Research

Role of cyclic nucleotides in Haloferax volcanii

Euryarchaeal genomes encode di-adeylate cyclases (DacZ). We showed that H. volcanii produces c-di-AMP and that under the tested laboratory conditions it is essential as we could not delete the gene encoding dacZ.

A mutant which expressed lower amounts of dacZ showed no changes in growth in normal conditions, but in low salt conditions the cells became large and could obviously not control their cell volume. Therefore we hypothesize, that in H.volcanii the c-di-AMP levels are important to control osmohomeostasis.

Chemotaxis and localization of the archaellum in Haloferax volcanii

Although archaea use a totally different motility machinery than bacteria, eg. euryarchaea have obtained the full chemotaxis system from bacteria via horizontal gene transfer.

However, the archaellum has no FliM, to which CheY binds in bacteria. Therefore archaea have adaptor proteins, like CheF, which interact with CheY and the archaellum motor.

Interestingly, the CheY structure between the archaeal and the bacterial one is very similar except for the FliM binding site.

Morever, we found that the archaella of H. volcanii are positioned at the poles. H. volcanii is mainly motile during the very early log phase when the cells are rod-shaped. When the cells round up during growth, the filament is disassembled and only the archaellar motor is still present.

Motility in Archaea – the Archaellum

Archaea use a unique structure for swimming motility which is not hoomologous to bacterial flagella, but instead resembles type IV pili. But in contrast to type IV pili, motion is not achieved by elongation and disassembly of the filament, but by rotation.

The smallest known archaellum operon is present in Sulfolobus acidocaldarius. Therefore we used the S. acidoalcarius archaellum as a model system to understand which role the different proteins play during assembly and rotation of the archaellum. To that end we use biochemical, genetic and structural approaches.

So far we have reported the structures of FlaI, FlaH and FlaF (follow the links to the publications). These structural analyses were done in a successful collaboration with the lab of John Tainer .

The image shows the graphical abstract of our recent paper about FlaH in Molecular Microbiology (Chaudhury et al, 2016). Although FlaH is not an active ATPase, nucleotide binding is essential for its interaction with FlaH and therefore archaellum assembly.

In our latest we show that FlaG forms a filament and interacts via FlaF with the S-layer. The interaction of FlaF with the S-layer is essential for torque-generation by the archaellum to allow rotation of the filament.

The UV induced pili system and DNA transfer

Almost all Sulfolobales contain an type IV pili operon named the UV inducible pilus operon (ups). This operon is highly induced after UV treatment of Sulfolobus cells or the occurence of DNA double strand breakages. As a result the cells express multiple pili all around the cell surface and start to aggregate (see image). In these aggregates the cells exchange DNA which is subsequently used to repair their genomic DNA by homologous recombination.

Recently, we have have identified the DNA importer, the Ced system (Crenarchaeal exchange of DNA), which takes up the DNA during the aggregation process and is depending on an ATPase (Van Wolferen et al., PNAS, 2016). We are now trying to understand how the cells aggregate and how the cell-cell-connection is established.

Recently we have shown that the cells interact species-specific. The species specificity is determined by the UpsA pilin component which interacts specifically with the N-glycan on the surface of the S-layer protein (van Wolferen et al., mBio. 2020).

Regulation of archaellum expression in Sulfolobus acidocaldarius

Already in 2007 Zalan Szabo showed that archaellum expression in Sulfolobus solfataricus is starvation induced. Lateron we showed that the same is true for S. acidocaldarius, but nothing was known about the mechanims behind this regulatory process.

To date we have identified 4 proteins that play a role in the control of archaellum expression. Most of them are termed Arn for Archaellum regulatory network:

ArnA and ArnB are repressor of archaellum expression as their deletion leads to hypermotile cells. ArnA contains a FHA domain and ArnB a vanWillebrand domain. Interestingly, both proteins were phosphorylated by two S. acidocaldarius kinases (Reimann et al, 2012).

The only serine/threonine phosphoatase (PP2A) in the S. acidocaldarius genome also seems to play an important role in this regulatory network, as its deletion also led to a hypermotile phenotype (Reimann et al., 2013).

ArnR is the so far only positive regulator of the archaellum. It is a membrane bound one component regulator that directly binds to two inverted repeats in the archaellum operon (Lassak et al, 2013). At the moment we are trying to understand how ArnR senses starvation stress to initiate archaellum expression. This work is currently funded by the CRC 746 in Freiburg.

In two recent papers (Bischof et al, 2019 a/b), we show an extensive analysis of the starvation response of S. acidocaldarius and a detailed analysis of ArnR and ArnR1.

Biofilm formation in Archaea

We studied how different Sulfolobus species form biofilms, how these are build and which components build their matrix. Moreover, we identified archaea specific regulators, the Lrs14 familiy, which are involved in the regulation of pathways that lead to biofilm formation such as EPS production.

The different show different phenotypes in biofilm with S. acidocaldarius showing the highest production of EPS during biofilm growth. AbfR1 was one of the regulators which were identified to be important for biofilm formation in S. acidocaldarius. This regulator is actually stimulating archaella expression and thereby inhibiting biofilm formation.

N-glycosylation in Sulfolobus acidocaldarius

Almost all extracellular proteins of archaea are modified by either N- or O-glycosylation or both. We have set out to analyze the N-glycosylation pathway in S. acidocaldarius and used the S-layer protein as a substrate protein. We identified a number of enzymes that play an important role in the N-glycosylation pathway in S. acidocaldarius.