Intrapopulation variability of antibiotic biosynthesis in streptomycetes

Spontaneous variants making up parallel series of hereditary variability inside the populations of antibiotic-producing actinomycetes differ in the level of their antibiotic activity. As a rule, spontaneous variants of the basic type possess the highest antibiotic activity. Other variants representing parallel series have a lower activity level. This raises the possibility to carry out a directed selection of previously known active colonies from populations on the basis of their easily discernible morphological properties. It enhances the efficiency of selection work both in the case of stabilization of the level of antibiotic activity and in the case of obtaining more productive commercial strains.     

Comparative study of a group of mutant strains of Trichoderma viride, producer of cellulolytic enzymes]

In the course of selection of Trichoderma viride, the producer of cellulolytic enzymes, the group of mutant strains characterized by a higher level of productivity are isolate. It is shown that the isolated mutants possess a number of common but differing them from original strains characters. These include: the small size of colonies ("dwarfs"), a lower capacity to carry out some biochemical reactions, and increased development rate and a higher resistance to lethal effect of nitrosoguanidine and nitrosoethyl urea. The data obtained indicate that in the series of populations of successively isolated mutants observed the stabilization of variability of the levels of C1 enzyme synthesis takes place. It is also shown that, unlike original cultures, the populations of mutant strains are characterized by a higher variability of Cx enzyme activity levels as compared with C1.     

Biological attributes of colony-type variants of Candida albicans

Twenty 'commensal' oral or 'pathogenic' vaginal isolates of Candida albicans were examined for colony morphology on malt/yeast-extract and serum-based agar media. Diverse and variable colony morphology was seen on serum agar. In 17 strains, selective subculture of morphologically atypical colonies produced progeny which had reverted to the morphology of the majority of parental colonies. However, in one strain, a highly stable colony variant was isolated which did not revert on subculture. In two further strains, variants were isolated which could be maintained with at least 99% homogeneous colony type by selective colony subculture, but reversion to the parental type or switching to other morphologies occurred at rates of 10(-2) to 10(-4): a rapid switching phenomenon. The relative proportions of mycelial or yeast forms were the main determinants of colony morphology. The variants were biotyped using a selection of biochemical tests. The stable variant differed from its parent in several characters, including rate of production of a proteinase enzyme. The pathogenicity of variants was compared in mice, and both stable and switching variants differed in virulence from their parental strains. Colony-type variation on suitable media is thus a powerful tool in the isolation of mutants or variants of C. albicans which differ from 'isogenic' parents in significant biological properties. Such variants may aid identification and characterization at the molecular level of determinants of, for example, pathogenicity and morphogenesis.     

Study of catalase and proteolytic activities of different variants of Bacillus mesentericus]

Catalase and proteolytic activity of the culures and morphological variants of Bacillus mesentericus fuscus, Bac. mesentericus vulgatus were studied. The variants were obtained as a result of prolonged cultivation of the stock strains in the potato mash under the layer of vaseline oil. The level of catalase activity varies in different morphological variants of the same culture, changes with age and depends on the storage conditions. The catalase activity in the rough, smooth and papillar variants that were freshly isolated from the potato mash was 1.5=2.5 times lower than that in the variants long kept on the agar medium. The quantitative indexes of the proteolytic activity of different variants also varied.     

Variability of colonial morphology in benomyl-induced morphological mutants from Candida albicans

Abstract Colonies of Candida albicans wild-type strain 1001 were white and glossy, and this character was rather stably maintained. In contrast, 2 benomyl (methyl benzimidazole-2-yl-carbamate)-induced mutant strains, B17 and B14, that grew as long filamentous forms and displayed a rough-wrinkled colonial phenotype, switched to other colonial morphologies at significant frequencies. Clonal populations of B17 segregated smooth or sectored (rough/smooth) colonies at a frequency of 1.85%, when plated in nutrient-agar. Strains derived from these rough or smooth segregants switched back to one or the other phenotype at similar frequencies. Colonial variability in C. albicans B14 was not restricted to spontaneous switching from rough to smooth or vice versa, but eventually other types of variants, characterized as 'wavy' and 'fuzzy' were obtained, and shown to have their own capacity to switch. Smooth variants, derived from B14, were essentiallt unicellular, whereas fuzzy strains consisted only of long thin filaments, wavy and rough clones apparently being intermediate in their degree of filamentation. It is concluded that the capacity for colonial variation shown to exist in natural isolates could be activated by benomyl in others, such as 1001, which are quite stable and do not switch colonial morphology spontaneously.     

Methodologic problems in the biotyping of Candida albicans

The biotyping of 284 C. albicans strains has been carried out in accordance with the system of three tests, proposed by F.C. Odds and A. B. Abbott. The reliability of the epidemiological conclusions made as the result of this work has been analyzed. The independence of the signs of C. albicans used in biotyping and the asymmetrical character of the test for its sensitivity to 5-fluorocytosine have been shown. The change of this test for a more symmetrical one is proposed. The study has shown that in the process of prolonged storage with periodic subculturing the proportion of C. albicans strains resistant to pH 1.40 and possessing proteolytic activity is decreased. The distribution of different biotypes among C. albicans strains isolated from candidiasis patients and from carriers has proved to be the same.     

Differences in extracellular enzymatic activity between Candida dubliniensis and Candida albicans isolates

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.     

Morphological and biosynthetic activity of Trichothecium roseum in relation to the polynuclear conidia of the fungus]

The effect of the number of the nuclei on the morphological and synthetic variability was studied with the conidia of Trichothecium roseum. Treatment of suspensions of the conidia with 0.5--1.0 percent solutions of colchicine during two hours increased the number of the nuclei in the conidia, induced the formation of giant colonies, and augmented the ability to produce antibiotics. No reliable differences have been found in the levels of proteolytic activity between the control strains and the strains treated with colchicine.     

Utility of a pre-optimized kit for random amplified polymorphic DNA in typing Candida albicans

The utility of a pre-optimized kit for random amplified polymorphic DNA (RAPD) was assessed in typing diverse strains of Candida albicans from epidemiologically unrelated inpatients (interpatient analysis) and in detecting clonal variations that maybe present within individual patient isolates (intrapatient analysis). Stool samples from inpatients were cultured on Inhibitory Mold agar. Nine individual colonies from all patients with > or =9 colonies of C. albicans (n = 18) were selected, frozen, and karyotyped using CHEF genomic DNA plug kits and CHEF-DRIII. Each of the selected colonies was then analyzed by RAPD, utilizing the selected kit, with 6 primers. Interpatient analysis revealed 9 karyotypes and 17 RAPD composites. RAPD discrimination was significantly better (p < 0.001). Intrapatient analysis revealed 34 (21%) and 33 (20.4%) variants among 162 colonies tested by RAPD and karyotyping, respectively. The results were discordant in 25 variants, all with differences of 1-3 bands. These results illustrate that this pre-optimized kit for RAPD provides excellent discrimination of genetically unrelated strains. Its performance in delineating subtle clonal differences was comparable with karyotyping; both methods failed to detect all minor genetic variations. The ease of use and quick turnaround time of this kit offer a practical and reliable method for typing diverse strains of C. albicans, but may be inadequate for assessing microevolution.     

Evaluation of the API ZYM system and RPMI agar plates supplemented with Tween 40 for detection of lipolytic enzymes of Candida spp

Lipolytic activity of 40 strains of Candida spp. was tested on API ZYM system and on RPMI agar plates supplemented with 1% Tween 40. Lipolytic activity was indicated by opaque zones around the inoculum cylindrical holes were punched in the medium. Clearing of the medium around the bacterial colonies indicated that an isolate produce lipase. Only 4 (21.1%) strains of C. albicans, and 3 (14.1%) strains of non-C. albicans which hydrolyzed 2-naftylomirystylan by use of the API ZYM system was observed. In contrast, 16 (78.9%) strains of C. albicans and 17 (80.7%) strains of non-C. albicans produced lipases on the agar plate using RPMI agar plates supplemented with 1.0% Tween 40. Determination oflipase activities with the API ZYM system were in no agreement with lipase tests in RPMI supplemented with Tween 40. Our study verify greater usefulness of RPMI supplemented with Tween 40 for detection of lipolytic enzymes of Candida species in comparison to the API ZYM.     

An investigation of the role of true hypha production in the pathogenesis of experimental oral candidosis

From 427 cases of human oral candidosis 2135 yeast clones were screened for the presence of germ tube-negative C. albicans and variants that formed only pseudohyphae in serum; one strain of each was found. The pathogenic potential of the serum-pseudohyphal and germ tube-negative C. albicans variants was investigated in the oral cavity of the rat; both variants failed to induce palatal candidosis, in contrast to a germ tube-positive C. albicans control strain. The pathogenic potential of C. albicans strains appears to be dependent on the formation of true germ tubes.     

Proteolytic mutants obtained from Clostridium botulinum type E.

Proteolytic mutants were isolated from toxigenic strains of Clostridium botulinum type E after several transfers. When these cultures were plated on blood agar, almost all of the colonies obtained were proteolytic, and there were fewer toxigenic colonies than nontoxigenic colonies. The proteolytic mutants and nonproteolytic original strains were different in their biological properties.     

A complementation analysis by parasexual recombination of Candida albicans morphological mutants

Benomyl treatment (at 100 micrograms ml-1) of Candida albicans 1001, and other strains derived from it, determined the appearance of morphological mutants similar to those derived from UV irradiation treatment. A permanent alteration in the morphogenesis of these mutant strains determined their inability to grow by budding, to form oval yeast cells or blastospores (Y-phenotype) and their growth as long filamentous forms, mostly with the appearance of pseudomycelium, giving rise to rough colonies (R phenotype). In order to carry out a genetic complementation analysis, we isolated morphological mutants that carried other genetic markers (nutritional, conditional lethal) adequate for crosses by means of protoplast fusion. Wild-type hybrids of regular mononuclear oval yeast cells and smooth colonies were obtained by crossing pairs of complementing mutants, whereas hybrids from crosses of non-complementing mutants still retained their morphological alterations. Our results define two complementation groups, which represent two genes relevant for dimorphism, whose alteration interferes with the correct transition from blastospores to mycelium.     

Proteinase production by halophilic isolates from marine sediments

Twenty-one strains of spore-forming bacteria were isolated from marine sediments of the Tyrrhenian seaboard (Livorno, Italy) and identified asBacillus licheniformis by their morphological, physiological and biochemical characteristics. All strains were able to grow at 15% NaCl and on media prepared with 100% seawater. In these conditions 75% of the strains were able to produce a bacitracin-like antibiotic and 100% of the strains showed proteolytic activity. Particularly, all strains showed proteinase production with an activity optimum at pH 8.5 and 60°C. Three strains produced high levels of proteolytic activity only when cultured in the presence of seawater.     

Fermenting Cucumber, a Potential Source for the Isolation of Pediocin-Like Bacteriocin Producers

Summary A strain of Pediococcus acidilactici CFR K7 isolated from cucumber, produced an antimicrobial peptide which acted against Leuconostoc mesenteroides, selected strains of Lactobacillus spp., Pediococcus spp. and Enterococcus spp. The partially purified bacteriocin had molecular weight of ~4.6 kDa, heat stability in a range of 40–121 °C and was active over a wide range of pH (2.0–9.0). This bacteriocin possessed strong antilisterial activity and was susceptible to proteolytic enzymes. Southern hybridization using the PCR-generated pedA probe established that the gene for the bacteriocin was plasmid-borne as in the case of pediocin PA-1. Nucleotide sequence of the pedAB gene indicated 100% homology to a pediocin AcH/PA-1. Certain bacteriocinogenic strains isolated from naturally fermented cucumber were tested by colony hybridization using the pedA gene probe. Nine out of twenty colonies reacted with the probe indicating their ability to produce the pediocin-like bacteriocin. These nine colonies were further tested for their antimicrobial spectrum, proteolytic inactivation and plasmid profile. It was found that a few of them were active against Bacillus cereus, Micrococcus luteus and Listeria monocytogenes. Their proteolytic inactivation showed that the antimicrobial compound was susceptible to proteinase K. Colony hybridization could thus enable rapid detection of pediocin and pediocin-like bacteriocin producers among a population of bacteriocinogenic strains.     

Screening of strains identified as extremely thermophilic bacilli for extracellular proteolytic activity and general properties of the proteinases from two of the strains

T. COOLBEAR, C.W. EAMES, Y. CASEY, R.M. DANIEL AND H.W. MORGAN. 1991. Forty-one strains isolated from thermal areas in New Zealand, Fiji and Antarctica were shown to be extremely thermophilic Bacillus spp. (growth optima > 65^.C) by comparison with reference strains with a series of standard tests. Some morphological and physiological variation between strains was noted. Various assay procedures were employed to assess the strains for their ability to produce extracellular proteolytic activity. The strain EA. 1 gave the highest yield of proteolytic activity under the conditions imposed. A second strain, OK3A.1, also gave high yields of activity but differed from the EA.1 activity in that it was more tolerant to both high pH and EDTA. The proteinases from these two strains were purified and characterized. Maximum activity was given by EA.1 proteinase over a narrow pH range with an optimum at pH 6.7 and 50% activity limits at pH 5.6 and 7.5. OK3A.1 had a similar pH optimum but was active over a broader range with 50% activity limits at pH 5.2 and 8.5. Both enzymes were endo-acting proteinases; neither showed activity against two small synthetic peptides. By SDS-polyacrylamide gel electrophoresis the molecular masses for EA.1 proteinase and OK3A.1 proteinase were 42 000 Da and 32 000 Da respectively. Both enzymes were resistant to 10 mmol/1 phenylmethylsulphonylfluoride and iodoacetic acid, but were deactivated by EDTA. Whereas EA.1 proteinase was inhibited by o -phenanthroline and activated by zinc ions, OK3A.1 proteinase was unaffected by either agent although some dependence on divalent metal ions for activity was apparent. The enzymes were stabilized by calcium ions, EA.1 proteinase exhibiting a half-life of 2 h at 85.C whilst OK3A.1 proteinase was less stable with a half-life of 40 min at this temperature.     

Natural variability of Actinomyces lavendulae, a producer of cholesterol oxidase

Five variants of Actinomyces lavendulae differing in the morphology of their colonies were found when natural variability of this organism was studied. A correlation was established between the colony morphology of the variants and their activity of cholesterol decomposition. The variants forming colonies of the basic and folded type had the highest activity. A variant with an elevated activity of cholesterol oxidase was selected. In order to maintain the high activity of the culture, it is necessary to examine its variability and to select variants corresponding to the basic type in their morphology.     

Correlation of the virulence of Bacillus anthracis with expression of signs, coded for by chromosomal genes

Clonal analysis of Bacillus anthracis strains showed heterogeneity of the majority of populations by the proteolytic, hemolytic, and pigment-adsorbing activities. Phenotypes isolated within each population were subdivided into 4 subpopulations. The appearance of sub-populations of virulent and vaccine strains (B. anthracis Sterne, Zenkowskii) was due to the ability of clones adsorbing Congo red and possessing high activities of proteolytic and hemolytic enzymes to dissociate with the formation of phenotypes characterized by different expression of proteolysis, hemolysis, pigment adsorption, pigment production, and sporulation signs. A characteristic feature of vaccine strains of B. anthracis CTIl, Wright, and Pasteur is a relative homogeneity of their populations consisting mainly of cells with low activities of proteolytic, hemolytic, and pigment-adsorbing enzymes. Study of the heterogeneity of di-, mono-, and plasmid-free derivatives of B. anthracis showed that the process of phenotype formation did not depend on the plasmids proper. Evaluation of the virulence of clones isolated from highly virulent B. anthracis strain 81/1 showed that the processes associated with loss of sporulation capacity notably decrease the virulence for laboratory animals.     

Ultrastructure of freshly isolated strains of Bordetella pertussis

The study of the electronograms of B. pertussis strains isolated in the foci of pertussis revealed the existence of the morphological variants of these cells, differing in the character of the cell wall, the state of the cytoplasm, the presence of amorphous inclusions of medium electron-optical density. The morphological variants did not correlate with the character of B. pertussis colonies isolated from blood-charcoal agar. The ultrastructure of the cells belonging to the second morphological variant was similar to that of the cells from the museum strain, altered by tetracycline treatment in the course of the experiment.