Incidence of Agrobacterium tumefaciens Biovar 1 in and on 'Paradox' (Juglans hindsii × Juglans regia) Walnut Seed Collected from Commercial Nurseries.

The walnut rootstock 'Paradox' (Juglans hindsii × J. regia) is susceptible to Agrobacterium tumefaciens, which often results in a high incidence of crown gall in nursery or walnut production orchards. Though A. tumefaciens is susceptible to the commonly used preplant soil fumigants, crown gall incidence can rise above acceptable levels. We examined the ability of Paradox seed to acquire A. tumefaciens as a function of harvest method used prior to planting. Over a 2-year period at two participating commercial nurseries, Paradox seed were collected directly from the mother tree without contacting the soil or gathered after sitting on the orchard floor for up to 28 days. A. tumefaciens was never detected in or on the 2,650 seeds collected directly from the mother tree. Both virulent and avirulent A. tumefaciens strains were detected in and on the husk of nuts incubated on the orchard floor at a frequency directly proportional to the time spent on the orchard floor. Regardless of A. tumefaciens contamination in or on the husk, A. tumefaciens was never detected in the seed interior. Avoiding soilborne populations of A. tumefaciens at the time of seed collection will play an important role in managing crown gall.

Cationic Surfactants: Potential Surface Disinfectants to Manage Agrobacterium tumefaciens Biovar 1 Contamination of Grafting Tools.

Nursery production of walnut seedlings is a 2-year process, during which crown gall, caused by Agrobacterium tumefaciens, often develops at grafting wounds. In this study, the spread of crown gall via contaminated tools and the efficacy of several disinfectants against A. tumefaciens were demonstrated. The cationic surfactants benzalkonium chloride (BC), cetyltrimethylammonium bromide (CTAB), and Physan 20 eliminated 100% of the A. tumefaciens population in water suspensions treated at 7, 5, and 2 ppm, respectively. Sodium hypochlorite eliminated 100% of the A. tumefaciens population at 0.5 ppm. Sodium hypochlorite efficacy, however, was reduced by 64% in the presence of total solids (0.7 g/ml) which are commonly found in field situations. At similar concentrations of total solids, the efficacy of cationic surfactants decreased, on average, by only 13%. The minimum effective treatment needed to eliminate A. tumefaciens on infested scalpels was a 5-s exposure to BC or CTAB at 5,000 ppm (0.5%). Infested scalpels treated with BC or CTAB at less than 5,000 ppm caused gall formation in 14 ± 7% of cuts made on Datura stramonium stems. This was significantly less than the tumor incidence (100%) in cuts made with inoculated blades not treated BC or CTAB.

Soil Solarization and Biological Control for Managing Mesocriconema xenoplax and Short Life in a Newly Established Peach Orchard.

The effects of soil solarization, with and without a Pseudomonas spp. cocktail or wheat rotation as alternatives to chemical control of Mesocriconema xenoplax, were investigated from 2004 to 2011. Preplant solarization and soil fumigation (67% methyl bromide + 33% chloropicrin mixture; henceforth, referred to as MBr) was initiated in 2004 in an orchard infested with M. xenoplax and a history of peach tree short life (PTSL). Plots consisted of nine treatments: (i) nonsolarized soil-alone, (ii) nonsolarized soil with bacteria cocktail (nonsolar-bacteria), (iii) nonsolarized soil with wheat (nonsolar-wheat), (iv) nonsolarized soil with bacteria cocktail and wheat (nonsolar-bacteria-wheat), (v) solarized soil-alone, (vi) solarized soil with bacteria cocktail solar-bacteria), (vii) solarized soil with wheat (solar-wheat), (viii) solarized soil with bacteria cocktail and wheat (solar-bacteria-wheat), and (ix) preplant MBr fumigation. Peach trees were planted into all plots in 2005. Nematode populations were suppressed 20 months longer after orchard establishment in solar-alone and solar-wheat plots than solar-bacteria and solar-bacteria-wheat plots. Pseudomonas spp. cocktails did not have a pronounced effect in suppressing M. xenoplax in this study. Fumigation effect on M. xenoplax population density dissipated 24 months after application. Solar-wheat-treated soil was as effective as preplant MBr fumigation in increasing tree survival from PTSL for at least 6 years after orchard establishment.

Role of Systemic Agrobacterium tumefaciens Populations in Crown Gall Incidence on the Walnut Hybrid Rootstock 'Paradox'.

Greater than 75% of English walnut production in the United States occurs on the walnut rootstock Juglans hindsii × J. regia 'Paradox', which is highly susceptible to infection by Agrobacterium tumefaciens. When seed were germinated and grown in the presence of A. tumefaciens, in the absence of wounding, 94% of the seedlings exhibited tumors while 89% contained systemic A. tumefaciens populations. When seedlings were wound inoculated, A. tumefaciens established endophytic populations in stem tissue and often migrated from the site of infection. Distribution of A. tumefaciens in the stem was random and may exhibit seasonal variation. A. tumefaciens populations in root tissue were more readily detected than in stem tissue and may serve as a reservoir for subsequent infection of the aerial portions of the tree. Importantly, 7% of inoculated, asymptomatic seedlings contained endophytic populations of A. tumefaciens. In all, 17% of seedlings inoculated as seeds developed galls at secondary stem-wound sites. These results provide an ecological and epidemiological foundation upon which to modify existing tree-handling practices in both nursery and orchard production environments to manage crown gall incidence.

Physical mapping, BAC-end sequence analysis, and marker tagging of the soilborne nematicidal bacterium, Pseudomonas synxantha BG33R.

A bacterial artificial chromosome (BAC) library was constructed for the genome of the rhizosphere-inhabiting fluorescent pseudomonad Pseudomonas synxantha BG33R. Three thousand BAC clones with an average insert size of 140 kbp and representing a 70-fold genomic coverage were generated and arrayed onto nylon membranes. EcoRI fingerprint analysis of 986 BAC clones generated 23 contigs and 75 singletons. Hybridization analysis allowed us to order the 23 contigs and condense them into a single contig, yielding an estimated genome size of 5.1 Mb for P. synxantha BG33R. A minimum-tile path of 47 BACs was generated and end-sequenced. The genetic loci involved in ring nematode egg-kill factor production in BG33R Tn5 mutants, 246 (vgrG homolog), 1122 (sensor kinase homolog), 1233 (UDP-galactose epimerase homolog), 1397 (ferrisiderophore receptor homolog), and 1917 (ribosomal subunit protein homolog), have been mapped onto the minimum-tile BAC library. Two of the genetic regions that flank Tn5 insertions in BG33R egg-kill-negative mutants 1233 and 1397 are separated by a single BAC clone. Fragments isolated by ligation-mediated PCR of the Tn5 mutagenized regions of 29 randomly selected, non-egg-kill-related, insertion mutants have been anchored onto the ordered physical map of P. synxantha.

Biological control of the phytoparasitic nematode Mesocriconema xenoplax on peach trees.

Seven fluorescent Pseudomonas spp. capable of inhibiting reproduction of Mesocriconema xenoplax have been isolated from soil sites that suppress both nematode multiplication and Peach Tree Short Life (PTSL). One of these seven strains, Pseudomonas sp. BG33R, inhibits M. xenoplax multiplication in vivo and egg hatch in vitro. Mesocriconema xenoplax populations on peach seedlings inoculated with BG33R and planted into soil-solarized field plots remained at or below the economic threshold for nematicide treatment in South Carolina for nearly 18 months. Soil solarization alone induced a shift toward a microbial community that was suppressive to nematode multiplication. Additionally, five Tn5 mutants of BG33R, lacking the ability to kill eggs, have been generated. The Tn5 insertion site in each mutant has been cloned and sequenced. DNA sequence analysis has revealed a high degree of homology to several genes of interest because of their potential involvement in the production of the egg-kill factor. These Tn5 egg-kill negative mutants also no longer produce protease or salicylic acid while producing nearly twice the amount of fluorescent siderophore as the wild type parent.

Substrate specificity in glycoside hydrolase family 10. Structural and kinetic analysis of the Streptomyces lividans xylanase 10A.

Endoxylanases are a group of enzymes that hydrolyze the beta-1, 4-linked xylose backbone of xylans. They are predominantly found in two discrete sequence families known as glycoside hydrolase families 10 and 11. The Streptomyces lividans xylanase Xyl10A is a family 10 enzyme, the native structure of which has previously been determined by x-ray crystallography at a 2.6 A resolution (Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D., and Derewenda, Z. S. (1994) J. Biol. Chem. 269, 20811-20814). Here, we report the native structure of Xyl10A refined at a resolution of 1.2 A, which reveals many features such as the rare occurrence of a discretely disordered disulfide bond between residues Cys-168 and Cys-201. In order to investigate substrate binding and specificity in glycoside hydrolase family 10, the covalent xylobiosyl enzyme and the covalent cellobiosyl enzyme intermediates of Xyl10A were trapped through the use of appropriate 2-fluoroglycosides. The alpha-linked intermediate with the nucleophile, Glu-236, is in a (4)C(1) chair conformation as previously observed in the family 10 enzyme Cex from Cellulomonas fimi (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The different interactions of Xyl10A with the xylobiosyl and cellobiosyl moieties, notably conformational changes in the -2 and -1 subsites, together with the observed kinetics on a range of aryl glycosides, shed new light on substrate specificity in glycoside hydrolase family 10.

Development of an Escherichia coli expression system and thermostability screening assay for libraries of mutant xylanase.

A thermostability screening assay was developed using an Escherichia coli expression system to express Streptomyces lividans xylanase A (XlnA). The screening system was tested using mutants randomized at position 49 of the S. lividans XlnA gene, a position previously shown to confer thermostability with a I49P point mutation. The library was cloned into an E. coli expression vector and transformed into XL1-blue bacteria. The resulting clones were screened for increased thermostability with respect to wild-type XlnA. Using this assay, we isolated the I49P mutant previously shown to be thermostable, as well as novel I49A and I49C mutants. The I49A and I49C mutants were shown to have 2.8- to 8-fold increase in thermostability over that of wild-type XlnA. The results show that the screening assay can selectively enrich for clones with increased thermostability and is suitable for screening small- to medium-sized libraries of 5000-20,000 clones.

Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans.

The role of four aromatic residues (W85, Y172, W266 and W274) in the structure-function relationship in xylanase A from Streptomyces lividans (XlnA) was investigated by site-directed mutagenesis where each residue was subjected to three substitutions (W85A/H/F; W266A/H/F; W274A/H/F and Y172A/F/S). These four amino acids are highly conserved among family 10 xylanases and structural data have implicated them in substrate binding at the active site. Far-UV circular dichroism spectroscopy was used to show that the overall structure of XlnA was not affected by any of these mutations. High-performance liquid chromatographic analysis of the hydrolysis products of birchwood xylan and xylopentaose showed that mutation of these aromatic residues did not alter the enzyme's mode of action. As expected, though, it did reduce the affinity of XlnA for birchwood xylan. A comparison of the kinetic parameters of different mutants at the same position demonstrated the importance of the aromatic nature of W85, Y172 and W274 in substrate binding. Replacement of these residues by a phenylalanine resulted in mutant proteins with a K(M) closer to that of the wild-type protein in comparison with the other mutations analyzed. The kinetic analysis of the mutant proteins at position W266 indicated that this amino acid is important for both substrate binding and efficient catalysis by XlnA. These studies also demonstrated the crucial role of these active site aromatic residues for the thermal stability of XlnA.

Site-directed mutagenesis study of a conserved residue in family 10 glycanases: histidine 86 of xylanase A from Streptomyces lividans.

Xylanases from family 10 glycanases contain three conserved histidine residues in their active site. The role of H86 in the structure-function of xylanase A from Streptomyces lividans (XlnA) was studied by site-directed mutagenesis. Six mutant proteins (H86A/E/F/K/Q/W) were produced, purified and characterized. The six mutations reduced the affinity of XlnA towards xylan without having any major effect on the catalytic constant. All these mutations also lowered the pKa of the acid-base catalyst by 0.46-1.94 pH units. The mutations decreased the enzyme stability at 60 degrees C by up to 95% and the transition temperature by 2.2-5.8 degrees C. Unfolding of the protein with guanidine hydrochloride (GdnxHCl) showed that five out of six mutations decreased the concentration required to denature 50% of the XlnA, confirming the importance of H86 for the stability of the enzyme. The increase in m value ¿m=d(deltaG)/d[GdnxHCl]¿ also suggested the involvement of residue H86 in the structure of the denatured state of XlnA. It can be concluded from this study that this active site residue was conserved in family 10 glycanases for its function in maintaining the elevated pKa of the acid-base catalyst and in the stability of the protein, while being of little importance for the activity.

Substrate-binding domains of glycanases from Streptomyces lividans: characterization of a new family of xylan-binding domains.

The substrate-binding domains of six glycanases from Streptomyces lividans were investigated to determine their specificity towards cellulose and xylan. Based upon amino acid sequence similarities, four of the six domains could be assigned to existing cellulose-binding domain families. However, the binding domains of xylanase A and arabinofuranosidase B could not be classified in any of the known families and should therefore be classified as members of a new family. Evidence is also presented that this new family is one of true xylan-binding domains.

Insect-Mediated Dispersal of the Rhizobacterium Pseudomonas chlororaphis.

ABSTRACT The southern corn rootworm, Diabrotica undecimpunctata subsp. howardi, a common and mobile insect pest, was shown to transmit the rhizobacte-rium Pseudomonas chlororaphis strain L11 between corn plants. Strain L11 has been genetically modified to contain the lacZY genes from Escherichia coli. It can reach high densities on roots and invade the roots and move into the foliage. D. undecimpunctata subsp. howardi became infested with L11 as larvae while feeding on roots of seed-inoculated corn and retained the bacteria through pupation, molting to the adult stage, and emergence from the soil. Bacterial densities on or in the insects increased 100-fold after they fed again as adults on L11-infested foliage. Adults retained the bacteria for at least 2 weeks after last exposure and could transmit L11 to new plants. The likelihood of transmission decreased with time since last exposure to L11, but increased with time spent on the new plants. This research demonstrates that rhizobacteria can escape the rhizosphere by moving in or onto foliage, where they can then be acquired and transmitted by insects. This transmission route may be common among naturally occurring rhizobacteria and facilitate the dispersal of both beneficial and harmful soilborne microorganisms.

Endo-beta-1,4-xylanase families: differences in catalytic properties.

Microbial endo-beta-1,4-xylanases (EXs, EC 3.2.1.8) belonging to glycanase families 10 (formerly F) and 11 (formerly G) differ in their action on 4-O-methyl-D-glucurono-D-xylan and rhodymenan, a beta-1,3-beta-1,4-xylan. Two high molecular mass EXs (family 10), the Cryptococcus albidus EX and XlnA of Streptomyces lividans, liberate from glucuronoxylan aldotetrauronic acid as the shortest acidic fragment, and from rhodymenan an isomeric xylotriose of the structure Xyl beta 1-3Xyl beta 1-4Xyl as the shortest fragment containing a beta-1,3-linkage. Low molecular mass EXs (family 11), such as the Trichoderma reesei enzymes and XlnB and XlnC of S. lividans, liberate from glucuronoxylan an aldopentauronic acid as the shortest fragment, and from rhodymenan an isomeric xylotetraose as the shortest fragment containing a beta-1,3-linkage. The structure of the oligosaccharides was established by: NMR spectroscopy, mass spectrometry of per-O-methylated compounds and enzymic hydrolysis by beta-xylosidase and EX, followed by analysis of products by chromatography. The structures of the fragments define in the polysaccharides the linkages attacked and non-attacked by the enzymes. EXs of family 10 require a lower number of unsubstituted consecutive beta-1,4-xylopyranosyl units in the main chain and a lower number of consecutive beta-1,4-xylopyranosyl linkages in rhodymenan than EXs of family 11. These results, together with a greater catalytic versatility of EXs of family 10, suggest that EXs of family 10 have substrate binding sites smaller than those of EXs of family 11. This suggestion is in agreement with the finding that EXs of family 10 show higher affinity for shorter linear beta-1,4-xylooligosaccharides than EXs of family 11. The results are discussed with relevant literature data to understand better the structure-function relationship in this group of glycanases.

Characterization of two important histidine residues in the active site of xylanase A from Streptomyces lividans, a family 10 glycanase.

The active site of xylanase A (XlnA) from Streptomyces lividans contains three histidine residues, two of which (H81 and H207) are almost completely conserved in family 10 glycanases. The structural analysis of the enzyme shows that H81 and H207 are part of an important hydrogen bond network in the vicinity of the two catalytic residues (E128 and E236). In order to investigate the role of these two histidine residues for the structure/function of XlnA, three mutant enzymes were produced at each position, namely, H81R/S/Y and H207E/K/R. The specific activity of these mutant enzymes is reduced by more than 95%, revealing the importance of these two residues for the catalytic function of XlnA. The kinetic parameters of the three more active enzymes were determined, of which mutation H207K increased the K(M) 3-fold. The k(cat) of the mutant enzymes is reduced proportionally to the specific activity. Furthermore, the pKa values of the two catalytic residues are decreased in all six mutations, demonstrating a role for H81 and H207 in the hydrogen bond network responsible for maintaining the ionization state of the two catalytic residues. In most cases, the unfolding of mutated XlnA in guanidine hydrochloride (Gdn-HCl) showed that the concentration required to denature 50% of the XlnA decreased, thus demonstrating the importance of those two residues for the stability of the enzyme. Moreover, the m value [m = d(deltaG)/d[Gdn-HCl]] for the unfolding of XlnA in Gdn-HCl is increased for each of the six mutations, suggesting that the mutant proteins have less residual structure in the denatured state than does the wild-type enzyme.

Asparagine-127 of xylanase A from Streptomyces lividans, a key residue in glycosyl hydrolases of superfamily 4/7: kinetic evidence for its involvement in stabilization of the catalytic intermediate.

Site-directed mutagenesis of asparagine-127 (N127) of xylanase A (XlnA) from Streptomyces lividans, belonging to family 10 and superfamily 4/7 of glycosyl hydrolases, was chosen to study the role of this conserved residue. The isosteric mutation N127D introduced did not affect the fold of XlnA as revealed by circular dichroism. Comparison of the kinetic constants of N127D and wild-type XlnA revealed a 70-fold decrease in the specificity constant (kcat/K(M)) towards birchwood xylan, which is attributed solely to the difference in the kcat value and indicates a role of N127 in stabilization of the catalytic intermediate. N127 also plays a role in maintaining the ionization states of the two catalytic residues, as shown by the modified pH profile of XlnA-N127D. Characterization of XlnA-N127D and the analysis of the three-dimensional structure of XlnA converge towards a stabilization role for N127 in the catalytic site of XlnA.

New alpha-L-arabinofuranosidase produced by Streptomyces lividans: cloning and DNA sequence of the abfB gene and characterization of the enzyme.

A fully secreted alpha-l-arabinofuranosidase was cloned from the homologous expression system of Streptomyces lividans. The gene, located upstream adjacent to the previously described xylanase A gene, was sequenced. It is divergently transcribed from the xlnA gene and the two genes are separated by an intercistronic region of 391nt which contains a palindromic AT-rich sequence. The deduced amino acid sequence of the protein shows that the enzyme contains a distinct catalytic domain which is linked to a specific xylan-binding domain by a linker region. The purified enzyme has a specific arabinofuranose-debranching activity on xylan from Gramineae, acts synergistically with the S. lividans xylanases and binds specifically to xylan. From small arabinoxylo-oligosides, it liberates arabinose and, after prolonged incubation, the purified enzyme exhibits some xylanolytic activity as well.

Protein secretion in streptomycetes.

Some aspects of the current knowledge on protein secretion in streptomycetes are presented including recent data on the identification of genes in the general secretory pathway, on the importance of the signal peptide structure and on the number of ribosome-binding sites inside signal peptides which can influence the production level of a gene product.

Improved Production of Mannanase by Streptomyces lividans.

Replacement of the natural promoter of the (beta)-mannanase gene of Streptomyces lividans by lacp resulted in a 15-fold increase in enzyme production over that of the previously reported clone S. lividans IAF36, a clone carrying multiple copies of manA, and a 350-fold increase over that of the wild-type strain S. lividans 1326. In addition, the use of lacp in the shuttle vector pIAF199 allowed synthesis of the enzymes on carbon sources that did not contain mannan, such as xylan and whey, which offers interesting possibilities for industrial production of the enzyme.

Substrate specificity and mode of action of acetylxylan esterase from Streptomyces lividans.

The substrate specificity of purified acetylxylan esterase (AcXE) from Streptomyces lividans was investigated on partially and fully acetylated methyl glycopyranosides. The enzyme exhibited deacetylation regioselectivity on model compounds which provided insights pertaining to its function in acetylxylan degradation. The enzyme catalyzed double deacetylation of methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside at positions 2 and 3. Two methyl xylopyranoside diacetates, which had a free hydroxyl group at position 2 or 3, i.e. the derivatives that most closely mimic monoacetylated xylopyranosyl residues in acetylxylan, were deacetylated 1 to 2 orders of magnitude faster than methyl 2,3,4-tri-O-acetyl-beta-D-xylopyranoside and methyl 2,3-di-O-acetyl-beta-D-xylopyranoside. These observations explain the double deacetylation. The second acetyl group is released immediately after the first one is removed from the fully acetylated methyl beta-D-xylo- and -glucopyranoside. The results suggest that in acetylxylan degradation the enzyme rapidly deacetylates monoacetylated xylopyranosyl residues, but attacks doubly acetylated residues much more slowly. Evidence is also presented that the St. lividans enzyme could be the first real substrate-specific AcXE.

Purification and characterization of an acetyl xylan esterase produced by Streptomyces lividans.

The acetyl xylan esterase cloned homologously from Streptomyces lividans [Shareck, Biely, Morosoli and Kluepfel (1995) Gene 153, 105-109] was purified from culture filtrate of the overproducing strain S. lividans IAF43. The secreted enzyme had a molecular mass of 34 kDa and a pI of 9.0. Under the assay conditions with chemically acetylated birchwood xylan the kinetic constants of the enzyme were: specific activity, 715 units/mg, Km 7.94 mg/ml and Vmax 1977 units/mg. Optimal enzyme activity was obtained at 70 degrees C and pH 7.5. Hydrolysis assays with different acetylated substrates showed that the enzyme is specific for deacetylating the O-acetyl group of polysaccharides and is devoid of N-deacetylation activity. Sequential hydrolysis shows that its action is essential for the complete degradation of acetylated xylan by the xylanases of S. lividans.