Microbial Library Research Articles

Use of photoswitchable fluorescent proteins for droplet-based microfluidic screening

Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a “green” to a “red” state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

S11.3 Use of whole genome sequencing to determine the probability of antimicrobial resistance inneisseria gonorrhoeae

Neisseria gonorrhoeae has demonstrated remarkable ability to rapidly develop antimicrobial resistance to each antimicrobial class used for therapy including the currently recommended class of antimicrobials, the cephalosporins. Emerging resistance to cephalosporins will severely complicate treatment of gonorrhoea. Advanced Molecular Detection approaches, such as whole genome sequencing to determine mechanisms of resistance, have the potential to advance our understanding of gonorrhoea. To date, we have sequenced >1,000 genomes from N. gonorrhoeae isolates with elevated cefixime or azithromycin minimum inhibitory concentrations (MICs) or resistance to previously recommended antimicrobials, and isolates collected from multiple geographic sites to accomplish the following objectives: (1) to identify mutations that confer antimicrobial resistance or increased MICs; (2) to inform the development of molecular assays for resistance determinants through insights gleaned from WG from which point-of-care assays for resistance markers will be developed. Such assays will change the paradigm of gonorrhoea treatment. Use of real-time results on the presence or absence of resistance determinants will allow clinicians to personalise treatment for patients, and prevent inadvertent use of agents that are no longer routinely recommended because of resistance. Such assays can also substantially expand the reach of surveillance of antimicrobial resistance and allow public health officials to rapidly detect and respond to outbreaks of resistant strains; and (3) to develop and maintain a microbial library, a web-accessible and searchable database that will include bacterial and viral genomic sequences and associated meta-data, such as basic de-identified demographic characteristics of patients and phenotypic susceptibility data. In the long term, knowledge of the genetic mechanisms responsible for resistance or deceased susceptibility in N. gonorrhoeae gained from sequencing may translate into the identification or development of new therapeutic agents.

Biosynthesis of nectrisine in Thelonectria discophora SANK 18292

Nectrisine, an iminosugar with a heterocyclic nitrogen-containing 5-membered ring, acts as a glycosidase inhibitor. Thelonectria discophora SANK 18292, a fungus, was identified as a nectrisine producer from its microbial library in our screening for nectrisine producing microorganisms. Biosynthesis of nectrisine produced by the fungus was studied using stable isotope tracer techniques. Incorporation of 13C-labeled d-ribose and d-xylose into nectrisine was confirmed by mass spectrometry and 13C NMR spectroscopy, which suggested that these were its precursors. Chromatographic separation of the hot water extract from the culture broth afforded not only nectrisine, but also substantial amounts of 4-amino-4-deoxyarabinitol. Incubation of the latter with the crude enzyme of the fungus at room temp. caused an increase in levels of nectrisine together with a decrease in amounts of the administered potential precursor suggesting that it is a biosynthetic intermediate. From these results, a biosynthetic pathway to nectrisine is proposed via d-xylulose 5-phosphate and 4-amino-4-deoxyarabinitol by the pentose phosphate pathway.

Identifying producers of antibacterial compounds by screening for antibiotic resistance

Microbially derived natural products are major sources of antibiotics and other medicines, but discovering new antibiotic scaffolds and increasing the chemical diversity of existing ones are formidable challenges. We have designed a screen to exploit the self-protection mechanism of antibiotic producers to enrich microbial libraries for producers of selected antibiotic scaffolds. Using resistance as a discriminating criterion we increased the discovery rate of producers of both glycopeptide and ansamycin antibacterial compounds by several orders of magnitude in comparison with historical hit rates. Applying a phylogeny-based screening filter for biosynthetic genes enabled the binning of producers of distinct scaffolds and resulted in the discovery of a glycopeptide antibacterial compound, pekiskomycin, with an unusual peptide scaffold. This strategy provides a means to readily sample the chemical diversity available in microbes and offers an efficient strategy for rapid discovery of microbial natural products and their associated biosynthetic enzymes.

Methane production pathway in biogas reservoirs via molecular biological technique

Abstract In order to investigate the microbes in formation water samples from Well Du 6-3 and Ao-7 in the Alaxin and Aonan gas fields, Songliao Basin, China, 16S rDNA was amplified by using the Polymerase Chain Reaction (PCR) technique with templates of the extracted DNA and primers of bacteria and archaea. The PCR products were also inserted into vectors and then ligated with competent cells for gene sequencing. Based on the obtained gene sequences, bacterial and archaeal libraries were constructed and microbiological phylogeny graphs were obtained for the samples to understand the structure of microbial community and deduce the possible pathways of methanogenesis. The result indicates that there are only a few types of microbes in the samples of the two wells. Pseudomonas and Methanobacterium are the dominant species in the microbial and archaeal libraries, respectively. It is concluded that the indigenous microbial community in Well Du 6-3 probably mainly generated methane by CO 2 reduction, but methanogenesis of acetic acid could not be completely excluded. The methane from Well Ao-7 is shown to have been definitely converted from CO 2 . Therefore, methane generation via indigenous microbial metabolism may be ultimately realized by using the right method under favourable conditions in the two fields.

ChemInform Abstract: Natural Products from Marine Derived Microorganisms

AbstractReview: 17 refs.

DNA Extraction and Construction of a Metagenomic Fosmid Library of Alpine Meadow Soil from the Mila Mountains in Tibet, China*

The Qinghai-Tibetan Plateau represents an extreme environment for special microbial resources. Extraction of total microbial DNA with cultured-in-dependent method and construction of metagenomic library is an alternative method to exploit uncultured microorganisms. The meadow soil from the Mila Mountains in Tibet was selected as alpine soil type and was collected. A pre-experiment indicated that the soil had high organic matter and humic acid content,which resulted in a great difficulty in DNA extraction. Our results showed that the DNA obtained by direct extraction from the soil was in small DNA fragments with high contamination,and it was not suitable for constructing big insert fragment library. Comparison with direct extraction,the DNA extracted from cells separated from combing low speed centrifugation and Nycodenz gradient centrifugation had larger molecular weigh and higher purity with great diversity. A microbial metagenomic library,which contained 30 624 clones and a total genomic storage capacity over 1 Gb,was constructed with the DNA fragments from indirect method. Bioassay demonstrated that the constructed Fosmid library had large capability of insert DNA with unknown species. The method for constructing library provides a powerful tool for soil samples from extreme environment and the constructed library provides resources for finding and utilizing new functional genes from the Tibetan Plateau. Fig 7,Tab 3,Ref 27

海洋微生物を用いた天然物化学

Marine microbial resources have been considered as potent sources for new and potent medicines. We have constructed marine microbial library consists of 50,000 strains. Among them, 26,000 strains have phylogenetically analyzed based on SSU–rRNA sequences. Number of biologically active substances have been isolated from marine microbes in this library using the screening based on marine phenomena. One typical example is thallusin (2), isolated in 2005 after pursuing for more than 50 years, showed foliaceous morphology–inducing activity at the exceptionally low concentration. Antimicroalgal substances HML (6) and Kailuin B (9) have been isolated from marine bacteria belongs to Vibrio. These compounds showed selective toxicity only against dinofragellates. Useful microalgae such as diatom was unaffected by these substances. Interestingly, these substances also showed potent anti–malarial activity. It is not strange, because dinofragellete and malaria are phylogenetically related to each other. As shown in the isolation of above mentioned substances and unnermicins, the screening methods based on marine phenomena seemed to be useful for the efficient isolation of novel biologically active compounds.

Novel technology for high throughput processing of microbial libraries for screening applications

Novel technology for high throughput processing of microbial libraries for screening applications

Novel method for high-throughput colony PCR screening in nanoliter-reactors

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20 000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100 000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies.

Building Partnership for Drug and Ag Discovery and Conservation of the Natural Resources in Brazil

Cerrado, Brazilian savanna, covers 2 million km2, representing 23% of the land surface of the country. It occupies the central part of Brazil, from the margin of the Amazonian forest to outlying areas in the southern states of Sao Paulo. According to Dias' [1] estimation, the Cerrado contains 160,000 species of plants, fungi and animals. This proposed research program will expand and upgrade the conservation effort. The project will: 1) build an International Partnership on Conservation and Natural Product Discovery; 2) map and protect the genetic resources by establishing germplasm bank of two endemic families Leguminosae and Combrestaceae; 3) search for new pharmaceuticals and agrochemicals to control tropical diseases, and agricultural pests and pathogens, 4) create an Eco-extract-library and ex situ collections for future studies; 5) establish a microbial library of plant associated microorganisms. As the establishment of in vitro germplasm bank progresses, endophytic microbes commonly associated with plants will outgrow the host tissues and allow us to detect and identify them. Some of these organisms are responsible for production of secondary metabolites [2,3]. Clonal propagation by in vitro methods will supply the biomass for fractionation and isolation of the active metabolite(s) and future developments. In addition, micropropagation will provide a unique opportunity to identify and evaluate the contribution of plant associated microorganism to the biological properties.

Mining for new enzymes

Mining for new enzymes

Metagenomic Analysis of Lysogeny in Tampa Bay: Implications for Prophage Gene Expression

Phage integrase genes often play a role in the establishment of lysogeny in temperate phage by catalyzing the integration of the phage into one of the host's replicons. To investigate temperate phage gene expression, an induced viral metagenome from Tampa Bay was sequenced by 454/Pyrosequencing. The sequencing yielded 294,068 reads with 6.6% identifiable. One hundred-three sequences had significant similarity to integrases by BLASTX analysis (e≤0.001). Four sequences with strongest amino-acid level similarity to integrases were selected and real-time PCR primers and probes were designed. Initial testing with microbial fraction DNA from Tampa Bay revealed 1.9×107, and 1300 gene copies of Vibrio-like integrase and Oceanicola-like integrase L−1 respectively. The other two integrases were not detected. The integrase assay was then tested on microbial fraction RNA extracted from 200 ml of Tampa Bay water sampled biweekly over a 12 month time series. Vibrio-like integrase gene expression was detected in three samples, with estimated copy numbers of 2.4-1280 L−1. Clostridium-like integrase gene expression was detected in 6 samples, with estimated copy numbers of 37 to 265 L−1. In all cases, detection of integrase gene expression corresponded to the occurrence of lysogeny as detected by prophage induction. Investigation of the environmental distribution of the two expressed integrases in the Global Ocean Survey Database found the Vibrio-like integrase was present in genome equivalents of 3.14% of microbial libraries and all four viral metagenomes. There were two similar genes in the library from British Columbia and one similar gene was detected in both the Gulf of Mexico and Sargasso Sea libraries. In contrast, in the Arctic library eleven similar genes were observed. The Clostridium-like integrase was less prevalent, being found in 0.58% of the microbial and none of the viral libraries. These results underscore the value of metagenomic data in discovering signature genes that play important roles in the environment through their expression, as demonstrated by integrases in lysogeny.

Novel Small-Molecules Isolated from a Marine Microbial Library Inhibit IL-5 Production In Vitro

Novel Small-Molecules Isolated from a Marine Microbial Library Inhibit IL-5 Production In Vitro

Evaluation of the anti-Trichophyton activity of a prodigiosin analogue produced by γ-proteobacterium, using stratum corneum epidermis of the Yucatan micropig

Prodigiosins (PGs) are known to be a family of natural red pigments, characterized by a common pyrrolydipyrrolylmethane skeleton structure with a C-4 methoxy group, and some of these pigments have been isolated from some microorganisms. Members of the PG family have been reported to show several biological activities, such as immunosuppressive and cytotoxic activities. Recently, we discovered a bacterial strain (MS-02-063), from our microbial library, that produces large amounts of a PG analogue (PG-L-1). In this study, we examined the anti-Trichophyton activity of PG-L-1 (produced by strain MS-02-063) against clinically isolated Trichophyton spp., by a method using stratum corneum epidermis (SCE) of the Yucatan micropig, which is suitable for estimating the antifungal activity of drugs in vitro. In the National Committee for Clinical Laboratory Standards (NCCLS) method, PG-L-1 showed potent antifungal activity against nine clinically isolated strains of Trichophyton spp., although the minimum inhibitory concentration (MIC) values were slightly higher than those of bifonazole. In spite of the lower efficiency of PG-L-1 transfer into SCE from medium than that of bifonazole, PG-L-1 transferred into SCE showed more potent antifungal activity than bifonazole, at lower concentrations.

Gongronella butleri, Schizosaccharomyces octosporus and Diplogelasinospora grovesii: novel microorganisms useful for the stereoselective reduction of ketones

A microbial library composed of 416 strains was selected from public collections. The selection was based on the criteria of the highest biodiversity achievable, being finally composed of 71 bacteria strains, 45 actinomycetes, 59 yeasts, 148 filamentous fungi, 33 marine fungi and 60 basidiomycetes. This set of microorganisms was used for a screening campaign searching for microbial biocatalysts for the stereoselective reduction of carbonylic compounds. All the microorganisms mentioned are mesophilic considering their optimal growth requirements. Gongronella butleri, Diplogelasinospora grovesii and Schizosaccharomyces octosporus were selected as the most interesting strains according to their productivity, their tolerance to high concentrations of ketones and the absence of secondary products in the reduction of cycloalkanones. The reduction of (4 R)- or (4 S)-carvones showed that the stereochemistry of the ketones determine the reduction pathway. Indeed, while (4 S)-carvone yields the (2 R)-alcohol, the (4 R)-carvone carries to dihydrocarveol. D. grovesii and S. octosporus were the strains that displayed better selectivity. The use of resting cells conditions or the lyophilised whole cells reduce the catalytic activity. We describe for the first time a strain, D. grovesii IMI 171018, that is able to produce (2 R,4 S)-carveol from (4 S)-carvone with good yields and enantiomeric excess (ee).

Rapid identification of enantioselective ketone reductions using targeted microbial libraries

A collection of about 300 microbes was surveyed for the ability to generate chiral secondary alcohols by enantioselective reduction of a series of alkyl aryl ketones. Microbial cultures demonstrating utility in reducing model ketones were arrayed in multi-well plates and used to rapidly identify specific organisms capable of producing chiral alcohols used as intermediates in the synthesis of several drug candidates. Approximately 60 cultures were shown to selectively reduce various ketones providing both the R and S enantiomers of the corresponding alcohols in 92–99% ee with yields up to 95% at 1–4 g/L. An alternative approach to chiral alcohols based on selective microbial oxidation of racemic alcohols is also reported. This study provides a useful reference for generating chiral alcohols by selective microbial bioconversion.

Implementation of a rapid microbial screening procedure for biotransformation activities

A rapid and efficient microbial screening procedure was developed utilizing a 24-well plate format in conjunction with an automated liquid handling system and an HPLC. For the evaluation of this miniaturized and automated screening system, we selected the bioreduction of 6-bromo-β-tetralone to 6-bromo-β-tetralol. This procedure employed both yeast and rhodococci libraries, representing a culture collection comprised of several hundred strains, from which to screen for desirable bioconversion activity. Most of these strains had demonstrated bioreducing activity during previous screens to insure a “hit rate” as high as possible. The cultivation of microbes in the plate format was facile, time saving, and efficient compared to the standard method of screening utilizing larger volumes, such as test tubes or shake flasks. This improved method of screening for bioconversion activity, employing pre-selected microbial libraries based on microtiter plates and a fully roboticized analytical system, proved to rapidly yield valuable leads which compared advantageously with a more classical approach. A total of 192 yeast strains and 48 rhodococci strains were screened using this procedure. Analytical data revealed that 78% of the strains tested bioconverted the tetralone to the desired alcohol.