Composition,secretion kinetics and radioactive labelling of the extracellular slime polysaccharide secreted during spherulation ofPhysarum polycephalum

The myxomycetePhysarum polycephalum synthesizes copious amounts of slime when differentiating into the hard walled resting stage. The chemical composition of slime obtained after introduction of spherulation in a nutrient and a non-nutrient salt-medium has been analysed and compared. The composition of slime is almost identical after the two different induction methods. This slime could be labelled with radioactived-[U-¹⁴C]glucose,³²PO₄ ^3–,³⁵SO₄ ^2– and⁷⁵SeO₄ ^2–. The kinetics of slime secretion after both induction methods has been followed using different criteria. The sulfate analog⁷⁵SeO₄ ^2– seems to be incorporated into the slime, partially replacing sulfate groups of the sulfated polysaccharide. Furthermore,d-[U-¹⁴C]glucose was used to labe the spherule walls. Determination of an alkali resistent polysaccharide component serves as a new method to follow wall formation.     

Achene morphology and slime structure in some taxa of Artemisia L. and Neopallasia L. (Asteraceae)

We have examined slime cell distribution on the surface of the achenes of some Artemisia and Neopallasia taxa, as well as slime composition, envelope formation during the hydration, and slime relation to different morphological features and environmental factors. The results of the studies show a characteristic pattern of slime cells distribution, which could differ between taxa. The slime in the taxa studied belongs to the cellulose type and consists of two components i.e., pectins and cellulose. Although all fruits contain slime cells, not all of them show the slime envelope formation. Plants occurring in dry habitats (such as A. barrelieri) or annual species (such as A. annua) are characterised by a large amount of slime and a fast process of slime envelope formation. Slime production has not been observed in some polyploid populations (A. campestris and A. campestris ssp. sericea) and in two species occurring in relatively fertile habitats (A. verlotiorum, A. vulgaris). A reason for this may be either the immaturity of polyploid fruits leading to the production of a scarce, not detectable slime amount or, alternatively, the occurrence of not functional slime cells. Slime facilitates and stimulates the germination, as well as the adherence of the fruits to the ground or to animals (for dispersal). The slime could play important role in the distribution and colonisation of new habitats in many Artemisia taxa.     

Proteomic characterization of the released/secreted proteins of Leishmania (Viannia) braziliensis promastigotes

Extracellular proteins secreted/released by protozoan parasites are key mediators of the host–parasite interaction. To characterise the profile of proteins secreted/released by Leishmania (Viannia) braziliensis promastigotes, a proteomic approach combining two-dimensional electrophoresis (2DE), tandem matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF) mass spectrometry, and data mining was carried out. The 2DE map revealed a set of 270 secreted protein spots from which 42 were confidently identified and classified into 11 categories according to Gene Ontology (GeneDB database) and KEEG Ontology annotation of biological processes. Parasite promastigotes were able to secrete/release proteins involved in immunomodulation, signal transduction, and intracellular survival, such as HSP70, acid phosphatase, activated protein kinase C receptor (LACK), elongation factor 1β, and tryparedoxin peroxidase. Data mining showed that ~ 5% of identified proteins present a classical secretion signal whereas ~ 57% were secreted following non-classical secretion mechanisms, indicating that protein export in this primitive eukaryote might proceed mainly by unconventional pathways. This study reports a suitable approach to identify secreted proteins in the culture supernatant of L. braziliensis and provides new perspectives for the study of molecules potentially involved in the early stages of infection.     

The ability of white-rot fungi to degrade the endocrine-disrupting compound nonylphenol

Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and Bjerkandera sp. BOL13 were tested for their ability to degrade the endocrine-disrupting compound nonylphenol at an initial concentration of 100 mg l^–1. The highest removals were achieved with T. versicolor and Bjerkandera sp. BOL13, which were able to degrade 97 mg l^–1 and 99 mg l^–1 of nonylphenol in 25 days of incubation, respectively. Nonylphenol removal was associated with the production of laccase by T. versicolor, but the levels of laccase, manganese peroxidase and lignin peroxidase produced by Bjerkandera sp. BOL13 were very low. At 14°C, T. versicolor and Bjerkandera sp. BOL13 sustained the removal of 88 mg l^–1 and 79 mg l^–1 of nonylphenol, respectively. No pollutant removal was recorded at 4°C, although both fungi could grow at this temperature in the absence of nonylphenol. A microtoxicity assay showed that the fungi produced compounds that were toxic to Vibrio fischerii; and thus a reduction in toxicity could not be correlated with nonylphenol metabolism. T. versicolor and Bjerkandera sp. BOL13 were capable of colonizing soil artificially contaminated with 430 mg kg^–1 of nonylphenol. Only 1.3±0.1% of nonylphenol remained in the soil after 5 weeks of incubation.     

Chemical Characterization of Polysaccharide from the Slime Layer of the Cyanobacterium Microcystis flos-aquae C3-40

Macromolecular material from the slime layer of the cyanobacterium Microcystis flos-aquae C3-40 was defined as material that adhered to cells during centrifugation in growth medium but was dislodged by washing with deionized water and retained within dialysis tubing with a molecular-weight cutoff of 3,500. At each step of this isolation procedure, the slime was observed microscopically. Cells in the centrifugal pellet were surrounded by large amounts of slime that excluded negative stain, whereas cells that had been washed with water lacked visible slime. Two independently isolated lots of slime contained no detectable protein (<1%, wt/wt) and consisted predominantly of anthrone-reacting polysaccharide. Sugars in a hydrolysate of slime polysaccharide were derivatized with trimethylsilylimidazole and examined by gas chromatography-mass spectrometry. The composition of the slime polysaccharide was 1.5% (wt/wt) galactose, 2.0% glucose, 3.0% xylose, 5.0% mannose, 5.5% rhamnose, and 83% galacturonic acid. This composition resembles that of the plant polysaccharide pectin, which was treated in parallel as a control. Consistent with earlier indications that M. flos-aquae slime preferentially binds certain cations, the ratio of Fe to Na in the dialyzed slime was 10⁴ times that in the growth medium. The composition of the slime is discussed with respect to possible mechanisms of cation binding in comparison with other cyanobacterial exopolysaccharides and pectin.     

Optimized Production and Purification of Bacillus anthracis Lethal Factor

Bacillus anthracis lethal factor (LF) is a 90-kDa zinc metalloprotease that plays an important role in the virulence of the organism. LF has previously been purified from Escherichia coli and Bacillus anthracis. The yields and purities of these preparations were inadequate for crystal structure determination. In this study, the genes encoding wild-type LF and a mutated, inactive LF (LF-E687C) were placed in an E. coli–Bacillus shuttle vector so that LF was produced with the protective antigen (PA) signal peptide at its N-terminus. The resulting vectors, pSJ115 and pSJ121, express wild-type and mutated LF fusion proteins, respectively. Expression of the LF genes is under the control of the PA promoter and, during secretion, the PA signal peptide is cleaved to release the 90-kDa LF proteins. The wild-type and mutated LF proteins were purified from the culture medium using three chromatographic steps (Phenyl–Sepharose, Q–Sepharose, and hydroxyapatite). The purified proteins were greater than 95% pure and yields (20–30 mg/L) were higher than those obtained in other expression systems (1–5 mg/L). These proteins have been crystallized and are being used to solve the crystal structure of LF. Their potential use in anthrax vaccines is also discussed.     

Evolution of NIN-Like Proteins in Arabidopsis, Rice, and Lotus japonicus

Genetic studies in Lotus japonicus and pea have identified Nin as a core symbiotic gene required for establishing symbiosis between legumes and nitrogen fixing bacteria collectively called Rhizobium. Sequencing of additional Lotus cDNAs combined with analysis of genome sequences from Arabidopsis and rice reveals that Nin homologues in all three species constitute small gene families. In total, the Arabidopsis and rice genomes encode nine and three NIN-like proteins (NLPs), respectively. We present here a bioinformatics analysis and prediction of NLP evolution. On a genome scale we show that in Arabidopsis, this family has evolved through segmental duplication rather than through tandem amplification. Alignment of all predicted NLP protein sequences shows a composition with six conserved modules. In addition, Lotus and pea NLPs contain segments that might characterize NIN proteins of legumes and be of importance for their function in symbiosis. The most conserved region in NLPs, the RWP-RK domain, has secondary structure predictions consistent with DNA binding properties. This motif is shared by several other small proteins in both Arabidopsis and rice. In rice, the RWP-RK domain sequences have diversified significantly more than in Arabidopsis. Database searches reveal that, apart from its presence in Arabidopsis and rice, the motif is also found in the algae Chlamydomonas and in the slime mold Dictyostelium discoideum. Thus, the origin of this putative DNA binding region seems to predate the fungus–plant divide.Reviewing Editor: Professor David Guttman     

Detection of capsule and slime polysaccharide layers in two strains of Rhodopseudomonas capsulata

A capsule and slime were visualized electronmicroscopically in Rhodopseudomonas capsulata strain St. Louis (=ATCC 23782) and strain Sp 11 after pre-incubation of the cells in the homologous O/K antisera. The slime consists of loosely associated material surrounding the cell in irregular distribution. The capsule is directly adjacent to the cell wall and has a constant thickness of 75–85 nm in strain St. Louis and 30–40 nm in strain Sp 11. The capsule has a fibrillar fine-structure with radial orientation to the cell surface. In contrast to the slime, it is not removed from the cells by washing with saline.An acidic polysaccharide fraction was obtained from both strains by cetavlon fractionation of hot phenol-water extracts. The composition is strain-specific: the relative amounts of the common sugars found, i.e. rhamnose, galactose, glucose, glucosamine and galacturonic acid are different, the fraction from strain Sp 11 contains additionally fucose, 3-amino-3,6-dideoxygalactose, an unknown amino sugar and an unknown acidic component. Whether the polysaccharides of these fractions are in fact the slime or capsular substances remains to be established.     

Biodegradation of nonylphenol in a continuous packed-bed bioreactor

A packed bed bioreactor, with 170 ml glass bead carriers and 130 ml medium, was tested for the removal of the endocrine disrupter, nonylphenol, with a Sphingomonas sp. The bioreactor was first continuously fed with medium saturated with nonylphenol in an attempt to simulate groundwater pollution. At best, nonylphenol was degraded by 99.5% at a feeding rate of 69 ml h^–1 and a removal rate of 4.3 mg nonylphenol day^–1, resulting in a 7.5-fold decrease in effluent toxicity according to the Microtox. The bioreactor was then fed with soil leachates at 69 ml h^–1 from artificially contaminated soil (1 g nonylphenol kg^–1 soil) and a real contaminated soil (0.19 g nonylphenol kg^–1 soil). Nonylphenol was always completely removed from the leachates of the two soils. It was removed by 99% from the artificial soil but only 62% from real contaminated soil after 18 and 20 d of treatment, respectively, showing limitation due to nonylphenol adsorption.     

Aerobic biodegradation of nonylphenol by cold-adapted bacteria

Three strains capable of mineralizing nonylphenol as sole carbon source were isolated from a sample of contaminated soil and characterized as two Pseudomonas spp. and a Stenotrophomonas sp. The two Pseudomonas spp. expressed characteristics typical of psychrophiles growing optimally of 10 °C and capable of growing at 0 °C. The Stenotrophomonas sp. was more likely psychrotrophic because it had an optimal temperature between 14 and 22 °C although it was not capable of growing at 4 °C. At 14 °C, one of the Pseudomonas spp. exhibited the highest rate of degradation of nonylphenol (4.4 mg l^–1 d^–1), when compared with axenic or mixed cultures of the isolates. This study represents, to the best of our knowledge, the first reported case of cold-adapted microorganisms capable of mineralizing nonylphenol.     

Proteomics of Protein Secretion by Bacillus subtilis: Separating the “Secrets” of the Secretome

Secretory proteins perform a variety of important “remote-control” functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which ~90 extracellular proteins were identified. Analysis of these proteins disclosed various “secrets of the secretome,” such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only ~50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.     

Mutant fs(1) 1163 of Drosophila melanogaster alters yolk protein secretion from the fat body

Summary The mutant fs(1) 1163 of Drosophila melanogaster, which was isolated by Gans et al. (1975) is a recessive homozygous female sterile at 18°C and a dominant female — sterile at 29°C. We reported previously that there are reduced quantities of the largest of the three yolk polypeptides in Drosophila melanogaster in the haemolymph and eggs of this mutant at 29°C (Bownes and Hames 1978 a). In this paper we show that the yolk protein defect maps within approximately 2.5 recombination units of the female sterility at 21±2.5 map units on the X-chromosome. The temperature-sensitive period of the yolk protein defect is after emergence. In vitro labelling of fs(1) 1163 ovaries and fat bodies showed that they were able to synthesise yolk polypeptide 1. Interestingly, studies on the proteins present in the various tissues indicate that the fat body tends to accumulate all three yolk polypeptides in the mutant. This phenotype is partially co-dominant in that an effect is seen in heterozygotes as well as homozygotes and is enhanced by increased temperature. This mutant could therefore have a defect (a) in the structural gene for yolk polypeptide 1, (b) in the processing and secretion enzyme systems; (c) in the fat body or all tissues leading to altered secretion properties.Mutants like fs(1) 1163 which alter specific steps in vitellogenesis should be of value for analysing the genetic and biochemical control of the synthesis, transport and sequestering of the yolk polypeptides during oogenesis.     

Evolutionary changes reflected by the cellular amino acid composition

Summary Comparison of the amino acid composition of cell-proteins using 17 amino acids has been used to investigate the biological evolution of organisms such as bacteria, blue-green alga, green alga, fungi, slime mold, protozoa and vertebrates. The degree of difference in the amino acid ratios between any two groups reflects the degree of divergency in biological evolution. The amino acid composition of the Gram-negative bacteria (Escherichia coli,Klebsiella,Proteus, andVibrio alginolyticus) was identical. However, the amino acid composition ofStaphylococcus aureus andBacillus subtilis, which are Gram-positive bacteria, differed from each other and from the Gram-negative bacteria. The amino acid composition of the blue-green alga (Cyanobacterium,Chroococidiopsis) was quite similar to that ofE. coli. A marked difference in the amino acid composition was observed betweenE. coli and green alga (Chlorella), and significant differences were observed betweenE. coli and other organisms, such as fungi, protozoa (Tetrahymena), slime mold (Dictyostelium discoideum) and vertebrates. In conclusion, the change in cellular amino acid composition reflects the divergence which has occurred during biological evolution, whereas a basic pattern of amino acid composition is maintained in spite of a long period of evolutional divergence among the various organisms. Thus, it is proposed that the primitive life forms established at the end of prebiotic evolution had a similar amino acid composition.     

Mutant invertase proteins accumulate in the yeast endoplasmic reticulum

Summary Intercompartmental transport of secreted proteins in yeast was analysed using invertase mutants. Deletions and insertions at the BamHI (position +787) or the Asp718 (position +1159) sites of the SUC2 gene led to mutant proteins with different behaviour regarding secretion, localization and enzyme activity. The deletion mutants showed accumulation of core glycosylated material in the endoplasmic reticulum (ER) a decrease of secreted protein by 5%–30% and loss of enzyme activity. The secreted material was localized in the culture medium and not — as is normal for invertase-in the cell wall. No delay in transport from the Golgi to the cell surface was observed, indicating that the rate-limiting step for secretion is at the ER-Golgi stage. Two insertion mutants, pIPA and pIPB, retained enzyme activity. Mutant pIPB showed 10% secretion, while 60%–70% secretion was observed for pIPA. While the non-secreted material accumulated in the ER, the secreted material was present in the cell wall. The results suggest that the presence of structures incompatible with secretion leads to ER accumulation of mutated invertase.     

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HIyB/HIyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hy1A from the 5 end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.     

Cell-specific synthesis and glycosylation of secretory proteins in larval salivary glands of Chironomus thummi

Synthesis and glycosylation of larval salivary gland secretory proteins of Chironomus thummi were analyzed with respect to cell specific differences in the Balbiani ring (BR) pattern and glycoprotein composition of secretion formerly detected by histochemical staining procedures. In the secretion of a special cell type in salivary glands, which is characterized by the appearance of an additional BR, an additional polypeptide with a relative molecular weight (M_r) of 160 kD was found differing in its antigenic properties and tryptic fingerprint pattern from main cell secretion proteins. This so-called ssp-160 component is preferentially synthesized and glycosylated in the special cells. In the same cells, both the synthesis and glycosylation of all other major secretory proteins was found to be diminished or even repressed. In contrast to the conspicuous cell-specific differences at the level of protein synthesis, RNA analyses show the prominent synthesis of 75 S RNA in both main and special cells and gave no clear indication of the synthesis of a smaller RNA fraction as expected from the size of ssp-160 component. — These and further data on synthesis and properties of secretory proteins as well as expression of BR DNA are discussed with regard to the assumption that at least some of the eight major secretory polypeptides are coded for by BR DNA. The BR gene(s) might have originated by manifold duplications and modifications of short repetitive prototype DNA sequences, which are coordinatively expressed.On the occasion of the 60th anniversary of his birth-day we wish to dedicate this paper to Professor Wolfgang Beermann who was the first to detect, by the discovery of cell specific expression of BR 4 of Chironomus pallidivittatus salivary gland chromosomes and the concomitant occurrence of cell specific secretion granules, a causual relationship between the activity of a Balbiani ring and the appearance of a secretion component (Beermann, 1961)addressee for reprint requests     

Structure and Composition of Biological Slimes on Paper and Board Machines

Biological slimes (biofilms) collected from the wet end of paper and board machines were examined by electron microscopy and analyzed for fatty acid composition, neutral sugar composition, and ATP. Electron microscopy revealed minuscule prokaryotic organisms (diameter, 0.2 to 0.4 μm). Larger cells morphologically resembling Sphaerotilus and Leptothrix spp. were found in slimes from machines using recycled fiber or unbleached pulp. The bacteria were embedded in a slimy matrix and often contained reserve materials microscopically resembling poly-β-hydroxybutyrate and glycogen. Fatty acid analysis of the slimes revealed bacterial signature fatty acids in concentrations equivalent to the presence of 2 × 10¹⁰ to 2.6 × 10¹² (average, 7 × 10¹¹) bacterial cells (live and dead) per g (dry weight) of slime. The slimes contained several known components of bacterial polysaccharides in addition to glucose, indicating that the slime body consisted of bacterial polysaccharides. The slimes contained uronic acids equivalent to a binding capacity of 12.5 to 50 μmol of divalent cations per g (dry weight) of slime. The uronic acid-containing polysaccharides may be responsible for the accumulation of heavy metals in the slime. Calculation of the ATP contents of the slimes resulted in an estimate of 5 × 10¹² cells per g (dry weight) of slime when calibrated with pure bacterial cultures isolated from the slimes. From electron micrographs, an estimate ranging from 1 × 10¹⁰ to 1.5 × 10¹² (average, 4 × 10¹¹) cells per g (dry weight) of slime was obtained.     

Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromolecular structures

Genes required for entry of Shigella flexneri into epithelial cells in vitro are clustered in two adjacent loci, one of which encodes secretory proteins, the IpaA–D proteins, and the other their dedicated secretion apparatus, the Mxi–Spa translocon. Ipa secretion, which is induced upon contact of bacteria with epithelial cells, is prevented during growth in vitro. Here, we show that ipaB and ipaD mutations lead to enhanced secretion of a set of about 15 proteins. These extracellular proteins and some Ipas associate in organized structures consisting of extended sheets. Growth of the wild-type strain in the presence of Congo red is shown to induce protein secretion through the Mxi–Spa translocon. Cultures grown to stationary phase in the presence of Congo red contain extracellular filaments whose composition and morphology are similar to those produced by the hyper-secreting ipaB and ipaD mutants.     

The association of Prototheca spp. with slime flux in Ulmus amencana and other trees

All known species of Prototheca and one new species were isolated from slime flux of woody stems. Fortyfive trees (11 species) harbored Prototheca spp. Monthly quantitative studies of slime flux from 10 trees of Ulmus amencana revealed that 6 harbored a new species, P. ulmea, often in large numbers (1×10⁵ colony forming units ml^–1 slime flux). In total, P. ulmea was isolated from 17 elms and one oak from three states in the U.S.A. In spite of this extensive association, no causal relationship has been established. Prototheca ulmea is a small, oval, capsulated species similar to P. stagnora, but differentiated by its morphology and inability to use fructose and galactose. Utilization of other carbohydrates is restricted and its only known habitat is slime flux.     

The influence of chlorsulfuron and metsulfuron methyl on root growth and on the ultrastructure of root tips of germinating maize seeds

Seeds of Zea mays L., germinating in soil, were exposed to very low doses of the sulfonylurea herbicides chlorsulfuron and metsulfuron methyl. At a concentration of 0.012 mg L^–1, chlorsulfuron caused 72% and metsulfuron methyl 55% growth reduction of the young primary roots. Both herbicides also caused obvious injuries to the root tips. Scanning electron microscopic observations of the root tip surfaces indicated an inhibition of slime secretion at a herbicide concentration of 1.5 mg L^–1. Transmission electron microscopy revealed obvious changes to the nuclei and deformation of radial cell walls in the primary root cortex at 0.012 and 1.5 mg L^–1 for both herbicides. Moreover, the secretory cells of the root cap periphery showed partially irregular deposition of premature cell wall or slime material at a concentration of 0.012 mg L^–1 of both herbicides.From the results of our electron microscopic observations we conclude that the primary roots of maize seedlings are seriously affected by extremely low concentrations of even those herbicides which (as chlorsulfuron and metsulfuron methyl) have been developed to inhibit the growth of dicotyledonous weeds. Moreover, we suggest that the frequently observed growth retardation of crop seedlings is a consequence of early root tip injuries caused by herbicide residues in the soil. ei]H Lambers