Identification of Mixed Organic Sulfur-Selenium and Sulfur-Tellurium Compounds in the Headspace Gases Above Genetically Modified Escherichia coli Jerry W. Swearingen Jr. and Thomas G. Chasteen Department of Chemistry Sam Houston State University

Geobacillus stearothermophilus V Resistant to TeO 3 -2 and TeO 4 -2 Grows at Thermophilic Temperature: 60º C Grows in an Aerobic Environment How the Genetic Material was Identified and Extracted  Pick restriction enzyme.  Carryout Polymerase Chain Reaction, then insert into plasmid.  Insert plasmid into host cell and grow in presence of tellurium.  Compare gel electrophoresis (protein separation) of control cultures and cultures amended with tellurium salts.  Look for presence of new or more concentrated proteins.  Protein sequencing.  Once the protein is sequenced the RNA and DNA coding can be determined.

Genetic Modification of E. coli JM109 Cells Open Reading Frames (ORF)  ORF 600 – UbiE methyltransferase  ORF 780 – uroporphyrin-III C-methyltransferase (cobA)  ORF 399 – BtuR protein pSP72 cloning vector  Plasmid is coded for ampicillin resistance.  DNA was inserted at the HindIII restriction site.

Organo-metalloid detection in headspace gases above bacteria Methods of Extraction  Manual Gas Syringe (1 mL)  Solid Phase Micro Extraction (SPME) Instrumentation  Gas Chromatography/F 2 -induced Chemiluminescence Detection (SCD)  Gas Chromatography/Mass Spectrometry (GC/MS)

GC- SCD Chromatogram of E. coli 1VH Headspace Amended with Tellurate

Methanetellurol Was the chromatographic peak dimethyl selenide or methanetellurol? If DMSe, that means the tellurium amendments were contaminated with selenium. Boiling Point of CH 3 SeCH º C Boiling Point of CH 3 TeH – 57º C

GC- MS Total Ion Chromatogram of E. coli 1VH Headspace

Mass Spectrum of dimethyl ditelluride - CH 3 TeTeCH 3

Mass Spectrum of dimethyl tellurenyl sulfide - CH 3 TeSCH 3 CH 3 TeSCH 3 + CH 3 TeS + TeS + CH 3 Te + Te +

Synthesis of CH 3 TeH and CH 3 TeSCH 3 There are no commercially available standards of CH 3 TeH and CH 3 TeSCH 3. A CAS registry number was only assigned last year based on our report. Reaction Mixture  25 µL CH 3 SSCH 3  25 µL CH 3 TeTeCH 3  1 mL of Reducing Agent Reducing Agents Used  1.0 M HCl + zinc  1.0 M H 2 SO 4 + zinc  0.5 mM DL-dithiothreitol (DTT)

GC-SCD Chromatogram of Headspace Above an Aqueous Reaction H 2 SO 4 + Zn + DMDS + DMDTe  The headspace gases detected are the products of either a solution phase or gas phase reaction.

Boiling Point Determination DMTeS GC-SCD Retention Time: minutes Boiling Point: 157° C The same method can be used with the GC-MS data to determine a boiling point.

Genetically Modified E. coli Amended with Tellurium Methanethiol and dimethyl tellurenyl sulfide was identified above modified E. coli cultures. Analysis showed that organo-tellurium was produced when ORF 600 was present. Experiments also determined that the oxidation state of tellurium affect the production or organo-tellurium compounds.  Tellurate ( TeO 4 -2 ) – dimethyl telluride (DMTe, CH 3 TeCH 3 ) was found in the headspace.  Tellurite ( TeO 3 -2 ) – DMTe was not found above the cultures; no organo-tellurium compounds were detected.

Genetically Modified E. coli Amended with Selenium What happens when the modified E. coli is amended with selenium salts.  Amendments with both SeO 3 -2 and SeO 4 -2 Did the difference oxidation states of selenium affect organo-selenium production?

GC- SCD Chromatogram of E. coli 1VH Headspace Amended with Selenite and Selenate Selenite AmendedSelenate Amended

What is DMSeDS? Dimethyl selenodisulfide (CH 3 SeSSCH 3 ) Could it be a compound other than DMSeDS?  Selectivity of the Detector  GC-MS Analysis

Mass Spectrum of dimethyl trisulfide - CH 3 SSSCH 3 CH 3 SSSCH 3 + CH 3 SSS + CH 3 SS + SS + CH 3 S +

Mass Spectrum of dimethyl selenodisulfide - CH 3 SeSSCH 3 CH 3 SeSSCH 3 + CH 3 Se + CH 3 SS + SS + CH 3 S +

Boiling Point Determination DMSeDS GC-SCD Retention Time: minutes Boiling Point: 192° C Retention Time (Minutes)

Work Still Needs To Be Done Because of the trace level of this analysis it is difficult to confirm the structure of DMSeDS.  CH 3 SeSSCH 3 Vs. CH 3 SSeSCH 3 CH 3 SeSCH 3 CH 3 SeSeCH 3 CH 3 SSSCH 3 CH 3 SeSSCH 3 Indole

Acknowledgements The genetic modifications were performed by the research group of Dr. Claudo C. Vásquez of the University of Santiago, Chile. This research was supported by the Robert A. Welch Foundation And by FONDECYT (Chile) And by DICYT grants from University of Santiago, Chile And by a doctoral fellowship from MECESUP (Chile)