Download Functional Control by Codon Bias in Magnetic Bacteria

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Journal of
Biomedical Nanotechnology
Vol.4,4,44-51,
1–8, 2008
Vol.
Functional Control by Codon Bias in Magnetic Bacteria
Megha Sharma, Vivek Hasija, Mohit Naresh, and Aditya Mittal∗
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi,
Hauz Khas, New Delhi 110016, India
Keywords: Magnetic Bacteria, Nanomagnets, Codon Bias, Protein Expression, Nano-Propellers,
Flagella, Gene Translation, Codon Evolution.
1. INTRODUCTION
Achieving nano-scale control of directed motion for
micron-scale bodies is a fascinating concept having a multitude of exciting possibilities in biomedical nanotechnology. Majority of prokaryotes are motile and bacteria are
no exception. Motile bacteria have enormous potential to
serve as model systems for understanding nano-scale control of motion of micron-scale bodies under different fluid
conditions. Bacterial motility is due to presence of flagella
(plural of “flagellum”) that work like nanopropellers on the
bacterial cell surface. These bacterial nanopropellers are
made up of multiple molecular components (proteins) to
perform a specific function on the surface of a living cell
contributing to its motility. Bacterial flagella are long, thin
helical filaments with one end free and other attached to
the cell, which rotate like screws and have been studied in
detail for the peritrichously flagellated bacteria, especially
E. coli.
Discovery of magnetotactic bacteria triggered great
interest in the possibility of extraterrestrial life.1–3 In more
∗
Author to whom correspondence should be addressed.
J. Biomed. Nanotechnol. 2008, Vol. 4, No. 1
recent times, investigations on the ability of these bacteria
to manufacture nanomagnets of controlled anisotropies and
highly uniform size distributions4 have opened up avenues
for fascinating applications including those for biomedical purposes.5 6 Magnetotactic bacteria are equipped with
their own nanopropellers (flagella) and nanomagnets that
allow them to undergo magneto-aerotaxis.7–9 Magnetotactic bacteria are also highly motile, however, not much is
known about the specifics of their nanopropeller systems.
While E. coli utilize their nanopropellers for chemotaxis,
the magnetotactic bacteria utilize their nanopropellers for
magneto-aerotaxis. To understand motility of magnetotactic bacteria (and hence nano-scale control of motion for
synthetic micron-scaled bodies by utilizing nano-magnets
manufactured by these bacteria), we initiated studies on
uncoupling the magnetic component of their motion from
the nano-propeller driven motion. Utilizing a simple functional assay with a 33 ms time resolution10 11 we measured the swimming speeds of E. coli (strain JM109) and
two strains of magnetotactic bacteria, Magnetospirillum
magnetotacticum and Magnetospirillum gryphiswaldense.
E. coli was found to swim with speed of 1863±064 m/s
(mean ± s.d., n = 3, n refers to independent experiments
1550-7033/2008/4/001/008
doi:10.1166/jbn.2008.001
441
RESEARCH ARTICLE
Directed and controlled motion of small (micron sized and smaller) objects in fluid systems is
an active area of research in biomedical nanotechnology. Magnetotactic bacteria undergo a kind
of directed and control motion called magneto-aerotaxis by utilizing nanopropellers (flagella) and
nanomagnets. While studying the role of nanomagnets in magnetic-bacterial motility to investigate
nano-scale control of motion, we have serendipitously discovered a major protein specific codon
bias residing in the Magnetospirillum magnetotacticum genome. While primary sequences of flagellar, iron uptake and the house-keeping RNA polymerase proteins are identical in magnetic bacteria
compared to E. coli and mammalian cells, there is no genetic similarity for flagellar and iron uptake
proteins. In contrast, the house-keeping RNA polymerase in magnetic bacteria shows complete
genetic similarity also. Surprisingly, the lack of any genetic homology in flagellar and iron uptake
proteins, in spite of identical primary amino acid sequences in different organisms, is a consequence
of a protein specific codon bias, thereby resulting in non-identical gene sequences. This codon bias
is directly correlated with differences in protein functions specific to magnetic bacteria. Based on
our findings, we propose a bold “Functional Control by Codon Bias” (FCCB) hypothesis suggesting
that protein function is controlled by codons responsible for its primary sequence in addition to the
primary sequence itself. The remarkable feature of our proposal is that even if primary sequences of
two proteins are identical, utilization of different codons to express those sequences directly affects
the function of the proteins.
FCCB Hypothesis
(a)
Sharma et al.
(b)
(c)
(d)
(e)
(f)
RESEARCH ARTICLE
Fig. 1. Differences in E. coli and magnetotactic bacteria. (a, b) Morphological differences observed in two bacterial cells with a 100× objective using
a glass slide and a cover slip. (c) TEM of a negatively stained sample of a magnetic bacterial cell with nanomagnets aligned inside the cell. The scale
bar represents 200 nm. (d) Alignment and connectivity of the nanomagnets is disturbed after lysing the bacterial cells. The scale bar represents 20 nm.
(e, f) Cartoon representations comparing the features of E. coli and magnetic bacteria. The magnetic bacterial cells (f), containing nanomagnets, are
twice as long as E. coli (e) with the magnetic bacterial flagellum being half the length of that in E. coli.
with each experiment measuring 10–40 individual bacterial
cells) where as the magnetic bacteria were found to swim
with speeds of 32 ± 1030 m/s (n = 4, two experiments
for each strain of the magnetic bacteria). A two fold
higher speed of magnetic bacteria compared to E. coli
was in agreement with separately reported results by other
groups.12 13 However, it is interesting to note that while
magnetic bacteria (length ∼ 3–4 m11 ) are about twice as
long as E. coli (length ∼ 1.5–2 m,11 14 Figs. 1(a, b)), with
chains of nano-magnets aligned inside them (Fig. 1(c))
that start losing their connectivity as well as perfect alignment after cell lysis (Fig. 1(d)), they consist of only
1–2 nanopropellers per bacterial cell compared to at least
4 nanopropellers per E. coli cell.12 Moreover, the magnetic bacterial nanopropeller is only 1–4 m long with a
diameter of 12–20 nm,13 compared to an E. coli nanopropeller that is 10–20 m long with a diameter of ∼50 nm.12
The differences in length of a bacterial cell and its flagellum, for E. coli and magnetic bacteria, are depicted in
Figures 1(e), (f), respectively. It is also interesting to note
that Pseudomonas aeruginosa, which has a single flagellum (like magnetic bacteria), but with dimensions similar to E. coli, swims at speeds similar to E. coli.15 16
These observations raise a very interesting question. How
do magnetic bacteria move with speeds twice as fast as
compared to other bacteria with at least half the number
of nanopropellers, each nanopropeller being significantly
smaller than other motile bacteria?
To answer the above, in absence of crystal structures of magnetic bacterial nanopropellers, we decided to
2
45
investigate protein sequences of the individual components
of the nanopropellers. We report very surprising findings that identical protein primary sequences, but with no
genetic homology, result in different functionality in different systems. In contrast, a house-keeping protein like
RNA polymerase, with identical protein primary sequence
as well as genetic homology, has the same functionality
regardless of the system.
2. MATERIALS AND METHODS
2.1. Chemicals
All reagents purchased from commercial sources were
used as received. Vitamin solution and trace element solution were prepared afresh. Vitamin solution components:
biotin, folic acid, pyridoxine-hydrochloride, thiaminehydrochloride dihydrate, riboflavin, nicotinic acid,
D-calcium-pantothenate, and Vitamin B12 were obtained
from Himedia. For trace element solution, nitrilotriacetate
acid, cobalt sulfate heptahydrate (CoSO4 × 7H2 O), and
disodium molybdate dihydrate (Na2 MoO4 × 2H2 O) were
obtained from Qualigen; Magnesium sulfate heptahydrate (MgSO4 × 7H2 O), manganese sulfate heptahydrate
(MnSO4 × 7H2 O), sodium chloride (NaCl), ferrous sulfate
heptahydrate (FeSO4 × 7H2 O), calcium sulfate heptahydrate (CaSO4 ×7H2 O), zinc sulfate heptahydrate (ZnSO4 ×
7H2 O), copper sulfate pentahydrate (CuSO4 × 5H2 O),
potassium aluminium sulfate dodecahydrate (KAl(SO4 2 ×
12H2 O), boric acid (H3 BO3 ), nickel chloride hexahydrate (NiCl2 × 6H2 O), and sodium selenite pentahydrate
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FCCB Hypothesis
Sharma et al.
(Na2 SeO3 × 5H2 O) were purchased from Merck. For
media 380 and 512, resazurin, sodium thioglycolate, and
yeast extract were obtained from Himedia; Monobasic
potassium phosphate (KH2 PO4 ), sodium nitrate (NaNO3 ),
L(+)-Tartaric acid, succinic acid, agar (for semi-solid
media), dibasic potassium phosphate (K2 HPO4 ), and
ammonium chloride (NH4 Cl) were obtained from Merck;
Ferric citrate was purchased from Lobachemie. For ferric
quinate solution, ferric chloride hexahydrate (FeCl3 ×
6H2 O) and quinic acid were purchased from Merck and
Spectrochem (India) respectively. Distilled water was used
for all solutions.
2.2. Growth Media
2.3. Inoculation
The bacteria were grown in 250 ml flasks, with custom
made rubber caps, and 30 ml anoxic vials, with screw caps
and air tight rubber lining. 10 ml of inoculum was added
to 150 ml media in 250 ml flasks and 1 ml inoculum
was added to 10 ml media in 30 ml vials. Inoculum was
added with a hypodermic syringe through the rubber cap
taking care not to introduce air. The bacteria were allowed
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2.4. Video Microscopy and Speed Measurements
1 ml of sample was withdrawn for video microscopy.
The cells were observed by placing a drop of sample on
a flat slide and observing under oil immersion at 100×
magnification. 10 seconds videos were captured at a 33
frames/ms resolution.10 17 For analysis, 300 frames, 33 ms
apart, were extracted from each 10 s videos. Using Scion
Image software, the average speed was measured. The
average speed was measured by measuring the distance
between the positions of the bacteria between two successive frames and dividing with the time (33 ms). The overall
speed was defined in terms of the total distance covered by
the bacteria divided by the total time. For Magnetospirillum magnetotacticum, 5–7 bacteria were observed and for
Magnetospirillum gryphiswaldense, 30–40 bacteria were
observed.10 17
2.5. Protein Similarity and Codon Specificity Analysis
Protein and nucleotide sequences of known mammalian iron regulatory proteins18–20 and flagellar proteins12
were obtained from NCBI. The genome blast tool
(http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) was
then used to search for these proteins in Magnetospirillum magnetotacticum MS-1. While comparing nucleotide
sequences, the default option of Mega BLAST was unselected. This is because Mega BLAST uses the greedy
algorithm for nucleotide sequence alignment search. This
program is optimized for aligning sequences that differ
slightly as a result of sequencing or other similar errors.
As a result if nucleotide sequences were compared using
mega BLAST “no match” was obtained for any of the
proteins. Sequences with e-value less than 10−5 were considered to be a match. To determine codon specificity, protein translation-table number 11 from NCBI was used. For
each protein the nucleotide and protein sequences were
compared for both the query and M. magnetotacticum. The
number of times a particular codon was used to code for
an amino acid was counted using MATLAB (Mathworks
Inc., USA). This was done for both iron regulatory and
flagellar proteins.
3. RESULTS
We searched for the flagellar proteins of E. coli12 in the
recently reported genome for the magnetotactic bacteria
M. magnetotacticum (being sequenced at the Joint Genome
Institute) using BLAST. This strain was specifically chosen
since our experimental work is being carried out with this
463
RESEARCH ARTICLE
Media 380 and 512, as per DSMZ (Germany) nomenclature, were used for Magnetospirillum magnetotacticum
and Magnetospirillum gryphiswaldense, respectively. The
media were prepared following standard protocol provided
by DSMZ. Medium 380, used for cultivation of M. magnetotacticum contained (per liter): 10 ml vitamin solution,
5 ml trace elements, 2 ml Fe(III) quinate solution, 0.5 mg
resazurin, 0.68 g KH2 PO4 , 0.12 g NaNO3 , 0.05 g Nathioglycolate, 0.37 g L(+)-Tartaric acid, 0.37 g succinic
acid, and 0.05 g Na-acetate. All the ingredients, except
Na-thioglycolate, were mixed and the medium was boiled
for 3 mins, after adjusting pH to 6.5 with NaOH. The
medium was autoclaved at 121 C for 15 mins. 0.01 M
Fe(III) quinate solution was prepared by dissolving 0.45 g
FeCl3 × 6H2 O and 0.19 g quinic acid in 100 ml distilled
water. This solution was autoclaved separately and added
to remaining autoclaved media components. TES and vitamin solution were filter sterilized and added to autoclaved
media. For cultivation of M. gryphiswaldense, medium 512
contained (per liter): 0.5 g KH2 PO4 , 1 g Na-acetate, 0.1 g
NH4 Cl, 0.1 g yeast extract, 20 m Fe(III) citrate, and
0.5 g Na-thioglycolate. The pH was adjusted to 6.8 and the
medium was prepared 2–3 days before use. For both media
freshly prepared Na-thioglycolate was filter sterilized and
added just before inoculation. The media was dispensed
in anoxic vials with screw caps. Anoxic conditions were
maintained by purging nitrogen for 15 minutes. Sterile air
was added with a hypodermic syringe through the rubber
cap to a concentration of 1% (v/v) in the vial.
To prepare semi-solid medium, agar was added to a concentration of 1.3 g/1000 ml media (only for medium 380).
to grow at 30 C for 2 weeks and growth was monitored
by carefully withdrawing small samples with a hypodermic needle and observing under a microscope. Quantitative
assessment of bacterial growth was not done as a part of
this study, since it was not required for the purpose of the
work done here.
FCCB Hypothesis
Sharma et al.
strain. We found homologous proteins, corresponding to
various components of the E. coli nanopropeller assembly in the magnetic bacterial genome (Table I). The
extremely small e-values (e 10−5 ) obtained for most
proteins, using BLAST, indicated nearly exact protein primary sequences for the nanopropeller components in both
the bacteria. However, very interestingly, no match was
found when the nucleotide sequences were compared (e 10−5 ) for the two bacteria (Table I). Was the astounding
lack of any homology in the nucleotide sequences of a single nanopropeller component proteins between E. coli and
magnetic bacteria a general feature of the latter or does the
magnetic bacteria have an altogether different nucleotide
coding arrangement, which still results in the same primary sequence assembly?
To answer the above question, we searched for a “house
keeping” protein of gram negative bacteria (both the
bacteria studied by us are gram negative) called RNA
polymerase. While the protein sequences, for different
subunits of RNA polymerase, present in E. coli and the
magnetic bacterial genome were expectedly identical, surprisingly there was very significant homology (in contrast
to a complete lack of homology for flagellar proteins) on
the nucleotide level also for the subunits (e 10−5 ). These
results, shown in Table I, led us to further ask whether
RESEARCH ARTICLE
Table I. Blast searches for various proteins in the M. magnetotacticum
genome.
Protein
Flagellar
proteins
IRPs
RNA
polymerase
Protein/
Subunit
Accession
number
Protein
e-value
NT
e-value
FlgB
FlgC
FlgD
FlgE
FlgF
FlgG
FlgH
FlgI
FlgK
FlhA
FlhB
FliA
FliC
FliF
FliG
FliI
FliM
FliN
FliP
FliR
IRP1/Aconitase
IRP2
subunit
subunit
subunit
subunit
BAB34874.1
P0ABX2
YP_852174.1
BAA35885.2
BAA35886.1
BAB34879.1
BAB34880.1
BAB34881.1
BAB34883.1
BAB33679.1
BAB36013.1
BAB36084.1
ABI23966.1
BAB36100.1
BAB36101.1
BAB36103.1
BAB36107.1
BAB36108.1
BAA15773.1
BAB36112.1
NP_002188.1
CAB62825.1
BAE77333.1
AAC76962.1
AP_004495.1
AAA24601.1
6 × 10−09
9 × 10−21
2 × 10−20
3 × 10−20
4 × 10−19
1 × 10−66
3 × 10−18
3 × 10−63
4 × 10−17
1 × 10−81
8 × 10−56
7 × 10−15
8 × 10−08
4 × 10−53
2 × 10−36
6 × 10−97
1 × 10−24
2 × 10−15
1 × 10−59
2 × 10−11
6 × 10−86
1 × 10−23
2 × 10−158
0
3 × 10−83
8 × 10−120
7 × 10−07
18
02
14
001
006
02
032
049
1 × 10−07
002
001
17
049
007
041
0005
012
085
091
10
62
7 × 10−36
3 × 10−41
n.d.∗
1 × 10−66
e-values obtained are given for both the protein as well as corresponding nucleotide
matching. ∗ Nucleotide sequence for this protein was not found and hence not
compared.
47
4
presence of identical primary sequences of proteins in
absence of any nucleotide level homology was a function
specific feature in magnetic bacteria. Thus, we carried out
similar blast searches for Iron Regulatory Proteins (IRPs:
IRP1 and IRP2) from mammalian18–20 as well as bacterial
sources (in bacteria, IRP1 is called aconitase) in the magnetic bacterial genome. The logic behind these searches
was that if IRPs are present in magnetic bacteria, then one
would expect primary sequences of these proteins to be
identical to the other sources, since iron transport is key
to nanomagnet assembly inside magnetic bacteria. However, based on the nucleotide dependent functional differences found in flagellar proteins, we would also expect no
nucleotide similarity for IRPs from other sources in the
magnetic bacterial genome. This is so since IRPs in magnetic bacteria are clearly expected to play a role in iron
transport towards nanomagnet formation, a functional feature absent in other sources. Once again, to our pleasant
surprise, we found that primary sequences of IRPs identical to those found in mammalian systems are present in the
magnetic bacterial genome however there is no nucleotide
homology (Table I, e 10−5 ).
Thus, we had clearly found a nucleotide level control of
function of proteins in magnetic bacteria. On one hand, the
magnetic bacterial nanopropellers providing unique motility characteristics and iron regulatory proteins providing
their unique roles in iron transport leading to nanomagnet production inside magnetic bacteria had no nucleotide
matches from other sources in spite of identical protein
primary sequences. On the other hand, RNA polymerase,
which has exactly the same functional outputs in magnetic
bacteria compared to E. coli had identical protein primary
sequences as well as nucleotide sequences in both the systems. This, to our knowledge, is the first time that a correlation has been found between functional differences of
identical proteins (i.e., identical primary sequences) differing on the nucleotide level.
What was the reason for these nucleotide dissimilarities,
in spite of the same primary sequence, that were correlating well with different protein functions? To investigate
this, we studied the codon preferences utilized in magnetic bacteria and the other sources for the proteins shown
in Table I. Soon it became apparent that magnetic bacteria had a particular codon bias for amino acid residues.
Except for methionine and tryptophan, that have only one
codon for each of them, we compared the codons for all
the other 18 amino acid residues (which have 2–6 codons
for each residue). Close inspection revealed a certain bias,
in magnetic bacteria, against utilizing “T” or “A” as the
third base in the codons for these 18 residues. These results
are more clearly shown in Figure 2. It can be seen from
Figure 2(a), that when we consider the percentage of times
“non T or A” in the third base of codons are plotted as a
function of different proteins, the magnetic bacteria (gray
bars) appear to have a higher percentage in all cases. Were
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FCCB Hypothesis
Sharma et al.
140
(a)
120
Percentage
100
80
60
40
20
σ
β’
β
IRP-2
IRP-1
FliR
FliP
FliN
FliM
FliI
FliG
FliF
FlhB
FlhA
FlgK
FlgL
FlgE
FlgD
FlgC
FlgB
0
0
–2
Log(p)
–4
–6
–8
–10
(b)
–12
these higher percentages statistically relevant? To check
this, we carried out t-tests between the non T or A codon
percentages for the 18 amino acids for magnetic bacteria
as well as the other sources. Figure 2(b) shows the log
of “p” values obtained from those t-tests. The dashed line
shows p = 001. Any p value smaller than this (i.e., more
negative in Fig. 2(b)) signifies statistically relevant difference in the codon usage while avoiding T or A as the third
base in the codons. Clearly, while the RNA polymerase
subunits do not show any statistically relevant differences
in codon preferences (all the three bars in Fig. 2(b) are
greater than p = 001), most of the flagellar (11 out of
15) proteins and both the iron regulatory proteins show
substantial statistical differences.
Therefore, the next question was where specifically
was this codon preference arising from? Grouping amino
acid residues based on conventional systems (e.g., polar:
neutral, negatively and positively charged or non-polar)
did not yield any specific insight. However, when we
divided the amino acid residues into two groups based on
the number of codons for each residue, very interesting
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results were observed. Figures 3(a and c) show groups
of amino acids having more than two codons for each
amino acid (Fig. 3(a)) and groups of amino acids having only two codons (Fig. 3(c)). For ease of visualization,
Figure 3 shows results for all combined subunits of individual proteins. For example, all the 15 flagellar subunits
shown in Table I and Figure 2 are taken together as flagellar proteins. Thus, if 10 amino acid residues have more
than two codons for each residue, the appearance of these
10 amino acids is evaluated for all 15 flagellar proteins,
and hence n = 150 for the pair of bars representing flagellar proteins in Figure 3(a). It is clear from Figures 3(a
and b) that magnetic bacteria (gray bars) have a significantly higher percentage of non T or A as the third base in
codons compared to other sources (p 001), regardless
of proteins. However, this result is in contrast to results in
Table I and Figure 2, where it is clear RNA polymerase
has nucleotide identity and similar codon usage in magnetic bacteria and other sources. This is easily explained by
considering the relative low frequency of these amino acid
residues in the protein sequences, along with the results
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RESEARCH ARTICLE
Fig. 2. Codon bias in magnetic bacteria. (a) Percentage of codons not containing T or A as the third base for all the amino acid residues in particular
proteins for E. coli (open bars) and magnetic bacteria (gray bars). Since methionine and tryptophan have only one codon, they were not included in
the comparisons. Thus, for each data set, n = 18. First a protein was found in the magnetic bacterial genome using BLAST. Then, occurrence of each
amino acid residue in a particular protein sequence was counted. Then codons for each of the amino acid residues were tabulated. Subsequently, the
percentage of codons with no T or A the third base was calculated for each amino acid. Finally, percentages of the 18 amino acids were pooled to
calculate the mean ± standard deviation of the percentage of codons without T or A in the third base. (b) log of p-values from t-tests performed on
data represented by each set of bars (i.e., open and gray) in (a). The dashed line corresponds to a p-value of 0.01. Any black bar below it (i.e., more
negative) shows a significant difference in the open and gray bars in (a) and any black bar above the dashed line shows no statistical difference. All
marked in yellow constitute the flagella, orange constitute the IRPs and blue constitute RNA polymerase.
FCCB Hypothesis
Sharma et al.
120
120
Percentage
(a)
(c)
100
100
80
80
60
60
40
40
20
20
0
0
Flagellar
IRPs
RNA Pol
0
RNA Pol
–4
–10
–6
–15
–8
– 20
–10
– 25
–12
(b)
– 30
RESEARCH ARTICLE
IRPs
–2
–5
Log(p)
Flagellar
0
(d)
–14
Fig. 3. Codon bias in magnetic bacteria, absent/minimal in a housekeeping protein like RNA polymerase, specifically arises from residues for which
there exist only two codons. (a, c) Percentage of codons not containing T or A as the third base for all the amino acid residues having more than two
codons (a), or two codons only (c), in particular proteins for E. coli (open bars) and magnetic bacteria (gray bars). Note that all the flagellar proteins
in Figure 2(a) and Table I (i.e., total 15) were pooled into one category called flagellar, both the IRPs were pooled as one and all the RNA polymerase
subunits were pooled into RNA pol. Once again, methionine and tryptophan have only one codon, so they were not included in the comparisons. Thus,
in (a) n = 150 for flagellar proteins (15 flagellar proteins, 10 residues having more than two codons), n = 20 for IRPs (2 IRPs), n = 30 for RNA
polymerase (3 subunits). Similarly in (c) n = 120 for flagellar (15 flagellar proteins, 8 residues having two codons), n = 16 for IRPs, n = 24 for RNA
polymerase. All data in (a) and (c) is mean ± standard deviation. (b, d) The log of p-values obtained for t-tests performed on the data shown in (a)
and (c) respectively. Note that the Y -axis scale is different in (b) and (d). While the RNA polymerase as the largest p-values in both the figures, using
a statistically significance criteria of p = 001 (dashed lines), there is no significant differences in the open and gray bars for RNA polymerase in (c)
only as shown by the corresponding black bar in (d).
shown in Figure 3(d). While the usage of non T or A
codons in flagellar as well as iron regulatory proteins, for
residues having only two codons corresponding to each of
them, is significantly higher in magnetic bacteria compared
to other sources (Fig. 3(d), p 001), there is no difference between codon preferences for magnetic bacteria and
other sources for RNA polymerase. Thus, it is clear that
when there is only a 50% chance of choosing T or A as
the third base in codons (out of two possible codons, one
has either T or A in the third base), somehow the magnetic
bacteria have evolved a bias against using these codons.
4. DISCUSSION AND CONCLUSIONS
We report functional dependence of proteins with identical primary sequences on usage of specific codons utilized
for expressing those primary sequences in magnetic bacteria. Here it is pertinent to mention that codon bias has
already been actively implicated in misfolding of proteins,
6
49
especially with attempts to over-express certain external
proteins in bacterial systems using plasmids.21–24 However,
the prime reason attributed to this misfolding has been
considered as overloading of the natural bacterial protein
expression machinery due to plasmids.
How can nucleotide sequences affect protein function
with identical primary sequences? We propose a novel
“Functional Control by Codon Bias” (FCCB) hypothesis as
a potential answer to this question. While our newly proposed (FCCB) hypothesis is sure to be quite controversial
since at this stage it is purely theoretical and challenges
the well accepted norm that only primary sequences, leading to particular protein structures under given conditions, control protein functionality, it indeed is bound
to open up new experimental avenues. Figure 4 shows
a cartoon representation of our FCCB hypothesis. For
convenience, we have utilized a cartoon of the bacterial
nanopropeller (flagellar protein) with its major molecular
components to conceptualize the FCCB hypothesis. It is
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FCCB Hypothesis
Sharma et al.
?
Fig. 4. Functional control by codon bias (FCCB) hypothesis. Assembly of bacterial nanopropellers (flagellar) is shown as an example. The
assembly results from transcription and translation of genetic codes using
the same machinery (shown in red). However, subtle differences in the
finally assembled protein structure (shown by arrows in the lower flagellar protein) can results from different genetic coding (due to codon bias)
leading to different kinetics of transcription/translation and hence assembly of the complex. This could lead to different protein functionality
in spite of the same primary sequences of amino acids.
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Acknowledgments: This work was supported by funding from SERC, Department of Science and Technology,
Government of India, awarded to Aditya Mittal. Megha
Sharma and Mohit Naresh acknowledge support from the
Department of Biochemical Engineering and Biotechnology IIT Delhi.
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RESEARCH ARTICLE
possible that while the machinery for transcription and
translation is identical for magnetic bacteria and other
species (e.g., RNA polymerase), shown in red in Figure 4,
if the nucleotide sequences to be transcribed or translated are different, then there would be completely different kinetics of expression and folding of these primary
sequences. This would lead to changes in the eventual protein structures, presumably subtle but significant enough
(shown by arrows in the lower flagellar structure) to cause
functional differences for those proteins. These changes
would be even more prominent for functional multi-protein
complexes, where kinetics and/or the order of assembling
several protein subunits would produce changes in the final
functional protein complex structure and function. Interestingly, the nucleotide level correlation with protein function in our study comes from a codon bias arising from
amino acid residues which have only two codon options,
one having T or A as the third base and the other without
T or A as the third base, where there is a 50% chance of
choosing one codon over the other. Assembly of a different nanopropeller system due to codon bias might lead to
the uniqueness of magnetic bacteria that are motile with
speeds twice as fast as compared to other bacteria with half
the number of nanopropellers, each nanopropeller being
significantly smaller than other motile bacteria. Further,
FCCB hypothesis may be a key link to understanding why
homologous iron uptake and transport proteins serve different roles in magnetotactic bacteria and other bacteria or
mammalian cells.
Our FCCB hypothesis has the potential to invoke a new
debate, if not a significant paradigm shift, in evolutionary
biology. While codon bias has been reported for several
different species from microbial to higher systems, we suggest altogether a new impact resulting out of the codon
bias. We propose that codon bias has its amplified effects
only on proteins whose functions are specific to a particular species and not on housekeeping proteins. Finally, our
results and the FCCB hypothesis clearly have profound
implications in general (i.e., not just magnetic bacteria) in
the post-genomic era especially for utilizing recombinant
strains for expressing foreign genetic codes (e.g., for biological production of nano-materials), and also in the field
of protein function predictions.
FCCB Hypothesis
Sharma et al.
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RESEARCH ARTICLE
Received: 29 September 2007. Revised/Accepted: 8 October 2007.
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J. Biomed. Nanotechnol. 4,
4,44-51,
1–8, 2008
J. Biomed. Nanotechnol. 4, 44-51, 2008
Graphical Abstract
Functional Control by Codon Bias in Magnetic Bacteria
Megha Sharma, Vivek Hasija, Mohit Naresh, Aditya Mittal
?
In this study, we propose a bold “Functional Control by Codon Bias” (FCCB) hypothesis
suggesting that protein function is controlled by codons responsible for its primary
sequence in addition to the primary sequence itself. The remarkable feature of our
proposal is that even if primary sequences of two proteins are identical, utilization of
different codons to express those sequences directly affects the function of the proteins.