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.

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.

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.

Increased xylanase yield in Streptomyces lividans: dependence on number of ribosome-binding sites.

The Streptomyces lividans xylanase A (XlnA) signal peptide (sp) was replaced with the signal peptides of either mannanase A (ManA) or cellulase A (CelA), two enzymes secreted by S. lividans. Depending on the location of the ribosome binding sites (RBS) with respect to a potential initiation codon, the length of the putative sps of ManA and CelA is either 34 or 43 amino acids and 27 or 46 amino acids, respectively. The sequence encoding these sps were fused to the xylanase A gene (xlnA). Clones harboring the short sps of ManA and CelA produced as much xylanase as the clone with the control wild-type sp sequence of XlnA. In clones containing the long sps of ManA and CelA, the XlnA production was enhanced 1.5- and 2.5-fold, respectively. These XlnA yields are reduced by half and one third respectively when the internal initiation codons of the long sp sequences of ManA and CelA are mutated. Since these clones exhibited the same transcription levels, the results indicate that both RBSs are used concomitantly in S. lividans to increase the enzyme production at the translational level. However, when the short and long sps of ManA were fused to the long CelA sp sequence, giving constructs containing respectively 3 and 4 RBSs, a decrease in xylanase production was observed.

Mode of action of three endo-beta-1,4-xylanases of Streptomyces lividans.

The mode of action of three genetically distinct endo-beta-1,4-xylanases (EXs) of Streptomyces lividans, XlnA, XlnB and XlnC, belonging to two different xylanase families, was investigated on a variety of polysaccharide and oligosaccharide substrates. Viscosimetric measurements showed that all three enzymes have about the same endo-acting character. Occurrence of multiple pathways of substrate degradation at high concentration of beta-1,4-xylooligosaccharides suggested that all three enzymes were retaining glycanases. The enzymes differed considerably in their mode of action on various heteroxylans and on rhodymenan. XlnA hydrolyzed all tested polysaccharides to a higher degree than XlnB or XlnC, through liberation of smaller hydrolysis products, both linear or branched. XlnA performed much better than XlnB or XlnC, particularly on acetylxylan, liberating large amounts of short acetylated and non-acetylated fragments. XlnB and XlnC liberated from acetylxylan only limited amounts of larger acetylated fragments. XlnA exhibited also much higher catalytic efficiency than the other two EXs on short beta-1,4-xylooligosaccharides. The kinetic parameters and bond-cleavage frequencies determined for xylotriose, xylotetraose and xylopentaose using 1-3H-reducing-end-labelled compounds suggested that the substrate binding site of XlnA is smaller and differently organized than those in XlnB or XlnC. In contrast to XlnB and XlnC, XlnA also exhibited significant aryl-beta-xylosidase activity. No distinctive catalytic properties of either XlnB or XlnC were found which were not inherent also to XlnA. High-molecular-mass EXs of the XlnA type show much greater catalytic versatility due than low-molecular-mass EXs of the XlnB or XlnC type.

Cloning of a secA homolog from Streptomyces lividans 1326 and overexpression in both S. lividans and Escherichia coli.

We cloned a gene encoding a SecA homolog from Streptomyces lividans 1326, a Gram-positive bacterium known to produce large amounts of extracellular proteins. A protein sequence alignment with the other bacterial SecA homologs revealed that S. lividans SecA shares from 39.5 to 44% identity with them, while it shares 34.2 to 37.2% identity with SecA homologs from plastids of algae and plants. We overexpressed the secA gene in S. lividans 1326 and Escherichia coli MM52 and in both cases we observed the production of a protein with an apparent molecular mass of 117.4 kDa. Although S. lividans SecA is similar to E. coli SecA, it does not complement a thermosensitive mutation in the E. coli secA gene. However, a hybrid polypeptide consisting of the N-terminal portion (first 242 amino acids) of the S. lividans SecA and the C-terminal portion (657 a.a.) of the wild-type E. coli SecA was able to complement this mutant.

Cloning of the xylanase gene of Streptomyces lividans.

The xylanase (xln) gene of Streptomyces lividans 1326 was cloned by functional complementation of the xylanase-negative and beta-1,4-glucan-glucanohydrolase-negative double mutant of S. lividans using the multicopy plasmid pIJ702. Three clones had a common 2-kb DNA fragment as determined by restriction mapping and Southern hybridization. These clones secreted a xylanase of Mr 43,000 which reacted with specific anti-xylanase antibodies and corresponded exactly to the enzyme previously isolated from the wild-type strain. The DNA fragment likely carried the full structural gene, the xln promoter and also the regulatory sequence, since the xylanase activity was inducible by xylan. Enzyme levels of up to 380 IU/ml of culture filtrate were obtained.

Analysis of DNA flanking the xlnB locus of Streptomyces lividans reveals genes encoding acetyl xylan esterase and the RNA component of ribonuclease P.

Nucleotide sequencing revealed the gene (axeA) encoding acetyl xylan esterase (AxeA) downstream from xlnB in the Streptomyces lividans DNA insert of plasmid pIAF42. AxeA consists of a catalytic- and a substrate-binding domain separated by a Gly-rich linker. The N terminus showed no significant homology with published esterases and acetyl xylan esterases, but some homology was found with the xylanases XylA and XylD and the NodB protein of Rhizobium species which is involved in the biosynthesis of root nodulation factors. The C terminus of AxeA is highly homologous to the C-termini of xylanases XlnB and TFXA, corresponding to the xylan-binding domain of these enzymes. Furthermore, the RNaseP RNA component was found immediately upstream from xlnB gene.

Cloning of a second xylanase-encoding gene of Streptomyces lividans 66.

A second xylanase-encoding gene (xln) of Streptomyces lividans 66 was cloned by functional complementation of a xylanase/endocellulase-negative double mutant of S. lividans, using the multicopy plasmid pIJ702. Three clones contained a common 3.1-kb DNA fragment encoding the biosynthesis of a 31-kDa xylanase and a 22-kDa protein which was immunorelated. The xylanase represented at least 80% of the total secreted proteins. These three clones also secreted a small amount of the previously reported 43-kDa xylanase which was detected only by using specific antibodies. Most likely, the DNA fragment contained the complete structural xln gene and some regulatory sequences, since the enzymatic activity was repressed by glucose. In the in vitro transcription-translation system, the plasmid pIAF42 encoded an immunoprecipitable 35-kDa xylanase indicating the presence of a 4-kDa signal peptide.

Sequences of three genes specifying xylanases in Streptomyces lividans.

The entire nucleotide (nt) sequences of three genes (xlnA, xlnB and xlnC) of Streptomyces lividans encoding three distinct xylanases (Xln) have been determined. The nt sequences were confirmed by comparing the deduced amino acid (aa) sequences with the ones derived from the N-terminal aa sequences of the mature purified proteins. The N-terminus of the XlnA showed some homology with either the N-termini or the C-termini of eight other Xln and of two exo-glucanases. The N-terminus of XlnB is homologous to that of XlnC and to Xln of seven other microorganisms.

Cloning and sequencing of the secY homolog from Streptomyces lividans 1326.

Two conserved regions of SecY proteins from six Gram+ bacteria were exploited in a PCR-based strategy for isolating a secY homolog from Streptomyces lividans (Sl). The nucleotide sequence of part of a 3.8-kb fragment showed that the secY homolog is flanked, at the 5' end, by the gene encoding ribosomal protein L15 and, at the 3' end, by an adenylate kinase-encoding gene. The deduced gene product of secY would have 437 amino acids (aa) and an M(r) of 47,200. Sl SecY shows 89.5, 56.1, 42 and 40% identity to its homologs from Streptomyces scabies, Brevibacterium flavum, Bacillus subtilis and Escherichia coli, respectively. Promoterprobe analyses indicated that the secY gene probably contains its own promoter.

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.

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.

Evidence for lysozyme-type mechanism of hydrolysis in xylanases.

In the last year several new xylanase three-dimensional structures were solved. Examination of these new structures in combination with recently obtained data from site-directed mutagenesis and kinetic analysis provided insights into the catalytic mechanism of xylanases. It is now possible to determine the type of mechanism by which xylanases hydrolyse a complex substrate such as xylan.

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.

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.

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.

Naphthyridinomycin, a new broad-spectrum antibiotic.

A new antibiotic, naphthyridinomycin, was isolated in crystalline form from the culture filtrate of Streptomyces lusitanus AY B-1026. The antibiotic is active against a large number of both gram-positive and gram-negative bacteria, and inactive against Candida albicans, Trichophyton granulosum and Microsporum gypseum. The antibiotic is toxic in mice.

Increase in catalytic activity and thermostability of the xylanase A of Streptomyces lividans 1326 by site-specific mutagenesis.

The xylanase A gene from Streptomyces lividans was modified by site-directed mutagenesis, selecting for mutations that improved the catalytic activity and thermostability of the enzyme. Mutant notation uses the one-letter abbreviation for amino acids. The first and the last letters represent, respectively, the residue to be changed and the replacing residue. The number indicates the position of the substitution. The mutant enzymes F155Y, R156E, R156K, and N173D were respectively 28, 10, 50, and 25% more active than the wild-type enzyme. In addition, the half-lives at 60 degrees C of the R156E and N173D xylanases were respectively 6 and 40 min longer than that of the wild-type enzyme even in the absence of substrate. The favorable single mutations were combined to generate the double mutants E156/173D and K156/173D, which were 22 and 47% less active than the wild type. However, the activity half-life of the E156/173D enzyme at 60 degrees C was twice that of the xylanase A. The pH-activity profiles of all the mutant xylanases were similar to that of the wild-type enzyme.