Copper-and zinc-containing superoxide dismutases ([Cu,Zn]-SODS) are generally considered almost exclusively eukaryotic enzymes, protecting the cytosol and extracellular compartments of higher organisms from damage by oxygen free-radicals. The recent description of a few examples of bacterial forms of the enzyme, located in the periplasm of different Gram-negative micro-organisms, prompted a re-evaluation of this general perception. A PCRbased approach has been developed and used successfully to identify bacterial genes encoding [Cu,Zn]

Rationally designed mariner vectors to allow functional genomic analysis of 1 Actinobacillus pleuropneumoniae and other bacteria by transposon-directed insertion-2 site sequencing (TraDIS) 3 4 Abstract 29 30Transposon Directed Insertion Sequencing (TraDIS) is a high-throughput 31 method for mapping insertion sites in large libraries of transposon mutants. The 32 Himar1 (mariner) transposon is ideal for generating near-saturating mutant 33 libraries, especially in AT-rich chromosomes, as the requirement for integration is 34 a TA dinucleotide. In this study, we generated two novel mariner vectors, 35 pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the 36 transposase gene), in order to facilitate TraDIS identification of conditionally 37 essential genes in Actinobacillus pleuropneumoniae and other bacteria. Using the 38 pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant 39 libraries in both A. pleuropneumoniae and Pasteurella multocida that showed a 40 near random distribution of insertions around the respective chromosomes. A 41 preliminary screen of 5000 mutants each identified 8 and 15 genes, respectively, 42 that are required for growth under anaerobic conditions. 43 44 Importance 45 46 Comprehensive identification of conditionally essential genes requires 47 efficient tools for generating high-density transposon libraries that, ideally, can be 48 analysed using next-generation sequencing methods. The mariner transposon 49 has been used for mutagenesis of a wide variety of bacteria, however plasmids for 50 delivery of this transposon do not necessarily work well in all bacteria. In 51 particular, there are limited tools for functional genomic analysis of 52Pasteurellaceae species of major veterinary importance, such as swine and cattle 53 pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, 54 respectively. Here, we have developed plasmids that allow delivery of mariner and 55 the production of genome saturated mutant libraries for both of these pathogens, 56 but which should also be applicable to a wider range of bacteria. High-throughput 57 screening of the generated libraries will identify mutants required for growth under 58 different conditions, including in vivo, highlighting key virulence factors and 59 pathways that can be exploited for development of novel therapeutics and 60 vaccines. 61 62

Neisseria meningitidis is a nasopharyngeal commensal of humans which occasionally invades the blood to cause septicaemia. The transcriptome of N. meningitidis strain MC58 grown in human blood for up to 4 hours was determined and around 10% of the genome was found to be differentially regulated. The nuo, pet and atp operons, involved in energy metabolism, were up-regulated, while many house-keeping genes were down-regulated. Genes encoding protein chaperones and proteases, involved in the stress response; complement resistant genes encoding enzymes for LOS sialylation and biosynthesis; and fHbp (NMB1870) and nspA (NMB0663), encoding vaccine candidates, were all up-regulated. Genes for glutamate uptake and metabolism, and biosynthesis of purine and pyrimidine were also up-regulated. Blood grown meningococci are under stress and undergo a metabolic adaptation and energy conservation strategy. The localisation of four putative outer membrane proteins encoded by genes found to be up-regulated in blood was assessed by FACS using polyclonal mouse antisera, and one (NMB0390) showed evidence of surface expression, supporting its vaccine candidacy.