Screening a wide host-range, waste-water metagenomic library in tryptophan auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different classes of cloned trp genes.

A metagenomic cosmid library was constructed, in which the insert DNA was derived from bacteria in a waste-water treatment plant and the vector was the wide host-range cosmid pLAFR3. The library was screened for clones that could correct defined tryptophan auxotrophs of the alpha-proteobacterium Rhizobium leguminosarum and of Escherichia coli. A total of 26 different cosmids that corrected at least one trp mutant in one or both of these species were obtained. Several cosmids corrected the auxotrophy of one or more R. leguminosarum trp mutants, but not the corresponding mutants in E. coli. Conversely, one cosmid corrected trpA, B, C, D and E mutants of E. coli but none of the trp mutants of R. leguminosarum. Two of the Trp+ cosmids were examined in more detail. One contained a trp operon that resembled that of the pathogen Chlamydophila caviae, containing the unusual kynU gene, which specifies kynureninase. The other, whose trp genes functioned in R. leguminosarum but not in E. coli, contained trpDCFBA in an operon that is likely co-transcribed with five other genes, most of which had no known link with tryptophan synthesis. The sequences of these TRP proteins, and the products of nine other genes encoded by this cosmid, failed to affiliate them with any known bacterial lineage. For one metagenomic cosmid, lac reporter fusions confirmed that its cloned trp genes were transcribed in R. leguminosarum, but not in E. coli. Thus, rhizobia, with their many sigma-factors, may be well-suited hosts for metagenomic libraries, cloned in wide host-range vectors.

[1]  J. Wingender,et al.  Metagenome Survey of Biofilms in Drinking-Water Networks , 2003, Applied and Environmental Microbiology.

[2]  J. Handelsman,et al.  Cloning the Soil Metagenome: a Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms , 2000, Applied and Environmental Microbiology.

[3]  W. Wood,et al.  Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA. , 1966, Journal of molecular biology.

[4]  D. M. Ward,et al.  16S rRNA sequences reveal numerous uncultured microorganisms in a natural community , 1990, Nature.

[5]  J. Beynon,et al.  High frequency transfer of nodulating ability between strains and species of Rhizobium , 1978, Nature.

[6]  Wolfgang R Streit,et al.  Metagenomics--the key to the uncultured microbes. , 2004, Current opinion in microbiology.

[7]  C. Napoli,et al.  Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea , 1987, Journal of bacteriology.

[8]  M. Wagner,et al.  Evidence for additional genus-level diversity of Chlamydiales in the environment. , 2001, FEMS microbiology letters.

[9]  I. Crawford,et al.  Regulation of tryptophan genes in Rhizobium leguminosarum , 1982, Journal of bacteriology.

[10]  D. Helinski,et al.  Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Bibb,et al.  Tryptophan genes in Rhizobium—Their organization and their transfer to other bacterial genera , 1978, Molecular and General Genetics MGG.

[12]  J. Beringer.  R factor transfer in Rhizobium leguminosarum. , 1974, Journal of general microbiology.

[13]  J. Handelsman,et al.  Toward functional genomics in bacteria: analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Nemat O. Keyhani,et al.  Ancient Origin of the Tryptophan Operon and the Dynamics of Evolutionary Change , 2003, Microbiology and Molecular Biology Reviews.

[15]  W. Martin.  Mosaic bacterial chromosomes: a challenge en route to a tree of genomes. , 1999, BioEssays : news and reviews in molecular, cellular and developmental biology.

[16]  P. Babitzke.  Regulation of transcription attenuation and translation initiation by allosteric control of an RNA-binding protein: the Bacillus subtilis TRAP protein. , 2004, Current opinion in microbiology.

[17]  O. White,et al.  Environmental Genome Shotgun Sequencing of the Sargasso Sea , 2004, Science.

[18]  Joern Hopke,et al.  Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts , 2004, Applied and Environmental Microbiology.

[19]  Pascale Jeannin,et al.  Recombinant Environmental Libraries Provide Access to Microbial Diversity for Drug Discovery from Natural Products , 2003, Applied and Environmental Microbiology.

[20]  J. Banfield,et al.  Community structure and metabolism through reconstruction of microbial genomes from the environment , 2004, Nature.

[21]  S. Giovannoni,et al.  The uncultured microbial majority. , 2003, Annual review of microbiology.

[22]  I. Crawford,et al.  The Rhizobium meliloti trpE(G) gene is regulated by attenuation, and its product, anthranilate synthase, is regulated by feedback inhibition , 1990, Journal of bacteriology.

[23]  S. Salzberg,et al.  Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae. , 2003, Nucleic acids research.

[24]  C. Roshick,et al.  Tryptophan recycling is responsible for the interferon‐γ resistance of Chlamydia psittaci GPIC in indoleamine dioxygenase‐expressing host cells , 2004, Molecular microbiology.

[25]  C. Bonner,et al.  Dynamic diversity of the tryptophan pathway in chlamydiae: reductive evolution and a novel operon for tryptophan recapture , 2002, Genome Biology.

[26]  J. Handelsman,et al.  Metagenomics: genomic analysis of microbial communities. , 2004, Annual review of genetics.

[27]  S. Salzberg,et al.  Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. , 2000, Nucleic acids research.

[28]  J. Beringer,et al.  Linkage Mapping in Rhizobium leguminosarum by means of R Plasmid-mediated Recombination , 1978 .

[29]  R. Simon,et al.  New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in gram-negative bacteria. , 1989, Gene.

[30]  J B Stevens,et al.  The Rhizobium leguminosarum tonB gene is required for the uptake of siderophore and haem as sources of iron , 2001, Molecular microbiology.