Transcript No Slide Title

Genomic Analyses of Transport Proteins in
Mycobacterium tuberculosis and Mycobacterium leprae
Ji-Won Youm and Milton H. Saier Jr.
Section of Molecular Biology, University of California, San Diego, La Jolla, CA 92093
Introduction
It had long been accepted that tuberculosis was eradicated, but recent
findings of virulent strains that are multi-drug resistant indicate
otherwise. Tuberculosis has resurfaced as a serious pandemic and has
thus drawn much attention from the scientific community. Leprosy has
likewise been a major public health problem.
Tuberculosis and leprosy are caused by the causative pathogens
Mycobacterium tuberculosis (Mtu) and Mycobacterium leprae (Mle),
respectively. While Mle is an intracellular bacterium, Mtu is an obligate
aerobe capable of intracellular and extracellular life. Previous genomic
analyses have revealed that Mle lost many genes through evolution,
some of which are undoubtedly required for extracellular life since Mle
is an intracellular parasite while Mtu is an extracellular parasite.
Various events such as deletions, missense & nonsense mutations, and
translocations are thought to have contributed to the inactivation of
genes. The remnants of the genes resulting from such evolutionary
events are what are pseudogenes – inactivated genes that no longer
produce functional proteins. Pseudogenes are known to be present in
Mtu, and very prevalent in Mle. We are interested in pseudogenes
because the presence of pseudogenes in Mle and their functional
counterparts in Mtu suggests that these particular genes are “on their
way out” because they are unnecessary for intracellular life.
My research focuses on the transport proteins of these two major
parasitic bacteria. Comparative analysis of the transport proteins
encoded within the two proteomes and the subsequent analysis of the
putative functions of pseudogenes may shed light onto the transport
proteins requisite to intracellular life and/or extracellular life. The
former discovery may open up possibilities of synthesizing drugs to
inhibit growth in its entirety, and the latter may aid in curbing the
contagious nature of tuberculosis.
My project is not yet complete. Table 1 reflects an incomplete set
of data, but the protein-specific figures accurately depict the selected
findings.
Discussion
Results
Assignment
hit TC #
#
1
2
5
1
3.A.1.1.7?
3.A.1.1.7
3.A.1.1.7
3.A.1.1.7
e-value
5.00E-35
2.00E-06
8.00E-30
8.00E-42
q. len s. len TMS:
query/
subject
317
426
274
290
330 0 (TC 1)
450
278
300 6 (TC 8)
Idnt query
ity align
Sco
re
1
6
35
24
30
35
56~313
59~369
20~274
7~276
subject
align
29~312
66~389
19~276
17~290
Pseudogenes
GENE FUSIONS
Nonetheless, the result of my scripts and subsequent analyses have already revealed that M.
tuberculosis abounds with examples of gene fusions. For example, a drug transporter (15843349,
2.A.1.3.12) was found to have fused a N-terminal fragment of the native transport protein with a
regulatory region D. Other transport proteins showing fusions include a virulence factor (15843543,
2.A.66.4.1), a threonine export carrier (15843358, 2.A.79.1.1), and an arsenical resistance
q.T s.TMS
ncbi annot
tcd q. s.
gi #
swissPro query
Syst co
MS location
b
se seq
t
Locations
ems mp
protein/arsenate reductase (15842183, 2.A.59.1.2). Further analyses revealed fusion of regulatory
loca
ann q
on
domains such as the cAMP binding domain, and various phosphatases as in the case of 15842183 to
tion
ot
ent
be rather prevalent in M. tuberculosis. This reinforces the notion that evolution tends toward
CONCLUSION
Description
[29,46] ABC transporter, ATP-binding protein
Daunorubicin
MDEPAHRARPKGNGANHDGAQPCCGIGTCGNRGDPRARAHLPLPKGGRAGGAWHGVTVGRGEIFGLLGPSGAGKSTTQKLLIGLLRDHGGQATVWDKEPAEWGPDYYERIGVSFELPNHYQKLTGYENLRFFASLYAGATADPMQLLAAVGLADDAHTLVGKYSKGMQMRLTFARSLINDPELLFLDEPTSGLDPV
MNTQPTRAIETSGLVKVYNGTRAVDGLDLNVPAGLVYGILGPNGAGKSTTIRMLATLLRPDGGTARVFGHDVTSEPDTVRRRISVTGQYASVDEGLTGTENLVMMGRLQGYSWARARERAAELIDGFGLGDARDRLLKTYSGGMRRRLDIAASIVVTPDLLFLDEPTTGLDPRSRNQVWDIVRALVDAGTTVLLT
resistance
15842226
ATP-binding
?
protein
2999689..3000642
drrA - Streptomyces 2
peucetius.
comp is this the C??
[7,30]
?
sugar ABC transporter, sugar-binding protein
TREHALOSE/MALTOSE
MTRPRQSTLVATALVLVAILLGVTAVLLGLSAEPRGGKIVVTVRLWDEPIAAAYRQSFAAFTRSHPDIEVRTNLVAYSTYFETLRTDVAGGSADDIFWLSNAYFAAYADSGRLMKIQTDAADWEPAVVDQFTRSGVLWGVPQLTDAGIAVFYNADLLAAAGVDPTQVDNLRWSRGDDDTLRPMLARLTVDADGRT
?
15841823
BINDING
O51923
PROTEIN
2586364..2587644
- Thermococcus litoralis, 3and
comp
Pyrococcus furiosus.
complexity and that like humans, mycobacteria fuse related genes together.
[11,35]
? [69,93] sugar
[106,129]
ABC transporter,
[138,156] [181,204]
permease[241,260]
proteinINNER
MSSPSRVSNTAVYAVLTIGAVITLSPFLLGLLTSFTSAHQFATGTPLQLPRPPTLANYADIADAGFRRAAVVTALMTAVILLGQLTFSVLAAYAFARLQFRGRDALFWVYVATLMVPGTVTVVPLYLMMAQLGLRNTFWALVLPFMFGSPYAIFLLREHFRLIPDDLINAARLDGANTLDVIVHVVIPSSRPVLAALAM
MEMBRANE
?
15841822
PROTEIN MALG
Q9R9Q5
(TREHALOSE/MALTOSE
2585543..2586367
TRANSPORT
3 comp
INNER MEMBRANE PROTEIN) - Thermococcus litoralis, and Pyrococcus furiosus.
There were other interesting discoveries as well. 3.A.1.5.2, which belongs to the
[14,38]
[23,47]
[69,93]
[81,105]
sugar
[106,127]
ABC
[114,131]
transporter,
[154,178]
[148,172]
[209,231]
permease
[181,203]
[252,276]
proteinINNER
[212,229]
MRDAPRRRTALAYALLAPSLVGVVAFLLLPILVVVWLSLHRWDLLGPLRYVGLTNWRSVLTDSGFADSLVVTAVFVAIVVPAQTVLGLLAASLLARRLPGTGLFRTLYVLPWICAPLAIAVMWRWILAPTDGAISTVLGHRIEWLTDPGLALPVVSAVVVWTNVGYVSLFFLAGLMAIPQDIHNAARTDGASAWQRF
MEMBRANE
MEGKIMDNNLTSKLKYREAKLGYLMILPLLTVVLVFIILPVMGTFWISLHRDVTFIPEKPFVGLRNYLRVLSAREFWYSTFVTVSFSFVSVSLETILGLSFALILNERLKGRGVLRAIVLIPWAVPTIISARTWELMYNYSYGLFNWILSILGVSPVNWLGTPISAFFAIVIADVWKTTPLMTLLLLAGLQAIPQDLYEA
[238,262]
15841821
PROTEIN
[271,295]
MALF
O51924
- Thermococcus
2584684..2585556
litoralis.
3 comp
Peptide/Opine/Nickel Uptake Transporter (PepT) Family, is known to be a 5-component transport
system. That is, the transport system requires 5 different proteins encoded by 5 different genes.
Generally, the genes encoding the proteins that work in concert towards a specific goal such as a
transport mechanism are usually embedded within the same operon. What was particularly
interesting is that while four of these five proteins showed significant similarity scores, one of them
(15843277) did not show significant similarity to its expected match: the dipeptide transport ATPbinding protein DppD, found in Bacillus subtilis. This suggests that there was an alternate gene
different from those ABC systems characterized, recruited for energization of this mycobacterial
transporter. However, it is not clear at this moment as to whether this 5th gene is essential for
proper function of this transporter system.
The most striking finding was regarding 3.A.2.1.2, a family of ATP synthases. It was found
that in M. tuberculosis, there is a fusion of a delta subunit to the c-terminus of a b subunit and in
contrast to all known F-type ATPases, there are 3 b-subunits. It is possible that evolutionary
pressure to make the genome more compact and the transcription/translation of genes to be more
efficient may have led to these fusional events.
PSEUDOGENES
A very interesting pseudogene that encodes for the protein of interest (gi: 15841200) was
found in M. tuberculosis, as shown in Fig. 1. Its origin appears to be that of the anaerobic,
respiratory, membrane-bound nitrate reductase (5.A.3.1.2) of The Prokaryotic Molybdopterincontaining Oxidoreductase (PMO) Family. The nitrate reductase system has 3 components; in the
order in which it is transcribed in the operon, we have the alpha chain (1245 aas, the hydrophilic
component that reduces NO3- to NO2-), the beta chain (512 aas, the hydrophilic component that has
4 iron-sulfur centers), and the gamma chain (225 aas, the 5 TMS hydrophobic component that
anchors the alpha and beta chains). Assembly of this transport system is aided by a chaperon
protein, the delta chain (narJ, 206 aas, located in between the two genes encoding the beta and
gamma chains). The first 214 aas of the protein of interest (gi: 15841200, 652 aas, 5 TMSs) shows
significant sequence similarity to the first 214 aas of the alpha chain, the last 250 aas (containing
the 5 TMSs) aligns to the entire delta chain, also with 5 TMSs. Since the middle region aligns to
the first 3/4th of the delta chain, there must have been at least two deletion events to give rise to this
putative pseudogene. It will be interesting to see if this pseudogene is still present in M. leprae or if
this gene was deleted in its entirety.
Computer Methods
Classes of Transporters Found In M. tuberculosis and M. leprae
The complete protein sequences of Mtu and Mle were
extracted from the NCBI non-redundant database. Computer-aided
analyses were conducted to retrieve all proteins encoded within the
genomes of Mtu and Mle that are recognizably homologous to
transport system constituents included in the transporter
classification database (TCDB) [1,2]. Briefly, all proteins were
blasted in an automated manner (using BLASTP, tBLASTn, NCBI
PSI-BLAST) against the Transporter Classification and NCBI
databases. Additional databases used for protein functional analysis
were the nonredundant SWISSPROT and TrEMBL protein
sequence databases. Several protein pattern databases (Conserved
Domain Database at NCBI and Pfam) were also used. Charge-bias
analyses of membrane protein topology were performed using the
TMHMM [6] and WHAT [7] programs. A side-by-side comparison
of transport proteins within the two genomes were performed using
The Integrated Microbial Genomes (IMG) system. Currently, we
are on the brink of incorporating PSI-FI to systematically search for
pseudogenes in a consistent manner.
Acknowledgement
Table 1
Categories of Recognized Transport Proteins and Pseudogenes Found in M.
tuberculosis and M. leprae
TC Class
1 Channels
2 Secondary carriers
3 Primary transporters
4 Group translocators (PTS)
5 Transmembrane electron carriers
8 Auxiliary transport proteins
9 Poorly defined systems
-' = not yet analyzed/assigned
( %) = relative percentage of
pseudogenes with respect to total
number of proteins
Number of Transport Proteins
Functional ?
M . tuberculosis
M . leprae
7
82
136
0
9
1
35+
0
26
-
Pseudogenes ?
M . tuberculosis
0 (0%)
0 (0%)
1 (< 0.37%)
-
I would like to thank Dr. Saier for his endless guidance, as
well as Dorjee Tamang and Ming Zheng for their technical
support. Also, I would like to thank Calit2 for their financial
support and general research advice
M . leprae
0 (0%)
4 (15.4%)
-
References
1. Busch W and Saier MH Jr. (2002) The Transporter Classification (TC) System,
2002. CRC Crit. Rev. Biochem. Mol. Biol. 37:287-337.
2. Tran CV, Yang NM, and Saier MH Jr. (2003) TC-DB: An architecture for membrane
transport protein analysis. Proc. 2nd Intl. IEEE Computer Society Computational
Systems Bioinformatics Conference, p. 658.
3. Lerat E and Ochman H (2004) Exploring the outer limits of bacterial pseudogenes.
Genome Research 14: 2273-2278.
4. Lerat E. and Ochman H (2005) Recognizing pseudogenes in bacterial genomes.
Nucleic Acids Research 33: 3125-3132.
5. Gerstein M and Zheng D (2006) The Real life of Pseudogenes. Scientific American
August: 49-55.
6. Krog A, Larsson B, von Heijne G, and Sonnhammer EL. (2001) Predicting
transmembrane protein topology with a hidden Markov model. Application to
complete genomes. J. Mol. Biol. 305:567-580.
7. Zhai Y and Saier MH Jr. (2001) A web-based program (WHAT) for the simultaneous
prediction of hydropathy, amphipathicity, secondary structure and transmembrane
topology for a single protein sequence. J. Mol. Microbiol. Biotechnol. 3:501-502.