Calcofluor white and Congo red inhibit chitin microfibril assembly of Poterioochromonas: evidence for a gap between polymerization and microfibril formation

The influence of the light microscopical stains, Calcofluor white and Congo red, on the process of chitin microfibril formation of the chrysoflagellate alga Poterioochromonas stipitata was studied with light and electron microscopy. There is a concentration-dependent inhibition of lorica formation with both dyes. In the presence of the inhibitors malformed loricae are made, which do not show the usual ultrastructure and arrangement of the chitin microfibrils. Instead of long, laterally associated microfibrils, short rods or irregular networks of subelementary (15-25 A) fibrils are found. Microfibril assembly obviously takes place on the accessible outside of the plasma membrane. There must be a gap between the polymerization and microfibril formation reactions, allowing the stains to bind to the polymerized subunits. Thus, later association of these units to form microfibrils is disturbed. The microfibril-orienting mechanism also depends on normal microfibril formation. A model summarizing these hypotheses is suggested.     

Cu,Zn-superoxide dismutase is required for cell wall structure and for tolerance to cell wall-perturbing agents in Saccharomyces cerevisiae

Here we report that deletion of SOD1, the Cu,Zn-superoxide dismutase in Saccharomyces cerevisiae is sensitive to cell wall-perturbing agents, such as Calcofluor white and Congo red. The sensitivity was restored by retransformation with wild type SOD1 or the addition of N-acetylcysteine or reduced glutathione to the medium. Additionally, the accumulation of reactive oxygen species was observed in sod1Δ mutant in the presence of Calcofluor white or Congo red. Cell wall analysis indicated an increase of cell wall chitin and cell wall thickness in sod1Δ mutant compared to wild type. These results indicate a novel direct connection between antioxidative functions and cell wall homeostasis.     

Development of cell wall appendages inAcanthosphaera zachariasi (Chlorococcales): Kinetics,site of cellulose synthesis and of microfibril assembly,and barb formation

Summary The investigation of the formation of cell wall appendages inAcanthosphaera by means of light and electron microscopy and by the use of dyes which interfere with microfibril assembly resulted in several observations which are helpful to an understanding of the formation of normal cell walls. The barbs are built up in the ER, pass through the Golgi apparatus, and are extruded exocytotically after cytokinesis, a remarkable example of the secretion of a structured product. Each cellulose microfibril in a spike develops in a distinct pit of the plasmalemma. The pits are aggregated in a pit field, generating one spike, and are closely adjacent to a basal vesicle which might have morphogenetic and/or regulatory functions. The pits are the site of cellulose synthesis; here the plasmalemma is conspicuously thickened. As shown directly and by the application of Calcofluor white and Congo red, the microfibrils assemble at a certain distance from the plasma membrane,i.e. cellulose synthesis and microfibril assembly are separated by a gap. It is discussed whether single glucan chains or small bundles of them are released from the plasmalemma. The elongation rate of the spikes indicates that about 1000 glycosidic linkages per glucan chain per minute are formed.     

A novel endoplasmic reticulum membrane protein Rcr1 regulates chitin deposition in the cell wall of Saccharomyces cerevisiae

Congo red binds to the cell wall and inhibits the growth of yeast. In a screening for multicopy suppressor genes of Congo red hypersensitivity of erd1Delta mutant, we found that a previously uncharacterized gene, YBR005w, makes most of the Saccharomyces cerevisiae strains resistant to Congo red. This gene was named RCR1 (resistance to Congo red 1). An rcr1Delta null mutant showed an increased sensitivity to Congo red. RCR1 encodes a novel ER membrane protein with a single transmembrane domain. Molecular dissection suggested that the transmembrane domain and a part of the C-terminal polypeptide are sufficient for the activity. We examined the effect of RCR1 in various null mutants of genes related to the cell wall. The resistance of mutants to Congo red correlates with a reduction of chitin content. Multicopy RCR1 caused a significant decrease in the chitin content while the amount of alkali-soluble glucan did not change. The binding of Calcofluor white to the cell wall significantly decreased in these cells. Our results show that RCR1 regulates the chitin deposition and add firm genetic and biochemical evidences that the primary target of Congo red is chitin in S. cerevisiae.     

Tunicamycin Prevents Cellulose Microfibril Formation in Oocystis solitaria

The effect of tunicamycin (TM) on the development of the cell wall in Oocystis solitaria has been investigated. It was found that 10 micromolar TM completely stops the assembly of new microfibrils as observed at the ultrastructural level. During cell wall formation, freeze fracture replicas of the E-face of the plasma membrane reveal two major substructures: the terminal complexes (TC), paired and unpaired, and the microfibril imprints extending from unpaired TCs. In cells treated for 3 hours or longer with TM, the TCs are no longer visible, whereas microfibril imprints are still present. Because of the reported highly selective mode of action of TM, our results implicate a role for lipid-intermediates in cellulose synthesis in O. solitaria. It is assumed that TM prevents the formation of a glycoprotein which probably is a fundamental part of the TCs and may act as a primer for the assembly of the microfibrils.     

Mutations in SNF1 complex genes affect yeast cell wall strength

The trimeric SNF1 complex from Saccharomyces cerevisiae, a homolog of mammalian AMP-activated kinase, has been primarily implicated in signaling for the utilization of alternative carbon sources to glucose. We here find that snf1 deletion mutants are hypersensitive to different cell wall stresses, such as the presence of Calcofluor white, Congo red, Zymolyase or the glucan synthase inhibitor Caspofungin in the growth medium. They also have a thinner cell wall. Caspofungin treatment triggers the phosphorylation of the catalytic Snf1 kinase subunit at Thr210 and removal of this phosphorylation site by mutagenesis (Snf1-T210A) abolishes the function of Snf1 in cell wall integrity. Deletion of the PFK1 gene encoding the α-subunit of the heterooctameric yeast phosphofructokinase suppresses the cell wall phenotypes of a snf1 deletion, which suggests a compensatory effect of central carbohydrate metabolism. Epistasis analyses with mutants in cell wall integrity (CWI) signaling confirm that the SNF1 complex and the CWI pathway independently affect yeast cell integrity.     

烟曲霉pbs2基因在热应激与胞壁应激的双重应激中的作用

目的 通过研究烟曲霉对细胞壁合成干扰物质及棘白菌素类药物的敏感性、烟曲霉在受热应激后HOG通路成分的表达变化探讨pbs2基因在热应激和胞壁应激的双重应激中的作用.方法 烟曲霉野生株AF293和pbs2突变株在含钙荧光白(Calcofluor white,CFW),刚果红(Congo red,CR)和十二烷基硫酸钠(So...     

Microfibril deposition on cultured protoplasts ofVicia hajastana

Summary Cell wall regeneration by protoplasts fromVicia hajastana suspension cultures was investigated with Calcofluor White ST staining and platinum-palladium surface replicas. Microfibril deposition was initiated after 10–20 minutes of culture and within 20 hours protoplasts were covered with a heavy mat of microfibrils. The early stages of microfibril formation could not be detected with Calcofluor staining.Supported by the National Research Council of Canada, Grant A6304.Supported by Deutsche Forschungsgemeinschaft.     

Evidence for an intramembrane component associated with a cellulose microfibril-synthesizing complex in higher plants

Freeze-fracture of rapidly frozen, untreated plant cells reveals terminal complexes on E-fracture faces and intramembrane particle rosettes on P-fracture faces. Terminal complexes and rosettes are associated with the ends of individual microfibril impressions on the plasma membrane. In addition, terminal complexes and rosettes are associated with the impressions of new orientations of microfibrils. These structures are sparse within pit fields where few microfibril impressions are observed, but are abundant over adjacent impressions of microfibrils. It is proposed that intramembrane rosettes function in association with terminal complexes to synthesize microfibrils. The presence of a cellulosic microfibril system in Zea mays root segments is confirmed by degradation experiments with Trichoderma cellulase.     

人类致病菌新型隐球酵母Snf1/AMPK 蛋白激酶调节细胞壁的完整性

摘要：【目的】Snf1/AMPK在真核生物中是重要的且高度保守的一类蛋白激酶。在新型隐球酵母中,SNF1 基因在调节致病因子的生物合成和细胞毒力方面具有重要作用。本文进一步报道了该基因在维持细胞壁完整方面的新功能,这一功能在其他微生物中未见报道。【方法】利用荧光增白剂染料（Calcofluor white dye）染色,荧光显微观察细胞分离、胞壁完整性;利用恒定流速和压力水流冲击菌落,测定细胞黏附琼脂糖表面能力;在含有十二烷基硫酸钠（Sodium dodecyl sulfate,SDS）,刚果红（Congo red）染料和增白剂（Fluorescent Brightener 28）的培养基上观察突变株的生长情况,以验证细胞壁完整性。【结果】SNF1 基因突变菌株对细胞壁抑制剂SDS等敏感,表明细胞壁完整性的损坏;在葡萄糖固体培养基上表现为细胞与琼脂间的黏附力丧失;在热击压力下,该菌株不能正常生长,而这种生长缺陷能够被渗透平衡抑制。【结论】新型隐球酵母SNF1 基因对于维持细胞壁完整性是非常重要的,并且影响细胞与琼脂间黏附作用以及细胞对抗热的能力。     

Identification and deletion of the major secreted protein of Pichia pastoris

A major contaminating host cell protein was identified in fed batch cultures of Pichia pastoris producing an antibody Fab fragment. Purification and peptide sequencing identified this protein to be related to the cysteine-rich secretory protein family. The same protein was also observed as one of the most abundantly secreted proteins in chemostat cultures of a wild type P. pastoris strain. It has an apparent molecular weight of 65 kDa, 2-fold higher than predicted from the amino acid sequence, which is due to high O-glycosylation. It was denominated extracellular protein X 1 (Epx1), as no clear function could be attributed to it. The EPX1 gene is upregulated in different stress situations, and the respective deletion strain was more susceptible than the wild type to the cell wall damaging agents Calcofluor white and Congo red. The EPX1 deletion strain (Δepx1) was evaluated for its suitability for recombinant protein production. No significant difference in growth and product formation was observed between the wild type and the Δepx1 strain. Batch purification of a Fab fragment produced in the Δepx1 strain highlighted its superior purity due to the decreased host cell protein load.     

Mutations in the csgD Promoter Associated with Variations in Curli Expression in Certain Strains of Escherichia coli O157:H7

Single-base-pair csgD promoter mutations in human outbreak Escherichia coli O157:H7 strains ATCC 43894 and ATCC 43895 coincided with differential Congo red dye binding from curli fiber expression. Red phenotype csgD::lacZ promoter fusions had fourfold-greater expression than white promoter fusions. Cloning the red variant csgDEFG operon into white variants induced the red phenotype. Substrate utilization differed between red and white variants.     

The control of cellulose microfibril deposition in the cell wall of higher plants

In maize (Zea mays L.) and pine (Pinus taeda L.) seedlings, cellulose microfibril impressions are present on freeze-fractured plasma membranes. It has been proposed that impressions of newly synthesized microfibrils are a record of the movement of terminal synthesizing complexes through the plasma membrane (Mueller and Brown, 1980, J. Cell Biol. 84, 315–326). The association of terminal complexes with the ends of microfibril impressions or with the ends of microfibrils torn through the membrane indicates the orientation of microfibril tips. Unidirectionally-oriented microfibril tips (all pointing in the same direction) are associated with the organized deposition of parallel arrays of microfibrils. Multidirectionally-oriented microfibril tips were observed in a cell in which microfibril deposition was unusually disorganized. Microfibril patterns around pit fields are asymmetric and resemble flow patterns. Unidirectionally-oriented tears are associated with these microfibrils. Although microfibril orientations are deflected around pit fields, the main axis of microfibril orientation is maintained across the surface of the cell. The hypothesis is proposed that the interaction of a flowing plasma membrane with microfibril synthesizing complexes in the plane of the membrane may result in unidirectional deposition and asymmetric microfibril impressions around pit fields.Some of this work has been published in preliminary form (Brown 1979)     

Understanding the degradation of Congo red and bacterial diversity in an air–cathode microbial fuel cell being evaluated for simultaneous azo dye removal from wastewater and bioelectricity generation

We investigated the mechanism of Congo red degradation and bacterial diversity in a single-chambered microbial fuel cell (MFC) incorporating a microfiltration membrane and air–cathode. The MFC was operated continuously for more than 4 months using a mixture of Congo red and glucose as fuel. We demonstrated that the Congo red azo bonds were reduced at the anode to form aromatic amines. This is consistent with the known mechanism of anaerobic biodegradation of azo dyes. The MFC developed a less dense biofilm at the anode in the presence of Congo red compared to its absence indicating that Congo red degradation negatively affected biofilm formation. Denaturing gradient gel electrophoresis and direct 16S ribosomal DNA gene nucleotide sequencing revealed that the microbial communities differed depending on whether Congo red was present in the MFC. Geobacter-like species known to generate electricity were detected in the presence or absence of Congo red. In contrast, Azospirillum, Methylobacterium, Rhodobacter, Desulfovibrio, Trichococcus, and Bacteroides species were only detected in its presence. These species were most likely responsible for degrading Congo red.     

Arrays of plasma-membrane “rosettes” involved in cellulose microfibril formation of Spirogyra

The cell-wall structure and plasma-membrane particle arrangement during cell wall formation of the filamentous chlorophycean alga Spirogyra sp. was investigated with the freeze-fracture technique. The cell wall consists of a thick outer slime layer and a multilayered inner wall with ribbon-like microfibrils. This inner wall shows three differing orientations of microfibrils: random orientation on its outside, followed by axial bundles of parallel microfibrils, and several internal layers of bands of mostly five to six parallel associated microfibrils with transverse to oblique orientation. The extraplasmatic fracture face of the plasma membrane shows microfibril imprints, relatively few particles, and “terminal complexes” arranged in a hexagonal package at the end of the imprint of a microfibril band. The plasmatic fracture face of the plasma membrane is rich in particles. In places, it reveals hexagonal arrays of “rosettes”. These rosettes are best demonstrable with the double-replica technique. These findings on rosette arrays of the zygnematacean alga Spirogyra are compared in detail with the published data on the desmidiacean algae Micrasterias and Closterium.     

Congo Red Interactions with Curli-Producing E. coli and Native Curli Amyloid Fibers

Microorganisms produce functional amyloids that can be examined and manipulated in vivo and in vitro. Escherichia coli assemble extracellular adhesive amyloid fibers termed curli that mediate adhesion and promote biofilm formation. We have characterized the dye binding properties of the hallmark amyloid dye, Congo red, with curliated E. coli and with isolated curli fibers. Congo red binds to curliated whole cells, does not inhibit growth, and can be used to comparatively quantify whole-cell curliation. Using Surface Plasmon Resonance, we measured the binding and dissociation kinetics of Congo red to curli. Furthermore, we determined that the binding of Congo red to curli is pH-dependent and that histidine residues in the CsgA protein do not influence Congo red binding. Our results on E. coli strain MC4100, the most commonly employed strain for studies of E. coli amyloid biogenesis, provide a starting point from which to compare the influence of Congo red binding in other E. coli strains and amyloid-producing organisms.     

Analysis of Congo red-induced changes in the cell surface and macrocolony structure of the bacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

Adsorption of the vital dye Congo red suppresses swarming of Azospirillum brasilense in a semiliquid medium, and the bacteria become able to spread with the formation of microcolonies. By using direct and stereoscopic light microscopy, the patterns of the front of Azospirillum spreading in a semiliquid medium containing the dye were analyzed. It was found that in a medium with Congo red, small motile colonies were formed among the individual cells, and once formed, they left the boundaries of the swarming front. The microcolonies produced by azospirilla in the presence of the dye were ordered bacterial structures, rather than random cell aggregates. Transmission electron microscopy revealed that the cells grown without the dye had polar flagella, whereas the cells from the medium with Congo red had no flagella and were covered with a layer of fibrillike material. Immunochemical data for the cell surface changes resulting from interaction with the dye make it possible to consider Azospirillum lipopolysaccharide as a probable Congo red receptor.     

In vivo accumulation of self-assembling dye Congo red in an area marked by specific immune complexes: possible relevance to chemotherapy

Supramolecular micellar structures have been proposed as carriers in aim-oriented drug transportation to a target marked by specific immune complexes. In this study, the self-assembling dye Congo red was used as a model supramolecular carrier and its accumulation in the target was studied in vivo. The target was created in vivo as the local specific inflammation provoked by subcutaneous injection of antigen to the ear of a previously immunized rabbit. The color caused by accumulation of Congo red after its intravenous injection was registered by pictures of the ear with suitably filtered visible light shining through it to distinguish Congo red against the background color of hemoglobin. The results confirmed the expected accumulation and retention of Congo red in the inflammation area marked by deposits of specific immune complexes. The role of albumin and its possible interference with transportation of drugs through the blood by supramolecular carriers was also subjected to preliminary examination. The results revealed that albumin collaborates rather than interferes with drug transportation; this is another factor making the use of supramolecular carriers for aim-oriented chemotherapy highly promising.     

Correlation between Congo red binding and contact haemolysin production in Shigella species

Haemolytic strains of Shigella dysenteriae type 1, Shigella flexneri, Shigella boydii and Shigella sonnei cultured on Congo red agar produced pigmented colonies (Pcr+) whereas nonhaemolytic strains produced white colonies and did not bind Congo red (Pcr-). S. flexneri-1 haemolysin negative mutant (lacking plasmid) of haemolysin positive prototroph also did not bind Congo red and produced nonpigmented colonies. Among the twelve strains of Shigella included in this study, the characteristics of Congo red binding, plasmid profile and haemolytic activity appeared to be correlated. Congo red binding occurred comparatively more by haemolysin-producing strains. Congo red binding can be used as a quick and reliable method for virulence traits of pathogens, including haemolysin activity.     

Enhancing Aniline Blue Fluorescent Staining of Cell Wall Structures

Prior staining with the periodic acid-Schiff reaction, toluidine blue O, Congo red, or Calcofluor White M2R New, or reduction by NaBH₄ do not interfere with aniline blue-induced fluorescence of sieve plates, new cell walls, pit fields or tracheids in compression wood of conifers. Detail of such fluorescent structures is improved by these treatments because of increased contrast, reduced flare, and a quenching of autofluorescence.