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Identification of a Gene Cluster in Klebsiella pneumoniae Which Includes citX, a Gene Required for Biosynthesis of the Citrate Lyase Prosthetic Group. Karin Schneider, 2002.The biosynthesis of the 2'-(5"-phosphoribosyl)-3'-dephospho-coenzyme A (CoA) prosthetic group of citrate lyase (EC 4.1.3.6), a key enzyme of citrate fermentation, proceeds via the initial formation of the precursor 2'-(5"-triphosphoribosyl)-3'-dephospho-CoA and subsequent transfer to apo-citrate lyase with removal of pyrophosphate . In Escherichia coli, the two steps are catalyzed by CitG and CitX, respectively, and the corresponding genes are part of the citrate lyase gene cluster, citCDEFXG . In the homologous citCDEFG operon of Klebsiella pneumoniae, citX is missing . A search for K . pneumoniae citX led to the identification of a second genome region involved in citrate fermentation which comprised the citWX genes and the divergent citYZ genes . The citX gene was confirmed to encode holo-citrate lyase synthase, whereas citW was shown to encode a citrate carrier, the third one identified in this species . The citYZ genes were found to encode a two-component system consisting of the sensor kinase CitY and the response regulator CitZ . Remarkably, both proteins showed  40% sequence identity to the citrate-sensing CitA-CitB two-component system, which is essential for the induction of the citrate fermentation genes in K . pneumoniae . A citZ insertion mutant was able to grow anaerobically with citrate, indicating that CitZ is not essential for expression of citrate fermentation genes . CitX synthesis was induced to a basal level under anaerobic conditions, independent of citrate, CitB, and CitZ, and to maximal levels during anaerobic growth with citrate as the sole carbon source . Similar to the other citrate fermentation enzymes, CitX synthesis was apparently subject to catabolite repression .
Characterization of divIVA and Other Genes Located in the Chromosomal Region Downstream of the dcw Cluster in Streptococcus pneumoniae. Daniela Fadda, 2003.We analyzed the chromosome region of Streptococcus pneumoniae located downstream of the division and cell wall (dcw) cluster that contains the homolog of the Bacillus subtilis cell division gene divIVA and some genes of unknown function . Inactivation of divIVA in S . pneumoniae resulted in severe growth inhibition and defects in cell shape, nucleoid segregation, and cell division . Inactivation of the ylm genes resulted in some morphological and/or division abnormalities, depending on the inactivated gene . Transcriptional analysis revealed a relationship between these genes and the ftsA and ftsZ cell division genes, also indicating that the connection between the dcw cluster and the divIVA region is more extensive than just chromosomal position and gene organization . Real-Time Reverse Transcription-PCR Analysis of Expression of Halobenzoate and Salicylate Catabolism-Associated Operons in Two Strains of Pseudomonas aeruginosa. M. E. Corbella, 2003.Pseudomonas aeruginosa JB2 can use 2-chlorobenzoate (2-CBa), 3-CBa, 2,3-dichlorobenzoate (2,3-DCBa), and 2,5-DCBa as sole carbon and energy sources, whereas strain 142 can only grow on 2-CBa and 2,4-DCBa . Both strains, however, harbor the same halobenzoate 1,2-dioxygenase (ohbAB) and chlorocatechol (clcABD) degradation genes necessary for the metabolism of ortho-CBas . In addition, the hybABCD operon, encoding a salicylate 5-hydroxylase, is also found in both strains . The expression of ohbAB, hybABCD, and clcABD operons was measured in cultures grown on different CBas as the sole carbon source and also in glucose-grown cells supplemented with CBas as inducers . A method to standardize real-time reverse transcription-PCR experimental data was used that allows the comparison of semiquantitative mRNA accumulation in different strains and culture conditions . In both strains, the ohb and hyb systems were induced in cells grown on 2-CBa or DCBas, whereas clc was induced only by DCBas . Repression by catabolite was observed both on ohb and clc systems in glucose-grown cells . Chlorocatechol 1,2-dioxygenase activity in JB2 was detected even in clc-repressed conditions, confirming the presence of additional isofunctional genes previously detected in P . aeruginosa 142 . Although similar levels of induction of ohbAB were observed in strain JB2 grown on either benzoate, monochlorobenzoates, or DCBas, the ohbAB operon of strain 142 was only strongly induced by growth on 2-CBa and, to a lesser extent, on 2,4-DCBa . This observation suggests that regulation of the ohbAB operon may be different in both strains . The concomitant induction of ohb and hyb by CBas may allow the formation of hybrid halobenzoate dioxygenase(s) composed of Ohb/Hyb dioxygenase subunits and Hyb ferredoxin/ferredoxin reductase components .
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