Genomic analysis of a microbial community by microarray comparative genomic hybridization, and, Genetic characterization of the pdt locus for the production of pyridine-2,6-dithiocarboxylic acid

This dissertation follows two lines of investigation. The first chapter is the development of an approach to comprehensively classifying DNA from a metagenomic library. With the use of a technique known as microarray comparative genomic hybridization (CGH), we constructed a microarray consisting of 672 cosmids from a groundwater-derived metagenomic library (COSMO). Using COSMO, we compared the unknown clones to the genomes of various groundwater isolates and reference strains. In this manner, we were able to identify strain specific DNA and conserved DNA. In addition, we used COSMO to probe metagenomic DNA samples from the environment, which allowed us to identify clones corresponding to organisms that are abundant in the microbial community but have not been characterized. Sequence analysis of the uncultured set of clones identified numerous functional genes including some potentially involved in hydrogen oxidation, nitrate reduction and transposition. We introduce “metagenomic profiling” as a means for identifying clones that correspond to uncultured organisms that may be relevant to a specific metabolic activity.; The remaining chapters follow the genetic characterization of the pdt locus from Pseudomonas stutzeri KC. Plasmid pT31 is a cosmid from a genomic library of P. stutzeri KC that encodes genes for the biosynthesis of a metal chelator pyridine-2,6-dithiocarboxylic acid (pdtc). Identification of pT31 and complete sequencing of its ∼26 Kb insert revealed a cluster of genes, some having similarity to genes involved in the biosynthesis of siderophores and antibiotics. Mutagenesis of pT31 with a mini-Tn5 transposon revealed that at least 6 genes are required for normal production of pdtc. These include genes homologous to the following: an AMP ligase, sulfurylase, oxidoreductase, methyl transferase, siderophore receptor and ABC transporter. We propose that pdtc is synthesized by a mechanism common to other siderophores.; Additional physiological studies of pdtc and pdt genes revealed that pdtc has significant antimicrobial properties primarily as a result of the sequestration of trace metals, which may be a key aspect of its function. In addition, pT31 enhances the pdtc resistance of E. coli. In order to identify the pdtc resistance factors, we screened the collection of mutagenized pT31 for the ability to enhance the pdtc resistance of E. coli. We identified 3 genes required for pdtc resistance, including a putative sugar permease, aminotransferase and an unknown protein. Sequence analysis revealed that the putative permease PdtE is a member of a family of AmpG-like proteins with greatest similarity to YbtX and Irp8, transporters from Yersinia pestis and Y. entercolitica that are present within the yersinia-bactin uptake operon but have no known function. The periplasmic loops of PdtE contain many hydrophobic and aromatic residues, and the transmembrane domains contain charged and polar residues (characteristics shared by some siderophore transporters). In addition a potential metal binding site is present within a 30 residue cytoplasmic loop. We propose the PdtE is responsible for the utilization of pdtc trace-metal complexes.