Loss of RND-type multidrug efflux pumps triggers iron starvation and lipid A modifications in Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Justyna W. Adamiak ◽  
Varsha Jhawar ◽  
Vincent Bonifay ◽  
Courtney E. Chandler ◽  
Inga V. Leus ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Lipid A ◽  
Cell Envelope ◽  
Iron Acquisition ◽  
Bacterial Species ◽  
Transport Systems ◽  
Permeability Barrier ◽  
Iron Starvation ◽  
Analytical Approaches

Transporters belonging to the Resistance-Nodulation-Division (RND) superfamily of proteins are invariably present in the genomes of Gram-negative bacteria and are largely responsible for the intrinsic antibiotic resistance of these organisms. The number of genes encoding RND transporters per genome vary from one to sixteen and correlates with environmental versatilities of bacterial species. Pseudomonas aeruginosa PAO1 strain, a ubiquitous nosocomial pathogen, possesses twelve RND pumps, which are implicated in development of clinical multidrug resistance and known to contribute to virulence, quorum sensing and many other physiological functions. In this study, we analyzed how P. aeruginosa physiology adapts to the lack of RND-mediated efflux activities. A combination of transcriptomics, metabolomics, genetic and analytical approaches showed that the P. aeruginosa PΔ6 strain lacking six best characterized RND pumps activates a specific adaptation response that involves significant changes in abundance and activities of several transport systems, quorum sensing, iron acquisition and lipid A modifications. Our results demonstrate that these cells accumulate large quantities of pseudomonas quorum signal (PQS), which triggers iron starvation and activation of siderophore biosynthesis and acquisition pathways. The accumulation of iron in turn activates lipid A modification and membrane protection pathways. A transcriptionally regulated RND pump MuxABC-OpmB contributes to these transformations by controlling concentrations of coumarins. Our results suggest that these changes reduce the permeability barrier of the outer membrane and are needed to protect the cell envelope of efflux-deficient P. aeruginosa .

scholarly journals Environmental modification via a quorum sensing molecule influences the social landscape of siderophore production

2017 ◽  
Vol 284 (1852) ◽  
pp. 20170200 ◽  
Author(s):  
Roman Popat ◽  
Freya Harrison ◽  
Ana C. da Silva ◽  
Scott A. S. Easton ◽  
Luke McNally ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Public Goods ◽  
Bacterial Species ◽  
Relative Fitness ◽  
Iron Starvation ◽  
Signal Molecule ◽  
Signalling Molecules ◽  
Signalling Molecule ◽  
The Impact ◽  
Social Trait

Bacteria produce a wide variety of exoproducts that favourably modify their environment and increase their fitness. These are often termed ‘public goods’ because they are costly for individuals to produce and can be exploited by non-producers (cheats). The outcome of conflict over public goods is dependent upon the prevailing environment and the phenotype of the individuals in competition. Many bacterial species use quorum sensing (QS) signalling molecules to regulate the production of public goods. QS, therefore, determines the cooperative phenotype of individuals, and influences conflict over public goods. In addition to their regulatory functions, many QS molecules have additional properties that directly modify the prevailing environment. This leads to the possibility that QS molecules could influence conflict over public goods indirectly through non-signalling effects, and the impact of this on social competition has not previously been explored. The Pseudomonas aeruginosa QS signal molecule PQS is a powerful chelator of iron which can cause an iron starvation response. Here, we show that PQS stimulates a concentration-dependent increase in the cooperative production of iron scavenging siderophores, resulting in an increase in the relative fitness of non-producing siderophore cheats. This is likely due to an increased cost of siderophore output by producing cells and a concurrent increase in the shared benefits, which accrue to both producers and cheats. Although PQS can be a beneficial signalling molecule for P. aeruginosa , our data suggest that it can also render a siderophore-producing population vulnerable to competition from cheating strains. More generally, our results indicate that the production of one social trait can indirectly affect the costs and benefits of another social trait.

scholarly journals Exogenous and Endogenous Phosphoethanolamine Transferases Differently Affect Colistin Resistance and Fitness in Pseudomonas aeruginosa

2021 ◽  
Vol 12 ◽  
Author(s):  
Matteo Cervoni ◽  
Alessandra Lo Sciuto ◽  
Chiara Bianchini ◽  
Carmine Mancone ◽  
Francesco Imperi
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Cell Envelope ◽  
Resistance Mechanism ◽  
Multidrug Resistant ◽  
Transferase Activity ◽  
Colistin Resistance ◽  
Phosphoethanolamine Transferases ◽  
Positively Charged ◽  
Inducible Gene

Colistin represents a last-line treatment option for infections caused by multidrug resistant Gram-negative pathogens, including Pseudomonas aeruginosa. Colistin resistance generally involves the modification of the lipid A moiety of lipopolysaccharide (LPS) with positively charged molecules, namely phosphoethanolamine (PEtN) or 4-amino-4-deoxy-L-arabinose (Ara4N), that reduce colistin affinity for its target. Several lines of evidence highlighted lipid A aminoarabinosylation as the primary colistin resistance mechanism in P. aeruginosa, while the contribution of phosphoethanolamination remains elusive. PEtN modification can be due to either endogenous (chromosomally encoded) PEtN transferase(s) (e.g., EptA in P. aeruginosa) or plasmid borne MCR enzymes, commonly found in enterobacteria. By individually cloning eptA and mcr-1 into a plasmid for inducible gene expression, we demonstrated that MCR-1 and EptA have comparable PEtN transferase activity in P. aeruginosa and confer colistin resistance levels similar to those provided by lipid A aminoarabinosylation. Notably, EptA, but not MCR-1, negatively affects P. aeruginosa growth and, to a lesser extent, cell envelope integrity when expressed at high levels. Mutagenesis experiments revealed that PEtN transferase activity does not account for the noxious effects of EptA overexpression, that instead requires a C-terminal tail unique to P. aeruginosa EptA, whose function remains unknown. Overall, this study shows that both endogenous and exogenous PEtN transferases can promote colistin resistance in P. aeruginosa, and that PEtN and MCR-1 mediated resistance has no impact on growth and cell envelope homeostasis, suggesting that there may be no fitness barriers to the spread of mcr-1 in P. aeruginosa.

scholarly journals Environmental modification via a quorum sensing molecule influences the social landscape of siderophore production

2016 ◽  
Author(s):  
Roman Popat ◽  
Freya Harrison ◽  
Ana C. da Silva ◽  
Scott A. S. Easton ◽  
Luke McNally ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Public Goods ◽  
Bacterial Species ◽  
Relative Fitness ◽  
Iron Starvation ◽  
Signal Molecule ◽  
Signalling Molecules ◽  
Signalling Molecule ◽  
The Impact ◽  
Social Trait

Bacteria produce a wide variety of exoproducts that favourably modify their environment and increase their fitness. These are often termed ‘public goods’ because they are costly for individuals to produce and can be exploited by non-producers (‘cheats’). The outcome of conflict over public goods is dependent upon the prevailing environment and the phenotype of the individuals in competition. Many bacterial species use quorum sensing (QS) signalling molecules to regulate the production of public goods. QS therefore determines the cooperative phenotype of individuals, and influences conflict over public goods. In addition to their regulatory functions, many QS molecules have additional properties that directly modify the prevailing environment. This leads to the possibility that QS molecules could influence conflict over public goods indirectly through nonsignalling effects, and the impact of this on social competition has not previously been explored. ThePseudomonas aeruginosaQS signal molecule PQS is a powerful chelator of iron which can cause an iron starvation response. Here we show that PQS stimulates a concentration-dependent increase in the cooperative production of iron scavenging siderophores, resulting in an increase in the relative fitness of non-producing siderophore cheats. This is likely due to an increased cost of siderophore output by producing cells and a concurrent increase in the shared benefits, which accrue to both producers and cheats. Although PQS can be a beneficial signalling molecule forP. aeruginosa, our data suggests that it can also render a siderophore-producing population vulnerable to competition from cheating strains. More generally our results indicate that the production of one social trait can indirectly affect the costs and benefits of another social trait.

scholarly journals Global and Targeted Lipid Analysis ofGemmata obscuriglobusReveals the Presence of Lipopolysaccharide, a Signature of the Classical Gram-Negative Outer Membrane

2015 ◽  
Vol 198 (2) ◽  
pp. 221-236 ◽  
Author(s):  
Rajendra Mahat ◽  
Corrine Seebart ◽  
Franco Basile ◽  
Naomi L. Ward
Keyword(s):  
Outer Membrane ◽  
Lipid A ◽  
Direct Evidence ◽  
Cell Envelope ◽  
Bacterial Species ◽  
Gas Chromatography Mass Spectrometry ◽  
Differential Staining ◽  
Gram Negative ◽  
Negative Cell ◽  
Cell Plan

ABSTRACTPlanctomycete bacteria possess many unusual cellular properties, contributing to a cell plan long considered to be unique among the bacteria. However, data from recent studies are more consistent with a modified Gram-negative cell plan. A key feature of the Gram-negative plan is the presence of an outer membrane (OM), for which lipopolysaccharide (LPS) is a signature molecule. Despite genomic evidence for an OM in planctomycetes, no biochemical verification has been reported. We attempted to detect and characterize LPS in the planctomyceteGemmata obscuriglobus. We obtained direct evidence for LPS and lipid A using electrophoresis and differential staining. Gas chromatography-mass spectrometry (GC-MS) compositional analysis of LPS extracts identified eight different 3-hydroxy fatty acids (3-HOFAs), 2-keto 3-deoxy-d-manno-octulosonic acid (Kdo), glucosamine, and hexose and heptose sugars, a chemical profile unique to Gram-negative LPS. Combined with molecular/structural information collected from matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS analysis of putative intact lipid A, these data led us to propose a heterogeneous hexa-acylated lipid A structure (multiple-lipid A species). We also confirmed previous reports ofG. obscuriglobuswhole-cell fatty acid (FA) and sterol compositions and detected a novel polyunsaturated FA (PUFA). Our confirmation of LPS, and by implication an OM, inG. obscuriglobusraises the possibility that other planctomycetes possess an OM. The pursuit of this question, together with studies of the structural connections between planctomycete LPS and peptidoglycans, will shed more light on what appears to be a planctomycete variation on the Gram-negative cell plan.IMPORTANCEBacterial species are classified as Gram positive or negative based on their cell envelope structure. For 25 years, the envelope of planctomycete bacteria has been considered a unique exception, as it lacks peptidoglycan and an outer membrane (OM). However, the very recent detection of peptidoglycan in planctomycete species has provided evidence for a more conventional cell wall and raised questions about other elements of the cell envelope. Here, we report direct evidence of lipopolysaccharide in the planctomyceteG. obscuriglobus, suggesting the presence of an OM and supporting the proposal that the planctomycete cell envelope is an extension of the canonical Gram-negative plan. This interpretation features a convoluted cytoplasmic membrane and expanded periplasmic space, the functions of which provide an intriguing avenue for future investigation.

scholarly journals Links between Anr and Quorum Sensing in Pseudomonas aeruginosa Biofilms

2015 ◽  
Vol 197 (17) ◽  
pp. 2810-2820 ◽  
Author(s):  
John H. Hammond ◽  
Emily F. Dolben ◽  
T. Jarrod Smith ◽  
Sabin Bhuju ◽  
Deborah A. Hogan
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcription Factor ◽  
Quorum Sensing ◽  
Cellular Response ◽  
Iron Acquisition ◽  
Laboratory Strain ◽  
Interspecies Interactions ◽  
Low Oxygen ◽  
Chronic Infections ◽  
Content Type

ABSTRACTInPseudomonas aeruginosa, the transcription factor Anr controls the cellular response to low oxygen or anoxia. Anr activity is high in oxygen-limited environments, including biofilms and populations associated with chronic infections, and Anr is necessary for persistence in a model of pulmonary infection. In this study, we characterized the Anr regulon in biofilm-grown cells at 1% oxygen in the laboratory strain PAO1 and in a quorum sensing (QS)-deficient clinical isolate, J215. As expected, transcripts related to denitrification, arginine fermentation, high-affinity cytochrome oxidases, and CupA fimbriae were lower in the Δanrderivatives. In addition, we observed that transcripts associated with quorum sensing regulation, iron acquisition and storage, type VI secretion, and the catabolism of aromatic compounds were also differentially expressed in the Δanrstrains. Prior reports have shown that quorum sensing-defective mutants have higher levels of denitrification, and we found that multiple Anr-regulated processes, including denitrification, were strongly inversely proportional to quorum sensing in both transcriptional and protein-based assays. We also found that in LasR-defective strains but not their LasR-intact counterparts, Anr regulated the production of the 4-hydroxy-2-alkylquinolines, which play roles in quorum sensing and interspecies interactions. These data show that Anr was required for the expression of important metabolic pathways in low-oxygen biofilms, and they reveal an expanded and compensatory role for Anr in the regulation of virulence-related genes in quorum sensing mutants, such as those commonly isolated from infections.IMPORTANCEPseudomonas aeruginosacauses acute ocular, soft tissue, and pulmonary infections, as well as chronic infections in the airways of cystic fibrosis patients.P. aeruginosauses quorum sensing (QS) to regulate virulence, but mutations in the gene encoding the master regulator of QS,lasR, are frequently observed in clinical isolates. We demonstrated that the regulon attributed to Anr, an oxygen-sensitive transcription factor, was more highly expressed inlasRmutants. Furthermore, we show that Anr regulates the production of several different secreted factors inlasRmutants. These data demonstrate the importance of Anr in naturally occurring quorum sensing mutants in the context of chronic infections.

scholarly journals Iron and Fur Regulation in Vibrio cholerae and the Role of Fur in Virulence

2005 ◽  
Vol 73 (12) ◽  
pp. 8167-8178 ◽  
Author(s):  
Alexandra R. Mey ◽  
Elizabeth E. Wyckoff ◽  
Vanamala Kanukurthy ◽  
Carolyn R. Fisher ◽  
Shelley M. Payne
Keyword(s):  
Gene Expression ◽  
Vibrio Cholerae ◽  
Iron Acquisition ◽  
Bacterial Species ◽  
Regulation Of Gene Expression ◽  
Transport Systems ◽  
Infant Mouse ◽  
Genes Encoding ◽  
Regulatory Functions

ABSTRACT Regulation of iron uptake and utilization is critical for bacterial growth and for prevention of iron toxicity. In many bacterial species, this regulation depends on the iron-responsive master regulator Fur. In this study we report the effects of iron and Fur on gene expression in Vibrio cholerae. We show that Fur has both positive and negative regulatory functions, and we demonstrate Fur-independent regulation of gene expression by iron. Nearly all of the known iron acquisition genes were repressed by Fur under iron-replete conditions. In addition, genes for two newly identified iron transport systems, Feo and Fbp, were found to be negatively regulated by iron and Fur. Other genes identified in this study as being induced in low iron and in the fur mutant include those encoding superoxide dismutase (sodA), fumarate dehydratase (fumC), bacterioferritin (bfr), bacterioferritin-associated ferredoxin (bfd), and multiple genes of unknown function. Several genes encoding iron-containing proteins were repressed in low iron and in the fur mutant, possibly reflecting the need to reserve available iron for the most critical functions. Also repressed in the fur mutant, but independently of iron, were genes located in the V. cholerae pathogenicity island, encoding the toxin-coregulated pilus (TCP), and genes within the V. cholerae mega-integron. The fur mutant exhibited very weak autoagglutination, indicating a possible defect in expression or assembly of the TCP, a major virulence factor of V. cholerae. Consistent with this observation, the fur mutant competed poorly with its wild-type parental strain for colonization of the infant mouse gut.

scholarly journals Surfing Motility: a Conserved yet Diverse Adaptation among Motile Bacteria

2018 ◽  
Vol 200 (23) ◽  
Author(s):  
Evelyn Sun ◽  
Sijie Liu ◽  
Robert E. W. Hancock
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Broad Spectrum ◽  
Proteus Mirabilis ◽  
Salmonella Enterica ◽  
Bacterial Species ◽  
Content Type ◽  
Adaptive Resistance ◽  
Sensing Systems

ABSTRACTBacterial rapid surfing motility is a novel surface adaptation ofPseudomonas aeruginosain the presence of the glycoprotein mucin. Here, we show that other Gram-negative motile bacterial species, includingEscherichia coli,Salmonella enterica,Vibrio harveyi,Enterobacter cloacae, andProteus mirabilis, also exhibit the physical characteristics of surfing on the surface of agar plates containing 0.4% mucin, where surfing motility was generally more rapid and less dependent on medium viscosity than was swimming motility. As previously observed inPseudomonas aeruginosa, all surfing species exhibited some level of broad-spectrum adaptive resistance, although the antibiotics to which they demonstrated surfing-mediated resistance differed. Surfing motility inP. aeruginosawas found to be dependent on the quorum-sensing systems of this organism; however, this aspect was not conserved in other tested bacterial species, includingV. harveyiandS. enterica, as demonstrated by assaying specific quorum-sensing mutants. Thus, rapid surfing motility is a complex surface growth adaptation that is conserved in several motile bacteria, involves flagella, and leads to diverse broad-spectrum antibiotic resistance, but it is distinct in terms of dependence on quorum sensing.IMPORTANCEThis study showed for the first time that surfing motility, a novel form of surface motility first discovered inPseudomonas aeruginosaunder artificial cystic fibrosis conditions, including the presence of high mucin content, is conserved in other motile bacterial species known to be mucosa-associated, includingEscherichia coli,Salmonella enterica, andProteus mirabilis. Here, we demonstrated that key characteristics of surfing, including the ability to adapt to various viscous environments and multidrug adaptive resistance, are also conserved. Using mutagenesis assays, we also identified the importance of all three known quorum-sensing systems, Las, Rhl, and Pqs, inP. aeruginosain regulating surfing motility, and we also observed a conserved dependence of surfing on flagella in certain species.

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 Pseudomonas aeruginosa Las quorum sensing autoinducer suppresses growth and biofilm production in Legionella species

Microbiology ◽  
2009 ◽  
Vol 155 (6) ◽  
pp. 1934-1939 ◽  
Author(s):  
Soichiro Kimura ◽  
Kazuhiro Tateda ◽  
Yoshikazu Ishii ◽  
Manabu Horikawa ◽  
Shinichi Miyairi ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Cell Signalling ◽  
Homoserine Lactone ◽  
Alcaligenes Faecalis ◽  
Suppressive Effect ◽  
Dependent Manner ◽  
A Cell

Bacteria commonly communicate with each other by a cell-to-cell signalling mechanism known as quorum sensing (QS). Recent studies have shown that the Las QS autoinducer N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) of Pseudomonas aeruginosa performs a variety of functions not only in intraspecies communication, but also in interspecies and interkingdom interactions. In this study, we report the effects of Pseudomonas 3-oxo-C12-HSL on the growth and suppression of virulence factors in other bacterial species that frequently co-exist with Ps. aeruginosa in nature. It was found that 3-oxo-C12-HSL, but not its analogues, suppressed the growth of Legionella pneumophila in a dose-dependent manner. However, 3-oxo-C12-HSL did not exhibit a growth-suppressive effect on Serratia marcescens, Proteus mirabilis, Escherichia coli, Alcaligenes faecalis and Stenotrophomonas maltophilia. A concentration of 50 μM 3-oxo-C12-HSL completely inhibited the growth of L. pneumophila. Additionally, a significant suppression of biofilm formation was demonstrated in L. pneumophila exposed to 3-oxo-C12-HSL. Our results suggest that the Pseudomonas QS autoinducer 3-oxo-C12-HSL exerts both bacteriostatic and virulence factor-suppressive activities on L. pneumophila alone.

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