|
|
|
|
|
|
Transcriptome Analysis of the ArgR Regulon in Pseudomonas aeruginosa. Chung-Dar Lu, 2004.Arginine metabolism in pseudomonads with multiple catabolic pathways for its utilization as carbon and nitrogen sources is of particular interest as the model system to study control of metabolic integration . We performed transcriptome analyses to identify genes controlled by the arginine regulatory protein ArgR and to better understand arginine metabolic pathways of P . aeruginosa . We compared gene expression in wild-type strain PAO1 with that in argR mutant strain PAO501 grown in glutamate minimal medium in the presence and absence of arginine . Ten putative transcriptional units of 28 genes were inducible by ArgR and arginine, including all known ArgR-regulated operons under aerobic conditions . The newly identified genes include the putative adcAB operon, which encodes a catabolic arginine decarboxylase and an antiporter protein, and PA0328, which encodes a hypothetical fusion protein of a peptidase and a type IV autotransporter . Also identified as members of the arginine network are the following solute transport systems: PA1971 (braZ) for branched-chain amino acids permease; PA2042 for a putative sodium:serine symporter; PA3934, which belongs to the family of small oligopeptide transporters; and PA5152-5155, which encodes components of an ABC transporter for a putative opine uptake system . The effect of arginine on the expression of these genes was confirmed by lacZ fusion studies and by DNA binding studies with purified ArgR . Only five transcriptional units of nine genes were qualified as repressible by ArgR and arginine, with three operons (argF, carAB, and argG) in arginine biosynthesis and two operons (gltBD and gdhA) in glutamate biosynthesis . These results indicate that ArgR is important in control of arginine and glutamate metabolism and that arginine and ArgR may have a redundant effect in inducing the uptake systems of certain compounds . Microbial Genomics—Challenges and Opportunities: The 9th International Conference on Microbial Genomes. Jizhong Zhou, 2002. Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain. Naeem Rashid, 2002.A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGTTk) was identified and characterized . The gene (cgtTk) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the  -amylase family . However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure . In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated . The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80°C and 5.5 to 6.0, respectively . The presence of Ca2+ enhanced the enzyme activity and elevated the optimum temperature to 85 to 90°C . With the addition of Ca2+, the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85°C, and a half-life of 20 min at 100°C . CGTTk could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan . Most importantly, although CGTTk harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with ß-cyclodextrin as the main product . In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGTTk C) with 23 C-terminal amino acid residues deleted . CGTTkC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGTTk . In contrast, the cyclization activity of CGTTkC was abolished . The results indicate that the presence of the structurally novel C-terminal domain is essential for CGTTk to properly catalyze the cyclization reaction .
Methanococcus jannaschii Coenzyme F420 Analogs Contain a Terminal  -Linked Glutamate.Marion Graupner, 2003.Analyses of the F420s present in Methanococcus jannaschii have shown that these cells contain a series of  -glutamyl-linked F420s capped with a single, terminal
-linked L-glutamate . The predominant form of F420 was designated as
-F420-3 and represented 86% of the F420s in these cells . Analyses of Methanosarcina thermophila, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, and Mycobacterium smegmatis showed that they contained only
-glutamyl-linked F420s .
Combining Culture-Dependent and -Independent Methodologies for Estimation of Richness of Estuarine Bacterioplankton Consuming Riverine Dissolved Organic Matter. Veljo Kisand, 2003.Three different methods for analyzing natural microbial community diversity were combined to maximize an estimate of the richness of bacterioplankton catabolizing riverine dissolved organic matter (RDOM) . We also evaluated the ability of culture-dependent quantitative DNA-DNA hybridization, a 16S rRNA gene clone library, and denaturing gradient gel electrophoresis (DGGE) to detect bacterial taxa in the same sample . Forty-two different cultivatable strains were isolated from rich and poor solid media . In addition, 50 unique clones were obtained by cloning of the bacterial 16S rDNA gene amplified by PCR from the community DNA into an Escherichia coli vector . Twenty-three unique bands were sequenced from 12 DGGE profiles, excluding a composite fuzzy band of the Cytophaga-Flavobacterium group . The different methods gave similar distributions of taxa at the genus level and higher . However, the match at the species level among the methods was poor, and only one species was identified by all three methods . Consequently, all three methods identified unique subsets of bacterial species, amounting to a total richness of 97 operational taxonomic units in the experimental system . The confidence in the results was, however, dependent on the current precision of the phylogenetic determination and definition of the species . Bacterial consumers of RDOM in the studied estuary were primarily both cultivatable and uncultivable taxa of the Cytophaga-Flavobacterium group, a concordant result among the methods applied . Culture-independent methods also suggested several not-yet-cultivated ß-proteobacteria to be RDOM consumers . Innovative Approach for Improvement of an Antibiotic-Overproducing Industrial Strain of Streptomyces albus. Norimasa Tamehiro, 2003.Working with a Streptomyces albus strain that had previously been bred to produce industrial amounts (10 mg/ml) of salinomycin, we demonstrated the efficacy of introducing drug resistance-producing mutations for further strain improvement . Mutants with enhanced salinomycin production were detected at a high incidence (7 to 12%) among spontaneous isolates resistant to streptomycin (Strr), gentamicin, or rifampin (Rifr) . Finally, we successfully demonstrated improvement of the salinomycin productivity of the industrial strain by 2.3-fold by introducing a triple mutation . The Strr mutant was shown to have a point mutation within the rpsL gene (encoding ribosomal protein S12) . Likewise, the Rifr mutant possessed a mutation in the rpoB gene (encoding the RNA polymerase ß subunit) . Increased productivity of salinomycin in the Strr mutant (containing the K88R mutation in the S12 protein) may be a result of an aberrant protein synthesis mechanism . This aberration may manifest itself as enhanced translation activity in stationary-phase cells, as we have observed with the poly(U)-directed cell-free translation system . The K88R mutant ribosome was characterized by increased 70S complex stability in low Mg2+ concentrations . We conclude that this aberrant protein synthesis ability in the Strr mutant, which is a result of increased stability of the 70S complex, is responsible for the remarkable salinomycin production enhancement obtained .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
