Orio Ciferri

The microbial degradation of silk: a laboratory investigation

Abstract A few bacterial species, mostly gram-negatives, were found to attach themselves and grow on silk buried in soil. On the contrary, no fungi were isolated in such experiments. Growth was more abundant on raw silk (composed of sericin and fibroin) than on degummed silk (fibroin only) indicating that the majority of these bacteria use sericin rather than fibroin for growth. Electron microscopy demonstrated that bacteria formed a biofilm on the fabric and caused extensive damage to the fibers resulting in considerable reduction in the mechanical properties. Of the three main bacterial species isolated from silk exposed to soil or by enrichment cultures of silk cocoons, only Pseudomonas (Burkholderia) cepacia appeared to be able to use fibroin as a sole source of carbon and nitrogen for growth. Indeed, in laboratory experiments, pure cultures of P. cepacia were found to form a well-developed biofilm on fibroin, to hydrolyze fibroin, and to produce an extracellular enzyme attacking this protein. The reported data indicate that bacteria but not fungi may attack and degrade silk proteins and thus cause irreversible damage to silk artifacts of artistic or historical interest.

Species specificity in protein synthesis

The phenomenon of species specificity in poly U-directed cell-free protein synthesis (whereby combinations of ribosomes of one organism with supernatant enzymes from a different one are sometimes inactive) was studied in the case of the amino acid-incorporating systems prepared from Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae , castor bean seedlings and rat liver. These organisms could be classified into at least two groups, one including B. subtilis and E. coli , and the other S. cerevisiae , castor bean seedlings and rat liver. It appears that within each group, the combination of ribosomes and supernatant enzymes extracted from different organisms gave active synthesis, whereas the permutations of ribosomes and enzymes prepared from organisms belonging to the two different groups gave very little if any synthesis. The mechanism of incompatibility was analyzed in detail in the E. coli -castor bean seedlings combination. It was found that the inability to synthesize polypeptides was not due to any of the following factors: the synthesis of phenylalanyl-sRNA; the source of the phenylalanyl-sRNA used; the selective degradation of heterologous phenylalanyl-sRNA; the binding of phenylalanyl-sRNA to ribosomes; the presence of specific inhibitory activities. The tentative conclusion is drawn that failure to observe protein synthesis in mixed systems is due to the lack of interaction between ribosomes of a given size (70 or 80 s) and polymerizing enzymes extracted from organisms containing ribosomes of different size.

Presence of two sets of ribosome-specific transfer factors in the cell-free extracts from the non-photosynthetic alga Prototheca zopfii

Abstract Preparations of polymerizing enzymes from the non-photosynthetic alga Prototheca zopfii possess two transfer factors G, one active on the ribosomes of the 70 s type ( Escherichia coli ) and one active on those of the 80 s type ( Saccharomyces cerevisiae ). Evidence is presented in favour of the presence also of two ribosome-specific transfer factors T. The G-like activity for E. coli ribosomes present in the polymerizing enzymes from the yeast S. cerevisiae (Ciferri, Parisi, Perani & Grandi, 1968) has been demonstrated to be due to the presence of a transfer factor G, specific for ribosomes of the 70 s type that may be separated from that active on ribosomes of the 80 s type.

The microbial colonization of oil paintings. A laboratory investigation

Only a few bacterial and fungal species present in a soil extract grew and survived on painted canvases prepared utilizing the materials and following the recipes traditionally used for paintings. When pure cultures of these microorganisms were used to contaminate such canvases, microbial growth and survival were even more reduced. On the other hand, on this substrate, the bacteria isolated from a heavily contaminated XVIth century fresco grew to a limited extent, but remained viable for a much longer period of time. Unlike the bacteria isolated from soil, those isolated from the fresco were able to hydrolyse cellulose and proteins. It is thus possible that the capacity to utilize these macromolecules may favour microbial survival on paintings. Furthermore, the addition of phosphate strongly stimulates growth of all microbial species indicating that this anion is the limiting nutrient factor.

Ribosomes and translation factors from isolated spinach chloroplasts

Abstract Chloroplasts isolated from spinach leaves carry on a light-driven, RNAase-insensitive incorporation of amino acids. From such chloroplasts an active S-20 fraction was prepared which was capable of incorporating amino acids under the direction of the endogenous mRNA and phenylalanine under that of polyuridylic acid (poly U). Initiation and elongation factors as well as ribosomes active in vitro were obtained from isolated chloroplasts.

RELATION OF BIOCHEMICAL MUTATIONS TO ACTINOMYCIN SYNTHESIS IN STREPTOMYCES ANTIBIOTICUS.

Biochemical mutants of Streptomyces antibioticus were isolated and tested for their capacity to produce actinomycin. The yield of actinomycin produced in minimal medium plus the required amino acid, by strains requiring an amino acid not present in the molecule of actinomycin, was not significantly different from that obtained from the wild-type strain. On the other hand, all the strains which required an amino acid, which was also a precursor of the antibiotic, showed on minimal medium a drastic decrease in the production of actinomycin. The results have been interpreted by assuming a different utilization by the cell of exogenous and endogenous amino acid pools for antibiotic and protein synthesis.

Cloning and expression of the genes for ribulose-1,5-bisphosphate carboxylase from Spirulina platensis

Abstract The genes for the large and small subunits of ribulose-1,5-bisphosphate carboxylase have been cloned from the filamentous cyanobacterium Spirulina platensis. The two genes, located very closely on a 4.6 kbp DNA fragment, appear to be expressed although to a different extent in minicells of Escherichia coli. The amount of large subunit produced in the bacterial host represents at least 10% of the total protein.

Incorporation of amino acids by a cell-free system prepared from human cells cultured in vitro

Abstract 1. A cell-free amino acid-polymerizing system has been prepared from human cells (EUE) cultured in vitro . The requirements and properties of the system are those expected for protein-synthesizing activity in vitro . 2. The system responds to endogenous mRNA, tobacco mosaic virus RNA and synthetic polynucleotides. 3. Of the compounds known to interfere with protein synthesis, pederin appears to be the most active, being inhibitory at concentrations as low as 0.01 μg/ml. 4. Ribosomes from EUE cells are active in the presence of polymerizing enzymes prepared from other organisms containing 80-S ribosomes but not from those endowed with 70-S ribosomes. Likewise, polymerizing enzymes from EUE cells are active only when added to 80-S ribosomes.

The chloroplast DNA mystery

Twenty years after the discovery that chloroplasts contain DNA we still do not know the significance of approximately 90% of this DNA.

Selective inhibition of the reactions catalyzed by ribosome-specific transfer factors G

It is well known that one of the transfer factors required for peptide chain elongation, transfer factor G (translocase) catalyzes the translocation of peptidyl-tRNA onto the ribosome [l] The translocation requires GTP and it has been calculated that one GTP is hydrolyzed to GDP and inorganic phosphate for every peptide bond formed [2]. In addition the factor, whether isolated from prokaryotic or eukaryotic organisms, is endowed with GTP-ase activity dependent on the presence of ribosomes [2-51. Albeit evident also under conditions in which there is no peptide chain elongation, it has been assumed that the hydrolytic activity is related to the synthetic activity [4,6] since the two activities are purified together and all the antibiotics so far tested that inhibit translocation also inhibit ribosome dependent GTP-ase. Indeed fusidic acid appears to inhibit both activities in the case of the factors G from prokaryotic and eukaryotic organisms [7, 81 , while siomycin exerts a similar effect on Escherichia coli transfer factor G and E. coli ribosomes [6] and diphtheria toxin and factors G (transfer factors II) * and ribosomes from mammalian cells and organs [9111. We have previously shown that the achloric alga Prototheca zopfii is endowed with two separated transfer factors G, one specific for 70 S ribosomes (such as are those present in prokaryotic organisms and in cellular organelles) and the other specific for ribosomes of the 80 S type as are those present in the cytoplasm of eukaryotic organisms [ 121. The availability of the two transfer factors G at a degree