The biosphere is dominated by microorganisms (32), yet most microbes in nature have not been studied. Traditional methods for culturing microorganisms limit analysis to those that grow under laboratory conditions (14,25). The recent surge of research in molecular microbial ecology provides compelling evidence for the existence of many novel types of microorganisms in the environment in numbers and varieties that dwarf those of the comparatively few microorganisms amenable to laboratory cultivation (7,13,31). Corroboration comes from estimates of DNA complexity and the discovery of many unique 16S rRNA gene sequences from numerous environmental sources (8,10,28). Collectively, the genomes of the total microbiota found in nature, which we termed the metagenome (11), contain vastly more genetic information than is contained in the culturable subset. Given the profound utility and importance of microorganisms to all biological systems, methods are needed to access the wealth of information within the metagenome.Cloning large fragments of DNA isolated directly from microbes in natural environments provides a method to access soil metagenomic DNA. Previously, we investigated the use of the bacterial artificial chromosome (BAC) vector to express Bacillus cereus genomic DNA (20). The advantage of BAC vectors is that they maintain very large DNA inserts (greater than 100 kb) stably in Escherichia coli (23), facilitating the cloning of large fragments of DNA. Our results demonstrated that expression of heterologous DNA from B. cereus in an E. coli BAC system was detectable at a reasonable frequency (20), validating the idea that the low-copy BAC vector (one to two per cell) (23) could be used to express foreign DNA from foreign promoters in E. coli.Here we describe the construction and initial screening of two BAC libraries made with DNA isolated directly from soil. We found detectable levels of several biochemical activities from BAC library clones. Sequence analysis of selected BAC plasmids encoding such activities and of 16S rRNA genes in one of the libraries confirms the novelty of the genomic information cloned in our libraries. The results show that DNA extracted directly from soil is a valuable source of new genetic information and is accessible by using BAC libraries. Our results demonstrate that both traditional and functional genomics of uncultured microorganisms can be carried out by this approach and that screening of metagenome libraries for activities or gene sequences can provide a basis for conducting genomic analyses of uncultured microorganisms. MATERIALS AND METHODSBacterial strains and plasmids. E. coli strain DH10B and the BAC vector pBeloBAC11 were provided by H. Shizuya (15). Bacillus subtilis strain BR151(pPL608) is strain 1E32 (lys-3 metB10 trpC2) from the Bacillus Genetic Stock Center, Ohio State University. -TnphoA was used as described before (20).

Summary An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its pathogenic mechanism remains unclear. Here we use human induced motor neurons (iMNs) to show that repeat-expanded C9ORF72 is haploinsufficient in ALS. We show that C9ORF72 interacts with endosomes and is required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduces C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms leads to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescues patient neuron survival and ameliorates neurodegenerative processes in both gain- and loss-of function C9ORF72 mouse models. Thus, modulating vesicle trafficking can rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN, and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS/FTD.

To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone "shotgun" environmental DNA library by using an Escherichia coli-Streptomyces lividans shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.

Rifamycin production in A. mediterranei is governed by a single gene cluster consisting of structural, resistance and export, and regulatory genes. The genes characterized here could be modified to produce novel forms of the rifamycins that may be effective against rifamycin-resistant microorganisms.

Myelination in the central nervous system is the process by which oligodendrocytes form myelin sheaths around the axons of neurons. Myelination enables neurons to transmit information more quickly and more efficiently and allows for more complex brain functions; yet, remarkably, the underlying mechanism by which myelination occurs is still not fully understood. A reliable in vitro assay is essential to dissect oligodendrocyte and myelin biology. Hence, we developed a protocol to generate myelinating oligodendrocytes from mouse embryonic stem cells and established a myelin formation assay with embryonic stem cell-derived neurons in microfluidic devices. Myelin formation was quantified using a custom semi-automated method that is suitable for larger scale analysis. Finally, early myelination was followed in real time over several days and the results have led us to propose a new model for myelin formation.

Using a tongue-inspired in vitro platform, Nesmith et al. demonstrate that DMD myoblasts fail to align and polarize with respect to extracellular matrix cues in the same manner as healthy myoblasts, resulting in diminished myotube formation and weaker contractile strength.

Two genes (mcrA and mcrB) from Streptomyces lavendulae that together confer resistance to mitomycin C were identified. This DNA appears to comprise a polycistronic operon with a drug-inducible leaderless mRNA. The deduced amino acid sequence of mcrA shows similarity to sequences of a special class of bacterial, plant, and animal oxygen oxidoreductases.

In an effort to characterize the diversity of mechanisms involved in cellular self-protection against the antitumor antibiotic mitomycin C (MC), DNA fragments from the producing organism (Streptomyces lavendulae) were introduced into Streptomyces lividans and transformants were selected for resistance to the drug. Subcloning of a 4.0-kb BclI fragment revealed the presence of an MC resistance determinant, mrd. Nucleotide sequence analysis identified an open reading frame consisting of 130 amino acids with a predicted molecular weight of 14,364. Transcriptional analysis revealed that mrd is expressed constitutively, with increased transcription in the presence of MC. Expression of mrd in Escherichia coli resulted in the synthesis of a soluble protein with an M r of 14,400 that conferred high-level cellular resistance to MC and a series of structurally related natural products. Purified MRD was shown to function as a drug-binding protein that provides protection against cross-linking of DNA by preventing reductive activation of MC.Streptomyces species are gram-positive soil bacteria known for their ability to produce a wide range of biologically active metabolites. In addition, many resistance genes have been cloned from these bacteria. Mechanisms of cellular self-protection include drug inactivation, target site modification, reduction of intracellular concentration via efflux, and drug binding (8). The presence of multiple modes of self-protection toward a single antibiotic is well documented (9), often with one or more resistance determinants located adjacent to the corresponding biosynthetic genes.Mitomycin C (MC) was identified in 1956 as an antibiotic produced by Streptomyces lavendulae (18) and subsequently established as an important antitumor agent (19,21). MC functions as a prodrug and requires enzymatic or chemical reduction to become a highly reactive alkylating agent (19,40). The intracellular activation of MC is specified by endogenous flavoreductases (34) and proceeds by single electron reduction to the MC semiquinone radical. The relatively long-lived semiquinone species either rearranges to an alkylating intermediate (by further reduction) or transfers an electron to molecular oxygen to generate superoxide (32). Therefore, the ability of MC to inhibit bacterial and mammalian cell growth involves the combined action of DNA alkylation and the formation of reactive oxygen species. Other naturally occurring compounds within this class include bleomycin (37), enediynes (10), and the more recently discovered dihydrobenzoxazines (25,42).Recently, a locus, mcr, that confers high-level MC resistance in S. lavendulae has been reported (2, 3). The resistance gene, mcrA, encodes a flavoenzyme (MCRA) that reoxidizes reductively activated MC (23). In another example involving a DNA damaging agent, bleomycin self-resistance has been determined by drug modification (Bat) and binding (BLMA) proteins in Streptomyces verticillus (38). Beyond these examples, little is known about bacterial resistance to the growing cl...