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PubMed2001

A comprehensive study into the molecular methodology and molecular biology of methanogenic Archaea.

Methanogens belong to the kingdom of Euryarchaeota in the domain of Archaea. The Archaea differ from Bacteria in many aspects important to molecular work. Among these are cell wall composition, their sensitivity to antibiotics, their translation and transcription machinery, and their very strict demands to anaerobic culture conditions. These differences may, at least partly, be responsible for the delay in availability of genetic research tools for methanogens. At present, however, the research within genetics of methanogens and their gene regulation and expression is in rapid progress. Two complete methanogenic genomes have been sequenced and published and more are underway. Besides, sequences are known from a multitude of individual genes from methanogens. Standard methods for simple DNA and RNA work can normally be employed, but permeabilization of the cell wall may demand special procedures. Efficient genetic manipulation systems, including shuttle and integration vector systems, have appeared for mesophilic, but not for thermophilic species within the last few years and will have a major impact on future investigations of methanogenic molecular biology.

Euryarchaeota↗
PubMed1998

Implication of cell wall constituents in the sensitivity of Kluyveromyces lactis strains to amphotericin B.

In Kluyveromyces lactis, the cell wall compositions of Kl (ATCC 96897), a wild sensitive strain, and Klm (ATCC 96896), a strain resistant to amphotericin B (AmB), were shown to be very different, since the walls in the latter were significantly enriched in hexosamine, but had a reduced content in phosphate and amino acid. In both strains, the cell walls limited their sensitivity to this antifungal agent. The absence of cell wall increased the sensitivity of the cells to this polyene by 5 to 10-fold. When the cells were treated with enzymes such as pronase and chitinase in order to change the cell wall structure just before inoculation, the yeasts appeared more resistant to the antibiotic. However, treatments with chymopapain and phospholipase C did not significantly change the sensitivity of the two strains to this agent. Cells treated with acid phosphatase displayed a longer lag phase than the control cells. In addition, when cultured in the presence of AmB, the cells were less sensitive to this agent. The present results reveal that both a change in the ionic charges of the cell wall and an alteration in the cell wall structure modified the sensitivity of these yeast strains to AmB.

Acid Phosphatase↗
PubMed1978

Streptoalloteichus, a new genus of the family Actinoplanaceae.

A new genus Streptoalloteichus is proposed in the family Actinoplanaceae to distinguish species of actinomycetes which form short or long spore-chains on aerial mycelium, bears oval sporangia with motile spores and has a characteristic cell-wall composition of strain C677-91 type. Strain C677-91 (ATCC 31217, FERM-P No. 4070) was named Streptoalloteichus hindustanus gen. nov. and sp. nov. The actinomycete strain C677-91 produces spore-chain clusters and sclerotia in the aerial mycelium which are morphologically similar to those found in some species of Streptomyces. The cultural characteristics of the strain on agar media also resemble those of Streptomyces species and the colonies have no distinct color. Strain C677-91 produces sporangia or sporangia-like vesicles which contain one to several spores in the vegetative mycelium. The sporangiospores possess a single long polar flagellum and are motile. The cell wall of strain C677-91 contains meso-alpha,epsilon-diaminopimelic acid, alanine, glutamic acid, galactose, mannose, rhamnose and glucosamine. Strain C677-91 has several important characteristics in common with Streptomyces tenebrarius including the production of nebramycin factors but the latter strain does not produce sporangia.

Actinomycetales↗
PubMed1992

Alterations in the cell wall of Saccharomyces cerevisiae induced by the alpha sex factor or a mutation in the cell cycle.

We performed experiments in parallel to study the rate of synthesis of cell wall polysaccharides and the activity of glycosyl transferases in Saccharomyces cerevisiae after arrest of a cdc 28 mutant in G1 phase by either addition of alpha-factor or transfer to the non-permissive temperature. Both effectors brought about similar time-dependent increases in the rate of synthesis and deposition of the cell wall polysaccharides chitin, glucan and mannan. These changes in cell wall composition were accompanied by an increase in the specific activities of glucan and chitin synthetases. This increase was inhibited by cycloheximide suggesting that it represented de novo enzyme biosynthesis and not enzyme activation. Our data are consistent with the notion that both alpha-factor and the cdc 28 mutation affect the same stage-specific function that controls the temporal expression of glycosyl transferases.

Cell Cycle↗
PubMed2000

Staphylococcus fleurettii sp. nov., isolated from goat's milk cheeses.

A new coagulase-negative and novobiocin-resistant species of the genus Staphylococcus, Staphylococcus fleurettii, isolated from raw-milk cheeses, is described. This species is differentiated from the other novobiocin-resistant staphylococci on the basis of ribotype and intergenic transcribed spacer patterns, DNA-DNA reassociation reactions, cell wall composition and phenotypic characteristics. S. fleurettii could be distinguished by its oxidase activity, by its ability to produce acid aerobically from D-trehalose, D-mannose, D-turanose and maltose and by its inability to produce acid from D-cellobiose. The type strain of S. fleurettii is CIP 106114T (= DSM 13212T).

Animals↗
PubMed1999

The mur4 mutant of arabidopsis is partially defective in the de novo synthesis of uridine diphospho L-arabinose.

To obtain information on the synthesis and function of arabinosylated glycans, the mur4 mutant of Arabidopsis was characterized. This mutation leads to a 50% reduction in the monosaccharide L-arabinose in most organs and affects arabinose-containing pectic cell wall polysaccharides and arabinogalactan proteins. Feeding L-arabinose to mur4 plants restores the cell wall composition to wild-type levels, suggesting a partial defect in the de novo synthesis of UDP-L-arabinose, the activated sugar used by arabinosyltransferases. The defect was traced to the conversion of UDP-D-xylose to UDP-L-arabinose in the microsome fraction of leaf material, indicating that mur4 plants are defective in a membrane-bound UDP-D-xylose 4-epimerase.

Arabidopsis↗
PubMed1971

Biochemical alterations in Bacillus megaterium as produced by aflatoxin B1.

Bacillus megaterium NRRL B-1368 cells and spores were produced on Trypticase Soy Broth (TSB) and Agar (TSA) containing 3.8 mug of aflatoxin B(1) per ml, analyzed for selected chemical constituents, and compared to cells and spores of B. megaterium produced on nontoxic Trypticase Soy Media. There was an initial 30% kill of cells after inoculation into toxic TSB and during the first 3.5 hr of incubation followed by a logarithmic growth phase in which the generation time was 75 min as compared to 20 min for the control culture. Chemical analyses revealed an increase in protein, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) on both a per cell basis and a per cent dry weight basis when B. megaterium was grown in toxic TSB. There was a concurrent decrease in the total amounts of cellular protein, DNA, and RNA synthesized in toxic TSB. Amino acid analyses of control and test cell walls showed little, if any, difference in cell wall composition. About 97% sporulation of B. megaterium occurred after 3 days on nontoxic TSA although 6 days were required to attain 65% sporulation on toxic TSA. Germination of spores was not inhibited by 4.0 mug of aflatoxin per ml but outgrowth was. No significant differences were observed in the heat resistance, protein, DNA, RNA, or dipicolinic acid content of spores formed on toxic TSA and nontoxic TSA.

Aflatoxins↗
PubMed1992

[Preparation and characteristics of protoplasts from various strains of Streptomyces roseoflavus var. roseofungini].

It was shown that the variants of the roseofungin-producing organism, which differ in their differentiation, antibiotic activity, structure and cell wall composition had different sensitivity to the protoplasting factors. The protoplasting increased the population heterogeneity: in strain 1128 it was evident from an increased frequency of the secondary colonies, in variant 1-68, folding of the colonies increased and their consistency become milder, sectorial colonies and colonies with coremia formed. It was in principle possible to transform the protoplasts of S. roseoflavus var. roseofungini by plasmid DNA, which suggests that the roseofungin-producing culture may be useful in genetic engineering.

Antifungal Agents↗
PubMed2002

Change of cell wall chitin content in amphotericin B resistant Kluyveromyces strains.

The culture of two Kluyveromyces species, Kluyveromyces lactis (ATCC 96897) and Kluyveromyces bulgaricus (ATCC 96631), in the presence of subinhibitory doses of amphotericin B leads to the selection of mutants which are resistant to this polyene. The mutants show an alteration of their cell wall composition with the main change corresponding to an increase of chitin. The enzyme activities involved in the metabolism of this polymer, i.e. chitin synthases and chitinase, were measured. The results demonstrate that in both mutants the activity of chitinase was drastically decreased by 99% in comparison with the activity measured in the corresponding wild-type strain while no significant change of the chitin synthase I, II and III activities could be detected.

Amphotericin B↗
PubMed1993

Coryneform bacteria isolated from middle ear fluid.

Nineteen strains of facultatively anaerobic gram-positive rods isolated in pure culture from middle ear fluids were identified. All effusions were collected by tympanocentesis in children with acute otitis media. Identification of microorganisms to the genus level was done by studying the cell wall composition. Sixteen strains contain meso-diaminopimelic acid and arabinogalactan polymer but lack mycolic acids; therefore, these strains do not belong to a previously described taxon. Because of similarities with Corynebacterium afermentans (Centers for Disease Control group ANF-1), we temporarily classified these mycolateless strains ANF-1 like. Isolation of these microorganisms in pure culture from middle ear fluids collected by tympanocentesis is a strong argument for their involvement in acute otitis media.

Actinomycetales↗
PubMed1986

[Production and regeneration of yeast protoplasts: a review].

The latest advance in protoplast technology results from a long and detailed study of cell wall composition and protoplast systems relating to their isolation and culture. The reports on protoplast preparation and fusion discussed in this review clearly demonstrate the applicability of technique for the genetic manipulation of microorganisms. These points have been demonstrated in Candida strains. The behaviour of protoplasts especially with respect to reversion could be an additional factor that operates during selection. Furthermore the fusion technique, in providing a mechanism which genetic recombination can be readily achieved, should be of great potential in empirical breeding and strain improvement. These aspects are reviewed.

Cell Wall↗
PubMed1988

Characterization of 15 selected coccal bacteria isolated from Antarctic rock and soil samples from the McMurdo-Dry Valleys (South-Victoria Land).

Approximately 1500 cultures of microorganisms were isolated from rocks and soils of the Ross Desert (McMurdo-Dry Valleys). From these, 15 coccoid strains were chosen for more detailed investigation. They were characterized by morphological, physiological and chemotaxonomical properties. All isolates were Gram-positive, catalase-positive and nonmotile. Six strains showed red pigmentation and could be identified as members of the genera Micrococcus (M. roseus, M. agilis) or Deinococcus. In spite of their coccoid morphology, the remaining nine strains had to be associated with coryneform bacteria (Arthrobacter, Brevibacterium), because of their cell wall composition and G+C ratios. Most of the strains were psychrotrophic, but one strain was even obligately psychrophilic, with a temperature maximum below 20 degrees C. Red cocci had in vitro pH optima above 9.0 although they generally originated from acid samples. Most isolates showed a preference for sugar alcohols and organic acids, compounds which are commonly known to be released by lichens, molds and algae, the other components of the cryptoendolithic ecosystem. These properties indicate that our strains are autochthonous members of the natural Antarctic microbial population.

Antarctic Regions↗
PubMed2002

Staphylococcus succinus subsp. casei subsp. nov., a dominant isolate from a surface ripened cheese.

A new subspecies of the species Staphylococcus succinus, isolated from a Swiss surface ripened cheese, is described. This subspecies is differentiated from the species Staphylococcus succinus ATCC 700337T on the basis of DNA-DNA hybridisation, cell wall composition and phenotypic characteristics. Staphylococcus succinus subsp. casei could be distinguished among other things by its ability to reduce nitrate, form acid from D-mannose and D-melezitose, ferment adenosine, inosine, D-sorbitol, and 2,3-butanediol, but not D-alanine. The type strain of Staphylococcus succinus subsp. casei is DSM 15096 (CIP no. pending). The GenBank accession numbers for the reference sequences of the 16S rDNA and the hsp60 gene used in this study are AJ320272 and AF527482, respectively.

Bacterial Typing Techniques↗
PubMed1992

Utilization of 1,3,5-trihydroxybenzene (phloroglucinol) by a soil isolate, Rhodococcus species BPG-8.

A Gram-positive bacterial strain was isolated from oil rich soil in Newfoundland and found to utilize various di- and trihydroxylated aromatic compounds as a source of carbon and energy. This bacterium exhibited rod/coccus dimorphism during its growth cycle. Chemical analysis of cell wall composition (amino acids, sugars, and fatty acids) was performed using gas chromatography-mass spectrophotometry and high pressure liquid chromatography. Comparison of both acid production and growth substrates showed complete homology with Rhodococcus erythropolis. Growth of the isolate on phloroglucinol (1,3,5 trihydroxybenzene) occurred in the pH range 5-8; with a substrate and temperature optima of 8.0 mM and 25 degrees C. The oxidation of PG was examined using whole cells as well as crude cell extracts. PG oxidation was shown to be due to an inducible enzyme system. Tentatively the isolate was identified as Rhodococcus species BPG-8 which is able to utilize phloroglucinol as the sole source of carbon and energy.

Acids↗
PubMed2005

Specific molecular features in the organization and biosynthesis of the cell wall of Aspergillus fumigatus.

The cell wall of Aspergillus fumigatus is composed of a branched beta1,3 glucan covalently bound to chitin, beta1,3, beta1,4 glucans, and galactomannan, that is embedded in an amorphous cement composed of alpha1,3 glucan, galactomannan and polygalactosamin. The mycelial cell wall of A. fumigatus is very different from the yeast Saccharomyces cerevisiae cell wall, and in particular lacks beta1,6 glucans and proteins covalently bound to cell wall polysaccharides. The differences in cell wall composition between the mould A. fumigatus and the yeast S. cerevisiae are also reflected at the genomic level where unique features have been identified in A. fumigatus. A single gene codes for the glucan synthase catalytic subumit; this finding has lead to the development of a RNAi methodology for the disruption of essential genes in A. fumigatus. In contrast to the glucan synthase, multiple genes have been found in the chitin synthase and the alpha glucan synthase families; in spite of homologous sequences, each gene in each family have very different function. Similarly homologous mannosyltransferase genes are found in yeast and moulds but they lead to the synthesis of very different N-mannan structures. This chemo-genomic comparative analysis has also suggested that GPI-anchored proteins do not have a role of linker in the three dimensional organization of the fungal cell wall.

Aspergillus fumigatus↗
PubMed1999

Taxonomic study and morphological differentiation of an actinomycete genus, Kitasatospora.

The history and taxonomic criteria of the actinomycete genus, Kitasatospora, are described. The reasons for a change of the position of the genus in the classification system are given and a restriction length polymorphism (RFLP) method for rapid identification is described. Although the bacterial genus, Kitasatospora, resembles the fungal one, Streptomyces, in its morphology, it is clearly different in its cell-wall composition which includes LL- and meso-DAP, glycine and galactose. Following a change in its taxonomic position as a result of insufficient gene analysis, it was re-established by phylogenetic analysis of the entire 16S rRNA gene. Most actinomycetes produce bioactive compounds but the relationship between antibiotic production and morphology has not been studied in detail. Kitasatospora strains may be used as a model because they grow in submerged culture like those actinomycetes strains which produce bioactive compounds. The authors suggest further studies of the correlation between metabolic and morphological differentiation and the mechanism of the production of submerged spores.

Actinomycetales↗
PubMed1986

Chemical Composition of the Edible Mushroom Pleurotus ostreatus Produced by Fermentation.

Pleurotus ostreatus ;Florida' was grown in submerged liquid culture. The biomass yield of the fungus, grown for 3 days in 2-liter fermentors, where the mycelial pellets measuring 5 mm in diameter were formed, was 11.7 g (dry weight)/liter. Comparing the chemical constituents of fruiting bodies produced on cotton straw and mycelial pellets revealed several similarities in total nitrogen, protein, glycogen, fatty acids, RNA, and ash content. Differences were observed in the contents of six amino acids. Although the total fatty acid content was similar, there were more saturated fatty acids in the mycelium. Cell wall composition, typical for basidiomycetes, was observed in both mycelium and fruiting bodies, with laminarin as the main polymer.

Journal Article↗
PubMed2001

Involvement of multiple genetic loci in Staphylococcus aureus teicoplanin resistance.

Teicoplanin resistance was transformed from a teicoplanin-resistant Staphylococcus aureus into the susceptible strain BB255 to give strain BB938. The cell wall composition, amidation of the iD-glutamate, and peptide crosslinking were identical in BB938 as in BB255 except for a 60% increased length of the glycan chain. Transductional crosses revealed that at least two distinct loci contributed in a cumulative fashion to teicoplanin resistance. One of these loci correlated with a mutation inactivating the anti-sigma factor RsbW. This mutation must have occurred during transformation and selection for teicoplanin resistance in BB938. Genetic manipulations involving the sigB operon showed that transcription factor SigB contributed to decreased teicoplanin susceptibility.

Animals↗