Abstract

1. Abstract In order to investigate the physiology and central metabolic pathways of Geobacter metallireducens Strain GS-15, a plasmid library of EcoRI-digested chromosomal DNA fragments was constructed in E. coli. A probe for a nirS homolog ­from Pseudomonas stutzeri and an oligonucleotide probe based on cytochrome c7 protein sequence data are being used to investigate cd and c7 cytochromes. Degenerate probe HEM1B, based on 7 amino acids including the heme 1 binding site of cytochrome c7 found in G. metallireducens hybridized with a clone containing a 1.83 kb insert (GenBank accession AY167567). This sequence contains an ORF for a hypothetical 130 amino acid protein of unknown function. Analysis of the amino acid sequence reveals a protein of 15,302 D, pI 9.89, with 3 hydrophobic domains. The sequence contains 9 cysteine residues, 6 of which occur pair-wise in the form of CXXC indicative of c-type cytochrome heme binding sites. Structural classification of the protein suggests it is in the super family cytochrome c, most closely resembling cyt. c552 of Nitrosomonas europaea and cyt. c549 of Synechocystis. This clone also includes the sequence of a transposase not found in other G. metallireducens sequence databases and the N-terminal sequence of a polyferredoxin similar to those found in Desulfovibrio. Another clone that hybridized with the same HEM1B probe reveals partial sequences for a putative metallo--lactamase and a complete gene sequence for a putative acetyl-coA hydrolase. Further investigation with other probes resulted in the cloning of a seryl-tRNA synthetase protein gene fragment (GenBank accession AY173026), as well as a fraction of a putative histidine kinase (GenBank accession AF503927). Cloning of the c-type cytochrome may prove useful in elucidating the electron transport chains resulting in the reduction of Fe(III) and NO3-. The detection of a ferredoxin gene is consistent with the hypothesis that the iron sulfur protein acts as the physiological electron acceptor for the coenzyme A-dependent 2-oxoglutarate oxidoreductase present in the cell.

2. Fe3+, NO3-, Mn4+, U6+ Ethanol Acetate kinase, TCA cycle, ?Fd, Mk, ?cyt. c Acetate CO2 Benzoate Toluene Fe2+, NH3, Mn2+, U4+ Physiology of Geobacter metallireducens

3. Hypothesis and Approach • The underlying hypothesis to this research is that identifying cytochrome genes in Geobacter will lead to a better understandingof anaerobic respiration by the organism. • Chromosomal DNA was extracted from Geobacter metallireducens GS-15, and digested with EcoRI. The fragments were ligated into pBluescript SK phagemid, and the mixture of plasmids was used to transform XL-10 Gold Ultracompetent E. coli cells. • A portion of the N-terminal aa sequence from a 3-heme c7-type cytochrome was used to design a probe (AAY-TGY-AAR-AAR-TGY-CAY-GA) complementary to a heme binding site (CKKCH) . The oligonucleotide probe was 3’-labeled with digoxigenin, and hybridized to colony material lifted from an E. coli plasmid library plated on LB-Ampicillin. • Clones displaying hybridization were purified, re-screened, and plasmid DNA was subjected to EcoRI digestion to verify the presence and size of inserted DNA. Plasmid was submitted for sequencing to the BioResource Center (Cornell University), using T3 and T7 universal primers. Internal primers were designed using WebGenetics software, and each sequence was verified with overlapping sequences on each strand. • Sequences were compared to those in the NCBI databases using the Blast protocols. All sequences matched those from the Geobacter metallireducens genomic sequences confirming that clones were in fact of Geobacter origin. ORFs were identified by BlastX analysis.

4. Clone Hem1B – AY167567 Fd Cyt Transposase Vector Vector Cloned Sequence ORF Hem1B MTRSTCLTYDDPMETFSNKQLKDVKGTRRLVRVYCHACHDRSLRAPFDLPPELQRRYSRGVELCPECAALLAHGIQKRRKCPLDPKPSCKSCRIHCYSKEYRAKIREVMGFSGKRMIMRGRLDYLLHFLF Incomplete Sequence

5. Inferred Properties of Protein Hem1B • Initial search using Superfamily reveals the protein as a cytochrome c based on homology with other proteins from Nitrosomonaseuropaeaand Synechocystis sp. • ORF Hem1B contains a cytochrome c heme-binding site of consensus sequence Cys-X-X-Cys-His. Despite multiple cysteine residues, it appears to be a monoheme c-type cytochrome. • Periplasmic consensus signaling sequencesa of Ala-Asp, Ala-Pro, Ala-Glu, Ala-Leu, Ala-Ile, and Val-Asp, are not found at the N-terminus. Signal Sequence computer search indicated that protein Hem1B does not contain a signal peptide. This suggests Hem1B is not located in the periplasm a LeGall and Peck. 1987. FEMS Microbio Rev. 46:122-126

6. Physical Properties of Cytochrome c Coded by ORF Hem1B • The ORF is 393 bases yielding a protein of 130 amino acids. • The molecular weight of the hypothetical protein is 15,302 Daltons. • This is a very basic protein noted by a high pI of 9.89, and 21% positively charged amino acids. • Protein Hem1B contains no significant hydrophobic regionsa (see below) and most likely does not have any transmembrane segments. This suggests it is a soluble cytochrome. a Hopp and Woods (PNAS USA 78, 3824-2828)

7. metallo-B-lactamase Acetyl-coA hydrolase Vector Vector Clone Hem2B – AY261456 MMLSMTFPYSPPHSDGELVEIGPDVRWLRMPVTYAPDHVNIYLVRVAGGWLIVDTGLDSPEARRIWEEVFSGPLAGEKVVGVYCTHYHVDHLGLAGYLTERWRVPLFMTYEEYYTLLGWPDLPQEVPWQHVEFFQRAGFPQELLPQTLVMFDFAREISPMPLSFVRLQDRSHLPLDEEWQVIVGRGHSPEHALLLSKVRKILISGDQLLPSISTNVSVSVMNPEDDPLSHWLASLDRLATIPDDVLVLPGHGLPFRGARKRVAELRGHHRRRSQVIVDACAGSELSAYELVKVLYSFSLGDFDLQLALGECLSHVRYLACRGRLEARLDGEGINRYRSVRGVRAVNGGAGFCRD

8. Penicillin Resistance in G. metallireducens • Effect of Penicillin-G on iron-reduction (A) and growth (B) by G. metallireducens • The MIC for growth appears at or near 10 g ml-1. Most bacteria are susceptible at concentrations below 5 g ml-1.

9. Characterization of metallo-β-lactamase • The ORF is 1065 bases yielding a protein of 354 amino acid of molecular weight 40,040 Daltons. • The protein has a pI of 6.21 and the hydropathy plot is below. • His residues at amino acid positions 86 and 88, and an Asp at 90 are consistent with a Zn-binding domaina described in B1 class metallo--lactamase enzymes. • Blast indicates putative conserved domains of a Zn-dependent hydrolase and a metallo--lactamase. a Page. 2002. ASM News 68:217-221.

10. Clone Hem13A – AY173026 • 184 base fragment of a seryl-tRNA synthetase. • Amino acid sequence: • EFAHTLNGSGLAVGRTLVAILENYQQEDGTVVIPEVLRPYMGGCSSIGR Seryl-tRNA synthetase

11. Clone 093 - AF503927 Partial Histidine Kinease • 119 base fragment of a Histidine Kinase • Amino acid sequence: • GVMKQGEKPVYFVRDNGAGFDMRYAGQLFTPFQRMHRSE

12. Clone SP3 – Submission not complete ORF serS ORF capA Vector Vector • ORF serS bases 1-1190 • Amino acid sequence homology with seryl-tRNA synthetase gene of E. coli • ORF capA 1280-2016 • Amino acid sequence homology with polyglutamate capsule synthetase, known as CapA protein. • Homologous sequences found in Bacillus cereus, Bacillus anthracis, and Myxococcus (another member of the delta subdivision of proteobacteria).

13. -500 -300 -100 +100 +300 Electron Transporta in G. metallireducensb Cyt c gene in Clone Heme 1B could be part of a cytochrome oxidase system 2-OG/Su Ferredoxin gene in Clone Heme 1B supports this model Cyt c7 Ox/Red Complex III Analog? Complex IV Cyt Oxidase? Fdox/Fdred Complex I Analog Quinol Oxidase? NAD+/NADH Mkox/Mkred Fe3+ /Fe2+ Complex II Analog Ictr/2-OG Mal/OA Su/Fum Membrane a After White. 2000. The Physiology and Biochemistry of Prokaryotes b Champine et al. 2000. Anaerobe6:187-196 Soluble

14. Summary • Probes failed to detect cytochrome c7 and nirS. This may be due to the EcoR1 digest step used to create the library. Hybridizations with HindIII digested chromosomal DNA show hybridization to nirS, but not EcoR1 digested (data not shown). • Clone Heme1B indicates presence of both ferredoxin and a cytochrome c. Hybridization was probably due to concensus heme aa sequence CXXCH. The cytochrome protein does not match size and features of those previously reported (Naik et al. 1993. FEMS Microbiol Lett 106:53-58. • Serendipity: A variety of additional features were found • Metallo-β-lactamase – This gene will be used to probe other, newly isolated ampicillin bacteria from agricultural samples. • Transposase – A significant finding with regards to the genetics of Geobacter

15. Acknowledgements • Principal funding for this project came from the Southeast Missouri State University Grants and Funding research Committee. Additional funding came from the Southeast Missouri State University Undergraduate Research Program. The presenting author (pictured above) would like to thank Dr. Allan Bornstein and Dr. Jane Stephens for their support of undergraduate research. Donna Kridelbaugh also received support from a Beta Beta Beta fellowship. • Ms. Kridelbaugh would like to thank members of her Graduation with Distinction committee: Drs. Walt Lilly, Bjorn Olesen, John Kraemer, and Sharon Coleman for their guidance. • Funding for student travel was made available through the Southeast Missouri State University Student Professional Development program (Drs. Rick Burns and Christina Frazier), and the Southeast Research Conference (Dr. Martha Zlokovich). • The authors would like to thank Maija Bluma and Laura Holman for their excellent technical assistance. Also, Vicki Howell and Joanna Kubik provided administrative support.