Exo Mutants Research Articles

Structures of Exopolysaccharides Involved in Receptor-mediated Perception of Mesorhizobium loti by Lotus japonicus

In the symbiosis formed between Mesorhizobium loti strain R7A and Lotus japonicus Gifu, rhizobial exopolysaccharide (EPS) plays an important role in infection thread formation. Mutants of strain R7A affected in early exopolysaccharide biosynthetic steps form nitrogen-fixing nodules on L. japonicus Gifu after a delay, whereas mutants affected in mid or late biosynthetic steps induce uninfected nodule primordia. Recently, it was shown that a plant receptor-like kinase, EPR3, binds low molecular mass exopolysaccharide from strain R7A to regulate bacterial passage through the plant's epidermal cell layer (Kawaharada, Y., Kelly, S., Nielsen, M. W., Hjuler, C. T., Gysel, K., Muszyński, A., Carlson, R. W., Thygesen, M. B., Sandal, N., Asmussen, M. H., Vinther, M., Andersen, S. U., Krusell, L., Thirup, S., Jensen, K. J., et al. (2015) Nature 523, 308–312). In this work, we define the structure of both high and low molecular mass exopolysaccharide from R7A. The low molecular mass exopolysaccharide produced by R7A is a monomer unit of the acetylated octasaccharide with the structure (2,3/3-OAc)β-d-RibfA-(1→4)-α-d-GlcpA-(1→4)-β-d-Glcp-(1→6)-(3OAc)β-d-Glcp-(1→6)-*[(2OAc)β-d-Glcp-(1→4)-(2/3OAc)β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-β-d-Galp]. We propose it is a biosynthetic constituent of high molecular mass EPS polymer. Every new repeating unit is attached via its reducing-end β-d-Galp to C-4 of the fourth glucose (asterisked above) of the octasaccharide, forming a branch. The O-acetylation occurs on the four glycosyl residues in a non-stoichiometric ratio, and each octasaccharide subunit is on average substituted with three O-acetyl groups. The availability of these structures will facilitate studies of EPR3 receptor binding of symbiotically compatible and incompatible EPS and the positive or negative consequences on infection by the M. loti exo mutants synthesizing such EPS variants.

Mechanism of ganciclovir-induced chain termination revealed by resistant viral polymerase mutants with reduced exonuclease activity.

Many antiviral and anticancer drugs are nucleoside analogs that target polymerases and cause DNA chain termination. Interestingly, ganciclovir (GCV), the first line of therapy for human cytomegalovirus (HCMV) infections, induces chain termination despite containing the equivalent of a 3'-hydroxyl group. Certain HCMV GCV resistance (GCV(r)) mutations, including ones associated with treatment failures, result in substitutions in the 3'-5' exonuclease (Exo) domain of the catalytic subunit of the viral DNA polymerase (Pol). To investigate how these mutations confer resistance, we overexpressed and purified wild-type (WT) HCMV Pol and three GCV(r) Exo mutants. Kinetic studies provided little support for resistance being due to effects on Pol binding or incorporation of GCV-triphosphate. The mutants were defective for Exo activity on all primer templates tested, including those with primers terminating with GCV, arguing against the mutations increasing excision of the incorporated drug. However, although the WT enzyme terminated DNA synthesis after incorporation of GCV-triphosphate and an additional nucleotide (N+1), the Exo mutants could efficiently synthesize DNA to the end of such primer templates. Notably, the Exo activity of WT Pol rapidly and efficiently degraded N+2 primer templates to N+1 products that were not further degraded. On N+1 primer templates, WT Pol, much more than the Exo mutants, converted the incoming deoxynucleoside triphosphate to its monophosphate, indicative of rapid addition and removal of incorporated nucleotides ("idling"). These results explain how GCV induces chain termination and elucidate a previously unidentified mechanism of antiviral drug resistance.

Exo1 independent DNA mismatch repair involves multiple compensatory nucleases.

Functional DNA mismatch repair (MMR) is essential for maintaining the fidelity of DNA replication and genetic stability. In hematopoiesis, loss of MMR results in methylating agent resistance and a hematopoietic stem cell (HSC) repopulation defect. Additionally MMR failure is associated with a variety of human malignancies, notably Lynch syndrome. We focus on the 5'→3' exonuclease Exo1, the primary enzyme excising the nicked strand during MMR, preceding polymerase synthesis. We found that nuclease dead Exo1 mutant cells are sensitive to the O6-methylguanine alkylating agent temozolomide when given with the MGMT inactivator, O6benzylguanine (BG). Additionally we used an MMR reporter plasmid to verify that Exo1(mut) MEFs were able to repair G:T base mismatches in vitro. We showed that unlike other MMR deficient mouse models, Exo1(mut) mouse HSC did not gain a competitive survival advantage post temozolomide/BG treatment in vivo. To determine potential nucleases implicated in MMR in the absence of Exo1 nuclease activity, but in the presence of the inactive protein, we performed gene expression analyses of several mammalian nucleases in WT and Exo1(mut) MEFs before and after temozolomide treatment and identified upregulation of Artemis, Fan1, and Mre11. Partial shRNA mediated silencing of each of these in Exo1(mut) cells resulted in decreased MMR capacity and increased resistance to temozolomide/BG. We propose that nuclease function is required for fully functional MMR, but a portfolio of nucleases is able to compensate for loss of Exo1 nuclease activity to maintain proficiency.

EXO modifies sucrose and trehalose responses and connects the extracellular carbon status to growth

Plants have the capacity to adapt growth to changing environmental conditions. This implies the modulation of metabolism according to the availability of carbon (C). Particular interest in the response to the C availability is based on the increasing atmospheric levels of CO2. Several regulatory pathways that link the C status to growth have emerged. The extracellular EXO protein is essential for cell expansion and promotes shoot and root growth. Homologous proteins were identified in evolutionarily distant green plants. We show here that the EXO protein connects growth with C responses. The exo mutant displayed altered responses to exogenous sucrose supplemented to the growth medium. Impaired growth of the mutant in synthetic medium was associated with the accumulation of starch and anthocyanins, altered expression of sugar-responsive genes, and increased abscisic acid levels. Thus, EXO modulates several responses related to the C availability. Growth retardation on medium supplemented with 2-deoxy-glucose, mannose, and palatinose was similar to the wild type. Trehalose feeding stimulated root growth and shoot biomass production of exo plants whereas it inhibited growth of the wild type. The phenotypic features of the exo mutant suggest that apoplastic processes coordinate growth and C responses.

Termination of exonuclease 1‐catalyzed mismatch excision requires physical interaction between exonuclease 1 and MutLα

The human mismatch repair (MMR) reaction was previously reconstituted in vitro using purified recombinant proteins that include MutSα or MutSβ, MutLα, RPA, EXO1, HMGB1, PCNA, RFC, polymerase δ, and DNA ligase I. Although MutLα is not essential for 5′ nick‐directed excision, it promotes termination of mismatch‐provoked EXO1‐ catalyzed DNA excision upon mismatch removal. However, the mechanism of the excision termination reaction is unknown. This study examines whether the physical interaction between the MLH1 subunit of MutLα and EXOI, which is mediated by a conserved motif in EXO1 known as an MLH1 interacting protein (MIP)‐box, is responsible for the excision termination. For this purpose, alanine substitution mutations were engineered at residues F506 and F507 in the MIP‐box of EXO1, and the resulting FF‐EXO‐AA mutant protein was tested for its role in MMR in vitro. As expected, FF‐EXO1‐AA behaves similarly as wild type EXO1 in catalyzing 5′‐directed excision and interacting with MutSα, but the EXO1 mutant protein fails to interact with MutLα. Our in vitro MMR assay show that mismatch‐provoked excision is efficiently terminated in reactions containing wild type EXO1, but not in reactions containing FF‐EXO1‐ AA. These observations strongly suggest that the termination of EXO1‐catalyzed mismatch excision is executed via physical interaction between MutLα and EXO1.

Potential Application in Animal Feed of Phytase Produced from Agro-Industrial Residues by Aspergillus japonicus

of the plant wall cell, as cellulose, hemicellulose, lignin and pectins are degraded by enzymatic systems produced by a wide variety of saprophytic and phythopathogenic microorganisms, including bacteria and fungi. The aim of this work was to produce avicelase, carboxymethylcellulase, xylanase and pectinase from the exo-1 mutantNeurospora crassa, FGSC 2256 and thewild typeNeurospora crassa, FGSC 424. The exo-1 mutant has a pleiotropic mutation which confers derepression of alpha-amylase, glucoamylase, betafructofuranosidase, and trehalase, as previously documented. This mutation appears to also affect the composition of the cell wall, since segregants resulting from the backcross of exo-1 to the wild-type strain from which it derived are altered in the ratio of galactosamine to glucosamine in the cell wall. Conidial cell walls of exo-1 mutant exhibit a marked decrease in the amount of galactosamine. In this work, increased levels (approximately sixfold) of pectinasewere found in conidia of exo-1 as comparedwith those of thewild type andalso thepolysaccharide-degrading activitieswere preferentially induced and secreted by the exo-1mutant compared with wild type cells, in the presence of inducers. Then, in culture media supplemented with 1% avicel from the exo-1 mutant were observed higher levels of avicelase (10.6-fold), carboxymethylcellulase (8.92-fold) and xylanase (4.74-fold) than in the wild type. Galactose was better inducer of pectinase than pectin and the exo1 mutant produced three-fold more polygalacturonase than the wild type. Constitutive levels of polygalacturonase and cellulolytic enzymes were detected in medium devoid of carbon source. This results show the importance of composition of the fungal cell wall in the regulation and secretion of industrial enzymes with application in the biomass treatment and biofuel biotechnology. Support: FAPESP and CNPq

Production of plant cell wall degrading enzymes by the exo-1 mutant and wild type Neurospora crassa

Production of plant cell wall degrading enzymes by the exo-1 mutant and wild type Neurospora crassa

The extracellular EXO protein mediates cell expansion in Arabidopsis leaves

BackgroundThe EXO (EXORDIUM) gene was identified as a potential mediator of brassinosteroid (BR)-promoted growth. It is part of a gene family with eight members in Arabidopsis. EXO gene expression is under control of BR, and EXO overexpression promotes shoot and root growth. In this study, the consequences of loss of EXO function are described.ResultsThe exo loss of function mutant showed diminished leaf and root growth and reduced biomass production. Light and scanning electron microscopy analyses revealed that impaired leaf growth is due to reduced cell expansion. Epidermis, palisade, and spongy parenchyma cells were smaller in comparison to the wild-type. The exo mutant showed reduced brassinolide-induced cotyledon and hypocotyl growth. In contrast, exo roots were significantly more sensitive to the inhibitory effect of synthetic brassinolide. Apart from reduced growth, exo did not show severe morphological abnormalities. Gene expression analyses of leaf material identified genes that showed robust EXO-dependent expression. Growth-related genes such as WAK1, EXP5, and KCS1, and genes involved in primary and secondary metabolism showed weaker expression in exo than in wild-type plants. However, the vast majority of BR-regulated genes were normally expressed in exo. HA- and GFP-tagged EXO proteins were targeted to the apoplast.ConclusionThe EXO gene is essential for cell expansion in leaves. Gene expression patterns and growth assays suggest that EXO mediates BR-induced leaf growth. However, EXO does not control BR-levels or BR-sensitivity in the shoot. EXO presumably is involved in a signalling process which coordinates BR-responses with environmental or developmental signals. The hypersensitivity of exo roots to BR suggests that EXO plays a diverse role in the control of BR responses in the root.

A mutation in EXO1 defines separable roles in DNA mismatch repair and post-replication repair

Replication forks stall at DNA lesions or as a result of an unfavorable replicative environment. These fork stalling events have been associated with recombination and gross chromosomal rearrangements. Recombination and fork bypass pathways are the mechanisms accountable for restart of stalled forks. An important lesion bypass mechanism is the highly conserved post-replication repair (PRR) pathway that is composed of error-prone translesion and error-free bypass branches. EXO1 codes for a Rad2p family member nuclease that has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity. In this report, we show EXO1 functions in the MMS2 error-free branch of the PRR pathway independent of the role of EXO1 in DNA mismatch repair (MMR). Consistent with the idea that EXO1 functions independently in two separate pathways, we defined a domain of Exo1p required for PRR distinct from those required for interaction with MMR proteins. We then generated a point mutant exo1 allele that was defective for the function of Exo1p in MMR due to disrupted interaction with Mlh1p, but still functional for PRR. Lastly, by using a compound exo1 mutant that was defective for interaction with Mlh1p and deficient for nuclease activity, we provide further evidence that Exo1p plays both structural and catalytic roles during MMR.

Is hEXO1 a Cancer Predisposing Gene?

Is <i>hEXO1</i> a Cancer Predisposing Gene?

Exopolysaccharide synthesis in Rhizobium leguminosarum bv. trifolii is related to various metabolic pathways

Rhizobium leguminosarum bv. trifolii synthesizes extracellular polysaccharide (EPS) that is postulated to be a biologically active signalling molecule in clover symbiosis. A group of seven exopolysaccharide-deficient (Exo), non-nitrogen-fixing mutants of R. leguminosarum bv. trifolii strain 24.1 isolated by transposon mutagenesis were complemented to mucoid phenotype by a low-copy plasmid carrying the pssA gene encoding the first glucosyl-IP-transferase. Some of these mutants were not corrected in their symbiotic defect by the pssA gene. Precise localization of Tn 5 insertion sites by subcloning and sequencing the adjacent genomic DNA in the Exo mutants identified the disrupted genes and their possible functions. Only one mutant (Rt74) was mutated in pssA gene; others were mutated in diverse genes that were not directly involved in EPS biosynthesis. The suppression of EPS deficiency in these mutants by additional copies of pssA indicated a possible connection between exopolysaccharide biosynthesis and various metabolic pathways.

Genetic characterization of a mutant of Sinorhizobium fredii strain USDA208 with enhanced competitive ability for nodulation of soybean, Glycine max (L.) Merr.

Sinorhizobium fredii strain USDA208 is a nitrogen-fixing bacterium that forms nodules on roots of soybean and other legume plants. We previously found that the Tn 5-containing mutant 208T3, which was derived from strain USDA208, is both deficient in production of exopolysaccharides and more competitive than the wild-type strain in competing against other rhizobia for nodulation of soybean. We now demonstrate that the transposon insertion of the mutant lies in a locus that is highly homologous to a portion of the exo region, which functions in exopolysaccharide biosynthesis by Sinorhizobium meliloti. We sequenced 2906 bp surrounding the insertion site and identified three genes: exoA, exoM, and exoO. The transposon lies within exoM, a glucosyl transferase. A cosmid containing exoHKLAMONP of S. meliloti restores exopolysaccharide production by mutant 208T3 to wild-type levels. Although exo mutants of S. meliloti are defective in their abilities to form indeterminate nodules, the capacities of mutant 208T3 and its wild-type parent to form such nodules on five legume species are indistinguishable. Thus the symbiotic function of exopolysaccharide in S. fredii appears to differ fundamentally from that in S. meliloti.

Genetic characterization of a mutant of Sinorhizobium fredii strain USDA208 with enhanced competitive ability for nodulation of soybean, Glycine max (L.) Merr.

Sinorhizobium fredii strain USDA208 is a nitrogen-fixing bacterium that forms nodules on roots of soybean and other legume plants. We previously found that the Tn5-containing mutant 208T3, which was derived from strain USDA208, is both deficient in production of exopolysaccharides and more competitive than the wild-type strain in competing against other rhizobia for nodulation of soybean. We now demonstrate that the transposon insertion of the mutant lies in a locus that is highly homologous to a portion of the exo region, which functions in exopolysaccharide biosynthesis by Sinorhizobium meliloti. We sequenced 2906 bp surrounding the insertion site and identified three genes: exoA, exoM, and exoO. The transposon lies within exoM, a glucosyl transferase. A cosmid containing exoHKLAMONP of S. meliloti restores exopolysaccharide production by mutant 208T3 to wild-type levels. Although exo mutants of S. meliloti are defective in their abilities to form indeterminate nodules, the capacities of mutant 208T3 and its wild-type parent to form such nodules on five legume species are indistinguishable. Thus the symbiotic function of exopolysaccharide in S. fredii appears to differ fundamentally from that in S. meliloti.

Regulation of pectic enzymes from theexo-1 mutant strain ofNeurospora crassa: effects of glucose, galactose, and galacturonic acid

The exo-1 mutant of Neurospora crassa produced and secreted pectolytic activities when incubated in the presence of pectin-containing biological materials. This study shows that polygalacturonase, pectate lyase and pectin lyase activities were induced in media supplemented with galactose or galacturonic acid, indicating that these sugars induced the synthesis of pectinases. Pectinesterase activity was undetectable. Polygalacturonase activity was better induced by galactose than by galacturonic acid. The reverse was true for lyase activities. The inducing effect of galactose and galacturonic acid seemed to be different: (i) a mixture of galactose and galacturonic acid synergistically increased the production of pectic enzymes, as compared to that in the presence of one of these sugars; (ii) the inducing effect of galacturonic acid was partially repressed by glucose; (iii) in contrast, the inducing effect of galactose, rather than repressed, was enhanced by the presence of glucose. Altogether, these data point out to a complex mechanism of regulation of pectolytic enzymes by pectin-containing organic substances.

Regulation of pectic enzymes from the exo-1 mutant strain of Neurospora crassa: effects of glucose, galactose, and galacturonic acid

Regulation of pectic enzymes from the exo-1 mutant strain of Neurospora crassa: effects of glucose, galactose, and galacturonic acid

Characterization of galactose-induced extracellular and intracellular pectolytic activities from the exo -1 mutant strain of Neurospora crassa

Pectolytic enzymes from the hyperproducer exo-1 mutant of Neurospora crassa are induced either by pectin or galactose. Galactose-induced pectinases, in contrast with pectin-induced enzymes, are not affected by glucose repression. Here, the pectolytic enzymes induced by galactose were purified and characterized. Extracellular pectolytic activities were separated into two main fractions. Pool I contained lyases, and a polygalacturonase (PG) copurifying as a complex of about 80 kDa (gel filtration). Pool II contained PG only. Under urea-SDS-PAGE the lyases and polygalacturonase from pool I migrated with an apparent MW of 56.2 kDa, and 34.3 kDa, respectively. PG from pool II exhibited an apparent MW of 44.7 kDa. Cell extracts contained PG free of lyase activities. Purified intracellular PG migrated (SDS-PAGE) as a single band of apparent MW of 31.5 kDa. All pectinases were glycoproteins (18.5–39% carbohydrate), with stability and optimum pH at 5–6 and 9–10 for PG and lyases, respectively. Temperature optima were 40–50°C, respectively. All enzymes were inactivated at 60°C, with a half-life from 1.5 to 5 min. Activation energy (Ea) values for extracellular and intracellular PG varied between 0.45 and 2.0 Kcal mol−1. Pool II and intracellular PG and lyases, exhibited a random mechanism of hydrolysis. Pool I PG exhibited an exo character.

Parafusin is a membrane and vesicle associated protein that cycles at exocytosis

In the unicellular eukaryote Paramecium tetraurelia, stimulation of exocytosis leads to Ca2+ activation of an alpha Glc-1-phosphodiesterase that dephosphoglucosylates the phosphoglycoprotein parafusin (PFUS). This process fails in exo mutant nd9 and also in the absence of Ca2+ influx upon stimulation suggesting that PFUS dephosphoglucosylation may be causally related to exocytosis. To further corroborate the hypothesis that PFUS is involved in the molecular events in exocytosis, we used laser confocal scanning microscopy and a PFUS specific peptide antibody to perform localization studies of PFUS in wild type (wt) and mutant Paramecium. In unstimulated wt cells, PFUS was associated both with the exocytic site of the cell membrane and with the membrane of the dense core secretory vesicles. Localization at these two sites was shown to be independent of each other since the exocytosis mutant (exo-) tam8, in which docking of its vesicles is blocked, still showed cell membrane staining. Immunofluorescence and immunoblotting of isolated intact secretory vesicles also revealed PFUS association. Upon stimulation of exocytosis, PFUS dissociated from both the dense core secretory vesicles and the cell membrane in a Ca(2+)-dependent manner. During recovery of exocytic capacity, PFUS reassociated with the newly formed secretory vesicles in the cytoplasm prior to their docking at the exocytic sites. Immunoblot analysis of PFUS during this time showed no changes in levels of the protein. Stimulation of exocytosis in wt in Mg2+ buffer or in the exo- temperature sensitive mutant (nd9) at the non-permissive temperature did not lead to dissociation of the PFUS. We conclude that PFUS is a novel critical component whose cycling probably participates in the molecular exocytic fusion machinery in these cells.

Iron-dependent transcription of the regulatory gene ros of Agrobacterium radiobacter.

Transcription of the regulatory gene ros of Agrobacterium radiobacter requires growth in the presence of Fe although this regulation was not mediated by ros itself. The ros gene repressed its own transcription, independently of the Fe status of the growth media. It was shown that the two cysteine residues in the Ros protein were essential for the complementation of the exopolysaccharide synthesis defect of ros mutant strains. It was found that the mutation in one exo mutant strain that was complemented both by ros and by the "structural" exoY gene was not, in fact, in ros but is in some other, unknown gene. The two cysteines were also essential for the correction of this mutant. This mutant affected the expression of exoY but not of ros.

Cloning and characterization of four genes of Rhizobium leguminosarum bv. trifolii involved in exopolysaccharide production and nodulation.

Four different genes of Rhizobium leguminosarum bv. trifolii strain RBL5599 involved in exopolysaccharide (EPS) production were identified by complementation of Tn5-induced EPS-deficient mutants (Exo mutants) with a cosmid bank. On one cosmid pssA was located, which was found to be almost identical to the pss4 gene from R. leguminosarum bv. viciae VF39 and highly homologous to a family of glycosyl transferases. Two pssA mutants, exo2 and exo4, were characterized and found to produce 19 and 1% of the wild-type amount of EPS, respectively. The three other genes were found to be closely linked on a different complementing cosmid. pssC revealed similarity to exoM and exoW of R. meliloti, both encoding glucosyl transferases involved in the synthesis of succinoglycan. A mutation in this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount. We found an operon closely linked to pssC, consisting of two overlapping genes, pssD and pssE, that is essential for EPS production. Homology of pssD and pssE was found with cps14F and cps14G of Streptococcus pneumoniae, respectively: two genes responsible for the second step in capsule polysaccharide synthesis. Furthermore, pssD and pssE were homologous to the 5' and 3' parts, respectively, of spsK of Sphingomonas S88, which encodes a putative glycosyl transferase. Structural analysis of EPS produced by Exo mutants exo2, exo4, and exo50 showed it to be identical to that of the parental strain RBL5599, with the exception of acetyl groups esterified to one of the glucose residues being absent.

Biochemical characterization of glucoamylase from the hyperproducer exo-1 mutant strain of Neurospora erassa

The Neurospora crassa exo-1 mutant produced maximum extracellular glucoamylase activity in media supplemented with starch as the sole carbon source. The apparent molecular mass of the enzyme was 82 kDa (SDS-PAGE and gel filtration). The enzyme was a glycoprotein with 5.1 % carbohydrate content and exhibited a temperature optimum of 60 °C. The pH optima were 5.4 and 5.0 for glucoamylase and maltase activities, respectively. Cu 2+ inhibited maltase activity while Mn 2+ stimulated glucoamylase activity. The purified enzyme hydrolyzed branched substrates more efficiently than linear substrates. Starch was the best substrate utilized and amylose was hydrolyzed faster than maltose. Kinetic experiments suggested that maltose and starch were hydrolyzed at the same catalytic site.