Photorhabdus luminescens genes induced upon insect infection

Background: Photorhabdus luminescens is a Gram-negative luminescent
enterobacterium and a symbiote to soil nematodes belonging to the
species Heterorhabditis bacteriophora. P. luminescens is
simultaneously highly pathogenic to insects. This bacterium
exhibits a complex life cycle, including one symbiotic stage
characterized by colonization of the upper nematode gut, and a
pathogenic stage, characterized by release from the nematode into
the hemocoel of insect larvae, resulting in rapid insect death
caused by bacterial toxins. P. luminescens appears to sense and
adapt to the novel host environment upon changing hosts, which
facilitates the production of factors involved in survival within
the host, host-killing, and -exploitation. Results: A differential
fluorescence induction (DFI) approach was applied to identify genes
that are up-regulated in the bacterium after infection of the
insect host Galleria mellonella. For this purpose, a P. luminescens
promoter-trap library utilizing the mCherry fluorophore as a
reporter was constructed, and approximately 13,000 clones were
screened for fluorescence induction in the presence of a G.
mellonella larvae homogenate. Since P. luminescens has a variety of
regulators that potentially sense chemical molecules, like
hormones, the screen for up-regulated genes or operons was
performed in vitro, excluding physicochemical signals like oxygen,
temperature or osmolarity as variables. Clones (18) were obtained
exhibiting at least 2.5-fold induced fluorescence and regarded as
specific responders to insect homogenate. In combination with a
bioinformatics approach, sequence motifs were identified in these
DNA-fragments that are similar to 29 different promoters within the
P. luminescens genome. By cloning each of the predicted promoters
upstream of the reporter gene, induction was verified for 27
promoters in vitro, and for 24 promoters in viable G. mellonella
larvae. Among the validated promoters are some known to regulate
the expression of toxin genes, including tccC1 (encoding an
insecticidal toxin complex), and others encoding putative toxins. A
comparably high number of metabolic genes or operons were observed
to be induced upon infection; among these were eutABC, hutUH, and
agaZSVCD, which encode proteins involved in ethanolamine, histidine
and tagatose degradation, respectively. The results reflect
rearrangements in metabolism and the use of other metabolites
available from the insect. Furthermore, enhanced activity of
promoters controlling the expression of genes encoding enzymes
linked to antibiotic production and/or resistance was observed.
Antibiotic production and resistance may influence competition with
other bacteria, and thus might be important for a successful
infection. Lastly, several genes of unknown function were
identified that may represent novel pathogenicity factors.
Conclusion: We show that a DFI screen is useful for identifying
genes or operons induced by chemical stimuli, such as diluted
insect homogenate. A bioinformatics comparison of motifs similar to
known promoters is a powerful tool for identifying regulated genes
or operons. We conclude that signals for the regulation of those
genes or operons induced in P. luminescens upon insect infection
may represent a wide variety of compounds that make up the insect
host. Our results provide insight into the complex response to the
host that occurs in a bacterial pathogen, particularly reflecting
the potential for metabolic shifts and other specific changes
associated with virulence.