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Identification of Carnobacterium Species by Restriction Fragment Length Polymorphism of the 16S-23S rRNA Gene Intergenic Spacer Region and Species-Specific PCR. Cinta Rachman, 2004.The genus Carnobacterium is currently divided into the following eight species: Carnobacterium piscicola, C . divergens, C . gallinarum, C . mobile, C . funditum, C . alterfunditum, C . inhibens, and C . viridans . An identification tool for the rapid differentiation of these eight Carnobacterium species was developed, based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR) . PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of this 16S-23S rDNA ISR was performed in order to obtain restriction profiles for all of the species . Three PCR amplicons, which were designated small ISR (S-ISR), medium ISR (M-ISR), and large ISR (L-ISR), were obtained for all Carnobacterium species . The L-ISR sequence revealed the presence of two tRNA genes, tRNAAla and tRNAIle, which were separated by a spacer region that varied from 24 to 38 bp long . This region was variable among the species, allowing the design of species-specific primers . These primers were tested and proved to be species specific . The identification method based on the 16S-23S rDNA ISR, using PCR-RFLP and specific primers, is very suitable for the rapid low-cost identification and discrimination of all of the Carnobacterium species from other phylogenetically related lactic acid bacteria . In Vivo and In Vitro Studies of Bacillus subtilis Ferrochelatase Mutants Suggest Substrate Channeling in the Heme Biosynthesis Pathway. Ulf Olsson, 2002.Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway . The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known . Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have . The effects of these changes were studied in vivo and in vitro . S54 and Q63 are both located at helix  3 . The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure . None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure . The exchange S54A, but not Q63A, reduced the growth rate of B . subtilis and resulted in the accumulation of coproporphyrin III in the growth medium . This was in contrast to the in vitro activity measurements with the purified enzymes . The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in Vmax . The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product .
PlmA, a New Member of the GntR Family, Has Plasmid Maintenance Functions in Anabaena sp . Strain PCC 7120. Martin H. Lee, 2003.The filamentous cyanobacterium Anabaena (Nostoc) sp . strain PCC 7120 maintains a genome that is divided into a 6.4-Mb chromosome, three large plasmids of more that 100 kb, two medium-sized plasmids of 55 and 40 kb, and a 5.5-kb plasmid . Plasmid copy number can be dynamic in some cyanobacterial species, and the genes that regulate this process have not been characterized . Here we show that mutations in an open reading frame, all1076, reduce the numbers of copies per chromosome of several plasmids . In a mutant strain, plasmids pCC7120  and pCC7120 are both reduced to less than 50% of their wild-type levels . The exogenous pDU1-based plasmid pAM1691 is reduced to less than 25% of its wild-type level, and the plasmid is rapidly lost . The peptide encoded by all1076 shows similarity to members of the GntR family of transcriptional regulators . Phylogenetic analysis reveals a new domain topology within the GntR family . PlmA homologs, all coming from cyanobacterial species, form a new subfamily that is distinct from the previously identified subfamilies . The all1076 locus, named plmA, regulates plasmid maintenance functions in Anabaena sp . strain PCC 7120 .
Diversity of Wolbachia Endosymbionts in Heteropteran Bugs. Yoshitomo Kikuchi, 2003.An extensive survey of Wolbachia endosymbionts in Japanese terrestrial heteropteran bugs was performed by PCR detection with universal primers for wsp and ftsZ genes of Wolbachia, cloning of the PCR products, restriction fragment length polymorphism analysis of infecting Wolbachia types, and molecular phylogenetic characterization of all the detected Wolbachia strains . Of 134 heteropteran species from 19 families examined, Wolbachia infection was detected in 47 species from 13 families . From the 47 species, 59 Wolbachia strains were identified . Of the 59 strains, 16 and 43 were assigned to A group and B group in the Wolbachia phylogeny, respectively . The 47 species of Wolbachia-infected bugs were classified into 8 species with A infection, 28 species with B infection, 2 species with AA infection, 3 species with AB infection, 5 species with BB infection, and 1 species with ABB infection . Molecular phylogenetic analysis showed little congruence between Wolbachia phylogeny and host systematics, suggesting frequent horizontal transfers of Wolbachia in the evolutionary course of the Heteroptera . The phylogenetic analysis also revealed several novel lineages of Wolbachia . Based on statistical analyses of the multiple infections, we propose a hypothetical view that, in the heteropteran bugs, interactions between coinfecting Wolbachia strains are generally not intense and that Wolbachia coinfections have been established through a stochastic process probably depending on occasional horizontal transfers .
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