Metagenomic applications in exploration and development of novel enzymes from nature: a review.

BACKGROUND: Microbial community has an essential role in various fields, especially the industrial sector. Microbes produce metabolites in the form of enzymes, which are one of the essential compounds for industrial processes. Unfortunately, there are still numerous microbes that cannot be identified and cultivated because of the limitations of the culture-based method. The metagenomic approach is a solution for researchers to overcome these problems. Metagenomics is a strategy used to analyze the genomes of microbial communities in the environment directly. Metagenomics application used to explore novel enzymes is essential because it allows researchers to obtain data on microbial diversity, reaching of 99% and various types of genes encoding an enzyme that has not yet been identified. Basic methods in metagenomics have been developed and are commonly used in various studies. A basic understanding of metagenomics for researchers is needed, especially young researchers to support the success of the research. SHORT CONCLUSION: Therefore, this review was done in order to provide a deep understanding of metagenomics. It also discussed the application and basic methods of metagenomics in the exploration of novel enzymes, especially in the latest research. Several types of enzymes, such as cellulases, proteases, and lipases, which have been explored using metagenomics, were reviewed in this article.

Diversity of Akanthomyces on moths (Lepidoptera) in Thailand.

Akanthomyces is a genus of invertebrate-pathogenic fungi from the family Cordycipitaceae (Ascomycota, Hypocreales). Its species occurs on two different types of hosts, spiders and insects, and in the latter case specifically Lepidoptera adults. Three new species of Akanthomyces, A. noctuidarum, A. pyralidarum, and A. tortricidarum occurring on adult moths from Thailand are proposed based on the differences of their morphological characteristics and molecular data. Phylogenetic analyses using a combined dataset, including the internal transcribed spacer regions, the large subunit of the ribosomal DNA, translation elongation factor 1-α, the largest subunit of RNA polymerase II, and the second largest subunit of RNA polymerase II, support the delimitation of these new species in Akanthomyces.

Complete Genome Sequence of Bacillus altitudinis P-10, a Potential Bioprotectant against Xanthomonas oryzae pv. oryzae, Isolated from Rice Rhizosphere in Java, Indonesia.

Bacillus altitudinis P-10 was isolated from the rhizosphere of rice grown in an organic rice field and provides strong antagonism against the bacterial blight caused by Xanthomonas oryzae pv. oryzae in rice. Herein, we provide the complete genome sequence and a possible explanation of the antibiotic function of the P-10 strain.

Transposon mutagenesis of the plant-associated Bacillus amyloliquefaciens ssp. plantarum FZB42 revealed that the nfrA and RBAM17410 genes are involved in plant-microbe-interactions.

Bacillus amyloliquefaciens ssp. plantarum FZB42 represents the prototype of Gram-positive plant growth promoting and biocontrol bacteria. In this study, we applied transposon mutagenesis to generate a transposon library, which was screened for genes involved in multicellular behavior and biofilm formation on roots as a prerequisite of plant growth promoting activity. Transposon insertion sites were determined by rescue-cloning followed by DNA sequencing. As in B. subtilis, the global transcriptional regulator DegU was identified as an activator of genes necessary for swarming and biofilm formation, and the DegU-mutant of FZB42 was found impaired in efficient root colonization. Direct screening of 3,000 transposon insertion mutants for plant-growth-promotion revealed the gene products of nfrA and RBAM_017140 to be essential for beneficial effects exerted by FZB42 on plants. We analyzed the performance of GFP-labeled wild-type and transposon mutants in the colonization of lettuce roots using confocal laser scanning microscopy. While the wild-type strain heavily colonized root surfaces, the nfrA mutant did not colonize lettuce roots, although it was not impaired in growth in laboratory cultures, biofilm formation and swarming motility on agar plates. The RBAM17410 gene, occurring in only a few members of the B. subtilis species complex, was directly involved in plant growth promotion. None of the mutant strains were affected in producing the plant growth hormone auxin. We hypothesize that the nfrA gene product is essential for overcoming the stress caused by plant response towards bacterial root colonization.

Amylocyclicin, a novel circular bacteriocin produced by Bacillus amyloliquefaciens FZB42.

Bacillus amyloliquefaciens FZB42 is a Gram-positive plant growth-promoting bacterium with an impressive capacity to synthesize nonribosomal secondary metabolites with antimicrobial activity. Here we report on a novel circular bacteriocin which is ribosomally synthesized by FZB42. The compound displayed high antibacterial activity against closely related Gram-positive bacteria. Transposon mutagenesis and subsequent site-specific mutagenesis combined with matrix-assisted laser desorption ionization-time of flight mass spectroscopy revealed that a cluster of six genes covering 4,490 bp was responsible for the production, modification, and export of and immunity to an antibacterial compound, here designated amylocyclicin, with a molecular mass of 6,381 Da. Peptide sequencing of the fragments obtained after tryptic digestion of the purified peptide revealed posttranslational cleavage of an N-terminal extension and head-to-tail circularization of the novel bacteriocin. Homology to other putative circular bacteriocins in related bacteria let us assume that this type of peptide is widespread among the Bacillus/Paenibacillus taxon.

The highly modified microcin peptide plantazolicin is associated with nematicidal activity of Bacillus amyloliquefaciens FZB42.

Bacillus amyloliquefaciens FZB42 has been shown to stimulate plant growth and to suppress the growth of plant pathogenic organisms including nematodes. However, the mechanism underlying its effect against nematodes remains unknown. In this study, we screened a random mutant library of B. amyloliquefaciens FZB42 generated by the mariner transposon TnYLB-1 and identified a mutant strain F5 with attenuated nematicidal activity. Reversible polymerase chain reaction revealed that three candidate genes RAMB_007470, yhdY, and prkA that were disrupted by the transposon in strain F5 potentially contributed to its decreased nematicidal activity. Bioassay of mutants impaired in the three candidate genes demonstrated that directed deletion of gene RBAM_007470 resulted in loss of nematicidal activity comparable with that of the F5 triple mutant. RBAM_007470 has been reported as being involved in biosynthesis of plantazolicin, a thiazole/oxazole-modified microcin with hitherto unknown function. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) analyses of surface extracts revealed that plantazolicin bearing a molecular weight of 1,354 Da was present in wild-type B. amyloliquefaciens FZB42, but absent in the ΔRABM_007470 mutant. Furthermore, bioassay of the organic extract containing plantazolicin also showed a moderate nematicidal activity. We conclude that a novel gene RBAM_007470 and its related metabolite are involved in the antagonistic effect exerted by B. amyloliquefaciens FZB42 against nematodes.

Bacterial Traits Involved in Colonization of Arabidopsis thaliana Roots by Bacillus amyloliquefaciens FZB42.

Colonization studies previously performed with a green-fluorescent-protein, GFP, labeled derivative of Bacillus amyloliquefaciens FZB42 revealed that the bacterium behaved different in colonizing surfaces of plant roots of different species (Fan et al., 2012). In order to extend these studies and to elucidate which genes are crucial for root colonization, we applied targeted mutant strains to Arabidopsis seedlings. The fates of root colonization in mutant strains impaired in synthesis of alternative sigma factors, non-ribosomal synthesis of lipopeptides and polyketides, biofilm formation, swarming motility, and plant growth promoting activity were analyzed by confocal laser scanning microscopy. Whilst the wild-type strain heavily colonized surfaces of root tips and lateral roots, the mutant strains were impaired in their ability to colonize root tips and most of them were unable to colonize lateral roots. Ability to colonize plant roots is not only dependent on the ability to form biofilms or swarming motility. Six mutants, deficient in abrB-, sigH-, sigD-, nrfA-, yusV and RBAM017410, but not affected in biofilm formation, displayed significantly reduced root colonization. The nrfA- and yusV-mutant strains colonized border cells and, partly, root surfaces but did not colonize root tips or lateral roots.

Efficient colonization of plant roots by the plant growth promoting bacterium Bacillus amyloliquefaciens FZB42, engineered to express green fluorescent protein.

A single copy of the gfp gene linked with the P(spac) promoter and flanked by the terminal FZB42 amyE sequences was stably integrated into the chromosome of plant growth promoting bacterium Bacillus amyloliquefaciens FZB42 via homologous recombination. A spontaneous mutant, FB01mut, emitting bright fluorescence was detected among the transformants and found suitable for colonization experiments performed with Zea mays, Arabidopsis thaliana and Lemna minor. Real-time RT-PCR revealed that FB01mut expressed 2.5 times more of the gfp transcript than the original GFP-labeled strain. Confocal laser scanning microscopy of plant roots infected with gfp+ tagged FZB42 revealed that the bacterium behaves different in colonizing surfaces of plant roots of different species. In contrast to maize, FZB42 colonized preferentially root tips when colonizing Arabidopsis. FZB42 colonized heavily Lemna fronds and roots by forming biofilms consisting of extracellular matrix and cells with altered morphology. Surfactin, but no other lipopeptide or polyketide synthesized by FZB42 under laboratory conditions, was detected in extracts of Lemna plantlets colonized by FZB42. Due to its stable and long-lasting emission of bright fluorescence without antibiotic pressure FB01mut is an excellent tool for studying plant colonization under competitive, environmental conditions.