Untersuchungen zur Molekularbiologie von Escherichia coli ...

1 Zusammenfassung, Summary
those bacteria induced cell cycle arrest and megalocytosis (cytopathic effect). Deletion of the
asnW island resulted in abolition of the cytopathic effect, which could be restored by intro-
duction of a BAC vector containing the gene cluster. The cytopathic effect was only observed
after direct contact of the bacteria with HeLa cells and could not be achieved using bacterial
lysates, killed bacteria or culture supernatants.
The PKS/NRPS gene cluster comprises 18 ORFs (clbA to clbR) of which 17 are involved
in the synthesis of the active compound. The number of gene products and the sequence of
putative domains differ from those of all PKS/NRPS systems described so far. Investigati-
ons on the transcription of the island revealed three monocistronically transcribed units and
four polycistrons of sizes up to 23 kb. In addition, a constitutive transcription of every ORF
was observed, albeit at variable levels. Upon cell contact, neither elevated transcription nor
different promoter activities of single ORFs were observed. Thus, the contact dependence of
the cytopathic effect does not seem to result from an induction of expression caused by HeLa
cells.
The contact dependence could not be overcome by induction or overexpression of either
a PKS/NRPS (clbB), the putative key enzymes thioesterase (clbQ) and phosphopantetheinyl
transferase (clbA), or the potential regulator clbR.
Using luciferase reporter gene fusions an influence of diverse media and culture conditions
on the promoter activities of singele genes was detected. This was ascribed to the influence
of the BarA/UvrY two-component system, which regulates the carbon metabolism of E. coli
on the post-transcriptional level via CsrA/CsrBC. The natural uvrY deletion mutant UPEC
536, despite containing the complete PKS/NRPS gene cluster, did not exhibit an cytopathic
effect. However, this feature could be restored by complementation with uvrY. This is the first
evidence for an regulatory mechanism of the PK/NRP synthesis located beyond the asnW
island. The in vivo function of the peptide-polyketide so far remains unclear, but it might
affect fitness as well as virulence of E. coli.
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