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Staphylococcus aureus is a major human pathogen causing a wide range of illnesses from the trivial to the life-threatening. S. aureus produces many surface-associated and exoproteins, several of which have been implicated in its virulence. Production of these virulence determinants is co-ordinately controlled by several global regulatory elements in a growth phase dependent manner. The best characterised of these regulators are the accessory gene regulator (agr) and staphylococcal accessory regulator (sar). The agr locus comprises a quorum sensing system and encodes a signalling pheromone that autoregulates agr in a density dependent manner. Upregulation of agr expression leads to production of an mRNA transcript, RNAHI which is the actual effector of virulence gene expression. The RNAIII molecule upregulates several extracellular toxins including haemolysins, toxic shock syndrome toxin 1 (TSST-1) and epidermolytic toxin A (Eta), and down-regulates surface proteins such as protein A and fibronectin binding protein (FnBP) during late exponential growth and stationary phase. The regulation of toxin production by S. aureus is extremely complex and it is not yet understood exactly how this organism responds to environmental stimuli in order to mediate changes in virulence gene expression. In order to determine whether environmental signals are transduced via agr, the effect of several stimuli on both agr expression and a-haemolysin production was examined using a ß-galactosidase reporter gene fusion to the hid gene, which is encoded as part of the RNAIII transcript. A number of environmental stimuli were identified which led to changes in agr expression. Several of these stimuli resulted in different effects on a- haemolysin activity when compared to RNAIII levels. This suggests the presence of novel regulatory elements involved in the control of Hla production, independently of agr. In order to identify other novel regulators which interact with, or control, agr, transposon libraries have been created using Tn917 and Tn551. Two Tn917 transposon mutants were isolated as deficient in production of ß-haemolysin, which is also positively controlled by agr. These mutants were found to contain novel transposon insertions in the agr locus. Five Tn551 mutants were isolated which showed pleiotropic effects on virulence determinant levels and did not contain the transposon in previously mapped regulators. The Tn551 insertions may have therefore occurred in novel regulators of virulence determinant production. The regulation of toxin production by S. aureus in response to environmental stimuli is discussed.

New methodologies have been developed for the determination of arsenic and selenium species in a variety of environmentally important matrices. A simple liquid chromatographic separation technique based upon mini-column technology was developed to obtain a simultaneous, fast, efficient and reliable separation of relatively toxic from relatively non-toxic arsenic and selenium species. The relatively toxic arsenic and selenium species studied were inorganic Asv, AsIII, SeVI and SeIV. The relatively non-toxic species of arsenic and selenium studied were AsBet, DMA and Se Met. Optimum conditions were found to be the use of a Hamilton PRP X100 12-20 µm anion-exchange resin with column dimensions of 100 x 3 mm. The mobile phase utilized a 10 mM K2S04 solution at pH 10.2 with a flow rate of 1 ml minˉ¹ and a sample injection loop of 100 µ1. Total analysis time was under 7 minutes with limits of detection in the range of 2.0 - 10 µg kgˉ¹ for arsenic and selenium species, respectively. Work was undertaken, using HPLC-ICP-MS instrumentation, as part of a feasibility study, into the production and certification of six new reference materials; these being analyzed for the species of arsenic, in chicken, fish, rice and soil samples, and selenium, in wheat and yeast samples. Enzyme extraction techniques were used throughout, except for soil where a microwave H3P04 extraction was used. Efficiencies were in the range 90-100%. The results obtained provided speciation information as well as total elemental concentrations with no operationally defined limits. Speciation analysis requires that the endogenous species are extracted without modification of their chemical form or disturbance to the equilibrium existing between the various species present. Work was undertaken to identify and quantify the selenium species present in two samples of novel, previously unstudied, bio-natured nutrients, these nutrients being: i) a selenized yeast from a new process and: ii) a probiotic bacteria-based dried milk sample (Biogurt®). Specific interest was directed towards enzyme, MeOH and KOH and TMAH extraction efficiencies together with retention of species information. Selenium speciation was performed using ion-exchange HPLC-ICP-MS. It was found that the selenium content, in the form of SeMet, was adequately extracted from the yeast (Pharma Nord) that was used for method validation using protease, which yielding 90% of the total selenium. However, the determination of selenium and selenium species in the bionatured nutrients proved to be quite problematic. Methods that avoided species conversion with the highest extraction efficiencies were found to be: i) the use of protease for the yeast sample (19%) and; ii) the use of 0.01 M HCl for the Biogurt® (71%). Information obtained from speciation of these samples by anion and cation-exchange HPLC-ICP-MS was limited due to the low extraction efficiencies of any procedure undertaken for the samples, by the retention of the analyte on-column and by the lack of standards available for matching of retention times. HPLC-ICP-MS has proved an efficient tool for the identification and determination of arsenic and selenium species providing detection limits at µg kgˉ¹ levels. However, a major concern with this instrumentation is the unambiguous assignment of peaks which relies on the chromatographic purity of the signal and the availability of standards. Anion-exchange chromatography employing Hamilton PRP X100 resin with NH4HC03 (10 mM, pH 10.2 for arsenic and 10-50 mM, pH 5 for selenium species) with methanol (10 %, v/v) as the mobile phase allowed separation of the arsenic and selenium species investigated under conditions that were compatible for both HPLC-ICP-MS and HPLC-ESMS. Molecular ions and structural fragmentation patterns of these by tandem MS have facilitated the identification of chromatographic peaks obtained using HPLC-ICP-MS. In the analysis of marine algae, arsenosugars were the major species found, and in yeast the dominant species was found to be selenomethionine.

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