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The Escherichia coli argU10(Ts) Phenotype Is Caused by a Reduction in the Cellular Level of the argU tRNA for the Rare Codons AGA and AGG. Kensaku Sakamoto, 2004.The Escherichia coli argU10(Ts) mutation in the argU gene, encoding the minor tRNAArg species for the rare codons AGA and AGG, causes pleiotropic defects, including growth inhibition at high temperatures, as well as the Pin phenotype at 30°C . In the present study, we first showed that the codon selectivity and the arginine-accepting activity of the argU tRNA are both essential for complementing the temperature-sensitive growth, indicating that this defect is caused at the level of translation . An in vitro analysis of the effects of the argU10(Ts) mutation on tRNA functions revealed that the affinity with elongation factor Tu-GTP of the argU10(Ts) mutant tRNA is impaired at 30 and 43°C, and this defect is more serious at the higher temperature . The arginine acceptance is also impaired significantly but to similar extents at the two temperatures . An in vivo analysis of aminoacylation levels showed that 30% of the argU10(Ts) tRNA molecules in the mutant cells are actually deacylated at 30°C, while most of the argU tRNA molecules in the wild-type cells are aminoacylated . Furthermore, the cellular level of this mutant tRNA is one-tenth that of the wild-type argU tRNA . At 43°C, the cellular level of the argU10(Ts) tRNA is further reduced to a trace amount, while neither the cellular abundance nor the aminoacylation level of the wild-type argU tRNA changes . We concluded that the phenotypic properties of the argU10(Ts) mutant result from these reduced intracellular levels of the tRNA, which are probably caused by the defective interactions with elongation factor Tu and arginyl-tRNA synthetase . The Arginine Regulatory Protein Mediates Repression by Arginine of the Operons Encoding Glutamate Synthase and Anabolic Glutamate Dehydrogenase in Pseudomonas aeruginosa. Shehab Hashim, 2004.The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions . ArgR also induces the operon that encodes a catabolic NAD+-dependent glutamate dehydrogenase (GDH), which converts L-glutamate, the product of the AST pathway, in  -ketoglutarate .
The studies reported here show that ArgR also participates in
the regulation of other enzymes of glutamate metabolism . Exogenous
arginine repressed the specific activities of glutamate synthase
(GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of
strain PAO1, and this repression was abolished in an argR
mutant . The promoter regions of the gltBD operon, which encodes
GltBD, and the gdhA gene, which encodes GdhA, were identified
by primer extension experiments . Measurements of ß-galactosidase
expression from gltB::lacZ and gdhA::lacZ
translational fusions confirmed the role of ArgR in mediating
arginine repression . Gel retardation assays demonstrated the binding
of homogeneous ArgR to DNA fragments carrying the regulatory regions
for the gltBD and gdhA genes . DNase I footprinting
experiments showed that ArgR protects DNA sequences in the control
regions for these genes that are homologous to the consensus sequence
of the ArgR binding site . In silica analysis of genomic information
for P . fluorescens, P . putida, and P.
stutzeri suggests that the findings reported here regarding
ArgR regulation of operons that encode enzymes of glutamate
biosynthesis in P . aeruginosa likely apply to other
pseudomonads .
Effects of Different Spices Used in Production of Fermented Sausages on Growth of and Curvacin A Production by Lactobacillus curvatus LTH 1174. Jurgen Verluyten, 2004.Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the listericidal bacteriocin curvacin A . The effect of different spices relevant for the production of fermented sausages was investigated in vitro through laboratory fermentations with a meat simulation medium and an imposed pH profile relevant for Belgian-type fermented sausages . The influence on the growth characteristics and especially on the kinetics of curvacin A production with L . curvatus LTH 1174 was evaluated . Pepper, nutmeg, rosemary, mace, and garlic all decreased the maximum specific growth rate, while paprika was the only spice that increased it . The effect on the lag phase was minor except for nutmeg and especially for garlic, which increased it, yet garlic was stimulatory for biomass production . The maximum attainable biomass concentration (Xmax) was severely decreased by the addition of 0.40% (wt/vol) nutmeg, while 0.35% (wt/vol) garlic or 0.80% (wt/vol) white pepper increased Xmax . Nutmeg decreased both growth and bacteriocin production considerably . Garlic was the only spice enhancing specific bacteriocin production, resulting in higher bacteriocin activity in the cell-free culture supernatant . Finally, lactic acid production was stimulated by the addition of pepper, and this was not due to the manganese present because an amount of manganese that was not growth limiting was added to the growth medium . Addition of spices to the sausage mixture is clearly a factor that will influence the effectiveness of bacteriocinogenic starter cultures in fermented-sausage manufacturing . Using a DNA Microarray To Investigate the Distribution of Insect Virulence Factors in Strains of Photorhabdus Bacteria. Judit Marokhazi, 2003.Photorhabdus is an insect-pathogenic bacterium in which oral toxicity to insects is found in two distinct taxonomic groups . Using a DNA microarray and comparative genomics, we show that oral toxicity is associated with toxin complex genes tcaABC and that this locus can be mobilized or deleted within different strains . Development and Application of an Assay for Uranyl Complexation by Fungal Metabolites, Including Siderophores. Joanna C. Renshaw, 2003.An assay to detect UO22+ complexation was developed based on the chrome azurol S (CAS) assay for siderophores (B . Schwyn and J . B . Neilands, Anal . Biochem . 160:47-56, 1987) and was used to investigate the ability of fungal metabolites to complex actinides . In this assay the discoloration of two dyed agars (one containing a CAS-Fe3+ dye and the other containing a CAS-UO22+ dye) caused by ligands was quantified . The assay was tested by using the siderophore desferrioxamine B (DFO), and the results showed that there was a regular, reproducible relationship between discoloration and the amount of siderophore added . The ratio of the discoloration on the CAS-UO22+ agar to the discoloration on the CAS-Fe3+ agar was independent of the amount of siderophore added . A total of 113 fungi and yeasts were isolated from three soil samples taken from the Peak District National Park . The fungi were screened for the production of UO22+ chelators by using the CAS-based assay and were also tested specifically for hydroxamate siderophore production by using the hydroxamate siderophore auxotroph Aureobacterium flavescens JG-9 . This organism is highly sensitive to the presence of hydroxamate siderophores . However, the CAS-based assay was found to be less sensitive than the A . flavescens JG-9 assay . No significant difference between the results for each site for the two tests was found . Three isolates were selected for further study and were identified as two Pencillium species and a Mucor species . Our results show that the new assay can be effectively used to screen fungi for the production of UO22+ chelating ligands . We suggest that hydroxamate siderophores can be produced by mucoraceous fungi .
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