Download Cloning and Sequencing of DNA from a Plasmid Library

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 ctype 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 NO 3-. 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.
Physiology of
Geobacter metallireducens
Fe3+, NO3-, Mn4+, U6+
Ethanol
Acetate
Benzoate
Toluene
Acetate kinase, TCA cycle,
?Fd, Mk, ?cyt. c
Fe2+, NH3, Mn2+, U4+
CO2
Hypothesis and Approach
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The underlying hypothesis to this research is that identifying cytochrome genes in Geobacter
will lead to a better understanding of 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.
Clone Hem1B – AY167567
Fd
Cyt
Transposase
Vector
Vector
Cloned Sequence
ORF Hem1B
MTRSTCLTYDDPMETFSNKQLKD
VKGTRRLVRVYCHACHDRSLRAP
FDLPPELQRRYSRGVELCPECAAL
LAHGIQKRRKCPLDPKPSCKSCRI
HCYSKEYRAKIREVMGFSGKRMI
MRGRLDYLLHFLF
Incomplete
Sequence
Inferred Properties of Protein
Hem1B
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Initial search using Superfamily reveals the protein as a cytochrome c based
on homology with other proteins from Nitrosomonas europaea and
Synechocystis sp.
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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.
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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
Physical Properties of Cytochrome c
Coded by ORF Hem1B
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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)
Clone Hem2B – AY261456
metallo-B-lactamase
Acetyl-coA hydrolase
Vector
MMLSMTFPYSPPHSDGELVEIGPDVRWLRMPVTYAPDHV
NIYLVRVAGGWLIVDTGLDSPEARRIWEEVFSGPLAGEKV
VGVYCTHYHVDHLGLAGYLTERWRVPLFMTYEEYYTLL
GWPDLPQEVPWQHVEFFQRAGFPQELLPQTLVMFDFARE
ISPMPLSFVRLQDRSHLPLDEEWQVIVGRGHSPEHALLLS
KVRKILISGDQLLPSISTNVSVSVMNPEDDPLSHWLASLDR
LATIPDDVLVLPGHGLPFRGARKRVAELRGHHRRRSQVIV
DACAGSELSAYELVKVLYSFSLGDFDLQLALGECLSHVRY
LACRGRLEARLDGEGINRYRSVRGVRAVNGGAGFCRD
Vector
Penicillin Resistance in
G. metallireducens
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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.
Characterization of metallo-βlactamase
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The ORF is 1065 bases yielding a protein of 354 amino acid of molecular
weight 40,040 Daltons.
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The protein has a pI of 6.21 and the hydropathy plot is below.
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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.
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Blast indicates putative conserved domains of a Zn-dependent hydrolase and
a metallo--lactamase.
a
Page. 2002. ASM News 68:217-221.
Clone Hem13A – AY173026
Seryl-tRNA synthetase
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184 base fragment of a seryl-tRNA synthetase.
Amino acid sequence:
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EFAHTLNGSGLAVGRTLVAILENYQQEDGTVVI
PEVLRPYMGGCSSIGR
Clone 093 - AF503927
Partial Histidine Kinease
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119 base fragment of a Histidine Kinase
Amino acid sequence:
 GVMKQGEKPVYFVRDNGAGFDMRYAG
QLFTPFQRMHRSE
Clone SP3 – Submission not complete
ORF serS
Vector
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ORF capA
Vector
ORF serS bases 1-1190
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Amino acid sequence homology with seryl-tRNA synthetase gene of E. coli
ORF capA 1280-2016
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Amino acid sequence homology with polyglutamate capsule synthetase, known
as CapA protein.
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Homologous sequences found in Bacillus cereus, Bacillus anthracis, and
Myxococcus (another member of the delta subdivision of proteobacteria).
Electron Transporta in G. metallireducensb
2-OG/Su
Ferredoxin gene in Clone Heme
1B supports this model
Cyt c gene in Clone Heme 1B could be
part of a cytochrome oxidase system
Cyt c7 Ox/Red
Complex III
Analog?
Fdox/Fdred
Complex I
Analog
NAD+/NADH
Mkox/Mkred
Complex IV
Cyt Oxidase?
Quinol Oxidase?
Fe3+ /Fe2+
Complex II Analog
Ictr/2-OG
-500
Mal/OA
-300
-100
Su/Fum
+100
+300
Membrane
a After
Soluble
b
White. 2000. The Physiology and Biochemistry of Prokaryotes
Champine et al. 2000. Anaerobe 6:187-196
Summary
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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
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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
Acknowledgements
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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.
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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.
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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).
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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.