Asuncion Martinez

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

ABSTRACT 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.

Proteorhodopsin lateral gene transfer between marine planktonic Bacteria and Archaea

Planktonic Bacteria, Archaea and Eukarya reside and compete in the oceans photic zone under the pervasive influence of light. Bacteria in this environment were recently shown to contain photoproteins called proteorhodopsins, thought to contribute to cellular energy metabolism by catalysing light-driven proton translocation across the cell membrane. So far, proteorhodopsin genes have been well documented only in proteobacteria and a few other bacterial groups. Here we report the presence and distribution of proteorhodopsin genes in Archaea affiliated with the order Thermoplasmatales, in the oceans upper water column. The genomic context and phylogenetic relationships of the archaeal and proteobacterial proteorhodopsins indicate its probable lateral transfer between planktonic Bacteria and Archaea. About 10% of the euryarchaeotes in the photic zone contained the proteorhodopsin gene adjacent to their small-subunit ribosomal RNA. The archaeal proteorhodopsins were also found in other genomic regions, in the same or in different microbial lineages. Although euryarchaeotes were distributed throughout the water column, their proteorhodopsins were found only in the photic zone. The cosmopolitan phylogenetic distribution of proteorhodopsins reflects their significant light-dependent fitness contributions, which drive the photoproteins lateral acquisition and retention, but constrain its dispersal to the photic zone.

Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts

ABSTRACT The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.

Widespread known and novel phosphonate utilization pathways in marine bacteria revealed by functional screening and metagenomic analyses

Phosphonates (Pn), compounds with a direct C-P bond instead of the more common C-O-P ester bond, constitute a significant fraction of marine dissolved organic phosphorus and recent evidence suggests that they may be an alternative source of P for marine microorganisms. To further characterize the microorganisms and pathways involved in Pn utilization, we screened bacterioplankton genomic libraries for their ability to complement an Escherichia coli strain unable to use Pns as a P source. Using this approach we identified a phosphonatase pathway as well as a novel pair of genes that allowed utilization of 2-aminoethylphosphonate (2-AEPn) as the sole P source. These pathways are present in diverse bacteria common in marine plankton including representatives of Proteobacteria, Planctomycetes and Cyanobacteria. Analysis of metagenomic databases for Pn utilization genes revealed that they are widespread and abundant among marine bacteria, suggesting that Pn metabolism is likely to play an important role in P-depleted surface waters, as well as in the more P-rich deep-water column.

Light-induced transcriptional responses associated with proteorhodopsin-enhanced growth in a marine flavobacterium

Proteorhodopsin (PR) is a photoprotein that functions as a light-driven proton pump in diverse marine Bacteria and Archaea. Recent studies have suggested that PR may enhance both growth rate and yield in some flavobacteria when grown under nutrient-limiting conditions in the light. The direct involvement of PR, and the metabolic details enabling light-stimulated growth, however, remain uncertain. Here, we surveyed transcriptional and growth responses of a PR-containing marine flavobacterium during carbon-limited growth in the light and the dark. As previously reported (Gómez-Consarnau et al., 2007), Dokdonia strain MED134 exhibited light-enhanced growth rates and cell yields under low carbon growth conditions. Inhibition of retinal biosynthesis abolished the light-stimulated growth response, supporting a direct role for retinal-bound PR in light-enhanced growth. Among protein-coding transcripts, both PR and retinal biosynthetic enzymes showed significant upregulation in the light. Other light-associated proteins, including bacterial cryptochrome and DNA photolyase, were also expressed at significantly higher levels in the light. Membrane transporters for Na+/phosphate and Na+/alanine symporters, and the Na+-translocating NADH-quinone oxidoreductase (NQR) linked electron transport chain, were also significantly upregulated in the light. Culture experiments using a specific inhibitor of Na+-translocating NQR indicated that sodium pumping via NQR is a critical metabolic process in the light-stimulated growth of MED134. In total, the results suggested the importance of both the PR-enabled, light-driven proton gradient, as well as the generation of a Na+ ion gradient, as essential components for light-enhanced growth in these flavobacteria.

Phosphite utilization by the marine picocyanobacterium Prochlorococcus MIT9301

Primary productivity in the oceans oligotrophic regions is often limited by phosphorus (P) availability. In low phosphate environments, the prevalence of many genes involved in P acquisition is elevated, suggesting that the ability to effectively access diverse P sources is advantageous for organisms inhabiting these regions. Prochlorococcus, the numerically dominant primary producer in the oligotrophic ocean, encodes high-affinity P transporters, P regulatory proteins and enzymes for organic phosphate utilization, but its ability to use reduced P compounds has not been previously demonstrated. Because Prochlorococcus strain MIT9301 encodes genes similar to phnY and phnZ, which constitute a novel marine bacterial 2-aminoethylphosphonate (2-AEPn) utilization pathway, it has been suggested that this organism might use 2-AEPn as an alternative P source. We show here that although MIT9301 was unable to use 2-AEPn as a sole P source under standard culture conditions, it was able to use phosphite. Phosphite utilization by MIT9301 appears to be mediated by an NAD-dependent phosphite dehydrogenase encoded by ptxD. We show that phosphite utilization genes are present in diverse marine microbes and that their abundance is higher in low-P waters. These results strongly suggest that phosphite represents a previously unrecognized component of the marine P cycle.

Metatranscriptomic and functional metagenomic analysis of methylphosphonate utilization by marine bacteria

Aerobic degradation of methylphosphonate (MPn) by marine bacterioplankton has been hypothesized to contribute significantly to the oceans methane supersaturation, yet little is known about MPn utilization by marine microbes. To identify the microbial taxa and metabolic functions associated with MPn-driven methane production we performed parallel metagenomic, metatranscriptomic, and functional screening of microcosm perturbation experiments using surface water collected in the North Pacific Subtropical Gyre. In nutrient amended microcosms containing MPn, a substrate-driven microbial succession occurred. Initially, the addition of glucose and nitrate resulted in a bloom of Vibrionales and a transcriptional profile dominated by glucose-specific PTS transport and polyhydroxyalkanoate biosynthesis. Transcripts associated with phosphorus (P) acquisition were also overrepresented and suggested that the addition of glucose and nitrate had driven the community to P depletion. At this point, a second community shift occurred characterized by the increase in C-P lyase containing microbes of the Vibrionales and Rhodobacterales orders. Transcripts associated with C-P lyase components were among the most highly expressed at the community level, and only C-P lyase clusters were recovered in a functional screen for MPn utilization, consistent with this pathway being responsible for the majority, if not all, of the methane accumulation we observed. Our results identify specific bacterioplankton taxa that can utilize MPn aerobically under conditions of P limitation using the C-P lyase pathway, and thereby elicit a significant increase in the dissolved methane concentration.

Environmental DNA fragment conferring early and increased sporulation and antibiotic production in Streptomyces species.

ABSTRACT Here we describe the rep gene, isolated from an environmental DNA library, which when transformed into Streptomyces species resulted in increased production of secondary metabolites and accelerated sporulation. We show that Streptomyces lividans strains bearing rep are particularly useful as expression hosts for heterologous antibiotic production.

Accessing the Genomes of Uncultivated Microbes for Novel Natural Products

Recent findings suggest that only 1% or less of the total number of soil microbial species can be easily cultivated. The fact that uncultured species represent spectacular microbial diversity has sparked great interest in these microorganisms as a potentially prolific source of untapped genetic diversity encoding novel natural products. Multiple approaches are being developed to access this diversity, such as methods to improve the ability to cultivate some of these organisms, most of which are not easily grown under standard laboratory conditions. Here we discuss an alternative approach, aimed at developing technologies for gaining access to the genomes of uncultivated microbes by creating environmental DNA libraries. This method involves isolating large DNA fragments (100–300 kb) from soil microorganisms (or from microorganisms derived from other environments), and inserting these fragments into a bacterial vector, thus generating recombinant DNA libraries. Such libraries are then used to identify novel natural products by various means, including expression of the DNA in a heterologous host strain and screening for activities, or by directly analyzing the DNA for genes of interest. The recombinant approach thus obviates the need for culturing diverse microorganisms and provides a relatively unbiased sampling of the vast untapped genetic diversity present in various microenvironments. As an additional advantage, the genes encoding a product of interest are already isolated and can be analyzed using the tools of bioinformatics, thus providing a potential boost to the efforts of analytical chemists to identify the product. Furthermore, the possibility of regulating the expression of isolated environmental gene clusters or combining them with genes for other pathways to obtain new compounds could furnish a further advantage over traditional naturalproduct discovery methodologies.

Preparation of fosmid libraries and functional metagenomic analysis of microbial community DNA.

One of the most important challenges in contemporary microbial ecology is to assign a functional role to the large number of novel genes discovered through large-scale sequencing of natural microbial communities that lack similarity to genes of known function. Functional screening of metagenomic libraries, that is, screening environmental DNA clones for the ability to confer an activity of interest to a heterologous bacterial host, is a promising approach for bridging the gap between metagenomic DNA sequencing and functional characterization. Here, we describe methods for isolating environmental DNA and constructing metagenomic fosmid libraries, as well as methods for designing and implementing successful functional screens of such libraries.