Diacylglycerol kinase A is essential for polymyxin resistance provided by EptA, MCR-1 and other lipid A phosphoethanolamine transferases.

2021 ◽  
Author(s):  
Alexandria B. Purcell ◽  
Bradley J. Voss ◽  
M. Stephen Trent
Keyword(s):  
Lipid A ◽  
Cell Envelope ◽  
Diacylglycerol Kinase ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Severe Reduction ◽  
Bacterial Fitness ◽  
Phosphoethanolamine Transferases ◽  
Kinase A

Gram-negative bacteria utilize glycerophospholipids (GPLs) as phospho-form donors to modify various surface structures. These modifications play important roles in bacterial fitness in diverse environments influencing cell motility, recognition by the host during infection, and antimicrobial resistance. A well-known example is the modification of the lipid A component of lipopolysaccharide by the phosphoethanolamine (pEtN) transferase EptA that utilizes phosphatidyethanoalmine (PE) as the phospho-form donor. Addition of pEtN to lipid A promotes resistance to cationic antimicrobial peptides (CAMPs), including the polymyxin antibiotics like colistin. A consequence of pEtN modification is the production of diacylglycerol (DAG) that must be recycled back into GPL synthesis via the diacylglycerol kinase A (DgkA). DgkA phosphorylates DAG forming phosphatidic acid, the precursor for GPL synthesis. Here we report that deletion of dgkA in polymyxin-resistant E. coli results in a severe reduction of pEtN modification and loss of antibiotic resistance. We demonstrate that inhibition of EptA is regulated post-transcriptionally and is not due to EptA degradation during DAG accumulation. We also show that the inhibition of lipid A modification by DAG is a conserved feature of different Gram-negative pEtN transferases. Altogether, our data suggests that inhibition of EptA activity during DAG accumulation likely prevents disruption of GPL synthesis helping to maintain cell envelope homeostasis.

scholarly journals Structural Modification of Lipopolysaccharide Conferred by mcr-1 in Gram-Negative ESKAPE Pathogens

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Yi-Yun Liu ◽  
Courtney E. Chandler ◽  
Lisa M. Leung ◽  
Christi L. McElheny ◽  
Roberta T. Mettus ◽  
...  
Keyword(s):  
Structural Changes ◽  
Lipid A ◽  
Colistin Resistance ◽  
Gram Negative ◽  
Content Type ◽  
Clinical Strains ◽  
E Coli ◽  
The Family ◽  
Phosphoethanolamine Transferases ◽  
Eskape Pathogens

ABSTRACT mcr-1 was initially reported as the first plasmid-mediated colistin resistance gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae in China and has subsequently been identified worldwide in various species of the family Enterobacteriaceae. mcr-1 encodes a phosphoethanolamine transferase, and its expression has been shown to generate phosphoethanolamine-modified bis-phosphorylated hexa-acylated lipid A in E. coli. Here, we investigated the effects of mcr-1 on colistin susceptibility and on lipopolysaccharide structures in laboratory and clinical strains of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, which are often treated clinically by colistin. The effects of mcr-1 on colistin resistance were determined using MIC assays of laboratory and clinical strains of E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa. Lipid A structural changes resulting from MCR-1 were analyzed by mass spectrometry. The introduction of mcr-1 led to colistin resistance in E. coli, K. pneumoniae, and A. baumannii but only moderately reduced susceptibility in P. aeruginosa. Phosphoethanolamine modification of lipid A was observed consistently for all four species. These findings highlight the risk of colistin resistance as a consequence of mcr-1 expression among ESKAPE pathogens, especially in K. pneumoniae and A. baumannii. Furthermore, the observation that lipid A structures were modified despite only modest increases in colistin MICs in some instances suggests more sophisticated surveillance methods may need to be developed to track the dissemination of mcr-1 or plasmid-mediated phosphoethanolamine transferases in general.

scholarly journals DpaA detaches Braun’s lipoprotein from peptidoglycan

2021 ◽  
Author(s):  
Matthias Winkle ◽  
Víctor M. Hernández-Rocamora ◽  
Karthik Pullela ◽  
Emily C. A. Goodall ◽  
Alessandra M. Martorana ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Stress Conditions ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Sequence Motifs ◽  
Gram Negative ◽  
E Coli ◽  
Impermeable Barrier ◽  
Unique Cell

ABSTRACTGram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun’s lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB and LdtC. LdtD and LdtE are members of the same family of LD-transpeptidases but they catalyse a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF remains unclear, although it has been shown to become essential in cells with inhibited LPS export to the outer membrane. We now show that LdtF hydrolyses the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product down-regulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated.IMPORTANCEGram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun’s lipoprotein (Lpp) is the most abundant protein in E. coli and about one third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from PG and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

scholarly journals DpaA Detaches Braun’s Lipoprotein from Peptidoglycan

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Matthias Winkle ◽  
Víctor M. Hernández-Rocamora ◽  
Karthik Pullela ◽  
Emily C. A. Goodall ◽  
Alessandra M. Martorana ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Stress Conditions ◽  
Permeability Barrier ◽  
Gram Negative Bacteria ◽  
Sequence Motifs ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Impermeable Barrier

ABSTRACT Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection between the outer membrane and peptidoglycan is needed to maintain the outer membrane as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp (Braun’s lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA, LdtB, and LdtC. LdtD and LdtE are members of the same family of ld-transpeptidases but they catalyze a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The function of the sixth homologue in E. coli, LdtF, remains unclear, although it has been shown to become essential in cells with inhibited lipopolysaccharide export to the outer membrane. We now show that LdtF hydrolyzes the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA). We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the lapB (yciM) gene, whose product downregulates lipopolysaccharide biosynthesis. DpaA-like proteins have characteristic sequence motifs and are present in many Gram-negative bacteria, of which some have no Lpp, raising the possibility that DpaA has other substrates in these species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic than previously appreciated. IMPORTANCE Gram-negative bacteria have a complex cell envelope with two membranes and a periplasm containing the peptidoglycan layer. The outer membrane is firmly connected to the peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun’s lipoprotein (Lpp) is the most abundant protein in E. coli, and about one-third of the Lpp molecules become covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability barrier for a range of toxic molecules and antibiotics. So far, the attachment of Lpp to peptidoglycan has been considered to be irreversible. We have now identified an amidase, DpaA, which is capable of detaching Lpp from peptidoglycan, and we show that the detachment of Lpp is important under certain stress conditions. DpaA-like proteins are present in many Gram-negative bacteria and may have different substrates in these species.

scholarly journals Nonclonal Emergence of Colistin Resistance Associated with Mutations in the BasRS Two-Component System in Escherichia coli Bloodstream Isolates

mSphere ◽  
2020 ◽  
Vol 5 (2) ◽  
Author(s):  
Axel B. Janssen ◽  
Toby L. Bartholomew ◽  
Natalia P. Marciszewska ◽  
Marc J. M. Bonten ◽  
Rob J. L. Willems ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lipid A ◽  
Gram Negative Bacteria ◽  
Colistin Resistance ◽  
Anionic Lipid ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Two Component ◽  
Resistant Strains

ABSTRACT Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen. IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.

scholarly journals Bacterial Metal Resistance: Coping with Copper without Cooperativity?

mBio ◽  
2021 ◽  
Author(s):  
Nicholas P. Greene ◽  
Vassilis Koronakis
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Cell Envelope ◽  
Metal Resistance ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Rnd Transporter ◽  
Entire Cell

In Escherichia coli and other Gram-negative bacteria, tripartite efflux pumps (TEPs) span the entire cell envelope and serve to remove noxious molecules from the cell. CusBCA is a TEP responsible for copper and silver detoxification in E. coli powered by the resistance-nodulation-cell division (RND) transporter, CusA.

scholarly journals Cardiolipin aids in lipopolysaccharide transport to the gram-negative outer membrane

2021 ◽  
Vol 118 (15) ◽  
pp. e2018329118
Author(s):  
Martin V. Douglass ◽  
François Cléon ◽  
M. Stephen Trent
Keyword(s):  
Outer Membrane ◽  
Protein Interactions ◽  
Lipid A ◽  
Cell Envelope ◽  
Transport Systems ◽  
Gram Negative ◽  
Membrane Asymmetry ◽  
Synthetic Lethal ◽  
Bacterial Fitness ◽  
Synthetically Lethal

In Escherichia coli, cardiolipin (CL) is the least abundant of the three major glycerophospholipids in the gram-negative cell envelope. However, E. coli harbors three distinct enzymes that synthesize CL: ClsA, ClsB, and ClsC. This redundancy suggests that CL is essential for bacterial fitness, yet CL-deficient bacteria are viable. Although multiple CL–protein interactions have been identified, the role of CL still remains unclear. To identify genes that impact fitness in the absence of CL, we analyzed high-density transposon (Tn) mutant libraries in combinatorial CL synthase mutant backgrounds. We found LpxM, which is the last enzyme in lipid A biosynthesis, the membrane anchor of lipopolysaccharide (LPS), to be critical for viability in the absence of clsA. Here, we demonstrate that CL produced by ClsA enhances LPS transport. Suppressors of clsA and lpxM essentiality were identified in msbA, a gene that encodes the indispensable LPS ABC transporter. Depletion of ClsA in ∆lpxM mutants increased accumulation of LPS in the inner membrane, demonstrating that the synthetic lethal phenotype arises from improper LPS transport. Additionally, overexpression of ClsA alleviated ΔlpxM defects associated with impaired outer membrane asymmetry. Mutations that lower LPS levels, such as a YejM truncation or alteration in the fatty acid pool, were sufficient in overcoming the synthetically lethal ΔclsA ΔlpxM phenotype. Our results support a model in which CL aids in the transportation of LPS, a unique glycolipid, and adds to the growing repertoire of CL–protein interactions important for bacterial transport systems.

scholarly journals LpxT-Dependent Phosphorylation of Lipid A in Escherichia coli Increases Resistance to Deoxycholate and Enhances Gut Colonization

2021 ◽  
Vol 12 ◽  
Author(s):  
Xudong Tian ◽  
Guillaume Manat ◽  
Elise Gasiorowski ◽  
Rodolphe Auger ◽  
Samia Hicham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Lipid A ◽  
Negative Charge ◽  
Polymyxin B ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Gram Negative ◽  
E Coli ◽  
Gut Colonization

The cell surface of Gram-negative bacteria usually exhibits a net negative charge mostly conferred by lipopolysaccharides (LPS). This property sensitizes bacterial cells to cationic antimicrobial peptides, such as polymyxin B, by favoring their binding to the cell surface. Gram-negative bacteria can modify their surface to counteract these compounds such as the decoration of their LPS by positively charged groups. For example, in Escherichia coli and Salmonella, EptA and ArnT add amine-containing groups to the lipid A moiety. In contrast, LpxT enhances the net negative charge by catalyzing the synthesis of tri-phosphorylated lipid A, whose function is yet unknown. Here, we report that E. coli has the intrinsic ability to resist polymyxin B upon the simultaneous activation of the two component regulatory systems PhoPQ and PmrAB by intricate environmental cues. Among many LPS modifications, only EptA- and ArnT-dependent decorations were required for polymyxin B resistance. Conversely, the acquisition of polymyxin B resistance compromised the innate resistance of E. coli to deoxycholate, a major component of bile. The inhibition of LpxT by PmrR, under PmrAB-inducing conditions, specifically accounted for the acquired susceptibility to deoxycholate. We also report that the kinetics of intestinal colonization by the E. coli lpxT mutant was impaired as compared to wild-type in a mouse model of infection and that lpxT was upregulated at the temperature of the host. Together, these findings highlight an important function of LpxT and suggest that a tight equilibrium between EptA- and LpxT-dependent decorations, which occur at the same position of lipid A, is critical for the life style of E. coli.

scholarly journals Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Hayley E. Young ◽  
Jinshi Zhao ◽  
Jeffrey R. Barker ◽  
Ziqiang Guan ◽  
Raphael H. Valdivia ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Biosynthetic Pathway ◽  
Lipid A ◽  
Genomic Library ◽  
Essential Gene ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Pyrophosphatase Activity

ABSTRACT Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, including Chlamydia trachomatis . Although nearly all of the enzymes in the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN (UDP-DAGn) to form lipid X is carried out by two unrelated enzymes: LpxH in beta- and gammaproteobacteria and LpxI in alphaproteobacteria. The intracellular pathogen C. trachomatis lacks an ortholog for either of these two enzymes, and yet, it synthesizes lipid A and exhibits conservation of genes encoding other lipid A enzymes. Employing a complementation screen against a C. trachomatis genomic library using a conditional-lethal lpxH mutant Escherichia coli strain, we have identified an open reading frame (Ct461, renamed lpxG ) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function in E. coli and catalyzes the conversion of UDP-DAGn to lipid X in vitro . LpxG shows little sequence similarity to either LpxH or LpxI, highlighting LpxG as the founding member of a third class of UDP-DAGn hydrolases. Overexpression of LpxG results in toxic accumulation of lipid X and profoundly reduces the infectivity of C. trachomatis , validating LpxG as the long-sought-after UDP-DAGn pyrophosphatase in this prominent human pathogen. The complementation approach presented here overcomes the lack of suitable genetic tools for C. trachomatis and should be broadly applicable for the functional characterization of other essential C. trachomatis genes . IMPORTANCE Chlamydia trachomatis is a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of many Chlamydia genes remain uncharacterized, including the essential gene encoding the UDP-DAGn pyrophosphatase activity for the biosynthesis of lipid A, the membrane anchor of lipooligosaccharide and the predominant lipid species of the outer leaflet of the bacterial outer membrane. We designed a complementation screen against the C. trachomatis genomic library using a conditional-lethal mutant of E. coli and identified the missing essential gene in the lipid A biosynthetic pathway, which we designated lpxG . We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activity in vitro . Overexpression of LpxG in C. trachomatis leads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating the in vivo function of LpxG and highlighting the importance of regulated lipid A biosynthesis in C. trachomatis .

scholarly journals Binding of surfactant protein A to the lipid A moiety of bacterial lipopolysaccharides

1994 ◽  
Vol 303 (2) ◽  
pp. 407-411 ◽  
Author(s):  
J F Van Iwaarden ◽  
J C Pikaar ◽  
J Storm ◽  
E Brouwer ◽  
J Verhoef ◽  
...  
Keyword(s):  
Lipid A ◽  
Magnetic Beads ◽  
Protein A ◽  
Surfactant Protein ◽  
Biologically Active ◽  
Surfactant Protein A ◽  
Carbohydrate Binding ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli

Surfactant protein A (SP-A) enhances the phagocytosis of opsonized and non-opsonized bacteria by alveolar macrophages, but it is not known with which component of the bacterial surface it associates. We investigated the interaction of SP-A with lipopolysaccharides (LPS), which are important biologically active constituents of the outer membranes of Gram-negative bacteria. Flow cytometry was used to study the binding of fluorescein isothiocyanate-labelled SP-A either to LPS of various chain lengths coupled to magnetic beads or to Gram-negative bacteria. The binding of SP-A to LPS-coated beads was saturable, both time- and concentration-dependent, and required both Ca2+ and Na+. SP-A bound to the lipid A moiety of LPS and to LPS from either the Re-mutant of Salmonella minnesota or the J5-mutant of Escherichia coli. In contrast, it did not bind to O111 LPS of E. coli, suggesting that SP-A binds only to rough LPS. The binding of SP-A to LPS was not affected by mannan and heparin or by deglycosylation of the SP-A, indicating that the carbohydrate-binding domain and the carbohydrate moiety of SP-A are not involved in its interaction with LPS. We also observed saturable and concentration-dependent binding of SP-A to the live J5 mutant of whole E. coli, but not to its O111 mutant. In addition, Re LPS aggregated in the presence of SP-A, Ca2+ and Na+. We conclude that SP-A associates with LPS via the lipid A moiety of rough LPS and may be involved in the anti-bacterial defences of the lung.

scholarly journals A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria

2002 ◽  
Vol 68 (12) ◽  
pp. 6343-6352 ◽  
Author(s):  
Yan Y. Goh ◽  
Bow Ho ◽  
Jeak L. Ding
Keyword(s):  
Lipid A ◽  
Fluorescent Protein ◽  
Computational Design ◽  
Site Directed Mutagenesis ◽  
Dual Function ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Fluorescent Biosensor ◽  
Wide Range

ABSTRACT Site-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computational design was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria. EGFP mutants (EGFPi) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in an Escherichia coli host. The EGFPi proteins were purified and tested for their efficacy as a novel fluorescent biosensor. After efficient removal of lipopolysaccharide from the E. coli lysates, the binding affinities of the EGFPi G10 and G12 to lipid A were established. The KD values of 7.16 × 10−7 M for G10 and 8.15 × 10−8 M for G12 were achieved. With high affinity being maintained over a wide range of pH and ionic strength, the binding of lipid A/lipopolysaccharide to the EGFPi biosensors could be measured as a concentration-dependent fluorescence quenching of the EGFP mutants. The EGFPi specifically tagged gram-negative bacteria like E. coli and Pseudomonas aeruginosa, as well as other gram-negative bacteria in contaminated water sampled from the environment. This dual function of the EGFPi in detecting both free endotoxin and live gram-negative bacteria forms the basis of the development of a novel fluorescent biosensor.

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