Transcript MamB and MamM: Genes Involved in

Magnetosirillum magneticum Genes MamB and MamM
and a Possible Agent for Bioremediation
Rachel Bolender
Biology Department, Colby College, Waterville, Maine, USA
Magnetotactic Bacteria
Synthesize Magnetic Crystals
Magnetotactic bacteria (MTB) are bacteria with the
ability to form prokaryotic organelles called
magnetosomes, magnetic crystals of magnetite
(Fe3O4) or greigite (S3O4) enveloped in a phospholid
bilayer. Magnetosomes are usually organized into
one or more chains parallel to the bacteria’s axis of
motility, allowing the bacteria to orient themselves
and swim passively along the earth’s geomagnetic
fields. This ability is termed magnetotaxis.
Magnetobacteria are Promising
Agents for Bioremediation
Prediction of Transmembrane
Proteins
Accumulation of heavy metals due to mining, smelting,
agriculture, electroplating, and nuclear power and
weapons has become a problem in the U.S.
Genes mamB and mamM, which are involved in
magnetosome formation, are thought to code for
proteins that allow for the uptake of metal ions. They
are thus expected to be located in the cell membrane.
Topology can be used to predict transmembrane
regions by observing the hydrophobicity of amino
acids encoded by a gene. Regions of hydrophobic
amino acids are likely incorporated into a membrane.
Transformation of E. coli with
Expression Vector
PCR products of mamB and mamM were inserted into an
expression vector with antibiotic resistance as the
selectable marker. These plasmids were then inserted
into E. coli and cultured on a plate containing an antibiotic.
Individual colonies of the transformed E. coli were
cultured. Restriction enzyme digests were performed and
gels were run to determine which colonies contained the
vector with the gene in the correct orientation.
Vector + mamB
Vector + mamM
Fig. 7 Visuals of restrictions digests for plasmids containing mamB or
mamM in the correct orientation. Expected band lengths shown.
http://faculty.virginia.edu/metals/cases/prescott3.html
Ba Bb Bc Bd Be
Ma Mb
Mc Md
Fig. 3 Map of U.S. with the worst toxic metal sites shown, according to the
EPA
http://magnetolab.bio.lmu.de/de/bilder/diversity_crystals.jpg
Fig. 1 Species of magnetobacteria display a wide range of crystal
morphology and chain arrangement
MTB typically reside in aqueous habitats or
sediments that are microaerophilic to anaerobic.
Magnetotaxis is hypothesized to aid the bacteria in
their pursuit of optimal levels of oxygen by reducing a
three dimensional search (such as that of nonmagnetotactic bacteria) to a one dimensional search.
Magnetospirillum
magneticum
M. magneticum is an aquatic, motile Gram-negative
magnetotactic bacteria that lives in microaerophilic
conditions in shallow fresh water and sediments. It is
one of the few MTB whose genome is fully
sequenced and available to the public.
magnetosome chain
Many studies have found MTB to be promising
biosorbants of heavy metal ions such as cadmium,
copper, gold, and arsenic among others. MTB are
especially appealing for bioremedation because they
can be easily separated from waste water using
magnetic fields.
One study found that M. gryhiswaldense, an MTB
closely related to M. magneticum, is capable of
reducing gold ions in solution to form nanoparticles on
the cell ’ s surface. The nanoparticles could be
collected and used for a variety of applications.
Genetic Control of
Magnetosome Formation
The genetic control of magnetosome formation is
poorly
understood.
Genes
involved
in
the
magnetosome formation process have been found in
the magnetosome island (MAI), a highly conserved
region among MTB. The mamAB operon located within
the MAI has been shown to be both necessary and
sufficient for magnetosome formation. Genes mamB
and mamM lie in this operon and are thought to be
cation diffusion facilitator (CDF) proteins involved in
the uptake of metals such as zinc, cobalt, and
cadmium.
Fig. 5 Topology predictions for mamB. Transmembrane regions indicate
that mamB encodes a membrane protein
Topology of mamM showed a similar pattern,
indicating that both mamB and mamM are membrane
proteins that may be involved in the transport of ions
into the cell.
Fig 8 Gels of restriction digests. Lanes Ba-Be represent colonies with the
vector containing mamB. Lanes Ma-Md represent colonies with the vector
containing mamM. Colonies Bd, Be, and Mc have bands showing that the
gene is in the correct orientation.
Tests for Metal Biosorption
Amplification of Genes mamB
and mamM
Template DNA was extracted from M. magneticum.
Forward and reverse primers for mamB and mamM
were designed and used to amplify these genes via
PCR. A gel was run on the PCR products to
determine whether the amplification of the genes was
successful. The sizes of PCR products matched the
expected sizes, indicating that the genes were
successfully amplified.
B1 B2 B3 B4 B5
M1 M2 M3 M4 M5
http://2011.igem.org/Team:NYMU-Taipei/chassis
Fig. 2 M. magneticum with single magnetosome chain visible
mamM
Acknowledgements
Thanks to Assistant Professor Ron Peck for his guidance
in this project and to Dan Sunderland ’14, Wei Ma ’16,
and Shayla Williiams ’16 for their feedback in weekly
meetings.
MamB
RB 2014
http://www.biomedsearch.com/attachments/00/20/16/17/20161777/pone.0009151.g001.jpg
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Fig. 4 Magnetosome island with genes mamB and mamM in the mamAB
operon
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Fig. 6 Gel of PCR products for mamB and mamM. Lanes B1-B5 are
mamB PCR products at different annealing temperatures. Lanes M1-M5
are mamM PCR products at different annealing temperatures.
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