Claude V. Déry

Characterization of bifidobacteria by random DNA amplification

RAPD conditions were optimized to generate reproducible banding patterns by testing primers, thermocyclers and overall reproducibility in repeat DNA analysis and separate DNA extractions. Five primers were chosen on the basis of band intensity and distribution (between 2 and 10 bands) which clearly distinguished among strains of Bifidobacterium adolescentis, B. animalis, B. bifidum, B. breve, B. infantis and B. longum. The use of five single-primer reactions under optimized conditions improved the resolution and accuracy of the RAPD method for the characterization of dairy-related bifidobacteria. The results indicated that this method was highly reproducible in repeated analysis. Similarity between bifidobacteria strains was evaluated based on their RAPD profiles. Using a set of five primers, it was demonstrated that it may be possible to distinguish three different species of Bifidobacterium (B. breve, B. bifidum and B. adolescentis), based on similarity of the RAPD profiles to known reference strains. Furthermore, application of the RAPD technique may also be useful and faster, than traditional systematics for placement of industrial strains into specific clusters (either B. longum/infantis or B. animalis/lactis).

The structure of adenovirus chromatin in infected cells.

The structure of adenovirus chromatin in infected cells was studied by micrococcal nuclease digestion and hybridization with virus-specific probes. In the early phase of infection (5 h) a significant proportion of viral molecules was organized like actively transcribed cellular chromatin. As expected for a transcriptionally active population of molecules, even at high multiplicity of infection the nucleosomal repeating pattern was less distinct than in a transformed cell which contained the corresponding but less active genomic region. The observed repeating pattern in infected cells was unlikely to be due to integrated molecules since less than 0.07% of input genomes became associated with cellular DNA. After the onset of viral DNA replication, the pool of viral chromatin organized like cellular chromatin rapidly increased. In addition, newly replicated molecules also maintained the cellular chromatin-like organization as measured by [3H]thymidine incorporation after the cessation of cellular DNA synthesis. These data suggest that newly replicated viral molecules are organized by histones into cell-like chromatin throughout the infection cycle. Coincident with the peak of viral DNA and core protein synthesis, and the decline of histone synthesis, the late, core-like non-repeating viral chromatin became dominant, increasingly obscuring the underlying repeating pattern. Experiments suggest that this late chromatin is destined for encapsidation, that the early chromatin persists and that viral core proteins do not displace histones on viral DNA. A model is proposed suggesting that transcription and type I replication occur on histone-condensed templates, while type II replication products late in infection are condensed by core proteins and are destined for encapsidation.

Chitinolytic properties of Streptomyces lividans

Streptomyces lividans TK24, an established host for genetic and molecular studies in actinomycetes, is able to use chitin as sole carbon and nitrogen source. Extracellular chitinase and N-acetyl-β-d-glucosamidinase (chitobiase) activities were detected in liquid cultures. Chitinase production was inducible by chitin and its low molecular weight derivatives. Low levels of chitinase were also produced in the absence of chitin. Production of extracellular N-acetylglucosaminidase was correlated with the beginning of the stationary phase of growth and was independent of the presence of chitin. Beside highly N-acetylated chitin, supernatants of chitin-induced cultures were able to hydrolyse chitosans with a wide range of degrees of N-acetylation.

Saccharopolyspora hirsuta 367 encodes clustered genes similar to ketoacyl synthase, ketoacyl reductase, acyl carrier protein, and biotin carboxyl carrier protein.

The actI gene, encoding a component of the actinorhodin polyketide synthase of Streptomyces coelicolor, was used to identify and clone a homologous 11.7 kb BamHI DNA fragment from Saccharopolyspora hirsuta 367. The cloned fragment complemented actinorhodin production in a strain of Streptomyces coelicolor bearing a mutant actI gene. The DNA sequence of a 5.1 kb fragment revealed 6 open reading frames (ORF). ORF1 does not resemble any known DNA or deduced protein sequence, while the deduced protein sequence of ORF2 resembles that of biotin carboxyl carrier proteins. Based on the similarity to deduced protein sequences from cloned genes of polyketide producers, ORF3 would code for a ketoreductase, ORF4 and ORF5 for the putative heterodimeric β-ketoacyl synthase, and ORF6 for an acyl carrier protein.

Possible role of ADP-ribosylation of adenovirus core proteins in virus infection

We have investigated the role of poly(ADP)-ribosylation of adenoviral proteins in virus infection. Viral core proteins V and the precursor to protein VII were shown to be in vivo and in vitro acceptors of ADP-ribose. In vivo ADP-ribosylation was restricted to viral proteins as the histones were not labeled during the late phase of infection. The ADP-ribosylated core proteins were assembled into mature virus particles. In vitro ADP-ribosylation of adenoviral core proteins performed with purified poly(ADP-ribose) polymerase led to relaxation of the chromatin structure of both ts1 and wild type pyridine cores and pentonless particles and triggered the complete dissociation of wild type particles. A critical role for poly(ADP)-ribosylation in virus infection was confirmed by measuring the effect of the inhibitors 3-aminobenzamide and nicotinamide on virus particle yield and infectivity. Both inhibitors depressed particle yield by up to 9-fold, but infectivity was reduced by up to 10(4)-fold. These results suggest that ADP-ribosylation of adenovirus core proteins may have a role in virus decapsidation.

Characterization of SE-3, a Virulent Bacteriophage of Saccharopolyspora erythraea

SE-3 is a virulent bacteriophage isolated from a large-scale culture of Saccharopolyspora erythraea, an erythromycin producer. The host range of the phage is narrow, limited to some strains of this species. Another strain of Sac. erythraea, and a strain of Sac. hirsuta, are able to adsorb phage particles but do not sustain their complete multiplication. SE-3 is closely related to the phage SE-5 as shown by DNA restriction mapping. The differences between SE-3 and SE-5 genomes are apparently limited to two DNA segments flanked by short inverted repeats, visualized by electron microscopy.

Biological characterization of induced phages from Saccharopolyspora hirsuta 367 and comparison with phage JHJ-1

Phages JHJ-2 and JHJ-3 were isolated from Saccharopolyspora hirsuta 367 UC 8106 following induction with mitomycin C and amplified on S. hirsuta NRRL B-5792. Their properties were compared with those of phage JHJ-1, isolated previously from S. hirsuta 367 NRRL 12045. The DNA restriction patterns appeared to be identical. One-step growth experiments showed no differences between the replication cycles. Burst sizes ranged from 100 to 110 p.f.u. per cell. However, the three phages showed some differences in their behaviour in different hosts. The host range of phage JHJ-1, on non-lysogenic strains, was emended to include all of the Saccharopolyspora strains tested; the host range of phage JHJ-2 was shown to be identical to JHJ-1. Phage JHJ-3 did not form detectable plaques on strains of S. rectivirgula or S. erythraea except S. erythraea NRRL 2359. Neither phage JHJ-2 nor JHJ-3 formed plaques on any lysogenic strains, while JHJ-1 formed plaques on all such strains except S. hirsuta 367 UC8106. Phage JHJ-3 was characterized as a temperate bacteriophage because it formed turbid, self-limiting plaques and lysogenized S. hirsuta NRRL B-5792. It was spontaneously released from UC8106. Both JHJ-1 and JHJ-2 formed clear and invasive (Inv+ phenotype: the property to grow on old mycelium) plaques on some Saccharopolyspora strains but clear and self-limiting plaques on others. Thus, the expression of the Inv+ phenotype encoded by JHJ-1 and JHJ-2 appears to be modulated by the host cell.

Activation of the major late promoter in adenovirus transformed cells by 5-azacytidine

The adenovirus major late promoter (MLP) is normally not active in transformed cells. We investigated if it could be activated with 5-azacytidine. Three days of treatment with 10 microM 5-azacytidine induced transient activation of the MLP as shown by hybridization with an L1 r-strand-specific probe. The po/III-transcribed VA-RNAs were not activated. L1 activation was not accompanied by detectable changes in methylation of HpaII sites at the promoter or in the body of the transcript. Stably activated cell clones could be obtained at 20% frequency after long-term drug treatment.

A cloned replicon of Saccharopolyspora phages JHJ-1 and JHJ-3 is stably maintained as a plasmid in various actinomycetes

A replicon of phage JHJ-1 (and JHJ-3) was cloned. The autonomously replicating phage element was maintained as a medium-copy-number shuttle plasmid in many actinomycetes, and was efficiently transmitted to spores without antibiotic selection. One gene was shown to be expressed in a vector containing the JHJ-3 replicon.