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Antibiotic tolerance and antibiotic resistance are the two major obstacles to the efficient and reliable treatment of bacterial infections. Identifying antibiotic adjuvants that sensitize resistant and tolerant bacteria to antibiotic killing may lead to the development of superior treatments with improved outcomes. Vancomycin, a lipid II inhibitor, is a frontline antibiotic for treating methicillin-resistant Staphylococcus aureus and other Gram-positive bacterial infections. However, vancomycin use has led to the increasing prevalence of bacterial strains with reduced susceptibility to vancomycin. Here, we show that unsaturated fatty acids act as potent vancomycin adjuvants to rapidly kill a range of Gram-positive bacteria, including vancomycin-tolerant and resistant populations. The synergistic bactericidal activity relies on the accumulation of membrane-bound cell wall intermediates that generate large fluid patches in the membrane leading to protein delocalization, aberrant septal formation, and loss of membrane integrity. Our findings provide a natural therapeutic option that enhances vancomycin activity against difficult-to-treat pathogens, and the underlying mechanism may be further exploited to develop antimicrobials that target recalcitrant infection.

Background Gram-negative bacterial bloodstream infections (GNB-BSI) are common and frequently lethal. Many patients experience multiple episodes of GNB-BSI for unclear reasons. This study examines the genetic differences between initial and subsequent isolates of recurrent GNB-BSI. Methods We used a prospective cohort of patients with GNB-BSI at Duke Hospital to identify patients with >1 episode of GNB-BSI due to the same bacterial species. We performed pulsed field gel electrophoresis (PFGE) on paired isolates to determine if the events were Reinfection (paired isolates different) or Relapse (paired isolates genetically identical). We then used whole genome sequencing (WGS) to verify the PFGE findings and explore the genetic similarity between initial and relapsed GNB-BSI isolates. Results Among 1,423 unique patients with GNB-BSI, 60 (4%) experienced recurrent GNB-BSI with the same bacterial species. We performed genotyping (PFGE, followed by WGS) on the paired bacterial isolates from the index and recurrent bloodstream infections for the four most common species in our study population (Escherichia coli, Klebsiella species, Pseudomonas aeruginosa, and Serratia marcescens) (n=48 pairs). We determined that 63% (30/48) of recurrent GNB-BSI episodes were due to relapse and 37% (18/48) were due to reinfection. PFGE correctly identified relapse versus reinfection in 98% (47/48) of cases. The WGS data illustrated the microevolution of relapsed GNB-BSI isolates within patients. When comparing initial to relapsed isolates, we calculated a median of 5 single-nucleotide polymorphisms (SNPs) in relapsed E. coli isolates and 2 SNPs in Klebsiella spp. isolates. Of these SNPs, 37% (E. coli) and 12% (Klebsiella) were non-synonymous changes in coding regions, most commonly genes associated with antimicrobial resistance (AMR), energy metabolism and stress response. Alterations in the AMR profile was common, with 20/30 relapsed isolates demonstrating either loss or acquisition of AMR. Figure 1:Time from initial to recurrent episode of gram-negative bloodstream infection. Relapse (i.e., same bacterial strain noted by green circle) versus reinfection (i.e., different bacterial strain noted by red circle) was determined by pulse field gel electrophoresis followed by WGS. Whole genome sequencing was performed on the initial and recurrent isolate from patients with relapsed GNB-BSI. We identified single nucleotide polymorphisms (SNPs) present in the recurrent isolate, which were not present in the initial isolate. We identified the function of genes containing SNPs in E. coli (n=28 SNPs) and Klebsiella spp. (n=13 SNPs). Synonymous mutations, insertions, deletions, and non-synonymous mutations in hypothetical proteins were excluded. Conclusion Most recurrent GNB-BSI with same species are due to relapse. PFGE has comparable accuracy to WGS to determine reinfection versus relapse. Multiple mutations in genes involved in stress response, AMR and metabolism were identified in relapsed isolates. Disclosures Vance G. Fowler, Jr, MD, MHS, Affinergy: Grant/Research Support|Affinergy: Honoraria|Affinium: Honoraria|Amphliphi Biosciences: Honoraria|ArcBio: Stocks/Bonds|Basilea: Grant/Research Support|Basilea: Honoraria|Bayer: Honoraria|C3J: Honoraria|Cerexa/Forest/Actavis/Allergan: Grant/Research Support|Contrafect: Grant/Research Support|Contrafect: Honoraria|Cubist/Merck: Grant/Research Support|Debiopharm: Grant/Research Support|Deep Blue: Grant/Research Support|Destiny: Honoraria|Genentech: Grant/Research Support|Genentech: Honoraria|Integrated Biotherapeutics: Honoraria|Janssen: Grant/Research Support|Janssen: Honoraria|Karius: Grant/Research Support|Medicines Co.: Honoraria|MedImmune: Grant/Research Support|MedImmune: Honoraria|NIH: Grant/Research Support|Novartis: Grant/Research Support|Novartis: Honoraria|Pfizer: Grant/Research Support|Regeneron: Grant/Research Support|Regeneron: Honoraria|Sepsis diagnostics: Sepsis diagnostics patent pending|UpToDate: Royalties|Valanbio: Stocks/Bonds.

Background The causes and clinical characteristics of recurrent gram-negative bacterial bloodstream infections (GNB-BSI) are poorly understood. Methods We used a cohort of patients with GNB-BSI to identify clinical characteristics, microbiology, and risk factors associated with recurrent GNB-BSI. Bacterial genotyping (pulsed-field gel electrophoresis [PFGE] and whole-genome sequencing [WGS]) was used to determine whether episodes were due to relapse or reinfection. Multivariable logistic regression was used to identify risk factors for recurrence. Results Of the 1423 patients with GNB-BSI in this study, 60 (4%) had recurrent GNB-BSI. Non-White race (odds ratio [OR], 2.35; 95% confidence interval [CI], 1.38–4.01; P = .002), admission to a surgical service (OR, 2.18; 95% CI, 1.26–3.75; P = .005), and indwelling cardiac device (OR, 2.73; 95% CI, 1.21–5.58; P = .009) were associated with increased risk for recurrent GNB-BSI. Among the 48 patients with recurrent GNB-BSI whose paired bloodstream isolates underwent genotyping, 63% were due to relapse (30 of 48) and 38% were due to reinfection (18 of 48) based on WGS. Compared with WGS, PFGE correctly differentiated relapse and reinfection in 98% (47 of 48) of cases. Median time to relapse and reinfection was similar (113 days; interquartile range [IQR], 35–222 vs 174 days; IQR, 69–599; P = .13). Presence of a cardiac device was associated with relapse (relapse: 7 of 27, 26%; nonrelapse: 65 of 988, 7%; P = .002). Conclusions In this study, recurrent GNB-BSI was most commonly due to relapse. PFGE accurately differentiated relapse from reinfection when compared with WGS. Cardiac device was a risk factor for relapse.

Antibiotic tolerance and antibiotic resistance are the two major obstacles to the efficient and reliable treatment of bacterial infections. Identifying antibiotic adjuvants that sensitize resistant and tolerant bacteria to antibiotic killing may lead to the development of superior treatments with improved outcomes. Vancomycin, a lipid II inhibitor, is of major clinical importance for the treatment of Gram-positive bacterial infections. Here we show that unsaturated fatty acids (UFAs) and vancomycin act synergistically to rapidly kill S. aureus, including vancomycin tolerant and resistant populations. Our results suggest that antibiotic-mediated accumulation of lipid II at the septum facilitates membrane invasion by antimicrobial UFAs. UFA-vancomycin dual treatment generates large fluid patches of flexible lipids in the membrane leading to protein delocalization, aberrant septal formation, and loss of membrane integrity. This mechanism of synergy may be exploited for the development of new antibiotic therapies that target lipid II to combat both antibiotic tolerance and resistance.

ImportanceObtaining follow-up blood cultures (FUBCs) in patients with Staphylococcus aureus bloodstream infection (BSI) is standard practice, although its utility in patients with gram-negative bacterial BSI (GN-BSI) is unclear.ObjectiveTo examine whether obtaining FUBCs is associated with decreased mortality (key question [KQ] 1) and whether positive vs negative FUBCs are associated with increased mortality (KQ2).Data SourcesMEDLINE, EMBASE, Cochrane Central Register of Controlled Trials, Web of Science, and gray literature were searched from inception to March 11, 2022.Study SelectionTwo investigators used predefined eligibility criteria to independently screen titles, abstracts, and relevant full texts. Randomized clinical trials or observational studies that matched or statistically adjusted for differences in, at minimum, level of acute illness between patients in the intervention (eg, FUBCs obtained) and control (eg, FUBCs not obtained) groups were included in primary analyses. Articles published in languages other than English were excluded.Data Extraction and SynthesisData abstraction and quality assessments were performed by one investigator and verified by a second investigator. Risk of bias was assessed with the Newcastle-Ottawa Scale. Effect sizes were pooled using random-effects models. The study followed the Meta-analysis of Observational Studies in Epidemiology (MOOSE) reporting guideline.Main Outcomes and MeasuresMortality before hospital discharge or up to 30 days from the index blood culture.ResultsFrom 3495 studies, 15 were included (all nonrandomized). In the 5 studies (n = 4378 patients) that met criteria for the KQ1 primary analysis, obtaining FUBCs was associated with decreased mortality (hazard ratio, 0.56; 95% CI, 0.45-0.71). For KQ2, 2 studies met criteria for the primary analysis (ie, matched or statistically adjusted for differences in patients with positive vs negative FUBCs), so an exploratory meta-analysis of all 9 studies that investigated KQ2 (n = 3243 patients) was performed. Positive FUBCs were associated with increased mortality relative to negative blood cultures (odds ratio, 2.27; 95% CI, 1.54-3.34). Limitations of the literature included a lack of randomized studies and few patient subgroup analyses.Conclusions and RelevanceIn this systematic review and meta-analysis, obtaining FUBCs in patients with GN-BSI was associated with decreased mortality. The benefit of FUBCs may stem from identification of patients with positive FUBCs, which was a poor prognostic marker.

Coagulase-negative staphylococci (CoNS) are a common etiology of serious and recurrent infections in immunocompromised patients. Although most isolates appear susceptible to vancomycin, a single strain might have a subpopulation of resistant bacteria. This phenomenon is termed heteroresistance and may adversely affect the response to treatment. A retrospective cohort study was performed of pediatric patients with leukemia treated at St. Jude Children’s Research Hospital who developed CoNS central line-associated bloodstream infection (CLABSI). Available isolates were sequenced and tested for vancomycin heteroresistance by population analysis profiling. Risk factors for heteroresistance and the association of heteroresistance with treatment failure (death or relapse of infection) or poor clinical response to vancomycin therapy (treatment failure or persistent bacteremia after vancomycin initiation) were evaluated. For 65 participants with CoNS CLABSI, 62 initial isolates were evaluable, of which 24 (39%) were vancomycin heteroresistant. All heteroresistant isolates were of Staphylococcus epidermidis and comprised multiple sequence types. Participants with heteroresistant bacteria had more exposure to vancomycin prophylaxis (P = 0.026) during the 60 days prior to infection. Of the 40 participants evaluable for clinical outcomes, heteroresistance increased the risk of treatment failure (P = 0.012) and poor clinical response (P = 0.001). This effect persisted after controlling for identified confounders. These data indicate that vancomycin heteroresistance is common in CoNS isolates from CLABSIs in pediatric patients with leukemia and is associated with poor clinical outcomes. Validation of these findings in an independent cohort and evaluation of alternative antibiotic therapy in patients with heteroresistant infections have the potential to improve care for serious CoNS infections.

Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were isolated. All mutants selected against one antibiotic were cross-resistant to the other two antibiotics. Mutations were not detected in fabF , but the resistant strains harbored missense mutations in fabH . The altered amino acids clustered in and around the FabH active-site tunnel. The mutant FabH proteins were catalytically compromised based on the low activities of the purified enzymes, a fatty acid-dependent growth phenotype, and elevated expression of the fabHF operon in the mutant strains. Independent manipulation of fabF and fabH expression levels showed that the FabH/FabF activity ratio was a major determinant of antibiotic sensitivity. Missense mutations that reduce FabH activity are sufficient to confer resistance to multiple antibiotics that bind to the FabF acyl-enzyme intermediate in S. aureus .

PlsX is an acyl-acyl carrier protein (ACP):phosphate transacylase that interconverts the two acyl donors in Gram-positive bacterial phospholipid synthesis. The deletion of plsX in Staphylococcus aureus results in a requirement for both exogenous fatty acids and de novo type II fatty acid biosynthesis. Deletion of plsX (SP0037) in Streptococcus pneumoniae did not result in an auxotrophic phenotype. The DeltaplsX S. pneumoniae strain was refractory to myristic acid-dependent growth arrest, and unlike the wild-type strain, was susceptible to fatty acid synthesis inhibitors in the presence of exogenous oleate. The DeltaplsX strain contained longer chain saturated fatty acids imparting a distinctly altered phospholipid molecular species profile. An elevated pool of 18- and 20-carbon saturated fatty acids was detected in the DeltaplsX strain. A S. pneumoniae thioesterase (TesS, SP1408) hydrolyzed acyl-ACP in vitro, and the DeltatesS DeltaplsX double knockout strain was a fatty acid auxotroph. Thus, the TesS thioesterase hydrolyzed the accumulating acyl-ACP in the DeltaplsX strain to liberate fatty acids that were activated by fatty acid kinase to bypass a requirement for extracellular fatty acid. This work identifies tesS as the gene responsible for the difference in exogenous fatty acid growth requirement of the DeltaplsX strains of S. aureus and S. pneumoniae.

Acyl-CoA and acyl-acyl carrier protein (ACP) synthetases activate exogenous fatty acids for incorporation into phospholipids in Gram-negative bacteria. However, Gram-positive bacteria utilize an acyltransferase pathway for the biogenesis of phosphatidic acid that begins with the acylation of sn-glycerol-3-phosphate by PlsY using an acyl-phosphate (acyl-PO4 ) intermediate. PlsX generates acyl-PO4 from the acyl-ACP end-products of fatty acid synthesis. The plsX gene of Staphylococcus aureus was inactivated and the resulting strain was both a fatty acid auxotroph and required de novo fatty acid synthesis for growth. Exogenous fatty acids were only incorporated into the 1-position and endogenous acyl groups were channeled into the 2-position of the phospholipids in strain PDJ39 (ΔplsX). Extracellular fatty acids were not elongated. Removal of the exogenous fatty acid supplement led to the rapid accumulation of intracellular acyl-ACP and the abrupt cessation of fatty acid synthesis. Extracts from the ΔplsX strain exhibited an ATP-dependent fatty acid kinase activity, and the acyl-PO4 was converted to acyl-ACP when purified PlsX is added. These data reveal the existence of a novel fatty acid kinase pathway for the incorporation of exogenous fatty acids into S. aureus phospholipids.

Inactivation of acetyl-coenzyme A (acetyl-CoA) carboxylase confers resistance to fatty acid synthesis inhibitors in Staphylococcus aureus on media supplemented with fatty acids. The addition of anteiso -fatty acids (1 mM) plus lipoic acid supports normal growth of Δ accD strains, but supplementation with mammalian fatty acids was less efficient. Mice infected with strain RN6930 developed bacteremia, but bacteria were not detected in mice infected with its Δ accD derivative. S. aureus bacteria lacking acetyl-CoA carboxylase can be propagated in vitro but were unable to proliferate in mice, suggesting that the acquisition of inactivating mutations in this enzyme is not a mechanism for the evasion of fatty acid synthesis inhibitors.

Membrane lipid homeostasis is a vital facet of bacterial cell physiology. For decades, research in bacterial lipid synthesis was largely confined to the Escherichia coli model system. This basic research provided a blueprint for the biochemistry of lipid metabolism that has largely defined the individual steps in bacterial fatty acid and phospholipids synthesis. The advent of genomic sequencing has revealed a surprising amount of diversity in the genes, enzymes and genetic organization of the components responsible for bacterial lipid synthesis. Although the chemical steps in fatty acid synthesis are largely conserved in bacteria, there are surprising differences in the structure and cofactor requirements for the enzymes that perform these reactions in Gram-positive and Gram-negative bacteria. This review summarizes how the explosion of new information on the diversity of biochemical and genetic regulatory mechanisms has impacted our understanding of bacterial lipid homeostasis. The potential and problems of developing therapeutics that block pathogen phospholipid synthesis are explored and evaluated. The study of bacterial lipid metabolism continues to be a rich source for new biochemistry that underlies the variety and adaptability of bacterial life styles.

This study examines the alteration in Staphylococcus aureus gene expression following treatment with the type 2 fatty acid synthesis inhibitor AFN-1252. An Affymetrix array study showed that AFN-1252 rapidly increased the expression of fatty acid synthetic genes and repressed the expression of virulence genes controlled by the SaeRS 2-component regulator in exponentially growing cells. AFN-1252 did not alter virulence mRNA levels in a saeR deletion strain or in strain Newman expressing a constitutively active SaeS kinase. AFN-1252 caused a more pronounced increase in fabH mRNA levels in cells entering stationary phase, whereas the depression of virulence factor transcription was attenuated. The effect of AFN-1252 on gene expression in vivo was determined using a mouse subcutaneous granuloma infection model. AFN-1252 was therapeutically effective, and the exposure (area under the concentration-time curve from 0 to 48 h [AUC 0–48 ]) of AFN-1252 in the pouch fluid was comparable to the plasma levels in orally dosed animals. The inhibition of fatty acid biosynthesis by AFN-1252 in the infected pouches was signified by the substantial and sustained increase in fabH mRNA levels in pouch-associated bacteria, whereas depression of virulence factor mRNA levels in the AFN-1252-treated pouch bacteria was not as evident as it was in exponentially growing cells in vitro . The trends in fabH and virulence factor gene expression in the animal were similar to those in slower-growing bacteria in vitro . These data indicate that the effects of AFN-1252 on virulence factor gene expression depend on the physiological state of the bacteria.

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 (18:1Δ9) were nontoxic, whereas palmitoleate (16:1Δ9) was a potent growth inhibitor. Cells treated with 16:1Δ9 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 18:1Δ9 was used for phospholipid synthesis, whereas toxic 16:1Δ9 was not and required elongation to 18:1Δ11 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 18:1Δ9, indicating that inefficient metabolism did not play a determinant role in fatty acid toxicity. Nontoxic 18:1Δ9 was as toxic as 16:1Δ9 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 .

The emergence of resistance against most current drugs emphasizes the need to develop new approaches to control bacterial pathogens, particularly Staphylococcus aureus. Bacterial fatty acid synthesis is one such target that is being actively pursued by several research groups to develop anti-Staphylococcal agents. Recently, the wisdom of this approach has been challenged based on the ability of a Gram-positive bacterium to incorporate extracellular fatty acids and thus circumvent the inhibition of de novo fatty acid synthesis. The generality of this conclusion has been challenged, and there is enough diversity in the enzymes and regulation of fatty acid synthesis in bacteria to conclude that there is not a single organism that can be considered typical and representative of bacteria as a whole. We are left without a clear resolution to this ongoing debate and await new basic research to define the pathways for fatty acid uptake and that determine the biochemical and genetic mechanisms for the regulation of fatty acid synthesis in Gram-positive bacteria. These crucial experiments will determine whether diversity in the control of this important pathway accounts for the apparently different responses of Gram-positive bacteria to the inhibition of de novo fatty acid synthesis in presence of extracellular fatty acid supplements.

The rationale for the pursuit of bacterial type 2 fatty acid synthesis (FASII) as a target for antibacterial drug discovery in Gram-positive organisms is being debated vigorously based on their ability to incorporate extracellular fatty acids. The regulation of FASII by extracellular fatty acids was examined in Staphylococcus aureus and Streptococcus pneumoniae , representing two important groups of pathogens. Both bacteria use the same enzymatic tool kit for the conversion of extracellular fatty acids to acyl-acyl carrier protein, elongation, and incorporation into phospholipids. Exogenous fatty acids completely replace the endogenous fatty acids in S. pneumoniae but support only 50% of phospholipid synthesis in S. aureus . Fatty acids overcame FASII inhibition in S. pneumoniae but not in S. aureus . Extracellular fatty acids strongly suppress malonyl-CoA levels in S. pneumoniae but not in S. aureus , showing a feedback regulatory system in S. pneumoniae that is absent in S. aureus . Fatty acids overcame either a biochemical or a genetic block at acetyl-CoA carboxylase (ACC) in S. aureus , confirming that regulation at the ACC step is the key difference between these two species. Bacteria that possess a stringent biochemical feedback inhibition of ACC and malonyl-CoA formation triggered by environmental fatty acids are able to circumvent FASII inhibition. However, if exogenous fatty acids do not suppress malonyl-CoA formation, FASII inhibitors remain effective in the presence of fatty acid supplements.

Background Heteroresistance to vancomycin in Staphylococcus aureus may be associated with poor response to therapy. Although CoNS are the most important CLABSI pathogens in children with leukemia, and treatment failure is common, little is known about the frequency or clinical significance of heteroresistance. This is a retrospective study to evaluate frequency, risk factors and clinical impact of heteroresistance in CoNS CLABSI in immunocompromised children. Methods The study was approved by the Institutional Review Board. All patients undergoing treatment for leukemia at St. Jude Children’s Research Hospital with CoNS isolated from blood between 2010 and 2016 were eligible. The first available isolate from each blood culture episode was obtained from the clinical laboratory and tested for vancomycin heteroresistance by population analysis profiling in comparison to the hVISA strain Mu3. Clinical data were collected from the medical record for up to 9 months after the episode. Episodes with ≥2 positive cultures or a single positive culture from a single lumen CVC were classified as CLABSI. Outcomes of interest included treatment failure (death or relapse of infection) or poor response to vancomycin therapy (persistence of bacteremia ≥1 day after initiation of vancomycin or treatment failure). Logistic regression was used to test associations between heteroresistance and exposures, and cumulative incidence analyses were used to test the effect on outcomes. Results A total of 74 CoNS isolates were obtained from 65 participants, 39 with ALL and 26 with AML; 25/74 (33.8%) of isolates showed showed heteroresistance. The strongest identified risk factor for infection with a heteroresistant organism was number of days of vancomycin in the preceding 60 days (OR = 1.05/day; P = 0.035). In the 40 CLABSI episodes, heteroresistant isolates had a higher cumulative incidence of poor response and of treatment failure (P = 0.006 and P = 0.003, respectively). Conclusion Vancomycin heteroresistance is common in CoNS causing CLABSI in children undergoing treatment for leukemia, and is associated with an increased risk of Treatment Failure. Further research should aim to validate this finding in an independent cohort and identify strategies to improve the diagnosis and treatment of these infections. Disclosures R. Hayden, Roche Molecular: Scientific Advisor, Consulting fee. Abbott Molecular: Scientific Advisor, Consulting fee. Quidel: Scientific Advisor, Consulting fee. J. Wolf, Karius Inc.: Investigator, Research support.

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.

The elongation condensing enzymes in the bacterial fatty acid biosynthesis pathway represent desirable targets for the design of novel, broad-spectrum antimicrobial agents. A series of substituted benzoxazolinones was identified in this study as a novel class of elongation condensing enzyme (FabB and FabF) inhibitors using a two-step virtual screening approach. Structure activity relationships were developed around the benzoxazolinone scaffold showing that N-substituted benzoxazolinones were most active. The benzoxazolinone scaffold has high chemical tractability making this chemotype suitable for further development of bacterial fatty acid synthesis inhibitors.

Background The balanced synthesis of membrane phospholipids, fatty acids and cell wall constituents is a vital facet of bacterial physiology, but there is little known about the biochemical control points that coordinate these activities in Gram-positive bacteria. In Escherichia coli, the glycerol-phosphate acyltransferase (PlsB) plays a key role in coordinating fatty acid and phospholipid synthesis, but pathogens like Staphylococcus aureus have a different acyltransferase (PlsY), and the headgroup of the major membrane phospholipid, phosphatidylglycerol (PtdGro), is used as a precursor for lipoteichoic acid synthesis. Results The PlsY acyltransferase in S. aureus was switched off by depriving strain PDJ28 (ΔgpsA) of the required glycerol supplement. Removal of glycerol from the growth medium led to the rapid cessation of phospholipid synthesis. However, the continued utilization of the headgroup caused a reduction in PtdGro coupled with the accumulation of CDP-diacylglycerol and phosphatidic acid. PtdGro was further decreased by its stimulated conversion to cardiolipin. Although acyl-acyl carrier protein (ACP) and malonyl-CoA accumulated, fatty acid synthesis continued at a reduced level leading to the intracellular accumulation of unusually long-chain free fatty acids. Conclusions The cessation of new phospholipid synthesis led to an imbalance in membrane compositional homeostasis. PtdGro biosynthesis was not coupled to headgroup turnover leading to the accumulation of pathway intermediates. The synthesis of cardiolipin significantly increased revealing a stress response to liberate glycerol-phosphate for PtdGro synthesis. Acyl-ACP accumulation correlated with a decrease in fatty acid synthesis; however, the coupling was not tight leading to the accumulation of intracellular fatty acids.

PduS is a corrin reductase and is required for the reactivation of the cobalamin-dependent diol dehydratase. It is one component encoded within the large propanediol utilisation (pdu) operon, which is responsible for the catabolism of 1,2-propanediol within a self-assembled proteinaceous bacterial microcompartment. The enzyme is responsible for the reactivation of the cobalamin coenzyme required by the diol dehydratase. The gene for the cobalamin reductase from Citrobacter freundii (pduS) has been cloned to allow the protein to be overproduced recombinantly in E. coli with an N-terminal His-tag. Purified recombinant PduS is shown to be a flavoprotein with a non-covalently bound FMN that also contains two coupled [4Fe-4S] centres. It is an NADH-dependent flavin reductase that is able to mediate the one-electron reductions of cob(III)alamin to cob(II)alamin and cob(II)alamin to cob(I)alamin. The [4Fe-4S] centres are labile to oxygen and their presence affects the midpoint redox potential of flavin. Evidence is presented that PduS is able to bind cobalamin, which is inconsistent with the view that PduS is merely a flavin reductase. PduS is also shown to interact with one of the shell proteins of the metabolosome, PduT, which is also thought to contain an [Fe-S] cluster. PduS is shown to act as a corrin reductase and its interaction with a shell protein could allow for electron passage out of the bacterial microcompartment.

It has been suggested that ethanol metabolism in the strict anaerobe Clostridium kluyveri occurs within a metabolosome, a subcellular proteinaceous bacterial microcompartment. Two bacterial microcompartment shell proteins [EtuA (ethanol utilization shell protein A) and EtuB] are found encoded on the genome clustered with the genes for ethanol utilization. The function of the bacterial microcompartment is to facilitate fermentation by sequestering the enzymes, substrates and intermediates. Recent structural studies of bacterial microcompartment proteins have revealed both hexamers and pentamers that assemble to generate the pseudo-icosahedral bacterial microcompartment shell. Some of these shell proteins have pores on their symmetry axes. Here we report the structure of the trimeric bacterial microcompartment protein EtuB, which has a tandem structural repeat within the subunit and pseudo-hexagonal symmetry. The pores in the EtuB trimer are within the subunits rather than between symmetry related subunits. We suggest that the evolutionary advantage of this is that it releases the pore from the rotational symmetry constraint allowing more precise control of the fluxes of asymmetric molecules, such as ethanol, across the pore. We also model EtuA and demonstrate that the two proteins have the potential to interact to generate the casing for a metabolosome.

Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required.