Functional evenness of N-to-P ratios of evergreen-deciduous mixtures predicts positive non-additive effect on leaf litter decomposition

Plant and Soil - To evaluate the functions of a new biocontrol bacterium, Lysobacter enzymogenes LE16, in the mineralization of soil organic phosphorus (P) and in the stimulation of plant P uptake...     

Molecular characterization and expression in Escherichia coli of three beta-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7

Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular beta-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with beta-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of beta-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product. Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with beta-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45 degrees C and that GluB is most active between pH 4.5 and 5.0 at 41 degrees C. Activity of recombinant proteins against various beta-1,3 glucan substrates indicates that GluA and GluC are most active against linear beta-1,3 glucans, while GluB is most active against the insoluble beta-1,3 glucan substrate zymosan A. These data suggest that the contribution of beta-1,3-glucanases to the biocontrol activity of L. enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of beta-1,3 glucans from fungal cell walls.     

'Lysobacter enzymogenes ssp. cookii ' Christensen 1978 should be recognized as an independent species, Lysobacter cookii sp. nov.

' Lysobacter enzymogenes ssp. cookii ' was proposed by Christensen and Cook in 1978; however, this subspecies name has not been cited in the Approved Lists of Bacterial Names and therefore the nomenclature has not been validated. In our genetic approach to clarify the relationships of the designated type strain of ' L. enzymogenes ssp. cookii ' PAGU 1119 (GenBank accession number ATCC29488 ) within the genus Lysobacter revealed that the strain was closely related to Lysobacter capsici YC5194 ^T (99.4%) rather than L. enzymogenes DSM2043 ^T (97.2%). The value for whole genome DNA–DNA relatedness between strain PAGU 1119 and L. enzymogenes DSM 2043^T or L. capsici YC5194 ^T was 20.7–26.1% or 60.9–62.0%, respectively. Although PAGU 1119 and L. capsici YC5194 ^T showed relatively high DNA relationships, the fatty acid profiles and some phenotypic characteristics were different, and we concluded that PAGU 1119 should be placed in a new species. We therefore propose a new species with the name Lysobacter cookii sp. nov. The type strain is PAGU 1119^T ( ATCC29488 ).     

Comparison of the phosphatases of Lysobacter enzymogenes with those of related bacteria

Lysobacter enzymogenes ATCC 29487 (UASM 495) produces an outer-membrane-associated phosphatase and an excreted phosphatase. The cell-associated enzyme was compared to phosphatases of nine other Gram-negative gliding bacteria and to that of Escherichia coli. The other three species of the genus Lysobacter also produce a particulate, cell-associated phosphatase. Antiserum prepared against the phosphatase from the outer membrane of L. enzymogenes effectively precipitated the phosphatases of two other L. enzymogenes strains and the enzymes of L. antibioticus, L. brunescens and L. gummosus. Some inhibition of the enzyme by the antiserum also was observed. No significant reaction could be detected between the antiserum and the cell-associated phosphatases of species of Cytophaga johnsonae, 'C. compacta', Myxococcus xanthus, E. coli and the excreted phosphatase of L. enzymogenes. The results indicate that the four species of the genus Lysobacter are closely related despite their physiological differences and that the outer-membrane-associated phosphatases of these organisms have different structural characteristics than the phosphatases of the other Gram-negative bacteria that were used. Furthermore, differences in the amino acid compositions of the cell-associated and the excreted phosphatase of L. enzymogenes confirm the immunological results and are in agreement with the physical and chemical differences noted between the two enzymes.     

Characterisation of Lysobacter enzymogenes (Christensen and Cook 1978) strain 3.1T8, a powerful antagonist of fungal diseases of cucumber

Isolate 3.1T8 of Lysobacter enzymogenes (Christensen and Cook 1978), originating from the rhizosphere of cucumber and shown to have the potential to control Pythium aphanidermatum, is described. The strain produces extracellular proteases and lipases and shows high levels of resistance against streptomycin, kanamycin and tetracycline, but not to chloramphenicol. It shows strong in vitro antibiosis against P. aphanidermatum and several other phytopathogenic fungi. In order to identify the isolate, a carbon substrate oxidation profile (Biolog) was generated, and fatty acid methyl ester (FAME) analysis was performed. Also, the 16S rRNA gene was cloned and sequenced. With Biolog and FAME analysis, no assignment to species level was possible, because the species was not in the respective databases. BLAST analysis of the obtained sequence, followed by phylogenetic analysis, using a number of related and unrelated sequences, showed that the isolate was most closely related to Lysobacter enzymogenes (Christensen and Cook 1978).     

Extracellular nucleases of Lysobacter enzymogenes: production of the enzymes and purification and characterization of an endonuclease

Lysobacter enzymogenes produced a nonspecific extracellular nuclease and an extracellular RNAase when grown in tryptone broth. Both enzyme activities appeared after the exponential growth phase of the organism. The addition of RNA to the medium specifically inhibited the production of the nuclease and the addition of phosphate prevented the synthesis of the RNAase. DNA had no effect on the enzyme production. The Lysobacter nuclease was purified 274-fold and its molecular weight was estimated to be between 22 000 and 28 000. Freshly purified nuclease showed one major protein band and one major activity band on polyacrylamide gels, whereas two major bands were seen after prolonged storage of the enzyme. The nuclease was most active at pH 8.0 and required Mg2+ or Mn2+. Little activity was obtained in the presence of Ca2+. The enzyme degraded double-stranded DNA more rapidly than single-stranded DNA or RNA and was essentially inactive with poly(A) or poly(C) as the substrate. Extensive hydrolysis of double-stranded DNA by the enzyme yielded oligodeoxyribonucleotides with terminal 5'-phosphate groups. The Lysobacter RNAase appeared to have a molecular weight approximately twice that of the nuclease and was specific for ribonucleotide polymers.     

Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control

A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonas maltophilia strain 34S1 by heterologous expression in Burkholderia cepacia. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of S. maltophilia strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by chiA. Marker exchange mutagenesis of chiA resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of S. maltophilia.     

Development and application of a TaqMan real-time PCR assay for rapid detection of Magnaporthe poae

In North America, one of the most important root diseases of Poa and Festuca turf is summer patch, caused by Magnaporthe poae. Detection and identification of M. poae in infected roots by conventional culture-based methods is difficult and time consuming, typically taking 3 wk or longer to accomplish. In this study, a culture-independent, TaqMan real-time PCR assay was developed for the detection of M. poae from the roots of fungicide treated and non-treated Kentucky bluegrass (Poa pratensis) turf. The assay was validated with the target pathogen, closely related fungal species and a number of other microorganisms that inhabit the same host and soil environment. This assay was more sensitive (could detect as little as 3.88 pg genomic DNA of M. poae), rapid and accurate compared to direct microscopic observation and isolation on a selective medium. The real-time PCR detection results corresponded closely to visual assessments of disease severity in the field. Utilization of this assay in diagnostic laboratories will enable turfgrass managers to more quickly and effectively detect and potentially reduce fungicide usage through early and accurate identification of the pathogen.     

The use of Tween 20 in a sensitive turbidimetric assay of lipolytic enzymes

A turbidimetric esterase assay was developed using a Tween 20 solution in the presence of CaCl2 and Lysobacter enzymogenes esterase (EC 3.1.1.1) as the enzyme source. The reaction was followed by measuring the increase in the optical density at 500 nm (OD500) due to the hydrolytic release of the fatty acids from Tween 20 and their precipitation as the calcium salts. Concentrations of 1.8% Tween and 3 mM CaCl2 were found to be optimal for the assay of 0.036 to 0.15 esterase units in a 4-mL reaction mixture over a 30-min period. The esterase reactions were linear with time at least up to 1.2 OD500 and the rate of increase in the OD500 was proportional to the enzyme concentration. Low initial reaction rates were seen with low esterase activity, presumably because of the limited solubility of the fatty acid - calcium salt in a 1.8% Tween solution. This turbidimetric method is much simpler and at least 36 times more sensitive than the titrimetric assay with Tween 20, and at least four times more sensitive than a spectrophotometric assay with p-nitrophenyl palmitate. This assay has been used to determine the activities of cell-associated and excreted esterases produced by Lysobacter enzymogenes and Pseudomonas aeruginosa, and of lipolytic enzymes from porcine liver, Chromobacterium viscosum, Candida cylindracea, and wheat germ.     

Analysis of prepro-alpha-lytic protease expression in Escherichia coli reveals that the pro region is required for activity.

The alpha-lytic protease of Lysobacter enzymogenes was successfully expressed in Escherichia coli by fusing the promoter and signal sequence of the E. coli phoA gene to the proenzyme portion of the alpha-lytic protease gene. Following induction, active enzyme was found both within cells and in the extracellular medium, where it slowly accumulated to high levels. Use of a similar gene fusion to express the protease domain alone produced inactive enzyme, indicating that the large amino-terminal pro region is necessary for activity. The implications for protein folding are discussed. Furthermore, inactivation of the protease by mutation of the catalytic serine residue resulted in the production of a higher-molecular-weight form of the alpha-lytic protease, suggesting that the enzyme is self-processing in E. coli.     

Selection of Bacterial Antagonists Using Enrichment Cultures for the Control of Summer Patch Disease in Kentucky Bluegrass

A continuous enrichment culture procedure was used to isolate bacteria from various soil sources capable of suppressing summer patch disease of turfgrass. Cultures consisted of a minimal salts medium supplemented with the mycelium of Magnaporthe poae, the causal agent of summer patch, as the sole carbon source. Percentages of chitinolytic bacteria increased in all enrichments over time. Approximately 30% of randomly selected isolates from enrichment cultures significantly reduced summer patch symptom development compared with untreated controls. Six isolates consistently suppressed summer patch in Kentucky bluegrass by more than 50% compared with controls in repeated experiments. All six isolates colonized the turfgrass rhizosphere in high populations. The six isolates ranged in the spectrum of extracellular enzymes that they expressed. The best disease-suppressing isolate, N4-7, expressed chitinase, glucanase, lipase, and protease activity, and inhibited the growth of M. poae in vitro.     

Characterization of a Chitinase Gene from Stenotrophomonas maltophilia Strain 34S1 and Its Involvement in Biological Control

A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonas maltophilia strain 34S1 by heterologous expression in Burkholderia cepacia. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of S. maltophilia strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by chiA. Marker exchange mutagenesis of chiA resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of S. maltophilia.     

Use of endoproteinase Lys-C from Lysobacter enzymogenes in protein sequence analysis

Endoproteinase Lys-C from Lysobacter enzymogenes, which is commercially available, proved to be useful in the determination of primary structures of proteins. The enzyme preferentially cleaves at the carboxyl side of lysine residues.     

Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

Restriction endonucleases (REases) with 8-base specificity are rare specimens in nature. NotI from Nocardia otitidis-caviarum (recognition sequence 5′-GCGGCCGC-3′) has been cloned, thus allowing for mutagenesis and screening for enzymes with altered 8-base recognition and cleavage activity. Variants possessing altered specificity have been isolated by the application of two genetic methods. In step 1, variant E156K was isolated by its ability to induce DNA-damage in an indicator strain expressing M.EagI (to protect 5′-NCGGCCGN-3′ sites). In step 2, the E156K allele was mutagenized with the objective of increasing enzyme activity towards the alternative substrate site: 5′-GCTGCCGC-3′. In this procedure, clones of interest were selected by their ability to eliminate a conditionally toxic substrate vector and induce the SOS response. Thus, specific DNA cleavage was linked to cell survival. The secondary substitutions M91V, F157C and V348M were each found to have a positive effect on specific activity when paired with E156K. For example, variant M91V/E156K cleaves 5′-GCTGCCGC-3′ with a specific activity of 8.2 × 10⁴ U/mg, a 32-fold increase over variant E156K. A comprehensive analysis indicates that the cleavage specificity of M91V/E156K is relaxed to a small set of 8 bp substrates while retaining activity towards the NotI sequence.     

辣椒溶杆菌X2-3 LC_Clp转录因子功能分析

辣椒溶杆菌(Lysobacter capsici) X2-3是从小麦根际分离的一株对多种病原真菌和卵菌有抑菌活性的菌株,目前对该菌株产生的抗菌物质及其产生调控机制尚不明确。【目的】明确转录因子LC_Clp对该菌株抗菌物质产生的调节作用,为深入了解L. capsici X2-3的生防机制提供依据。【方法】从转座子EZ-Tn5随机插入突变体库中筛选获得X2-3的LC_clp基因突变体M356,通过恢复性克隆获得功能互补菌株,分析LC_clp基因在拮抗活性、胞外酶分泌以及调控基因表达方面的差异。【结果】与X2-3相比,M356对测试病原真菌、卵菌的抑菌活性和产生体外抗菌物质的能力完全丧失,蛋白酶和纤维素酶产生量明显减少,几乎不产几丁质酶;所检测的转录调节因子、次生代谢及胞外酶等相关基因的表达量均显著低于野生株X2-3,而互补菌株MCS28和X2-3水平相当。【结论】LC_Clp不仅与菌株的抗菌物质合成及抑菌活性有关,还影响胞外酶的产生,并调控多种基因的表达,具有广泛的调节作用。     

Structural Investigations and Identification of the Extracellular Bacteriolytic Endopeptidase L1 from Lysobacter sp. XL1

The N-terminal amino acid sequence (23 amino acid residues) and the amino acid composition of the extracellular bacteriolytic enzyme L1 of 21 kD from the bacterium Lysobacter sp. XL1 have been determined. The enzyme was hydrolyzed by trypsin, the resulting peptides were isolated, and their primary structures were determined. A high extent of homology (92%) of the N-terminal amino acid sequence and the primary structure of isolated peptides of the enzyme L1 (62 amino acid residues or 31% of protein sequence) to the corresponding sites of alpha-lytic proteinases (EC 3.4.21.12) of Lysobacter enzymogenes and Achromobacter lyticus was found. These data allowed identification of the endopeptidase L1 of Lysobacter sp. XL1 as alpha-lytic proteinase EC 3.4.21.12.     

Behaviour of α-aminoadipylcysteine and glutamylcysteine in the presence of intact and disrupted mycelium of a Cephalosporium sp

1. delta-(l-alpha-Aminoadipyl)-l-cysteine, the corresponding d- and dl-alpha-aminoadipyl isomers, delta-(dl-alpha-amino[6-(14)C]adipyl)-l-cysteine and gamma- and alpha-l-glutamyl-l-cysteine were synthesized. 2. The behaviour of delta-(l-aminoadipyl)-l-cysteine and the corresponding d- and dl-alpha-aminoadipyl isomers was studied in the presence of suspensions of intact mycelium of a Cephalosporium sp., suspensions treated ultrasonically and extracts obtained by grinding with alumina. 3. With intact mycelium the l-alpha-aminoadipyl isomer was removed more rapidly from the extracellular fluid than the corresponding d-isomer. 4. Addition of delta-(dl-alpha-amino[6-(14)C]adipyl)-l-cysteine to suspensions of intact mycelium led to the labelling of extracellular and intracellular penicillin N and cephalosporin C, but also to extensive hydrolysis of the dipeptide. 5. Broken-cell systems hydrolysed delta-(l-alpha-aminoadipyl)-l-cysteine and the corresponding d-alpha-aminoadipyl isomer, but the former was hydrolysed more readily than the latter. 6. gamma- and alpha-l-Glutamyl-l-cysteine were also hydrolysed but delta-(l-alpha-aminoadipyl)-l-cysteinyl-l-valine was not. 7. Only part of the enzyme activity in broken-cell systems responsible for the hydrolysis of delta-(alpha-aminoadipyl)-l-cysteine was present in the supernatant obtained on centrifugation at 20000g. 8. Possible implications of these findings are discussed.