Type II fatty acid biosynthesis in bacteria can be broadly classified into the initiation and elongation phases. The biochemical functions defining each step in the two phases have been studied in vitro. Among the β-ketoacyl-acyl carrier protein (ACP) synthases, FabH catalyzes the initiation reaction, while FabB and FabF, which primarily catalyze the elongation reaction, can also drive initiation as side reactions. A role for FabB and FabF in the initiation of fatty acid biosynthesis would be supported by the viability of the ΔfabH mutant. In this study, we show that the ΔfabH and ΔyiiD mutations were synthetically lethal and that ΔfabH ΔrelA ΔspoT and ΔfabH ΔdksA synthetic lethality was rescued by the heterologous expression of yiiD. In the ΔfabH mutant, the expression of yiiD was positively regulated by (p)ppGpp. The growth defect, reduced cell size, and altered fatty acid profile of the ΔfabH mutant and the growth defect of the ΔfabH ΔfabF fabB15(Ts) mutant in oleate- and palmitate-supplemented medium at 42°C were rescued by the expression of yiiD from a multicopy plasmid. Together, these results indicate that the yiiD-encoded function supported initiation of fatty acid biosynthesis in the absence of FabH. We have renamed yiiD as fabY. IMPORTANCE Fatty acid biosynthesis is an essential process conserved across life forms. β-Ketoacyl-ACP synthases are essential for fatty acid biosynthesis. FabH is a β-ketoacyl-ACP synthase that contributes to the initiation of fatty acid biosynthesis in Escherichia coli. In this study, we present genetic and biochemical evidence that the yiiD (renamed fabY)-encoded function contributes to the biosynthesis of fatty acid in the absence of FabH activity and that under these conditions, the expression of FabY was regulated by the stringent response factors (p)ppGpp and DksA. Combined inactivation of FabH and FabY resulted in growth arrest, possibly due to the loss of fatty acid biosynthesis. A molecule(s) that inhibits the two activities can be an effective microbicide.
Stringent response mediated by modified guanosine nucleotides is conserved across bacteria and is regulated through the Rel/Spo functions. In Escherichia coli , RelA and SpoT proteins synthesize the modified nucleotides ppGpp and pppGpp, together referred to as (p)ppGpp. SpoT is also the primary (p)ppGpp hydrolase. In this study, using hypomorphic relA alleles, we provide experimental evidence for SpoT-mediated negative regulation of the amplification of RelA-dependent stringent response. We investigated the kinetics of ppGpp degradation in cells recovering from stringent response in the complete absence of SpoT function. We found that, although greatly diminished, there was slow ppGpp degradation and growth resumption after a lag period, concomitant with decrease in ppGpp pool. We present evidence for reduction in the ppGpp degradation rate following an increase in pppGpp pool, during recovery from stringent response. From a genetic screen, the nudix hydrolases MutT and NudG were identified as over-expression suppressors of the growth defect of Δ spoT and Δ spoT Δ gppA strains. The effect of over-expression of these hydrolases on the stringent response to amino acid starvation and basal (p)ppGpp pool was studied. Over-expression of each hydrolase reduced the strength of the stringent response to amino acid starvation, and additionally, perturbed the ratio of ppGpp to pppGpp in strains with reduced SpoT hydrolase activity. In these strains that do not accumulate pppGpp during amino acid starvation, the expression of NudG or MutT supported pppGpp accumulation. This lends support to the idea that a reduction in the SpoT hydrolase activity is sufficient to cause the loss of pppGpp accumulation and therefore the phenomenon is independent of hydrolases that target pppGpp, such as GppA.
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