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The Pseudomonas putida Crc Global Regulator Controls the Expression of Genes from Several Chromosomal Catabolic Pathways for Aromatic Compounds. Gracia Morales, 2004.The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium [catabolic repression] . Crc appears to be a componentof a signal transduction pathway modulating carbon metabolismin pseudomonads, although its mode of action is unknown . Tobetter understand the role of Crc, the proteome profile of twootherwise isogenic P . putida strains containing either a wild-typeor an inactivated crc allele was compared . The results showedthat Crc is involved in the catabolic repression of the hpdand hmgA genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons . This led us to analyze whetherCrc also regulates the expression of the other central catabolicpathways for aromatic compounds present in P . putida . It wasfound that genes required to assimilate benzoate through thecatechol pathway [benA and catBCA] and 4-OH-benzoate throughthe protocatechuate pathway [pobA and pcaHG] are also negativelymodulated by Crc . However, the pathway for phenylacetate appearedto be unaffected by Crc . These results expand the influenceof Crc to pathways used to assimilate several aromatic compounds,which highlights its importance as a master regulator of carbonmetabolism in P . putida. Inactivation of sll1556 in Synechocystis Strain PCC 6803 Impairs Isoprenoid Biosynthesis from Pentose Phosphate Cycle Substrates In Vitro. Kelly Poliquin, 2004.In cyanobacteria many compounds, including chlorophylls, carotenoids, and hopanoids, are synthesized from the isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate . Isoprenoid biosynthesis in extracts of the cyanobacterium Synechocystis strain PCC 6803 grown under photosynthetic conditions, stimulated by pentose phosphate cycle substrates, does not appear to require methylerythritol phosphate pathway intermediates . The sll1556 gene, distantly related to type 2 IPP isomerase genes, was disrupted by insertion of a Kanr cassette . The mutant was fully viable under photosynthetic conditions although impaired in the utilization of pentose phosphate cycle substrates . Compared to the parental strain the  sll1556
mutant (i) is deficient in isoprenoid biosynthesis in vitro with
substrates including glyceraldehyde-3-phosphate,
fructose-6-phosphate, and glucose-6-phosphate; (ii) has smaller cells
(diameter ca . 13% less); (iii) has fewer thylakoids (ca . 30% less);
and (iv) has a more extensive fibrous outer wall layer . Isoprenoid
biosynthesis is restored with pentose phosphate cycle substrates plus
the recombinant Sll1556 protein in the
sll1556
supernatant fraction . IPP isomerase activity could not be
demonstrated for the purified Sll1556 protein under our in vitro
conditions . The reduction of thylakoid area and the effect on outer
wall layer components are consistent with an impairment of isoprenoid
biosynthesis in the mutant, possibly via hopanoid biosynthesis . Our
findings are consistent with an alternate metabolic shunt for
biosynthesis of isoprenoids .
Differences between Betaproteobacterial Ammonia-Oxidizing Communities in Marine Sediments and Those in Overlying Water. Thomas E. Freitag, 2004.To assess links between betaproteobacterial ammonia-oxidizing bacteria (AOB) in marine sediment and in overlying water, communities in Loch Duich, Scotland, were characterized by analysis of clone libraries and denaturant gradient gel electrophoresis of 16S rRNA gene fragments . Nitrosospira cluster 1-like sequences were isolated from both environments, but different sequence types dominated water and sediment samples . Detailed phylogenetic analysis of marine Nitrosospira cluster 1-like sequences in Loch Duich and surrounding regions suggests the existence of at least two different phylogenetic subgroups, potentially indicative of new lineages within the betaproteobacterial AOB, representing different marine ecotypes . Regulation of the Bacillus subtilis fur and perR Genes by PerR: Not All Members of the PerR Regulon Are Peroxide Inducible. Mayuree Fuangthong, 2002.PerR is a ferric uptake repressor (Fur) homolog that functions as the central regulator of the inducible peroxide stress response in Bacillus subtilis . PerR has been previously demonstrated to regulate the mrgA, katA, ahpCF, hemAXCDBL, and zosA genes . We now demonstrate that PerR also mediates both the repression of its own gene and that of fur . Whereas PerR-mediated repression of most target genes can be elicited by either manganese or iron, repression of perR and fur is selective for manganese . Genetic studies indicate that repression of PerR regulon genes by either manganese or iron requires PerR and is generally independent of Fur . Indeed, in a fur mutant, iron-mediated repression is enhanced . Unexpectedly, repression of the fur gene by manganese appears to require both PerR and Fur, but only PerR binds to the fur regulatory region in vitro . The fur mutation appears to act indirectly by affecting cellular metal ion pools and thereby affecting PerR-mediated repression . While many components of the perR regulon are strongly induced by hydrogen peroxide, little, if any, induction of fur and perR could be demonstrated . Thus, not all components of the PerR regulon are components of the peroxide stimulon . We suggest that PerR exists in distinct metallated forms that differ in DNA target selectivity and in sensitivity to oxidation . This model is supported by the observation that the metal ion composition of the growth medium can greatly influence the transcriptional response of the various PerR regulon genes to hydrogen peroxide . A Green Nonsulfur Bacterium, Dehalococcoides ethenogenes, with the LexA Binding Sequence Found in Gram-Positive Organisms. Antonio R. Fernández de Henestrosa, 2002.Dehalococcoides ethenogenes is a member of the physiologically diverse division of green nonsulfur bacteria . Using a TBLASTN search, the D . ethenogenes lexA gene has been identified, cloned, and expressed and its protein has been purified . Mobility shift assays revealed that the D . ethenogenes LexA protein specifically binds to both its own promoter and that of the uvrA gene, but not to the recA promoter . Our results demonstrate that the D . ethenogenes LexA binding site is GAACN4GTTC, which is identical to that found in gram-positive bacteria . In agreement with this fact, the Bacillus subtilis DinR protein binds specifically to the D . ethenogenes LexA operator . This constitutes the first non-gram-positive bacterium exhibiting a LexA binding site identical to that of B . subtilis. Introduction of Culex Toxicity into Bacillus thuringiensis Cry4Ba by Protein Engineering. Mohd Amir F. Abdullah, 2003.Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC50], >80,000 and >20,000 ng/ml, respectively) . We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba . The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C . quinquefasciatus, and C . pipiens . Putative loop 1 and 2 exchanges eliminated activity towards A . aegypti and A . quadrimaculatus . Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C . quinquefasciatus (LC50  114 ng/ml) and C . pipiens (LC50
37 ng/ml), respectively . The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or Aedes . These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B . thuringiensis Cry toxins .
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