Biomedical subjects

C Hardisson

Publications and source records attributed to C Hardisson.

At least 19 recordsLinked to original sources

PubMed1999

Hyphal death during colony development in Streptomyces antibioticus: morphological evidence for the existence of a process of cell deletion in a multicellular prokaryote.

During the life cycle of the streptomycetes, large numbers of hyphae die; the surviving ones undergo cellular differentiation and appear as chains of spores in the mature colony. Here we report that the hyphae of Streptomyces antibioticus die through an orderly process of internal cell dismantling that permits the doomed hyphae to be eliminated with minimum disruption of the colony architecture. Morphological and biochemical approaches revealed progressive disorganization of the nucleoid substructure, followed by degradation of DNA and cytoplasmic constituents with transient maintenance of plasma membrane integrity. Then the hyphae collapsed and appeared empty of cellular contents but retained an apparently intact cell wall. In addition, hyphal death occurred at specific regions and times during colony development. Analysis of DNA degradation carried out by gel electrophoresis and studies on the presence of dying hyphae within the mycelium carried out by electron microscopy revealed two rounds of hyphal death: in the substrate mycelium during emergence of the aerial hyphae, and in the aerial mycelium during formation of the spores. This suggests that hyphal death in S. antibioticus is somehow included in the developmental program of the organism.

Cell Membrane↗

PubMed1997

Plasmid-encoded fosfomycin resistance in bacteria isolated from the urinary tract in a multicentre survey.

Sixty out of 219 fosfomycin-resistant bacteria selected from more than 7400 urinary pathogens in an epidemiological multicentre survey performed in Italy were screened for plasmid genes fosA and fosB conferring fosfomycin resistance. Only five strains, three enterobacteria and two staphylococci, carried plasmids harbouring, respectively, fosA and fosB genes. Fosfomycin resistance in the other isolates was caused by an alteration of the chromosomally encoded GlpT transport system. One strain, Morganella morganii 279, incorporated alpha-glycerolphosphate and its mechanism of fosfomycin resistance needs to be further investigated. Our study showed that PCR amplification is the most accurate, simple and rapid method for epidemiological studies of plasmid-encoded fosfomycin resistance, and that fosfomycin resistance conferred by plasmid genes (both fosA and fosB) accounts for only a low percentage of the fosfomycin-resistant strains.

Alkyl and Aryl Transferases↗

PubMed1997

A glgC gene essential only for the first of two spatially distinct phases of glycogen synthesis in Streptomyces coelicolor A3(2).

By using a PCR approach based on conserved regions of ADP-glucose pyrophosphorylases, a glgC gene was cloned from Streptomyces coelicolor A3(2). The deduced glgC gene product showed end-to-end relatedness to other bacterial ADP-glucose pyrophosphorylases. The glgC gene is about 1,000 kb from the leftmost chromosome end and is not closely linked to either of the two glgB genes of S. coelicolor, which encode glycogen branching enzymes active in different locations in differentiated colonies. Disruption of glgC eliminated only the first of two temporal peaks of ADP-glucose pyrophosphorylase activity and glycogen accumulation and prevented cytologically observable glycogen accumulation in the substrate mycelium of colonies (phase I), while glycogen deposition in young spore chains (phase II) remained readily detectable. The cloned glgC gene therefore encodes an ADP-glucose pyrophosphorylase essential only for phase I (and it is therefore named glgCI). A second, phase II-specific, glgC gene should also exist in S. coelicolor, though it was not detected by hybridization analysis.

Amino Acid Sequence↗

PubMed1996

Functional analysis of pneumolysin by use of monoclonal antibodies.

We have produced a panel of monoclonal antibodies to pneumolysin, the membrane-damaging toxin from Streptococcus pneumoniae. We have used these antibodies to identify three regions of the toxin sequence that are involved in the lytic mechanism of this toxin. Two of these sites probably form the cell binding site of this toxin. Antibodies to the third site inhibit the lytic action of this toxin but not the binding of this toxin to cells. This site is engaged in the oligomerization process involved in the formation of pores in cell membranes. Two of these epitopes are also present in the related toxin perfringolysin O.

Animals↗

PubMed1994

Structural changes induced by glycine on Streptomyces antibioticus.

Germination and vegetative growth of Streptomyces antibioticus in liquid medium with different concentrations of glycine was examined. Both processes proved to be sensitive to the amino acid, being inhibited by 5 and 2.5% glycine, respectively. At concentrations of 5% or more, lysis of the vegetative mycelium occurred. Subinhibitory concentrations of glycine induced structural changes on germinating spores. These included an increase in the number of germ tubes produced by spore, in relation to the control. Moreover, soon after outgrowth the tubes bifurcate, giving rise to germinated spores with a characteristic aspect, and anomalous formation of cross-walls that appear both within the spores and in the newly formed germinative tubes, at or close to the region of outgrowth. The branching effect of glycine was also observed during vegetative growth of S. antibioticus.

Dose-Response Relationship, Drug↗

PubMed1994

Autoradiographic study of hyphal growth during aerial mycelium development in Streptomyces antibioticus.

The pattern of growth of aerial mycelium in Streptomyces species was investigated by autoradiography. Colonies of Streptomyces antibiotics were labeled with N-acetyl-D-[1-3H] glucosamine to localize the sites of hyphal growth during the development of aerial mycelium. Autoradiographs obtained with sections of the colonies revealed that hyphal growth occurs not only at the top of the colony but also in the inner zones of the aerial mycelium.

Acetylglucosamine↗

PubMed1993

Synchronous germination of Streptomyces antibioticus spores: tool for the analysis of hyphal growth in liquid cultures.

We have devised a method for obtaining synchronous and dispersed growth of Streptomyces antibioticus in liquid cultures. After ultrasonic treatment, most of the spores germinated at the same time, yielding hyphae very similar in length. Dispersed growth was achieved in media without Ca2+ and in which the levels of Fe2+ and Mg2+ were carefully controlled. Studies on the kinetics of growth carried out with synchronous cultures of young hyphae revealed a multiphasic pattern of hyphal elongation, with successive periods of linear growth and changes in growth rate at defined intervals.

Culture Media↗

PubMed1992

Growth and morphogenesis in Streptomyces.

In this report we propose a model of apical growth for streptomycetes. The apical tip is considered as a multilayered wall that expands by an inside-to-outside mechanism of growth. It is also assumed that each layer is made up of peptidoglycan blocks, each of them being the result of the biosynthetic activity of a wall-synthesizing unit or membrane-associated growth zone. According to our model, apical growth occurs as follows: as a consequence of the hydrostatic pressure and the cleavage of some bonds, the layers are pushed and forced to slide (one with respect to the other), migrating from the center of the tip (at the inside of the wall) towards a peripheral location (at the outside of the wall). The model also incorporates a mechanism by which apical growth can be regulated and coordinated with the replication of the chromosome.

Cell Wall↗

PubMed1992

Purification and characterization of a nutritionally controlled endodeoxyribonuclease from Streptomyces glaucescens.

Streptomyces glaucescens has a DNAase whose synthesis is under nutritional control. We have purified this enzyme to apparent homogeneity by phosphocellulose chromatography followed by heparin-agarose, Cibacron Blue F3-GA-Sepharose and Sephadex G-75 chromatography and MonoQ f.p.l.c. The enzyme had an apparent Mr of 39,600 and a pI of approx. 8.15. The Mr of the native enzyme estimated by gel chromatography was 49,000. The DNAase had a pH optimum of 7.5 and an absolute requirement for bivalent cations in the reaction buffer. It was inhibited by high salt concentrations, chelating agents or phosphate-containing compounds and was stimulated by dimethyl sulphoxide. The activity was greatly diminished unless dithiothreitol or 2-mercaptoethanol was included in the reaction mixture. Reagents such as Hg2+ or iodoacetate strongly inhibited the enzyme. The nuclease hydrolysed both double-stranded and single-stranded DNA, showing greater affinity for double-stranded DNA, and no detectable hydrolysis of RNA. The enzyme produced nicks in double-stranded DNA, generating 3'-hydroxy and 5'-phosphate termini, and degraded circular DNA.

Chromatography, Affinity↗

PubMed1992

Yersinia enterocolitica serotype 0:3-induced arthritis in mice: microbiological and histopathological information.

Gross anatomical and histopathological changes in arthritic joints resulting from oral challenge with Yersinia enterocolitica serotype 0:3, upon pretreatment with desferrioxamine, were always more severe than those induced by intravenous infection of immunized animals. In all the acute inflammation episodes studied, live Yersiniae were isolated from the arthritic region. Invariably, a heavy mixed infiltration of synovia, joint spaces and soft tissues was observed at this stage. Concurrent fibrous thickening and vascular proliferation, along with erosion of articular cartilages and anomalous bone regeneration, were also apparent. In spite of these significant facts, the bacterium could be histopathologically identified only in bone marrow where it developed microcolonies and caused significant necrosis as well. The live bacterium was also retrieved from two- and six-month-old arthritic ankles/paws examined, but it could not be seen in histological sections of joints. By this time, no cellular infiltration was evident, but there was extensive fibrosis. Bones were at times greatly enlarged, showing a spongeous-like structure. Additionally, articular cartilages could be completely lost and were substituted by an anomalous ossification filling the joint spaces. This situation led to bone fusion, resembling articular ankylosing traits. In summary, we present the first experimental evidence that Y. enterocolitica serotype 0:3 is a causal agent of osteoarthritis and osteomyelitis, and that it may survive for prolonged periods of time in osseous structures.

Animals↗

PubMed1991

Biological characterization of the lytic cycle of actinophage phi A7 in Streptomyces antibioticus.

Some basic parameters of the lytic development of phage phi A7 in Streptomyces antibioticus are described. One-step growth experiments demonstrated that at 28 degrees C phi A7 has a latent period of about 60 min and an exponential growth period of about 35 min. The average burst size ranged from 70-100 plaque forming units per infected cell. At the same temperature 50% of the virions were adsorbed to germ tubes of S. antibioticus in about 10 min. This corresponds to an adsorption constant of 6.5 x 10(-10) ml/min. The phage was unable to adsorb the host at other stages of the life cycle (spores or mycelium). Divalent cations are not required for phi A7 stability but Ca2+ proved to be essential for adsorption and also for a later stage of the vegetative development of the phage.

Adsorption↗

PubMed1991

Characterization of the temperate actinophage phi A7 DNA and its deletion derivatives.

A restriction map of phi A7 DNA (46.7 kb) was established for nine endonucleases (BclI, ClaI, EcoRI, EcoRV, HpaI, PvuI, SacII, SphI and XbaI) which cut the phage genome up to 11 times. There was no sites for BamHI, BglII, HindIII, PstI, PvuII, SacI or SalI. phi A7 DNA, circularized through its cohesive ends, could integrate into the genome of several Streptomyces hosts, to form stable lysogens. Integration occurred by recombination between unique attachment sites on the phage (attP) and the host (attB) genomes. The attP site has been located on the phi A7 restriction map. Deletion mutants of phi A7 DNA were obtained by selecting for pyrophosphate- or EDTA-resistant clones. The deletions occurred either near the left-hand end of the conventional restriction map, or about 18 kb from the right-hand end, close to, but not affecting the unique SacII site. Together, the deletions defined at least 7.9 kb of DNA (16.9% of the phage genome) non-essential for plaque formation. phi A7 DNA was introduced into S. lividans protoplasts by liposome-assisted transfection. Since the phage does not adsorb to intact cells of this strain, and therefore does not form plaques, an overlay of S. antibioticus spores was used to detect the infectious progeny released by the protoplasts. Using this technique, phi A7 could be introduced into S. antibioticus with an efficiency of about 6 x 10(6) p.f.u. per micrograms DNA (equivalent to 3 x 10(-4) p.f.u. per DNA molecule).

Attachment Sites, Microbiological↗

PubMed1991

Nutritional regulation of differentiation and synthesis of an exocytoplasmic deoxyriboendonuclease in Streptomyces antibioticus.

Streptomyces antibioticus produces a cell-wall-located deoxyriboendonuclease (DNAase) the synthesis of which in submerged and surface cultures is related to the growth rate. DNAase synthesis always preceded aerial mycelium formation in surface cultures. Production of aerial mycelium began at the end of exponential growth or in the early stationary phase; it was absent in cultures grown on nutrient agar/glucose or in media with a high concentration of casein hydrolysate. These nutritional conditions also impaired production of the DNAase. External DNA substrates were not degraded by mycelium producing the DNAase. These observations lead us to suggest a role for the enzyme in the developmental cycle of S. antibioticus.

Amino Acids↗

PubMed1991

Characterization of Rrh4273I, a restriction-modification system of Rhodococcus rhodochrous ATCC 4273 (Nocardia corallina) which recognizes the same sequence as the Streptomyces albus G SalI restriction-modification system.

Rhodococcus rhodochrous ATCC 4275 (Nocardia corallina) has a restriction-modification system with the same recognition sequence, methylation site and cleavage site as the SalI restriction-modification system. Both the restriction endonuclease and the DNA-methyltransferase (DNA-MTase) have been partially purified and characterized. The nuclease has requirements of activity similar to SalI, and a native Mr of about 46,000. The DNA-MTase is a protein with an Mr of about 67,000. No DNA homology was detected between the cloned salI restriction-modification genes of Streptomyces albus and R. rhodochrous chromosomal DNA.

Base Sequence↗

PubMed1991

Stringent response and initiation of secondary metabolism in Streptomyces clavuligerus.

Cephalosporin biosynthetic activity and extracellular protease production increased during growth of Streptomyces clavuligerus in defined medium, while the level of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) remained very low and stable. Cephalosporin biosynthesis (measured in resting cell systems) was initiated during early exponential growth in complex media, without appreciable change in the small ppGpp pool. Nutritional shift-down induced by withdrawal of Casamino acids caused a transient increase in ppGpp and a reduction of RNA accumulation. The increase in ppGpp was small in very young cultures, but increased as the culture aged. Twenty-seven spontaneous thiostrepton-resistant mutants were isolated and partially characterized. Most of them had a reduced ppGpp-forming ability and gave normal titres of cephalosporin. However, in complex medium, some mutants did not produce cephalosporins or extracellular protease, whereas others overproduced cephalosporins. The results indicate that, in S. clavuligerus, there is no obligatory relationship between the initiation of secondary metabolism and the stringent response.

Amino Acids↗

PubMed1991

Protection against the lethal effect and arthritogenic capacity of Yersinia enterocolitica serovar O:3 for mice.

Protection against the intravenous lethal effect of Yersinia enterocolitica serovar O:3 for mice can be achieved by oral immunization with bacteria expressing the O:3 lipopolysaccharide (LPS). Under similar experimental conditions, Ca(2+)-dependent cells are more protective than their Ca(2+)-independent counterparts, live vaccines are more efficacious than killed ones, and parenteral immunization is more efficient than the oral route. Antibodies induced by enzyme-treated LPS from an O:3 strain are able to mediate protection against challenge with one homologous strain, but they do not prevent induction of arthritis.

Administration, Oral↗

PubMed1991

The effect of sinefungin and synthetic analogues on RNA and DNA methyltransferases from Streptomyces.

Sinefungin is an antibiotic structurally related to S-adenosylmethionine. It has been described as an inhibitor of RNA transmethylation reactions in viruses and eukaryotic organisms, but not in bacteria. We show here that sinefungin strongly inhibits RNA methyltransferase activity, but not the biosynthesis of these enzymes in Streptomyces. All the methylated bases found in Streptomyces RNA (1-methyladenine, N6-methyladenine, N6,N6-dimethyladenine and 7-methylguanine) are inhibited by this antibiotic. Experiments with sinefungin analogues show that specific changes in the ornithine radical of the molecule still preserve its inhibitory capability. The substitution of the adenine radical by uridine causes the loss of the inhibitory effect. These results and our former studies on Streptomyces DNA methylation, suggest that nucleic acid modification is the main target of sinefungin in Streptomyces.

Adenosine↗

PubMed1990

Effects of sinefungin and S-adenosylhomocysteine on DNA and protein methyltransferases from Streptomyces and other bacteria.

Sinefungin is a naturally occurring nucleoside isolated from cultures of Streptomyces griseolus and S. incarnatus. It is structurally related to S-adenosyl-methionine (SAM) and S-adenosyl-L-homocysteine (SAH). Its effect and level of action on prokaryotes has not been studied with the same detail as with eukaryotic cells. In this report we describe the effect of sinefungin and SAH on several Streptomyces methyltransferases (DNA and protein MTases) and on other bacterial DNA-MTases. Protein MTases are resistant to sinefungin, whereas DNA-MTases are inhibited. Adenine MTases however, seem more sensitive to this analogue than cytosine MTases.

Adenosine↗