Colletotrichum acutatum var. fioriniae (teleomorph: Glomerella acutata var. fioriniae var. nov.) infection of a scale insect

An epizootic has been reported in Fiorinia externa populations in New York, Connecticut, Pennsylvania and NewJersey. Infected insects have profuse sclerotial masses enclosing their bodies. The most commonly isolated microorganism from infected F. externa was Colletotrichum sp. A morphological and molecular characterization of this fungus indicated that it is closely related to phytopathogenic C. acutatum isolates. Isolates of Colletotrichum sp. from F. externa in areas of the epizootic were similar genetically and were named Colletotrichum acutatum var. fioriniae var. nov, based on our findings. In vitro and in planta mating observed between isolates of C. acutatum var. fioriniae could serve as a possible source of genetic variation and might give rise to new biotypes with a propensity to infect insects. Only one other strain, C. gloeosporioides f. sp. ortheziidae, has been reported to show entomopathogenic activity.     

Effect of storage conditions on population variability and activity of Streptomyces recifensis var. lyticus

The results of the study on survival and variation of Streptomyces recifensis var. lyticus 2435 producing lytic enzymes are presented. The culture was maintained for 2.5 years under a layer of vaseline oil, at a temperature of -20 degrees C and in lyophilized state. It was shown that irrespective of the storage method strain 2435 preserved its viability. However, the most intensive growth was observed in the lyophilized cultures. During the storage the content of the productive colonies characteristic of the morphological type culture in the population decreased while the number of the low active variants increased. Lyophilization of the strain spores in the sucrose-gelatine medium provided insignificant morphological variation of the culture and preservation of the initial level of its lytic activity against a number of test-microbes except S. aureus and M. lysodeikticus. Storage of the culture under vaseline oil and at a temperature of -20 degrees C resulted in lowering of its lytic activity against all the test-microbes used. For long-term maintenance of Streptomyces recifensis var. lyticus 2435 the method of lyophilization in the sucrose-gelatine medium is recommended.     

Genetic Recombination in Streptomyces bikiniensis var. zorbonensis

A genetic recombination system in Streptomyces bikiniensis var. zorbonensis is described. This strain produces a mixture of antibiotics including zorbamycin and zorbonomycin B and C. A genetic map has been constructed from data obtained from an analysis of haploid recombinants which shows linkage relationships of 17 marker loci. Determination of map location has been made for three different loci affecting antibiotic biosynthesis in this strain.     

Diversity among strains of Cryptococcus neoformans var. gattii as revealed by a sequence analysis of multiple genes and a chemotype analysis of capsular polysaccharide.

We demonstrated the diversity of Cryptococcus neoformans var. gattii strains by a sequence analysis of multiple genes: (i) the intergenic spacer (IGS) 1 and 2 regions of the rRNA gene; (ii) the internal transcribed spacer (ITS) region, including 5.8S of the rRNA gene; (iii) TOP1 (topoisomerase); and (iv) CAP59. In these studies, we compared C. neoformans var. gattii with varieties grubii, and neoformans of C. neoformans. Phylogenetic analysis indicated that both C. neoformans var. grubii and C neoformans var. neoformans are monophyletic, but C. neoformans var. gattii showed polyphyletic. C. neoformans var. gattii can be divided into three phylogenetic groups, I, II, and III, with high bootstrap support. Phylogenetic group I contains serotype B and C strains, and groups II and III include serotype B strains. Because the serotype B strains of C. neoformans var. gattii exhibited more genetic divergence, the serological characteristics and chemotypes of their capsular polysaccharide were further investigated. No remarkable difference among the serotype B strains was found in the reactivities to factor serum 5, which is specific for serotype B. The NMR spectra of the capsular polysaccharide from serotype B strains could be divided into three characteristic patterns, but the chemical shifts were very similar. These results suggested that the serotype B strain of C. neoformans var. gattii has more genetic diversity than the serotype C strain of C. neoformans var. gattii or the varieties grubii and neoformans of C. neoformans, but there was no correlation between genotype and chemotype.     

Palynological study of Cupressus sempervirens L. var. pyramidalis and var. horizontalis

ABSTRACT

Cupressus sempervirens L. var. pyramidalis and C. sempervirens var. horizontalis were examined for their pollen morphometry and ultrastructure to verify whether, in addition to phenotype, pollen could also show characteristics useful to discriminate the two infraspecific taxa. C. sempervirens var. pyramidalis compared to C. sempervirens var. horizontalis has mainly larger, subcircular pollen with a higher percentage of apolar and germinating grains, and a wall with thicker intine; the cytoplasm has a greater number of callose-containing vesicles. C. sempervirens var. horizontalis has pollen distributed in five classes of shape. In addition it is smaller and shows a higher percentage of ruptured grains, and a lower percentage of germination in vitro. The cytoplasm contains vesicles rich in callose and also a higher amount of osmophilic bodies. Pollen grains in both varietas contain only one cell at dispersal. The reported differences in pollen morphometry, structure and biology, together with the phenotypic characteristics of the tree, support the validity of two infraspecific taxa.     

Cephamycin C production is regulated by relA and rsh genes in Streptomyces clavuligerus ATCC27064

The effects of growth rate and nutrient uptake rate on the production of cephamycin C were determined in the parental strain, deltarelA mutant, and deltarsh null mutant of S. clavuligerus. Production of cephamycin C was inversely related to mycelium growth and the phosphate feed rate was more critical for the production of cephamycin C. On the contrary, the production of cephamycin C was completely abolished in the deltarelA mutant, but not in deltarsh mutant. The changes in the cephamycin C production by disruption of the relA and rsh genes are presumably associated with the consequent ability of the mutants to accumulate (p)ppGpp under nutrient starvation. Therefore, it is concluded that the stringent response of S. clavuligerus to starvation for nutrients is governed mainly by RelA rather than Rsh and that the response is more apparently regulated by the limitation of phosphate.     

Applications of Gene Replacement Technology to Streptomyces clavuligerus Strain Development for Clavulanic Acid Production

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine -aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.     

A broad-spectrum cytolytic toxin from Bacillus thuringiensis var. kyushuensis.

Bacillus thuringiensis (Bt) var. kyushuensis synthesizes a mosquitocidal crystalline inclusion containing several proteins ranging from 140 to 14 kDa. We have identified a 25 kDa protein protoxin in this inclusion which is not cytolytic, but when activated proteolytically to 23-22 kDa products is cytolytic to mosquito, lepidopteran and mammalian cells, can release entrapped glucose from liposomes and forms cation-selective channels in a planar lipid bilayer. This broad-spectrum cytolytic toxin is related antigenically to the 23 kDa toxin from Bt var. darmstadiensis strain 73-E10-2, but not to the 25 kDa CytA toxin of Bt var. israelensis. The cytolytic activity of these Bt var. kyushuensis toxins, like that of the latter two toxins, can be neutralized by incubation with liposomes containing phospholipids.     

In vitro antifungal susceptibility of clinical isolates of Cryptococcus neoformans var. neoformans and C. neoformans var. gattii

One of the differences observed between the two varieties of Cryptococcus neoformansis the greater difficulty to achieve an adequate therapeutical response in patients affected by C. neoformans var. gattii, an observation that has been validated in vitro only rarely. The aim of this work was to study the susceptibility patterns of 35 Colombian clinical isolates of C. neoformans, 20 of which belonged to the var. neoformans and 15 to the var. gattii. The minimal inhibitory concentration (MIC) was determined by broth microdilution, according to a modification of the methodology proposed by the National Committee for Clinical Laboratory Standards (NCCLS), using the breakpoints recently suggested by Nguyen et al. (Antimicrob Agents Chemother 1998; 42: 471-472). The antifungals tested were amphotericin B, fluconazole and itraconazole. Most of the isolates were susceptible to the three antimycotics tested regardless of the variety. Resistance to amphotericin B (MIC=2 microg/ml) was documented in two (10%) C. neoformans var. neoformans isolates; additionally, five (33%) C. neoformans var. gattii isolates felt in the category of fluconazole susceptible but dose dependent (MIC 16 microg/ml). In general, all C. neoformans var. gattii isolates proved susceptible only to the higher concentrations of the antifungals tested. For amphotericin B, seven (47%) isolates of this variety had MICs of 1 microg/ml, for fluconazole there were seven (47%) with MICs of 8 microg/ml and in the case of itraconazole, 10 isolates (66%) had MICs > 0.03 microg/ml. The data showed that although these isolates would be classified as susceptible, they actually require greater concentrations of the antifungals to be inhibited. This finding may well correlate both with the difficulty to attain therapeutic success in patients affected with C. neoformans var. gattii and with the need for more prolonged treatment courses in such cases.     

Cryptococcus neoformans var. gattii isolates from two Peruvian patients.

The ecology of Cryptococcus neoformans var. gatti is restricted to tropical and subtropical areas whereas C. neoformans var. neoformans is cosmopolitan. Perú is a country with three different well defined geographic areas, some of them have the conditions for the presence of the variety gattii. In order to determine the presence of the two varieties of C. neoformans in Perú, we made the C. neoformans differentiation from the clinical isolates. C. neoformans var. gattii was identified by their ability to assimilate D-proline. We tested 68 strains; only two of them were recognized as the variety gattii and were recovered from two patients without any predisposing factor. We described the clinical spectrum of these two patients, who were diagnosed with disseminated cryptococcosis and cryptococcal meningitis. Neither the presence of Eucalyptus camaldulensis nor Eucalyptus tereticornis has been reported in Perú. So there should exist other ecologic niches for the presence of C. neoformans var. gattii in our country, different from those mentioned.     

Site-specific restriction endonuclease BtcI from Bacillus thuringiensis var. canadensis

Efficiency of bacteriophage Tp4 plating on Bacillus thuringiensis var. canadensis H5 (Can) is decreased 10(7)-fold as compared with the efficiency of plating on Bacillus thuringiensis var. galleriae H5 (Gal). Bacteriophage Tp4 having propagated for one cycle in Can cells might be further grown in this strain without restriction. The sitespecific restriction endonuclease BtcI isolated from Bacillus thuringiensis var. canadensis recognises the same nucleotide sequence GATC in DNA as recognised by restriction endonuclease Sau3A.     

Time-lapsed confocal microscopy reveals temporal and spatial expression of the lysine epsilon-aminotransferase gene in Streptomyces clavuligerus

To investigate the temporal and spatial expression patterns of the gene (lat ) encoding lysine epsilon-aminotransferase (LAT) for cephamycin C biosynthesis, a mutant form of green fluorescent protein (mut1GFP) was integrated into the Streptomyces clavuligerus chromosome (strain LH369), resulting in a translational fusion with lat. LAT activity and fluorescence profiles of the recombinant protein paralleled the native LAT enzyme activity profile in wild-type S. clavuligerus, which peaked during exponential growth phase and decreased slowly towards stationary phase. These results indicate that the LAT-Mut1GFP fusion protein retains both LAT and GFP functionality in S. clavuligerus LH369. LH369 produced wild-type levels of cephamycin C in minimal medium culture conditions supplemented with lysine. Time-lapsed confocal microscopy of the S. clavuligerus LH369 strain revealed the temporal and spatial characteristics of lat gene expression and demonstrated that physiological development of S. clavuligerus colonies leading to cephamycin C biosynthesis is limited to the substrate mycelia.     

金瓜和白瓜花叶病毒病病原的初步鉴定

崇明是上海出口蔬菜生产基地之一,也是国家级的绿色食品园区之一,具有生产绿色蔬菜商品的生态环境和资源优势。特色出口蔬菜金瓜(Cucurbita pepo L.var.ovifera)、白瓜(Cucumis melo L.var.conomon,又名盐渍菜瓜)1997～1999统计病毒病发病率为23％,被感染的金瓜、白瓜田     

Dimorphism and hydrocarbon metabolism in Yarrowia lipolytica var. indica

Yarrowia lipolytica is able to metabolize high Mr hydrophobic natural compounds such as fatty acids and hydrocarbons. Characteristically, strains of Y. lipolytica can grow as populations with variable proportions of yeast and filamentous forms. In the present study, we describe the dimorphic characteristics of a variant designated as Y. lipolytica var. indica isolated from petroleum contaminated sea water and the effect of cell morphology on hydrocarbon metabolism. The variant behaved as a yeast monomorphic strain, under conditions at which terrestrial Y. lipolytica strain W29 and its derived strains, grow as almost uniform populations of mycelial cells. Using organic nitrogen sources and N-acetylglucosamine as carbon source, var. indica was able to form mycelial cells, the proportion of which increased when incubated under semi-anaerobic conditions. The cell surface characteristics of var. indica and W29 were found to be different with respect to contact angle and percent hydrophobicity. For instance, percent hydrophobicity of var. indica was 89.93 ± 1.95 while that of W29 was 70.78 ± 1.1. Furthermore, while all tested strains metabolize hydrocarbons, only var. indica was able to use it as a carbon source. Yeast cells of var. indica metabolized hexadecane with higher efficiency than the mycelial form, whereas the mycelial form of the terrestrial strain metabolized the hydrocarbon more efficiently, as occurred with the mycelial monomorphic mutant AC11, compared to the yeast monomorphic mutant AC1.     

Site-specific restrictases from Bacillus thuringiensis var. Kumantoensis]

The efficiency of bacteriophages CP-54 and CP-55 plating on Bacillus thuringiensis var. kumantoensis H18 (Kum) is decreased about 10-fold as compared with the efficiency of plating on Bacillus thuringiensis var. galleriae H5 (Gal). Bacteriophages having propagated for one cycle in Kum cells might be further grown in this strain without growth restriction. Two site-specific restriction enzymes isolated from Bacillus thuringiensis var. kumantoensis were designated BtkI and BtkII. The endonuclease BtkI recognises the same nucleotide sequence CGCG in DNA as recognised by the restriction endonuclease FnuDII; BtkII recognises the same nucleotide sequence GATC as the endonuclease Sau3A.     

少根根霉原变种发酵生产薯蓣皂苷元

从穿龙薯蓣Dioscorea nipponica中分离得到的一少根根霉原变种Rhizopus arrhizus var. arrhizus菌株,能实现薯蓣皂苷的生物转化。用该菌株发酵穿龙薯蓣D. nipponica生产薯蓣皂苷元,采用高效液相色谱法测定薯蓣皂苷元的含量,其总得率可达3.00%。运用该菌株发酵制备薯蓣皂苷元,操作简单,环保,且得率高。     

Production of a New Acidic Polysaccharide,Succinoglucan by Alcaligenes faecalis var. myxogenes

A slimy non-spore-forming bacterium strain 10C3 isolated from soil was motile with peritrichous flagella and named Alcaligenes faecalis var. myxogenes. Studies were made on the conditions necessary for maximal production of a new acidic succinoglucan polysaccharide by this strain in shaken cultures. Much production was observed with sucrose, glucose, xylose, galactose, cellobiose, maltose, fructose, mannose and rhamnose. The yield was greatest with sucrose and decreased in order with the above sugars from about 36 to 23 per cent. The most suitable medium contained 4 per cent sugar, 0.5 per cent yeast extract and one per cent calcium carbonate in tap water. The optimum temperature was 28°C.     

Homologous expression of aspartokinase (ask) gene in Streptomyces clavuligerus and its hom-deleted mutant: effects on cephamycin C production

In this study, the effect of homologous multiple copies of the ask gene, which encodes aspartokinase catalyzing the first step of the aspartate pathway, on cephamycin C biosynthesis in S. clavuligerus NRRL 3585 and its hom mutant was investigated. The intracellular pool levels of aspartate pathway amino acids accorded well with the Ask activity levels in TB3585 and AK39. When compared with the control strain carrying vector alone without any gene insert, amplification of the ask gene in the wild strain resulted in a maximum of 3.1- and 3.3-fold increase in specific, 1.7- and 1.9-fold increase in volumetric cephamycin C production when grown in trypticase soy broth (TSB) and a modified chemically defined medium (mCDM), respectively. However, expression of multicopy ask gene in a hom-deleted background significantly decreased cephamycin C yields when the cells were grown in either TSB or mCDM, most probably due to physiological disturbance resulting from enzyme overexpression and high copy number plasmid burden in an auxotrophic host, respectively.     

Recognition of cytoplasmic yeast antigens of Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii by immune human sera

The humoral immune response of patients infected with Cryptococcus neoformans var. neoformans and C. neoformans var. gattii to cytoplasmic (non-capsular) antigens from the two varieties of Cryptococcus has been investigated. Cytoplasmic antigens from C. neoformans (one clinical isolate and one acapsular mutant of var. neoformans and two clinical isolates from var. gattii) were subject to isoelectric focusing, SDS-PAGE and Western blotting; patients sera was then used in the immunoenzyme development of the Western blots. The humoral response from the 20 patients (all HIV+) infected with var. neoformans against the var. neoformans antigens was predominantly IgG based, with a large number of bands recognised; the most commonly recognised bands were at 26, 52, 74, 100, 115 and 144 kDa. The IgM response was less pronounced and the IgA response was practically non-existent. The humoral response of the sera from the 15 patients (all but one HIV-) infected with var. gattii against var. gattii antigens was also predominantly IgG based with bands at 37, 55, 65, 74, 94 and 115 kDa being most commonly recognised. Periodate treatment of cytoplasmic antigens reduced the intensity of antigen recognition, though it did not absolutely destroy reactivity to any individual antigen. Comparison of immunodevelopment of cytoplasmic antigens from both varieties grown at 25°C and 37°C revealed that culture temperature made no differences in the number of bands recognised although there were differences in the intensity of recognition. This is the first report on the pattern of serological recognition of the non-capsular antigens from the two varieties of Cryptococcus and it identifies a number of major antigenic components.