The identification of the arcAB regulon by two fundamentally different screening approaches emphasizes Doxorubicin concentration the key role of ArcAB in GI colonisation and furthermore underscores the validity of the screening approaches. Our screening assay also identified a Klebsiella two-gene cluster of unknown function, here designated kpn_01507 and kpn_01508, which conferred enhanced GI colonisation
ability to EPI100. KPN_01507 is a putative membrane protein, whereas the use of SignalP 4.0 predicted the presence of a secretory signal peptide in KPN_01508, a signal targeting its passenger domain for translocation across the bacterial cytoplasmic membrane . These findings, therefore, suggest that KPN_01508 may be translocated and/or secreted from the cell. Interestingly, homologues of both genes are found among several sequenced strains of K. pneumoniae but do not appear to be present in E. coli. Future studies may reveal the function of these genes in GI colonisation. The fact that genes associated with metabolism were selected in the in vivo screening buy GPCR Compound Library assay is not surprising since the ability to obtain nutrients for growth is essential for any GI colonizing organism. However, many highly conserved proteins involved in metabolism are increasingly recognized as having additional roles, some of which are related
to bacterial virulence . The GalET cluster may be viewed as an example of such so-called moon-lighting proteins as the colonisation enhancing effect was not associated with galactose fermentation per se but was due to increased resistance against bile salt possibly mediated by the modification
of LPS core synthesis. A key limitation of the library-based technique is its inability to identify interactions among distant genetic Nutlin-3 cell line loci. This limitation could be circumvented by using co-expressed plasmid- and fosmid-based genomic libraries as recently described . Thus, future studies combining the C3091 fosmid library with a co-expressed plasmid-based C3091 library may lead to the selection of more GI-enhancing genes than those obtained in this study. The fact that our screening method is based on a laboratory E. coli strain, as opposed to a commensal E. coli isolate, raises another important point. Genes mutated in the laboratory strain, e.g. recA, would most likely not have been selected if the screening had been carried out using a commensal strain. However, since commensal E. coli are already excellent GI colonisers, it is possible that genes which are important for K. pneumoniae GI colonisation but also present in E. coli commensal strains will not be selected in the screening. However, if the objective is to specifically identify K. pneumoniae virulence genes, using a commensal E. coli strain as a host in the screening will be a favourable approach. Using E. coli as a host has several advantages when it comes to construction, cloning, and expression of the fosmid library.