Synthesis of the penicillin precursor, δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV), by an immobilized enzyme preparation

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase activity has been partially-purified from cell-free extracts of Streptomyces clavuligerus by ammonium sulfate precipitation. The salt precipitated enzyme was immobilized on an anion exchange resin and synthesis of ACV was observed by exposing the immobilized enzyme preparation to a reaction mixture containing l--aminoadipic acid, l-valine and l-cysteine in the presence of appropriate cofactors. Reaction mixtures containing l--aminobutyric acid(aB) in place of l-valine synthesized the ACV analog ACaB. Immobilized ACV synthetase can be reused, and after six cycles of reaction, 28.9% of original activity remains.     

An ultraviolet resonance Raman study of dehydrogenase enzymes and their interactions with coenzymes and substrates

Ultraviolet resonance Raman (UVRR) spectra, with 260-nm excitation, are reported for oxidized and reduced nicotinamide adenine dinucleotides (NAD+ and NADH, respectively). Corresponding spectra are reported for these coenzymes when bound to the enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and liver and yeast alcohol dehydrogenases (LADH and YADH). The observed differences between the coenzyme spectra are interpreted in terms of conformation, hydrogen bonding, and general environment polarity differences between bound and free coenzymes and between coenzymes bound to different enzymes. The possibility of adenine protonation is discussed. UVRR spectra with 220-nm excitation also are reported for holo- and apo-GAPDH (GAPDH-NAD+ and GAPDH alone, respectively). In contrast with the 260-nm spectra, these show only bands due to vibrations of aromatic amino acid residues of the protein. The binding of coenzyme to GAPDH has no significant effect on the aromatic amino acid bands observed. This result is discussed in the light of the known structural change of GAPDH on binding coenzyme. Finally, UVRR spectra with 240-nm excitation are reported for GAPDH and an enzyme-substrate intermediate of GAPDH. Perturbations are reported for tyrosine and tryptophan bands on forming the acyl enzyme.     

Recognition of the P1 plasmid centromere analog involves binding of the ParB protein and is modified by a specific host factor.

The P1 plasmid partition system is responsible for segregation of daughter plasmids during division of the Escherichia coli host cell. The P1-encoded elements consist of two essential proteins, ParA and ParB, and the cis-acting incB region. The incB region determines partition-mediated incompatibility and contains the centromere-like site parS. We have isolated and purified the two proteins. ParB binds specifically to the incB region in vitro. DNase I footprinting assays place a strong binding site over the 35-bp parS sequence previously shown to be sufficient for partition when the Par proteins are supplied in trans. A weaker site lies within the incB region in sequences that are important for specifying incompatibility, but are not essential for partition. Gel band retardation assays show that a host factor binds specifically to the incB sequence. The factor strongly stimulates binding of ParB. Cutting the region at a site between the two ParB binding sites yields two fragments that can bind ParB but not host factor. Thus, information for host-factor binding lies in the region determining the specificity of plasmid incompatibility. The roles of parB and the host factor in partition and the specificity of plasmid incompatibility are discussed.     

Plasmid content and homology of 16 strains of filamentous,nonheterocystous cyanobacteria

We have developed several strain-specific, rapid, small-scale plasmid isolation procedures in order to characterize the plasmid profiles of 16 filamentous, nonheterocstous cyanobacteria. At least one distinct plasmid was found in eight strains, with seven of these containing two or more different plasmids. Eight strains were found to be without plasmid DNA. Both the large, 12.9 kb, and the small, 1.6 kb, plasmids fromPlectonema boryanum 581 were isolated, purified, and cloned. Southern blots of plasmid DNAs from the eight strains were probed with these cloned DNAs and also with ultra-pure plasmid DNA fromPhormidium liridum 426. Four strains ofP. boryanum (485, 581, 594, 1542) andP. luridum 426 have identical plasmid profiles, and plasmid homology is extensive.     

Production of the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) by cell-free extracts from Streptomyces clavuligerus

Abstract Glycerol-stabilised cell extracts of Streptomyces clavuligerus contain an enzyme activity which synthesises ACV from the individual amino acids L -α-aminoadipic acid, L -cysteine and L -valine. Enzyme activity was optimum in reaction mixtures containing 1 mM ATP together with an ATP regenerating system. The ACV synthetase enzyme formed ACV analogs when provided with L - carboxymethylcysteine in place of L -α-aminoadipic acid or when provided with L - allo isoleucine or L -α-aminobutyrate in place of L -valine. Multistep conversion of individual amino acids to penicillin and cephalosporin antibiotics was restricted as a result of the inhibitory effects of L -α-aminoadipic acid and L -cysteine on isopenicillin N synthetase.     

Fate of bacteria ingested by larvae of the freshwater mayfly,Ephemera danica

The fate of bacteria in the food of a common freshwater invertebrate has been studied both in controlled laboratory experiments and in a stream sediment. The animal chosen was the larva of the burrowing mayfly,Ephemera danica. It ingested all available bacteria nonselectively. More bacteria were found associated with the hindgut than with the mesenteron despite continuous plug flow of food through the alimentary canal. Species of bacteria were affected in different ways.Aeromonas hydrophila andCitrobacter freundii were both digested, the former selectively.Flavobacterium sp. and other unidentified species appeared to attach to the hindgut wall. Digestion of bacteria was not due to a sudden change in pH.     

Nanoplanktonic coccolithophorids (Prymnesiophyceae, Haptophyceae) from the Weddell Sea, Antarctica

The examination of whole mounts prepared for transmission electron microscopy has resulted in the finding of thirteen taxa of nanoplanktonic coccolithophorids from the Weddell Sea, Antarctica. The material was collected as part of the AMERIEZ programme, March 1986. Cold-water adapted nanoplanktonic coccolithophorids have previously been shown to constitute a recurrent plankton element at subarctic and arctic localities. Three of the Weddell Sea species, Wigwamma annulifera, W. arctica , and Papposphaera sagittifera , are conspecific with northern hemisphere material, while two species, Calciarcus alaskensis and Turrisphaera arctica , are possibly identical with previously described arctidsubarctic material. Six taxa new to science have been described from the Weddell Sea, Wigwamma antarctica, W. triradiata, Trigonaspis melvillea, Pappomonas weddellensis, Papposphaera obpyramidalis , and P. simplicissima . The cooccurrence of identical forms at the two poles, and the fact that the species described are allocated to "arctic" genera, indicate a geologically relatively recent exchange of biological material between the poles.