Timothy S. Charlton

Biotechnological uses of enzymes from psychrophiles

The bulk of the Earths biosphere is cold (e.g. 90% of the oceans waters are ≤ 5°C), sustaining a broad diversity of microbial life. The permanently cold environments vary from the deep ocean to alpine reaches and to polar regions. Commensurate with the extent and diversity of the ecosystems that harbour psychrophilic life, the functional capacity of the microorganisms that inhabitat the cold biosphere are equally diverse. As a result, indigenous psychrophilic microorganisms provide an enormous natural resource of enzymes that function effectively in the cold, and these cold‐adapted enzymes have been targeted for their biotechnological potential. In this review we describe the main properties of enzymes from psychrophiles and describe some of their known biotechnological applications and ways to potentially improve their value for biotechnology. The review also covers the use of metagenomics for enzyme screening, the development of psychrophilic gene expression systems and the use of enzymes for cleaning.

Evidence for Acyl Homoserine Lactone Signal Production in Bacteria Associated with Marine Sponges

ABSTRACT We report for the first time the production of acyl homoserine lactones (AHLs) by bacteria associated with marine sponges. Given the involvement of AHLs in bacterial colonization of many higher organisms, we speculate that such quorum sensing signals could play a part in interactions between sponges and the dense bacterial communities living within them.

The Role of Regulators in the Expression of Quorum-Sensing Signals in Pseudomonas aeruginosa

Quorum-sensing systems provide Pseudomonas aeruginosa with a sensitive regulatory mechanism that allows for the induction of several phenotypic genes in a cell density fashion. In this work, a mathematical model of the acylated homoserine lactones regulatory network system in P. aeruginosa has been developed. It is the first integrated model to consider both quorum-sensing systems. The model has allowed us to disentangle the complex behavior exhibited by the system as the concentration of extracellular OdDHL is increased. At either low or high levels of extracellular OdDHL, the bacterium remains in an uninduced or induced state, respectively. At moderate levels, the behavior is characterized by several states. Here, the bacteria can switch suddenly from an uninduced to an induced phenotype in response to small changes in the concentration of extracellular OdDHL. Additionally, we have been able to address the roles of RsaL and Vfr as regulators of the quorum-sensing system. An important result from this analysis suggests that RsaL will increase the concentration of extracellular OdDHL required to induce the system, and it is a key regulator of the inhibition of the quorum-sensing system under low cell densities. Most importantly, our results suggest that Vfr has strong regulatory effects on the system as an increased affinity between the LasR/OdDHL complex, and the lasR promoter leads to significant qualitative changes in induction patterns. We also show experimental data that demonstrate that Vfr is required for signal production in the early phase of growth, but that in the latter stages of growth, the vfr mutant is able to synthesize wild-type levels of signal.

Dynamics of Algal Secondary Metabolites in Two Species of Sea Hare

The function of acquired algal secondary metabolites in sea hares is the subject of debate, in part because the dynamics/processing of metabolites by sea hares is poorly understood. This study investigates the dynamics of red algal secondary metabolites in two sea hares, Aplysia parvula and Aplysia dactylomela. Secondary metabolite levels were quantified for the dietary red algae Laurencia obtusa and Delisea pulchra and for sea hares collected from these seaweeds in the field. The patterns and dynamics of algal secondary metabolites were further investigated in the laboratory by quantitative analysis of secondary metabolites in sea hares grown on diets of L. obtusa, D. pulchra, or the green alga Ulva sp. Sea hares accumulated the most abundant metabolites from each red alga, the terpene palisadin A from L. obtusa, and the halogenated furanone 3 from D. pulchra, and stored a greater proportion of these metabolites than other algal metabolites. A. parvula accumulated D. pulchra metabolites at much higher levels than L. obtusa metabolites. A. dactylomela accumulated similar concentrations of L. obtusa metabolites to A. parvula. The loss of L. obtusa metabolites by A. dactylomela matched that expected for dilution of metabolites via growth of the sea hares. However, the loss of L. obtusa metabolites by A. parvula was faster than predicted for growth alone, suggesting that metabolites were actively metabolized or excreted. Data for the loss of D. pulchra metabolites by A. parvula was equivocal. The secretions of A. parvula fed D. pulchra or L. obtusa were analyzed for the presence of algal secondary metabolites to investigate one possible path of excretion. L. obtusa secondary metabolites were detected in the mucous and opaline secretions of A. parvula, but D. pulchra metabolites were not detected in any secretions. The deployment of L. obtusa secondary metabolites in secretions by A. parvula may explain the more rapid rate of loss of these compounds and is consistent with a possible defensive role for acquired metabolites.

Quantification of the neurotoxin 2-amino-3-(methylamino)-propanoic acid (BMAA) in cycadales

Abstract Cycads have been recognized as toxic for many years and are known to cause hind limb ataxia in grazing animals. The l -isomer of 2-amino-3-(methylamino)propanoic acid (BMAA; β-methyldiaminopropanonic acid), a constituent of cycads, has recently been implicated in the onset of human neurologic disorders. We have used combined gas chromatography-mass spectrometry (GC-MS) to determine the BMAA content of the leaves ( n = 30) and female gametophyte ( n = 11) of a variety of cycads. A stable isotopomer of BMAA (i.e. [ 2 H 3 ] BMAA) was used as an internal standard to optimize specificity and precision. BMAA content is greater in members of the genus Cycas (i.e. up to 1800 μg g −1 fr. wt) whereas smaller amounts (i.e. −1 ) are present in members of the six other genera tested. Where BMAA content of both leaves and seeds was determined in the same species ( n = 4), values were comparable. Based on our results we estimate that grazing animals are exposed to exceedingly low doses of BMAA. These data would tend to exclude BMAA as the etiologic agent in hind limb ataxia in grazing animals.

Versatile peroxidase degradation of humic substances: Use of isothermal titration calorimetry to assess kinetics, and applications to industrial wastes

The kinetic constants of a hybrid versatile-peroxidase (VP) which oxidizes complex polymeric humic substances (HS) derived from lignin (humic and fulvic acids) and industrial wastes were determined for the first time using isothermal titration calorimetry (iTC). The reaction conditions were manipulated to enable manganese-peroxidase (MnP) and/or lignin-peroxidase (LiP) activities to be evaluated. The peroxidase reactions exhibited varying degrees of product inhibition or activation; properties which have not previously been reported for VP enzymes. In contrast to previous work (Ertan et al., 2012) on small non-polymeric substrates (MnSO4, veratryl alcohol and dyes), all kinetic plots for polymeric HS were sigmoidal, lacked Michaelis-Menten characteristics, and were indicative of positive cooperativity. Under conditions when both LiP and MnP were active, the kinetic data fitted to a novel biphasic Hill Equation, and the rate of enzymatic reaction was significantly greater than the sum of individual LiP plus MnP activities implying synergistic activation. By employing size-exclusion chromatography and electrospray ionization mass spectrometry, the characteristics of the oxidative degradation products of the HS were also monitored. Our study showed that the allosteric behaviour of the VP enzyme promotes a high level of regulation of activity during the breakdown of model and industrial ligninolytic substrates. The work was extended to examine the kinetics of breakdown of industrial wastes (effluent from a pulp and paper plant, and fouled membrane solids extracted from a ground water treatment membrane) revealing unique, VP-mediated, kinetic responses. This work demonstrates that iTC can be successfully employed to study the kinetic properties of VP enzymes in order to devise reaction conditions optimized for oxidative degradation of HS present in materials used in a wide range of industries.

A covalent thymine-tyrosine adduct involved in DNA-protein crosslinks: synthesis, characterization, and quantification

A thymine-tyrosine adduct, (3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]-L-tyrosine), was synthesized using a simple, single-step condensation between 5-(hydroxymethyl)uracil and L-tyrosine. This approach provides access to useful quantities (mg-g) of analytically pure reference material, and with minor modification, to stable isotope-labeled analogues (isotopomers). With reference material and a suitable internal standard available, isotope-dilution liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/MS/MS) was used to assay the adduct in a model system purged of oxygen, i.e., a gamma-irradiated N2O-saturated aqueous solution of thymine and tyrosine. The convenient synthetic route to standards and the method for quantification reported here will prove useful in assessing the significance of the adduct in biological systems. These studies also highlight the potential for artefactual adduct formation if the appropriate substrates are present under acidic conditions.

Genetic and chemical tools for investigating signaling processes in biofilms.

Publisher Summary This chapter discusses genetic and chemical tools for investigating signaling processes in biofilms. The vast majority of microbial interactions in the environment center on surfaces, where it has been estimated that approximately 99% of all bacteria reside. Of those surface-associated organisms, many are found in large aggregates or communities that are recognized as biofilms. The formation of biofilms appears to offer cells several advantages over planktonic cells in that the biofilm protects the cells from many adverse conditions including, but not limited to, changes in pH, low nutrients, predation, and antimicrobial compounds. Communication systems based on N-acylhomoserine lactones (AHLs) have been described in a range of gram-negative bacteria and are proposed to be involved in causing such diverse problems as food spoilage and infection of the lungs. This bacterial communication system functions via small diffusible AHL signal molecules. This chapter discusses the various genetic and chemical tools used for investigating signaling processes in biofilms.

A tuneable switch for controlling environmental degradation of bioplastics: addition of isothiazolinone to polyhydroxyalkanoates

Controlling the environmental degradation of polyhydroxybutyrate (PHB) and polyhydroxyvalerate (P(HB-co-HV)) bioplastics would expand the range of their potential applications. Combining PHB and P(HB-co-HV) films with the anti-fouling agent 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOI, <10% w/w) restricted microbial colonisation in soil, but did not significantly affect melting temperature or the tensile strength of films. DCOI films showed reduced biofouling and postponed the onset of weight loss by up to 100 days, a 10-fold increase compared to unmodified films where the microbial coverage was significant. In addition, the rate of PHA-DCOI weight loss, post-onset, reduced by about 150%; in contrast a recorded weight loss of only 0.05% per day for P(HB-co-HV) with a 10% DCOI loading was observed. This is in stark contrast to the unmodified PHB film, where a recorded weight loss of only 0.75% per day was made. The ‘switch’ that initiates film weight loss, and its subsequent reduced rate, depended on the DCOI loading to control biofouling. The control of biofouling and environmental degradation for these DCOI modified bioplastics increases their potential use in biodegradable applications.

A repeatable biofilm removal assay and its application in the assessment of commercial cleaning formulations for medical devices

Bacterial biofilms are an important target of cleaning protocols for reusable medical devices. One approach to assess the efficacy of chemical cleaning formulations against this challenging type of soil is to grow biofilm of a specific strain on test coupons and measure the amount of biofilm removed after coupons have been immersed in a cleaning solution or control solution. This study reports on a comparison of four commercial formulations and two defined solutions in a biofilm removal assay. Biofilms of Pseudomonas aeruginosa (ATCC 25619) were grown on polytetrafluoroethylene coupons in a stirred reactor. A crystal violet assay was used to measure percent reduction of biofilm from test coupons by four commercial formulations and two defined solutions (sodium hydroxide and Tween 20). There was a significant pair-wise difference between all treatments (P < 0.05), other than one of the commercial formulations (3M RMEC 70505) and sodium hydroxide. A high level of repeatability was achieved with coefficients of variation from 1 to 19% for the different treatments. The repeatability of this method may allow an objective assessment of biofilm removal efficacy of commercial formulations used for cleaning medical devices.