Carlos Hardisson

Fine structure, physiology and biochemistry of arthrospore germination in Streptomyces antibioticus.

During germination, Streptomyces antibioticus arthrospores passed through stages: darkening, swelling and germ tube emergence. The first stage, darkening, whose main features were a decrease in absorbance and a loss of refractility, only required exogenous divalent cations (Ca2+, Mg2+ or Fe2+) and energy that can be obtained from the spore reserves. This stage was blocked by agents that inhibit ATP formation but not by antibiotics that inhibit macromolecular synthesis. The second stage, swelling, needed an exogenous carbon source and was not blocked by mitomycin C. In this stage, the spores exhibited the highest cytochrome oxidase and catalase activities and respiratory quotient. The last stage, germ tube emergence, required additional carbon and nitrogen sources. Ammonium compounds were superior to nitrate. Dry weight remained constant during the stages of darkening and swelling, with a rapid increase from the moment of germ tube emergence. Optimum pH and temperature for germination were 8.0 and 45 degrees C, respectively. Heat treatment (55 degrees C for 10 min) had no effect on germination. The fine structure of the spore underwent important changes during germination. The wall of the swollen spore became stratified and the inner layer was continuous with the germ tube wall. Macromolecular synthesis occurred in the sequence RNA, protein and then DNA. Rifampicin, streptomycin and mitomycin C prevented synthesis when added at the start of incubation. The same effect was obtained if the addition was made during germination, except with mitomycin C which inhibited DNA, but not RNA and protein synthesis.

Formation of an adduct between fosfomycin and glutathione: a new mechanism of antibiotic resistance in bacteria.

Plasmid-borne resistance to fosfomycin in bacteria is due to modification of the antibiotic molecule by a glutathione S-transferase that catalyzes the formation of a covalent bond between the sulfhydryl residue of the cysteine in glutathione and the C-1 of fosfomycin. This reaction results in opening of the epoxide ring of the antibiotic to form an inactive adduct, the structure of which was confirmed by nuclear magnetic resonance. Dialyzed extracts prepared from resistant Escherichia coli strains were unable to modify fosfomycin unless exogenous glutathione was added to the reaction mixtures. Similarly, mutants defective in glutathione biosynthesis were susceptible to fosfomycin, despite harboring a resistance plasmid. Extracts of resistant but not susceptible strains could join glutathione to 1-chloro-2,4-dinitrobenzene, confirming the nature of the enzymatic activity. Adduct formation appeared to be specific for glutathione: none of the other thiols tested (cysteine, N-acetylcysteine, and dithiothreitol) could modify fosfomycin.

Purification of a glutathione S-transferase that mediates fosfomycin resistance in bacteria.

The enzyme that modifies fosfomycin by formation of an adduct with glutathione was purified 12-fold with a 56% activity yield by passage through DEAE Sephacel and high-performance liquid chromatography molecular exclusion columns. Its functional form was a homodimer of two 16,000-dalton polypeptides, which possibly showed an antiparallel alpha tertiary structure and which lacked marked hydrophobic regions. Visualization of the reaction was achieved by precolumn derivatization of glutathione and the adduct, separation by high-performance liquid chromatography, and fluorescence detection of both compounds. Temperature and pH optima were 20 to 30 degrees C and 8.25, respectively; Mn2+, Fe2+, and Co2+ enhanced the rate of modification; and Km values were 9.4 and 11 mM for fosfomycin and glutathione, respectively. Phosphoenolpyruvate did not interfere with fosfomycin modification. The enzyme was stable at 4 degrees C for at least 6 months but progressively lost its activity upon being heated for 60 min at temperatures over 30 degrees C. Images

Glycogen and Trehalose Accumulation during Colony Development in Streptomyces antibioticus

Streptomyces antibioticus accumulated glycogen and trehalose in a characteristic way during growth on solid medium. Glycogen storage in the substrate mycelium took place during development of the aerial mycelium. The concentration of nitrogen source in the culture medium influenced the time at which accumulation started as well as the maximum levels of polysaccharide stored. Degradation of these glycogen reserves was observed near the beginning of sporulation. The onset of sporogenesis was always accompanied by a new accumulation of glycogen in sporulating hyphae. During spore maturation the accumulated polysaccharide was degraded. No glycogen was observed in aerial non-sporulating hyphae or in mature spores. Trehalose was detected during all phases of colony development. A preferential accumulation was found in aerial hyphae and spores, where it reached levels up to 12% of the cell dry weight. The possible roles of both carbohydrates in the developmental cycle of Streptomyces are discussed.

Biosynthesis of oleandomycin by Streptomyces antibioticus: influence of nutritional conditions and development of resistance.

The influence of different nutritional compounds on oleandomycin biosynthesis by Streptomyces antibioticus was studied, resulting in the design of a chemically defined medium for production of the antibiotic. Of the variety of carbon and nitrogen compounds tested, fructose and aspartic acid (carbon and nitrogen sources, respectively) supported the highest oleandomycin titres. Addition of propionate but not acetate, both precursors of the skeleton of the macrolide lactone ring, stimulated the biosynthesis of the antibiotic. Oleandomycin biosynthesis was repressed by glucose but not by phosphate. S. antibioticus develops oleandomycin resistance shortly before the antibiotic begins to be synthesized, showing a triphasic pattern of resistance: spores and producing mycelium are resistant, while non-producing mycelium is sensitive.

Plasmid-determined resistance to fosfomycin in Serratia marcescens.

Multiple-antibiotic-resistant strains of Serratia marcescens isolated from hospitalized patients were examined for their ability to transfer antibiotic resistance to Escherichia coli by conjugation. Two different patterns of linked transferable resistance were found among the transconjugants. The first comprised resistance to carbenicillin, streptomycin, and fosfomycin; the second, and more common, pattern included resistance to carbenicillin, streptomycin, kanamycin, gentamicin, tetracycline, chloramphenicol, sulfonamide, and fosfomycin. The two types of transconjugant strains carried a single plasmid of either 57 or 97 megadaltons in size. Both of these plasmids are present in parental S. marcescens strains resistant to fosfomycin. The 57-megadalton plasmid was transformed into E. coli. Images

Regulation of extracellular protease production in Streptomyces clavuligerus

SummaryThe production of extracellular protease by Streptomyces clavuligerus was strongly influenced by the nature of the nitrogen source. Production took place in batch cultures during growth at low or intermediate growth rates, but was delayed to the post-exponential phase in media supporting high growth rates. Protease formation could be initiated by a nutritional shift-down induced by casamino acids deprivation. Under both types of conditions maximal production was related to the growth rates of the cultures and was stimulated by low concentrations of casamino acids or yeast extract. Some purine compounds also influenced production in shift-down conditions. Ammonium interfered with protease formation whenever it was added to the medium. Some mutants with ltered nitrogen control of primary metabolism were also affected in the production of protease. A partial characterization of the activity indicated that it was due to a single metalloprotease with an apparent molecular mass of 41,700 Da.

Regulation of Nitrogen Catabolic Enzymes in Streptomyces clavuligerus

The levels of several enzymes involved in assimilation of different nitrogen compounds were investigated in Streptomyces clavuligerus in relation to the nitrogen source supplied to the cultures. Threonine dehydratase, serine dehydratase, proline dehydrogenase, histidase and urocanase were not decreased in the presence of ammonium. The latter two enzymes were induced by histidine in the culture medium, while proline dehydrogenase was induced by proline. Glutamine synthetase, urease and ornithine aminotransferase levels were higher with poor nitrogen sources and were repressed by ammonium. Arginase was induced by arginine and repressed by ammonium. Glutamine synthetase was rapidly inactivated upon addition of ammonium to the culture, and could be reactivated in vitro by treatment with snake venom phosphodiesterase, which suggested that adenylylation is involved in the inactivation. Three previously isolated mutants with abnormal glutamine synthetase activities showed pleiotropic effects on urease formation. All these data point to a mechanism controlling preferential utilization of some nitrogen sources in this species.

Plasmid-mediated fosfomycin resistance is due to enzymatic modification of the antibiotic.

The molecular mechanism of plasmid-mediated resistance to fosfomycin is described. The antibiotic was inactivated intracellularly and remained inside the cells. Modification was also obtained from cell extracts and was not energy dependent. The modifying enzyme seems to have sulfhydryl groups in its active center.

Purification and study of a bacterial glutathione S-transferase.

A glutathione S‐transferase from Escherichia coli has been purified approximately 800‐fold with an 11% activity yield by passage through DEAE Sephacel and glutathione‐agarose affinity columns. Its functional form is a homodimer of two 24 000 Da polypeptides that catalyzes the binding of glutathione and 1‐chloro‐2,4‐dinitrobenzene withK m values of 0.25 and 1.5 mM, respectively. Optima of pH and temperature were 7.5 and 35°C. The activity was stimulated (30%) by ethylenediaminetetraacetic add. The N‐tenninal amino acid sequence was: Met‐Leu‐Leu‐Phe‐Ile‐Leu‐Pro‐Gly‐Ala.