Novel bioactive natural products from bacteria via bioprospecting,genome mining and metabolic engineering

For over seven decades, bacteria served as a valuable source of bioactive natural products some of which were eventually developed into drugs to treat infections, cancer and immune system-related diseases. Traditionally, novel compounds produced by bacteria were discovered via conventional bioprospecting based on isolation of potential producers and screening their extracts in a variety of bioassays. Over time, most of the natural products identifiable by this approach were discovered, and the pipeline for new drugs based on bacterially produced metabolites started to run dry. This mini-review highlights recent developments in bacterial bioprospecting for novel compounds that are based on several out-of-the-box approaches, including the following: (i) targeting bacterial species previously unknown to produce any bioactive natural products, (ii) exploring non-traditional environmental niches and methods for isolation of bacteria and (iii) various types of ‘genome mining’ aimed at unravelling genetic potential of bacteria to produce secondary metabolites. All these approaches have already yielded a number of novel bioactive compounds and, if used wisely, will soon revitalize drug discovery pipeline based on bacterial natural products.     

The complexity of the Rab and Rho GTP-binding protein subfamilies revealed by a PCR cloning approach.

Partial sequences corresponding to eleven novel Rab proteins and one new Rho protein have been isolated using a PCR-based cloning approach. These results confirm that the overall diversity of the Rab and Rho protein subfamilies account for more than thirty different members in mammalian cells.     

Quantification of mesocosm fish and amphibian species diversity via environmental DNA metabarcoding

Freshwater fauna are particularly sensitive to environmental change and disturbance. Management agencies frequently use fish and amphibian biodiversity as indicators of ecosystem health and a way to prioritize and assess management strategies. Traditional aquatic bioassessment that relies on capture of organisms via nets, traps and electrofishing gear typically has low detection probabilities for rare species and can injure individuals of protected species. Our objective was to determine whether environmental DNA (eDNA) sampling and metabarcoding analysis can be used to accurately measure species diversity in aquatic assemblages with differing structures. We manipulated the density and relative abundance of eight fish and one amphibian species in replicated 206‐L mesocosms. Environmental DNA was filtered from water samples, and six mitochondrial gene fragments were Illumina‐sequenced to measure species diversity in each mesocosm. Metabarcoding detected all nine species in all treatment replicates. Additionally, we found a modest, but positive relationship between species abundance and sequencing read abundance. Our results illustrate the potential for eDNA sampling and metabarcoding approaches to improve quantification of aquatic species diversity in natural environments and point the way towards using eDNA metabarcoding as an index of macrofaunal species abundance.     

Experimental approaches to identify non-coding RNAs

Cellular RNAs that do not function as messenger RNAs (mRNAs), transfer RNAs (tRNAs) or ribosomal RNAs (rRNAs) comprise a diverse class of molecules that are commonly referred to as non-protein-coding RNAs (ncRNAs). These molecules have been known for quite a while, but their importance was not fully appreciated until recent genome-wide searches discovered thousands of these molecules and their genes in a variety of model organisms. Some of these screens were based on biocomputational prediction of ncRNA candidates within entire genomes of model organisms. Alternatively, direct biochemical isolation of expressed ncRNAs from cells, tissues or entire organisms has been shown to be a powerful approach to identify ncRNAs both at the level of individual molecules and at a global scale. In this review, we will survey several such wet-lab strategies, i.e. direct sequencing of ncRNAs, shotgun cloning of small-sized ncRNAs (cDNA libraries), microarray analysis and genomic SELEX to identify novel ncRNAs, and discuss the advantages and limits of these approaches.     

Using rarity to infer how dendritic network structure shapes biodiversity in riverine communities

Dispersal of organisms connects physical localities, but the strength of connection varies widely. Variability in the influence of dispersal can be predictable in sharply defined networks like river systems because some sections of the network are more isolated, leading to different balances of local (i.e. environmental filtering, species interactions) and regional (i.e. dispersal‐driven) processes in structuring communities. We examined the influence of spatial isolation on the relative contributions of α‐ and β‐diversity to regional (γ) diversity, and examined how that influence differed between common and rare species in stream macroinvertebrate communities. One explanation for rarity on a regional scale is that common species are habitat generalists while rare species are specialists. Therefore, common species should be influenced more by dispersal‐driven processes while rare species should be more influenced by local processes. We predicted that for rare taxa, β‐diversity should represent a higher fraction of γ‐diversity in isolated headwaters but that differences between rare and common taxa with regard to the contribution of β‐diversity to γ‐diversity should be less distinct in well‐connected mainstem habitats. To test these predictions, we used macroinvertebrate communities from 634 sites across 22 watersheds. Regardless of rarity, β‐ and γ‐diversity were higher in headwaters compared to mainstems. However, α‐diversity was similar regardless of isolation for rare assemblages. But contrary to our predictions, common assemblages of predators and herbivores did exhibit differences in α‐diversity between locations. Our predictions were strongly supported for two guilds of consumers, the detritivores and collectors, but less so for herbivores and predators. However, these results make sense considering differences in life histories between the groups. For detritivores and collectors, species turnover (β‐diversity) was higher in isolated regions in river networks, and rarity exacerbated this effect, resulting in higher regional diversity of rare species, supporting the general theory that rarity reflects habitat specialization.     

Rare genera of actinomycetes as potential producers of new antibiotics

A literature survey covering more than twenty-three thousand bioactive microbial products including eight thousand antiinfectives demonstrated the increasing relevance of the so called 'rare' actinomycetes as a source of new antibiotics. Past and present efforts in the isolation of rare actinomycetes have enriched the Biosearch Italia Strain Collection with more than twenty thousand strains, showing that, when selective isolation methods are developed and extensively applied, some genera, such as Actinomadura, Actinoplanes, Micromonospora, Microtetraspora, are not rare at all and can be recovered from many soil samples. The current focus is on the isolation of members of Streptosporangiaceae family, given their promising chemical diversity.     

Winogradsky columns as a strategy to study typically rare microbial eukaryotes

Winogradsky columns have been widely used to study soil microbial communities, but the vast majority of those investigations have focused on the ecology and diversity of bacteria. In contrast, microbial eukaryotes (ME) have been regularly overlooked in studies based on experimental soil columns. Despite the recognized ecological relevance of ME in soil communities, investigations focused on ME diversity and the abundance of certain groups of interest are still scarce. In the present study, we used DNA metabarcoding (high-throughput sequencing of the V4 region of the 18S rRNA locus) to survey the ME diversity and abundance in an experimental Winogradsky soil column. Consistent with previous surveys in natural soils, our survey identified members of Cercozoa (Rhizaria; 31.2%), Apicomplexa and Ciliophora (Alveolata; 12.5%) as the predominant ME groups, but at particular depths we also detected the abundant presence of ME lineages that are typically rare in natural environments, such as members of the Vampyrellida (Rhizaria) and Breviatea (Amorphea). Our survey demonstrates that experimental soil columns are an efficient enrichment-culture approach that can enhance investigations about the diversity and ecology of ME in soils.     

PCR克隆新基因策略研究进展

自从PCR被开发及其基本反应条件被优化以来,这项技术已被应用于分子生物学研究的各个方面。其中应用最多的是从给定的有机体中扩增和克隆新基因：那些从事新基因的结构和功能分析研究的工作者必须获得第一手资料,也就是新基因的序列。而且快速方便地克隆这些新基因序列在很大程度上依赖于那些由PCR技术演变而来的方法。本文综述概括了建立在PCR基础上的方法在新基因分离克隆中的各种应用.     

A global census of nitrogenase diversity

The global diversity of nitrogen-fixing microorganisms was assessed through construction and analysis of an aligned database of 16,989 nifH sequences. We conclude that the diversity of diazotrophs is still poorly described and that many organisms remain to be discovered. Our analyses indicate that diversity is not distributed evenly across phylogenetic groups or across environments and that some of the most diverse assemblages and environments remain the most poorly characterized. The majority of OTUs were rare, falling in the long tail of the frequency distribution. The most dominant OTUs fell into either the Cyanobacteria or the α, β, and γ Proteobacteria, and five of these dominant OTUs do not have any representatives cultivated in isolation. Soils contained the greatest diversity of nifH sequences of all of the environments surveyed. Cluster III, which is dominated by nifH sequences from obligate anaerobes, was found to contain the greatest diversity of all nifH lineages and is also the group for which diversity is the least sampled. Our findings provide context for ongoing efforts to explore diazotroph diversity, indicating specific groups and environments that remain poorly characterized.     

Rare genera of actinomycetes as potential producers of new antibiotics

A literature survey covering more than twenty-three thousand bioactive microbial products including eight thousand antiinfectives demonstrated the increasing relevance of the so called 'rare' actinomycetes as a source of new antibiotics. Past and present efforts in the isolation of rare actinomycetes have enriched the Biosearch Italia Strain Collection with more than twenty thousand strains, showing that, when selective isolation methods are developed and extensively applied, some genera, such as Actinomadura, Actinoplanes, Micromonospora, Microtetraspora, are not rare at all and can be recovered from many soil samples. The current focus is on the isolation of members of Streptosporangiaceae family, given their promising chemical diversity.     

Rare taxa and dark microbial matter: novel bioactive actinobacteria abound in Atacama Desert soils

An “in house” taxonomic approach to drug discovery led to the isolation of diverse actinobacteria from hyper-arid, extreme hyper-arid and very high altitude Atacama Desert soils. A high proportion of the isolates were assigned to novel taxa, with many showing activity in standard antimicrobial plug assays. The application of more advanced taxonomic and screening strategies showed that strains classified as novel species of Lentzea and Streptomyces synthesised new specialised metabolites thereby underpinning the premise that the extreme abiotic conditions in the Atacama Desert favour the development of a unique actinobacterial diversity which is the basis of novel chemistry. Complementary metagenomic analyses showed that the soils encompassed an astonishing degree of actinobacterial ‘dark matter’, while rank-abundance analyses showed them to be highly diverse habitats mainly composed of rare taxa that have not been recovered using culture-dependent methods. The implications of these pioneering studies on future bioprospecting campaigns are discussed.     

Ecosystem productivity is associated with bacterial phylogenetic distance in surface marine waters

Understanding the link between community diversity and ecosystem function is a fundamental aspect of ecology. Systematic losses in biodiversity are widely acknowledged but the impact this may exert on ecosystem functioning remains ambiguous. There is growing evidence of a positive relationship between species richness and ecosystem productivity for terrestrial macro‐organisms, but similar links for marine micro‐organisms, which help drive global climate, are unclear. Community manipulation experiments show both positive and negative relationships for microbes. These previous studies rely, however, on artificial communities and any links between the full diversity of active bacterial communities in the environment, their phylogenetic relatedness and ecosystem function remain hitherto unexplored. Here, we test the hypothesis that productivity is associated with diversity in the metabolically active fraction of microbial communities. We show in natural assemblages of active bacteria that communities containing more distantly related members were associated with higher bacterial production. The positive phylogenetic diversity–productivity relationship was independent of community diversity calculated as the Shannon index. From our long‐term (7‐year) survey of surface marine bacterial communities, we also found that similarly, productive communities had greater phylogenetic similarity to each other, further suggesting that the traits of active bacteria are an important predictor of ecosystem productivity. Our findings demonstrate that the evolutionary history of the active fraction of a microbial community is critical for understanding their role in ecosystem functioning.     

Metagenomic exploration of antibiotic resistance in soil

The ongoing development of metagenomic approaches is providing the means to explore antibiotic resistance in nature and address questions that could not be answered previously with conventional culture-based strategies. The number of available environmental metagenomic sequence datasets is rapidly expanding and henceforth offer the ability to gain a more comprehensive understanding of antibiotic resistance at the global scale. Although there is now evidence that the environment constitutes a vast reservoir of antibiotic resistance gene determinants (ARGDs) and that the majority of ARGDs acquired by human pathogens may have an environmental origin, a better understanding of their diversity, prevalence and ecological significance may help predict the emergence and spreading of newly acquired resistances. Recent applications of metagenomic approaches to the study of ARGDs in natural environments such as soil should help overcome challenges concerning expanding antibiotic resistances.     

Metagenomics: Concept,methodology, ecological inference and recent advances

Microorganisms constitute two third of the Earth's biological diversity. As many as 99% of the microorganisms present in certain environments cannot be cultured by standard techniques. Culture-independent methods are required to understand the genetic diversity, population structure and ecological roles of the majority of organisms. Metagenomics is the genomic analysis of microorganisms by direct extraction and cloning of DNA from their natural environment. Protocols have been developed to capture unexplored microbial diversity to overcome the existing barriers in estimation of diversity. New screening methods have been designed to select specific functional genes within metagenomic libraries to detect novel biocatalysts as well as bioactive molecules applicable to mankind. To study the complete gene or operon clusters, various vectors including cosmid, fosmid or bacterial artificial chromosomes are being developed. Bioinformatics tools and databases have added much to the study of microbial diversity. This review describes the various methodologies and tools developed to understand the biology of uncultured microbes including bacteria, archaea and viruses through metagenomic analysis.     

Diploid/polyploid syntenic shuttle mapping and haplotype-specific chromosome walking toward a rust resistance gene (Bru1) in highly polyploid sugarcane (2n approximately 12x approximately 115)

The genome of modern sugarcane cultivars is highly polyploid ( approximately 12x), aneuploid, of interspecific origin, and contains 10 Gb of DNA. Its size and complexity represent a major challenge for the isolation of agronomically important genes. Here we report on the first attempt to isolate a gene from sugarcane by map-based cloning, targeting a durable major rust resistance gene (Bru1). We describe the genomic strategies that we have developed to overcome constraints associated with high polyploidy in the successive steps of map-based cloning approaches, including diploid/polyploid syntenic shuttle mapping with two model diploid species (sorghum and rice) and haplotype-specific chromosome walking. Their applications allowed us (i) to develop a high-resolution map including markers at 0.28 and 0.14 cM on both sides and 13 markers cosegregating with Bru1 and (ii) to develop a physical map of the target haplotype that still includes two gaps at this stage due to the discovery of an insertion specific to this haplotype. These approaches will pave the way for the development of future map-based cloning approaches for sugarcane and other complex polyploid species.     

Are cladoceran diversity and community structure linked to spatial heterogeneity in urban landscapes and pond environments?

Biodiversity patterns in cladoceran communities were investigated in urban waterbodies in relation with residential land use, pond management, and waterbody environments. We evaluated species richness in the pelagic and littoral zones of eighteen waterbodies of a large Canadian city. Gamma diversity (26 species) observed at a small scale in the urban survey was important comparatively to large-scale surveys of lakes. Beta diversity ranged from 1 to 8 species among waterbodies. We tested if littoral species greatly contributed to regional diversity in urban waterbodies. Littoral species (Chydoridae, Ilyocryptidae, Macrothricidae, Polyphemidae) accounted for 58% of the total species pool. We distinguished five cladoceran assemblages associated to different waterbodies (temporary ponds, permanent lakes, and wetlands). Cladoceran communities were more diverse and variable in permanent lakes than in temporary ponds. Changes in cladoceran species assemblages among waterbodies were driven by variations in waterbody size and phosphorus enrichment, macrophyte and algal biomass, urban density, pond management practices, and the presence of potential predators as fish and macroinvertebrates. Our study indicates that both artificial ponds and lakes and natural wetlands are valuable habitats for the conservation of cladoceran biodiversity and rare endemic species in urban regions. Further research on pond management strategies promoting urban aquatic biodiversity should be undertaken.     

Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes.

PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of chimeric molecule formation, mixed genomic DNAs from eight actinomycete species whose 16S rRNA sequences had been determined were used for PCR coamplification of 16S rRNA genes. A large number of cloned 16S ribosomal DNAs were examined by sequence analysis, and chimeric molecules were identified by multiple-sequence alignment with reference species. Here, we report that the level of occurrence of chimeric sequences after 30 cycles of PCR amplification was 32%. We also show that PCR-induced chimeras were formed between different rRNA gene copies from the same organism. Because of the wide use of PCR for direct isolation of 16S rRNA sequences from environmental DNA to assess microbial diversity, the extent of chimeric molecule formation deserves serious attention.     

Exploring the bioprospecting and biotechnological potential of white-rot and anaerobic Neocallimastigomycota fungi: peptidases,esterases, and lignocellulolytic enzymes

Fungi constitute an invaluable natural resource for scientific research, owing to their diversity; they offer a promising alternative for bioprospecting, thus contributing to biotechnological advances. For a long time, extensive information has been exploited and fungal products have been tested as a source of natural compounds. In this context, enzyme production remains a field of interest, since it offers an efficient alternative to the hazardous processes of chemical transformations. Owing to their vast biodiversity and peculiar biochemical characteristics, two fungal categories, white-rot and anaerobic Neocallimastigomycota, have gathered considerable attention for biotechnological applications. These fungi are known for their ability to depolymerize complex molecular structures and are used in degradation of lignocellulosic biomass, improvement of animal feed digestibility, biogas and bioethanol production, and various other applications. However, there are only limited reports that describe proteolytic enzymes and esterases in these fungi and their synergistic action with lignocellulolytic enzymes on degradation of complex polymers. Thus, in this minireview, we focus on the importance of these organisms in enzyme technology, their bioprospecting, possibility of integration of their enzyme repertoire, and their prospects for future biotechnological innovation.