CRISPR-mediated gene silencing reveals involvement of the archaeal S-layer in cell division and virus infection

Isabelle Anna Zink,Erika Wimmer,Uwe B Sleytr,Christa Schleper,Kevin Pfeifer,Bernhard Schuster

doi:10.1038/s41467-019-12745-x

Abstract

The S-layer is a proteinaceous surface lattice found in the cell envelope of bacteria and archaea. In most archaea, a glycosylated S-layer constitutes the sole cell wall and there is evidence that it contributes to cell shape maintenance and stress resilience. Here we use a gene-knockdown technology based on an endogenous CRISPR type III complex to gradually silence slaB, which encodes the S-layer membrane anchor in the hyperthermophilic archaeon Sulfolobus solfataricus. Silenced cells exhibit a reduced or peeled-off S-layer lattice, cell shape alterations and decreased surface glycosylation. These cells barely propagate but increase in diameter and DNA content, indicating impaired cell division; their phenotypes can be rescued through genetic complementation. Furthermore, S-layer depleted cells are less susceptible to infection with the virus SSV1. Our study highlights the usefulness of the CRISPR type III system for gene silencing in archaea, and supports that an intact S-layer is important for cell division and virus susceptibility.

Highlights

The S-layer is a proteinaceous surface lattice found in the cell envelope of bacteria and archaea
For silencing of the slaB gene, three 37-bp target sites on the slaB mRNA were chosen such that their 3′ flanking sequence (PAS, protospacer adjacent sequence) matched at least 5 nt of the 5′ handle of the targeting CRISPR RNAs (crRNAs) expressed from a miniCRISPR loci (miniCRs) locus, in order to avoid CRISPRmediated DNA degradation (Fig. 1a)[34]
Two miniCRs carrying the targeting spacer SB3 two times (SB3×2) and six times (SB3×6), respectively, were designed with the attempt to yield stronger silencing by increasing the dosage of targeting crRNAs (Fig. 1a)

Summary

Introduction

The S-layer is a proteinaceous surface lattice found in the cell envelope of bacteria and archaea. We use a gene-knockdown technology based on an endogenous CRISPR type III complex to gradually silence slaB, which encodes the S-layer membrane anchor in the hyperthermophilic archaeon Sulfolobus solfataricus. Our study highlights the usefulness of the CRISPR type III system for gene silencing in archaea, and supports that an intact S-layer is important for cell division and virus susceptibility. Loss or alteration of the N-glycan structure caused growth retardation under elevated salt conditions and compromised protein secretion of archaeal glycosylation mutant cells in Haloferax volcanii and Sulfolobus acidocaldarius[10,11,12,13] In some of these mutants, an aberrant or perturbed S-layer lattice was observed, which seemed more susceptible to proteolytic degradation, indicating a protective and stabilizing role of the glycosylation[10,12]. Our study reveals important functions of the S-layer in S. solfataricus, and highlights the usefulness of the CRISPR-based silencing technology in archaea

Methods

Results

Conclusion