Suppression of fabB Mutation by fabF1 Is Mediated by Transcription Read-through in Shewanella oneidensis

Abstract

As type II fatty acid synthesis is essential for the growth of Escherichia coli, its many components are regarded as potential targets for novel antibacterial drugs. Among them, ␤-ketoacyl-acyl carrier protein (ACP) synthase (KAS) FabB is the exclusive factor for elongation of the cis-3-decenoyl-ACP (cis-3-C 10 -ACP). In our previous study, we presented evidence to suggest that this may not be the case in Shewanella oneidensis, an emerging model gammaproteobacterium renowned for its respiratory versatility. Here, we identified FabF1, another KAS, as a functional replacement for FabB in S. oneidensis. In fabB ؉ or desA ؉ (encoding a desaturase) cells, which are capable of making unsaturated fatty acids (UFA), FabF1 is barely produced. However, UFA auxotroph mutants devoid of both fabB and desA genes can be spontaneously converted to suppressor strains, which no longer require exogenous UFAs for growth. Suppression is caused by a TGTTTT deletion in the region upstream of the fabF1 gene, resulting in enhanced FabF1 production. We further demonstrated that the deletion leads to transcription read-through of the terminator for acpP, an acyl carrier protein gene immediately upstream of fabF1. There are multiple tandem repeats in the region covering the terminator, and the TGTTTT deletion, as well as others, compromises the terminator efficacy. In addition, FabF2 also shows an ability to complement the FabB loss, albeit substantially less effectively than FabF1. IMPORTANCEIt has been firmly established that FabB for UFA synthesis via type II fatty acid synthesis in FabA-containing bacteria such as E. coli is essential. However, S. oneidensis appears to be an exception. In this bacterium, FabF1, when sufficiently expressed, is able to fully complement the FabB loss. Importantly, such a capability can be obtained by spontaneous mutations, which lead to transcription read-through. Therefore, our data, by identifying the functional overlap between FabB and FabFs, provide new insights into the current understanding of KAS and help reveal novel ways to block UFA synthesis for therapeutic purposes. The de novo fatty acid synthesis (FAS) pathway, namely, type II, is the predominant, if not exclusive, route for endogenous production of fatty acids (1). The FAS pathway, the current knowledge of which derives mainly from the model organism Escherichia coli, is highly conserved in bacteria. Central to the pathway are reactions catalyzed by ␤-ketoacyl-acyl carrier protein (ACP) synthases (KAS), including FabH, FabB, and FabF. FabH is responsible for the condensation of an acyl coenzyme A (acylCoA) unit and a malonyl-acyl carrier protein (malonyl-ACP) unit, but cannot work with acetyl-ACP, the substrate of FabB and FabF; as a consequence, FabH could not function as a replacement for FabB or FabF (1). While FabB and FabF are exchangeable in elongation of saturated intermediates, each catalyzes a reaction with the unsaturated branch that the other cannot or at least much less effectively (2) (Fig. 1). The key reaction catalyze...

Loss of OxyR reduces efficacy of oxygen respiration in Shewanella oneidensis

“…3B ). When IPTG was added to 0.05 mM, which confers the promoter ~120 Miller units according to previous calibration 13 42 , expression of cyd slowed growth of ∆ oxyR Δ cyd to extent comparable to that the ∆ oxyR strain ( Fig. 3B ).…”

“…We then manipulated production levels of cytochrome bd by IPTG-controlled promoter P tac within pHGE-Ptac to assess effect of the enzyme in varying amounts on growth of the Δ cyd and ∆ oxyR Δ cyd strains. The expression system is highly reliable, producing protein of interest proportional to IPTG up to 1 mM 13 42 . Despite this, in order to precisely interpret data, levels of cytochrome bd proteins produced in these strains by IPTG induction were estimated by nitrite sensitivity assay.…”

A cryptic long-chain 3-ketoacyl-ACP synthase in the Pseudomonas putida F1 unsaturated fatty acid synthesis pathway

“…Shewanella oneidensis MR has one fabB and two fabF genes where fabF1 has the functions of both 3-ketoacyl ACP synthase I and II (14). Increased expression of fabF1 can restore the synthesis of UFAs when the pathway is blocked by inactivation of fabB (15).…”

“…The P. putida mechanism is different from that of the ∆fabB suppressors of Shewanella. In Shewanella fabF1 is located in the fatty acid synthesis gene cluster and mutations that eliminate an upstream transcriptional terminator allow increased transcription of fabF1 that compensates for loss of fabB (15).…”

Introducing Broadened Antibacterial Activity to Rhodanine Derivatives Targeting Enoyl-Acyl Carrier Protein Reductase

“…5) Among present methods on controlling bacterial pathogens, blocking their biosynthesis of fatty acids has been convinced practicable. 6,7) In this pathway, the enoyl-acyl carrier protein reductase (InhA) is highly conserved and significant. 8) InhA has been validated as a potent target for tuberculosis treatment by the first-line drug isoniazid.…”