School of Engineering and Science - Jacobs University

Results
While the function of module 1 is unclear, modules 2-4 are responsible for
activation and condensation of glutamate, tyrosine, and diaminobutyric acid,
respectively (Mossialos et al., 2002). It is well-known that the modular structure
of NRPS allows some genetical modifications. If one of the modules is deleted
completely the rest of the enzyme remains functional, producing a similar
peptide just missing the respective amino acid residue (Finking & Marahiel,
2004). Taking this into account, deletion of the complete adenylation domain
and part of the condensation domain of module 2 in the marker exchange
construct should block chromophore synthesis. We could identify a homologue
of pvsA in the genome sequence of PssB728a.
This strain is closely related to Pss22d and primers derived from the nucleotide
sequence of PssB728a were used to amplify a PCR product of Pss22d. A first
PCR fragment of 1.4 kb was amplified by primer pair pvsA_fwd1_Spe and
pvsA_rev6_Kpn. The amplificate was purified and subcloned in the T/A cloning
vector pGEM-T Easy (Promega) resulting in plasmid pPVSA1. The second
PCR fragment, 1.6 kb in size, was amplified by primers pvsA_fwd7_Kpn and
pvsA_rev4_Bam. It was purified and ligated into pGEM-T Easy, resulting in
plasmid pPVSA2. Subsequently, the SpeI/KpnI fragment of pPVSA 1 was
cleaved, purified and inserted into pPVSA2, resulting in plasmid pPVSA3.
Finally, a 1.7 kb KpnI fragment of plasmid pMKm, carrying the kanamycin
resistance cassette, was inserted into the unique KpnI site of pPVSA3. The
resulting plasmid, pPVSA4, carries the entire marker exchange cassette
consisting of the kanamycin cassette flanked by the two PCR fragments.
pPVSA4 was transferred into Pss22d by electroporation. As the pGEM-T Easy
vector backbone is not able to replicate in Pss22d, the plasmid acts as a
suicide plasmid. Consequently, the resistance against kanamycin is transferred
to Pss22d only if the plasmid recombines into the chromosome. If integration of
the resistance cassette occurs due to a single cross-over, the complete plasmid
integrates and the transformants carry the resistance of the plasmid backbone
(i.e. ampicillin), too.
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