Induction and repression of DAN1 and the family of anaerobic mannoprotein genes in Saccharomyces cerevisiae occurs through a complex array of regulatory sites

The DAN/TIR mannoprotein genes of Saccharomyces cerevisiae (DAN1, DAN2, DAN3, DAN4, TIR1, TIR2, TIR3 and TIR4) are expressed in anaerobic cells while the predominant cell wall proteins Cwp1 and Cwp2 are down-regulated. Elements involved in activation and repression of the DAN/TIR genes were defined in this study, using the DAN1 promoter as a model. Nested deletions in a DAN1/lacZ reporter pinpointed regions carrying activation and repression elements. Inspection revealed two consensus sequences subsequently shown to be independent anaerobic response elements (AR1, consensus TCGTTYAG; AR2, consensus AAAAATTGTTGA). AR1 is found in all of the DAN/TIR promoters; AR2 is found in DAN1, DAN2 and DAN3. A 120 bp segment carrying two copies of AR1 preferentially activated transcription of lacZ under anaerobic conditions. A fusion of three synthetic copies of AR1 to MEL1 was also expressed anaerobically. Mutations in either AR1 site within the 120 bp segment caused a drastic loss of expression, indicating that both are necessary for activation and implying cooperativity between adjacent transcriptional activation complexes. A single AR2 site carried on a 46 bp fragment from the DAN1 promoter activated lacZ transcription under anaerobic conditions, as did a 26 bp synthetic AR2 fragment fused to MEL1. Nucleotide substitutions within the AR2 sequence eliminated the activity of the 46 bp segment. Ablation of the AR2 sequences in the full promoter caused a partial reduction of expression. The presence of the ATTGTT core (recognized by HMG proteins) in the AR2 sequence suggests that an HMG protein may activate through AR2. One region was implicated in aerobic repression of DAN1. It contains sites for the heme-induced Mot3 and Rox1 repressors.

Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme

Bacteriophage lytic enzymes quickly destroy the cell wall of the host bacterium to release progeny phage. Because such lytic enzymes specifically kill the species in which they were produced, they may represent an effective way to control pathogenic bacteria without disturbing normal microflora. In this report, we studied a murein hydrolase from the streptococcal bacteriophage C1 termed lysin. This enzyme is specific for groups A, C, and E streptococci, with little or no activity toward several oral streptococci or other commensal organisms tested. Using purified lysin in vitro, we show that 1,000 units (10 ng) of enzyme is sufficient to sterilize a culture of ≈107 group A streptococci within 5 seconds. When a single dose of lysin (250 units) is first added to the oral cavity of mice, followed by 107 live group A streptococci, it provides protection from colonization (28.5% infected, n = 21) compared with controls without lysin (70.5% infected, n = 17) (P < 0.03). Furthermore, when lysin (500 units) was given orally to 9 heavily colonized mice, no detectable streptococci were observed 2 h after lysin treatment. In all, these studies show that lysin represents a unique murein hydrolase that has a rapid lethal effect both in vitro and in vivo on group A streptococci, without affecting other indigenous microorganisms analyzed. This general approach may be used to either eliminate or reduce streptococci from the upper respiratory mucosal epithelium of either carriers or infected individuals, thus reducing associated disease.

Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome

Clostridium cellulovorans uses not only cellulose but also xylan, mannan, pectin, and several other carbon sources for its growth and produces an extracellular multienzyme complex called the cellulosome, which is involved in plant cell wall degradation. Here we report a gene for a cellulosomal subunit, pectate lyase A (PelA), lying downstream of the engY gene, which codes for cellulosomal enzyme EngY. pelA is composed of an ORF of 2,742 bp and encodes a protein of 914 aa with a molecular weight of 94,458. The amino acid sequence derived from pelA revealed a multidomain structure, i.e., an N-terminal domain partially homologous to the C terminus of PelB of Erwinia chrysanthemi belonging to family 1 of pectate lyases, a putative cellulose-binding domain, a catalytic domain homologous to PelL and PelX of E. chrysanthemi that belongs to family 4 of pectate lyases, and a duplicated sequence (or dockerin) at the C terminus that is highly conserved in enzymatic subunits of the C. cellulovorans cellulosome. The recombinant truncated enzyme cleaved polygalacturonic acid to digalacturonic acid (G2) and trigalacturonic acid (G3) but did not act on G2 and G3. There have been no reports available to date on pectate lyase genes from Clostridia.

Characterization of Two Divergent Endo-β-1,4-Glucanase cDNA Clones Highly Expressed in the Nonclimacteric Strawberry Fruit

Two cDNAs clones (Cel1 and Cel2) encoding divergent endo-β-1,4-glucanases (EGases) have been isolated from a cDNA library obtained from ripe strawberry (Fragaria x ananassa Duch) fruit. The analysis of the amino acid sequence suggests that Cel1 and Cel2 EGases have different secondary and tertiary structures and that they differ in the presence of potential N-glycosylation sites. By in vitro translation we show that Cel1 and Cel2 bear a functional signal peptide, the cleavage of which yields mature proteins of 52 and 60 kD, respectively. Phylogenetic analysis revealed that the Cel2 EGase diverged early in evolution from other plant EGases. Northern analysis showed that both EGases are highly expressed in fruit and that they have different temporal patterns of accumulation. The Cel2 EGase was expressed in green fruit, accumulating as the fruit turned from green to white and remaining at an elevated, constant level throughout fruit ripening. In contrast, the Cel1 transcript was not detected in green fruit and only a low level of expression was observed in white fruit. The level of Cel1 mRNA increased gradually during ripening, reaching a maximum in fully ripe fruit. The high levels of Cel1 and Cel2 mRNA in ripe fruit and their overlapping patterns of expression suggest that these EGases play an important role in softening during ripening. In addition, the early expression of Cel2 in green fruit, well before significant softening begins, suggests that the product of this gene may also be involved in processes other than fruit softening, e.g. cell wall expansion.

Change in Apoplastic Aluminum during the Initial Growth Response to Aluminum by Roots of a Tolerant Maize Variety1

Root elongation, hematoxylin staining, and changes in the ultrastructure of root-tip cells of an Al-tolerant maize variety (Zea mays L. C 525 M) exposed to nutrient solutions with 20 μm Al (2.1 μm Al3+ activity) for 0, 4, and 24 h were investigated in relation to the subcellular distribution of Al using scanning transmission electron microscopy and energy-dispersive x-ray microanalysis on samples fixed by different methods. Inhibition of root-elongation rates, hematoxylin staining, cell wall thickening, and disturbance of the distribution of pyroantimoniate-stainable cations, mainly Ca, was observed only after 4 and not after 24 h of exposure to Al. The occurrence of these transient, toxic Al effects on root elongation and in cell walls was accompanied by the presence of solid Al-P deposits in the walls. Whereas no Al was detectable in cell walls after 24 h, an increase of vacuolar Al was observed after 4 h of exposure. After 24 h, a higher amount of electron-dense deposits containing Al and P or Si was observed in the vacuoles. These results indicate that in this tropical maize variety, tolerance mechanisms that cause a change in apoplastic Al must be active. Our data support the hypothesis that in Al-tolerant plants, Al can rapidly cross the plasma membrane; these data clearly contradict the former conclusions that Al mainly accumulates in the apoplast and enters the symplast only after severe cell damage has occurred.

Regulation of Growth Anisotropy in Well-Watered and Water-Stressed Maize Roots. II. Role of Cortical Microtubules and Cellulose Microfibrils1

We tested the hypothesis that the degree of anisotropic expansion of plant tissues is controlled by the degree of alignment of cortical microtubules or cellulose microfibrils. Previously, for the primary root of maize (Zea mays L.), we quantified spatial profiles of expansion rate in length, radius, and circumference and the degree of growth anisotropy separately for the stele and cortex, as roots became thinner with time from germination or in response to low water potential (B.M. Liang, A.M. Dennings, R.E. Sharp, T.I. Baskin [1997] Plant Physiol 115:101–111). Here, for the same material, we quantified microtubule alignment with indirect immunofluorescence microscopy and microfibril alignment throughout the cell wall with polarized-light microscopy and from the innermost cell wall layer with electron microscopy. Throughout much of the growth zone, mean orientations of microtubules and microfibrils were transverse, consistent with their parallel alignment specifying the direction of maximal expansion rate (i.e. elongation). However, where microtubule alignment became helical, microfibrils often made helices of opposite handedness, showing that parallelism between these elements was not required for helical orientations. Finally, contrary to the hypothesis, the degree of growth anisotropy was not correlated with the degree of alignment of either microtubules or microfibrils. The mechanisms plants use to specify radial and tangential expansion rates remain uncharacterized.

Phenotypic variation and intracellular parasitism by Histoplasma capsulatum

The success of Histoplasma capsulatum as an intracellular pathogen depends completely on successful conversion of the saprophytic mycelial (mold) form of this fungus to a parasitic yeast form. It is therefore not surprising that yeast phase-specific genes and gene products are proving to be important for survival and proliferation of H. capsulatum within macrophages. In this study, we have focused on the role and regulation of two yeast-specific characteristics: α-(1,3)-glucan, a cell wall polysaccharide modulated by cell-density (quorum) sensing, and a secreted calcium-binding protein (CBP) that is essential for pathogenicity.

VanX, a bacterial d-alanyl-d-alanine dipeptidase: Resistance, immunity, or survival function?

The zinc-containing d-alanyl-d-alanine (d-Ala-d-Ala) dipeptidase VanX has been detected in both Gram-positive and Gram-negative bacteria, where it appears to have adapted to at least three distinct physiological roles. In pathogenic vancomycin-resistant enterococci, vanX is part of a five-gene cluster that is switched on to reprogram cell-wall biosynthesis to produce peptidoglycan chain precursors terminating in d-alanyl-d-lactate (d-Ala-d-lactate) rather than d-Ala-d-Ala. The modified peptidoglycan exhibits a 1,000-fold decrease in affinity for vancomycin, accounting for the observed phenotypic resistance. In the glycopeptide antibiotic producers Streptomyces toyocaensis and Amylocatopsis orientalis, a vanHAX operon may have coevolved with antibiotic biosynthesis genes to provide immunity by reprogramming cell-wall termini to d-Ala-d-lactate as antibiotic biosynthesis is initiated. In the Gram-negative bacterium Escherichia coli, which is never challenged by the glycopeptide antibiotics because they cannot penetrate the outer membrane permeability barrier, the vanX homologue (ddpX) is cotranscribed with a putative dipeptide transport system (ddpABCDF) in stationary phase by the transcription factor RpoS (σs). The combined action of DdpX and the permease would permit hydrolysis of d-Ala-d-Ala transported back into the cytoplasm from the periplasm as cell-wall crosslinks are refashioned. The d-Ala product could then be oxidized as an energy source for cell survival under starvation conditions.

Biochemical evolution III: Polymerization on organophilic silica-rich surfaces, crystal–chemical modeling, formation of first cells, and geological clues

Catalysis at organophilic silica-rich surfaces of zeolites and feldspars might generate replicating biopolymers from simple chemicals supplied by meteorites, volcanic gases, and other geological sources. Crystal–chemical modeling yielded packings for amino acids neatly encapsulated in 10-ring channels of the molecular sieve silicalite-ZSM-5-(mutinaite). Calculation of binding and activation energies for catalytic assembly into polymers is progressing for a chemical composition with one catalytic Al–OH site per 25 neutral Si tetrahedral sites. Internal channel intersections and external terminations provide special stereochemical features suitable for complex organic species. Polymer migration along nano/micrometer channels of ancient weathered feldspars, plus exploitation of phosphorus and various transition metals in entrapped apatite and other microminerals, might have generated complexes of replicating catalytic biomolecules, leading to primitive cellular organisms. The first cell wall might have been an internal mineral surface, from which the cell developed a protective biological cap emerging into a nutrient-rich “soup.” Ultimately, the biological cap might have expanded into a complete cell wall, allowing mobility and colonization of energy-rich challenging environments. Electron microscopy of honeycomb channels inside weathered feldspars of the Shap granite (northwest England) has revealed modern bacteria, perhaps indicative of Archean ones. All known early rocks were metamorphosed too highly during geologic time to permit simple survival of large-pore zeolites, honeycombed feldspar, and encapsulated species. Possible microscopic clues to the proposed mineral adsorbents/catalysts are discussed for planning of systematic study of black cherts from weakly metamorphosed Archaean sediments.

Benzothiadiazole-Mediated Induced Resistance to Fusarium oxysporum f. sp. radicis-lycopersici in Tomato1

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic chemical, was applied as a foliar spray to tomato (Lycopersicon esculentum) plants and evaluated for its potential to confer increased resistance against the soil-borne pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL). In nontreated tomato plants all root tissues were massively colonized by FORL hyphae. Pathogen ingress toward the vascular stele was accompanied by severe host cell alterations, including cell wall breakdown. In BTH-treated plants striking differences in the rate and extent of fungal colonization were observed. Pathogen growth was restricted to the epidermis and the outer cortex, and fungal ingress was apparently halted by the formation of callose-enriched wall appositions at sites of fungal penetration. In addition, aggregated deposits, which frequently established close contact with the invading hyphae, accumulated in densely colonized epidermal cells and filled most intercellular spaces. Upon incubation of sections with gold-complexed laccase for localization of phenolic-like compounds, a slight deposition of gold particles was observed over both the host cell walls and the wall appositions. Labeling was also detected over the walls of fungal cells showing signs of obvious alteration ranging from cytoplasm disorganization to protoplasm retraction. We provide evidence that foliar applications of BTH sensitize susceptible tomato plants to react more rapidly and more efficiently to FORL attack through the formation of protective layers at sites of potential fungal entry.

Expression Analysis of a Ripening-Specific, Auxin-Repressed Endo-1,4-β-Glucanase Gene in Strawberry

A cDNA (Cel1) encoding an endo-1,4-β-glucanase (EGase) was isolated from ripe fruit of strawberry (Fragaria × ananassa). The deduced protein of 496 amino acids contains a presumptive signal sequence, a common feature of cell wall-localized EGases, and one potential N-glycosylation site. Southern- blot analysis of genomic DNA from F. × ananassa, an octoploid species, and that from the diploid species Fragaria vesca indicated that the Cel1 gene is a member of a divergent multigene family. In fruit, Cel1 mRNA was first detected at the white stage of development, and at the onset of ripening, coincident with anthocyanin accumulation, Cel1 mRNA abundance increased dramatically and remained high throughout ripening and subsequent fruit deterioration. In all other tissues examined, Cel1 expression was invariably absent. Antibodies raised to Cel1 protein detected a protein of 62 kD only in ripening fruit. Upon deachenation of young white fruit to remove the source of endogenous auxins, ripening, as visualized by anthocyanin accumulation, and Cel1 mRNA accumulation were both accelerated. Conversely, auxin treatment of white fruit repressed accumulation of both Cel1 mRNA and ripening. These results indicate that strawberry Cel1 is a ripening-specific and auxin-repressed EGase, which is regulated during ripening by a decline in auxin levels originating from the achenes.

Localized Changes in Peroxidase Activity Accompany Hydrogen Peroxide Generation during the Development of a Nonhost Hypersensitive Reaction in Lettuce1

Peroxidase activity was characterized in lettuce (Lactuca sativa L.) leaf tissue. Changes in the activity and distribution of the enzyme were examined during the development of a nonhost hypersensitive reaction (HR) induced by Pseudomonas syringae (P. s.) pv phaseolicola and in response to an hrp mutant of the bacterium. Assays of activity in tissue extracts revealed pH optima of 4.5, 6.0, 5.5 to 6.0, and 6.0 to 6.5 for the substrates tetramethylbenzidine, guaiacol, caffeic acid, and chlorogenic acid, respectively. Inoculation with water or with wild-type or hrp mutant strains of P. s. pv phaseolicola caused an initial decline in total peroxidase activity; subsequent increases depended on the hydrogen donor used in the assay. Guaiacol peroxidase recovered more rapidly in tissues undergoing the HR, whereas changes in tetramethylbenzidine peroxidase were generally similar in the two interactions. In contrast, increases in chlorogenic acid peroxidase were significantly higher in tissues inoculated with the hrp mutant. During the HR, increased levels of Mn2+/2,4-dichlorophenol-stimulated NADH and NADPH oxidase activities, characteristic of certain peroxidases, were found in intercellular fluids and closely matched the accumulation of H2O2 in the apoplast. Histochemical analysis of peroxidase distribution by electron microscopy revealed a striking, highly localized increase in activity within the endomembrane system and cell wall at the sites of bacterial attachment. However, no clear differences in peroxidase location were observed in tissue challenged by the wild-type strain or the hrp mutant. Our results highlight the significance of the subcellular control of oxidative reactions leading to the generation of reactive oxygen species, cell wall alterations, and the HR.

petit1, a Conditional Growth Mutant of Arabidopsis Defective in Sucrose-Dependent Elongation Growth1

The hypocotyl of Arabidopsis is well suited for the analysis of cell elongation because it elongates without cell division. We have isolated a new class of recessive mutants, petit1 (pet1), which are defective in aspects of hypocotyl elongation. The short-hypocotyl phenotype of pet1 is caused by shortened cells. The cells of the elongation zone of the hypocotyl are often deformed. pet1 also shows defects in elongation of the roots, flower stalk, leaves, petals, pedicels, and siliques, and these defects cannot be repaired by the application of auxin, gibberellin, brassinolide, or an inhibitor of ethylene biosynthesis. The short-hypocotyl phenotype of pet1 is pronounced only in growth medium supplemented with sucrose, which has promotive effects on hypocotyl elongation. In pet1 this effect is much reduced, causing the sucrose-dependent short-hypocotyl phenotype of pet1. pet1 accumulates more soluble sugars than the wild type and also shows more intensive iodo-starch staining in the cotyledon and hypocotyl. These results indicate that PETIT1 is involved in a sugar-dependent elongation process that may include correct assembly of expanding cell wall architecture.

Acid-Growth Response and α-Expansins in Suspension Cultures of Bright Yellow 2 Tobacco1

The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of α-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple α-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.

Purification and Characterization of Peroxidases Correlated with Lignification in Poplar Xylem1

Lignin is an integral cell wall component of all vascular plants. Peroxidases are widely believed to catalyze the last enzymatic step in the biosynthesis of lignin, the dehydrogenation of the p-coumaryl alcohols. As the first stage in identifying lignin-specific peroxidase isoenzymes, the classical anionic peroxidases found in the xylem of poplar (Populus trichocarpa Trichobel) were purified and characterized. Five different poplar xylem peroxidases (PXP 1, PXP 2, PXP 3–4, PXP 5, and PXP 6) were isolated. All five peroxidases were strongly glycosylated (3.6% to 4.9% N-glucosamine), with apparent molecular masses between 46 and 54 kD and pI values between pH 3.1 and 3.8. Two of the five isolated peroxidases (PXP 3–4 and PXP 5) could oxidize the lignin monomer analog syringaldazine, an activity previously correlated with lignification in poplar. Because these isoenzymes were specifically or preferentially expressed in xylem, PXP 3–4 and PXP 5 are suggested to be involved in lignin polymerization.