Preliminary characterization of an antibiotic produced by Xanthomonas albilineans which inhibits DNA synthesis in Escherichia coli

Chlorosis-inducing isolates of Xanthomonas albilineans, the sugarcane leaf scald pathogen, produced a mixture of antibacterial compounds in culture. The antibiotic mixture, which eluted as a single strongly retarded peak from Sephadex LH-20 in methanol, was bactericidal to Escherichia coli. Inhibition of E. coli was not reversed by added nutrients, and affected cells were not lysed but many accumulated polyphosphate granules. The major antibacterial component, isolated in crystalline form after HPLC, is given the trivial name albicidin. Near the minimum inhibitory concentration, albicidin caused a complete block to DNA synthesis, followed by partial inhibition of RNA and protein synthesis, as assessed by incorporation of radioactive precursors. Spontaneous antibiotic-resistant mutants of E. coli showed no cross-resistance between albicidin and inhibitors of either subunit of DNA gyrase. Mixing albicidin with purified DNA from E. coli did not alter the thermal denaturation behaviour of the DNA, or the absorption spectrum of the antibiotic. PolA+ and PolA - strains of E. coli were equally sensitive to albicidin, indicating that the antibiotic does not bind to or modify DNA. Selective inhibition of DNA synthesis without evidence of DNA binding suggests a specific interaction of albicidin with an essential replication protein.     

Role of Sugars and Phenols in Charcoalrot Resistance of Sorghum

Total sugars, reducing sugars, and phenols were estimated in internodes of tolerant and highly susceptible sovhum genotypes to charcoal, ln tolerant genotypes the sugar level was 2 to 3 times higher than in susceptible ones; phenol concentration also was higher in tolerant genotypes. In tolerant genotypes, sugar and phenol concentration was high in the lower most and in the upper most mternode. This may be used to identify sources of resistance.     

Regulation of `malic'' enzyme of Solanum tuberosum by metabolites

A purification of ;malic' enzyme from potato is described. The purified enzyme is specific for NADP and requires a bivalent cation for activity. At pH values below 7 the plot of rate versus malate concentration approximates to normal Michaelis-Menten kinetics. At pH values above 7 the plot of rate versus malate concentration is sigmoid. A number of dicarboxylic acids activate the enzyme and remove the sigmoidicity. The enzyme is inhibited by phosphate, triose phosphates and AMP. In general, effectors of the oxidative decarboxylation of malate behave in the same manner in the reductive carboxylation of pyruvate. The response of the enzyme to energy charge is reported and the physiological significance of the response to metabolites is discussed in relation to the proposed role of the enzyme in the control of pH.     

Amino acid sequence of the regulatory-site glyoxylate peptide of biodegradative threonine dehydratase of Escherichia coli.

Incubation of purified Escherichia coli biodegradative threonine dehydratase with glyoxylate resulted in covalent binding of 1 mol of glyoxylate per mol of protein with concomitant loss of enzyme activity. The glyoxylate-binding site was identified as a heptapeptide representing amino acid residues Ser-33-Asn-Tyr-Phe-Ser-Glu-Arg-39 in the protein primary structure. Addition of glyoxylate to a culture of E. coli cells led to time-dependent enzyme inactivation. Immunoprecipitation with anti-dehydratase antibody of extract from [14C]glyoxylate-treated cells revealed labeled dehydratase polypeptide. These results are interpreted to mean that enzyme inactivation by glyoxylate in E. coli cells is associated with covalent protein modification.     

EPR studies with 77Se-enriched (NiFeSe) hydrogenase of Desulfovibrio baculatus. Evidence for a selenium ligand to the active site nickel

The periplasmic hydrogenase containing equivalent amounts of nickel and selenium plus non-heme iron [NiFeSe) hydrogenase) has been purified from cells of the sulfate reducing bacterium Desulfovibrio baculatus (DSM 1748) grown on a lactate/sulfate medium containing natural Se isotopes and the nuclear isotope, 77Se. Both the 77Se-enriched and unenriched hydrogenases were shown to be free of other hydrogenases and characterized with regard to their Se contents. EPR studies of the reduced nickel signal generated by redox titrations of the enriched and unenriched (NiFeSe) hydrogenases demonstrated that the gx = 2.23 and gy = 2.17 resonances are appreciably broadened by the spin of the 77Se nucleus (I = 1/2). This observation demonstrates unambiguously that the unpaired electron is shared by the Ni and Se atoms and that Se serves as a ligand to the nickel redox center of the (NiFeSe) hydrogenase.     

Molecular cloning and restriction enzyme analysis of a long repetitive DNA sequence in rice

Rice long repetitive DNA (9–20 kbp) reassociating at Cot 50 M.s was cloned in pBR325. Out of several recombinants (Cam^r Amp^r Tet^s), only a few were selected randomly for further characterization. The insert size in all these clones was 3–4 kbp. Restriction enzyme analysis showed the absence ofEcoRI andBclI sites, presence of a singlePstI andPvuII site and multiple sites forAluI in 3 clones namely pRLl, pRL7 and pRL10. TheBamHI-PstI fragment of about 0.4 kbp in the pRL7 insert DNA (pRL7-0.4 kbp) was subcloned in M13mpl8 and partially sequenced using Sanger’s dideoxynucleotide chain termination method. Dot matrix comparison of this sequence with rice rDNA sequences revealed low homology with the 25 S rDNA sequence of rice, however, hybridisation did not indicate any homology.     

Alterations in calcium homeostasis on lead exposure in rat synaptosomes

The effects of lead on Ca²⁺ homeostasis in nerve terminals was studied. Incubation with leadin vitro stimulated the activity of calmodulin and the maximum effect was observed at 30 M lead, higher concentrations had an inhibitory effect.In vivo exposure to lead increased the activity of calmodulin by 45%. Lead had an inhibitory effect on Ca²⁺ ATPase activity in both calmodulin-rich and calmodulin-depleted synaptic plasma membranes, the IC₅₀ values for inhibition being 13.34 and 16.69 M respectively. Exogenous addition of calmodulin (5 g) and glutathione (1 mM) to calmodulin rich synaptic plasma membranes reversed the inhibition by IC₅₀ concentration of lead.In vivo exposure of lead also significantly reduced the Ca²⁺ ATPase activity, resulting in an increase in intrasynaptosomal calcium. Concomitant with the increase in intrasynaptosomal calcium, lipid peroxidation values also increased significantly in lead-treated animals. In addition lead also had an inhibitory effect on depolarization induced Ca²⁺ uptake and the inhibition was found to be a competitive one. The results sugest that lead exerts its toxic effects by modifications of the intracellular calcium messenger system which would have serious consequences on neuronal functioning.     

RasG protein accumulation occurs just prior to amoebae emergence during spore germination in Dictyostelium discoideum

Abstract RasG protein levels in dormant and germinating spores of Dictyostelium discoideum strains JC1 and SG1 were estimated by Western blotting. Ras Glevels were very low in dormant spores and remained low during the lag period, regardless of whether spores were heat activated or treated with autoactivator during the early stages of spore germination. RasG levels increased late during spore swelling just prior to the emergence stage of germination. These data are consistent with a requirement for RasG during vegetative growth.     

Genetic organization of a cluster of genes involved in the production of phaseolotoxin, a toxin produced by Pseudomonas syringae pv. phaseolicola.

Phaseolotoxin [N delta(N'-sulfo-diaminophosphinyl)-ornithyl-alanyl- homoarginine] produced by Pseudomonas syringae pv. phaseolicola, the bean halo blight pathogen, is a potent inhibitor of ornithine carbamoyltransferase (OCT). Inhibition of OCT in infected plants leads to chlorosis and growth inhibition. A genomic cosmid clone, pHK120, containing a 25-kb fragment of DNA from a wild-type strain of P. syringae pv. phaseolicola restores toxin production in Tox- mutants. Tn5 mutagenesis of pHK120 and marker exchange of pHK120::Tn5 plasmids in the wild-type strain resulted in the isolation of 39 chromosomal mutants that harbor Tn5 insertions at known positions. Toxin bioassays revealed that 28 of the mutants, with Tn5 insertions distributed throughout the insert of pHK120, were Tox-, indicating that a functional locus for toxin production in each mutant was inactivated. Complementation analysis was done by testing for toxin production strains that carried a genomic Tn5 at one location and a plasmid-borne Tn5 at another location (pair complementation). Pair complementation analysis of nine marker exchange mutants and a random genomic Tn5 mutant revealed that there are a minimum of eight toxin loci (phtA through phtH) in pHK120. Mutants carrying Tn5 insertions in the phtA, phtD, and phtF loci were complemented by deletion subclones containing fragments from pHK120; mutants carrying Tn5 insertions in the phtC locus were partially complemented by a subclone, and mutants carrying Tn5 insertions in the phtB, phtE, phtG, and phtH loci were not complemented by any of the available subclones. A comparison of the insert from pHK120 with that from pRCP17, a clone reported previously (R. C. Peet, P. B. Lindgren, D. K. Wills, and N. J. Panopoulos, J. Bacteriol. 166:1096-1105, 1986) by another laboratory to contain some of the phaseolotoxin genes and the phaseolotoxin-resistant OCT gene, revealed that the inserts in these two cosmids overlap but differ in important respects.