Long-chain N-acyltyrosine synthases from environmental DNA

The heterologous expression of DNA extracted directly from environmental samples (environmental DNA [eDNA]) in easily cultured hosts provides access to natural products produced by previously inaccessible microorganisms. When eDNA cosmid libraries were screened in Escherichia coli for antibacterially active clones, long-chain N-acyltyrosine-producing clones were found in every eDNA library. These apparently common natural products have not been previously described from screening extracts of cultured bacteria for biologically active natural products. Of the 11 long-chain N-acyl amino acid synthases (NASs) that were characterized, 10 are unique sequences. A predicted protein of previously unknown function from Nitrosomonas europaea, a gram-negative nitrifying beta-proteobacterium, is 14 to 37% identical to eDNA NASs. When cloned into E. coli, this open reading frame confers the production of long-chain N-acyltyrosines to the host and is therefore the first NAS from a cultured bacterium to be functionally characterized. Understanding the role that long-chain N-acyl amino acids play in soil microbial communities should now be feasible with the identification of a cultured organism that has the genetic capacity to produce these compounds.     

Long-Chain N-Acyltyrosine Synthases from Environmental DNA

The heterologous expression of DNA extracted directly from environmental samples (environmental DNA [eDNA]) in easily cultured hosts provides access to natural products produced by previously inaccessible microorganisms. When eDNA cosmid libraries were screened in Escherichia coli for antibacterially active clones, long-chain N-acyltyrosine-producing clones were found in every eDNA library. These apparently common natural products have not been previously described from screening extracts of cultured bacteria for biologically active natural products. Of the 11 long-chain N-acyl amino acid synthases (NASs) that were characterized, 10 are unique sequences. A predicted protein of previously unknown function from Nitrosomonas europaea, a gram-negative nitrifying beta-proteobacterium, is 14 to 37% identical to eDNA NASs. When cloned into E. coli, this open reading frame confers the production of long-chain N-acyltyrosines to the host and is therefore the first NAS from a cultured bacterium to be functionally characterized. Understanding the role that long-chain N-acyl amino acids play in soil microbial communities should now be feasible with the identification of a cultured organism that has the genetic capacity to produce these compounds.     

Biocatalysts and small molecule products from metagenomic studies

The vast majority of bacteria present in environmental samples have never been cultured and therefore have not been exploited for the ability to produce useful biocatalysts or collections of biocatalysts generating interesting small molecules. Metagenomic libraries constructed using DNA extracted directly from natural bacterial communities offer access to the genetic information present in the genomes of these as yet uncultured bacteria. This review highlights recent efforts to recover both discrete enzymes and small molecules from metagenomic libraries.     

Metagenomic analysis of Streptomyces lividans reveals host-dependent functional expression

Most functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA in Escherichia coli, which has been the predominant surrogate host to date. To expand the range of expressed genes, we developed tools for construction and functional screening of metagenomic libraries in Streptomyces lividans. We expanded on previously published protocols by constructing a system that enables retrieval and characterization of the metagenomic DNA from biologically active clones. To test the functionality of these methods, we constructed and screened two metagenomic libraries in S. lividans. One was constructed with pooled DNA from 14 bacterial isolates cultured from Alaskan soil and the second with DNA directly extracted from the same soil. Functional screening of these libraries identified numerous clones with hemolytic activity, one clone that produces melanin by a previously unknown mechanism, and one that induces the overproduction of a secondary metabolite native to S. lividans. All bioactive clones were functional in S. lividans but not in E. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.     

Recent application of metagenomic approaches toward the discovery of antimicrobials and other bioactive small molecules

Bacteria grown in pure culture have been the starting point for the discovery of many of the antibacterials now in use. Metagenomics, which utilizes culture-independent methods to access the collective genomes of natural bacterial populations, provides a means of exploring the antimicrobials produced by the large collections of bacteria that are known to be present in the environment but remain recalcitrant to culturing. Both novel small molecule antibiotics and new antibacterially active proteins have been identified using metagenomic approaches. The recent application of metagenomics to the discovery of bioactive small molecules, small molecule biosynthetic gene clusters and antibacterially active enzymes is discussed here.     

Denaturing Gradient Gel Electrophoresis Can Rapidly Display the Bacterial Diversity Contained in 16S rDNA Clone Libraries

Two different strategies for molecular analysis of bacterial diversity, 16S rDNA cloning and denaturing gradient gel electrophoresis (DGGE), were combined into a single protocol that took advantage of the best attributes of each: the ability of cloning to package DNA sequence information and the ability of DGGE to display a community profile. In this combined protocol, polymerase chain reaction products from environmental DNA were cloned, and then DGGE was used to screen the clone libraries. Both individual clones and pools of randomly selected clones were analyzed by DGGE, and these migration patterns were compared to the conventional DGGE profile produced directly from environmental DNA. For two simple bacterial communities (biofilm from a humics-fed laboratory reactor and planktonic bacteria filtered from an urban freshwater pond), pools of 35–50 clones produced DGGE profiles that contained most of the bands visible in the conventional DGGE profiles, indicating that the clone pools were adequate for identifying the dominant genotypes. However, DGGE profiles of two different pools of 50 clones from a lawn soil clone library were distinctly different from each other and from the conventional DGGE profile, indicating that this small number of clones poorly represented the bacterial diversity in soil. Individual clones with the same apparent DGGE mobility as prominent bands in the humics reactor community profiles were sequenced from the clone plasmid DNA rather than from bands excised from the gel. Because a longer fragment was cloned (∼1500 bp) than was actually analyzed in DGGE (∼350 bp), far more sequence information was available using this approach that could have been recovered from an excised gel band. This clone/DGGE protocol permitted rapid analysis of the microbial diversity in the two moderately complex systems, but was limited in its ability to represent the diversity in the soil microbial community. Nonetheless, clone/DGGE is a promising strategy for fractionating diverse microbial communities into manageable subsets consisting of small pools of clones.     

Uncultured soil bacteria are a reservoir of new antibiotic resistance genes

Antibiotic resistance genes are typically isolated by cloning from cultured bacteria or by polymerase chain reaction (PCR) amplification from environmental samples. These methods do not access the potential reservoir of undiscovered antibiotic resistance genes harboured by soil bacteria because most soil bacteria are not cultured readily, and PCR detection of antibiotic resistance genes depends on primers that are based on known genes. To explore this reservoir, we isolated DNA directly from soil samples, cloned the DNA and selected for clones that expressed antibiotic resistance in Escherichia coli. We constructed four libraries that collectively contain 4.1 gigabases of cloned soil DNA. From these and two previously reported libraries, we identified nine clones expressing resistance to aminoglycoside antibiotics and one expressing tetracycline resistance. Based on the predicted amino acid sequences of the resistance genes, the resistance mechanisms include efflux of tetracycline and inactivation of aminoglycoside antibiotics by phosphorylation and acetylation. With one exception, all the sequences are considerably different from previously reported sequences. The results indicate that soil bacteria are a reservoir of antibiotic resistance genes with greater genetic diversity than previously accounted for, and that the diversity can be surveyed by a culture-independent method.     

Screening cultured marine microalgae for anticancer-type activity

Marine Cyanophyceae, and to a lesser extent other marine microalgae, are a promising source of new anticancer-type natural products. Microscopic forms that do not form mats or tufts in nature must be cultured in order to obtain sufficiently sized samples. Field collections of microalgae in intertidal and shallow subtidal tropical environments utilize hand collection and manipulation techniques into small-volume wide-mouth jars. Acclimation times in the laboratory environment are important in bringing new cultures into cultivation. Manipulation on agar plates has given the best success rate for obtaining unialgal cultures; sometimes these are obtained directly as axenic clones. These pure strains are cultured in an initial volume of 3 L to give sufficient material for pharmacological screening. The extracts are evaluated in a series of mechanism-based anticancer screens, including protein kinase C (PKC), protein tryosine kinase (PTK) and inosine monophosphate dehydrogenase (IMPDH). More that 501 extracts have been screened in these three assay areas, and have yielded 23 active to IMPDH, 9 to PKC, and 9 to PTK. The extract of one cultured microalga which was active to PTK,Poteriochromonas malhamensis, has yielded a novel chlorosulfolipid, whose structure is discussed. Future efforts will (a) target less well explored groups of microalgae including several orders of cyanophyceae as well as field collections of cryptophytes and chrysophytes, and (b) complement mechanism-based screening with cancer cell cytotoxicity screening.     

Expression and isolation of antimicrobial small molecules from soil DNA libraries

Natural products have been a critically important source of clinically relevant small molecule therapeutics. However, the discovery rate of novel structural classes of antimicrobial molecules has declined. Recently, increasing evidence has shown that the number of species cultivated from soil represents less than 1% of the total population, opening up the exciting possibility that these uncultured species may provide a large untapped pool from which novel natural products can be discovered. We have constructed and expressed in E. coli a BAC (bacterial artificial chromosome) library containing genomic fragments of DNA (5-120kb) isolated directly from soil organisms (S-DNA). Screening of the library resulted in the identification of several antimicrobial activities expressed by different recombinant clones. One clone (mg1.1) has been partially characterized and found to express several small molecules related to and including indirubin. These results show that genes involved in natural product synthesis can be cloned directly from S-DNA and expressed in a heterologous host, supporting the idea that this technology has the potential to provide novel natural products from the wealth of environmental microbial diversity and is a potentially important new tool for drug discovery.     

Bioactive Compounds from Marine Bacteria and Fungi

Marine bacteria and fungi are of considerable importance as new promising sources of a huge number of biologically active products. Some of these marine species live in a stressful habitat, under cold, lightless and high pressure conditions. Surprisingly, a large number of species with high diversity survive under such conditions and produce fascinating and structurally complex natural products. Up till now, only a small number of microorganisms have been investigated for bioactive metabolites, yet a huge number of active substances with some of them featuring unique structural skeletons have been isolated. This review covers new biologically active natural products published recently (2007–09) and highlights the chemical potential of marine microorganisms, with focus on bioactive products as well as on their mechanisms of action.     

Species Diversity of Uncultured and Cultured Populations of Soil and Marine Ammonia Oxidizing Bacteria

Although molecular techniques are considered to provide a more comprehensive view of species diversity of natural microbial populations, few studies have compared diversity assessed by molecular and cultivation-based approaches using the same samples. To achieve this, the diversity of natural populations of ammonia oxidising bacteria in arable soil and marine sediments was determined by analysis of 16S rDNA sequences from enrichment cultures, prepared using standard methods for this group, and from 16S rDNA cloned from DNA extracted directly from the same environmental samples. Soil and marine samples yielded 31 and 18 enrichment cultures, respectively, which were compared with 50 and 40 environmental clones. There was no evidence for selection for particular ammonia oxidizer clusters by different procedures employed for enrichment from soil samples, although no culture was obtained in medium at acid pH. In soil enrichment cultures, Nitrosospira cluster 3 sequences were most abundant, whereas clones were distributed more evenly between Nitrosospira clusters 2, 3, and 4. In marine samples, the majority of enrichment cultures contained Nitrosomonas, whereas Nitrosospira sequences were most abundant among environmental clones. Soil enrichments contained a higher proportion of identical sequences than clones, suggesting laboratory selection for particular strains, but the converse was found in marine samples. In addition, 16% of soil enrichment culture sequences were identical to those in environmental clones, but only 1 of 40 marine enrichments was found among clones, indicating poorer culturability of marine strains represented in the clone library, under the conditions employed. The study demonstrates significant differences in species composition assessed by molecular and culture-based approaches but indicates also that, employing only a limited range of cultivation conditions, 7% of the observed sequence diversity in clones of ammonia oxidizers from these environments could be obtained in laboratory enrichment culture. Further studies and experimental approaches are required to determine which approach provides better representation of the natural community.     

Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products

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.     

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge <Emphasis Type="Italic">Gelliodes carnosa</Emphasis> Collected from the Hainan Island Coastal Waters of the South China Sea

Several molecular techniques were employed to document the bacterial diversity associated with the marine sponge Gelliodes carnosa. Cultivation-dependent and cultivation-independent methods were used to obtain the 16S rRNA gene sequences of the bacteria. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the bacterial community structure was highly diverse with representatives of the high G + C Gram-positive bacteria, cyanobacteria, low G + C Gram-positive bacteria, and proteobacteria (α-, β-, and γ-), most of which were also found in other marine environments, including in association with other sponges. Overall, 300 bacterial isolates were cultivated, and a total of 62 operational taxonomic units (OTUs) were identified from these isolates by restriction fragment length polymorphism (RFLP) analysis and DNA sequencing of the 16S rRNA genes. Approximately 1,000 16S rRNA gene clones were obtained by the cultivation-independent method. A total of 310 clones were randomly selected for RFLP analysis, from which 33 OTUs were acquired by further DNA sequencing and chimera checking. A total of 12 cultured OTUs (19.4% of the total cultured OTUs) and 13 uncultured OTUs (39.4% of the total uncultured OTUs) had low sequence identity (≤97%) with their closest matches in GenBank and were probably new species. Our data provide strong evidence for the presence of a diverse variety of unidentified bacteria in the marine sponge G. carnosa. A relatively high proportion of the isolates exhibited antimicrobial activity, and the deferred antagonism assay showed that over half of the active isolates exhibited a much stronger bioactivity when grown on medium containing seawater. In addition to demonstrating that the sponge-associated bacteria could be a rich source of new biologically active natural products, the results may have ecological implications. This study expands our knowledge of the diversity of sponge-associated bacteria and contributes to the growing database of the bacterial communities within sponges.     

Rapid screening of a large insert BAC library for specific 16S rRNA genes using TRFLP

It is widely believed that the vast majority of microbes in the environment have-yet-to-be cultured using standard techniques. Bulk DNA from microbial communities is therefore often cloned into large insert vectors (e.g. bacterial artificial chromosomes [BAC] or cosmids) in order to study the genetic properties of these as yet (un)-cultured bacteria. In a typical BAC experiment, tens of thousands of clones are generated with only a small fraction of colonies containing the target(s) of interest. Efficient screening methodologies are therefore needed to allow targeted clone isolation. In this paper, we describe a rapid, inexpensive protocol that allows for the identification of specific 16S ribosomal RNA genes in a metagenomic library arrayed into 384-well microtiter plates. The rapid screening protocol employs Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis to identify wells containing specific T-RF peaks. A nested approach using multiplexed samples of 384, 48, 8, and single colony analysis is described and applied in order to survey a BAC library generated from a marine microbial community off the coast of New Jersey. Screening revealed a total of 50 different 16 rRNA genes within the BAC library. Overall, the multiplexing format provided a simple, cost effective methodology for detecting clones bearing a target gene of interest in a large clone library. However, the limitations of screening BAC libraries using PCR methodologies and recommendations for improved screening efficiency using this approach are also discussed.     

Recombinant Environmental Libraries Provide Access to Microbial Diversity for Drug Discovery from Natural Products

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.     

Culture-independent discovery of natural products from soil metagenomes

Bacterial natural products have proven to be invaluable starting points in the development of many currently used therapeutic agents. Unfortunately, traditional culture-based methods for natural product discovery have been deemphasized by pharmaceutical companies due in large part to high rediscovery rates. Culture-independent, or “metagenomic,” methods, which rely on the heterologous expression of DNA extracted directly from environmental samples (eDNA), have the potential to provide access to metabolites encoded by a large fraction of the earth’s microbial biosynthetic diversity. As soil is both ubiquitous and rich in bacterial diversity, it is an appealing starting point for culture-independent natural product discovery efforts. This review provides an overview of the history of soil metagenome-driven natural product discovery studies and elaborates on the recent development of new tools for sequence-based, high-throughput profiling of environmental samples used in discovering novel natural product biosynthetic gene clusters. We conclude with several examples of these new tools being employed to facilitate the recovery of novel secondary metabolite encoding gene clusters from soil metagenomes and the subsequent heterologous expression of these clusters to produce bioactive small molecules.     

碱性土壤微生物基因的克隆和多样性分析

从碱性土壤样品中直接抽提和分离宏基因组DNA, 首先构建了包含5 562个阳性克隆的碱性土壤16S rDNA文库, 随机抽取9个克隆测序后构建的系统进化树表明了碱性土壤环境微生物种群基因的多样性。纯化土壤宏基因组DNA后采用EcoRⅠ酶部分酶切处理, 我们又构建了以pGEM-3Zf(+)为载体的DNA部分文库AL01。AL01文库包含23650个克隆, 随机插入载体的外源DNA片段平均大小为3.2 kb左右, DNA文库的总容量为75.68 Mb。建库效率为从每克环境样品中获得6 000个左右的含随机外源DNA片段插入载体的克隆。采用酶活筛选策略, 我们从AL01文库中筛选到一个编号为pGXAA2011的阳性克隆携带有一个完整的碱性蛋白酶基因。蛋白酶活性检测其酶活作用最佳温度为40℃, 最适作用pH 值为9.5。另外, 我们还克隆和表达了一个新型b-葡萄糖苷酶基因unglu01, 该基因和现有数据库中的b-葡萄糖苷酶基因没有任何DNA或者氨基酸水平的同源性。将unglu01基因的ORF与表达载体pETBlue-2连接后导入宿主菌株Tuner(DE3)pLacI中, 该重组表达克隆在含柠檬酸高铁铵和七叶苷的LA平板上表现清晰的b-葡萄糖苷酶活性, SDS-PAGE电泳可以检测到29 kDa大小的目的蛋白。     

Intracellular screen to identify metagenomic clones that induce or inhibit a quorum-sensing biosensor

The goal of this study was to design and evaluate a rapid screen to identify metagenomic clones that produce biologically active small molecules. We built metagenomic libraries with DNA from soil on the floodplain of the Tanana River in Alaska. We extracted DNA directly from the soil and cloned it into fosmid and bacterial artificial chromosome vectors, constructing eight metagenomic libraries that contain 53,000 clones with inserts ranging from 1 to 190 kb. To identify clones of interest, we designed a high throughput "intracellular" screen, designated METREX, in which metagenomic DNA is in a host cell containing a biosensor for compounds that induce bacterial quorum sensing. If the metagenomic clone produces a quorum-sensing inducer, the cell produces green fluorescent protein (GFP) and can be identified by fluorescence microscopy or captured by fluorescence-activated cell sorting. Our initial screen identified 11 clones that induce and two that inhibit expression of GFP. The intracellular screen detected quorum-sensing inducers among metagenomic clones that a traditional overlay screen would not. One inducing clone carries a LuxI homologue that directs the synthesis of an N-acyl homoserine lactone quorum-sensing signal molecule. The LuxI homologue has 62% amino acid sequence identity to its closest match in GenBank, AmfI from Pseudomonas fluorescens, and is on a 78-kb insert that contains 67 open reading frames. Another inducing clone carries a gene with homology to homocitrate synthase. Our results demonstrate the power of an intracellular screen to identify functionally active clones and biologically active small molecules in metagenomic libraries.     

Identification of active bacterial communities in a model drinking water biofilm system using 16S rRNA-based clone libraries

Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rRNA gene clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, an annular reactor was used to generate model drinking water biofilms grown on polycarbonate slides. High-quality RNA was extracted from 2-month-old biofilms and used to generate 16S rRNA-based clones. Sequencing analyses of 16S rRNA-based clones suggested that the active bacterial fraction consisted of a few dominant bacterial groups related to Nevskia ramosa and to uncultured bacteria. Several of these bacterial groups were closely related to clones characterized in a DNA-based clone library also generated in this study. Altogether, these results suggest that some of the predominant drinking water bacteria identified using DNA-based techniques are indeed active.