Cyclic AMP Directly Activates NasP, an
            <i>N</i>
            -Acyl Amino Acid Antibiotic Biosynthetic Enzyme Cloned from an Uncultured β-Proteobacterium

Clardy, Jon

doi:10.1128/jb.00457-07

Cited by 16 publications

(21 citation statements)

References 27 publications

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“…Two NasYs (NasY6 and NasY10, both from group 2) show low-level similarity (ϳ15% identity) to individual N-acylhomoserine lactone synthases (4); however, the majority of NAS enzymes do not show similarity to known enzymes, and none is predicted to contain the Pfam "autoinducer synthetase" domain PF00765 that is commonly found in N-acylhomoserine lactone synthases. There are two known N-acylphenylalanine-producing NASs, NasP1 and NasP2 (NasA), that are nonhomologous to one another and lack similarity to the NasYs (12,13). There are also singular examples of N-acyltryptophanproducing (NasW) and N-acylarginine-producing (NasR) NASs, neither of which resembles other previously described NAS enzymes (7).…”

Section: Resultsmentioning

confidence: 99%

“…Extracts of antibacterially active eDNA clones have yielded several distinct varieties of N-acyl amino acids, including N-acyl derivatives of tyrosine, phenylalanine, tryptophan, and arginine ( Fig. 1, compounds 1 to 4, respectively) (6,7,12). eDNA clones that produce N-acyl amino acids have been identified in metagenomic studies spanning geographically and compositionally distinct soils, suggesting that N-acyl amino acid-producing bacteria may be common to the majority of soil ecosystems.…”

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confidence: 99%

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Long-Chain N -Acyl Amino Acid Synthases Are Linked to the Putative PEP-CTERM/Exosortase Protein-Sorting System in Gram-Negative Bacteria

Craig

Cherry

2011

J Bacteriol

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Clones that encode the biosynthesis of long-chain N-acyl amino acids are frequently recovered from activitybased screens of soil metagenomic libraries. Members of a diverse set of enzymes referred to as N-acyl amino acid synthases are responsible for the production of all metagenome-derived N-acyl amino acids characterized to date. Based on the frequency at which N-acyl amino acid synthase genes have been identified from metagenomic samples, related genes are expected to be common throughout the global bacterial metagenome. Homologs of metagenome-derived N-acyl amino acid synthase genes are scarce, however, within the sequenced genomes of cultured bacterial species. Toward the goal of understanding the role(s) played by N-acyl amino acids in environmental bacteria, we looked for conserved genetic features that are positionally linked to metagenome-derived N-acyl amino acid synthase genes. This analysis revealed that N-acyl amino acid synthase genes are frequently found adjacent to genes predicted to encode PEP-CTERM motif-containing proteins and, in some cases, other conserved elements of the PEP-CTERM/exosortase system. Although relatively little is known about the PEP-CTERM/exosortase system, its core components are believed to represent the putative Gram-negative equivalent of the LPXTG/sortase protein-sorting system of Gram-positive bacteria. During the course of this investigation, we were able to provide evidence that an uncharacterized family of hypothetical acyltransferases, which had previously been linked to the PEP-CTERM/exosortase system by bioinformatics, is a new family of N-acyl amino acid synthases that is widely distributed among the PEP-CTERM/exosortase system-containing Proteobacteria.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Long-Chain N -Acyl Amino Acid Synthases Are Linked to the Putative PEP-CTERM/Exosortase Protein-Sorting System in Gram-Negative Bacteria

Craig

Cherry

2011

J Bacteriol

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“…Binding of c-di-GMP to a PilZ domain usually affects the cellular physiology through modulation of protein-protein or protein-DNA interactions, but a direct effect on the catalytic activity of enzymes involved in alginate and cellulose biosynthesis was also shown 56 57 . NasP, a metagenome-derived NAS, was shown to be activated by c-AMP, another widespread bacterial secondary messenger 58 . Therefore, we propose a role of the c-di-GMP binding PilZ domains for the regulation of Nas354 and Nas343 activity; the detailed mechanism needs to be further explored.…”

Section: Discussionmentioning

confidence: 99%

Metagenomic discovery of novel enzymes and biosurfactants in a slaughterhouse biofilm microbial community

Thies

Rausch

Kovačić

et al. 2016

Sci Rep

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DNA derived from environmental samples is a rich source of novel bioactive molecules. The choice of the habitat to be sampled predefines the properties of the biomolecules to be discovered due to the physiological adaptation of the microbial community to the prevailing environmental conditions. We have constructed a metagenomic library in Escherichia coli DH10b with environmental DNA (eDNA) isolated from the microbial community of a slaughterhouse drain biofilm consisting mainly of species from the family Flavobacteriaceae. By functional screening of this library we have identified several lipases, proteases and two clones (SA343 and SA354) with biosurfactant and hemolytic activities. Sequence analysis of the respective eDNA fragments and subsequent structure homology modelling identified genes encoding putative N-acyl amino acid synthases with a unique two-domain organisation. The produced biosurfactants were identified by NMR spectroscopy as N-acyltyrosines with N-myristoyltyrosine as the predominant species. Critical micelle concentration and reduction of surface tension were similar to those of chemically synthesised N-myristoyltyrosine. Furthermore, we showed that the newly isolated N-acyltyrosines exhibit antibiotic activity against various bacteria. This is the first report describing the successful application of functional high-throughput screening assays for the identification of biosurfactant producing clones within a metagenomic library.

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“…HPLC-MS analysis coupled with 1 H NMR studies of the two most abundant chemical species isolated from these ethyl acetate extracts indicated that the major clone-specific compounds produced by EC5 are spectroscopically and chromatographically identical to the 12-carbon N-acyl derivative of phenylalanine (observed m/z ϭ 348.2) and to the 14-carbon N-acyl derivative of tryptophan (observed m/z ϭ 415.3) (see Fig. S1 in the supplemental material) (7,9).…”

Section: Resultsmentioning

confidence: 99%

Expanding Small-Molecule Functional Metagenomics through Parallel Screening of Broad-Host-Range Cosmid Environmental DNA Libraries in Diverse Proteobacteria

Craig

Chang

Kim

et al. 2010

Appl Environ Microbiol

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163

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The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.Uncultured bacteria are predicted to be a significant reservoir of novel small-molecule biosynthetic machinery (19,34). One means by which to access the biosynthetic potential contained within the genomes of uncultured bacteria is functional metagenomics (19). This approach involves cloning DNA directly from naturally occurring microbial populations (environmental DNA [eDNA]) and screening the resulting clone libraries for phenotypes traditionally associated with the production of secondary metabolites. A major limitation of functional metagenomics is its reliance on a foreign host to facilitate the expression of eDNA-derived genes and gene clusters (17). Codon bias, missing substrates, and the inability to recognize foreign regulatory elements, including promoters and ribosomal binding sites, are just some of the obstacles that are likely to limit the success of expression-dependent studies with any single host organism. Circumventing these obstacles through an expansion of the collection of hosts available for functional metagenomic studies should increase the efficacy of this approach.Soil ecosystems are rich in bacterial diversity, and the majority of soil-dwelling bacteria remain recalcitrant to standard microbial culture methods (33, 39). Large-scale metagenomic sequencing studies indicate that soil microbiomes are often dominated by five bacterial phyla: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Acidobacteria, and Actinobacteria (14). Bacteria from these common phyla are therefore appealing hosts for use in functional metagenomic studies of soil-derived eDNA libraries.In this study, six unique bacterial hosts [Agrobacterium tumefaciens (Alphaproteobacteria), ...

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Cyclic AMP Directly Activates NasP, an N -Acyl Amino Acid Antibiotic Biosynthetic Enzyme Cloned from an Uncultured β-Proteobacterium

Cited by 16 publications

References 27 publications

Long-Chain N -Acyl Amino Acid Synthases Are Linked to the Putative PEP-CTERM/Exosortase Protein-Sorting System in Gram-Negative Bacteria

Long-Chain N -Acyl Amino Acid Synthases Are Linked to the Putative PEP-CTERM/Exosortase Protein-Sorting System in Gram-Negative Bacteria

Metagenomic discovery of novel enzymes and biosurfactants in a slaughterhouse biofilm microbial community

Expanding Small-Molecule Functional Metagenomics through Parallel Screening of Broad-Host-Range Cosmid Environmental DNA Libraries in Diverse Proteobacteria

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