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Serratia species are an important source of antimicrobial compounds with numerous studies concerning their production of secondary metabolites with applications in medicine, pharmaceutics and biotechnology. This study continued investigations on the antibacterial activity of an environmental isolate of Serratia plymuthica. This activity could be detected on agar plates against Gram-positive bacteria. A previous study reported the isolation of KmR transposon mutants deficient in the activity identified on agar plates but appeared to possess another activity secreted in liquid media. Those mutants had Tn5 insertions into genes encoding polyketide synthases (PKSs). The multiple antibacterial activities of S. plymuthica indicate the production of antimicrobials that can diffuse into aqueous environments in addition to the ones closely bound to the outer cell surface and secreted into solid mediums. This raised the question about the relationship between the two activities, were they distinct or related? This study investigated the secreted antibacterial activity of S. plymuthica using transposon mutagenesis to create mutants deficient in the secreted antibacterial activity and to identify the genes involved in antibacterial production. Cultivation parameters such as; culture medium, aeration and temperature had a clear impact on the production of the secreted antibacterial activity. Growing S. plymuthica in MRS broth at 16oC for 48h-72h produced the highest amount of inhibitory activity detected in the CFCS. The secreted antibacterial activity was thermo-stable and remained active even after sterilization (121°C/15mins) and was insensitive to methanol, Proteinase K, Tween and pH. However, the antibacterial activity faded after few days even when stored at (4oC). The antibacterial activity was bactericidal and targeted a sub-set of Gram-postive bacteria especially Bacillaceae. The CFCS precipitated in the presences of solvents such as methanol, Tris-buffer and Tween. Structural characterization of the antibacterial compound(s) was confirmed via NMR and COSY spectrometry and showed the presence of two polyketide compounds resembling the macrolide antibiotics erythromycin and rapamycin produced by type I PKSs. Erythromycin and rapamycin are anti-bacterial and immune-suppressant compounds. Results of this study indicate that the secreted antibacterial activity of S. plymuthica is synthesized by PKSs that are distinct from other known Serratia PKSs. Results are also consistent with the possibility of S. plymuthica possessing separate biochemical pathways leading to two distinct antibacterial activities or a branched biochemical pathway. However, future studies are needed to reveal if the genes responsible for the two activities are in separate genomic locations and regulated in different manners to prove that the strain possesses two separate biochemical pathways leading to the two distinct activities. Further work is needed to unlock these unanswered questions. This includes genomic studies in order to identify new secondary metabolites with future importance that might be produced by this novel strain of S. plymuthica.

Butylated hydroxyanisole (BHA) is a synthetic substance commonly used as dietary antioxidant which plays an important role in protecting foods from oxidation. It has been reported that this compound caused an increase in glutathione-s-transferase specific activity, which is responsible for resistance to oxidative stress, i.e., there was some evidence that it could act as a pro-oxidant. In the present study, BHA was found to be toxic towards yeast and this was investigated further by determining its effect on the viability of a variety of yeast mutants lacking key genes required for resistance to oxidative stress. The results showed that the cell viability of the mutants was not significantly different from the wild type parental strains. This suggests that BHA toxicity was unlikely to involve reactive oxygen species (O2 - and H2O2) and might not directly involve the classical oxidant stress responses found in yeast cells. Interestingly, the pre-treatment of yeast cells with low concentrations of BHA (0.2 mM) and subsequent exposure to higher concentrations of either BHA or hydrogen peroxide (H2O2) resulted in pre-treated yeast cells becoming more resistant to both toxic levels of BHA and H2O2 than non pre-treated cells. These findings indicated that low level of BHA could induce an adaptive response to BHA and induce cross-protection against H2O2. A genetic approach was adopted to identify genes involved in this process, involving the isolation and characterisation of BHA sensitive mutants. The results demonstrated that the yeast genes TYR1, KRE6 and GPH1 play role in the cells response towards BHA.

During a screening of seaweed associated marine micro organisms, for their ability to produce antimicrobial compounds, an organism was isolated, producing antibacterial metabolite(s) and was found to be active against gram positive pathogens, multi-drug resistant pathogenic bacteria, and food borne spoilage fungi. The strain was identified as a gram-negative, short rod, which produced red-pigmented colonies and proved to be both catalase and oxidase positive. Ribotyping, indicated that this strain demonstrated a high sequence identity (99%) to Serratia plymuthica. Tests with a type strain of Serratia plymuthica, revealed that the strain did not produce a similar spectrum of antimicrobial activity. Preliminary characterisation of the inhibitory molecule(s) produced by this marine isolate, demonstrated that it was heat stable, low molecular weight, with pH resistant compound(s). Moreover, the activity was unaffected by prolonged incubation with enzymes such as Proteinase K. Transposon, mutagenesis was utilised, in an attempt to identify the genes involved, in antimicrobial production, to hopefully gain an insight into the nature of the antimicrobial compound. Fourteen mutants, unable to produce antimicrobial compound(s) were isolated, and the preliminary sequence analysis of three of the mutants, highlighted that they had transposon insertions within gene(s) homologous to polyketide synthase. Size exclusion chromatography (Gel filtration) was utilised in an attempt to purify and characterise the antimicrobial compound(s).

The widely distributed marine bivalve Mytilus edulis is used as a sentinel organism for ecological and toxicological assessments. As a filter feeder, it has the potential to bio-accumulate pollutants. It has been assumed that the cell concentration and cell type ratio of its circulating immune cells, haemocytes, could become complementary sub-lethal indicators of toxicology. These two parameters are respectively referred to as total haemocyte count (THC) and differential cell count (DCC). This study examines these commonly used methods, quantifies their limitations, and develops alternative techniques. The circulating immune cells are investigated to assess their fluctuations. Finally, impacts of environmental challenges on the circulating haemocytes are examined. Despite its importance in the field of Mytilus edulis immunology, THC evaluation is present in only 20% of publications in this field, and DCC in 10%. Ultimately, only 9% of papers consider both THC and DCC before further analyses. The remaining studies disregard THC and DDC, or regard these parameters as being constant and homogenous in M. edulis populations. This study initially quantifies the systematic error induced by sampling, and suggests improvements. For example, a systematic error of 26% is attributed to the use of low precision syringes, and can be reduced with use of higher precision sampling equipment. While the systematic errors in visual count and image analysis of THC and DCC evaluations are equivalent, the computerised methods allow the throughput of larger data sets, reduce workload, and avoid tedious eye counts. Flow cytometry was found to be the most accurate method in THC and DCC evaluation. Furthermore, repeated bleedings influence DCC, triggering the decrease of circulating eosinophils (up to 20%) and the increase of hyaline cells (up to 30%). To mitigate this reaction to sampling, a maximum volume of 50μl using a permanent cannulation is recommended. However, even with improved methods reducing systematic error by half, this study still reports variations as high as 20-fold in the haemocyte concentration in populations of healthy individuals. In addition, over a 2-hour period in a single cannulated individual, fluctuation of the DDC is observed to be as high as 30% for eosinophils, 10% for hyaline cells, and 20% for basophils. These measured variations are explained by haemocyte reservoirs in the tissues. Eosinophils are found in large numbers in epithelial association in the gills, guts and in the mantle, where their numbers have been evaluated at 3x10 7 As a case study, Mytilus edulis challenged with barium sulphate smothering, used by the oil industry in drilling muds, shows gill damage and their subsequent infiltration by eosinophils. In bacterial outbreak, basophilic cells are observed to infiltrate the tissue surrounding the stomach and eosinophils are depleted from the epithelium of the digestive tissues. THC is 10-fold lower than in healthy organisms and eosinophils are depleted from the haemolymph. cells. In conclusion, the THC and DDC methods are shown to be unreliable despite the use of more accurate methods. In addition, Mytilus edulis circulating haemocytes present large variations and the assumption of their homogeneity in terms of time, individuals or methods used cannot be made. These results challenge the conclusions of many past publications regarding causalities established between potential stresses and measured effects. Further research is necessary to understand the mechanisms regulating the circulating haemocytes, the inter-individual variability and to improve investigation methods.