Rumen bacterial degradation of forage cell walls investigated by electron microscopy

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues.     

Mode of Attack on Orchardgrass Leaf Blades by Rumen Protozoa

Leaf blade sections of orchardgrass were incubated with rumen fluid and examined by scanning and transmission electron microscopy for the mode of attack on tissues by rumen protozoa. Rumen protozoa resembling Epidinium ecaudatum from caudatum degraded forage tissue in diluted, whole rumen fluid suspensions of microbes containing 1.6 mg of streptomycin per ml, which inhibited bacterial fiber-digesting activity. Cell walls of mesophyll, parenchyma bundle sheath, and epidermis became swollen and frayed to reveal a microfibrillar network and loss of electron density, indicating partial degradation. Then the protozoa ingested whole cells and fragments of cell walls with the aid of their cilia. Plant cells with partially degraded walls as well as chloroplasts without walls were present within the protozoa. These entodiniomorphs digested orchardgrass leaves by partially degrading the plant cell walls apparently by extracellular enzymes and then ingestion of the plant cells and cell wall fragments.     

Ultrastructure of rumen bacterial attachment to forage cell walls.

The degradation of forage cell walls by rumen bacteria was investigated with critical-point drying/scanning electron microscopy and ruthenium red staining/transmission electron microscopy. Differences were observed in the manner of attachment of different morphological types of rumen bacteria to plant cell walls during degradation. Cocci, constituting about 22% of the attached bacteria, appeared to be attached to degraded plant walls via capsule-like substances averaging 58 nm in width (range, 21 to 84 nm). Many bacilli appeared to adhere to forage substrates without distinct capsule-like material, although unattached bacteria with capsules were observed occasionally. Certain bacili appeared to be attached to degraded tissue via small amounts of extracellular material, but others apparently had no extracellular material. Bacilli with a distinct morphology due to an irregularly folded, electron-dense outer layer or layers (about 15 nm thick) and without fibrous extracellular material consituted about 37% of the attached bacteria and were observed to adhere so closely to degraded plant walls that the bacterial shape conformed to the shape of the degraded zone. In the rumen ecosystem, bacteria appeared to adhere to plant substrates during degradation by capsule-like material and by small amounts of extracellular material, as well as by the other means not observable by electron microscopy.     

Degradation of Bermuda and Orchard Grass by Species of Ruminal Bacteria

Fiber degradation in Bermuda grass and orchard grass was evaluated gravimetrically and by scanning and transmission electron microscopy after incubation with pure cultures of rumen bacteria. Lachnospira multiparus D-32 was unable to degrade plant cell wall components. Butyrivibrio fibrisolvens 49 degraded 6 and 14.9% of the fiber components in Bermuda grass and orchard grass, respectively, and Ruminococcus albus 7 degraded 11.4% orchard grass fiber but none in Bermuda grass. Both B. fibrisolvens and R. albus lacked capsules, did not adhere to fiber, and degraded only portions of the more easily available plant cell walls. R. flavefaciens FD-1 was the most active fiber digester, degrading 8.2 and 55.3% of Bermuda and orchard grass fiber, respectively. The microbe had a distinct capsule and adhered to fiber, especially that which is slowly degraded, but was able to cause erosion and disorganization of the more easily digested cell walls, apparently by extracellular enzymes. Results indicated that more digestible cell walls could be partially degraded by enzymes disassociated from cellulolytic and noncellulolytic bacteria, and data were consistent with the hypothesis that the more slowly degraded plant walls required attachment. Microbial species as well as the cell wall architecture influenced the physical association with and digestion of plant fiber.     

An electron microscope study of wheat straw composted as a substrate for the cultivation of the edible mushroom (Agaricus bisporus)

The degradation of wheat straw, during composting, to produce the growth substrate for the edible mushroom ( Agaricus bisporus ), and subsequent colonisation by the fungal hyphae, was studied by electron microscopy which revealed an ecological succession of micro-organisms, initially dominated by a largely bacterial flora with few fungi. Later in the composting process actinomycetes were dominant. The initial rise in numbers of vegetative bacterial cells was followed by a steady decline and the appearance of spore forms. Several modes of microbial attack were observed. The most rapid degradation occurred initially on the cuticle and in the phloem and spread to a general degradation of all the plant tissue types present. Microbial attachment on the plant cell walls was non-uniform. As a result of these processes many of the plant fibres became separated but the final material still retained considerable structural integrity. Agaricus bisporus mycelium rapidly covered the surface of the straw but colonised the internal straw tissues more slowly. Surface-growing hyphal cells were encrusted with needle-like crystals presumed to be calcium oxalate.     

Purification and partial characterization of a glycoprotein from pea (Pisum sativum) with receptor activity for rhicadhesin,an attachment protein of Rhizobiaceae

Attachment of Rhizobium and Agrobacterium bacteria to cells of their host plants is a two-step process. The first step, direct attachment of bacteria to the plant cell wall, is mediated by the bacterial protein rhicadhesin. A putative plant receptor molecule for rhicadhesin was purified from cell walls of pea roots using a bioassay based on suppression of rhicadhesin activity. This molecule appeared to be sensitive to treatments with pronase or glycosidase. Its isoelectric point is 6.4, and its apparent molecular mass was estimated to be 32 kDa before and 29 kDa after glycosidase treatment, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ultrafiltration. The sequence of the first 29 N-terminal amino acids was determined: A-D-A-D-A-L-Q-D-L-C(?)-V-A-D-Y-A-S-V-I-L-V-N-G-F-A-S-K(Q)-(P/Q)-(L)-(I). No homology with known proteins was found. In the course of this research project the extracellular matrix protein vitronectin was reported to inhibit attachment of A. tumefaciens to carrot cells [29]. A variety of adhesive proteins, including vitronectin, contain a common cell attachment determinant with the sequence R-G-D. Since we could not detect other cell wall components able to suppress rhicadhesin activity, and since an R-G-D containing hexapeptide was also active as a receptor, we speculate that the plant receptor for rhicadhesin is a glycoprotein containing an R-G-D attachment site.     

Pectin and Xyloglucan Influence the Attachment of Salmonella enterica and Listeria monocytogenes to Bacterial Cellulose-Derived Plant Cell Wall Models

Minimally processed fresh produce has been implicated as a major source of foodborne microbial pathogens globally. These pathogens must attach to the produce in order to be transmitted. Cut surfaces of produce that expose cell walls are particularly vulnerable. Little is known about the roles that different structural components (cellulose, pectin, and xyloglucan) of plant cell walls play in the attachment of foodborne bacterial pathogens. Using bacterial cellulose-derived plant cell wall models, we showed that the presence of pectin alone or xyloglucan alone affected the attachment of three Salmonella enterica strains (Salmonella enterica subsp. enterica serovar Enteritidis ATCC 13076, Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028, and Salmonella enterica subsp. indica M4) and Listeria monocytogenes ATCC 7644. In addition, we showed that this effect was modulated in the presence of both polysaccharides. Assays using pairwise combinations of S. Typhimurium ATCC 14028 and L. monocytogenes ATCC 7644 showed that bacterial attachment to all plant cell wall models was dependent on the characteristics of the individual bacterial strains and was not directly proportional to the initial concentration of the bacterial inoculum. This work showed that bacterial attachment was not determined directly by the plant cell wall model or bacterial physicochemical properties. We suggest that attachment of the Salmonella strains may be influenced by the effects of these polysaccharides on physical and structural properties of the plant cell wall model. Our findings improve the understanding of how Salmonella enterica and Listeria monocytogenes attach to plant cell walls, which may facilitate the development of better ways to prevent the attachment of these pathogens to such surfaces.     

Attachment of phenoloxidase to fungal cell walls in arthropod immunity

In crayfish, phenoloxidase was located in the hemocytes. The plasma had infinitesimal enzyme activity. A phenoloxidase preparation from hemocytes precipitated spontaneously after approximately 1.5 hr at 22°C, which became attached spontaneously to glass, Plexiglas, and polystyrene plastic. The enzyme preparation could also become attached to Saccharomyces cerevisiae cell walls. Attachment was mediated by a proteinaceous substance, since trypsin significantly decreased the degree of attachment. Calcium ions were also necessary for attachment. A β-1,3-glucan, laminaran, partially prevented attachment to the fungal cell walls. Heparin caused precipitation of the phenoloxidase preparation from hemocytes. In crayfish cuticle, proteins with associated phenoloxidase activity were attached to cell walls of Aphanomyces astaci as well as to those of S. cerevisiae.     

Intra- and Extracellular Localization of Lignin Peroxidase during the Degradation of Solid Wood and Wood Fragments by Phanerochaete chrysosporium by Using Transmission Electron Microscopy and Immuno-Gold Labeling

The distribution of lignin peroxidase during degradation of both wood and woody fragments by the white rot fungus Phanerochaete chrysosporium was investigated by using anti-lignin peroxidase in conjunction with postembedding transmission electron microscopy and immuno-gold labeling techniques. The enzyme was localized in the peripheral regions of the fungal cell cytoplasm in association with the cell membrane, fungal cell wall, and extracellular slime materials. In solid wood, lignin peroxidase was detected in low concentrations associated with both superficial and degradation zones within secondary cell walls undergoing fungal attack. A similar but much greater level of extracellular peroxidase activity was associated with wood fragments degraded by the fungus grown under liquid culture conditions optimal for production of the enzyme. Efforts to infiltrate degraded wood pieces with high levels of lignin peroxidase showed the enzyme to be restricted to superficial regions of wood decay and to penetrate wood cell walls only where the wall structure had been modified. In this respect the enzyme was able to penetrate characteristic zones of degradation within the secondary walls of fibers to sites of lignin attack. This suggests a possibility for a close substrate-enzyme association during wood cell wall degradation.     

Caenorhabditis elegans BAH-1 Is a DUF23 Protein Expressed in Seam Cells and Required for Microbial Biofilm Binding to the Cuticle

The cuticle of Caenorhabditis elegans, a complex, multi-layered extracellular matrix, is a major interface between the animal and its environment. Biofilms produced by the bacterial genus Yersinia attach to the cuticle of the worm, providing an assay for surface characteristics. A C. elegans gene required for biofilm attachment, bah-1, encodes a protein containing the domain of unknown function DUF23. The DUF23 domain is found in 61 predicted proteins in C. elegans, which can be divided into three distinct phylogenetic clades. bah-1 is expressed in seam cells, which are among the hypodermal cells that synthesize the cuticle, and is regulated by a TGF-β signaling pathway.     

Ruminococcus flavefaciens Cell Coat and Adhesion to Cotton Cellulose and to Cell Walls in Leaves of Perennial Ryegrass (Lolium perenne)

Ruminococcus flavefaciens was shown to possess a prominent glycoprotein coat, which contained rhamnose, glucose, and galactose as its principal carbohydrates. Periodate-reactive carbohydrate occurred as a surface layer of the coat. The ruminococci adhered strongly by means of this coat to cotton cellulose and to cell walls in leaf sections of Lolium perenne L. (perennial ryegrass). The coat was diffuse at the point of contact so that the bacterial cell wall was in close contact with the substrate. Adhesion was influenced by the availability of damaged plant cell walls and by the cell wall type and occurred most rapidly to cell walls of the epidermis and sclerenchyma, followed by the phloem and mesophyll. Plaques of bacteria with filamentous coat extensions developed on all these tissues. The bacteria did not readily adhere to the walls of the bundle sheath cells or metaxylem or protoxylem vessels and did not adhere to the cuticle or chloroplasts. The epidermal and phloem cell walls were more rapidly digested than the walls of other cell types.     

Immunocytochemical localization of laccase L1 in wood decayed by Rigidoporus lignosus.

The cellular distribution of laccase L1 during degradation of wood chips by Rigidoporus lignosus, a tropical white rot fungus, was investigated by using anti-laccase L1 polyclonal antisera in conjunction with immunolabeling techniques. The enzyme was localized in the fungal cytoplasm and was associated with the plasmalemma and the fungal cell wall. An extracellular sheath, often observed around fungal cells, often contained laccase molecules. Diffusion of laccase within apparently unaltered wood was seldom observed. The enzyme penetrated all degraded cell walls, from the secondary wall toward the primary wall, including the middle lamella. Xylem cells showing advanced stages of decay were sometimes devoid of significant labeling. These data suggest that the initial attack on wood was not performed by laccase L1 of R. lignosus. Previous alteration of the lignocellulose complex may facilitate the movement of laccase within the wood cell walls. This immunogold study revealed that laccase localization during wood degradation seems limited not in space but in time.     

Immunocytochemical localization of laccase L1 in wood decayed by Rigidoporus lignosus.

The cellular distribution of laccase L1 during degradation of wood chips by Rigidoporus lignosus, a tropical white rot fungus, was investigated by using anti-laccase L1 polyclonal antisera in conjunction with immunolabeling techniques. The enzyme was localized in the fungal cytoplasm and was associated with the plasmalemma and the fungal cell wall. An extracellular sheath, often observed around fungal cells, often contained laccase molecules. Diffusion of laccase within apparently unaltered wood was seldom observed. The enzyme penetrated all degraded cell walls, from the secondary wall toward the primary wall, including the middle lamella. Xylem cells showing advanced stages of decay were sometimes devoid of significant labeling. These data suggest that the initial attack on wood was not performed by laccase L1 of R. lignosus. Previous alteration of the lignocellulose complex may facilitate the movement of laccase within the wood cell walls. This immunogold study revealed that laccase localization during wood degradation seems limited not in space but in time.     

Multiplication of Pseudomonas syringae pv. phaseolicola"in planta"

In the compatible combination of the halo blight disease of bean Pseudomonas phaseolicola was able to colonize large areas of the intercellular space of leaves, such that these confluent water congested areas became visible as water-soaked spots. Most of the plant cell walls in the infected region maintained their normal shape, even when the cytoplasm had collapsed. Some inward bending of plant cell walls preceded their rather slow degradation and final replacement by bacterial masses. Neighbouring plant cells appeared to be metabolically active. In resistant leaves no indications of active bacterial attachment or encapsulation could be observed. However, bacteria appeared to be more densely packed in resistant leaves, and relatively more plant cells completely collapsed as compared with susceptible leaves. From 8—14 days after inoculation, the bacterial concentration did not change much in susceptible or resistant leaves, indicating the absence of bactericidal components. Even Pseudomonas pisi snowed some multiplication in bean leaves (immune reaction), but its growth stopped earlier than that of P. phaseolicola. in the resistant cultivars, probably due to a different mechanism of resistance. Although less bacteria were determined in the intercellular washing fluid (IF) compared with leaf homogenates, the high bacterial concentrations in the IF supported our observation that an effective encapsulation of bacteria in resistant leaves did not occur.     

Adherence of Agrobacterium tumefaciens to the cells of immature wheat embryos

Agrobacterium attached to wheat embryos in vitro. This attachment was plasmid independent, and occurred on both wounded and unwounded cell surfaces. The pattern of attachment clearly demonstrated that bacterial attachment to cereal cells follows the same trends observed for dicotyledonous plants. During the inoculation period the bacterial cells attach to the plant cell walls either with lateral or polar orientation. Wounding (mechanical or enzymatic) preferentially promoted adherence of the bacteria at the wound site, however, attachment was not wound dependent.     

Effects of epiphyton onPotamogeton crispus L. leaves

Potamogeton crispus L. grows as a winter producing annual in the shallow lakes of the Pongolo Floodplain, South Africa. Colonization of leaves by algal and bacterial epiphytes, as seen by scanning electron microscopy, followed the established pattern of increasing diversity and density with leaf age. It was apparent from the micrographs that the primary and subsequent colonizers were present even after death of the host leaf. Cross sections of leaves, viewed by transmission electron microscopy, illustrated that bacterial attachment did not damage the surface of young leaves. There was, however, extensive inward swelling and disorganization of the epidermal walls, characteristic of a reaction to invasion by pathogens. In older leaves the swelling was also present in mesophyll cells, while bacteria had invaded and degraded the epidermal cell wall. The bacterial invasion was concomitant with signs of senescence, and in dead leaves the organisms had penetrated and degraded the epidermis and mesophyll cell walls. The epiphyton/ host relationship may therefore be considered necrotrophic with important consequences for the transfer of energy from producers to consumers during decomposition.     

Interaction of Pseudomonas solanacearum with Suspension-Cultured Tobacco Cells and Tobacco Leaf Cell Walls In Vitro

Attachment of radiolabeled Pseudomonas solanacearum cells to suspension-cultured tobacco cells and tobacco leaf cell walls was measured in vitro by a filtration technique that allowed separation of attached and unattached bacteria. An avirulent strain (B1) attached more rapidly to suspension-cultured cells than did the virulent parent strain (K60), and B1 attachment was less sensitive to inhibition by high ionic strength than was K60. Attachment of B1 bacteria to suspension-cultured cells and to leaf cell walls was comparable (50 to 70%), but only a small proportion (10 to 20%) of K60 bacteria attached to leaf cell walls under optimal conditions. With high bacterial populations (10⁸ bacteria per ml), attachment of K60 to suspension-cultured cells was greatly reduced. Attachment of both strains was completely inhibited by pretreating bacterial cells with heat (41°C) or azide and was partially inhibited by EDTA and kanamycin. The mechanism of attachment is not known, but ionic forces may be involved.     

Pollen tube reuses intracellular components of nucellar cells undergoing programmed cell death in Pinus densiflora

Through the process known as programmed cell death (PCD), nucelli of Pinus densiflora serve as the transmitting tissue for growth of the pollen tube. We sought to clarify the processes of degradation of nucellar cell components and their transport to the pollen tube during PCD in response to pollen tube penetration of such nucelli. Stimulated by pollination, synthesis of large amounts of starch grains occurred in cells in a wide region of the nucellus, but as the pollen tube penetrated the nucellus, starch grains were degraded in amyloplasts of nucellar cells. In cells undergoing PCD, electron-dense vacuoles with high membrane contrast appeared, assumed a variety of autophagic structures, expanded, and ultimately collapsed and disappeared. Vesicles and electron-dense amorphous materials were released inside the thickened walls of cells undergoing PCD, and those vesicles and materials reaching the pollen tube after passing through the extracellular matrix were taken into the tube by endocytosis. These results show that in PCD of nucellar cells, intracellular materials are degraded in amyloplasts and vacuoles, and some of the degraded material is supplied to the pollen tube by vesicular transport to support tube growth.     

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.     

Ultrastructure of rigid and lignified forage tissue degradation by a filamentous rumen microorganism.

A small (less than 1 mum)-filamentous, branching microorganism was observed in Gram-stained smears of the rumen microflora and was found to degrade tissues in forage samples incubated in vitro and in vivo with rumen fluid and observed by scanning and transmission electron microscopy. The microbe had prokaryotic cytoplasmic features and a gram-positive type of cell wall structure. Round to oval bodies apparently attached to hyphae resembled the sporulation pattern reported for Micromonospora. Filaments and rod and coccal forms of the microbe degraded rigid forage cell walls and lignified, thick-walled sclerenchymal cells. Location of the microbe at a slight distance from the degraded zones suggested the action of extracellular enzymes. The presence of a microbe with the capability of degrading lignified tissue represents an important and unique function in the rumen ecosystem.