Elisha Orr

Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K12

SummaryA strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth was isolated from Escherichia coli K12. Genetic mapping and the molecular weight of the gene product suggest that the mutation is in the cou gene, specifying a sub-unit of DNA gyrase. Nuclear organisation and segregation and placement of septa are grossly abnormal in the mutant at 42°C. RNA synthesis and initiation of DNA replication are also affected at the restrictive temperature but the rate of DNA chain elongation continues almost undisturbed.

A proline-rich protein, verprolin, involved in cytoskeletal organization and cellular growth in the yeast Saccharomyces cerevisiae

A gene (VRP1) encoding a novel proline‐rich protein (verprolin) has been isolated from the yeast Saccharomyces cerevisiae as a result of its hybridization to a chick vinculin cDNA probe. The deduced protein sequence contains 24% proline residues present as proline‐rich motifs throughout the verprolin sequence. Several of these motifs resemble recently identified sequences shown to bind Src homology 3 (SH3) domains in vitro. Replacement of the wild‐type VRP1 allele with a mutant allele results in strains that grow slower than wild‐type strains and are temperature sensitive. The vrp1 mutants are impaired in both cell shape and size and display aberrant chitin and actin localization. We propose that verprolin is involved in the maintenance of the yeast actin cytoskeleton, through interactions with other proteins, possibly containing SH3 domains.

Superoxide dismutase is a virulence factor produced by the coral bleaching pathogen Vibrio shiloi

Coral bleaching is a disease that threatens coral reefs throughout the world. The disease is correlated with higher-than-normal seawater temperatures. Data have been reported showing that bleaching of the coral Oculina patagonica during the summer in the Mediterranean Sea is the result of an infection with Vibrio shiloi. The summer temperatures induce the expression of virulence factors in the pathogen. We report here that V. shiloi produces an extracellular superoxide dismutase (SOD) at 30°C, but not at 16°C. An SOD− mutant was avirulent. The mutant adhered to corals, penetrated into coral cells, multiplied intracellularly for a short time, and then died. These data support the hypothesis that SOD protects the intracellular V. shiloi from oxidative stress caused by the high concentration of oxygen produced by intracellular zooxanthellae photosynthesis.

The Active Component of the Bioemulsifier Alasan from Acinetobacter radioresistens KA53 Is an OmpA-Like Protein

The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. Recently, one of the alasan proteins, with an apparent molecular mass of 45 kDa, was purified and shown to constitute most of the emulsifying activity. The N-terminal sequence of the 45-kDa protein showed high homology to an OmpA-like protein from Acinetobacter spp. In the research described here the gene coding for the 45-kDa protein was cloned, sequenced, and expressed in Escherichia coli. Recombinant protein AlnA (35.77 kDa without the leader sequence) had an amino acid sequence homologous to that of E. coli OmpA and contained 70% of the specific (hydrocarbon-in-water) emulsifying activity of the native 45-kDa protein and 2.4 times that of the alasan complex. In addition to their emulsifying activity, both the native 45-kDa protein and the recombinant AlnA were highly effective in solubilizing phenanthrene, ca. 80 microg per mg of protein, corresponding to 15 to 19 molecules of phenanthrene per molecule of protein. E. coli OmpA had no significant emulsifying or phenanthrene-solubilizing activity. The production of a recombinant surface-active protein (emulsification and solubilization of hydrocarbons in water) from a defined gene makes possible for the first time structure-function studies of a bioemulsan.

The Identification and Purification of a Mammalian-Like Protein Kinase C in the Yeast Saccharomyces cerevisiae

We have purified a yeast protein kinase that is phospholipid-dependent and activated by Diacylglycerol (DAG) in the presence of Ca2+ or by the tumour-promoting agent tetradecanoyl-phorbol acetate (TPA). The properties of this enzyme are similar to those of the mammalian protein kinase C (PKC). The enzyme was purified using chromatography on DEAE-cellulose followed by hydroxylapatite. The latter chromatography separated the activity to three distinguishable sub-species, analogous to the mammalian PKC isoenzymes. The fractions enriched in PKC activity contain proteins that specifically bind TPA, are specifically phosphorylated in the presence of DAG and recognized by anti-mammalian PKC antibodies.

Genetic and functional analysis of genes required for the post-modification of the polyketide antibiotic TA of Myxococcus xanthus

The antibiotic TA of Myxococcus xanthus is a complex macrocyclic polyketide, produced through successive condensations of acetate by a type I PKS (polyketide synthase) mechanism. The genes encoding TA biosynthesis are clustered on a 36 kb DNA fragment, which has been cloned and analysed. The chemical structure of TA and the mechanism by which it is synthesized indicate the need for several post-modification steps, which are introduced into the carbon chain of the polyketide to form the final bioactive molecule. These include the addition of several carbon atoms originating from acetate carbonyl, three C-methylations, O-methylation and a specific hydroxylation. This paper reports the analysis of five genes which are involved in the post-modification of TA. Their functional analysis, by specific gene disruption, suggests that they may be essential for the production of the active antibiotic. The characteristics and organization of the genes suggest that they may be involved in the addition of the carbon atoms which arise from acetate.

An Escherichia coli mutant thermosensitive in the B subunit of DNA gyrase: Effect on the structure and replication of the colicin E1 plasmid in vitro

SummaryAn E. coli strain which carries a mutation conferring clorobiocin resistance and temperature sensitivity for growth has recently been described and evidence has been presented suggesting that the mutation is located in the gyrB gene (Orr et al. 1979). The replication of the ColE1 plasmid was analysed in cell-free extracts from this thermosensitive strain. These extracts were totally deficient in the replication of exogenous plasmid DNA and were unable to maintain the superhelical structure of the plasmid DNA. Both defects could be fully complemented by addition of purified gyrB protein.

Chloramphenicol releases a block in initiation of chromosome replication in a dnaA strain of Escherichia coli K12

SummaryDNA-DNA hybridisation experiments show that chloramphenicol induces a burst of initiation from the oriC region of a dnaA46 mutant of Escherichia coli at 36.5° C but not from the isogenic dnaA+ strain. Following this stimulation of initiation, DNA replication proceeds normally towards the terminus. The temporal pattern of the extra initiation is in parallel with the induced stimulation of RNA synthesis caused by chloramphenicol in the same strain. This is consistent with the hypothesis that the stimulation of initiation in the dnaA mutant is the result of the stimulation of the synthesis of an RNA species.

The yeast type II myosin heavy chain: analysis of its predicted polypeptide sequence.

SummaryWe have completed the nucleotide sequence of the yeastMYO1 gene and deduced its amino acid sequence. The gene is 5553 bp long and contains no introns. Analysis of the sequence, as well as its comparison with other myosins, demonstrate that the yeast protein is a type II myosin heavy chain with characteristic head and tail regions. The latter domain contains six proline residues in two clusters of three, at approximately two thirds from the start of the gene.

Cloning and characterization of a Myxococcus xanthus cytochrome P-450 hydroxylase required for biosynthesis of the polyketide antibiotic TA.

The antibiotic TA, a complex macrocyclic polyketide of Myxococcus xanthus, is produced, like many other polyketides, through successive condensations of acetate by a type I polyketide synthase (PKS) mechanism. The chemical structure of this antibiotic and the mechanism by which it is synthesized indicate the need for several post-modification steps, such as a specific hydroxylation at C-20. Previous studies have shown that several genes, essential for TA biosynthesis, are clustered in a region of at least 36kb, which was subsequently cloned and analyzed. In this study, we report the analysis of a DNA fragment, containing a specific cytochrome P-450 hydroxylase, presumably responsible for the sole non-PKS hydroxylation at position C-20. Functional analysis of the cytochrome P-450 hydroxylase gene through specific gene disruption confirms that it is essential for the production of an active TA molecule.