QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa

The opportunistic pathogenic bacterium Pseudomonas aeruginosa uses quorum-sensing signaling systems as global regulators of virulence genes. There are two quorum-sensing signal receptor and signal generator pairs, LasR–LasI and RhlR–RhlI. The recently completed P. aeruginosa genome-sequencing project revealed a gene coding for a homolog of the signal receptors, LasR and RhlR. Here we describe a role for this gene, which we call qscR. The qscR gene product governs the timing of quorum-sensing-controlled gene expression and it dampens virulence in an insect model. We present evidence that suggests the primary role of QscR is repression of lasI. A qscR mutant produces the LasI-generated signal prematurely, and this results in premature transcription of a number of quorum-sensing-regulated genes. When fed to Drosophila melanogaster, the qscR mutant kills the animals more rapidly than the parental P. aeruginosa. The repression of lasI by QscR could serve to ensure that quorum-sensing-controlled genes are not activated in environments where they are not useful.

Generation of cell-to-cell signals in quorum sensing: acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein.

Many bacteria use acyl homoserine lactone signals to monitor cell density in a type of gene regulation termed quorum sensing and response. Synthesis of these signals is directed by homologs of the luxi gene of Vibrio fischeri. This communication resolves two critical issues concerning the synthesis of the V. fischeri signal. (i) The luxI product is directly involved in signal synthesis-the protein is an acyl homoserine lactone synthase; and (ii) the substrates for acyl homoserine lactone synthesis are not amino acids from biosynthetic pathways or fatty acid degradation products, but rather they are S-adenosylmethionine (SAM) and an acylated acyl carrier protein (ACP) from the fatty acid biosynthesis pathway. We purified a maltose binding protein-LuxI fusion polypeptide and showed that, when provided with the appropriate substrates, it catalyzes the synthesis of an acyl homoserine lactone. In V. fischeri, luxi directs the synthesis of N-(3-oxohexanoyl) homoserine lactone and hexanoyl homoserine lactone. The purified maltose binding protein-LuxI fusion protein catalyzes the synthesis of hexanoyl homoserine lactone from hexanoyl-ACP and SAM. There is a high level of specificity for hexanoyl-ACP over ACPs with differing acyl group lengths, and hexanoyl homoserine lactone was not synthesized when SAM was replaced with other amino acids, such as methionine, S-adenosylhomocysteine, homoserine, or homoserine lactone, or when hexanoyl-SAM was provided as the substrate. This provides direct evidence that the LuxI protein is an auto-inducer synthase that catalyzes the formation of an amide bond between SAM and a fatty acyl-ACP and then catalyzes the formation of the acyl homoserine lactone from the acyl-SAM intermediate.

Manipulation of the Quorum Sensing Signal AI-2 Affects the Antibiotic-Treated Gut Microbiota

The mammalian gut microbiota harbors a diverse ecosystem where hundreds of bacterial species interact with each other and their host. Given that bacteria use signals to communicate and regulate group behaviors (quorum sensing), we asked whether such communication between different commensal species can influence the interactions occurring in this environment. We engineered the enteric bacterium, Escherichia coli, to manipulate the levels of the interspecies quorum sensing signal, autoinducer-2 (AI-2), in the mouse intestine and investigated the effect upon antibiotic-induced gut microbiota dysbiosis. E. coli that increased intestinal AI-2 levels altered the composition of the antibiotic-treated gut microbiota, favoring the expansion of the Firmicutes phylum. This significantly increased the Firmicutes/Bacteroidetes ratio, to oppose the strong effect of the antibiotic, which had almost cleared the Firmicutes. This demonstrates that AI-2 levels influence the abundance of the major phyla of the gut microbiota, the balance of which is known to influence human health.

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhIRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown OS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique 'hypothetical' protein (PA0572), which could not be detected in the culture supernatants of Delta/as mutants, although they were unaffected in Deltarhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-1) could not be detected when any of the lasRI or rhIRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of OS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a CS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.

Sub-inhibitory concentrations of ceftazidime and tobramycin reduce the quorum sensing signals of Pseudomonas aeruginosa

Aim: Concentrations of antimicrobials below minimum inhibitory concentration (subMIC) may reduce the production by Pseudomonas aeruginosa of virulence factors such as elastase. We sought to determine whether the reduction in elastase production may be mediated by a reduction in acyl-homoserine lactones. Methods: Pseudomonas aeruginosa in broth was exposed to three conditions for ceftazidime and tobramycin: control, 6% MIC and 25% MIC. Elastase was assayed using elastin congo red. N-(3-Oxododecanoyl)-homoserine lactone (C12-HSL) and N-butyryl-homoserine lactone (C4-HSL) were assayed using biosensor Escherichia coli. Results: Elastase was unchanged with ceftazidime. Elastase was reduced by 16% at 6% MIC tobramycin and reduced by 70% at 25% MIC tobramycin (P

Quorum sensing, bacterial communication and new antibiotics [Editorial]

No Abstract

The detection of environmental autoinducing peptide quorum-sensing genes from an uncultured Clostridium sp in landfill leachate reactor biomass

Aims: To elucidate whether a dominant uncultured clostridial (Clostridium thermocellum-like) species in an environmental sample (landfill leachate), possesses an autoinducing peptide (AIP) quorum-sensing (QS) gene, although it may not be functional. Methods and Results: A modified AIP accessory gene regulator (agr)C PCR protocol was performed on extracted DNA from a landfill leachate sample (also characterized by 16S rRNA gene cloning) and the PCR products were cloned, sequenced and phylogenetically analysed. It appeared that two agrC gene phylotypes existed, most closely related to the C. thermocellum agrC gene, differing by only 1 bp. Conclusions: It is possible to specifically identify and characterize the agrC AIP QS gene from uncultured Firmicutes (C. thermocellum-like) bacteria derived from environmental (landfill leachate) sample. Significance and Impact of the Study: This is the first successful attempt at identifying AIP QS genes from a cellulolytic environment (landfill). The agrC gene was identified as being most closely related to the C. thermocellum agrC gene, the same bacterium identified as being dominant, according to 16S rRNA gene cloning and subsequently fluorescence in situ hybridization analyses, in the same biomass.

Helicopter automation using a low-cost sensing system

This paper details the design of an autonomous helicopter control system using a low cost sensor suite. Control is maintained using simple nested PID loops. Aircraft attitude, velocity, and height is estimated using an in-house designed IMU and vision system. Information is combined using complimentary filtering. The aircraft is shown to be stabilised and responding to high level demands on all axes, including heading, height, lateral velocity and longitudinal velocity.

A strain sensing system using a novel optical fibre Bragg grating sensor and a synthetic heterodyne interrogation technique

A potential low cost novel sensing scheme for monitoring absolute strain is demonstrated. The scheme utilizes a synthetic heterodyne interrogation technique working in conjunction with a linearly chirped, sinusoidally tapered, apodized Bragg grating sensor. The interrogation technique is relatively simple to implement in terms of the required optics and the peripheral electronics. This scheme generates an output signal that has a quasi-linear response to absolute strain with a static strain resolution of ~±20 με and an operating range of ~1000 με.

Novel hardwired distributive tactile sensing system for medical applications

This thesis described the research carried out on the development of a novel hardwired tactile sensing system tailored for the application of a next generation of surgical robotic and clinical devices, namely a steerable endoscope with tactile feedback, and a surface plate for patient posture and balance. Two case studies are examined. The first is a one-dimensional sensor for the steerable endoscope retrieving shape and ‘touch’ information. The second is a two-dimensional surface which interprets the three-dimensional motion of a contacting moving load. This research can be used to retrieve information from a distributive tactile sensing surface of a different configuration, and can interpret dynamic and static disturbances. This novel approach to sensing has the potential to discriminate contact and palpation in minimal invasive surgery (MIS) tools, and posture and balance in patients. The hardwired technology uses an embedded system based on Field Programmable Gate Arrays (FPGA) as the platform to perform the sensory signal processing part in real time. High speed robust operation is an advantage from this system leading to versatile application involving dynamic real time interpretation as described in this research. In this research the sensory signal processing uses neural networks to derive information from input pattern from the contacting surface. Three neural network architectures namely single, multiple and cascaded were introduced in an attempt to find the optimum solution for discrimination of the contacting outputs. These architectures were modelled and implemented into the FPGA. With the recent introduction of modern digital design flows and synthesis tools that essentially take a high-level sensory processing behaviour specification for a design, fast prototyping of the neural network function can be achieved easily. This thesis outlines the challenge of the implementations and verifications of the performances.

A low-cost remote sensing system for agricultural applications

This research develops a low cost remote sensing system for use in agricultural applications. The important features of the system are that it monitors the near infrared and it incorporates position and attitude measuring equipment allowing for geo-rectified images to be produced without the use of ground control points. The equipment is designed to be hand held and hence requires no structural modification to the aircraft. The portable remote sensing system consists of an inertia measurement unit (IMU), which is accelerometer based, a low-cost GPS device and a small format false colour composite digital camera. The total cost of producing such a system is below GBP 3000, which is far cheaper than equivalent existing systems. The design of the portable remote sensing device has eliminated bore sight misalignment errors from the direct geo-referencing process. A new processing technique has been introduced for the data obtained from these low-cost devices, and it is found that using this technique the image can be matched (overlaid) onto Ordnance Survey Master Maps at an accuracy compatible with precision agriculture requirements. The direct geo-referencing has also been improved by introducing an algorithm capable of correcting oblique images directly. This algorithm alters the pixels value, hence it is advised that image analysis is performed before image georectification. The drawback of this research is that the low-cost GPS device experienced bad checksum errors, which resulted in missing data. The Wide Area Augmented System (WAAS) correction could not be employed because the satellites could not be locked onto whilst flying. The best GPS data were obtained from the Garmin eTrex (15 m kinematic and 2 m static) instruments which have a highsensitivity receiver with good lock on capability. The limitation of this GPS device is the inability to effectively receive the P-Code wavelength, which is needed to gain the best accuracy when undertaking differential GPS processing. Pairing the carrier phase L1 with the pseudorange C/A-Code received, in order to determine the image coordinates by the differential technique, is still under investigation. To improve the position accuracy, it is recommended that a GPS base station should be established near the survey area, instead of using a permanent GPS base station established by the Ordnance Survey.

Ultra small integrated optical fiber sensing system

This paper introduces a revolutionary way to interrogate optical fiber sensors based on fiber Bragg gratings (FBGs) and to integrate the necessary driving optoelectronic components with the sensor elements. Low-cost optoelectronic chips are used to interrogate the optical fibers, creating a portable dynamic sensing system as an alternative for the traditionally bulky and expensive fiber sensor interrogation units. The possibility to embed these laser and detector chips is demonstrated resulting in an ultra thin flexible optoelectronic package of only 40 µm, provided with an integrated planar fiber pigtail. The result is a fully embedded flexible sensing system with a thickness of only 1 mm, based on a single Vertical-Cavity Surface-Emitting Laser (VCSEL), fiber sensor and photodetector chip. Temperature, strain and electrodynamic shaking tests have been performed on our system, not limited to static read-out measurements but dynamically reconstructing full spectral information datasets.

Ultra small integrated optical fiber sensing system

This paper introduces a revolutionary way to interrogate optical fiber sensors based on fiber Bragg gratings (FBGs) and to integrate the necessary driving optoelectronic components with the sensor elements. Low-cost optoelectronic chips are used to interrogate the optical fibers, creating a portable dynamic sensing system as an alternative for the traditionally bulky and expensive fiber sensor interrogation units. The possibility to embed these laser and detector chips is demonstrated resulting in an ultra thin flexible optoelectronic package of only 40 µm, provided with an integrated planar fiber pigtail. The result is a fully embedded flexible sensing system with a thickness of only 1 mm, based on a single Vertical-Cavity Surface-Emitting Laser (VCSEL), fiber sensor and photodetector chip. Temperature, strain and electrodynamic shaking tests have been performed on our system, not limited to static read-out measurements but dynamically reconstructing full spectral information datasets.

SNR enhanced distributed vibration fiber sensing system employing polarization OTDR and ultraweak FBGs

A distributed fiber sensing system based on ultraweak FBGs (UWFBGs) assisted polarization optical time-domain reflectometry (POTDR) is proposed for load and vibration sensing with improved signal-to-noise ratio (SNR) and sensitivity. UWFBGs with reflectivity higher than Rayleigh scattering coefficient per pulse are induced into a POTDR system to increase the intensity of the back signal. The performance improvement of the system has been studied. The numerical analysis has shown that the SNR and sensitivity of the system can be effectively improved by integrating UWFBGs along the whole sensing fiber, which has been clearly proven by the experiment. The experimental results have shown that by using UWFBGs with 1.1 x 10-5 reflectivity and 10-m interval distance, the SNR is improved by 11 dB, and the load and vibration sensitivities of the POTDR are improved by about 10.7 and 9 dB, respectively.