Arinthip Thamchaipenet

Plant growth enhancing effects by a siderophore-producing endophytic streptomycete isolated from a Thai jasmine rice plant (Oryza sativa L. cv. KDML105).

An endophytic Streptomyces sp. GMKU 3100 isolated from roots of a Thai jasmine rice plant (Oryza sativa L. cv. KDML105) showed the highest siderophore production on CAS agar while phosphate solubilization and IAA production were not detected. A mutant of Streptomyces sp. GMKU 3100 deficient in just one of the plant growth promoting traits, siderophore production, was generated by inactivation of a desD-like gene encoding a key enzyme controlling the final step of siderophore biosynthesis. Pot culture experiments revealed that rice and mungbean plants inoculated with the wild type gave the best enhancement of plant growth and significantly increased root and shoot biomass and lengths compared with untreated controls and siderophore-deficient mutant treatments. Application of the wild type in the presence or absence of ferric citrate significantly promoted plant growth of both plants. The siderophore-deficient mutant clearly showed the effect of this important trait involved in plant–microbe interaction in enhancement of growth in rice and mungbean plants supplied with sequestered iron. Our results highlight the value of a substantial understanding of the relationship of the plant growth promoting properties of endophytic actinomycetes to the plants. Endophytic actinomycetes, therefore, can be applied as potentially safe and environmentally friendly biofertilizers in agriculture.

Maklamicin, an Antibacterial Polyketide from an Endophytic Micromonospora sp.

A new spirotetronate-class polyketide, maklamicin (1), was isolated from the culture extract of an endophytic actinomycete of the genus Micromonospora. The structure and relative configuration of 1 were elucidated by interpretation of NMR and other spectroscopic data, and the absolute configuration was determined using the modified Mosher method. Maklamicin (1) showed strong to modest antimicrobial activity against Gram-positive bacteria.

Optimisation of synergistic biomass-degrading enzyme systems for efficient rice straw hydrolysis using an experimental mixture design

Synergistic enzyme system for the hydrolysis of alkali-pretreated rice straw was optimised based on the synergy of crude fungal enzyme extracts with a commercial cellulase (Celluclast™). Among 13 enzyme extracts, the enzyme preparation from Aspergillus aculeatus BCC 199 exhibited the highest level of synergy with Celluclast™. This synergy was based on the complementary cellulolytic and hemicellulolytic activities of the BCC 199 enzyme extract. A mixture design was used to optimise the ternary enzyme complex based on the synergistic enzyme mixture with Bacillus subtilis expansin. Using the full cubic model, the optimal formulation of the enzyme mixture was predicted to the percentage of Celluclast™: BCC 199: expansin=41.4:37.0:21.6, which produced 769 mg reducing sugar/g biomass using 2.82 FPU/g enzymes. This work demonstrated the use of a systematic approach for the design and optimisation of a synergistic enzyme mixture of fungal enzymes and expansin for lignocellulosic degradation.

Pseudonocardia acaciae sp. nov., isolated from roots of Acacia auriculiformis A. Cunn. ex Benth.

A novel Gram-positive-staining actinomycete designated strain GMKU095(T) was isolated from surface-sterilized roots of Acacia auriculiformis A. Cunn. ex Benth. (earpod wattle). The organism produced branching mycelium. The spores were non-motile and had a spiny surface. Growth of strain GMKU095(T) occurred at 18-42 degrees C, pH 5.0-8.0 and at NaCl concentrations up to 5 %. Whole-cell hydrolysates contained arabinose and galactose as major characteristic sugars. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The glycan moiety of the murein contained acetyl residues. The menaquinone was MK-8(H(4)); mycolic acids were not detected. The G+C content of the DNA was 71.6 mol%. iso-C(16 : 0) was detected as the major cellular fatty acid. Comparative studies of 16S rRNA gene sequences indicated that the strain was phylogenetically related to members of the genus Pseudonocardia. The most closely related type strain is Pseudonocardia spinosispora IMSNU 50581(T), which is 96.2 % similar in 16S rRNA gene sequence. On the basis of the genotypic and phenotypic properties presented, a novel species of the genus Pseudonocardia is proposed, Pseudonocardia acaciae sp. nov. The type strain is strain GMKU095(T) (=NRRL B-24609(T) =BCC 28481(T) =TISTR 1862(T) =NBRC 104274(T)).

Nonomuraea syzygii sp. nov., an endophytic actinomycete isolated from the roots of a jambolan plum tree (Syzygium cumini L. Skeels).

A novel endophytic actinomycete, designated strain GKU 164(T), was isolated from the roots of a jambolan plum tree (Syzygium cumini L. Skeels), collected at Khao Khitchakut National Park, Chantaburi province, Thailand. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain formed a distinct clade within the genus Nonomuraea , and was most closely related to Nonomuraea monospora PT708(T) (98.77% 16S rRNA gene sequence similarity) and Nonomuraea thailandensis KC-061(T) (98.73%). Strain GKU 164(T) formed a branched substrate and aerial hyphae that generated single spores with rough surfaces. The cell wall contained meso-diaminopimelic acid. The whole-cell sugars were madurose, galactose, mannose, ribose, rhamnose and glucose. The N-acyl type of muramic acid was acetyl. The predominant menaquinone was MK-9(H4) with minor amounts of MK-9(H6), MK-9(H2) and MK-9(H0). The phospholipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositolmannosides, phosphatidylmonomethylethanolamine, hydroxy-phosphatidylmonomethylethanolamine, an unidentified aminophosphoglycolipid and four unknown phospholipids. The major fatty acids were iso-C(16 : 0) and 10-methyl C(17 : 0). The genomic DNA G+C content was 70.4 mol%. Significant differences in the morphological, chemotaxonomical, and biochemical data together with DNA-DNA relatedness values between strain GKU 164(T) and type strains of closely related species, clearly demonstrated that strain GKU 164(T) represents a novel species of the genus Nonomuraea , for which the name Nonomuraea syzygii sp. nov. is proposed. The type strain is GKU 164(T) ( = BCC 70457(T) = NBRC 110400(T)).

Binding characteristics and synergistic effects of bacterial expansins on cellulosic and hemicellulosic substrates

Expansins are non-catalytic proteins which loosen plant cell wall structure. In this study, binding kinetics and synergistic action of five bacterial expansins on cellulosic and hemicellulosic polysaccharides were studied. The expansins differed in binding capacity (Bmax) and affinity (Kd) for different substrates. A common pattern of binding efficiency (Bmax/Kd) was found among the expansins tested, in which efficiency was greatest for the phosphoric acid-swollen cellulose (PASC), then the hemicellulose arabinoxylan followed by the microcrystalline cellulose (Avicel PH101). The expansins enhanced the action of Trichoderma reesei cellulase/hemicellulase mixture for degrading all three substrates to varying degrees. Among the substrates and expansins tested, BpEX from Bacillus pumilus and CmEX from Clavibacter michiganensis showed the greatest enhancement effect on arabinoxylan with 11.4 and 12.2-fold greater reducing sugar yield than the reaction with enzyme alone. The work gives insights into the wider application of expansins on enhancing polysaccharide hydrolysis, particularly on hemicellulosic substrates.

Kineococcus gynurae sp. nov., isolated from a Thai medicinal plant.

A novel, Gram-positive, motile, coccus-shaped, orange-pigmented organism, designated strain KKD096(T), was isolated from the roots of a Thai medicinal plant, Gynura pseudochina DC. var. hispida Thwaites. Growth of strain KKD096(T) occurred at temperatures of 14-34 degrees C, at pH 5.0-9.0 and at NaCl concentrations up to 7 % (w/v). Whole-cell hydrolysates contained arabinose and galactose as the characteristic sugars. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. The glycan moiety of the murein contained acetyl residues. The predominant menaquinone was MK-9(H2); mycolic acids were not detected. The genomic DNA G+C content was 73.3 mol%. The major cellular fatty acid was anteiso-C(15 : 0) (81.42 % of the total). Strain KKD096(T) was assigned to the genus Kineococcus on the basis of 16S rRNA gene sequence analysis; it was most closely related to Kineococcus radiotolerans DSM 14245(T) (97.1 % similarity). DNA-DNA hybridization revealed 39.4 % relatedness between these two taxa. On the basis of the genotypic and phenotypic data presented, strain KKD096(T) is considered to represent a novel species of the genus Kineococcus, for which the name Kineococcus gynurae sp. nov. is proposed. The type strain is KKD096(T) (=TISTR 1856(T)=NRRL B-24568(T)=BCC 26245(T)=NBRC 103943(T)).

Diversity of Culturable Plant Growth-Promoting Bacterial Endophytes Associated with Sugarcane Roots and Their Effect of Growth by Co-Inoculation of Diazotrophs and Actinomycetes

Application of environmentally friendly agents to reduce the use of chemicals and to enhance growth of plants is an ultimate goal of sustainable agriculture. The use of plant growth-promoting endophytes has become of great interest as a way to enhance plant growth and additionally protect plants from phytopathogens. In this study, 135 isolates of endophytic bacteria including actinomycetes were isolated from roots of commercial sugarcane plants cultivated in Thailand and were characterized for plant growth-promoting (PGP) traits. Based on morphological and 16S rRNA sequence analysis, the endophytes were distributed into 14 genera of which the most dominant species belong to Bacillus, Enterobacter, Microbispora, and Streptomyces. Two strains of endophytic diazotrophs, Bacillus sp. EN-24 and Enterobacter sp. EN-21; and two strains of actinomycetes, Microbispora sp. GKU 823 and Streptomyces sp. GKU 895, were selected based on their PGP traits including 1-aminocyclopropane-1-decarboxylate deaminase, indole-3-acetic acid, nitrogen fixation, phosphate solubilization, and siderophore production for evaluation of sugarcane growth enhancement by individual and co-inoculation. Sixty days after co-inoculation by endophytic diazotrophs and actinomycetes, the growth parameters of sugarcane plants were significantly greater than that of individual and un-inoculated plants. The results indicated that these endophytes have high potential as PGP agents that could be applied to promote sugarcane growth and could be developed as active added value biofertilizers in the future.

Amycolatopsis samaneae sp. nov., isolated from roots of Samanea saman (Jacq.) Merr.

A novel actinomycete, designated strain RM287(T), was isolated from surface-sterilized roots of Samanea saman (Jacq.) Merr., collected from Bangkok, Thailand. The status of the novel strain was determined using a polyphasic taxonomic approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that the organism formed a distinct phyletic line within the radiation of the genus Amycolatopsis. The 16S rRNA gene sequence similarity indicated that strain RM287(T) was most closely related to Amycolatopsis mediterranei IMSNU 20056(T) (97.4 %), A. rifamycinica DSM 46095(T) (97.2 %), A. kentuckyensis NRRL B-24129(T) (97.2 %), A. pretoriensis DSM 44654(T) (97 %) and A. australiensis DSM 44671(T) (97 %). The novel organism was found to have chemical properties typical of members of the genus Amycolatopsis such as meso-diaminopimelic acid as the dignostic diamino acid in the cell-wall peptidoglycan and arabinose and galactose as the diagnostic sugars. The major menaquinone was MK-9(H(4)). The major fatty acids were iso-C(16 : 0) iso-C(15 : 0), iso 2-OH-C(16 : 0) and iso-C(17 : 0). The DNA G+C content was 71.7 mol%. Phenotypic data clearly distinguished the novel isolate from its closest relatives. The combined genotypic and phenotypic data indicated that strain RM287(T) represented a novel species of the genus Amycolatopsis. The proposed name for this organism is Amycolatopsis samaneae sp. nov., with the type strain RM287(T) ( = TISTR 1919(T) = BCC 35842(T) = NBRC 106095(T)).

Conjugation in Escherichia coli

Bacterial conjugation is a genetic transfer that involves cell‐to‐cell between donor and recipient cells. With the current method used to teach students in genetic courses at the undergraduate level, the transconjugants are identified using bacterial physiology and/or antibiotic resistance. Using physiology, however, is difficult for both first‐year undergraduates and special science students at the high school levels, who do not have the basic knowledge. We have developed a laboratory exercise that comprises a simple and rapid technique for transferring bacterial DNA by conjugation and examining the transconjuants using only antibiotic resistance on agar. The identity of the right transconjuants is confirmed by agarose gel electrophoresis. This exercise is designed to help students understand how horizontal gene transfer occurs in bacteria by conjugation using Escherichia coli as a hands‐on learning model. Students should be able to draw concept maps of three DNA transfer methods on their own after carrying out the experiment and getting some additional information.