Characterization of a novel antibacterial N-acyl amino acid synthase from soil metagenome.

In an effort to isolate novel natural antibiotics, we searched for antibacterial long-chain N-acyl amino acid synthase (NAS) genes from 70,000 soil metagenome clones by Bacillus subtilis-overlaying screening. In an antibacterial cosmid clone, YS92B, a single gene nasYPL was responsible for the production of the Nas. nasYPL was 903 bp long, and the deduced amino acid sequence showed the highest 71% identity with a hypothetical protein from Massilia niastensis. Phylogenetic analysis demonstrated that NasYPL belongs to Group 1 Nas. Heterologous expression of the same nasYPL gene in Escherichia coli and two Pseudomonas strains (P. putida and P. koreensis) conferred antibacterial activities against Listeria monocytogenes, Staphylococcus epidermidis, and Bacillus subtilis. Mass spectral analysis of the antibacterial fractions identified 7 peaks corresponding to long-chain N-acyl tyrosine, 5 peaks to N-acyl phenylalanine, and 3 peaks to N-acyl leucine (or isoleucine) derivatives linked with 7 fatty acids, indicating enzymatic products derived by NasYPL. Therefore, NasYPL expression by host-specific manner may provide applicable antibacterial characteristics to biotechnologically important Pseudomonas strains.

Identification of a novel alkaline amylopullulanase from a gut metagenome of Hermetia illucens.

A novel pullulanase gene, PulSS4, was identified from the gut microflora of Hermetia illucens by a function-based metagenome screening. The PulSS4 gene had an open reading frame of 4455 base pairs, and encoded a mature protein of 1484 amino acids, with a signal peptide sequence of 44 amino acids. The deduced amino acid sequence of PulSS4 gene showed 51% identity with that of the amylopullulanase of Amphibacillus xylanus, exhibiting no significant sequence homology to already known pullulanases. A conserved domain analysis revealed it to be a pullulanase type II with respective active sites at the N-terminal pullulanase and C-terminal amylase domain. PulSS4 was active in the temperature range of 10-50°C, with an optimum activity at 40°C. It was active in the pH range of 6.5-10.5, with optimum pH at 9.0, and retained more than 80% of its original activity in a broad pH range of 5-11 for 24h at 30°C. Also, PulSS4 was highly stable against many different chemical reagents, including 10% polar organic solvents and 1% non-ionic detergents. Overall, PulSS4 is expected to have the strong potential for application in biotechnological industries that require high activity at moderate temperature and alkaline conditions.

Screening and characterization of a novel cellulase gene from the gut microflora of Hermetia illucens using metagenomic library.

A metagenomic fosmid library was constructed using genomic DNA isolated from the gut microflora of Hermetia illucens, a black soldier fly. A cellulase-positive clone, with the CS10 gene, was identified by extensive Congo-red overlay screenings for cellulase activity from the fosmid library of 92,000 clones. The CS10 gene was composed of a 996 bp DNA sequence encoding the mature protein of 331 amino acids. The deduced amino acids of CS10 showed 72% sequence identity with the glycosyl hydrolase family 5 gene of Dysgonomonas mossii, displaying no significant sequence homology to already known cellulases. The purified CS10 protein presented a single band of cellulase activity with a molecular mass of approximately 40 kDa on the SDS-PAGE gel and zymogram. The purified CS10 protein exhibited optimal activity at 50°C and pH 7.0, and the thermostability and pH stability of CS10 were preserved at the ranges of 20~50°C and pH 4.0~10.0. CS10 exhibited little loss of cellulase activity against various chemical reagents such as 10% polar organic solvents, 1% non-ionic detergents, and 0.5 M denaturing agents. Moreover, the substrate specificity and the product patterns by thinlayer chromatography suggested that CS10 is an endo-ß-1,4-glucanase. From these biochemical properties of CS10, it is expected that the enzyme has the potential for application in industrial processes.

Characterization of a novel cold-active esterase isolated from swamp sediment metagenome.

A functional screen of a metagenomic library from "Upo" swamp sediment in Korea identified a gene EstL28, the product of which displayed lipolytic properties on a tributyrin-supplemented medium. The EstL28 sequence encodes a 290 amino acid protein (designated as EstL28), with a predicted molecular weight of 31.3 kDa. The encoded EstL28 protein exhibited the highest sequence similarity (45 %) to a hydrolase found in Streptococcus sanguinis. Phylogenetic analysis indicated that EstL28 belongs to a currently uncharacterized family of esterases. Within the conserved α/ß-hydrolase 6 domain, the EstL28 retains the catalytic triad Ser103-Asp248-His268 that is typical of esterases. The Ser103 residue in the catalytic triad is located in the consensus pentapeptide motif GXSXG. The purified EstL28 enzyme worked optimally at 35 °C and pH 8.5 and remained stable at temperatures lower than 20 °C. The catalytic activity of EstL28 was maximal with p-nitrophenyl butyrate, indicating that it was an esterase. This enzyme also exhibited stable activity in the presence of methanol, ethanol, isopropanol, and dimethyl sulfoxide. Therefore, the level of stability in organic solvents and cold temperature suggests that EstL28 has potential for many biotechnological applications.

Burkholderia denitrificans sp. nov., isolated from the soil of Dokdo Island, Korea.

A novel, Gram-negative, bacterial strain KIS30-44(T) was identified from wet forest soil collected on the Korean island of Dokdo. Growth of the strain was observed at 15-30°C, pH 5-9, 0-3% NaCl, and 950 mM KNO(3). KIS30-44(T) reduced nitrate to nitrogen gas. Analysis of the 16S rRNA gene sequence showed that KIS30-44(T) was phylogenetically related to Burkholderia sacchari, Burkholderia mimosarum, and Burkholderia oxyphila (98.1%, 98.0%, and 98.0% sequence similarity, respectively). The genomic G+C content was 63.5 mol%. KIS30-44(T) exhibited less than 52% DNA-DNA relatedness with the type strains of 9 closely related Burkholderia species. The major isoprenoid quinone was Q-8. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unknown aminolipids. The major fatty acids in KIS30-44(T) were C(16:0), C(18:1) ω7c and summed feature 3 (iso-C(15:0) 2-OH and C(16:1) ω7c), and the strain contained half the amount of C(17:0) cyclo found in the 9 closely related Burkholderia species. The results of these phenotypic, 16S rRNA gene sequence, DNA-DNA hybridization, and chemotaxonomic data indicate that KIS30-44(T) represents a novel species within the genus Burkholderia, for which the name Burkholderia denitrificans (Type strain KIS30-44(T) =KACC 12733(T) =DSM 24336(T)) is proposed.

Novel metagenome-derived carboxylesterase that hydrolyzes ß-lactam antibiotics.

It has been proposed that family VIII carboxylesterases and class C ß-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze ß-lactam antibiotics except nitrocefin, a nonclinical chromogenic substrate. Here, we describe the first example of a novel carboxylesterase derived from a metagenome that is able to cleave the amide bond of various ß-lactam substrates and the ester bond of p-nitrophenyl esters. A clone with lipolytic activity was selected by functional screening of a metagenomic library using tributyrin agar plates. The sequence analysis of the clone revealed the presence of an open reading frame (estU1) encoding a polypeptide of 426 amino acids, retaining an S-X-X-K motif that is conserved in class C ß-lactamases and family VIII carboxylesterases. The gene was overexpressed in Escherichia coli, and the purified recombinant protein (EstU1) was further characterized. EstU1 showed esterase activity toward various chromogenic p-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstU1 could hydrolyze ß-lactam antibiotics. EstU1 was able to hydrolyze first-generation ß-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin, and cefazolin. In a kinetic study, EstU1 showed a similar range of substrate affinities for both p-nitrophenyl butyrate and first-generation cephalosporins while the turnover efficiency for the latter was much lower. Furthermore, site-directed mutagenesis studies revealed that the catalytic triad of EstU1 plays a crucial role in hydrolyzing both ester bonds of p-nitrophenyl esters and amide bonds of the ß-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities.

Characterization of Capsicum annuum recombinant alpha- and beta-tubulin.

There are several conditions which might modulate polymerization to produce polymers having normal lattice structure. In the absence of 1 mM MgCl(2) the assembly was reduced by 36% in Capsicum annuum tubulin (CAnm tubulin). There was no significant difference in the final assembly formation in the presence of 5% to 10% glycerol. However, nucleation rate was slow and apparent study state was achieved lately in the presence of 10% glycerol. Taxol at 100 microM concentration increased 23% tubulin assembly. One millimolar CaCl(2), >or=1% dimethyl sulfoxide (DMSO) and physiologically low temperature reduced CAnm tubulin assembly. A value of 0.089 mg/ml was obtained as critical concentration for polymerization. Benomyl significantly reduced the number of cysteine residues accessible to 5,5'-dithiobis-(2-nitrobenzoic acid); there were 4.77 +/- 0.21 and 3.49 +/- 0.35 residues accessible per tubulin dimer in the presence of 50 and 100 microM benomyl respectively.

Aquitalea denitrificans sp. nov., isolated from a Korean wetland.

A novel bacterium, designated strain 5YN1-3(T), was isolated from wetland peat collected from Yongneup, Korea. The bacterium was facultatively anaerobic, Gram-negative, yellow-coloured, rod-shaped, mesophilic and motile with one polar flagellum. The strain grew optimally at 30 degrees C, at pH 6.0-9.0 and with 0-1 % NaCl (w/v). 16S rRNA gene sequence analysis showed the highest similarity to the sequence from Aquitalea magnusonii TRO-001DR8(T), with 98.7 % sequence similarity. However, strain 5YN1-3(T) showed DNA-DNA relatedness of 43 % (40 % in a reciprocal experiment) with A. magnusonii LMG 23054(T). The strain contained summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c) and C(16 : 0) as major cellular fatty acids. On the basis of DNA-DNA relatedness and physiological and biochemical characterization, strain 5YN1-3(T) should be assigned to a novel species of the genus Aquitalea, for which the name Aquitalea denitrificans sp. nov. is proposed. The type strain is 5YN1-3(T) (=KACC 12729(T) =DSM 21300(T)).

Alpha- and beta-tubulin from Phytophthora capsici KACC 40483: molecular cloning, biochemical characterization, and antimicrotubule screening.

Internal fragments of alpha- and beta-tubulin genes were generated using reverse transcription polymerase chain reaction (RT-PCR), and the termini were isolated using 5'- and 3'-rapid amplification of cDNA ends. Phytophthora capsici alpha- and beta-tubulin specific primers were then used to generate full-length cDNA by RT-PCR. The recombinant alpha- and beta-tubulin genes were expressed in Escherichia coli BL21 (DE3), purified under denaturing conditions, and average yields were 3.38-4.5 mg of alpha-tubulin and 2.89-4.0 mg of beta-tubulin, each from 1-l culture. Optimum conditions were obtained for formation of microtubule-like structures. A value of 0.12 mg/ml was obtained as the critical concentration of polymerization of P. capsici tubulin. Benomyl inhibited polymerization with half-maximal inhibition (IC(50)) = 468 +/- 20 microM. Approximately 18.66 +/- 0.13 cysteine residues per tubulin dimer were accessible to 5,5'-dithiobis-(2-nitrobenzoic acid), a quantification reagent of sulfhydryl and 12.43 +/- 0.12 residues were accessible in the presence of 200 microM benomyl. The order of preference for accessibility to cysteines was benomyl > colchicine > GTP > taxol, and cysteine accessibility changes conformed that binding sites of these ligands in tubulin were folding correctly. Fluorescence resonance energy transfer technique was used for high throughput screening of chemical library in search of antimitotic agent. There was significant difference in relative fluorescence by 210-O-2 and 210-O-14 as compared to colchicine.

Identification of a novel 4-hydroxyphenylpyruvate dioxygenase from the soil metagenome.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a Fe(II)-dependent, non-heme oxygenase that converts 4-hydroxyphenylpyruvate to homogentisate. Essential cofactors, such as plastoquinone and tocopherol, are produced by HPPD-dependent anabolic pathways in plants. To isolate a novel hppd using culture-independent method, a cosmid metagenomic library was constructed from soil in Korea. Screening of Escherichia coli metagenomic libraries led to the identification of a positive clone, YS103B, producing dark brown pigment in Luria-Bertani medium supplemented with l-tyrosine. In vitro transposon mutagenesis of YS103B showed that the 1.3kb insert was sufficient to produce the hemolytic brown pigment. Sequence analysis of YS103B disclosed one open reading frame encoding a 41.4kDa protein with the well-conserved prokaryotic oxygenase motif of the HPPD family of enzymes. The HPPD-specific beta-triketone herbicide, sulcotrione, inhibited YS103B pigmentation. The recombinant protein expressed in E. coli generated homogentisic acid. Thus, we present the successful heterologous expression of a previously uncharacterized hppd gene from an uncultured soil bacterium.

Characterization of a gene encoding cellulase from uncultured soil bacteria.

To detect cellulases encoded by uncultured microorganisms, we constructed metagenomic libraries from Korean soil DNAs. Screenings of the libraries revealed a clone pCM2 that uses carboxymethyl cellulose (CMC) as a sole carbon source. Further analysis of the insert showed two consecutive ORFs (celM2 and xynM2) encoding proteins of 226 and 662 amino acids, respectively. A multiple sequence analysis with the deduced amino acid sequences of celM2 showed 36% sequence identity with cellulase from the Synechococcus sp., while xynM2 had 59% identity to endo-1,4-beta-xylanase A from Cellulomonas pachnodae. The highest enzymatic CMC hydrolysis was observable at pH 4.0 and 45 degrees C with recombinant CelM2 protein. Although the enzyme CelM2 additionally hydrolyzed avicel and xylan, no substrate hydrolysis was observed on oligosaccharides such as cellobiose, pNP-beta-cellobioside, pNP-beta-glucoside, and pNP-beta-xyloside. These results showed that CelM2 is a novel endo-type cellulase.

Methylobacterium iners sp. nov. and Methylobacterium aerolatum sp. nov., isolated from air samples in Korea.

Two bacterial strains isolated from air samples, 5317S-33(T) and 5413S-11(T), were characterized by determining their phenotypic characteristics, cellular fatty acid profiles and phylogenetic positions based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that these isolates belonged to the genus Methylobacterium. Strain 5317S-33(T) was most closely related to Methylobacterium adhaesivum AR27(T) (97.9% sequence similarity). Strain 5413S-11(T) was most closely related to Methylobacterium fujisawaense DSM 5686(T) (97.3% sequence similarity), Methylobacterium oryzae CBMB20(T) (97.1% similarity) and Methylobacterium radiotolerans JCM 2831(T) (97.0% similarity). Cells of both strains were strictly aerobic, Gram-negative, motile and rod-shaped. The major fatty acid was C(18:1)omega7c. The G+C contents of the genomic DNA were 68.0 mol% for strain 5317S-33(T) and 73.2 mol% for strain 5413S-11(T). According to DNA-DNA hybridization data, strain 5317S-33(T) showed a level of DNA-DNA relatedness of 33 % with M. adhaesivum DSM 17169(T), and strain 5413S-11(T) showed low levels of DNA-DNA relatedness (<35%) with M. fujisawaense DSM 5686(T), M. oryzae CBMB20(T) and M. radiotolerans DSM 1819(T). On the basis of this polyphasic analysis, it was concluded that strains 5317S-33(T) and 5413S-11(T) represent two novel species within the genus Methylobacterium, for which the names Methylobacterium iners sp. nov. (type strain 5317S-33(T) =KACC 11765(T) =DSM 19015(T)) and Methylobacterium aerolatum sp. nov. (type strain 5413S-11(T) =KACC 11766(T) =DSM 19013(T)) are proposed.

Screening and characterization of an enzyme with beta-glucosidase activity from environmental DNA.

A novel beta-glucosidase gene, bglA, was isolated from uncultured soil bacteria and characterized. Using genomic libraries constructed from soil DNA, a gene encoding a protein that hydrolyzes a fluorogenic analog of cellulose, 4-methylumbelliferyl beta-D-cellobioside (MUC), was isolated using a microtiter plate assay. The gene, bglA, was sequenced using a shotgun approach, and expressed in E. coli. The deduced 55-kDa amino acid sequence for bglA showed a 56% identity with the family 1 glycosyl hydrolase Chloroflexus aurantiacus. Bg1A included two conserved family 1 glycosyl hydrolase regions. When using p-nitrophenyl-beta-D-glucoside (pNPG) as the substrate, the maximum activity of the purified beta-glucosidase exhibited at pH 6.5 and 55 degrees C, and was enhanced in the presence of Mn2+. The Km and Vmax values for the purified enzyme with pNPG were 0.16 mM and 19.10 micromol/min, respectively. The purified BglA enzyme hydrolyzed both pNPG and p-nitrophenyl-beta-D-fucoside. The enzyme also exhibited substantial glycosyl hydrolase activities with natural glycosyl substrates, such as sophorose, cellobiose, cellotriose, cellotetraose, and cellopentaose, yet low hydrolytic activities with gentiobiose, salicin, and arbutin. Moreover, Bg1A was able to convert the major ginsenoside Rb1 into the pharmaceutically active minor ginsenoside Rd within 24 h.

Surfactin from Bacillus subtilis displays anti-proliferative effect via apoptosis induction, cell cycle arrest and survival signaling suppression.

The effect of surfactin on the proliferation of LoVo cells, a human colon carcinoma cell line, was examined. Surfactin strongly blocked the proliferation of LoVo cells by inducing pro-apoptotic activity and arresting the cell cycle, according to several lines of evidence on DNA fragmentation, Annexin V staining, and altered levels of poly (ADP-ribose) polymerase, caspase-3, p21(WAF1/Cip1), p53, CDK2 and cyclin E. The anti-proliferative activity of surfactin was mediated by inhibiting extracellular-related protein kinase and phosphoinositide 3-kinase/Akt activation, as assessed by phosphorylation levels. Therefore, our data suggest that surfactin may have anti-cancer properties as a result of its ability to downregulate the cell cycle and suppress its survival.

Pseudomonas koreensis sp. nov., Pseudomonas umsongensis sp. nov. and Pseudomonas jinjuensis sp. nov., novel species from farm soils in Korea.

Among Pseudomonas strains isolated from Korean agricultural soils, four strains (Ps 9-14 group: Ps 1-2, Ps 1-10, Ps 5-5 and Ps 9-14T) from the Suwon, Goesan and Samchok regions, three strains (Ps 3-10 group: Ps 2-22, Ps 3-1 and Ps 3-10T) from Umsong Region and four strains (Pss 26 group: Pss 14, Pss 25, Pss 26T and Pss 27) from Jinju Region were identified as three independent groups on the basis of 16S rDNA sequence analysis. While, on the basis of 16S rDNA sequence analysis, Ps 9-14T and Ps 3-10T form a phyletic line with Pseudomonas jessenii CIP 105274T, 'Pseudomonas pavonaceae' IAM 1155 and Pseudomonas graminis DSM 11363T, Pss 26T is grouped with Pseudomonas citronellolis ATCC 13674T and Pseudomonas nitroreducens IAM 1439T. According to DNA-DNA hybridization studies, strain Ps 9-14T shows high DNA relatedness to strain Ps 3-10T (52%) and Pseudomonas migulae CIP 105470T (49%) and strain Ps 3-10T reveals high relatedness to strain Ps 9-14T (48%) and P. jessenii CIP 105274T (45%). Strain Pss 26T shows high relatedness to P. citronellolis LMG 18378T (54%), P. nitroreducens ATCC 33634T (48%) and Pseudomonas aeruginosa LMG 1242T (48%). On the basis of phenotypic and genotypic analyses, three novel species of the genus Pseudomonas are proposed: Pseudomonas koreensis sp. nov. (type strain Ps 9-14T =LMG 21318T =KACC 10848T) for the Ps 9-14 group, Pseudomonas umsongensis sp. nov. (type strain Ps 3-10T =LMG 21317T =KACC 10847T) for the Ps 3-10 group and Pseudomonas jinjuensis sp. nov. (type strain Pss 26T =LMG 21316T =KACC 10760T) for the Pss 26 group.