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A Novel Evolutionary Lineage of Carbonic Anhydrase ( 
Class) Is a Component of the Carboxysome Shell.Anthony K.-C. So, 2004.A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes . In cyanobacteria and many chemolithoautotrophic bacteria, CO2 fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes . These structures play an integral role in a cellular CO2-concentrating mechanism and are essential components for autotrophic growth . Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase ( -class
CA) that has an evolutionary lineage distinct from those previously
recognized in animals, plants, and other prokaryotes . Functional CAs
encoded by csoS3 homologues were also identified in the
cyanobacteria Prochlorococcus sp . and Synechococcus
sp., which dominate the oligotrophic oceans and are major
contributors to primary productivity . The location of the
carboxysomal CA in the shell suggests that it could supply the active
sites of RuBisCO in the carboxysome with the high concentrations of
CO2 necessary for optimal RuBisCO activity and efficient
carbon fixation in these prokaryotes, which are important
contributors to the global carbon cycle .
S-Adenosylmethionine Transport in Rickettsia prowazekii. Aimee M. Tucker, 2003.Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate, intracellular, parasitic bacterium that grows within the cytoplasm of eucaryotic host cells . Rickettsiae exploit this intracellular environment by using transport systems for the compounds available in the host cell's cytoplasm . Analysis of the R . prowazekii Madrid E genome sequence revealed the presence of a mutation in the rickettsial metK gene, the gene encoding the enzyme responsible for the synthesis of S-adenosylmethionine (AdoMet) . Since AdoMet is required for rickettsial processes, the apparent inability of this strain to synthesize AdoMet suggested the presence of a rickettsial AdoMet transporter . We have confirmed the presence of an AdoMet transporter in the rickettsiae which, to our knowledge, is the first bacterial AdoMet transporter identified . The influx of AdoMet into rickettsiae was a saturable process with a KT of 2.3 µM . Transport was inhibited by S-adenosylethionine and S-adenosylhomocysteine but not by sinfungin or methionine . Transport was also inhibited by 2,4-dinitrophenol, suggesting an energy-linked transport mechanism, and by N-ethylmaleimide . AdoMet transporters with similar properties were also identified in the Breinl strain of R . prowazekii and in Rickettsia typhi . By screening Escherichia coli clone banks for AdoMet transport, the R . prowazekii gene coding for a transporter, RP076 (sam), was identified . AdoMet transport in E . coli containing the R . prowazekii sam gene exhibited kinetics similar to that seen in rickettsiae . The existence of a rickettsial transporter for AdoMet raises intriguing questions concerning the evolutionary relationship between the synthesis and transport of this essential metabolite . Growth Phase-Dependent Regulation of Target Gene Promoters for Binding of the Essential Orphan Response Regulator HP1043 of Helicobacter pylori. Isabel Delany, 2002.Helicobacter pylori encodes three two-component systems and two orphan response regulators (RRs) that are predicted to be involved in transcriptional regulation . The HP1043 gene encodes an essential OmpR-like RR, 1043RR, for which no histidine kinase has been identified . Gel filtration and cross-linking experiments on the purified 1043RR protein reveals that this protein is a dimer and in vivo dimerization assays localize the dimerization to the N-terminal regulatory domain . DNA-binding studies have revealed two targets for specific binding of the 1043RR protein and moreover, phosphorylation of the protein was not needed for the activation of binding . Footprinting analysis demonstrated that the 1043RR protein binds to its own promoter, P1043, overlapping the -35 promoter element from positions -17 to -45, suggesting that this protein is autoregulatory . In addition, it binds at a similar location, spanning nucleotides from positions -22 to -51 at the promoter of the methyl-accepting chemotaxis tlpB gene, PtlpB . A possible inverted repeat was identified in the binding sites of both promoters . In an attempt to overexpress 1043RR in H . pylori, the 10-fold induction in transcription of a second copy of HP1043 with use of an inducible promoter failed to increase cellular levels of the RR protein, suggesting that 1043RR is tightly regulated at a posttranscriptional level . The P1043 and PtlpB promoters were demonstrated to be coordinately regulated in response to growth phase in H . pylori . The essential role of HP1043 in encoding a cell cycle regulator is discussed . ClpE from Lactococcus lactis Promotes Repression of CtsR-Dependent Gene Expression. Pekka Varmanen, 2003.The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level . In this study we found that after a shift to a high temperature the Clp ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR) . Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in expression of clpP encoding the proteolytic component of the Clp protease complex, this decrease was delayed in the absence of ClpE . Site-directed mutagenesis of the zinc-binding motif conserved in ClpE ATPases interfered with the ability to repress CtsR-dependent expression . Quantification of ClpE by Western blot analysis revealed that at a high temperature ClpE is subjected to ClpP-dependent processing and that disruption of the zinc finger domain renders ClpE more susceptible . Interestingly, this domain resembles the N-terminal region of McsA, which was recently reported to interact with the CtsR homologue in Bacillus subtilis . Thus, our data point to a regulatory role of ClpE in turning off clpP gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR . Characterization of an Exo-ß-D-Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1. Takeshi Tanaka, 2003.We previously clarified that the chitinase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 produces diacetylchitobiose (GlcNAc2) as an end product from chitin . Here we sought to identify enzymes in T . kodakaraensis that were involved in the further degradation of GlcNAc2 . Through a search of the T . kodakaraensis genome, one candidate gene identified as a putative ß-glycosyl hydrolase was found in the near vicinity of the chitinase gene . The primary structure of the candidate protein was homologous to the ß-galactosidases in family 35 of glycosyl hydrolases at the N-terminal region, whereas the central region was homologous to ß-galactosidases in family 42 . The purified protein from recombinant Escherichia coli clearly showed an exo-ß-D-glucosaminidase (GlcNase) activity but not ß-galactosidase activity . This GlcNase (GlmATk), a homodimer of 90-kDa subunits, exhibited highest activity toward reduced chitobiose at pH 6.0 and 80°C and specifically cleaved the nonreducing terminal glycosidic bond of chitooligosaccharides . The GlcNase activity was also detected in T . kodakaraensis cells, and the expression of GlmATk was induced by GlcNAc2 and chitin, strongly suggesting that GlmATk is involved in chitin catabolism in T . kodakaraensis. These results suggest that T . kodakaraensis, unlike other organisms, possesses a novel chitinolytic pathway where GlcNAc2 from chitin is first deacetylated and successively hydrolyzed to glucosamine . This is the first report that reveals the primary structure of GlcNase not only from an archaeon but also from any organism . Regio- and Stereoselective Metabolism of 7,12-Dimethylbenz[a]anthracene by Mycobacterium vanbaalenii PYR-1. Joanna D. Moody, 2003.The degradation of 7,12-dimethylbenz[a]anthracene (DMBA), a carcinogenic polycyclic aromatic hydrocarbon, by cultures of Mycobacterium vanbaalenii PYR-1 was studied . When M . vanbaalenii PYR-1 was grown in the presence of DMBA for 136 h, high-pressure liquid chromatography (HPLC) analysis showed the presence of four ethyl acetate-extractable compounds and unutilized substrate . Characterization of the metabolites by mass and nuclear magnetic resonance spectrometry indicated initial attack at the C-5 and C-6 positions and on the methyl group attached to C-7 of DMBA . The metabolites were identified as cis-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA cis-5,6-dihydrodiol), trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA trans-5,6-dihydrodiol), and 7-hydroxymethyl-12-methylbenz[a]anthracene, suggesting dioxygenation and monooxygenation reactions . Chiral stationary-phase HPLC analysis of the dihydrodiols showed that DMBA cis-5,6-dihydrodiol had 95% 5S,6R and 5% 5R,6S absolute stereochemistry . On the other hand, the DMBA trans-5,6-dihydrodiol was a 100% 5S,6S enantiomer . A minor photooxidation product, 7,12-epidioxy-7,12-dimethylbenz[a]anthracene, was also formed . The results demonstrate that M . vanbaalenii PYR-1 is highly regio- and stereoselective in the degradation of DMBA .
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