Cell-cell communication via LuxR solos in Photorhabdus species

Bacteria constantly need to monitor their environment and adapt the
bacterial group-coordinated behaviour to changing habitats like
nutrition alterations or host variations. Commonly cell-cell
communication via acyl homoserine lactones (AHLs)is used to
synchronise the behaviour of a bacterial population dependent on
cell size. This process is referred to as quorum sensing (QS) and
predominantly occurs in Gram-negative bacteria. The typical QS
system consists of a LuxI-synthase that synthesises AHLs, and a
LuxR-type receptor, which then responds to these AHLs. Upon
AHL-binding, the LuxR-type receptor regulates the expression of
different target genes and thus influences several processes, like
biofilm formation, virulence, antibiotic production or cell-cell
interaction. Interestingly, many proteobacteria possess additional
LuxR homologs, but lack a cognate LuxI-type synthase. Those
LuxR-type receptors are referred to as LuxR orphans or LuxR solos
and can expand the regulatory QS network. Photorhabdus species are
insect pathogenic bacteria, living in symbiosis with
entomopathogenic nematodes. They all possess an exceptionally high
number of LuxR solos, but lack LuxI homologs and therefore do not
produce AHLs. The function of these LuxR solos, their role in
cell-cell communication and the identification of their cognate
signalling molecules in Photorhabdus species is the main focus of
this work. In this thesis a novel signalling molecule used for QS
could be identified for the first time in P. luminescens. This
novel QS molecule is an α-pyrone named photopyrone (PPY) and
produced endogenously by the photopyrone synthase (PpyS). The PPYs
are specifically recognized by the LuxR solo regulator PluR, which
then activates expression of the pcf (Photorhabdus clumping factor)
operon leading to cell clumping of P. luminescens cells. Moreover,
the PpyS/PluR quorum sensing system and its induced cell clumping
contribute to the overall toxicity of P. luminescens. Furthermore,
a second novel signalling molecule sensed by a LuxR solo of
Photorhabdus species could be identified besides PPYs. The insect
and human pathogenic bacteria P. asymbiotica lacks a PpyS homolog
as well as a LuxI homolog, but harbours a pcf operon and a
homologue to PluR, which is named PauR. The signalling molecule
sensed by the LuxR-type receptor PauR could be identified, which is
neither an AHL nor a PPY. PauR recognises a 2,5-dialkylresorcinol
(DAR) produced by the DarABC pathway. Upon binding of the cognate
signalling molecule, Summary XII PauR activates expression of the
pcf operon. This also leads to cell clumping in P. asymbiotica.
Furthermore, the DarABC/PauR QS system also contributes to the
overall pathogenicity of P. asymbiotica against Galleria mellonella
insect larvae. A bioinformatics approach revealed a high number of
LuxR solos present in P. temperata and P. asymbiotica like in P.
luminescens. Thereby, several conserved motives of amino acids
could be identified, which are potentially important for
signalbinding and -specificity. Variations in these amino acid
motifs are assumed to reflect the overall variety of signals that
can be sensed by LuxR solos. Furthermore, the specificity of the
two LuxR solos PluR and PauR towards their cognate signalling
molecules, PPYs and DARs, respectively, was analysed. Thereby, it
could be shown that the previously identified conserved amino acid
motives in the signal-binding domain (SBD), the TYDQCS-motif of
PluR and the TYDQYI-motif of PauR, are essential but not sufficient
for ligand-binding. Similar as for AHLs, it was unclear how the
signalling molecules PPYs and DARs can cross the bacterial cell
membrane. In the last part of this thesis the import mechanism for
the Photorhabdus-specific signalling compounds PPYs and DARs were
identified. Initial evidence could be provided that the
membrane-integrated transporter FadL is mainly involved in the
import of these hydrophobic compounds, and that they are not
transported via simple diffusion across the cell membrane, which is
assumed for AHLs. In conclusion, the data that is compiled presents
two LuxR solos of Photorhabdus species adapted to sense and respond
to novel non-AHL signalling molecules used for QS. Therefore, this
thesis reveals that cell-cell communication via LuxR-type receptors
goes far beyond AHL-signalling in nature.