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Background: The purpose of this study was to elucidate the community structure of rhizosphere soil bacteria and endophytic bacteria during the growth of moso bamboo (Phyllostachys edulis) shoots. Methods: This study collected the rhizospheric soil samples, tissue samples of rhizome roots, shoot buds, winter bamboo shoots, spring bamboo shoots, and samples of forest soil. Their metagenomic DNA was extracted, and the bacterial community structure and diversity characteristics were compared and analyzed using high-throughput sequencing technology. Results: These samples enabled the identification of 32 phyla, 52 classes, 121 orders, 251 families, and 593 genera of bacteria. The phyla primarily included Proteobacteria, Acidobacteria, and Cyanobacteria among others. Proteobacteria was the dominant phylum in the samples of bamboo shoots and rhizome roots, whereas Acidobacteria was dominant in the rhizosphere and forest soil samples. The predominant genera of the rhizome root samples were Acidothermus, Bradyrhizobium and Acidobacterium, and the predominant genera of the soil samples were Acidothermus and Acidobacterium. Conclusions: This study preliminarily revealed the regularity between the growth and development of bamboo shoots and the changes in the community structure of rhizosphere soil and endophytic bacteria, which provides insights into the relationship between growth and the bacterial community structure in different stages of bamboo shoots.

Culture-independent metagenomic approaches offer a promising solution to the discovery of therapeutically relevant compounds such as antibiotics by enabling access to the hidden biosynthetic potential of microorganisms. These strategies, however, often entail laborious, multi-step, and time-consuming procedures to recover the biosynthetic gene clusters (BGCs) from soil metagenomes for subsequent heterologous expression. Here, we developed an efficient method we called single Nanopore read cluster mining (SNRCM), which enables the fast recovery of complete BGCs from a soil metagenome using long- and short-read sequencing. A metagenomic fosmid library of 83,700 clones was generated and sequenced using Nanopore as well as Illumina technologies. Hybrid assembled contigs of the sequenced fosmid library were subsequently analyzed to identify BGCs encoding secondary metabolites. Using SNRCM, we aligned the identified BGCs directly to Nanopore long-reads and were able to detect complete BGCs on single fosmids. This enabled us to select for and recover BGCs of interest for subsequent heterologous expression attempts. Additionally, the sequencing data of the fosmid library and its corresponding metagenomic DNA enabled us to assemble and recover a large nonribosomal peptide synthetase (NRPS) BGC from three different fosmids of our library and to directly amplify and recover a complete lasso peptide BGC from the high-quality metagenomic DNA. Overall, the strategies presented here provide a useful tool for accelerating and facilitating the identification and production of potentially interesting bioactive compounds from soil metagenomes. Key points: • An efficient approach for the recovery of BGCs from soil metagenomes was developed to facilitate natural product discovery. • A fosmid library was constructed from soil metagenomic HMW DNA and sequenced via Illumina and Nanopore. • Nanopore long-reads enabled the direct identification and recovery of complete BGCs on single fosmids. [ABSTRACT FROM AUTHOR]

Abstract The study performed on the stone materials from the Convent of Christ revealed the presence of a complex microbial ecosystem, emphasizing the determinant role of microorganisms on the biodecay of this built cultural heritage. In this case study, the presence of Rubrobacter sp., Arthrobacter sp., Roseomonas sp., and Marinobacter sp. seems to be responsible for colored stains and biofilm formation while Ulocladium sp., Cladosporium sp., and Dirina sp. may be related to structural damages. The implementation of high‐throughput sequencing approaches on the Convent of Christ's biodecay assessment allowed us to explore, compare, and characterize the microbial communities, overcoming the limitations of culture‐dependent techniques, which only identify the cultivable population. The application of these different tools and insights gave us a panoramic view of the microbiota thriving on the Convent of Christ and signalize the main biodeteriogenic agents acting on the biodecay of stone materials. This finding highlighted the importance of performing metagenomic studies due to the improvements and the reduced amount of sample DNA needed, promoting a deeper and more detailed knowledge of the microbiota present on these dynamic repositories that support microbial life. This will further enable us to perform prospective studies in quarry and applied stone context, monitoring biogenic and nonbiogenic agents, and also to define long‐term mitigation strategies to prevent biodegradation/biodeterioration processes.

The world economy is currently moving towards more sustainable approaches. Lignocellulosic biomass has been widely used as a substitute for fossil sources since it is considered a low-cost bio-renewable resource due to its abundance and continuous production. Compost habitats presenting high content of lignocellulosic biomass are considered a promising source of robust lignocellulose-degrading enzymes. Recently, several novel biocatalysts from different environments have been identified using metagenomic techniques. A key point of the metagenomics studies is the extraction and purification of nucleic acids. Nevertheless, the isolation of high molecular weight DNA from soil-like samples, such as compost, with the required quality for metagenomic approaches remains technically challenging, mainly due to the complex composition of the samples and the presence of contaminants like humic substances. In this work, a rapid and cost-effective protocol for metagenomic DNA extraction from compost samples composed of lignocellulosic residues and containing high content of humic substances was developed. The metagenomic DNA was considered as representative of the global environment and presented high quality (> 99% of humic acids effectively removed) and sufficient quantity (10.5–13.8 µg g−1 of compost) for downstream applications, namely functional metagenomic studies. The protocol takes about 4 h of bench work, and it can be performed using standard molecular biology equipment and reagents available in the laboratory. Key points/Highlights: • Metagenomic DNA was successfully extracted from compost samples rich in humic acids • The improved protocol was established by optimizing the cell lysis method and buffer • Complete removal of humic acids was achieved through the use of activated charcoal • The suitability of the DNA was proven by the construction of a metagenomic library [ABSTRACT FROM AUTHOR]

Shotgun sequencing was employed to explore the community structure (phylotyping of rRNA genes) and functional potential of Cabernet Sauvignon grape must microbiome. A metagenomic library, representing 92.6 Mb of genetic information, was generated from DNA obtained from Cabernet Sauvignon grape must. Fungi were identified as the dominant domain (59.5%) followed by Streptophyta (39%). Among the 84 fungal species, 22 were yeasts of various genera. Additionally, grapevine endophytes such as Davidiella sp., Botryotinia fuckeliana, Alternaria sp., and Cladosporium sp. were identified. An unusually high prevalence of Mucor spp. was evidenced. Functional annotation revealed sequences of genes involved in metabolism (35.6%), followed by poorly characterized categories (28.3%), cellular processes and signalling (18.4%), and finally information storage (17.8%). Among the former, glycosidases were abundant followed by glycogen debranching enzyme, 6-phosphofructokinase and trehalose-6-phosphate synthase. Furthermore, the taxonomic analysis of the functional sequence data exhibited the eukaryotic gene pool that predominantly contains sequences derived from Streptophyta (mainly Vitis vinifera) 60% > Ascomycota (32%) > Basidiomycota (5%) > Bacteria (2.5%). Finally, sequences of a variety of hydrolytic enzymes of potential oenological relevance were retrieved, thereby confirming that grape juice is a rich reservoir for valuable biocatalysts that should be explored further. [ABSTRACT FROM AUTHOR]

Metagenomics is the study of gene pool of an entire community in a particular niche. This provides valuable information about the functionality of host-microbe interaction in a biological ecosystem. Efficient metagenomic DNA extraction is a critical pre-requisite for a successful sequencing run in a metagenomic study. Although isolation of human stool metagenomic DNA is fairly standardized, the same protocol does not work as efficiently in fecal DNA from other organisms. In this study, we report a comparison of manual and commercial DNA extraction methods for diverse samples such as human stool, fish gut and soil. Fishes are known to have variable microbial diversity based on their food habits, so the study included two different varieties of fishes. A modified protocol for effective isolation of metagenomic DNA from human milk samples is also reported, highlighting critical precautions. Recent studies have emphasized the importance of studying functionality of human milk metagenome to understand its influence on infants' health. While manual method works well with most samples and therefore can be a method of choice for testing new samples, broad-range commercial kit offers advantage of high purity and quality. DNA extraction of different samples would go a long way in unraveling the unexplored association between microbes and host in a biological system. [ABSTRACT FROM AUTHOR]

Deep sea is an extreme environment which harbours diverse microbial communities. The deep sea ecosystem of the northeastern part of Indian Ocean is poorly studied. In this study, to explore the bacterial diversity of the unexplored environment, we designed culture independent (denaturant gradient gel electrophoresis DGGE) and culture dependent (16S rDNA) methods. The samples for this study were taken from the depths of 1850 m off Barren Island of the Andaman Sea, and 2000 and 1400 m off Chennai and Cudallore of the Bay of Bengal. The phylogenetic analysis, based on 16S rDNA sequence data, has suggests their affiliation to three major phyla viz. Firmicutes (48%), Proteobacteria (30%, alpha and gamma-class) and Actinobacteria (22%). Bacillus was the most frequently isolated genus. This is the first report on the isolation of Brucella, Fictibacillus, Mesorhizobium and Cobetia spp. from deep sea sediments. Eleven of the 34 operational taxonomic units probably represents new species. To investigate the metabolic potential, the isolates were screened for production of the extracellular hydrolytic enzyme and antibacterial compound. Almost 91% of isolates showed production of at least one of the extracellular hydrolytic enzymes, such as caesinase, alpha-amylase, urease, gelatinase, lipase and DNase. Streptomyces was the only genus which showed antibacterial activity. This study highlights that the examined deep sea environment could be a hot spot for microbial derived natural products. [ABSTRACT FROM AUTHOR]

Background: The purpose of this study was to elucidate the community structure of rhizosphere soil bacteria and endophytic bacteria during the growth of moso bamboo ( Phyllostachys edulis ) shoots., Methods: This study collected the rhizospheric soil samples, tissue samples of rhizome roots, shoot buds, winter bamboo shoots, spring bamboo shoots, and samples of forest soil. Their metagenomic DNA was extracted, and the bacterial community structure and diversity characteristics were compared and analyzed using high-throughput sequencing technology., Results: These samples enabled the identification of 32 phyla, 52 classes, 121 orders, 251 families, and 593 genera of bacteria. The phyla primarily included Proteobacteria, Acidobacteria, and Cyanobacteria among others. Proteobacteria was the dominant phylum in the samples of bamboo shoots and rhizome roots, whereas Acidobacteria was dominant in the rhizosphere and forest soil samples. The predominant genera of the rhizome root samples were Acidothermus , Bradyrhizobium and Acidobacterium , and the predominant genera of the soil samples were Acidothermus and Acidobacterium ., Conclusions: This study preliminarily revealed the regularity between the growth and development of bamboo shoots and the changes in the community structure of rhizosphere soil and endophytic bacteria, which provides insights into the relationship between growth and the bacterial community structure in different stages of bamboo shoots., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s). Published by IMR Press.)

Microbial based cleaning products (MBCPs) are a new generation of cleaning products that are gaining greater use in household, institutional, and industrial settings. Little is known about the exact microbial composition of these products because they are not identified in detail on product labels and formulations are often proprietary. To gain a better understanding of their microbial and fungal composition towards risk assessment, the cultivable microorganisms and rDNA was surveyed for microbial content in five different MBCPs manufactured and sold in North America. Individual bacterial and fungal colonies were identified by ribosequencing and fatty acid methyl ester (FAME) gas chromatography. Metagenomic DNA (mDNA) corresponding to each of the products was subjected to amplification and short read sequencing of seven of the variable regions of the bacterial 16S ribosomal DNA. Taken together, the cultivable microorganism and rDNA survey analyses showed that three of the products were simple mixtures of Bacillus species. The two other products featured a mixture of cultivable fungi with Bacilli , and by rDNA survey analysis, they featured greater microbial complexity. This study improves our understanding of the microbial composition of several MBCPs towards a more comprehensive risk assessment. [ABSTRACT FROM AUTHOR]

A simple, reliable method for genomic DNA extraction from sediments with minimum contaminants was developed to address the risk of poor quality DNA in metagenomic studies. Nine DNA extraction methods using 20% cetyl-trimethyl-ammonium bromide (CTAB) were performed and compared to develop an extraction protocol that can offer humic acid-free metagenomic DNA from marine and saltpan sediments. Community DNA extraction was executed via., Zhou et al. modified protocol using 20% CTAB treatment at different steps to compare the efficacy of humic acid removal. Out of nine DNA extraction methods, method 6 significantly improved the quality of DNA with efficient removal of humic substances. 16S rRNA gene amplification and spectrophotometric analysis confirmed the efficiency of method 6 to remove DNA inhibitors from marine sediments as well as saltpan samples. Inhibitors extracted along with metagenomic DNA outcome increased DNA yield and PCR inhibition in method 1 and 3. However, repeated 20% CTAB wash in method 6 ensured 16S amplification and least yield and concentration. Current study explains a detailed protocol based on 20% CTAB wash for the extraction of humic acid-free DNA from diverse sediment samples. [ABSTRACT FROM AUTHOR]

Haloalkane dehalogenases (HLDs) are environmentally relevant enzymes cleaving a carbon-halogen bond in a wide range of halogenated pollutants. PCR with degenerate primers and genome-walking was used for the retrieval of four HLD-encoding genes from groundwater-derived environmental DNA. Using specific primers and the environmental DNA as a template, we succeeded in generating additional amplicons, resulting altogether in three clusters of sequences with each cluster comprising 8-13 closely related putative HLD-encoding genes. A phylogenetic analysis of the translated genes revealed that three HLDs are members of the HLD-I subfamily, whereas one gene encodes an enzyme from the subfamily HLD-II. Two metagenome-derived HLDs, eHLD-B and eHLD-C, each from a different subfamily, were heterologously produced in active form, purified and characterized in terms of their thermostability, pH and temperature optimum, quaternary structure, substrate specificity towards 30 halogenated compounds, and enantioselectivity. eHLD-B and eHLD-C showed striking differences in their activities, substrate preferences, and tolerance to temperature. Profound differences were also determined in the enantiopreference and enantioselectivity of these enzymes towards selected substrates. Comparing our data with those of known HLDs revealed that eHLD-C exhibits a unique combination of high thermostability, high activity, and an unusually broad pH optimum, which covers the entire range of pH 5.5-8.9. Moreover, a so far unreported high thermostability for HLDs was determined for this enzyme at pH values lower than 6.0. Thus, eHLD-C represents an attractive and novel biocatalyst for biotechnological applications. [ABSTRACT FROM AUTHOR]

Standardization of metagenomic DNA extraction protocol is a pre-requisite for a successful metagenomic study aiming to screen and exploit the variety of microorganisms inhabiting a particular soil environment. Six methods reported earlier were used for isolation of metagenomic DNA in the present study. These methods suffered with regard to either poor yield or quality of DNA. Therefore, we developed an improved method for isolation of high-molecular weight and good quality metagenomic DNA from different soil samples. Our protocol combines the enzymatic (lysozyme and proteinase K) and chemical (CTAB and CaCl) strategies to ensure efficient cell lysis and use of PEG and isopropanol for precipitation of humic impurities-free DNA. Our improved method gave high yield of good quality metagenomic DNA from diverse soils collected from garden, domestic waste dumping site, cellulose waste dumping site, sewage site, and tannery waste site. The good quality of the metagenomic DNA was evident by spectrophotometry data, PCR amplification of 16S rRNA gene and restriction digestion. [ABSTRACT FROM AUTHOR]

A functional gene containing an Open Reading Frame (ORF) encoding a β-1, 4-endoxylanase glycosyl hydrolase family 11 was cloned directly using metagenomic PCR-cloning method from Pawan Hot Spring sample in Riau. The gene consisted of 642 nucleotides, encoded for 213 amino acids. The amino acid sequence analysis using BLAST showed that the gene has high homology (93%) with xylanase gene from Bacillus subtilis. The gene showed its function when it was subcloned into an expression vector and overexpressed in E. coli. The crude extract of the recombinant enzyme had activity for 170 U/ml at 50 °C. The result of this work showed that metagenomic approach was a powerful short cut method to obtain recombinant biocatalyst that was useful for industrial application.

Ocean iron fertilization is an approach to increase CO2 sequestration. The Indo-German iron fertilization experiment LOHAFEX was carried out in the Southern Ocean surrounding Antarctica in 2009 to monitor changes in bacterial community structure following iron fertilization-induced phytoplankton bloom of the seawater from different depths. 16S rRNA gene libraries were constructed using metagenomic DNA from seawater prior to and after iron fertilization and the clones were sequenced for identification of the major bacterial groups present and for phylogenetic analyses. A total of 4439 clones of 16S rRNA genes from ten 16S rRNA gene libraries were sequenced. More than 97.35% of the sequences represented four bacterial lineages i.e. Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Firmicutes and confirmed their role in scavenging of phytoplankton blooms induced following iron fertilization. The present study demonstrates the response of Firmicutes due to Iron fertilization which was not observed in previous southern ocean Iron fertilization studies. In addition, this study identifies three unique phylogenetic clusters LOHAFEX Cluster 1 (affiliated to Bacteroidetes), 2 and 3 (affiliated to Firmicutes) which were not detected in any of the earlier studies on iron fertilization. The relative abundance of these clusters in response to iron fertilization was different. The increase in abundance of LOHAFEX Cluster 2 and Papillibacter sp. another dominant Firmicutes may imply a role in phytoplankton degradation. Disappearance of LOHAFEX Cluster 3 and other bacterial genera after iron fertilization may imply conditions not conducive for their survival. It is hypothesised that heterotrophic bacterial abundance in the Southern Ocean would depend on their ability to utilise algal exudates, decaying algal biomass and other nutrients thus resulting in a dynamic bacterial succession of distinct genera.

Recent advances in culture-independent studies of microbes had proved to be more reliable and efficient than the conventional ones. The isolation of good quality and quantity of total community DNA are one of the major hurdles in this endeavour. Shearing of DNA during the extraction process and the co-extraction of inhibitory compounds reduce the quality of the isolated nucleic acids making it unsuitable for the construction of large insert metagenomic libraries. In the present study, a multi-level filtration step was brought in which efficiently isolated total bacterial DNA from three different environment samples. The preprocessing method could efficiently improve the 260/230 ratio of the isolated DNA by 2.3-45 % and decreased the protein contamination by 22.5-34.5 % on saltpan and arctic sediment samples, respectively. The more significant part of the experiment was that the DNA obtained was of high quality with minimal shearing making it most suitable for the construction of large insert genomic libraries. PCR amplification of 16S rRNA gene confirmed that the filtration method was effective in the isolation of high-quality DNA. [ABSTRACT FROM AUTHOR]

In contrast to the traditional culturing techniques and microscopy that have led to the identification and characterization of only about 15-20% of the rumen microbes till date, nucleic acid-based molecular approaches are rapid, reproducible, and allow both the qualitative and quantitative assessment of microbial diversity. The aim of this study was to develop a simple, rapid and effective extraction protocol for the recovery of high-molecular-weight and cloneable metagenomic DNA (mDNA) from goat rumen contents. An efficient method was devised to isolate highmolecular- weight mDNA (>23kb) that was pure and cloneable after isolation in a relatively short period (3.5 h). This is the first report wherein purification of isolated mDNA could be passed. The purity and cloneability of mDNA was found to be possible with the successful restriction digestion, 16S rDNA PCR amplification of the isolated mDNA and mDNA library construction. The screening of 1600 clones from the metagenomic library revealed one clone with adistinct hydrolytic activity on carboxymethyl cellulose (CMC) agar suggesting its endoglucanase activity. Agarose gel electrophoresis showed aDNA insert of ~1.5kb size on digestion with BamH 1. The metagenomic clones offer a prodigious non-conventional means to explore the genetically untapped resources from nature. [ABSTRACT FROM AUTHOR]

Objectives: To acquire a thermostable xylanase, that is suitable for xylooligosaccharide production from pretreated corncobs, the metagenomic method was used to obtain the gene from an uncultured environmental microorganism. Results: A thermostable xylanase-encoding gene ( xyn10CD18) was cloned directly from the metagenomic DNA of cow dung compost. When xyn10CD18 was expressed in Bacillus megaterium MS941, extracellular xylansae activity at 106 IU/ml was achieved. The purified recombinant Xyn10CD18 was optimally active at pH 7 and 75 °C as measured over 10 min. It retained over 55 % of its initial activity at 70 °C and pH 7 after 24 h. Its action on birchwood xylan for 18 h liberated xylooligosaccharides with 2°-4° of polymerization, with xylobiose and xylotetraose as the main products. When pretreated corncobs were hydrolyzed by Xyn10CD18 for 18 h, the xylooligosaccharides (DP 2-4) products increased to 80 % and the xylose was just increased by 3 %. Conclusion: Xyn10CD18 is a thermostable endoxylanase and is a promising candidate for biomass conversion and xylooligosaccharide production. [ABSTRACT FROM AUTHOR]

Background: Heterologous expression of psychrophilic enzymes in E. coli is particularly challenging due to their intrinsic instability. The low stability is regarded as a consequence of adaptation that allow them to function at low temperatures. Recombinant production presents a significant barrier to their exploitation for commercial applications in industry. Methods: As part of an enzyme discovery project we have investigated the utility of a cold-shock inducible promoter for low-temperature expression of five diverse genes derived from the metagenomes of marine Arctic sediments. After evaluation of their production, we further optimized for soluble production by building a vector suite from which the environmental genes could be expressed as fusions with solubility tags. Results: We found that the low-temperature optimized system produced high expression levels for all putatively cold-active proteins, as well as reducing host toxicity for several candidates. As a proof of concept, activity assays with one of the candidates, a putative chitinase, showed that functional protein was obtained using the lowtemperature optimized vector suite. Conclusions: We conclude that a cold-shock inducible system is advantageous for the heterologous expression of psychrophilic proteins, and may also be useful for expression of toxic mesophilic and thermophilic proteins where properties of the proteins are deleterious to the host cell growth. [ABSTRACT FROM AUTHOR]

Ocean iron fertilization is an approach to increase CO2 sequestration. The Indo-German iron fertilization experiment "LOHAFEX" was carried out in the Southern Ocean surrounding Antarctica in 2009 to monitor changes in bacterial community structure following iron fertilization-induced phytoplankton bloom of the seawater from different depths. 16S rRNA gene libraries were constructed using metagenomic DNA from seawater prior to and after iron fertilization and the clones were sequenced for identification of the major bacterial groups present and for phylogenetic analyses. A total of 4439 clones of 16S rRNA genes from ten 16S rRNA gene libraries were sequenced. More than 97.35% of the sequences represented four bacterial lineages i.e. Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Firmicutes and confirmed their role in scavenging of phytoplankton blooms induced following iron fertilization. The present study demonstrates the response of Firmicutes due to Iron fertilization which was not observed in previous southern ocean Iron fertilization studies. In addition, this study identifies three unique phylogenetic clusters LOHAFEX Cluster 1 (affiliated to Bacteroidetes), 2, and 3 (affiliated to Firmicutes) which were not detected in any of the earlier studies on iron fertilization. The relative abundance of these clusters in response to iron fertilization was different. The increase in abundance of LOHAFEX Cluster 2 and Papillibacter sp. another dominant Firmicutes may imply a role in phytoplankton degradation. Disappearance of LOHAFEX Cluster 3 and other bacterial genera after iron fertilization may imply conditions not conducive for their survival. It is hypothesized that heterotrophic bacterial abundance in the Southern Ocean would depend on their ability to utilize algal exudates, decaying algal biomass and other nutrients thus resulting in a dynamic bacterial succession of distinct genera. [ABSTRACT FROM AUTHOR]

Background: Heterologous expression of psychrophilic enzymes in E. coli is particularly challenging due to their intrinsic instability. The low stability is regarded as a consequence of adaptation that allow them to function at low temperatures. Recombinant production presents a significant barrier to their exploitation for commercial applications in industry. Methods: As part of an enzyme discovery project we have investigated the utility of a cold-shock inducible promoter for low-temperature expression of five diverse genes derived from the metagenomes of marine Arctic sediments. After evaluation of their production, we further optimized for soluble production by building a vector suite from which the environmental genes could be expressed as fusions with solubility tags. Results: We found that the low-temperature optimized system produced high expression levels for all putatively cold-active proteins, as well as reducing host toxicity for several candidates. As a proof of concept, activity assays with one of the candidates, a putative chitinase, showed that functional protein was obtained using the lowtemperature optimized vector suite. Conclusions: We conclude that a cold-shock inducible system is advantageous for the heterologous expression of psychrophilic proteins, and may also be useful for expression of toxic mesophilic and thermophilic proteins where properties of the proteins are deleterious to the host cell growth. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the bacterial diversity in soils of the southern region of the Western Ghats, a 'biodiversity hotspot', and thereby futher our understanding of the microbial communities in this ecological niche. The diversity and phylogeny of bacterial populations in soil samples collected from various locations of the Tamil Nadu and Kerala regions of Western Ghats were compared using both cultivation-dependent and cultivation-independent methods. A total of 171 bacterial strains were isolated based on their morphological characteristics and their diversity indices calculated. The distinctive amplified ribosomal DNA restriction analysis (ARDRA) pattern of each isolate was determined, and representative isolates were then subjected to 16S rRNA gene sequencing. On the basis of their sequence similarity, the isolates were distributed among three different genera belonging to Firmicutes (83.3 %), Proteobacteria (8.3 %) and high G+C Gram-positive bacteria (8.3 %). The highest and the lowest values for the diversity indices were obtained for metagenomic DNA extracted from isolates BWGA and BVP, respectively; these were used for 16S rRNA gene library construction and analysis. Based on their phylogenetic analysis, the predominant members of the habitat were found to belong to the phylum Firmicutes (84.62 %). Firmicutes was the dominant bacterial phylum detected by both approaches, but the culture-independent approach detected a considerably higher number of uncultivable bacteria. In conclusion, in our study of the bacterial diversity of this Western Ghats region, we fund that the genus Bacillus was predominant among the samples assessed by both cultivation-dependent and cultivation-independent methods. [ABSTRACT FROM AUTHOR]

A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils. [ABSTRACT FROM AUTHOR]

In this work we adapted a protocol for the extraction of metagenomic DNA (ADNmg) bacteria in the digestive system (intestines, stomach and hepatopancreas) of Macrobrachium carcinus L., with reference to the method of extracting bacterial DNA from soils and sediments (Rojas-Herrera et al., 2008). This methodology consisted of enzymatic, physics, mechanics and chemistry after a series of tests was abolished enzymatic lysis. However, the success ADNmg extraction was influenced mainly by the preparation of the samples, in particular the hepatopancreas, where it was necessary to remove the fat by thermal shock temperature and phase separation by centrifugation with the sample frozen.The effectiveness of isolated DNA fragmentation was verified by gel electrophoresis in denaturing gradient (DGGE) after amplification with universal primers. In general, it had a low diversity (19 phylotypes) between the different organs analyzed of 13.5 ± 1 (intestines) to 11.7 ± 0.96 (stomach). The Shannon-Weaver index (2.45), Simpsons (10.88) and equity (0972) obtained from the digitization of the image of the gel, suggested that the phylotypes that form the gut microflora M. carcinus, is distributed unevenly between the different organs analyzed.

Ring-hydroxylating dioxygenases (RHDs) catalyze the initial oxidation step of a range of aromatic hydrocarbons including polycyclic aromatic hydrocarbons (PAHs). As such, they play a key role in the bacterial degradation of these pollutants in soil. Several polymerase chain reaction (PCR)-based methods have been implemented to assess the diversity of RHDs in soil, allowing limited sequence-based predictions on RHD function. In the present study, we developed a method for the isolation of PAH-specific RHD gene sequences of Gram-negative bacteria, and for analysis of their catalytic function. The genomic DNA of soil PAH degraders was labeled in situ by stable isotope probing, then used to PCR amplify sequences specifying the catalytic domain of RHDs. Sequences obtained fell into five clusters phylogenetically linked to RHDs from either Sphingomonadales or Burkholderiales. However, two clusters comprised sequences distantly related to known RHDs. Some of these sequences were cloned in-frame in place of the corresponding region of the phnAIa gene from Sphingomonas CHY-1 to generate hybrid genes, which were expressed in Escherichia. coli as chimerical enzyme complexes . Some of the RHD chimeras were found to be competent in the oxidation of two- and three-ring PAHs, but other appeared unstable. Our data are interpreted in structural terms based on 3D modeling of the catalytic subunit of hybrid RHDs. The strategy described herein might be useful for exploring the catalytic potential of the soil metagenome and recruit RHDs with new activities from uncultured soil bacteria. [ABSTRACT FROM AUTHOR]

Large-scale cultivation of Chroogomphus rutilus is too inefficient to be commercially feasible. In addition, isolating C. rutilus mycelia in the wild is difficult. Thus, determining the natural habitat of its fruiting body is important. The present study focused on the ecology of the C. rutilus habitat to facilitate its large-scale cultivation. A culture-independent molecular approach-a powerful technology for microbiological ecology studies-was used to investigate the diversity of soil fungal communities in samples surrounding C. rutilus from the Beijing region of China. Metagenomic DNA was isolated from soil samples collected around C. rutilus, and an internal transcribed spacer (ITS) gene library was constructed. Subsequently, polymerase chain reaction products were digested with HinfI, HaeIII, MspI, TaqI, or MboI. Clones were selected and sequenced based on their restriction fragment length polymorphisms. The diversity of the fungi represented by their ITS sequences was analyzed. Our results indicate the presence of numerous fungi in the C. rutilus habitat. This study is the first demonstration of the fungal ecology surrounding C. rutilus using a culture independent method. [ABSTRACT FROM AUTHOR]

Pharmaceutical wastewaters are recognized as reservoirs of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB), and also as hotspots for their horizontal gene transfer (HGT) using mobile genetic elements. Our study employed the use of PCR analysis of metagenomic DNA samples obtained from four pharmaceutical wastewaters using known primers to study the prevalence of thirty-six ARGs and four MGEs active against the commonly used antibiotics in Nigeria. The ARGs most frequently detected from the metagenomic DNA samples in each of the antibiotic classes under study include tetracycline [tet(G)], aminoglycoside [aadA, strA and strB], chloramphenicol [catA1], sulphonamides [sulI and sulII], and β-lactams and penicillins [blaOXA]. The ARGs showed a 100% prevalence in their various environmental sources. The pharmaceutical facility PFIV showed the highest concentration of ARGs in this study. The highest concentration for MGEs was shown by pharmaceutical facility PFIII, positive for intl1, intl2, and IFS genes. This study highlights the wide distribution of ARGs to the antibiotics tested in the wastewater, making pharmaceutical wastewater reservoirs of ARGs which could potentially be transferred from commensal microorganisms to human pathogens. [ABSTRACT FROM AUTHOR]