Genetic engineering of Acinetobacter baumannii.

A. baumannii mutants or variants were constructed using a standard allelic exchange approach with the counterselectable suicide vectors pGP704-Sac28 or pGP704Sac-kan (71, 72). Derivatives of these plasmids containing deletion constructs of the respective genes or site-directed allele exchanges (of pilA) were transferred to A. baumannii through mating with E. coli S17-1λpir. The deletion constructs were based on the PCR amplification of the flanking regions of the desired genetic regions and, when needed, the aph cassette (Kan^r) as the selection marker using PWO polymerase (Roche) or Q5 polymerase (BioLabs). Site-directed changes in the pilA allele were inserted using modified primers. The amplified fragments were joined by overlap extension PCR or Golden Gate assembly (73) and were cloned inside the suicide plasmids. The correct cloning products were screened for by colony PCR of the E. coli transformants (with GoTaq polymerase; Promega) and ultimately confirmed by Sanger sequencing (Microsynth, Switzerland).

Construction of inducible genes-of-interest was accomplished by placing the respective gene under the control of the arabinose-inducible promoter P_BAD inside the miniTn7 transposon TnAraC (23, 63). The resulting transposons were transferred to the respective A. baumannii strains through a standard triparental mating approach (74).