LINKAGE OF A NITRILASE-CONTAINING Nit1C GENE CLUSTER TO CYANIDE UTILIZATION IN Pseudomonas fluorescens NCIMB 11764 Pallab Ghosh Thesis Prepared for the Degree of MASTER OF SCIENCE UNIVERSITY OF NORTH TEXAS May 2009 APPROVED: Daniel A. Kunz, Major Professor Barney J. Venables, Committee Member Robert C. Benjamin, Committee Member Arthur J. Goven, Chair of the Department of Biological Sciences Michael Monticino, Interim Dean of the Robert B. Toulouse School of Graduate Studies Ghosh, Pallab. Linkage of a nitr ilase-containing Nit1C gene cluster to cyanide utilization in Pseudomonas fluorescens NCIMB 11764. Master of Science (Molecular Biology), May 2009, 49 pp., 7 tables, 9 figures, references, 62 titles. Pseudomonas fluorescens NCIMB 11764 (Pf11764) is unique ly able to grow on the poison cyanide as its sole nitrogen source. It does so by converting cyanide oxidatively to carbon dioxide and ammonia, the latter b eing ass imilated into c ellular m olecules. Th is requires a complex enzymatic machinery that includes nitrilase and oxygenase enzymes the nature of which are not well understood. In the course of a pr oteomics analysis aim ed at achieving a better understanding of the proteins that m ay be required for cyanide degradation by Pf11764, an unknown protein of 17.8 kDa was detected in cells e xposed to cyanide. Analysis of this protein by ESI-coupled m ass spectrom etry and bioinfor matics searches gave evidence of strong homology with a protein (Hyp1) of unknown function (hypothetical) present in the bacterium Photorhabdus luminescens subsp. laumondii TTO1 (locus plu_1232). A search of available microbial genom es revealed a number of Hyp1 orthologs the genes of which are found in a conserved gene cluster known as Nit1C. Independe nt studies revealed that in addition to Hyp1, Pf11764 possesses a gene ( nit) specifying a nitrilase enzym e whose closest homologue is a nitrilase found in Nit1C gene clusters (77% amino acid identity). DNA sequence analysis has further revealed that indeed, hyp1Pf11764 and nitPf11764 are contained in a cluster tha t includes also a gene specifying an oxygenase. Given the possi ble connection of Nit1 C-endoded nitrilase and oxygenase enzymes to enzymatic cyanide degradation, there is strong reason for thinking that the genes specifying these enzym es contribute to b acterial g rowth on cyanide in those bacteria containing the Nit1C cluster. Because the biolog ical function of the H yp1 protein is currently unknown, it was cloned and the protein expressed in E. coli so that its properties could further be explored. U nfortunately, the expr ession of the protein in an in soluble for m com plicated these analyses. However, at least two lines of evidence suggest a possible role as a regulator of gene expression. First, over-expression of the protein was accompanied by the parallel elevation of the putative vector-encoded -lactamase, implying that Hyp1Pf11764 can affect the expression of other genes. Second, a com parison of the am ino acid sequence of se lect peptide fragm ents of Hyp1Pf11764, by conducting searches for hom ology with proteins in the existing nonredundant protein database, consistently revealed m otifs in comm on with thos e presen t in bacte rial response regulators that are pa rt of two-component signal transduction system s widely distributed in bacteria. Copyright 2009 by Pallab Ghosh ii ACKNOWLEDGEMENTS It is a privilege to register my profound regards and deep sense of gratitude to Dr. Daniel A. Kunz for his benevolent guidance, valuable suggestion, constructive criticism and sharing his deep knowledge during the entire course of this project. I would like to express my sincere gratitude to my committee members, Dr. Barney J. Venables and Dr. Robert C. Benjamin for reviewing this thesis. Special word of thanks to Dr. J-H Lee, a visiting scientist from South Korea in our laboratory, for his kind assistance and cooperation. I am grateful to Prem, Trevor, Chiani for all their friendly support and help. Last, but not the least I would like to express my reverential thanks to my beloved parents, elder brother, sister in law and also to my fiancée for their unfailing support and encouragement. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ............................................................................................................. iii LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ...................................................................................................................... vii LIST OF ABBREVIATIONS ...................................................................................................... viii Chapters I. INTRODUCTION ...................................................................................................1 Physicochemical Properties of Cyanide and Its Occurrence .......................1 Cyanide Tolerance .......................................................................................2 Nutritional Utilization of Cyanide as a Growth Substrate ...........................3 II. MATERIALS AND METHODS .............................................................................6 Bacterial Strains, Plasmids and Cultivation Conditions ..............................6 Preparation of Cellular Extracts ...................................................................8 Protein Determination ..................................................................................8 Discontinuous SDS-PAGE of Proteins ........................................................8 Electroblotting..............................................................................................9 N-terminal Amino Acid Analysis ..............................................................10 Two Dimensional Gel Electrophoresis ......................................................10 Sample Cleanup for 2D Gels .....................................................................10 First Dimension Isoelectric Focusing ........................................................11 Second Dimension Electrophoresis and Gel Staining ...............................11 Image Analysis ...........................................................................................12 In-gel Trypsin Digestion and Peptide Extraction .......................................12 Electro Spray Tandem Mass Spectrometry (ESI-MS/MS) ........................13 Isolation of DNA ........................................................................................14 iv PCR Methodology .....................................................................................14 DNA Sequencing .......................................................................................16 Cloning and Expression of hyp1 ................................................................17 Sequence Analysis .....................................................................................18 III. RESULTS ..............................................................................................................19 The 17 kDa Polypeptide Made by Pf11764 in Response to Cyanide is a Protein with No Known Function Found in a Select Number of Bacteria 19 Hyp1 Protein is Part of a Unique Gene Cluster that Includes The Putative Pf11764 CynD Nitrilase .............................................................................25 Cloning of hyp1 for Expression in E. coli..................................................33 IV. DISCUSSION ........................................................................................................41 REFERENCES ..............................................................................................................................45 v LIST OF TABLES Page 1. Strains and Plasmids Used in this Study ..............................................................................7 2. ESI-MS/MS Protein Identity Matches with a Cyanide Upregulated ~19 kDa Polypeptide in P. fluorescens 11764 ..................................................................................................…22 3. Single Identity Match for the Induced 19kDa Polypeptide Made by P. fluorescens 11764 following Resolution by 2D Gel Electrophoresis and Peptide Analysis by Mass Spectrometry ......................................................................................................................24 4. Oligonucleotide Primers Used to PCR Amplify hyp1 and nit DNA from Pf11764 ..........28 5. Oligonucleotide Primers Used to Clone hyp1 ....................................................................35 6. ESI-MS/MS Identity Matches with a 28 kDa Polypeptide Expressed in Parallel with Hyp1 in E. coli Cells (DK1290) Carrying pGK1 ..............................................................40 7. Peptide Fragments of Hyp1Pf11764 Showing High Homology to Bacterial Response Regulator Motifs ................................................................................................................44 vi LIST OF FIGURES Page 1. SDS-PAGE (16%) of Pf11764 Cell Extracts .....................................................................21 2. Two-dimensional Gel Electrophoresis of Cell Extract Protein (250 μg) from Cyanide Induced (Panel A) and Uninduced Cells (Panel B) of P. fluorescens 11764 ....................23 3. Organization of Nit1C Cluster in Photorhabdus luminescence subsp. laumondii TT01 ..27 4. Physical Arrangement of hyp1 & nit Pf11764 Open Reading Frame and Region