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Genome Organization and Localization of the pufLM Genes of the Photosynthesis Reaction Center in Phylogenetically Diverse Marine Alphaproteobacteria. Silke Pradella, 2004.Genome organization, plasmid content and localization of the pufLM genes of the photosynthesis reaction center were studied by pulsed-field gel electrophoresis (PFGE) in marine phototrophic Alphaproteobacteria . Both anaerobic phototrophs (Rhodobacter veldkampii and Rhodobacter sphaeroides) and strictly aerobic anoxygenic phototrophs from the Roseobacter-Sulfitobacter-Silicibacter clade (Roseivivax halodurans, Roseobacter litoralis, Staleya guttiformis, Roseovarius tolerans, and five new strains isolated from dinoflagellate cultures) were investigated . The complete genome size was estimated for R . litoralis DSM6996T to be 4,704 kb, including three linear plasmids . All strains contained extrachromosomal elements of various conformations (linear or circular) and lengths (between 4.35 and 368 kb) . In strain DFL-12, a member of a putative new genus isolated from a culture of the toxic dinoflagellate Prorocentrum lima, seven linear plasmids were found, together comprising 860 kb of genetic information . Hybridization with probes against the pufLM genes of the photosynthesis gene cluster after Southern transfer of the genomic DNAs showed these genes to be located on a linear plasmid of 91 kb in R . litoralis and on a linear plasmid of 120 kb in S . guttiformis, theoretically allowing their horizontal transfer . In all other strains, the pufLM genes were detected on the bacterial chromosome . The large number and significant size of the linear plasmids found especially in isolates from dinoflagellates might account for the metabolic versatility and presumed symbiotic association with eukaryotic hosts in these bacteria . Evolving Insights: Symbiosis Islands and Horizontal Gene Transfer. Turlough M. Finan, 2002. Characterization of the Depletion of 2-C-Methyl-D-Erythritol-2,4-Cyclodiphosphate Synthase in Escherichia coli and Bacillus subtilis. Tracey L. Campbell, 2002.The ispF gene product in Escherichia coli has been shown to catalyze the formation of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEC) in the deoxyxylulose (DOXP) pathway for isoprenoid biosynthesis . In this work, the E . coli gene ispF and its Bacillus subtilis orthologue, yacN, were deleted and conditionally complemented by expression of these genes from distant loci in the respective organisms . In E . coli, complementation was achieved through integration of ispF at the araBAD locus with control from the arabinose-inducible araBAD promoter, while in B . subtilis, yacN was placed at amyE under control of the xylose-inducible xylA promoter . In both cases, growth was severely retarded in the absence of inducer, consistent with these genes being essential for survival . E . coli cells depleted of MEC synthase revealed a filamentous phenotype . This was in contrast to the depletion of MEC synthase in B . subtilis, which resulted in a loss of rod shape, irregular septation, multicompartmentalized cells, and thickened cell walls . To probe the nature of the predominant deficiency of MEC synthase-depleted cells, we investigated the sensitivity of these conditionally complemented mutants, grown with various concentrations of inducer, to a wide variety antibiotics . Synthetic lethal behavior in MEC synthase-depleted cells was prevalent for cell wall-active antibiotics . Phenotypic Mutants of the Intracellular Actinomycete Rhodococcus equi Created by In Vivo Himar1 Transposon Mutagenesis. Joseph Ashour, 2003.Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses . An effective live attenuated vaccine would be extremely useful in the prevention of R . equi disease in horses . Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R . equi mutants . We show that Himar1 transposition in R . equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide . The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes . One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R . equi . We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis . This mutant cannot grow in minimal medium in the absence of riboflavin supplementation . Experimental murine infection studies showed that, in contrast to wild-type R . equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo . The mutagenesis methodology we have developed will allow the characterization of R . equi virulence mechanisms and the creation of other attenuated strains with vaccine potential .
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