School of Engineering and Science - Jacobs University

Results
Fig. 15. Construction of the marker exchange mutant Pss22d∆Ach.
acsD is the first of three genes encoding for a siderophore synthetase. In mutant Pss22d∆Ach,
this ORF has been exchanged by a kanamycin cassette. For construction of this mutant, a 1,3 kb
PCR fragment was amplified by the primers Achr1_fwd (1) and Achr2_KpnI_rev. A second PCR
fragment, 2 kb in size, was amplified by the primer pair Achr5_EcoRV_fwd (4) and Achr6_rev (4).
The PCR fragments were subsequently fused to a 1.7-kb fragment containing a kanamycin
resistance cassette and incorporated into the chromosome by double homologous recombination.
Dashed arrow indicate the flanking genes acr and acsA.
The resulting plasmid, carrying the complete marker exchange construct was
designated pACS4. This plasmid was transferred into Pss22d by
electroporation and the resulting tranformants were screened for kanamycin
resistance and absence of ampicillin resistance. The correct genotype of the
candidate transformantes was verified by PCR (data not show). The resulting
mutant was designated Pss22d∆Ach.
5.1.5 In vitro comparison of Pss22d and its siderophore mutants
The phenotype of the Pss22d and the single siderophore mutants was
compared on CAS agar (Fig.16).
A
B
C
D
Figure 16. Siderophore production on CAS-agar.
Bacterial suspensions of A: Pss22d; B: Pss22d∆Ach;
C: Pss22d∆Sid; and D: Pss22d∆Pvd were applied on
a CAS-agar plate and incubated at 28°C for 48h.
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