Dr John George

The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.

Lipid A is a highly conserved component of lipopolysaccharide (LPS), itself a major component of the outer membrane of Gram-negative bacteria. Lipid A is essential to cells and elicits a strong immune response from humans and other animals. We developed a quantitative model of the nine enzyme-catalyzed steps of Escherichia coli lipid A biosynthesis, drawing parameters from the experimental literature. This model accounts for biosynthesis regulation, which occurs through regulated degradation of the LpxC and WaaA (also called KdtA) enzymes. The LpxC degradation signal appears to arise from the lipid A disaccharide concentration, which we deduced from prior results, model results, and new LpxK overexpression results. The model agrees reasonably well with many experimental findings, including the lipid A production rate, the behaviors of mutants with defective LpxA enzymes, correlations between LpxC half-lives and cell generation times, and the effects of LpxK overexpression on LpxC concentrations. Its predictions also differ from some experimental results, which suggest modifications to the current understanding of the lipid A pathway, such as the possibility that LpxD can replace LpxA and that there may be metabolic channeling between LpxH and LpxB. The model shows that WaaA regulation may serve to regulate the lipid A production rate when the 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) concentration is low and/or to control the number of KDO residues that get attached to lipid A. Computation of flux control coefficients showed that LpxC is the rate-limiting enzyme if pathway regulation is ignored, but that LpxK is the rate-limiting enzyme if pathway regulation is present, as it is in real cells. Control also shifts to other enzymes if the pathway substrate concentrations are not in excess. Based on these results, we suggest that LpxK may be a much better drug target than LpxC, which has been pursued most often.

© 2014 International Union of Biochemistry and Molecular Biology, Inc. Adenosine deaminase (ADA) deficiency, where a deleterious mutation in the ADA gene of patients results in a dysfunctional immune system, is ultimately caused by an absence of ADA. Over the last 25 years the disease has been treated with PEG-ADA, made from purified bovine ADA coupled with polyethylene glycol (PEG). However, it is thought that an enzyme replacement therapy protocol based on recombinant human ADA would probably be a more effective treatment. With this end in mind, a human ADA cDNA was inserted into plant expression vectors used to transform tobacco plant cell suspensions. Transgenic calli expressing constructs containing apoplast-directing signals showed significantly higher levels of recombinant ADA expression than calli transformed with cytosolic constructs. The most significant ADA activities, however, were measured in the media of transgenic cell suspensions prepared from high expressing transformed calli: where incorporation of a signal for arabinogalactan addition to ADA led to a recombinant protein yield of approximately 16 mg L -1 , a 336-fold increase over ADA produced by cell suspensions transformed with a cytosolic construct.

The development of novel antimicrobials provides additional treatment options for infectious diseases, including antimicrobial resistant infections. There are many hurdles to antimicrobial development and identifying an antimicrobial’s mechanism of action is a crucial step in progressing candidate molecules through the drug discovery pipeline. We used the genome wide screening method TraDIS-Xpressto identify genes in two model Gram-negative bacteria that affected sensitivity to three analogues of a novel antimicrobial compound (OPT-2U1). TraDIS-Xpressidentified that all three analogues targeted the lipid IVAbiosynthetic pathway inE. coliandSalmonellaTyphimurium. Specifically, we determined that the antimicrobial target was likely to be LpxD, and validated this by finding a 5 log2-fold increase in the MIC of the OPT-2U1 analogues inE. coliwhenlpxDwas overexpressed. Synergies were identified between OPT-2U1 analogues combined with rifampicin or colistin, to varying strengths, in bothE. coliandS. Typhimurium. LPS composition was a likely reason for differences betweenE. coliand S.Typhimurium, as perturbation of LPS synthesis affected synergy between antibiotics and OPT-2U1 analogues. Finally, genes involved in ATP synthesis and membrane signalling functions were also found to affect the synergy between colistin and OPT-2U1 analogues. TraDIS-Xpresshas proven a powerful tool to rapidly assay all genes (and notably, essential genes) within a bacterium for roles in dictating antimicrobial sensitivity. This study has confirmed the predicted target pathway of OPT-2U1 and identified synergies which could be investigated for development of novel antimicrobial formulations. Data Summary Nucleotide sequence data supporting the analysis in this study has been deposited in ArrayExpress under the accession number E-MTAB-13250. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

Lipopolysaccharide (LPS) is an essential structural component found in Gram-negative bacteria. The molecule is comprised of a highly conserved lipid A and a variable outer core consisting of various sugars. LPS plays important roles in membrane stability in the bacterial cell and is also a potent activator of the human immune system. Despite its obvious importance, little is understood regarding the regulation of the individual enzymes involved or the pathway as a whole. LpxA and LpxC catalyze the first two steps in the LPS pathway. The reaction catalyzed by LpxA possesses a highly unfavourable equilibrium constant with no evidence of coupling to an energetically favourable reaction. In our model the presence of the second enzyme LpxC was sufficient to abate this unfavourable reaction and confirming previous studies suggesting that this reaction is the first committed step in LPS synthesis. It is believed that the protease FtsH regulates LpxC activity via cleavage. It is also suspected that the activity of FtsH is regulated by a metabolite produced by the LPS pathway; however, it is not known which one. In order to investigate these mechanisms, we obtained kinetic parameters from literature and developed estimates for other simulation parameters. Our simulations suggest that under modest increases in LpxC activity, FtsH is able to regulate the rate of product formation. However, under extreme increases in LpxC activities such as over-expression or asymmetrical cell division then FtsH activation may not be sufficient to regulate this first stage of synthesis.

An inherited disorder, adenosine deaminase deficiency is a form of severe combined immunodeficiency, which is ultimately caused by an absence of adenosine deaminase (ADA), a key enzyme of the purine salvage pathway. The absence of ADA-activity in sufferers eventually results in a dysfunctional immune system due to the build-up of toxic metabolites. To date, this has been treated with mixed success, using PEG-ADA, made from purified bovine ADA coupled to polyethylene glycol. It is likely, however, that an enzyme replacement therapy protocol based on recombinant human ADA would be a more effective treatment for this disease. Therefore, as a preliminary step to produce biologically active human ADA in transgenic tobacco plants a human ADA cDNA has been inserted into a plant expression vector under the control of the CaMV 35S promoter and both human and TMV 5′ UTR control regions. Plant vector expression constructs have been used to transform tobacco plants via Agrobacterium-mediated transformation. Genomic DNA, RNA and protein blot analyses have demonstrated the integration of the cDNA construct into the plant nuclear genome and the expression of recombinant ADA mRNA and protein in transgenic tobacco leaves. Western blot analysis has also revealed that human and recombinant ADA have a similar size of approximately 41 kDa. ADA-specific activities of between 0. 001 and 0. 003 units per mg total soluble protein were measured in crude extracts isolated from transformed tobacco plant leaves. © 2012 Springer Science+Business Media Dordrecht.

A computational systems approach has been used to investigate the biogenesis of the outer envelope of Escherichia coli. Specifically, a deterministic model incorporating the lipopolysaccharide, peptidoglycan and phospholipid components. The expanded model now contains 195 reacting species, making up 145 biochemical reactions, making it the largest dynamic model published. The model was used to understand key regulatory elements underpinning pathway regulation and its findings extrapolated to account for phenotypic effects such as growth rates and cell envelope health. The model was capable of producing the known amounts of endpoint metabolites for each of the relevant pathways- Indicating that the parametrisation is reasonable for this network with steady state achieved for most reactions. However, there are numerous nodes which are fitted which can be a source of error. In order to test model predictions a strain library of tuneable CRISPR recombinant E. coli, which can downregulate specific genes (between 5-30%) within this network was generated. During strain validation it was found that genetic perturbation resulted in an associated accumulation at the metabolic level. This strongly suggests that the effect observed in these recombinant strains is likely a result of the perturbation of the specific gene of interest, making it an appropriate method to constrain the metabolic model. Intriguingly, one of the recombinant strains (MsbA- LPS flippase), when repressed caused a build up of DSMP which is the substrate for LpxK. This association has not been published previously and potentially represents a novel regulatory mechanism. However, more work is required in order to constrain and test the model accuracy.

Aflatoxin contamination poses a significant risk to all nuts, including pistachios, during harvest, storage, and processing. Dietary exposure to aflatoxins can lead to severe toxic and carcinogenic effects in humans. To safeguard human and animal health, aflatoxin legislation sets maximum permissible levels for aflatoxins in food products, including pistachios. Consequently, imported pistachios undergo rigorous aflatoxin contamination testing. Traditional methods for measuring aflatoxin levels, such as High-Performance Liquid Chromatography (HPLC), HPLC with Mass Spectrometry, and Enzyme-Linked Immunosorbent Assay (ELISA), although precise, are destructive, costly, and time-consuming. This paper investigates the application of emerging technologies, including Hyperspectral Imaging, Chromatographic Test Strips, Luminescent Metal-Organic Frameworks, spectroscopic methods, machine vision, and advanced artificial intelligence models, to develop a non-intrusive, real-time system for aflatoxin detection in pistachio nuts. Additionally, it outlines a comprehensive strategy to protect public health, mitigate economic losses estimated at \$932 million annually, and sustain the pistachio industry.

Aflatoxin contamination in pistachios, caused by Aspergillus flavus and Aspergillus parasiticus, poses significant risks to food safety and global trade due to its carcinogenic properties. This review examines traditional detection methods such as High-Performance Liquid Chromatography and Enzyme-Linked Immunosorbent Assay. Although these techniques are highly precise, they are costly, destructive, and impractical for smallholder farmers. Emerging nondestructive technologies enable rapid, accurate detection without destroying the sample, particularly when Hyperspectral Imaging (HSI) is combined with machine learning. Regulatory thresholds such as the European Union (EU) 8 µg/kg limit for AFB1 create challenges for producers and exporters, especially since HSI methods often lack the precision required for validated quantitative regression at this level on naturally contaminated pistachio kernels. High implementation costs, limited regulatory guidance, and calibration demands hinder its adoption. Climate change heightens contamination risks, calling for predictive models that integrate HSI with environmental data. To support equitable access, especially for smallholder farmers, reducing costs, standardizing protocols, and enhancing global cooperation are essential. These measures will strengthen food safety and regulatory compliance in pistachio production.