Stephan Heeb

• PhD
• University of Nottingham

Iron is an essential nutrient for microbial growth. The opportunistic pathogen Pseudomonas aeruginosa can survive under diverse conditions, including iron‐depleted environments with the aid of small non‐coding RNAs (sRNAs). P. aeruginosa also uses three quorum sensing (QS) systems: Las, Rhl and Pqs, to coordinate virulence and infection establishme...

Iron is an essential nutrient for microbial growth. The opportunistic pathogen Pseudomonas aeruginosa can survive under diverse conditions, including iron-depleted environments with the aid of small non-coding RNAs (sRNAs). P. aeruginosa also uses three quorum sensing (QS) systems Las, Rhl, and Pqs to coordinate virulence and infection establishmen...

Purpose Biofilm formation and biomineralization on urinary catheters may cause severe complications including infection and obstruction. Here, we describe an in vitro evaluation and prospective pilot clinical study of a silicone catheter coated with a biofilm-resistant polymer. Materials and Methods Biofilm biomass and biomineralization on uncoate...

Pseudomonas aeruginosa is one of the top priority pathogens that requires immediate attention according to the World Health Organisation (WHO). Due to the alarming shortage of novel antimicrobials, targeting quorum sensing (QS), a bacterial cell to cell signaling system controlling virulence, has emerged as a promising approach as an antibiotic adj...

In its canonical interpretation, quorum sensing (QS) allows single cells in a bacterial population to synchronize gene expression and hence perform specific tasks collectively once the quorum cell density is reached. However, growing evidence in different bacterial species indicates that considerable cell-to-cell variation in the QS activation stat...

Pseudomonas aeruginosa is a model quorum sensing (QS) pathogen with three interconnected QS circuits that control the production of virulence factors and antibiotic tolerant biofilms. The pqs QS system of P. aeruginosa is responsible for the biosynthesis of diverse 2-alkyl-4-quinolones (AQs), of which 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-...

Pseudomonas aeruginosa uses multiple protein regulators that work in tandem to control the production of a wide range of virulence factors and facilitate rapid adaptation to diverse environmental conditions. In this opportunistic pathogen, ToxR was known to positively regulate the production of the major virulence factor exotoxin A and now, through...

Pseudomonas aeruginosa uses multiple protein regulators that work in tandem to control the production of a wide range of virulence factors and facilitate rapid adaptation to diverse environmental conditions. In this opportunistic pathogen, ToxR was known to positively regulate the production of the major virulence factor exotoxin A and now, through...

There is currently a need to develop simple biofilm models that facilitate investigation of the architecture/biology of mature bacterial biofilms in a consistent/standardized manner given their environmental and clinical importance and the need for new anti‐biofilm interventions. This study introduces a novel biofilm culture system termed the rolli...

Non-toxic, biocompatible materials that inhibit bacterial biofilm formation on implanted medical devices and so prevent infection are urgently required. Weakly amphiphilic acrylate polymers with rigid hydrocarbon pendant groups resist bacterial biofilm formation in vitro and in vivo but the biological mechanism involved is not known. By comparing b...

As part of a screening programme for antibiotic-producing bacteria, a novel Actinomadura species was discovered from a soil sample collected in Santorini, Greece. Preliminary 16S rRNA gene sequence comparisons highlighted Actinomadura macra as the most similar characterised species. However, whole-genome sequencing revealed an average nucleotide id...

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine t...

P. aeruginosa (PA) continues to pose a threat to global public health due to its high levels of antimicrobial resistance (AMR). The ongoing AMR crisis has led to an alarming shortage of effective treatments for resistant microbes, and hence there is a pressing demand for the development of novel antimicrobial interventions. The potential use of ant...

The selective isolation of bacteria from mixed populations has been investigated in varied applications ranging from differential pathogen identification in medical diagnostics and food safety to the monitoring of microbial stress dynamics in industrial bioreactors. Selective isolation techniques are generally limited to the confinement of small po...

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-argini...

Post-transcriptional regulation is an important step in the control of bacterial gene expression in response to environmental and cellular signals. Pseudomonas putida KT2440 harbors three known members of the CsrA/RsmA family of post-transcriptional regulators: RsmA, RsmE and RsmI. We have carried out a global analysis to identify RNA sequences bou...

Current treatments for Pseudomonas aeruginosa infections are becoming less effective because of the increasing rates of multi-antibiotic resistance. Pharmacological targeting of virulence through inhibition of quorum sensing (QS) dependent virulence gene regulation has considerable therapeutic potential. In P. aeruginosa, the pqs QS system regulate...

The ability to selectively sequester bacteria from a mixed population is a desirable aim across a range of fields. Already some technologies exist to attempt to meet these aims spanning microfluidics and automated flow cytometry to antibody conjugated magnetic nanoparticle precipitation. However, these technologies are generally limited to the conf...

The Pseudomonas aeruginosa quorum sensing (QS) network plays a key role in the adaptation to environmental changes and the control of virulence factor production in this opportunistic human pathogen. Three interlinked QS systems, namely las, rhl, and pqs, are central to the production of pyocyanin, a phenazine virulence factor which is typically us...

Pseudomonads typically carry multiple non-identical alleles of the post-transcriptional regulator rsmA. In P. aeruginosa, RsmN is notable in that its structural rearrangement confers distinct and overlapping functions with RsmA. However, little is known about the specificities of RsmN for its target RNAs and overall impact on the biology of this pa...

The antibiotic pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite that plays an important role in the biocontrol of plant diseases due to its broad-spectrum of antimicrobial activities. The PRN biosynthetic gene cluster remains to be characterised in Serratia plymuthica, though it is highly conserved in PRN-producing bacteria. To bette...

uthors and contributions as published: Jonathan Naylor, Harold Fellermann, Yuchun Ding, Waleed K. Mohammed, Nicholas S. Jakubovics, Joy Mukherjee, Catherine A. Biggs, Phillip C. Wright, and Natalio Krasnogor. JN wrote the manuscript, developed the software and models and performed data analysis. HF supervised the work, designed the overall study, s...

Surface motility and biofilm formation are behaviours which enable bacteria to infect their hosts and are controlled by different chemical signals. In the plant symbiotic alpha-proteobacterium Sinorhizobium meliloti, the lack of long-chain fatty acyl-coenzyme A synthetase activity (FadD) leads to increased surface motility, defects in biofilm devel...

Pseudomonas aeruginosa is a major opportunistic pathogen in cystic fibrosis, wound and nosocomial infections, posing a serious burden to public health, due to its antibiotic resistance. The P. aeruginosa Pseudomonas Quinolone System (pqs) quorum sensing system, driven by the activation of the transcriptional regulator, PqsR (MvfR) by alkylquinolone...

The potential for siderophore mutants of Pseudomonas aeruginosa to attenuate virulence during infection, and the possibility of exploiting this for clinical ends, have attracted much discussion. This has largely been based on the results of in vitro experiments conducted in iron-limited growth medium, in which siderophore mutants act as social ‘che...

Expression of cfcR, encoding the only GGDEF/EAL response regulator in Pseudomonas putida, is transcriptionally regulated by RpoS, ANR and FleQ, and the functionality of CfcR as a diguanylate cyclase requires the multisensor CHASE3/GAF hybrid histidine kinase named CfcA. Here an additional level of cfcR control, operating post-transcriptionally via...

DNA methylation is widespread among prokaryotes, and most DNA methylation reactions are catalyzed by adenine DNA methyltransferases, which are part of restriction-modification (R-M) systems. R-M systems are known for their role in the defense against foreign DNA; however, DNA methyltransferases also play functional roles in gene regulation. In this...

The pqs quorum sensing (QS) system is crucial for Pseudomonas aeruginosa virulence both in vitro and in animal models of infection and is considered an ideal target for the development of anti-virulence agents. However, the precise role played by each individual component of this complex QS circuit in the control of virulence remains to be elucidat...

Genes whose transcription is controlled by HHQ, PQS and/or PqsE. (PDF)

PqsE does not affect anthranilate production and PphzA2 activity, while it positively controls PphzA1 activity. (A) Anthranilate was quantified by LC-MS/MS analysis in P. aeruginosa wild type and ∆4AQ grown to an OD600 of 1.5 in LB or in LB supplemented with 1 mM IPTG. Standard deviations are based on the mean values of three parallel cultures. (B)...

Impact of Fur and PvdS on the PpqsA and PpqsR promoter regions. (A-D) Maximal promoter activity in the strains carrying the transcriptional fusions PpqsA::lux (A and B) or PpqsR::lux (C and D). White bars indicate the pvdS-proficient genetic backgrounds (∆4AQ and ∆5AQ); grey bars indicate the pvdS-mutant genetic backgrounds (∆4AQ∆pvdS and ∆5AQ∆pvdS...

Bacterial strains and plasmids. (PDF)

Oligonucleotides. (PDF)

AQs and PqsE do not affect PpqsH and PpqsL activity. Maximal promoter activity measured in P. aeruginosa ∆4AQ strains carrying the transcriptional fusions (A) PpqsH::lux or (B) PpqsL::lux. Strains were grown in LB or in LB supplemented with 40 μM AQs or 1 mM IPTG, as indicated below the graphs. Promoter activity is reported as Relative Light Units...

The iron-chelators 2,2’-dipyridyl and deferiprone do not increase PpqsA activity. Maximal promoter activity in strains carrying the transcriptional fusions PpqsA::lux (A and B) or PpqsR::lux (C and D). Strains were grown in LB or in LB supplemented with 40 μM PQS, 500 μM 2,2’-dipyridyl (DIP), or 160 μM deferiprone (DEF), as indicated. Diamonds indi...

Validation of the P. aeruginosa ∆4AQ strain. (A) Levels of HHQ (white bars), PQS (light-grey bars) and HQNO (dark-grey bars) quantified by LC-MS/MS analysis in culture supernatants of P. aeruginosa ∆4AQ grown in LB or in LB supplemented with 40 μM HHQ, PQS, or HQNO, or with 1 mM IPTG, as indicated. AQ levels were quantified in supernatants from thr...

The pqsE RNA transcript does not promote pyocyanin production. Pyocyanin production (A) and pqsE RNA levels measured by Real Time PCR (B) in P. aeruginosa ∆pqsA ∆pqsE double mutant strains carrying the pME6032 empty vector or pME6032-derivative plasmids for IPTG-inducible expression of wild type pqsE (pME-pqsE), or pqsE mutated variants lacking the...

RT-PCR analysis showing co-transcription of pqsE and phnA. Amplification of cDNAs retro-transcribed from RNA extracted from (A) P. aeruginosa PAO1 wild type grown in LB and (B) P. aeruginosa ∆4AQ grown in LB supplemented with 1 mM IPTG, to an OD600 of 1.5. A 200 bp DNA region within the pqsE gene (pqsE), a 280 bp DNA region spanning from 97 bp upst...

The pqs quorum sensing (QS) system is crucial for Pseudomonas aeruginosa virulence both in vitro and in animal models of infection and is considered an ideal target for the development of anti-virulence agents. However, the precise role played by each individual component of this complex QS circuit in the control of virulence remains to be elucidat...

Background Biofilm development, specifically the fundamentally adaptive switch from acute to chronic infection phenotypes, requires global regulators and small non-coding regulatory RNAs (sRNAs). This work utilized RNA-sequencing (RNA-seq) to detect sRNAs differentially expressed in Pseudomonas aeruginosa biofilm versus planktonic state. Results A...

Dickeya solani and Pectobacterium carotovorum subsp. brasiliense are recently established species of bacterial plant pathogens causing black leg and soft rot of many vegetables and ornamental plants. Pseudomonas sp. strain P482 inhibits the growth of these pathogens, a desired trait considering the limited measures to combat these diseases. In this...

Dickeya solani and Pectobacterium carotovorurn subsp. brasiliense are recently established species of bacterial plant pathogens causing black leg and soft rot of many vegetables and ornamental plants. Pseudornonas sp. strain P482 inhibits the growth of these pathogens, a desired trait considering the limited measures to combat these diseases. In th...

In Pseudomonas aeruginosa, quorum sensing (QS) regulates the production of secondary metabolites, many of which are antimicrobials that impact on polymicrobial community composition. Consequently, quenching QS modulates the environmental impact of P. aeruginosa. To identify bacteria capable of inactivating the QS signal molecule 2-heptyl-3- hydroxy...

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we...

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consor...

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consor...

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consor...

In Pseudomonas aeruginosa quorum sensing (QS) regulates the production of secondary metabolites, many of which are antimicrobials that impact on polymicrobial community composition. Consequently, quenching QS modulates the environmental impact of P.aeruginosa. To identify bacteria capable of inactivating the QS signal molecule 2-heptyl-3-hydroxy-4(...

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune respons...

Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4...

CsrA/RsmA homologs are an extensive family of ribonucleic acid (RNA)-binding proteins that function as global post-transcriptional regulators controlling important cellular processes such as secondary metabolism, motility, biofilm formation and the production and secretion of virulence factors in diverse bacterial species. While direct messenger RN...

In bacteria, the highly conserved RsmA/CsrA family of RNA-binding proteins functions as global posttranscriptional regulators acting on mRNA translation and stability. Through phenotypic complementation of an rsmA mutant in Pseudomonas aeruginosa, we discovered a family member, termed RsmN. Elucidation of the RsmN crystal structure and that of the...

Electron density maps are shown for bound ligands displayed within a 2 Å radius. (A) Left panel shows the final refined 2.7 Å electron density map (blue) with SigmaA m2Fo-DFc coefficients and phases c of the PqsR-MPD structure contoured at 1.0 r.m.s (REFMAC, CCP4). The modelled MPD molecules are shown as stick and the figure was generated using PyM...

Strains and plasmids used in this study. (PDF)

Supplemental Materials and Methods and 1H NMR spectra. (PDF)

(A) Gel filtration chromatogram of PqsRCBD (residues 94-332). Absorbance elution profiles are shown PqsRCBD in blue overlapped with the elution profiles of protein standards (red) using a Superdex 75 10/300 analytical column. The standards include I - ovalbumin (43 kDa), II - carbonic anhydrase (29 kDa) and III - ribonuclease A (13.7 kDa). (B) The...

Unique dimer organisation of PqsR. Cartoon diagrams of the central dimer organisation for LTTRs PqsR, OxyR (pdb: 1I69), BenM (pdb: 2F78) and TsaR (pdb: 3FXQ). β-strands are colored magenta and α-helices in cyan. β-strands involved in the hinge region are indicated by a blue arrow showing the centrally located hinge regions in PqsR compared to the p...

Oligonucleotides used in this study. (PDF)

Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4...

Pseudomonas aeruginosa is a versatile bacterial pathogen capable of occupying diverse ecological niches. To cope with iron limitation, P. aeruginosa secretes two siderophores, pyoverdine and pyochelin, whose ability to deliver iron to the cell is crucial for biofilm formation and pathogenicity. In this study, we describe a link between iron uptake...

Bacteria are able to recognize and respond to various molecules produced by the host in stressful situations. Although there is a growing interest on deciphering pathogen-host communication during infection and other stressful situations, there is little information on the impact of catecholamine stress hormones on the opportunistic pathogen Pseudo...

AaaA does not remove methionine or Leucine from p-nitroanilide. Panel A. SGAP was incubated with either leucine-p-nitroanilide (solid circle) or arginine-p-nitroanilide (open circle) and the resultant changes in A405 nm are shown against time compared with a buffer blank (crosses). Panel B. The P. aeruginosa ΔaaaA mutant (open circles) or WT PAO1 c...

Insertion of MiniCTX does not significantly alter the growth profile of P. aeruginosa in LB. Wild type P. aeruginosa PAO1 (closed triangles), the ΔaaaA mutant (open circles) and the ΔaaaA mutant with insertion of ctx::aaaA PAJL (closed circles) were inoculated at an OD of 0.06 in LB, and the growth over 18 h as monitored in the automated plate read...

An in-frame deletion mutant of aaaA grows similarly to its parent in rich medium. (Panel A) The cartoon indicates the strategy used to generate the ΔaaaA mutant. Primer positions are indicated as aaaAfa (a), aaaArb (b), aaaAfb (c), and aaaArc (d). Genomic DNA from the parental PAO1 and ΔaaaA mutant was digested with XmnI, and hybridised to a probe...

Author Summary We present a new Pseudomonas aeruginosa virulence factor that promotes chronic skin wound infections. We propose the name AaaA for this cell-surface tethered autotransporter. This arginine-specific aminopeptidase confers a growth advantage upon P. aeruginosa, providing a fitness advantage by creating a supply of arginine in chronic w...

We report the identification of fatty acids as mediators of intercellular signalling in Pseudomonas putida, and between Pseudomonas aeruginosa and P. putida. Tetradecanoic acid and fatty acids of similar chain length are present in supernatants of these strains and activate population density-dependent expression of ddcA, a gene involved in corn se...

The GacS/GacA two-component system functions mechanistically in conjunction with global post-transcriptional regulators of the RsmA family to allow pseudomonads and other bacteria to adapt to changing environmental stimuli. Analysis of this Gac/Rsm signal transduction pathway in phytotoxin-producing pathovars of Pseudmonas syringae is incomplete, p...

We report the identification of fatty acids as mediators of intercellular signalling in Pseudomonas putida, and between Pseudomonas aeruginosa and P. putida. Tetradecanoic acid and fatty acids of similar chain length are present in supernatants of these strains and activate population density-dependent expression of ddcA, a gene involved in corn se...

Introduction Potent effects of human mesenchymal stem cells (MSCs) on immune responses have been demonstrated in vitro studies and animal models, encouraging further investigations into their clinical value. Studies of MSCs in fistula healing have emphasised the immunosuppressive mechanisms of action however, there has been little focus on their an...

Introduction Potent effects of human mesenchymal stem cells (MSCs) on immune responses have been demonstrated in vitro studies and animal models, encouraging further investigations into their clinical value. Studies of MSCs in fistula healing have emphasised the immunosuppressive mechanisms of action however, there has been little focus on their an...

Acute bacterial infections are associated with motility and cytotoxicity via the type III secretion system (T3SS), while chronic infections are linked to biofilm formation and reduced virulence. In Pseudomonas aeruginosa, the transition between motility and sessility involves regulatory networks including the RetS/GacS sensors, as well as the secon...

Since quinine was first isolated, animals, plants and microorganisms producing a wide variety of quinolone compounds have been discovered, several of which possess medicinally interesting properties ranging from antiallergenic and anticancer to antimicrobial activities. Over the years, these have served in the development of many synthetic drugs, i...

Quorum sensing, a cell-to-cell communication system based on small signal molecules, is employed by the human pathogen Pseudomonas aeruginosa to regulate virulence and biofilm development. Moreover, regulation by small trans-encoded RNAs has become a focal issue in studies of virulence gene expression of bacterial pathogens. In this study, we have...

Pyoverdine biosynthesis in fluorescent Pseudomonas spp. and especially in the opportunistic human pathogen Pseudomonas aeruginosa has been extensively studied. The acylase PvdQ is required for a maturation step in pyoverdine biosynthesis but also has been proven to be effective in degrading long-chain N-acyl homoserine lactones (AHLs). These molecu...

The quorum sensing (QS) system of Pseudomonas aeruginosa constitutes a sophisticated genome-wide gene regulatory network employing both N-acylhomoserine lactone and 2-alkyl-4-quinolone (AQ) signal molecules. AQ signalling utilizes 2-heptyl-3-hydroxy-4-quinolone (PQS) and its immediate precursor, 2-heptyl-4-quinolone (HHQ). AQ biosynthesis requires...

Pseudomonas putida KT2440 does not produce any of the common molecules involved in quorum sensing signalling described in other bacteria. However, as is the case in other microorganisms, the genome of this strain contains an open reading frame (PP_4647) coding for a transcriptional regulator belonging to the LuxR protein family. In this article, we...

This paper proposes a new methodology for the automated design of cell models for systems and synthetic biology. Our modelling framework is based on P systems, a discrete, stochastic and modular formal modelling language. The automated design of biological models comprising the optimization of the model structure and its stochastic kinetic constant...

In addition to the las and rhl qorum sensing (QS) systems, Pseudomonas aeruginosa utilises a non-acyl homoserine lactone (AHL)-based systems which relies on the production of 2-alkyl-4(1H)-quinolone (AQ) molecules. Although this organism produces a large range of AQ derivatives, the two main AQ signal molecules described are 2-heptyl-3-hydroxy-4(1H...

Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Ta...

Pseudomonas putida KT2440 does not produce any of the common molecules involved in quorum sensing signalling described in other bacteria. However, as is the case in other microorganisms, the genome of this strain contains an open reading frame (PP_4647) coding for a transcriptional regulator belonging to the LuxR protein family. In this article, we...