Synthesis and evaluation of 1,3,4-oxadiazole derivatives for development as broad-spectrum antibiotics.

The reality and intensity of antibiotic resistance in pathogenic bacteria calls for the rapid development of new antimicrobial drugs. In bacteria, trans-translation is the primary quality control mechanism for rescuing ribosomes arrested during translation. Because trans-translation is absent in eukaryotes but necessary to avoid ribosomal stalling and therefore essential for bacterial survival, it is a promising target either for novel antibiotics or for improving the activities of the protein synthesis inhibitors already in use.

Characterization of HicAB toxin-antitoxin module of Sinorhizobium meliloti.

Background: Toxin-antitoxin (TA) systems are little genetic units generally composed of two genes encoding antitoxin and toxin. These systems are known to be involved in many functions that can lead to growth arrest and cell death. Among the different types of TA systems, the type II gathers together systems where the antitoxin directly binds and inhibits the toxin.

A Genetic Tool to Quantify trans-Translation Activity in Vivo.

In bacteria, trans-translation is the main quality control mechanism for rescuing ribosomes arrested during translation. This key process is universally conserved and plays a critical role in the viability and virulence of many pathogens. We developed a reliable in vivo double-fluorescence reporter system for the simultaneous quantification of both trans-translation and the associated proteolysis activities in bacteria.

Resistance to organic hydroperoxides requires ohr and ohrR genes in Sinorhizobium meliloti.

Background: Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Medicago sativa. During nodule formation bacteria have to withstand oxygen radicals produced by the plant. Resistance to H2O2 and superoxides has been extensively studied in S.

RpoE2 of Sinorhizobium meliloti is necessary for trehalose synthesis and growth in hyperosmotic media.

Adaptation to osmotic stress can be achieved by the accumulation of compatible solutes that aid in turgor maintenance and macromolecule stabilization. The genetic regulation of solute accumulation is poorly understood, and has been described well at the molecular level only in enterobacteria. In this study, we show the importance of the alternative sigma factor RpoE2 in Sinorhizobium meliloti osmoadaptation.

The Sinorhizobium meliloti RNA chaperone Hfq mediates symbiosis of S. meliloti and alfalfa.

There exist commonalities between symbiotic Sinorhizobium meliloti and pathogenic Brucella bacteria in terms of extensive gene synteny and the requirements for intracellular survival in their respective hosts. The RNA chaperone Hfq is essential for virulence for several bacterial groups, including Brucella; however, its role in S. meliloti has not been investigated.

Proteomic alterations explain phenotypic changes in Sinorhizobium meliloti lacking the RNA chaperone Hfq.

The ubiquitous bacterial RNA-binding protein Hfq is involved in stress resistance and pathogenicity. In Sinorhizobium meliloti, Hfq is essential for the establishment of symbiosis with Medicago sativa and for nitrogen fixation. A proteomic analysis identifies 55 proteins with significantly affected expression in the hfq mutant; most of them are involved in cell metabolism or stress resistance.

Sinorhizobium meliloti rpoE2 is necessary for H(2)O(2) stress resistance during the stationary growth phase.

RpoE2 is an extracytoplasmic sigma factor produced by Sinorhizobium meliloti during stationary growth phase. Its inactivation affected the synthesis of the superoxide dismutase, SodC, and catalase, KatC. The absence of SodC within the cell did not result in an increased sensitivity to extracellular superoxides.

Characterization and expression patterns of Sinorhizobium meliloti tmRNA (ssrA).

tmRNA (ssrA) in Sinorhizobium meliloti is a small RNA annotated by homology with the Bradyrhizobium japonicum sra molecule. Here, this molecule is described in Sinorhizobium meliloti as a model for such molecules in Alphaproteobacteria subgroup-2. Northern blot analysis and mapping of both 5' and 3' ends of this tmRNA allow the identification of two pieces: a 214 nt mRNA-like domain and an 82 nt tRNA-like domain, both highly stable, whereas the premature form is unstable.

Interrelations between glycine betaine catabolism and methionine biosynthesis in Sinorhizobium meliloti strain 102F34.

Methionine is produced by methylation of homocysteine. Sinorhizobium meliloti 102F34 possesses only one methionine synthase, which catalyzes the transfer of a methyl group from methyl tetrahydrofolate to homocysteine. This vitamin B(12)-dependent enzyme is encoded by the metH gene.

Glucose 6-phosphate dehydrogenase is required for sucrose and trehalose to be efficient osmoprotectants in Sinorhizobium meliloti.

Inactivation of the zwf gene in Sinorhizobium meliloti induces an osmosensitive phenotype and the loss of osmoprotection by trehalose and sucrose, but not by ectoine and glycine betaine. This phenotype is not linked to a defect in the biosynthesis of endogenous solutes. zwf expression is induced by high osmolarity, sucrose and trehalose, but is repressed by betaine.

Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli.

Osmoprotectants exogenously supplied to a hyperosmotic culture medium are efficiently imported and amassed by stressed cells of Escherichia coli. In addition to their evident role in the recovery and maintenance of osmotic balance, these solutes should play an important role on the behavior of cellular macromolecules, for example in the process of protein folding. Using a random chemical mutagenesis approach, a conditional lysine auxotrophic mutant was obtained.

Site-specific integration of corynephage phi16: construction of an integration vector.

Phi16, a temperate phage induced from Corynebacterium glutamicum ATCC 21792, lysogenizes its host via site-specific recombination. The phage attachment site, attP, was located to a 6.5 kb BamHI fragment of the phi16 genome.

Analysis of the integration functions of phi304L: an integrase module among corynephages.

Plasmid p12929 was shown to integrate into the chromosome of Corynebacterium glutamicum RM3 and BL15. The minimal integrating fragment was subsequently defined. The arms flanking the integrated plasmid (attL and attR) were identified, allowing for the determination of the attP and the attB attachment sites.

pBLA8, from Brevibacterium linens, belongs to a gram-positive subfamily of ColE2-related plasmids.

A 3.1 kb DNA fragment from pBLA8, a Brevibacterium linens cryptic plasmid, containing all the information required for autonomous replication was cloned and sequenced. Using deletion analysis, the fragment essential and sufficient for autonomous replication was delimited to 1.

Cloning and sequence determination of a phi AAU2 gene whose product aborts the phage lytic cycle.

This communication describes the cloning of a 1.8-kb fragment from the genome of the corynephage phi AAU2, which aborts the phage lytic cycle when cloned on a high-copy shuttle vector. The associated phenotype, called Apld (aborting phage lytic development), was revealed by noting the reduced plaque size and lower efficiencies of plaquing of phi AAU2 cp, a virulent derivative of phi AAU2, on "Arthrobacter aureus"-C70 Apld+ cells.

Genetic characterization of site-specific integration functions of phi AAU2 infecting "Arthrobacter aureus" C70.

All the essential genetic determinants for site-specific integration of corynephage phi AAU2 are contained within a 1,756-bp DNA fragment, carried on the integrative plasmid p5510, and are shown to be functional in Escherichia coli. One open reading frame, ORF4, encoding a protein of 266 amino acids was shown to represent the phi AAU2 integrase. The nucleotide sequence of the phi AAU2 attachment site, attP, and the attB, attL, and attR sequences in the host "Arthrobacter aureus" C70 were determined.

Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA.

Growth of Erwinia chrysanthemi in media of elevated osmolarity can be achieved by the uptake and accumulation of various osmoprotectants. This study deals with the cloning and sequencing of the ousA gene-encoded osmoprotectant uptake system A from E. chrysanthemi 3937.

Prophage distribution in coryneform bacteria.

Four temperate bacteriophages of corynebacteria were isolated after UV induction. Phages phi 304L and phi 304S were both induced from Corynebacterium glutamicum ATCC 13058, ATCC 21488, ATCC 21649 and ATCC 21650 strains, and have no known sensitive host. Phages phi 15 and phi 16 were both induced from ATCC 14020 and ATCC 21792.

Structural organization of the Corynebacterium glutamicum plasmid pCG100.

pCG100, a 3 kb cryptic plasmid of Corynebacterium glutamicum ATCC 13058, probably identical with pSR1 from C. glutamicum ATCC 19223, was characterized. The minimum region for autonomous replication was shown to be contained on a 1.

Isolation and characterization of a restriction and modification deficient mutant of Brevibacterium lactofermentum.

In order to facilitate genetic engineering in amino-acid producing bacteria we have isolated two restriction-deficient Brevibacterium lactofermentum strains. They have been selected for their ability to obtain a high yield of plaques from CL31 phage which was grown on Corynebacterium lilium. These mutant strains do not restrict either phage DNA by transfection or DNA from the shuttle vector pBLA extracted from Escherichia coli by protoplast transformation.

Transfer of plasmid DNA to Brevibacterium lactofermentum by electrotransformation.

The Escherichia coli-Brevibacterium lactofermentum shuttle vector pBLA was introduced into intact cells of B. lactofermentum by electrotransformation. Several parameters of this procedure such as voltage and cell concentration were analysed.

Isolation and preliminary characterization of twenty bacteriophages infecting either brevibacterium or arthrobacter strains.

Thirty-seven bacteriophages plaquing on Corynebacterium, Brevibacterium, or Arthrobacter strains were isolated from soil or vegetation samples. Restriction analysis of phage DNA indicated that 20 phages were unique; one of them produced entirely turbid plaques on Brevibacterium ketoglutamicum and was characterized as temperate. All these phages were assigned to group B of the classification of Bradley (Bacteriol.

Plasmids with the uidA reporter gene for the detection of promoters and transcription signals.

We have constructed promoter-probe plasmids, pNB4 and pNB5, based on the promoterless gene for beta-glucuronidase (uidA) of Escherichia coli. Unique restriction sites for EcoRI, SacI, KpnI, SmaI, XmaI, XbaI, SalI, SphI and HindIII in pNB4 and for HindIII, PstI and BglII in pNB5 were included upstream of the uidA structural gene. The usefulness of these plasmids was demonstrated by cloning the promoter-operator region of the E.