Group B streptococcal opacity variants.

Colony opacity variants were detected for type III group B streptococci (GBS). Transparent colonies predominate in the parent GBS, with occasional colonies having opaque portions. Two stable opaque variants (1.1 and 1.5) were compared with three transparent clones (1.2, 1.3, and 1.4). All grew well on blood agar and on GC medium, but variant 1.1 failed to grow on Todd-Hewitt medium. Scanning and transmission electron microscopy demonstrated that colony opacity correlated with bacterial aggregation status, with opaque variants forming longer and more organized chains. Opaque-transparent switches were observed in both directions for most variants, with transparent to opaque noted most frequently, but 1.5 did not switch at all. Switching of the opacity phenotype was observed both in vitro and in neonatal mice. Relationships between colony opacity and several cell surface phenomena were explored. (i) Opaque variant 1.1 had two surface proteins (46 and 75 kDa) that were either unique or greatly overexpressed. (ii) Variant 1.1 was deficient in type III polysaccharide, while 1.5 lacked group B antigen. Diminished capsular polysaccharide of variant 1.1 was reflected in reduced negative electrophoretic mobility and in increased buoyant density. (iii) Transparent variant colonies growing closest to a penicillin disk were opaque, but colonial variants did not differ in their sensitivity to penicillin. These data indicate that GBS can exist in both opaque and transparent forms, with opaque appearance occurring by multiple routes. Opaque variants grow poorly on Todd-Hewitt medium generally used for isolation of GBS, so any possible relationships between opacity variation and pathogenesis of GBS infection are unknown.     

Differences in membrane fluidity and fatty acid composition between phenotypic variants of Streptococcus pneumoniae

Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in the pathogenesis of pneumococcal disease. This study examined the relationship between membrane characteristics and colony morphology in a few selected opaque-transparent couples of S. pneumoniae strains carrying different capsular types. Membrane fluidity was determined on the basis of intermolecular excimerization of pyrene and fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH). A significant decrease, 16 to 26% (P < or = 0.05), in the excimerization rate constant of the opaque variants compared with that of the transparent variants was observed, indicating higher microviscosity of the membrane of bacterial cells in the opaque variants. Liposomes prepared from phospholipids of the opaque phenotype showed an even greater decrease, 27 to 38% (P < or = 0.05), in the pyrene excimerization rate constant compared with that of liposomes prepared from phospholipids of bacteria with the transparent phenotype. These findings agree with the results obtained with DPH fluorescence anisotropy, which showed a 9 to 21% increase (P < or = 0.001) in the opaque variants compared with the transparent variants. Membrane fatty acid composition, determined by gas chromatography, revealed that the two variants carry the same types of fatty acids but in different proportions. The trend of modification points to the presence of a lower degree of unsaturated fatty acids in the opaque variants compared with their transparent counterparts. The data presented here show a distinct correlation between phase variation and membrane fluidity in S. pneumoniae. The changes in membrane fluidity most probably stem from the observed differences in fatty acid composition.     

小花棘豆(Oxytropis glabra DC.)内生真菌的培养与鉴定

小花棘豆(Oxytropis glabra DC.)是内蒙古草原上的重要毒草,实验结合微生物学和分子生物学手段进行了其内生真菌研究.结果表明:体外培养的小花棘豆内生真菌生长缓慢,呈圆形、隆起、边缘整齐、辐射状生长的白色菌落,后菌体分泌黑褐色的色素物质,分生孢子近圆柱形,有粗且比孢子壁厚的暗色横隔膜,隔膜数1～5个.10个菌株的5.8S rDNA/ITS序列与内生真菌Embellisia sp.L12株的序列高度相似.推测该内生真菌属于Embellisia.     

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

Chitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.     

Signal strains that can detect certain DNA replication and membrane mutants of Escherichia coli: isolation of a new ssb allele, ssb-3.

Mutations in several dna genes of Escherichia coli, when introduced into a strain with a lac fusion in the SOS gene sulA, resulted in formation of blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal). Unexpectedly, several lines of evidence indicated that the blue colony color was not primarily due to induction of the SOS system but rather was due to a membrane defect, along with the replication defect, making the cell X-Gal extrasensitive (phenotypically Xgx), possibly because of enhanced permeability to X-Gal or leakage of beta-galactosidase. (i) In most cases, beta-galactosidase specific activity increased only two- to threefold. (ii) Mutations conferring tolerance to colicin E1 resulted in blue colony color with no increase in beta-galactosidase specific activity. (iii) Mutations in either the dnaA, dnaB, dnaC, dnaE, dnaG, or ssb gene, when introduced into a strain containing a bioA::lac fusion, produced a blue colony color without an increase in beta-galactosidase synthesis. These lac fusion strains can serve as signal strains to detect dna mutations as well as membrane mutations. By localized mutagenesis of the 92-min region of the chromosome of the sulA::lac signal strain and picking blue colonies, we isolated a novel ssb allele that confers the same extreme UV sensitivity as a delta recA allele, which is a considerably greater sensitivity than that conferred by the two well-studied ssb alleles, ssb-1 and ssb-113. The technique also yielded dnaB mutants; fortuitously, uvrA mutants were also found.     

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.     

Patterns of reporter gene expression in the phase diagram of Bacillus subtilis colony forms.

Factors governing the morphogenesis of Bacillus subtilis colonies as well as the spatial-temporal pattern of expression of a reporter gene during colony development were examined by systematically varying the initial nutrient levels and agar concentrations (wetness), the relative humidity throughout incubation, and the genotype of the inoculum. A relationship between colony form and reporter gene expression pattern was found, indicating that cells respond to local signals during colony development as well as global conditions. The most complex colony forms were produced by motile strains grown under specific conditions such that cells could swim within the colony but not swarm outward uniformly from the colony periphery. The wetness of the growth environment was found to be a critical factor. Complex colonies consisted of structures produced by growth of finger-like projections that expanded outward a finite distance before giving rise to a successive round of fingers that behaved in a similar fashion. Finger tip expansion occurred when groups of cells penetrated the peripheral boundary. Although surfactin production was found to influence similar colony forms in other B. subtilis strains, the strains used here to study reporter gene expression do not produce it. The temporal expression of a reporter gene during morphogenesis of complex colonies by motile strains such as M18 was investigated. Expression arose first in cells located at the tips of fingers that were no longer expanding. The final expression pattern obtained reflects the developmental history of the colony.     

Candida albicans MTLalpha tup1Delta mutants can reversibly switch to mating-competent, filamentous growth forms

Candida albicans strains that are homozygous at the mating type locus (MTLa or MTLalpha) can spontaneously switch from the normal round-to-oval yeast cell morphology to an elongated, so-called opaque cell form that can mate with opaque cells of the opposite mating type. In response to environmental signals, C. albicans also undergoes a transition from yeast to filamentous growth, which is negatively regulated by the general repressor Tup1p. Therefore, C. albicans mutants in which the TUP1 gene is inactivated grow constitutively in the filamentous form. We found that tup1Delta mutants of the MTLalpha strain WO-1 are still able to undergo phenotypic switching. Although the mutants had lost the capacity to grow in the normal yeast (white) or opaque forms, they could still reversibly switch between four different cell and colony phenotypes (designated as fuzzy, frizzy, wrinkled and smooth) at a frequency of about 10(-3) to 10(-4). Deletion of TUP1 resulted in deregulated expression of phase-specific genes. While the white-specific WH11 gene was constitutively expressed in all four cell types, the opaque-specific SAP1 gene remained repressed and the opaque-specific OP4 gene was weakly induced in all phase variants. In spite of the loss of white- and opaque-specific cell morphology and gene expression, the tup1Delta mutants retained an important characteristic of their wild-type parent, the ability to switch to a mating-competent form. The three filamentous phase variants (fuzzy, frizzy and wrinkled) all were able to mate and produce recombinant progeny with opaque cells of an MTLa strain at frequencies that were somewhat lower than those of normal opaque cells, whereas the smooth phase variant was unable to do so. Therefore, although deletion of TUP1 in C. albicans MTLalpha cells affects cellular morphology and gene expression patterns, the mutants can still reversibly switch between mating-competent and -incompetent cell types and mate with a partner of the opposite mating type.     

Mechanism of the Selective Action of Eosin-Methylene-Blue Agar on the Enteric Group

The actual mechanism of the differentiation of lactose-fermenting and non-lactose-fermenting organisms on eosin-methylene-blue medium is not reported in the literature. The present study is an attempt to elucidate this problem.

The color of colon forms on E.M.B. agar was found to depend on two factors: (1) the reaction of eosin with methylene blue to form a dye compound of either acidic or neutral nature, and (2) the production, by lactose-fermenting colonies, of a sufficiently low pH so that this dye compound is taken up by individual cells of the colony. Non-lactose-fermenting organisms are not colored because the compound is not taken up in alkaline reaction.

An explanation is offered to account for the occasional blue colonies found on E.M.B. medium. It is suggested that these colonies form a relatively high pH and thus cause slight dissociation of the compound. This dissociation would allow independent staining of the colonies by methylene blue.     

"White-opaque transition": a second high-frequency switching system in Candida albicans

A second high-frequency switching system was identified in selected pathogenic strains in the dimorphic yeast Candida albicans. In the characterized strain WO-1, cells switched heritably, reversibly, and at a high frequency (approximately 10(-2] between two phenotypes readily distinguishable by the size, shape, and color of colonies formed on agar at 25 degrees C. In this system, referred to as the "white-opaque transition," cells formed either "white" hemispherical colonies, which were similar to the ones formed by standard laboratory strains of C. albicans, or "opaque" colonies, which were larger, flatter, and grey. At least three other heritable colony phenotypes were generated by WO-1 and included one irregular-wrinkle and two fuzzy colony phenotypes. The basis of the white-opaque transition appears to be a fundamental difference in cellular morphology. White cells were similar in shape, size, and budding pattern to cells of common laboratory strains. In dramatic contrast, opaque cells were bean shaped and exhibited three times the volume and twice the mass of white cells, even though these alternative phenotypes contained the same amount of DNA and a single nucleus in the log phase. In addition to differences in morphology, white and opaque cells differed in their generation time, in their sensitivity to low and high temperatures, and in their capacity to form hypae. The possible molecular mechanisms involved in high-frequency switching in the white-opaque transition are considered.     

放线共生放线杆菌粗糙型与光滑型菌落的主要外膜蛋白

目的：观察放线共生放线杆菌粗糙型与光滑型菌株菌体蛋白表达上的差异。方法：聚丙稀酶胺凝胶电泳观察两型细菌全细胞蛋白及超高速离心提取的细菌主要外膜蛋白差异。结果：全细胞蛋白电泳两型细菌蛋白带无明显差异;提取的主要外膜蛋白电泳粗糙型存在18、29、45kDa蛋白带,实验室参考菌株不存在,临床光滑型菌株存在少量,光滑型实验室参考菌株存在的蛋白带在所有实验菌株中均存在。结论：18、29、45kDa蛋白带可能与放线共生放线杆菌粗糙型菌株相关。     

Characterization of Free Exopolysaccharides Secreted by Mycoplasma mycoides Subsp. mycoides

Contagious bovine pleuropneumonia is a severe respiratory disease of cattle that is caused by a bacterium of the Mycoplasma genus, namely Mycoplasma mycoides subsp. mycoides (Mmm). In the absence of classical virulence determinants, the pathogenicity of Mmm is thought to rely on intrinsic metabolic functions and specific components of the outer cell surface. One of these latter, the capsular polysaccharide galactan has been notably demonstrated to play a role in Mmm persistence and dissemination. The free exopolysaccharides (EPS), also produced by Mmm and shown to circulate in the blood stream of infected cattle, have received little attention so far. Indeed, their characterization has been hindered by the presence of polysaccharide contaminants in the complex mycoplasma culture medium. In this study, we developed a method to produce large quantities of EPS by transfer of mycoplasma cells from their complex broth to a chemically defined medium and subsequent purification. NMR analyses revealed that the purified, free EPS had an identical β(1−>6)-galactofuranosyl structure to that of capsular galactan. We then analyzed intraclonal Mmm variants that produce opaque/translucent colonies on agar. First, we demonstrated that colony opacity was related to the production of a capsule, as observed by electron microscopy. We then compared the EPS extracts and showed that the non-capsulated, translucent colony variants produced higher amounts of free EPS than the capsulated, opaque colony variants. This phenotypic variation was associated with an antigenic variation of a specific glucose phosphotransferase permease. Finally, we conducted in silico analyses of candidate polysaccharide biosynthetic pathways in order to decipher the potential link between glucose phosphotransferase permease activity and attachment/release of galactan. The co-existence of variants producing alternative forms of galactan (capsular versus free extracellular galactan) and associated with an antigenic switch constitutes a finely tuned mechanism that may be involved in virulence.     

On the adaptive nature of the dissociation process in Bacillus thuringiensis

The process of dissociation into variants differing in colony morphology occurring in batch cloned cultures of two Bacillus thuringiensis strains belonging to different subspecies was studied at optimal and elevated temperatures. An increase in the cultivation temperature to 40 degrees C resulted in an increase in the fraction of R variants to 100% after 72 h of cultivation of either of the strains. This increase was not due to the selection of forms with greater resistance to elevated temperature. The level of resistance to elevated temperature was determined by the strain genotype and did not correlate with morphological characteristics of the colonies.     

Chemical basis of rough and smooth variation in mycobacteria.

Rough and smooth colony variants of Mycobacterium kansasii were compared with respect to surface glycolipid composition. Thin-layer chromatography of the native glycolipid antigens, gas chromatography of the constituent sugars, and in situ probing with an appropriate monoclonal antibody by colony dot blot enzyme-linked immunosorbent assay and immunogold labeling demonstrated that all M. kansasii strains of smooth colony morphology contain on their surfaces the recently described trehalose-containing lipooligosaccharides, whereas all rough variants were devoid of such surface antigens. Yet all strains, rough and smooth, contained another glycolipid, the M. kansasii-specific phenolic glycolipid. Previous studies by others had shown that the rough forms of M. kansasii persist longer than smooth variants in experimentally infected mice. Therefore, this study may provide some insight into the question of the chemical basis of pathogenesis in certain mycobacteria.     

Isolation and characterization of Pseudomonas putida PpF1 mutants defective in the toluene dioxygenase enzyme system.

Pseudomonas putida PpF1 degraded toluene via a dihydrodiol pathway to tricarboxylic acid cycle intermediates. The initial reaction was catalyzed by a multicomponent enzyme, toluene dioxygenase, which oxidized toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol). The enzyme consisted of three protein components: NADH-ferredoxintol oxidoreductase (reductasetol), ferredoxintol, and a terminal oxygenase which is an iron-sulfur protein (ISPtol). Mutants blocked in each of these components were isolated after mutagenesis with nitrosoguanidine. Mutants occurred as colony morphology variants when grown in the presence of toluene on indicator plates containing agar, mineral salts, a growth-supporting nutrient (arginine), 2,3,5-triphenyltetrazolium chloride (TTC), and Nitro Blue Tetrazolium (NBT). Under these conditions, wild-type colonies appeared large and red as a result of TTC reduction. Colonies of reductasetol mutants were white or white with a light blue center, ferredoxintol strains were light blue with a dark blue center, and strains that lacked ISPtol gave dark blue colonies. Blue color differences in the mutant colonies were due to variations in the extent of NBT reduction. Strains lacking all three components appeared white. Toluene dioxygenase mutants were characterized by assaying toluene dioxygenase activity in crude cell extracts which were complemented with purified preparations of each protein component. Between 40 and 60% of the putative mutants selected from the NBT-TTC indicator plates were unable to grow with toluene as the sole source of carbon and energy. This method should prove extremely useful in isolating mutants in other multicomponent oxygenase enzyme systems.     

Lack of correlation between colony morphology and lipooligosaccharide content in the Mycobacterium tuberculosis complex.

Rough and smooth colony variants of the Mycobacterium tuberculosis complex were compared with respect to their composition in trehalose-containing glycolipid antigens in view of the results of a recent investigation suggesting that the chemical basis of rough and smooth colony morphology in mycobacteria may reside in the occurrence of lipooligosaccharides. A careful chemical characterization of the individual glycolipids of the selected strains allowed the identification of the major glycolipids. The comparative study of the glycolipid content of the smooth Canetti strain, its spontaneous rough variant, and 16 additional strains of M. tuberculosis, M. bovis and M. africanum showed that the presence of lipooligosaccharides was not related to the morphology of the colonies.     

Colonial and Cellular Polymorphism in Xenorhabdus luminescens

A highly polymorphic Xenorhabdus luminescens strain was isolated. The primary form of X. luminescens was luminescent and nonswarming and produced a yellow pigment and antimicrobial substances. The primary form generated a secondary form that had a distinct orange pigmentation, was weakly luminescent, and did not produce antimicrobial substances. Both the primary and secondary forms generated a set of colony variants at frequencies that exceeded normal rates for spontaneous mutation. The variant forms include nonswarming and swarming forms that formed large colonies and a small-colony (SC) form. The primary and secondary forms generated their SC forms at frequencies of between 1 and 14% and 1 and 2%, respectively. The SC forms were distinct from their parental primary and secondary forms in colony and cellular morphology and in protein composition. The cellular morphology and protein patterns of the nonswarming and swarming colony variants were all very similar. The DNA fingerprints of all forms were similar. Each SC-form colony reverted at high frequency to the form from which it was derived. The proportion of parental-type cells in the SC-form colonies varied with age, with young colonies containing as few as 0.0002% parental-type cells. The primary-to-secondary switch was stable, but all the other colony forms were able to switch at high frequencies to the alternative colony phenotypes.     

On the Adaptive Nature of the Dissociation Process in Bacillus thuringiensis

The process of dissociation into variants that differ in colony morphology occurring in batch cloned cultures of two Bacillus thuringiensis strains belonging to different subspecies was studied at optimal and elevated temperatures. An increase in the cultivation temperature to 40°C resulted in an increase in the fraction of R variants to 100% after 72 h of cultivation of either of the strains. This increase was not due to the selection of forms with greater resistance to elevated temperature. The level of resistance to elevated temperature was determined by the strain genotype and did not correlate with morphological characteristics of the colonies.