Volvocine cell walls and their constituent glycoproteins: An evolutionary perspective

Summary Similarities in the composition of the extracellular matrix suggest that only some species of the unicellularChlamydomonas are closely related to the colonial and multicellular flagellated members of the family Volvocaceae. The cell walls from all of the algae in this volvocine group contain a crystalline layer. This lattice structure can be used as a phylogenetic marker to divideChlamydomonas species into distinct classes, only one of which includes the volvocacean algae. Similarly, not all species ofChlamydomonas are sensitive to each other's cell wall lytic enzymes, implying divergence of the enzyme's inner wall substrate. Interspecific reconstitution of the crystalline layer is possible betweenC. reinhardtii and the multicellularVolvox carteri, but not betweenC. reinhardtii andC. eugametos. The hydroxyproline-rich glycoproteins (HRGPs) which make up the crystalline layer in genera which have a similar crystal structure exhibit many homologies. Interestingly, the evolutionarily distant cell walls ofC. reinhardtii andC. eugametos also contain some HRGPs displaying a few morphological and amino acid sequence homologies. The morphological similarities between the flagellar agglutinins (HRGPs responsible for sexual recognition and adhesion during the mating reaction) and the cell wall HRGPs leads to the proposal of a superfamily from which novel HRGPs (designed for self-assembly/recognition) can constantly evolve. Just as variations in the wall HRGPs can lead to unique wall structures, new agglutinins facilitate sexual isolation of new species. Thus, the HRGPs could emerge as valuable phylogenetic markers.Abbreviations GLE gametic lytic enzyme - GP glycoprotein - HRGP hydroxyproline-rich glycoprotein - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - VLE vegetative lytic enzyme - VSP vegetative serine/proline-rich - WP wall protein - ZSP zygotic serine/proline-rich     

Electrophoretic and immunological comparisons of chloroplast and prokaryotic ribosomal proteins reveal that certain families of large subunit proteins are evolutionarily conserved

Summary Antibodies to individual chloroplast ribosomal (r-)proteins ofChlamydomonas reinhardtii synthesized in either the chloroplast or the cytoplasm were used to examine the relatedness ofChlamydomonas r-proteins to r-proteins from the spinach (Spinacia oleracea) chloroplast,Escherichia coli, and the cyanobacteriumAnabaena 7120. In addition,³⁵S-labeled chloroplast r-proteins from large and small subunits ofC. reinhardtii were coelectrophoresed on 2-D gels with unlabeled r-proteins from similar subunits of spinach chloroplasts,E. coli, andAnabaena to compare their size and net charge. Comigrating protein pairs were not always immunologically related, whereas immunologically related r-protein pairs often did not comigrate but differed only slightly in charge and molecular weight. In constrast, when³⁵S-labeled chloroplast r-proteins from large and small subunits of a closely related speciesC. smithii were coelectrophoresed with unlabeledC. reinhardtii chloroplast r-proteins, only one pair of proteins from each subunit showed a net displacement in mobility.Analysis of immunoblots of one-dimensional SDS and two-dimensional urea/SDS gels of large and small subunit r-proteins from these species revealed more antigenic conservation among the four species of large subunit r-proteins than small subunit r-proteins.Anabaena r-proteins showed the greatest immunological similarity toC. reinhardtii chloroplast r-proteins. In general, antisera made against chloroplast-synthesized r-proteins inC. reinhardtii showed much higher levels of cross-reactivity with r-proteins fromAnabaena, spinach, andE. coli than did antisera to cytoplasmically synthesized r-proteins. All spinach r-proteins that cross-reacted with antisera to chloroplast-synthesized r-proteins ofC. reinhardtii are known to be made in the chloroplast (Dorne et al. 1984b). FourE. coli r-proteins encoded by the S10 operon (L2, S3, L16, and L23) were found to be conserved immunologically among the four species. Two of the large subunit r-proteins, L2 and L16, are essential for peptidyltransferase activity. The third (L23) and two otherE. coli large subunit r-proteins (L5 and L27) that have immunological equivalents among the four species are functionally related to but not essential for peptidyltransferase activity.     

On the cell wall composition of the apiculate yeasts

Summary Sonic oscillation was used for the purpose of obtaining clean, chemically intact cell walls. The rate of disruption was determined for cells ofHanseniaspora uvarum andSaccharomyces cerevisiae. The carbohydrate fractions of cell walls ofHanseniaspora uvarum, H. valbyensis, Kloeckera apiculata, Saccharomycodes ludwigii andSaccharmyces cerevisiae were shown to be similar. Chromatography of cell wall hydrolysates of all these species demonstrated that glucose and mannose were the only sugars present (in about equal amounts) besides traces of glucosamine. The cell walls ofH. uvarum contained 78.1 per cent carbohydrates, 7 per cent protein and approximately 0.05 per cent of chitin. Fractionation of the polysaccharides lead to a recovery of 83.3 per cent of the carbohydrates present (30.4 per cent glucan and 34.9 per cent mannan). Saccharomyces cerevisiae cell walls were found to have a carbohydrate content of 82.8 per cent, 6.5 per cent protein and a trace of chitin (0.04 per cent). Nadsonia elongata contained a relatively large amount of chitin (ca. 5 per cent) and lacked mannan in its cell walls. It was concluded thatHanseniaspora andSaccharomycodes are closely related to theSaccharomyceteae but they have little in common with species ofNadsonia.     

Mating type specific induction of cell wall lytic factor by agglutination of gametes in Chlamydomonas reinhardtii

When mt⁺ and mt^– gametes of Chlamydomonas reinhardtiiwere mixed, shedding of cell walls took place in both matingtypes during massive agglutination and/or pairing. This wascaused by a cell wall lytic factor that had been induced byflagellar agglutination and excreted into the medium by cellsconcurrently with their cell wall release. When glutaraldehyde-fixed gametes and isolated flagella of onemating type caused isoagglutination of live gametes of the othermating type, the live mt⁺ gametes induced the lytic factor andshed their walls, whereas none of the live mt^– did this.The cell walls of mt^– gametes were lost only when thelytic factor, which had been excreted by mt⁺ gametes into themedium, acted from the outside. These data imply that mt⁺ gametesare responsible for the induction of the lytic factor by agglutination,which acts on cell walls of both mating types either endogenouslyor exogenously. (Received February 28, 1978; )     

Cell wall formation in yeast

Summary Incorporation of tritiated glucose into cell walls of growingSaccharomyces cerevisiac andSchizosaccharomyces pombe was studied using electron microscopic autoradiography. The pattern and the extent of labelling ofS. cerevisiae cell walls depended on the cell stage in the cell cycle. Quantitative evaluation of autoradiographs showed that the highest rate of wall synthesis took place during bud growth. The incorporation of new material into the wall of growing bud showed an increasing rate with the magnitude of the bud. The incorporation into the mother cell wall was almost negligible during bud growth. The rate of wall synthesis in double cells decreased during cell division. This period and that before new bud initiation was found to be the time of substantially reduced rate of wall replication inS. cerevisiae. A significant random incorporation was observed into the walls of post-division adult cells, both parental and daughter. The cell walls ofS. pombe were labelled almost exclusively at growing tips. The incorporation of tritiated carbohydrates into non-extensile regions ofS. pombe cell walls was found to be only about 5% of the total wall labelling.     

Domain conservation in several volvocalean cell wall proteins

Based on our previous work demonstrating that (SerPro)_x epitopes are common to extensin-like cell wall proteins in Chlamydomonas reinhardtii, we looked for similar proteins in the distantly related species C. eugametos. Using a polyclonal antiserum against a (SerPro)₁₀ oligopeptide, we found distinct sets of stage-specific polypeptides immunoprecipitated from in vitro translations of C. eugametos RNA. Screening of a C. eugametos cDNA expression library with the antiserum led to the isolation of a cDNA (WP6) encoding a (SerPro)_x-rich multidomain wall protein. Analysis of a similarly selected cDNA (VSP-3) from a C. reinhardtii cDNA expression library revealed that it also coded for a (SerPro)_x-rich multidomain wall protein. The C-terminal rod domains of VSP-3 and WP6 are highly homologous, while the N-terminal domains are dissimilar; however, the N-terminal domain of VSP-3 is homologous to the globular domain of a cell wall protein from Volvox carteri. Exon shuffling might be responsible for this example of domain conservation over 350 million years of volvocalean cell wall protein evolution.     

Evolution of fragmented mitochondrial ribosomal RNA genes inChlamydomonas

The fragmented mitochondrial ribosomal RNAs (rRNAs) of the green algaeChlamydomonas eugametos andChlamydomonas reinhardtii are discontinuously encoded in subgenic modules that are scrambled in order and interspersed with protein coding and tRNA genes. The mitochondrial rRNA genes of these two algae differ, however, in both the distribution and organization of rRNA coding information within their respective genomes. The objectives of this study were (1) to examine the phylogenetic relationships between the mitochondrial rRNA gene sequences ofC. eugametos andC. reinhardtii and those of the conventional mitochondrial rRNA genes of the green alga,Prototheca wickerhamii, and land plants and (2) to attempt to deduce the evolutionary pathways that gave rise to the unusual mitochondrial rRNA gene structures in the genusChlamydomonas. Although phylogenetic analysis revealed an affiliation between the mitochondrial rRNA gene sequences of the twoChlamydomonas taxa to the exclusion of all other mitochondrial rRNA gene sequences tested, no specific affiliation was noted between theChlamydomonas sequences andP. wickerhamii or land plants. Calculations of the minimal number of transpositions required to convert hypothetical ancestral rRNA gene organizations to the arrangements observed forC. eugametos andC. reinhardtii mitochondrial rRNA genes, as well as a limited survey of the size of mitochondrial rRNAs in other members of the genus, lead us to propose that the last common ancestor ofChlamydomonas algae contained fragmented mitochondrial rRNA genes that were nearly co-linear with conventional rRNA genes.     

cDNA and deduced amino acid sequences of cytochrome c fromChlamydomonas reinhardtii: Unexpected functional and phylogenetic implications

Summary We have isolated complementary DNA (cDNA) clones for apocytochrome c from the green algaChlamydomonas reinhardtii and shown that they are encoded by a single nuclear gene termedcyc.Cyc mRNA levels are found to depend primarily on the presence of acetate as a reduced carbon source in the culture medium. The deduced amino acid sequence shows that, apart from the probable removal of the initiating methionine,C. reinhardtii apocytochrome c is syntheszed in its mature form. Its structure is generally similar to that of cytochromes c from higher plants. Several punctual deviations from the general pattern of cytochrome c sequences that is found in other organisms have interesting structural and functional implications. These include, in particular, valines 19 and 39, asparagine 78, and alanine 83. A phylogenetic tree was constructed by the matrix method from cytochrome c data for a representative range of species. The results suggest thatC. reinhardtii diverged from higher plants approximately 700–750 million years ago; they also are not easy to reconcile with the current attribution ofChlamydomonas reinhardtii andEnteromorpha intestinalis to a unique phylum, because these two species probably diverged from one another at about the same time as they diverged from the line leading to higher plants.     

Surface structures of peridial cells ofGymnosporangium andRoestelia (Uredinales)

The surface structures of peridial cell walls (outer, side, and inner walls) of 40Gymnosporangium and 7Roestelia species were examined by scanning electron microscopy. These surface structures were classified into 10 types based on shape, size, and density of the processes. Surface structural types of each wall were stable within a species. Therefore, it is suggested that surface structure types of peridial cell walls could be used as important diagnostic characteristics ofGymnosporangium andRoestelia species. Contribution No. 140, Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Potential of genes and gene products fromTrichoderma sp. andGliocladium sp. for the development of biological pesticides

Fungal cell wall degrading enzymes produced by the biocontrol fungiTrichoderma harzianum andGliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes fromT. harzianum were able to improve substantially the antifungal ability of a biocontrol strain ofEnterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library ofT. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.     

The cell wall of the chlamydomonad flagellate,Gloeomonas kupfferi (Volvocales,Chlorophyta)

Summary The large unicellular flagellate,Gloeomonas kupfferi, has recently been used as an important tool in chlamydomonad cell biology research, especially in studies dealing with the structure and function of the endomembrane system. However, little is known about the main secretory product, the cell wall. This study presents structural, chemical and immunological information about this wall. This 850–900 nm thick matrix is highly elaborate and consists of three distinct layers: an inner stratum (325 nm thick) consisting of tightly interwoven fibers, a medial crystalline layer consisting of 22–23 nm subunits and an outer wall layer (500 nm thick) of outwardlyradiating fibrils. Rapid freeze-deep etch analysis reveals that the 35–40 nm fibers of the outer layer form a quasi-lattice of 160 nm subunits. The outer wall can be removed from whole pellets using the chelator, CDTA. The medial wall complex can be solubilized by perchlorate. SDS-gel electrophoresis reveals that the perchlorate soluble-material consists of five high molecular weight glycoproteins and five major low molecular weight glycoproteins. The electrophoretic profile is roughly similar to that ofChlamydomonas reinhardtii. Antibodies were successfully raised against the outer wall component and were shown to label the outer wall layer.     

Purification and Characterization of a Vegetative Lytic Enzyme Responsible for Liberation of Daughter Cells during the Proliferation ofChlamydomonas reinhardtii

A vegetative lytic enzyme (VLE) of Chlamydomonas reinhardtiimediates digestion of the cell walls of mother cells (sporangia)to allow release of daughter cells after mitotic cell divisionin the vegetative cell cycle. This enzyme is secreted into theculture medium concurrently with the appearance of daughtercells in synchronized cultures. Using an assay that monitorsdigestion of the mother cell wall, we purified VLE by ion-exchangeand gel-filtration chromatography from the medium of synchronizedcultures. The purified enzyme was a basic glycoprotein withan apparent molecular mass of 120 kDa on gel filtration and130 kDa on SDS-PAGE. Thus, VLE appeared to behave as a monomer.The enzyme acted specifically on the mother cell wall and wasunable to digest the cell walls derived from single vegetativecells. The enzymatic activity was inhibited by PMSF, p-APMSF,TLCK, HgCl₂, iodoacetate, EGTA, EDTA and 1,10-phenanthroline.VLE cleaved several synthetic model peptides on the carboxylside of a Lys or Arg residue, indicating that it is a proteasethat acts on protein in the mother cell wall in vivo to releasethe daughter cells. (Received November 30, 1994; Accepted March 22, 1995)     

Chemical and ultrastructural studies on the cell wall ofStaphylococcus simulans biovarstaphylolyticus

Cell walls of strains ofStaphylococcus simulans biovarstaphylolyticus andS. aureus FDA 209P were compared ultrastructurally and chemically to investigate the mechanism of resistance of this strain ofS. simulans to its own staphylolytic endopeptidase. Chemical analysis of the peptidoglycans of the various strains examined showed that cells that were more resistant to lysis by the endopeptidase had lower glycine/serine ratios in their cross bridges and that, within a species, the more resistant cells had either fewer residues in these cross bridges or fewer cross bridges. Ultrastructural studies showed that cell wall thickness was not involved in resistance to the enzyme. Comparisons of the endopeptidase susceptibility of intact cells and isolated peptidoglycans from these cells suggested that the three-dimensional structure of the cell wall may play a role in resistance to lysis by the endopeptidase.     

Zero order kinetics of cell wall turnover inStaphylococcus aureus

Cells exponentially grown from four strains ofS. aureus (SG 511, H, 52A5G, and248 PN-1) and uniformly labeled in their walls with³H-N-acetylglucosamine, were found to turn over their old walls at constant rates of up to 25% per generation. Wall turnover was not observed to follow first order kinetics, thus ruling out the implication that maintenance of normal wall thickness was achieved by a random distribution of new wall components in the old wall. Instead, wall turnover in all cases strictly followed zero order kinetics, indicating that newly synthesized wall material was placed layer by layer beneath the inner surface of the old cell wall. This finding correlates with evidence obtained from earlier electron microscopic investigations into the regeneration of the staphylococcal cell wall after chloramphenicol treatment. Based on the experimental data presented, a simplified model for wall turnover of the growing staphylococcal cell was proposed. The model also takes into account the finding, derived from additional experiments with strainSG 511, that the total cell wall turned over at a somewhat higher rate than the old portions of the wall. The rates of cell wall turnover found inS. aureus SG 511 are the highest reported to date for pathogenic bacteria. The medical implications of this finding were discussed.     

Cell wall lytic enzyme released by mating gametes of Chlamydomonas reinhardtii is a metalloprotease and digests the sodium perchlorate-insoluble component of cell wall

Chlamydomonas lytic enzyme of the cell wall, which is released during agglutination of gametes of opposite mating types, has been characterized as a metalloprotease. The purified enzyme contains zinc. Removal of zinc with EDTA results in an inactive, metal-free apoenzyme, and Co2+ restores the activity most effectively. Among various protease inhibitors of microbial origin, pepstatin A, chymostatin, antipain, leupeptin, and E-64 do not inactivate the enzyme, whereas phosphoramidon causes a complete loss of lytic activity. Cysteine, histidine, aspartic acid, and glutamic acid also inhibit the activity. The lytic enzyme splits casein and RNase A into several polypeptides of lower molecular masses. To determine which polypeptides of the cell wall are sensitive to the lytic enzyme, we first separated the intact cell walls into sodium perchlorate-soluble and -insoluble components, treated them with enzyme, and then analyzed them by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. We conclude that only 2 of 16 polypeptides are digested by exposure to the enzyme and that the sensitive polypeptides belong to the salt-insoluble component of the cell wall. The mechanism of cell wall digestion with the lytic enzyme is discussed.     

Substrate specificity of thePenicillium lilacinum enzyme lytic to the cell wall ofRhodotorula glutinis and the structure of theRhodotorula cell wall glucomannan

The lytic enzyme active on cell walls ofRhodotorula, which was produced byPenicillium lilacinum ATCC 36010, decomposed an extracellular mannan fromRhodotorula glutinis IFO 0695 and was confined to the β-1,3-mannoside bond on the reducing side of the β-1,4-linkedd-mannopyranose. Based on these results, the structure of the glucomannan, which was a major component ofRhodotorula cell walls, was proposed.     

Some observations on cell wall structure and taxonomy ofPhymatodocis nordstedtiana (Conjugatophyceae,Chlorophyta)

Filaments ofPhymatodocis nordstedtiana Wolle were isolated from a sample of a Texan lake. Cultures were established and examined by light and scanning electron (SEM) as well as transmission electron microscopy (TEM). It is shown that the pores apparent on light microscopical examination are not of the cosmaroid type as expected. TEM examination disclosed that they are similar to those found in the generaClosterium Ralfs andPenium Bréb. Furthermore, it could be demonstrated by light and SEM microscopy that the primary cell wall is shed during cell division. The remaining secondary cell wall of the mature cell consists of interwoven bands of parallel microfibrils. A conspicuous overlap of the semicell walls clearly denotes the isthmus region. The significance of these deviations unusual for desmids is discussed. Suggestions are made that the taxonomic position ofPh. nordstedtiana should be re-evaluated.     

The Isolation and Properties of the Lytic Enzyme of the Cell Wall Released by Mating Gametes of Chlamydomonas reinhardtii

The lytic enzyme of the cell wall, which is excreted into theculture medium by mating gametes of Chlamydomonas reinhardtii,was characterized through its purification, and a convenientquantitative test for activity which uses glutaraldehyde-fixedzoosporangia was devised. The enzyme was stabilized at a highsalt concentration (200 mM NaCl) then purified by gel filtrationand sucrose density gradient centrifugation. The results showthat the lytic enzyme is a molecule(s) of about 130,000 daltons. (Received August 28, 1980; Accepted December 18, 1980)     

Cell wall degradation ofStaphylococcus aureus by lysozyme

In contrast to former findings lysozyme was able to attack the cell walls ofStaphylococcus aureus under acid conditions. However, experiments with¹⁴C-labelled cell walls and ribonuclease indicated that, under these conditions, lysozyme acted less as an muralytic enzyme but more as an activator of pre-existing autolytic wall enzymes. Electron microscopic studies showed that under these acid conditions the cell walls were degraded by a new mechanism (i.e. attack from the inside). This attack on the cell wall started asymmetrically within the region of the cross wall and induced the formation of periodically arranged lytic sites between the cytoplasmic membrane and the cell wall proper. Subsequently, a gap between the cell wall and the cytoplasmic membrane resulted and large cell wall segments became detached and suspended in the medium. The sequence of lytic events corresponded to processes known to take place during wall regeneration and wall formation. In the final stage of lysozyme action at pH 5 no cell debris but stabilized protoplasts were to be seen without detectable alterations of the primary shape of the cells. At the same time long extended ribbon-like structures appeared outside the bacteria. The origin as well as the chemical nature of this material is discussed. Furthermore, immunological implications are considered.     

Biological Control ofMonilinia laxaandFusarium oxysporumf. sp.lycopersiciby a Lytic Enzyme-ProducingPenicillium purpurogenum

Biological control experiments were conducted with the lytic enzyme-producing fungusPenicillium purpurogenumagainst the plant pathogensMonilinia laxaandFusarium oxysporumf. sp.lycopersici.Applications ofP. purpurogenumto peach shoots previously inoculated withM. laxareduced lesion length and extent of pathogen colonization of shoots by 90 and 80% (P ≤ 0.05), respectively, comparable to the level of disease control obtained with the fungicide captan. Disease severity in tomato plants inoculated withF. oxysporumf. sp.lycopersiciwas decreased by 30% (P ≤ 0.05) with the biological treatment. The fungusP. purpurogenumproduced β-1,3-glucanase and chitinase activities in liquid culture that were inducible by cell walls and live mycelium ofM. laxabut not ofF. oxysporumf. sp.lycopersici.Crude filtrates or crude enzyme preparations ofP. purpurogenumcultures with lytic enzyme activities produced lysis of hyphae and spores ofM. laxaandF. oxysporumf. sp.lycopersici.These lytic effects were strong inM. laxaand ended in complete dissolution of mycelium. The induction of lytic enzymes byM. laxaand the effects of lytic enzymes on mycelia of the pathogens in relation to the different degrees of biological control obtained are discussed.