Since E. coli fabZ null strains are nonviable [15, 16], we first introduced pHW22 into strain
DY330, a “”recombineering”" strain . We then expressed the C. acetobutylicium FabZ in this strain and used standard phage γ recombinase manipulations to delete the host fabZ gene. These manipulations gave strain HW7, which grew well in VX-689 solubility dmso presence of arabinose but failed to grow in the presence of fucose, an anti-inducer of C59 wnt order arabinose promoter expression (Fig. 4). The fatty acid composition of the complemented mutant strain grown in presence of arabinose was similar to that of the parental strain, DY330, indicating that C. acetobutylicium FabZ functionally replaced E. coli FabZ (Table 3). The lack of fabA and fabM homologues in C. acetobutylicium raised the possibility that the FabZ of this organism might function as both an isomerase and a
dehydratase as does the E. faecalis FabZ-like protein, FabN . To test this possibility plasmid pHW22 was introduced into both the fabA(Ts) E. coli strain CY57 and the fabA null mutant strain MH121. Neither stain grew in the absence of unsaturated fatty acid supplementation (data not shown) indicating that C. acetobutylicium FabZ lacks isomerase function and thus was unable to functionally replace FabA. However, it remained possible that C. acetobutylicium FabZ catalyzed UFA synthesis, but that the levels of UFA produced were too low to support growth. This possibility was tested by [14C]-acetate labeling of the fatty acids synthesized by strain CY57 carrying pHW22 and analysis of the resulting BIBF 1120 solubility dmso radioactive fatty acids for traces of UFA (Fig. 5). No UFA synthesis was detected. Another possible explanation for the observed lack of UFA synthesis was that FabI, the enoyl-ACP reductase of E. coli, converted
the intermediate trans-2-decenoyl-ACP to decanoyl-ACP before the putative isomerase activity of C. acetobutylicium FabZ could act. Thus, we repeated the labeling experiment in the presence acetylcholine of a low dose of triclosan, a specific E. coli FabI inhibitor , in order to give the putative isomerase a better opportunity to act on the trans-2-decenoyl-ACP intermediate. Again no synthesis of unsaturated fatty acid was observed (data not shown). These in vivo results argued strongly that that C. acetobutylicium FabZ was unable to isomerize trans-2-decenoyl-ACP. Table 3 Composition of fatty acids of strain HW7 Fatty acid composition (% by weight) C14:0 C16:1 C16:0 C18:1 DY330 3.2 41.0 29.7 26.0 HW7 <0.5 49.6 29.2 21.2 Figure 4 Growth of E. coli fabZ mutant strain HW7 carrying plasmid pHW22 encoding C. acetobutylicium fabZ. The plates were of RB medium ei ther unsupplemented or supplemented with the inducer, L-arabinose, or supplemented with the anti-inducer, D-fucose, as shown. The plates were incubated at 30°C. Strain DY330 has the wild type fabZ locus whereas strain HW7 is ΔfabZ.