How do hybrid two-component systems regulate gene expression in Bacteroides?

Abstract

The human gut microbiota (HGM) is a synergistic collection of symbiotic microorganisms. A key metabolic function of the bacterial genus Bacteroides in the HGM is the breakdown of a diverse range of complex polysaccharides that cannot be degraded by the host. Bacteroides often regulate their polysaccharide degradation systems using hybrid two-component systems (HTCSs) which directly bind glycan derivatives to upregulate the transcription of genes encoding various catabolic proteins. Unlike canonical two-component systems (CTCSs) composed of two partner proteins, HTCSs exists as a single dimeric polypeptide that spans the cytoplasmic membrane. They consist of a periplasmic ligand-binding sensor domain, linked by transmembrane helices to the cytoplasmic histidine kinase (HK) and response regulator (RR), with an AraC-type DNA binding domain. We investigate the regulatory activities of HTCSs in Bacteroides thetaiotaomicron to answer questions regarding how and why a membrane bound regulator interacts with DNA. Electromobility shift assays show that inactive HTCS RRs bind to computationally predicted ~37bp target DNA sites within 300bp probes, with a loss of binding to shorter fragments of DNA. Likewise, a luciferase reporter assay showed that moving or mutating the ~37bp site dramatically affects gene expression in vivo, indicating there are other undiscovered binding elements required. Surprisingly, some HTCSs do not appear to be dependent on phosphorylation for activation, unlike CTCS. Structural studies of the full-length HTCS BT4663 through cryogenic electron microscopy and visualisation of the HTCS-DNA interaction in the Bacteroides cell through fluorescence microscopy, were attempted and will require further exploration. Our investigation revealed that HTCSs exhibit varied signalling transduction mechanisms, linked to their dependence on phosphorylation. We propose a model where some HTCSs are primed to adopt a more active conformation, with ligand binding being the main prerequisite for activation. This may facilitate a higher background expression of specific PULS, possibly supporting Bacteroides glycan prioritisation.