Signal Mimics Derived from a Metagenomic Analysis of the Gypsy Moth Gut Microbiota

Guan, Changhui; Ju, Jianhua; Borlee, Bradley R.; Williamson, Lillie D.; Shen, Ben; Raffa, Kenneth F.; Handelsman, Jo

doi:10.1128/aem.02617-06

Cited by 65 publications

(47 citation statements)

References 52 publications

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“…Metagenomics based on this approach has proven to be a powerful tool for the identification of novel biocatalyst (7,9,14,43,44,53). Many of the thereby encountered genes and gene products were derived from screens of small-insert metagenomic libraries, which have been generated by direct isolation and cloning of DNA from environmental samples (12,16,30,32,53,58). Usually, the environmental DNA is inserted in a high-copy plasmid by using a T4 DNA ligase reaction.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of Metalloproteases with a Novel Domain Structure by Construction and Screening of Metagenomic Libraries

Waschkowitz

Rockstroh

Daniel

2009

Appl Environ Microbiol

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Small-insert metagenomic libraries from four samples were constructed by a topoisomerase-based and a T4 DNA ligase-based approach. Direct comparison of both approaches revealed that application of the topoisomerase-based method resulted in a higher number of insert-containing clones per g of environmental DNA used for cloning and a larger average insert size. Subsequently, the constructed libraries were partially screened for the presence of genes conferring proteolytic activity. The function-driven screen was based on the ability of the library-containing Escherichia coli clones to form halos on skim milk-containing agar plates. The screening of 80,000 E. coli clones yielded four positive clones. Two of the plasmids (pTW2 and pTW3) recovered from positive clones conferred strong proteolytic activity and were studied further. Analysis of the entire insert sequences of pTW2 (28,113 bp) and pTW3 (19,956 bp) suggested that the DNA fragments were derived from members of the genus Xanthomonas. Each of the plasmids harbored one gene (2,589 bp) encoding a metalloprotease (mprA, pTW2; mprB, pTW3). Sequence and biochemical analyses revealed that MprA and MprB are similar extracellular proteases belonging to the M4 family of metallopeptidases (thermolysin-like family). Both enzymes possessed a unique modular structure and consisted of four regions: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The architecture of the latter region, which was characterized by the presence of two prepeptidase C-terminal domains and one proprotein convertase P domain, is novel for bacterial metalloproteases. Studies with derivatives of MprA and MprB revealed that the C-terminal extension is not essential for protease activity. The optimum pH and temperature of both proteases were 8.0 and 65°C, respectively, when casein was used as substrate.Proteolytic enzymes catalyze the hydrolytic cleavage of peptide bonds. These enzymes are present in all living organisms and are essential for cell growth and differentiation. Microorganisms produce a variety of intracellular and/or extracellular proteases. Intracellular microbial proteases are highly specific and are involved in several cellular and metabolic processes such as activation of inactive precursors, maintenance of the cellular protein pool, and sporulation. Extracellular proteases degrade proteins in cell-free environments. The resulting hydrolytic products (small peptides and amino acids) can be transported into the cells and utilized as carbon or nitrogen sources (13, 39). Especially, extracellular proteases are of industrial importance and are used as cleaning agents and food and feed additives.Proteases can be divided into various groups with respect to the functional group present at the active site and the pH profile of the activity. Microbial alkaline proteases, which are defined to be active in a neutral to alkaline pH range (13), possess either a serine center (serine proteases) or are of metal-type (metalloproteases). Extracellular...

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Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of Metalloproteases with a Novel Domain Structure by Construction and Screening of Metagenomic Libraries

Waschkowitz

Rockstroh

Daniel

2009

Appl Environ Microbiol

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“…Moreover, functional metagenomics couples a specific biological activity to genetic information, facilitating rapid identification and characterization of the genes and gene products involved in the biological activity. Among the diverse biological activities revealed using functional metagenomics are antimicrobial activity (8,15,24,34), antibiotic resistance determinants (2), quorum-sensing mimics (16,42), and enzymes with diverse functions (19,20,27). Many of these metabolites and activities would have been difficult to predict based on sequence information alone.…”

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confidence: 99%

Metagenomic Analysis of Streptomyces lividans Reveals Host-Dependent Functional Expression

McMahon

Guan

Handelsman

et al. 2012

Appl Environ Microbiol

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ABSTRACTMost functional metagenomic studies have been limited by the poor expression of many genes derived from metagenomic DNA inEscherichia 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 inStreptomyces 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 inS. 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 toS. lividans. All bioactive clones were functional inS. lividansbut not inE. coli, demonstrating the advantages of screening metagenomic libraries in more than one host.

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“…This approach was also used to identify signal synthases in a metagenomic library of the gypsy moth gut, revealing a monooxygenase that produces an acyl-HSL mimic compound (57). Theoretically, this type of approach may be used to identify acyl-HSL synthases among the mammalian intestinal microbiota, but to the best of our knowledge, this type of study has not been published.…”

Section: Potentially Positive Results From Genomics and Metagenomicsmentioning

confidence: 99%

Are There Acyl-Homoserine Lactones within Mammalian Intestines?

2013

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Many Proteobacteria are capable of quorum sensing using N-acyl-homoserine lactone (acyl-HSL) signaling molecules that are synthesized by LuxI or LuxM homologs and detected by transcription factors of the LuxR family. Most quorum-sensing species have at least one LuxR and one LuxI homolog. However, members of the Escherichia, Salmonella, Klebsiella, and Enterobacter genera possess only a single LuxR homolog, SdiA, and no acyl-HSL synthase. The most obvious hypothesis is that these organisms are eavesdropping on acyl-HSL production within the complex microbial communities of the mammalian intestinal tract. However, there is currently no evidence of acyl-HSLs being produced within normal intestinal communities. A few intestinal pathogens, including Yersinia enterocolitica, do produce acyl-HSLs, and Salmonella can detect them during infection. Therefore, a more refined hypothesis is that SdiA orthologs are used for eavesdropping on other quorum-sensing pathogens in the host. However, the lack of acyl-HSL signaling among the normal intestinal residents is a surprising finding given the complexity of intestinal communities. In this review, we examine the evidence for and against the possibility of acyl-HSL signaling molecules in the mammalian intestine and discuss the possibility that related signaling molecules might be present and awaiting discovery.

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Signal Mimics Derived from a Metagenomic Analysis of the Gypsy Moth Gut Microbiota

Cited by 65 publications

References 52 publications

Isolation and Characterization of Metalloproteases with a Novel Domain Structure by Construction and Screening of Metagenomic Libraries

Isolation and Characterization of Metalloproteases with a Novel Domain Structure by Construction and Screening of Metagenomic Libraries

Metagenomic Analysis of Streptomyces lividans Reveals Host-Dependent Functional Expression

Are There Acyl-Homoserine Lactones within Mammalian Intestines?

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