Bacterial fatty acid synthesis (FASII) is an attractive target for treatment of Gram‐positive pathogens. The value of FASII inhibitors was recently questioned based on the ability of Streptococcus agalactiae to circumvent inhibition by utilizing exogenous fatty acids (FA) for phospholipid synthesis. This study compares the efficacy of FASII inhibitors against Staphylococcus aureus and Streptococcus pneumoniae grown in media containing FA. Exogenous FA overcame inhibition in S. pneumoniae, but not S. aureus. Neither the substrate specificities of the acyltransferases, nor the divergent modes of transcriptional regulation accounted for this difference. Exogenous FA strongly suppressed malonyl‐CoA production in S. pneumoniae but not S. aureus, indicating biochemical regulation at the acetyl‐CoA carboxylase in S. pneumoniae is the key difference between the two species. The lack of feedback regulation of malonyl‐CoA formation results in the accumulation of short chain acyl‐ACP and a block in exogenous FA uptake in S. aureus treated with FASII inhibitors. The ability of exogenous FA to biochemically inhibit the initiation of FASII determines whether FASII inhibitors will be effective against Gram‐Positive bacteria in the presence of exogenous FA. Supported by NIH GM034496 and ALSAC.

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Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coli cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse structures can be generated by changing the expression profile of these genes, including the formation of large axial filaments that interfere with septation. Fusing GFP to PduC, PduD, or PduV, none of which are shell proteins, allows regiospecific targeting of the reporter group to the empty BMC. Live cell imaging provides unexpected evidence of filament-associated BMC movement within the cell in the presence of PduV.

Extracellular fatty acid incorporation into the phospholipids of Staphylococcus aureus occurs via fatty acid phosphorylation. We show that fatty acid kinase (Fak) is composed of two dissociable protein subunits encoded by separate genes. FakA provides the ATP binding domain and interacts with two distinct FakB proteins to produce acyl-phosphate. The FakBs are fatty acid binding proteins that exchange bound fatty acid/acyl-phosphate with fatty acid/acyl-phosphate presented in detergent micelles or liposomes. The ΔfakA and ΔfakB1 ΔfakB2 strains were unable to incorporate extracellular fatty acids into phospholipid. FakB1 selectively bound saturated fatty acids whereas FakB2 preferred unsaturated fatty acids. Affymetrix array showed a global perturbation in the expression of virulence genes in the ΔfakA strain. The severe deficiency in α-hemolysin protein secretion in ΔfakA and ΔfakB1 ΔfakB2 mutants coupled with quantitative mRNA measurements showed that fatty acid kinase activity was required to support virulence factor transcription. These data reveal the function of two conserved gene families, their essential role in the incorporation of host fatty acids by Gram-positive pathogens, and connects fatty acid kinase to the regulation of virulence factor transcription in S. aureus.T he pathway for the uptake and incorporation of host fatty acids (FA) by bacterial pathogens is important to understanding their physiology and determining if type II fatty acid synthesis (FASII) inhibitors will be useful antibacterial therapeutics. FASII is an energy-intensive process, and the advantage of being able to use host FA for membrane assembly obviously allows ATP to be diverted to the synthesis of other macromolecules. Gram-positive pathogens are capable of incorporating extracellular FA into their phospholipids. In species related to Streptococcus agalactiae, extracellular FA can completely replace endogenously synthesized FA, rendering the FASII inhibitors ineffective growth inhibitors if sufficient FA are present (1, 2). In contrast, FASII inhibitors exemplified by the enoyl-acyl carrier protein (ACP) reductase therapeutic AFN-1252 are effective against Staphylococcus aureus, even when extracellular FA are abundant (2, 3). A major impediment to our understanding of this diversity is that the pathway and enzymes responsible for the incorporation of extracellular FA is not established in the Firmicutes. A recent analysis of a ΔplsX knockout strain of S. aureus ruled out a role for either acyl-CoAs or acyl-ACP as intermediates in host FA metabolism and instead provided evidence for the existence of a new enzyme that phosphorylates FA, called FA kinase (Fak) (4) (Fig. 1A). Host FA are phosphorylated by Fak, and the acyl-PO 4 formed is either used by the PlsY glycerol-3-phosphate acyltransferase or converted to acyl-ACP by PlsX. The acyl-ACP may be either elongated by FASII or used by PlsC. Despite the strong evidence for their existence, the genes/proteins that encode this novel enzyme in FA metabolism were previously unidentif...

The skin represents an important barrier for pathogens and is known to produce fatty acids that are toxic toward Gram-positive bacteria. A screen of fatty acids as growth inhibitors of Staphylococcus aureus revealed structure-specific antibacterial activity. Fatty acids like oleate (18:1⌬9) were nontoxic, whereas palmitoleate (16:1⌬9) was a potent growth inhibitor. Cells treated with 16:1⌬9 exhibited rapid membrane depolarization, the disruption of all major branches of macromolecular synthesis, and the release of solutes and low-molecular-weight proteins into the medium. Other cytotoxic lipids, such as glycerol ethers, sphingosine, and acyl-amines blocked growth by the same mechanisms. Nontoxic 18:1⌬9 was used for phospholipid synthesis, whereas toxic 16:1⌬9 was not and required elongation to 18:1⌬11 prior to incorporation. However, blocking fatty acid metabolism using inhibitors to prevent acyl-acyl carrier protein formation or glycerol-phosphate acyltransferase activity did not increase the toxicity of 18:1⌬9, indicating that inefficient metabolism did not play a determinant role in fatty acid toxicity. Nontoxic 18:1⌬9 was as toxic as 16:1⌬9 in a strain lacking wall teichoic acids and led to growth arrest and enhanced release of intracellular contents. Thus, wall teichoic acids contribute to the structure-specific antimicrobial effects of unsaturated fatty acids. The ability of poorly metabolized 16:1 isomers to penetrate the cell wall defenses is a weakness that has been exploited by the innate immune system to combat S. aureus. Staphylococcus aureus is a common cutaneous pathogen responsible for serious infections that are becoming increasingly dangerous due to the prevalence of antibiotic-resistant organisms (8). Lipids have an important role in innate immunity. Human skin deploys a variety of innate defenses against S. aureus colonization that include antimicrobial peptides and fatty acids (9,18,47,49,51). In mice, 16:1⌬9 is the most potent antibacterial fatty acid, whereas humans synthesize a different isomer, 16:1⌬6 (49). It has been known for decades that these skin fatty acids block the growth of S. aureus (21,27,44). Humans (49) and mice (18) deficient in the production of these 16-carbon monounsaturated fatty acids are more susceptible to S. aureus skin infections. However, it is much less clear how these specific fatty acids produce their antibacterial effect. Ideas include the destabilization of the bacterial membrane due to their surfactant properties (19), uncoupling of ATP synthesis (17), the formation of fatty acid hydroperoxides that elicit oxidative stress (29), increased membrane fluidity due to the incorporation in unsaturated fatty acids in phospholipid (5, 7), and the inhibition of de novo fatty acid synthesis at the FabI step (44, 54). These toxic properties of fatty acids stand in contrast to the observations that S. aureus readily incorporates exogenous fatty acids into membrane phospholipids (1, 3, 4, 41) and that acetyl-coenzyme A (CoA) carboxylase knockout mutants can be isolated a...