Microsomal metabolism of dictamnine: identification of metabolites and evaluation of their mutagenicity in Salmonella typhimurium.
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Incubation of the furoquinoline alkaloid dictamnine with microsomal fractions from phenobarbital-induced rat liver resulted in a metabolite mixture from which demethyl-dictamnine and dictamnic acid were identified by a GCMS technique. The identity of the two compounds was confirmed by synthesis. Other metabolites were characterized by their mass spectra only. The pattern of the metabolites suggested a possible pathway of metabolism in vitro. We assume that the metabolism of dictamnine is analogous to the metabolism of the related 8-methoxypsoralen and takes place via an unstable epoxide and subsequent oxidative opening of the furan ring. Direct evidence for the formation of an epoxide was, however, not obtained. The identified compounds as well as some putative metabolites were shown to be non-mutagenic in Salmonella typhimurium TA98 except for 8-hydroxydictamnine, which, however, was not detected in the metabolite mixture. (+info)
Mutant strains (nit) of Salmonella typhimurium with a pleiotropic defect in nitrogen metabolism.
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We have isolated mutant strains (nit) of Salmonella typhimurium that are defective in nitrogen metabolism. They have a reduced ability to use a variety of compounds including glutamate, proline, arginine, N-acetyl-glucosamine, alanine, and adenosine as sole nitrogen source. In addition, although they grow normally on high concentrations of ammonium chloride (greater than 1 mM) as nitrogen source, they grow substantially more slowly than wild type at low concentrations (less than 1 mM). We postulated that the inability of these strains to utilize low concentrations of ammonium chloride accounts for their poor growth on other nitrogen sources. The specific biochemical lesion in strains with a nit mutation is not known; however, mutant strains have no detectable alteration in the activities of glutamine synthetase, glutamate synthetase, or glutamate dehydrogenase, the enzymes known to be involved in assimilation of ammonia. A nit mutation is suppressed by second-site mutations in the structural gene for glutamine synthetase (glnA) that decrease glutamine synthetase activity. (+info)
An essential role for DNA adenine methylation in bacterial virulence.
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Salmonella typhimurium lacking DNA adenine methylase (Dam) were fully proficient in colonization of mucosal sites but showed severe defects in colonization of deeper tissue sites. These Dam- mutants were totally avirulent and were effective as live vaccines against murine typhoid fever. Dam regulated the expression of at least 20 genes known to be induced during infection; a subset of these genes are among those activated by the PhoP global virulence regulator. PhoP, in turn, affected Dam methylation at specific genomic sites, as evidenced by alterations in DNA methylation patterns. Dam inhibitors are likely to have broad antimicrobial action, and Dam- derivatives of these pathogens may serve as live attenuated vaccines. (+info)
Identification of a cis-acting regulatory sequence responsible for the repression of brnQ in Salmonella typhimurium.
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brnQ is the gene encoding the LIV-II transport system for branched-chain amino acids in Salmonella typhimurium. The expression of the gene is transcriptionally repressed by an excess of glycyl-l-leucine added to the bacterial culture. To investigate the mechanism of regulation, we constructed brnQ-lacZ translational fusions with various deletions upstream from the promoter of brnQ, and examined the effects of the deletions on the regulation. We found a cis-acting region, 5'-GTGTTTTA-3', for the repression of brnQ expression, which was located 94 base pairs upstream from the transcription start site. Removal of the sequence resulted in derepression of brnQ. Two homologous sequences were found 45 base pairs downstream and 42 base pairs upstream from the sequence. We designated these sequences as O1, O2, and O3, in the order from the sequence proximal to the promoter to that distal to the promoter, respectively. The gleR1 mutation, which we reported previously to be a regulatory mutation enhancing transcription of brnQ, was a G-to-T transversion in the O1 sequence 50 base pairs upstream from the transcription start site. Insertion of five nucleotides between O1 and O2 resulted in derepression of brnQ. Further insertion of five nucleotides did not restore the original regulation of brnQ, indicating the importance of the proper spacing of these sequences. We also showed that the protein product of livS, the gene responsible for regulation of the LIV-I transport system, may bind to the O2 sequence. Furthermore, LivS was shown to be an allele of Lrp based on complementation experiments. (+info)
A HilA-independent pathway to Salmonella typhimurium invasion gene transcription.
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Salmonella typhimurium invasion of nonphagocytic cells requires the expression of a type III secretion system (TTSS) encoded within Salmonella pathogenicity island 1 (SPI1). TTSS gene transcription is activated in response to environmental signals and requires transcriptional regulators encoded within (HilA) and outside (SirA) SPI1. Two unique loci, sirB and sirC, which contribute to SPI1 gene transcription were defined. sirC is an SPI1-encoded transcription factor of the AraC family that contributes to the invasive phenotype. sirB is required for maximal expression of sirC and consists of two open reading frames located near kdsA, a gene involved in lipopolysaccharide biosynthesis. sirC expression, unlike expression of other SPI1 genes, does not require HilA. Overexpression of sirC or sirA restores expression of a subset of SPI1 genes, including invF and sspC, in the absence of HilA. These data define roles for SirC and SirA as part of a HilA-independent pathway to SPI1 gene expression. We postulate that HilA-independent activation of inv expression is important for efficient assembly and function of the SPI1 TTSS. (+info)
Th1 dominance in the immune response to live Salmonella typhimurium requires bacterial invasiveness but not persistence.
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Factors responsible for the predictable generation of Th1 or Th2 immune responses to microorganisms in vivo are not well characterized, although the ability of antigen presenting cells (APC) to provide co-stimulation, the kinetics of MHC-peptide ligand generation as well as the cytokine environment are all considered important factors for the differential Th1/Th2 priming of T cells. Our earlier findings of an IFN-gamma-dominant, Th1-type response to live Salmonella typhimurium (Stm) and a Th2-type response to killed Stm suggested that persistence of viable bacteria might be an important factor in the generation of IFN-gamma-dominant responses. Using genetically susceptible and resistant strains of mice to limit bacterial replication and persistence in vivo, we show that mice of the lty(r) genotype, capable of a 10-fold better clearance of Stm, mount an IFN-gamma-dominant immune response following immunization with live Stm similar to that in the lty(s) strain. Further, metabolically defective mutants of Stm, aroA and purA, when used in the live form, also elicit IFN-gamma-dominant immune responses similar to the wild-type Stm strain despite their inability to proliferate in vivo. While a laboratory strain of Escherichia coli, which is antigenically cross-reactive but non-invasive, elicits hardly any IFN-gamma in immune responses, an invasive strain of E. coil induces an IFN-gamma-dominant response. These data together indicate that, while entry of bacteria into macrophages is likely to be critical for the generation of IFN-gamma-dominant immune responses, their persistence is not. (+info)
Mutations which alter the elbow region of tRNA2Gly reduce T4 gene 60 translational bypassing efficiency.
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Translating ribosomes bypass a 50 nucleotide coding gap in bacteriophage T4 gene 60 mRNA between codons 46 and 47 in order to synthesize the full-length protein. Bypassing of the coding gap requires peptidyl-tRNA2Gly detachment from a GGA codon (codon 46) followed by re-pairing at a matching GGA codon just before codon 47. Using negative selection, based on the sacB gene from Bacillus subtilis, Escherichia coli mutants were isolated which reduce bypassing efficiency. All of the mutations are in the gene for tRNA2Gly. Most of the mutations disrupt the hydrogen bonding interactions between the D- and T-loops (G18*psi55 and G19*C56) which stabilize the elbow region in nearly all tRNAs. The lone mutation not in the elbow region destabilizes the anticodon stem at position 40. Previously described Salmonella typhimurium mutants of tRNA2Gly, which reduce the stability of the T-loop, were also tested and found to decrease bypassing efficiency. Each tRNA2Gly mutant is functional in translation (tRNA2Gly is essential), but has a decoding efficiency 10- to 20-fold lower than wild-type. This suggests that rigidity of the elbow region and the anticodon stem is critical for both codon-anticodon stability and bypassing. (+info)
CRITICA: coding region identification tool invoking comparative analysis.
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Gene recognition is essential to understanding existing and future DNA sequence data. CRITICA (Coding Region Identification Tool Invoking Comparative Analysis) is a suite of programs for identifying likely protein-coding sequences in DNA by combining comparative analysis of DNA sequences with more common noncomparative methods. In the comparative component of the analysis, regions of DNA are aligned with related sequences from the DNA databases; if the translation of the aligned sequences has greater amino acid identity than expected for the observed percentage nucleotide identity, this is interpreted as evidence for coding. CRITICA also incorporates noncomparative information derived from the relative frequencies of hexanucleotides in coding frames versus other contexts (i.e., dicodon bias). The dicodon usage information is derived by iterative analysis of the data, such that CRITICA is not dependent on the existence or accuracy of coding sequence annotations in the databases. This independence makes the method particularly well suited for the analysis of novel genomes. CRITICA was tested by analyzing the available Salmonella typhimurium DNA sequences. Its predictions were compared with the DNA sequence annotations and with the predictions of GenMark. CRITICA proved to be more accurate than GenMark, and moreover, many of its predictions that would seem to be errors instead reflect problems in the sequence databases. The source code of CRITICA is freely available by anonymous FTP (rdp.life.uiuc.edu in/pub/critica) and on the World Wide Web (http:/(/)rdpwww.life.uiuc.edu). (+info)