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PubMed2000

Delayed ripening of banana fruit by salicylic acid.

Salicylic acid treatment has been found to delay the ripening of banana fruits (Musa acuminata). Fruit softening, pulp:peel ratio, reducing sugar content, invertase and respiration rate have been found to decrease in salicylic acid treated fruits as compared with control ones. The activities of major cell wall degrading enzymes, viz. cellulase, polygalacturonase and xylanase were found to be decreased in presence of salicylic acid. The major enzymatic antioxidants namely, catalase and peroxidase, were also found to be decreased in presence of salicylic acid during banana fruit ripening.

Journal Article↗
PubMed1998

EXG1p, a novel exo-beta1,3-glucanase from the fungus Cochliobolus carbonum, contains a repeated motif present in other proteins that interact with polysaccharides.

Genomic and cDNA copies of EXG1, a gene encoding an exo-beta1, 3-glucanase from the plant pathogenic fungus Cochliobolus carbonum, were isolated. The gene contains two introns of 50 and 53 bp, and the mRNA has a 5'-untranslated region of 90 nt and a 3'-untranslated region of 159 nt. The deduced protein product, EXG1p, has a predicted signal peptide of 17 amino acids, but based on the known N-terminus of the mature protein is further processed to remove an additional 25 amino acids. The sequence of EXG1p is not closely related to any other known protein, but has a low similarity (29% overall amino acid identity) to BGN13.1, an endo-beta1,3-glucanase from the mycoparasitic fungus Trichoderma harzianum. EXG1p contains two imperfect copies of a 23-amino acid motif that is found in several other proteins that interact with polysaccharides, including plant and bacterial polygalacturonases, phage neck appendage protein, phage endoneuramidase, and bacterial mannuronan epimerase.

Amino Acid Sequence↗
PubMed2000

Physiological aspects of fungi isolated from root nodules of faba bean (Vicia faba L.).

The present study was made to isolate and assess some physiological characteristics of root nodule-colonizing fungi. During this study, 17 fungal species were isolated from root nodule samples taken from faba bean plants (Vicia faba L.) collected from different sites at Assiut area (Egypt). The growth of faba bean plants in pots was significantly promoted by soil inoculation with most fungi. Growth was checked in pots with inocula of Cladosporium cladosporioides, Fusarium moniliforme, F: oxysporium, F solani, Macrophominia phaseolina and Rhizoctonia solani which were added separately. All growth-promoting fungi were capable of producing cellulase, pectin lyase, polygalacturonase, protease, urease, amidase, acid phosphatase, alkaline phosphatase and arylsulfatase in growth medium supplemented with the corresponding substrates. Four fungal species, Aspergillus awamori, A. flavus, Penicillium chrysogenum and Trichoderma koningii showed the highest rates of enzyme formation. The effect of the addition of six trace elements to the growth media at 30 micromol/ml on enzyme production revealed some dependency on species, enzyme and metal ion. Cd2+, Hg2+ and Zn2+ generally inhibited enzyme activity. Cu(1+), Fe3+ and Al3+ showed a stimulatory effect. Fungicides (afugan and tilt) and herbicides (brominal and fusilade) at 50 ppm generally promoted enzyme activity, but insecticides (kelthane and fenvalerate) caused some inhibition to enzyme activities. Salinization of the growth media with NaCl strongly inhibited the enzymatic activity of all fungi at concentrations between 0.5 and 1.5%.

Fabaceae↗
PubMed1999

Functional expression in bacteria and plants of an scFv antibody fragment against tospoviruses.

BACKGROUND: Recombinant antibodies expressed in plants ('plantibodies'), directed against crucial antigens and addressed to the right cell compartment, may be able to protect against viral diseases. Moreover, antibody fragments produced in bacteria or plants may provide low cost reagents for immunodiagnosis. OBJECTIVES: In an attempt to develop genetic immunisation against tomato spotted wilt tospovirus (TSWV), we engineered an scFv fragment starting from a monoclonal antibody (mAb) able to recognise an epitope of the glycoprotein G1 conserved among a large number of tospoviruses. After establishing functional expression in bacteria, we aimed to drive expression of this molecule in the secretory pathway of plants. STUDY DESIGN: An antibody phage display expression system was used to isolate the correct VH and VL binding regions from the hybridoma secreting the original mAb. To assess functional expression in plant, we first used an epichromosomal expression vector derived from potato virus X (PVX). In this vector the scFv gene was cloned to produce a cytosolic or a secretory protein. For secretion, the signal sequence derived from the polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris was used. Subsequently, the gene encoding the secretory scFv, was used to transform Nicotiana benthamiana plants. RESULTS: High expression levels of fully active molecule were obtained in Escherichia coli. The engineered molecule retained the binding specificity and dissociation rate constant (k(off)) of the cognate monoclonal antibody. Both PVX-infected and transformed plants expressed fully functional scFv molecules in the secretory pathway. CONCLUSION: This engineered scFv may be valuable for inexpensive diagnosis, for studying the role of the glycoproteins in virus transmission and, possibly, for a 'plantibody'-mediated resistance to tospoviruses.

Amino Acid Sequence↗
PubMed1999

Degradation of pectic substances by two pectate lyases from a human intestinal bacterium, Clostridium butyricum-beijerinckii group.

Clostridium butyricum-beijerinckii group, an anaerobic human intestinal bacterium, produced exo-pectate lyase, endo-pectate lyase, and pectin methylesterase, but not polygalacturonase. The production of the two pectate lyases was strongly influenced by the composition of the carbon sources added to the medium. The enzymes secreted by the bacterium into the human large intestine cooperatively digested pectic substances, producing mainly 4,5-unsaturated digalacturonic acid with the participation of the pectin methylesterase. The findings suggest that in the human intestine the bacterium contributes to the digestion of ingested pectic substances.

Journal Article↗
PubMed2002

Xylanase, beta-glucanase, and other side enzymatic activities have greater effects on the viscosity of several feedstuffs than xylanase and beta-glucanase used alone or in combination.

This study was carried out to evaluate the effects of a pure xylanase, a pure beta-glucanase, a mix of the two pure enzymes, and a commercial enzyme preparation (Quatrazyme HP, Nutri-Tomen Les Ulis, France) on the viscosity exhibited by water-soluble nonstarch polysaccharides of several feedstuffs (Rialto wheat, Sidéral wheat, Isengrain wheat, triticale, rye, barley, oats, corn, wheat bran, rice bran, wheat screenings, soybean meal, rapeseed meal, sunflower meal, and peas). The viscosity depended on the feedstuffs and varieties of the same feedstuff. There was a correlation (R (2) = 0.86) between viscosity of cereals and their arabinoxylan and beta-glucan contents. The correlation was greater (R (2) = 0.99) when the type of cereal was taken into account. The addition of pure xylanase significantly decreased the viscosity of all feedstuffs except sunflower meal (P < or = 0.05). However, pure beta-glucanase was unable significantly to decrease the viscosity of Isengrain wheat, corn, rice bran, wheat screenings, soybean meal, and sunflower meal. There was a greater decrease in viscosity with the combination of xylanase and beta-glucanase than with addition of xylanase or beta-glucanase alone. This synergistic action of xylanase and beta-glucanase was observed only in Rialto wheat, Sidéral wheat, triticale, rye, barley, oats, and peas. Finally, the commercial enzyme preparation produced a greater reduction (P < or = 0.05) in viscosity for all feedstuffs compared to xylanase or beta-glucanase used alone or in combination. The greater effectiveness of the commercial enzyme preparation was due to the presence of side enzymatic activities (arabinofuranosidase, xylosidase, glucosidase, galactosidase, cellulase, and polygalacturonase).

Animal Feed↗
PubMed2006

Mode of de-esterification of alkaline and acidic pectin methyl esterases at different pH conditions.

Highly esterified citrus pectin was de-esterified at pH 4.5 and 8.0 by a fungal pectin methyl esterase (PME) that was shown to have an acidic isoelectric pH (pI) and an acidic pH optimum and by a plant PME that was characterized by an alkaline pI and an alkaline pH optimum. Interchain and intrachain de-esterification patterns were studied by digestion of the pectin products with endo-polygalacturonase and subsequent analysis using size exclusion and anion-exchange chromatography. No effect of pH was observed on the de-esterification mode of either of the two enzymes. Acidic, fungal PME converted pectin according to a multiple-chain mechanism, with a limited degree of multiple attack at the intrachain level, both at pH 4.5 and at pH 8.0. A multiple-attack mechanism, with a high degree of multiple attack, was more appropriate to describe the action mode of alkaline, plant PME, both at pH 4.5 and at pH 8.0.

Aspergillus↗
PubMed2006

Methyl jasmonate induces defense responses in grapevine and triggers protection against Erysiphe necator.

Grapevine is subject to a number of diseases that affect yield and wine quality. To limit the excessive use of phytochemicals in the vineyard, alternative strategies have to be developed. Plant treatment with signaling molecules like elicitors stimulates their natural defense mechanisms. To improve grapevine tolerance against fungal pathogens, Vitis vinifera plants were treated with a natural exogenous elicitor, methyl jasmonate (MeJA). MeJA-treated leaves (Cabernet Sauvignon foliar cuttings) reacted by increasing transcript levels coding pathogenesis-related proteins (acidic class IV chitinase, serine protease inhibitor, polygalacturonase-inhibiting protein, and beta-1,3-glucanase) and coding enzymes involved in phytoalexin biosynthesis (one phenylalanine ammonia lyase and one stilbene synthase). This was correlated with the accumulation of stilbenes (antimicrobial compounds). The eliciting activity of MeJA was confirmed by enhanced tolerance of grapevine foliar cuttings and vineyard against powdery mildew (75% and 73%, respectively). On the basis of these original results, MeJA can therefore act as an efficient elicitor in an alternative strategy of grapevine protection.

Acetates↗
PubMed1998

Induction of AGAMOUS gene expression plays a key role in ripening of tomato sepals in vitro.

In vitro culture of VFNT Cherry tomato sepals (calyx) at 16-21 degrees C results in developmental changes that are similar to those that occur in fruit tissue [10]. Sepals become swollen, red, and succulent, produce ethylene, and have increased levels of polygalacturonase RNA. They also produce many flavor volatiles characteristic of ripe tomato fruit and undergo similar changes in sugar content [11]. We examined the expression of the tomato AGAMOUS gene, TAG1, in ripening, in vitro sepal cultures and other tissues from the plant and found that TAG1 RNA accumulates to higher levels than expected from data from other plants. Contrary to reports on the absence of AGAMOUS in sepals, TAG1 RNA levels in green sepals from greenhouse-grown plants is detectable, its concentration increasing with in vitro ripening to levels that were even higher than in red, ripe fruit. Sepals of fruit on transgenic tomato plants that expressed TAG1 ectopically were induced by low temperature to ripen in vivo, producing lycopene and undergoing cell wall softening as is characteristic of pericarpic tissue. We therefore propose that the induction of elevated TAG1 gene expression plays a key role in developmental changes that result in sepal ripening.

AGAMOUS Protein, Arabidopsis↗
PubMed1998

A maize pectin methylesterase-like gene, ZmC5, specifically expressed in pollen.

Pectin methylesterase (PME) is responsible for the demethylation of pectin prior to pectin's degradation by the combined activities of polygalacturonase and pectate lyase. We have differentially screened a maize pollen cDNA library to detect cDNA clones whose genes are specifically expressed in pollen. One group of clones resulting from this screen showed similarity (between 18% and 41% identity) with plant and fungal PMEs. The full-length clone from this group, ZmC5, identifies a small gene family (at least 2 members) when used as a probe on genomic Southern blots. Northern analysis reveals that the ZmC5 transcript is expressed specifically in late pollen development. This tissue-specific gene expression programme is further confirmed in transgenic tobacco plants harbouring ZmC5 promoter/GUS chimeric gene constructs.

Base Sequence↗
PubMed1999

A leucine-rich repeat receptor-like protein kinase (LRPKm1) gene is induced in Malus x domestica by Venturia inaequalis infection and salicylic acid treatment.

A cDNA clone encoding a leucine-rich repeat (LRR) receptor-like protein kinase (LRPKm1) of Malus x domestica cv. Florina has been isolated using as a heterologous probe a cloned gene encoding a polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L. A genomic clone containing the 5'-regulatory region and a 5' portion of the open reading frame of the LRPKm1 gene has also been isolated. An open reading frame of 2997 nt (999 amino acids) was present in the cDNA clone, encoding a receptor-like protein comprising a 21 amino acid signal peptide for secretion, a leucine zipper, 23 LRRs, a putative membrane-spanning region and a serine/threonine protein kinase domain. LRPKm1 shows homology to the A. thaliana receptor-like protein kinase RLK5 and, to a minor extent, to PGIP. The LRPKm1 region from +5 to +600 exhibits an alternative reading frame that encodes a product corresponding to a proline-rich protein fragment homologous to several hydroxyproline-rich proteins. Southern blot analysis showed that LRPKm1 belongs to a multigene family and that there is length polymorphism of the hybridizing restriction fragments among different M. x domestica cultivars. Northern blot analysis was carried out on mRNA extracted from infected leaves of either cv. Florina (resistant to Venturia inaequalis) or cv. Golden Delicious (susceptible to V. inaequalis), and from tissues treated with salicylic acid. A 3500 bp transcript hybridizing at high stringency with the LRPKm1 cDNA accumulated in response to infection or salicylic acid treatment. Transcript accumulation was more intense in the incompatible interaction than in the compatible one. The possible involvement of this receptor-like protein kinase in resistance of apple to phytopathogenic fungi is discussed.

Amino Acid Sequence↗
PubMedDate not supplied

[Isolation and characterization of polysaccharides from Tansy].

Using extraction with 0.75% aqueous ammonium oxalate, the following polysaccharide fractions were isolated: tanacetans TVF, TVS, and TVR from floscules, sprouts, and roots, respectively, of Tanacetum vulgare L., spread throughout the European North of Russia. The sugar chain of tanacetan TVF consists of D-galacturonic acid (61.4%), arabinose (14.7%), galactose (10.2%), and rhamnose (3.7%) as the main constituents as well as xylose, glucose, mannose, apiose, and 2-O-methylxylose in trace amounts. Tanacetans TVS and TVR were shown to differ in the sugar quantitative composition. They contain 67 and 28% galacturonic acid, respectively. A partial acid hydrolysis of the tanacetan TVF gave a polysaccharide fragment TVF1, alpha-1,4-D-galacturonan (GalA 98.2%). Digestion with pectinase (alpha-1,4-D-polygalacturonase) resulted in fragment TVF3, containing residues of arabinose (27.1%) and galactose (17.3%). NMR spectroscopy allowed detection of the terminal residues of alpha-Araf and beta-Galp as well as of the residues of alpha-Araf substituted in 3,5- and 5-positions. Thus, tanacetan TVF was proved to be a pectic polysaccharide.

Asteraceae↗
PubMed2002

Pectin-bound beta-galactosidase present in cell walls of carrot cells under the different calcium status.

Glycosyl-hydrolytic enzymes from suspension-cultured carrot (Daucus carota L. cv. Kintoki) cells grown in calcium (Ca2+)-deficient and normal liquid media were studied after extraction successively by K-phosphate (pH 7.0) and Na-acetate (pH 5.2) containing 3 M LiCl. The same activities were detected in two protein fractions from control and Ca2+-deprived cells. The specific activities of alpha-galactosidase and polygalacturonase decreased under Ca2+ deprivation, but beta-galactosidase activity in the buffer-soluble protein from Ca2+-deprived cells increased 1.7-fold compared to control cells. Upon ion exchange and size-exclusion chromatography the fraction (Ca-Ia-I) in the buffer-soluble protein from Ca2+-deprived cells represented beta-galactosidase activity associated with a galacturonic acid-rich polysaccharide peak, whereas the corresponding fraction could hardly be detected in the buffer-soluble protein from control cells. Several of the same glycosidase activities were detected in the extract solubilized with cyclohexane-trans-1,2-diaminetetra-acetate (CDTA) from active cell walls of Ca2+-deprived cells as in the extract of control cells, but the beta-galactosidase activity was considerably reduced under Ca2+ deprivation. Following the same chromatography the fraction (CDTA-Ca-1) of beta-galactosidase activity in the extract solubilized with CDTA from active cell walls of Ca2+-deprived cells was also completely overlapping with the peak of galacturonic acid-rich polysaccharide. The molecular mass of fractions Ca-Ia-I and CDTA-Ca-1 was 300 kDa, and the polysaccharides in these two fractions were composed of approximately equal amounts of rhamnosyl and galacturonosyl residues. These results suggest that the increase of beta-galactosidase in the buffer-soluble protein fraction from Ca2+-deprived cells is the result of solubilization of a part of the acidic pectic polymer-bound beta-galactosidase due to the structural changes in the cell walls that occur during Ca2+ deprivation.

Journal Article↗
PubMed2006

A new group of exo-acting family 28 glycoside hydrolases of Aspergillus niger that are involved in pectin degradation.

The fungus Aspergillus niger is an industrial producer of pectin-degrading enzymes. The recent solving of the genomic sequence of A. niger allowed an inventory of the entire genome of the fungus for potential carbohydrate-degrading enzymes. By applying bioinformatics tools, 12 new genes, putatively encoding family 28 glycoside hydrolases, were identified. Seven of the newly discovered genes form a new gene group, which we show to encode exoacting pectinolytic glycoside hydrolases. This group includes four exo-polygalacturonan hydrolases (PGAX, PGXA, PGXB and PGXC) and three putative exo-rhamnogalacturonan hydrolases (RGXA, RGXB and RGXC). Biochemical identification using polygalacturonic acid and xylogalacturonan as substrates demonstrated that indeed PGXB and PGXC act as exo-polygalacturonases, whereas PGXA acts as an exo-xylogalacturonan hydrolase. The expression levels of all 21 genes were assessed by microarray analysis. The results from the present study demonstrate that exo-acting glycoside hydrolases play a prominent role in pectin degradation.

Acetylesterase↗
PubMed1997

Purification and characterization of a feruloyl esterase from the fungus Penicillium expansum.

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and rho-coumaric acid from methyl esters of the acids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1-->3)-O-beta- D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and O-[5-O-((E)-rho-coumaroyl)-alpha-L-arabinofuranosyl]- (1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (PAXX). The esterase was purified 360-fold in successive steps involving ultrafiltration and column chromatography by gel filtration, anion exchange and hydrophobic interaction. These chromatographic methods separated the phenolic acid esterase from alpha-L-arabinofuranosidase, pectate and pectin lyase, polygalacturonase, xylanase and beta-D-xylosidase activities. The phenolic acid esterase had an apparent mass of 65 kDa under non-denaturing conditions and a mass of 57.5 kDa under denaturing conditions. Optimal pH and temperature were 5.6 and 37 degrees C, respectively and the metal ions Cu2+ and Fe3+ at concentrations of 5 mmol 1-1 inhibited feruloyl esterase activity by 95% and 44%, respectively, at the optimum pH and temperature. The apparent Km and Vmax of the purified feruloyl esterase for methyl ferulate at pH 5.6 and 37 degrees C were 2.6 mmol 1-1 and 27.1 mumol min-1 mg-1. The corresponding constants of rho-coumaroyl esterase for methyl coumarate were 2.9 mmol 1-1 and 18.6 mumol min-1 mg-1.

Carboxylic Ester Hydrolases↗
PubMed2003

Characterization and in vitro expression patterns of an exopolygalacturonase encoding gene from Fusarium oxysporum f.sp. radicis lycopersici.

AIMS: In this work, we report the isolation, characterization and expression pattern in in vitro cultures of an EXOPG encoding gene (pgx2), a novel EXOPG encoding gene of Fusarium oxysporum f.sp. radicis lycopersici, responsible for foot crown and root rot disease in tomato plants. The gene was compared with other fungal polygalacturonases (PGs) previously reported. METHODS AND RESULTS: Partial sequences of the purified EXOPG native protein were used to design primers that amplified a genomic fragment by PCR. The amplified genomic fragment was used as a probe to screen a genomic library. One isolated clone was analysed. The complete genomic, cDNA and the deduced amino acid sequences were compared with other fungal EXOPGs and ENDOPGs. Regulation of pgx2 expression was analysed by Northern blot in in vitro cultures supplemented with different carbon sources. CONCLUSIONS: Pgx2 was present as single copy in the haploid genome of several Fusarium species. PGX2 showed the conserved amino acid motifs typical of PGs and those reported for fungal EXOPGs. Pgx2 was regulated at transcriptional level showing similar expression pattern to other EXOPG encoding gene (pgx1) when the fungus was cultured on different carbon sources suggesting a coordinate expression of both genes. This similarity would be supported by the presence of common putative regulatory motifs in the upstream regions of both genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the analysis of a novel EXOPG gene of the tomato pathogen F. oxysporum f.sp. radicis lycopersici, a contribution to the understanding of the role of cell-wall-degrading enzymes produced by fungi during pathogenesis.

Amino Acid Sequence↗
PubMed1997

Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937.

Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. The structural complexity of pectin requires the combined action of several pectinases for its efficient breakdown. Three types of pectinases have so far been identified in E. chrysanthemi: two pectin methyl esterases (PemA, PemB), a polygalacturonase (PehX), and eight pectate lyases (PelA, PelB, PelC, PelD, PelE, PelL, PelZ, PelX). We report in this paper the analysis of a novel enzyme, the pectin acetyl esterase encoded by the paeY gene. No bacterial form of pectin acetyl esterases has been described previously, while plant tissues and some pectinolytic fungi were found to produce similar enzymes. The paeY gene is present in a cluster of five pectinase-encoding genes, pelA-pelE-pelD-paeY-pemA. The paeY open reading frame is 1650 bases long and encodes a 551-residue precursor protein of 60704Da, including a 25-amino-acid signal peptide. PaeY shares one region of homology with a rhamnogalacturonan acetyl esterase of Aspergillus aculeatus. To characterize the enzyme, the paeY gene was overexpressed and its protein product was purified. PaeY releases acetate from sugar-beet pectin and from various synthetic substrates. Moreover, the enzyme was shown to act in synergy with other pectinases. The de-esterification rate by PaeY increased after previous demethylation of the pectins by PemA and after depolymerization of the pectin by pectate lyases. In addition, the degradation of sugar-beet pectin by pectate lyases is favoured after the removal of methyl and acetyl groups by PemA and PaeY, respectively. The paeY gene was first identified on the basis of its regulation, which shares several characteristics with that of other pectinases. Analysis of the paeY transcription, using gene fusions, revealed that it is induced by pectic catabolic products and is affected by growth phase, oxygen limitation and catabolite repression. Regulation of paeY expression appears to be dependent on the KdgR repressor, which controls all the steps of pectin catabolism, and on the catabolite regulatory protein (CRP), the global activator of sugar catabolism. The contiguous pelD, paeY and pemA genes are transcribed as an operon from a promoter proximal to pelD which allows the regulation by KdgR and CRP. However, transcription can be interrupted at the intra-operon Rho-independent terminator situated between pelD and paeY. The paeY mutant inoculated into Saintpaulia plants was less invasive than the wild-type E. chrysanthemi strain 3937, demonstrating the important role of PaeY in the soft-rot disease.

Acetylation↗
PubMed1998

Characterization of a novel RNA regulator of Erwinia carotovora ssp. carotovora that controls production of extracellular enzymes and secondary metabolites.

The enterobacterium Erwinia carotovora ssp. carotovora strain 71 (hereafter Ecc71) produces extracellular enzymes such as pectate lyase isozymes (Pels), cellulase (Cel), polygalacturonase (Peh) and protease (Prt). These enzymes degrade plant cell wall components and are largely responsible for the elicitation of soft-rot diseases in plants and plant products. Ecc71 also produces HarpinEcc, the elicitor of hypersensitive reaction (HR) and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (OHL). OHL controls extracellular enzyme and HarpinEcc production. The levels of these enzymes, as well as the expression of hrpNEcc, the structural gene for HarpinEcc, and ohll, the gene specifying OHL synthesis, are negatively regulated by RsmaA. rsmB, formerly aepH, on the other hand, positively regulates extracellular enzyme production. 6His-RsmA recombinant protein purified from E. coli binds rsmB RNA as indicated by gel mobility shift assays. rsmB comprises 547 bp DNA, which is transcribed from a single start site immediately after a sigma70-like promoter. In Ecc71, two rsmB RNA species are detected: a full-length 479 base rsmB RNA and a 259 base rsmB' RNA. rsmB' DNA hybridizes with the 259 base and the 479 base transcripts. A 3' RNase protection assay revealed that the 259 base and the 479 base RNA species end at the same position immediately after the putative rho-independent terminator. The expression of rsmB-lacZ transcriptional fusions established that the rsmB' RNA is not produced because of the activation of an internal promoter. These data strongly suggest that the 259 base rsmB' RNA is derived by processing of the primary rsmB RNA. In Ecc71, rsmB' expression driven by the lac promoter causes overproduction of Pel, Peh, Cel and Prt, and accumulation of pel-1, peh-1, hrpNEcc and ohll transcripts. By contrast, a plasmid with the rsmB' DNA sequence deleted fails to cause overproduction of the extracellular enzymes in Ecc71. The rsmB' effect also occurs in Escherichia coli as glycogen accumulation is stimulated in the presence of rsmB'. In vivo and in vitro translation as well as mutational analysis of rsmB' have established that rsmB' RNA does not yield a translational product. Therefore, we concluded that the rsmB' RNA itself functions as the regulator. Indeed, the expression rsmB' DNA leads to neutralization of the negative effects of the RNA-binding protein, RsmA, in Ecc71 and Serratia marcescens strain SM274. We propose a model that explains how RsmA and rsmB control the expression of genes for extracellular enzymes.

Bacterial Outer Membrane Proteins↗