provides evidence that considerable post-transcriptional and post-translational regulation is occurring upon heat shock. Functional enrichment analysis indicates that nearly all biological processes are affected by heat shock. An overall downregulation is observed for transcription, translation, DNA replication and cell division. However, quality control of translation and of the protein landscape is increased. Many DNA repair pathways are differentially expressed, accompanied with an increase in a DNA import system. Furthermore, expression of DNA topoisomerases is investigated, coinciding with a significant differential expression of nucleoid-associated proteins. Based on these findings and considering the absence of a conserved DNA-binding motif, we propose that regulation of the heat shock response in Sulfolobales is most likely not established by a classical transcription factor, but is rather caused by changes in chromosome organization or established at the post-transcriptional level.

Dataset Information

Agl16, a thermophilic glycosyltransferase mediating the last step of N-Glycan biosynthesis in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

Publications

Agl16, a thermophilic glycosyltransferase mediating the last step of N-Glycan biosynthesis in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

Similar Datasets

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