Linkage, Mobility, and Selfishness in the MazF Family of Bacterial Toxins: A Snapshot of Bacterial Evolution
Identifieur interne : 000546 ( Pmc/Corpus ); précédent : 000545; suivant : 000547Linkage, Mobility, and Selfishness in the MazF Family of Bacterial Toxins: A Snapshot of Bacterial Evolution
Auteurs : Nikita Chopra ; Abhinandan Pathak ; Rakesh Bhatnagar ; Sonika BhatnagarSource :
- Genome Biology and Evolution [ 1759-6653 ] ; 2013.
Abstract
Prokaryotic MazF family toxins cooccur with cognate antitoxins having divergent DNA-binding folds and can be of chromosomal or plasmid origin. Sequence similarity search was carried out to identify the Toxin–Antitoxin (TA) operons of MazF family followed by sequence analysis and phylogenetic studies. The genomic DNA upstream of the TA operons was searched for the presence of regulatory motifs. The MazF family toxins showed a conserved hydrophobic pocket in a multibinding site and are present in pathogenic bacteria. The toxins of the MazF family are associated with four main types of cognate antitoxin partners and cluster as a subfamily on the branches of the phylogenetic tree. This indicates that transmission of the entire operon is the dominant mode of inheritance. The plasmid borne TA modules were interspersed between the chromosomal TA modules of the same subfamily, compatible with a frequent interchange of TA genes between the chromosome and the plasmid akin to that observed for antibiotic resistance gens. The split network of the MazF family toxins showed the AbrB-linked toxins as a hub of horizontal gene transfer. Distinct motifs are present in the upstream region of each subfamily. The presence of MazF family TA modules in pathogenic bacteria and identification of a conserved binding pocket are significant for the development of novel antibacterials to disrupt the TA interaction. However, the role of TAs in stress resistance needs to be established. Phylogenetic studies provide insight into the evolution of MazF family TAs and effect on the bacterial genome.
Url:
DOI: 10.1093/gbe/evt175
PubMed: 24265503
PubMed Central: 3879964
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Linkage, Mobility, and Selfishness in the MazF Family of Bacterial Toxins: A
Snapshot of Bacterial Evolution</title><author><name sortKey="Chopra, Nikita" sort="Chopra, Nikita" uniqKey="Chopra N" first="Nikita" last="Chopra">Nikita Chopra</name><affiliation><nlm:aff id="evt175-AFF1">Computational and Structural Biology Laboratory, Division of Biotechnology, Netaji Subhas Institute of Technology, Dwarka, New Delhi, India</nlm:aff></affiliation></author><author><name sortKey="Pathak, Abhinandan" sort="Pathak, Abhinandan" uniqKey="Pathak A" first="Abhinandan" last="Pathak">Abhinandan Pathak</name><affiliation><nlm:aff id="evt175-AFF1">Computational and Structural Biology Laboratory, Division of Biotechnology, Netaji Subhas Institute of Technology, Dwarka, New Delhi, India</nlm:aff></affiliation></author><author><name sortKey="Bhatnagar, Rakesh" sort="Bhatnagar, Rakesh" uniqKey="Bhatnagar R" first="Rakesh" last="Bhatnagar">Rakesh Bhatnagar</name><affiliation><nlm:aff id="evt175-AFF2">Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India</nlm:aff></affiliation></author><author><name sortKey="Bhatnagar, Sonika" sort="Bhatnagar, Sonika" uniqKey="Bhatnagar S" first="Sonika" last="Bhatnagar">Sonika Bhatnagar</name><affiliation><nlm:aff id="evt175-AFF1">Computational and Structural Biology Laboratory, Division of Biotechnology, Netaji Subhas Institute of Technology, Dwarka, New Delhi, India</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24265503</idno><idno type="pmc">3879964</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879964</idno><idno type="RBID">PMC:3879964</idno><idno type="doi">10.1093/gbe/evt175</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">000546</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Linkage, Mobility, and Selfishness in the MazF Family of Bacterial Toxins: A
Snapshot of Bacterial Evolution</title><author><name sortKey="Chopra, Nikita" sort="Chopra, Nikita" uniqKey="Chopra N" first="Nikita" last="Chopra">Nikita Chopra</name><affiliation><nlm:aff id="evt175-AFF1">Computational and Structural Biology Laboratory, Division of Biotechnology, Netaji Subhas Institute of Technology, Dwarka, New Delhi, India</nlm:aff></affiliation></author><author><name sortKey="Pathak, Abhinandan" sort="Pathak, Abhinandan" uniqKey="Pathak A" first="Abhinandan" last="Pathak">Abhinandan Pathak</name><affiliation><nlm:aff id="evt175-AFF1">Computational and Structural Biology Laboratory, Division of Biotechnology, Netaji Subhas Institute of Technology, Dwarka, New Delhi, India</nlm:aff></affiliation></author><author><name sortKey="Bhatnagar, Rakesh" sort="Bhatnagar, Rakesh" uniqKey="Bhatnagar R" first="Rakesh" last="Bhatnagar">Rakesh Bhatnagar</name><affiliation><nlm:aff id="evt175-AFF2">Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India</nlm:aff></affiliation></author><author><name sortKey="Bhatnagar, Sonika" sort="Bhatnagar, Sonika" uniqKey="Bhatnagar S" first="Sonika" last="Bhatnagar">Sonika Bhatnagar</name><affiliation><nlm:aff id="evt175-AFF1">Computational and Structural Biology Laboratory, Division of Biotechnology, Netaji Subhas Institute of Technology, Dwarka, New Delhi, India</nlm:aff></affiliation></author></analytic><series><title level="j">Genome Biology and Evolution</title><idno type="eISSN">1759-6653</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Prokaryotic MazF family toxins cooccur with cognate antitoxins having divergent
DNA-binding folds and can be of chromosomal or plasmid origin. Sequence similarity search
was carried out to identify the Toxin–Antitoxin (TA) operons of MazF family followed
by sequence analysis and phylogenetic studies. The genomic DNA upstream of the TA operons
was searched for the presence of regulatory motifs. The MazF family toxins showed a
conserved hydrophobic pocket in a multibinding site and are present in pathogenic
bacteria. The toxins of the MazF family are associated with four main types of cognate
antitoxin partners and cluster as a subfamily on the branches of the phylogenetic tree.
This indicates that transmission of the entire operon is the dominant mode of inheritance.
The plasmid borne TA modules were interspersed between the chromosomal TA modules of the
same subfamily, compatible with a frequent interchange of TA genes between the chromosome
and the plasmid akin to that observed for antibiotic resistance gens. The split network of
the MazF family toxins showed the AbrB-linked toxins as a hub of horizontal gene transfer.
Distinct motifs are present in the upstream region of each subfamily. The presence of MazF
family TA modules in pathogenic bacteria and identification of a conserved binding pocket
are significant for the development of novel antibacterials to disrupt the TA interaction.
However, the role of TAs in stress resistance needs to be established. Phylogenetic
studies provide insight into the evolution of MazF family TAs and effect on the bacterial
genome.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Abascal, F" uniqKey="Abascal F">F Abascal</name></author><author><name sortKey="Zardoya, R" uniqKey="Zardoya R">R Zardoya</name></author><author><name sortKey="Posada, D" uniqKey="Posada D">D Posada</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Acebo, P" uniqKey="Acebo P">P Acebo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Agarwal, S" uniqKey="Agarwal S">S Agarwal</name></author><author><name sortKey="Mishra, Nk" uniqKey="Mishra N">NK Mishra</name></author><author><name sortKey="Bhatnagar, S" uniqKey="Bhatnagar S">S Bhatnagar</name></author><author><name sortKey="Bhatnagar, R" uniqKey="Bhatnagar R">R Bhatnagar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Altekar, G" uniqKey="Altekar G">G Altekar</name></author><author><name sortKey="Dwarkadas, S" 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Genome Biol Evol</journal-id><journal-id journal-id-type="iso-abbrev">Genome Biol Evol</journal-id><journal-id journal-id-type="publisher-id">gbe</journal-id><journal-id journal-id-type="hwp">gbe</journal-id><journal-title-group><journal-title>Genome Biology and Evolution</journal-title></journal-title-group><issn pub-type="epub">1759-6653</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24265503</article-id><article-id pub-id-type="pmc">3879964</article-id><article-id pub-id-type="doi">10.1093/gbe/evt175</article-id><article-id pub-id-type="publisher-id">evt175</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Linkage, Mobility, and Selfishness in the MazF Family of Bacterial Toxins: A
Snapshot of Bacterial Evolution</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Chopra</surname><given-names>Nikita</given-names></name><xref ref-type="aff" rid="evt175-AFF1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Saumitra</surname><given-names></given-names></name><xref ref-type="aff" rid="evt175-AFF1"><sup>1</sup></xref><xref ref-type="aff" rid="evt175-AFF3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Pathak</surname><given-names>Abhinandan</given-names></name><xref ref-type="aff" rid="evt175-AFF1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Bhatnagar</surname><given-names>Rakesh</given-names></name><xref ref-type="aff" rid="evt175-AFF2"><sup>2</sup></xref><xref ref-type="corresp" rid="evt175-COR1">*</xref></contrib><contrib contrib-type="author"><name><surname>Bhatnagar</surname><given-names>Sonika</given-names></name><xref ref-type="aff" rid="evt175-AFF1"><sup>1</sup></xref><xref ref-type="corresp" rid="evt175-COR1">*</xref></contrib><aff id="evt175-AFF1"><sup>1</sup>Computational and Structural Biology Laboratory, Division of Biotechnology, Netaji Subhas Institute of Technology, Dwarka, New Delhi, India</aff><aff id="evt175-AFF2"><sup>2</sup>Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India</aff><aff id="evt175-AFF3"><sup>3</sup>Present address: Neuro-Electronic Laboratory, Center for Nanoscience and Engineering, Indian Institute of Science, Bangalore, Karnataka, India</aff></contrib-group><author-notes><corresp id="evt175-COR1">*Corresponding author: E-mail:
<email>ecc999@gmail.com</email>; <email>rakeshbhatnagar@mail.jnu.ac.in</email>.</corresp><fn><p><bold>Associate editor:</bold> John McCutcheon</p></fn></author-notes><pub-date pub-type="ppub"><year>2013</year></pub-date><pub-date pub-type="epub"><day>20</day><month>11</month><year>2013</year></pub-date><pub-date pub-type="collection"><month>12</month><year>2013</year></pub-date><pub-date pub-type="pmc-release"><day>20</day><month>11</month><year>2013</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on the
<volume>5</volume><issue>12</issue><fpage>2268</fpage><lpage>2284</lpage><history><date date-type="accepted"><day>31</day><month>10</month><year>2013</year></date></history><permissions><copyright-statement>© The Author(s) 2013. Published by Oxford University Press on
behalf of the Society for Molecular Biology and Evolution.</copyright-statement><copyright-year>2013</copyright-year><license license-type="creative-commons" xlink:href="http://creativecommons.org/licenses/by-nc/3.0/"><license-p><pmc-comment>CREATIVE COMMONS</pmc-comment>This is an Open Access article distributed under the terms of the Creative Commons
Attribution Non-Commercial License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/3.0/">http://creativecommons.org/licenses/by-nc/3.0/</ext-link>),
which permits non-commercial re-use, distribution, and reproduction in any medium,
provided the original work is properly cited. For commercial re-use, please contact
journals.permissions@oup.com</license-p></license></permissions><abstract><p>Prokaryotic MazF family toxins cooccur with cognate antitoxins having divergent
DNA-binding folds and can be of chromosomal or plasmid origin. Sequence similarity search
was carried out to identify the Toxin–Antitoxin (TA) operons of MazF family followed
by sequence analysis and phylogenetic studies. The genomic DNA upstream of the TA operons
was searched for the presence of regulatory motifs. The MazF family toxins showed a
conserved hydrophobic pocket in a multibinding site and are present in pathogenic
bacteria. The toxins of the MazF family are associated with four main types of cognate
antitoxin partners and cluster as a subfamily on the branches of the phylogenetic tree.
This indicates that transmission of the entire operon is the dominant mode of inheritance.
The plasmid borne TA modules were interspersed between the chromosomal TA modules of the
same subfamily, compatible with a frequent interchange of TA genes between the chromosome
and the plasmid akin to that observed for antibiotic resistance gens. The split network of
the MazF family toxins showed the AbrB-linked toxins as a hub of horizontal gene transfer.
Distinct motifs are present in the upstream region of each subfamily. The presence of MazF
family TA modules in pathogenic bacteria and identification of a conserved binding pocket
are significant for the development of novel antibacterials to disrupt the TA interaction.
However, the role of TAs in stress resistance needs to be established. Phylogenetic
studies provide insight into the evolution of MazF family TAs and effect on the bacterial
genome.</p></abstract><kwd-group><kwd>MazF</kwd><kwd>toxin–antitoxin systems</kwd><kwd>multibinding site</kwd><kwd>phylogenetic studies</kwd><kwd>linkage</kwd><kwd>mobility</kwd></kwd-group><counts><page-count count="17"></page-count></counts></article-meta></front><body><sec><title>Introduction</title><p>Toxin–Antitoxin (TA) systems are widely found in free-living prokaryotes and Archaea.
Five distinct types of TA encoding loci are known. The TA systems are classified as Type
I–V depending on the chemical nature and mode of action of their two components (<xref ref-type="bibr" rid="evt175-B27">Gerdes et al. 2005</xref>; <xref ref-type="bibr" rid="evt175-B84">Wang et al. 2012</xref>). The Type II TA systems consist of two genes
encoded by an operonic structure, wherein the upstream gene codes for a labile antitoxin
protein and the downstream gene codes for a more stable toxin protein. The Type II TA
systems are found to be distributed in both plasmid and chromosomal genetic elements. The
toxins of the plasmid-encoded Type II TA systems have been attributed to mediate post
segregational killing consequent to plasmid loss, while the chromosome-encoded toxins
mediate a wide variety of different cellular functions related to cell stasis, genetic
stabilization, stress response, and quorum sensing. (<xref ref-type="bibr" rid="evt175-B84">Wang et al. 2012</xref>). The Type II proteic antitoxin usually serves two functions;
firstly, it binds to and prevents the toxin from mediating its harmful effects. Secondly,
the antitoxin alone, or in complex with the toxin, binds to its own operon to bring about
transcriptional autoregulation. As the antitoxin protein is highly susceptible to
proteolytic degradation by the action of Lon or Clp proteases, a continuous supply of the
antitoxin protein in the cell is required to circumvent the lethal action of the toxin.
Therefore, the TA systems are also termed as addiction modules. Exceptions to the
generalized mode of action of TA systems described are evident in three-component systems
(<xref ref-type="bibr" rid="evt175-B17">de la Hoz et al. 2000</xref>; <xref ref-type="bibr" rid="evt175-B38">Hallez et al. 2010</xref>). TA modules are important drug
targets as it has been hypothesized that the drug-induced disruption of their interaction
would set the toxin free to exert its toxic activity in the bacterial cell.</p><p>The known TA type II systems have been classified into 12 toxin and 20 antitoxin
superfamilies based on sequence homology and association with toxin/antitoxin-like sequences
(<xref ref-type="bibr" rid="evt175-B49">Leplae et al. 2011</xref>). Toxins having diverse
sequence, upstream antitoxin partners, and functions may belong to the same superfamily of
toxins (<xref ref-type="bibr" rid="evt175-B39">Hayes and Van Melderen 2011</xref>).
Additionally, they may occur as a TA complex with a number of different antitoxins having
divergent DNA-binding folds (<xref ref-type="bibr" rid="evt175-B49">Leplae et al.
2011</xref>). As an example, the toxins of the HigB/RelE/ParE superfamily adopt a unique
RelE-like fold and are associated with antitoxins having divergent folds. The RelE and ParE
families of toxins have been unified into one superfamily having a common evolutionary
origin despite their divergent activity/cellular targets as well as the lack of significant
sequence similarity (<xref ref-type="bibr" rid="evt175-B6">Anantharaman and Aravind
2003</xref>). Similarly, the toxins of the MazF/PemK/CcdB superfamily share a ribonuclease
SH3 fold and have several different types of cognate antitoxins. In previous studies,
co-occurrence of PemK-type toxins with a number of different types of antitoxins has also
been observed (<xref ref-type="bibr" rid="evt175-B56">Makarova et al. 2009</xref>).</p><p>In general, the antitoxin has a modular structure, wherein the N-terminal DNA-binding
domain is responsible for autoregulation, while the C-terminal domain is responsible for
neutralization of the toxin. Many of the known antitoxins have a
helix–turn–helix (HTH) or a ribbon–helix–helix (RHH) DNA-binding
motif at the N-terminus. Functional dimers of CcdA form a β-sheet or ribbon that
inserts into the major groove of DNA (<xref ref-type="bibr" rid="evt175-B54">Madl et al.
2006</xref>), much like the mode of binding seen in the transcriptional repressors
MetJ/Arc/CopG (<xref ref-type="bibr" rid="evt175-B33">Gomis-Ruth et al. 1998</xref>). The
mode of binding of RelB to DNA is also expected to be similar to that of CopG (<xref ref-type="bibr" rid="evt175-B51">Li et al. 2008</xref>). It has been proposed that the
RelE2 corepressor of <italic>Streptococcus pneumoniae</italic> serves to bridge the
antitoxin dimers bound at two distinct sites of the operator region, leading to greater
avidity of binding of the TA complex when compared with the RelB2 alone (<xref ref-type="bibr" rid="evt175-B13">Chan et al. 2013</xref>). HipB and MsqA antitoxins bind
to DNA via an HTH motif, containing a “recognition helix” that penetrates into
the major groove of DNA and makes base-specific interactions, whereas additional backbone
contacts stabilize the complex (<xref ref-type="bibr" rid="evt175-B75">Schumacher et al.
2009</xref>; <xref ref-type="bibr" rid="evt175-B11">Brown et al. 2011</xref>). Though MsqA
is a dimer, each of the MsqA recognition helices individually binds to one palindromic
half-site of its promoter. Further, DNA recognition by MsqA can be attributed completely to
specific residues of the recognition helix, which mediate a direct readout of the promoter
DNA sequence (<xref ref-type="bibr" rid="evt175-B11">Brown et al. 2011</xref>). HipB and
MsqA bear significant sequence and structural similarity to the 434 and 434 cro repressors,
thus establishing them as members of the Xre-HTH family of transcriptional regulators (<xref ref-type="bibr" rid="evt175-B75">Schumacher et al. 2009</xref>). Structural studies have
shown that the organization of the C-terminal helices of RHH motif is identical to that of
the classical HTH domain (<xref ref-type="bibr" rid="evt175-B33">Gomis-Ruth et al.
1998</xref>). A CopG-like transcription factor from the streptococcal plasmid pMV158
shared structural similarity with both HTH- and RHH-type DNA-binding proteins (<xref ref-type="bibr" rid="evt175-B2">Acebo et al. 1998</xref>). Mutagenesis studies have shown
that even small changes in the strand forming the ribbon are sufficient to induce a packing
close to the HTH domain (<xref ref-type="bibr" rid="evt175-B16">Cordes et al. 1999</xref>).
Thus, evolutionary unification of the HTH and RHH domains is possible (<xref ref-type="bibr" rid="evt175-B7">Aravind et al. 2005</xref>). MazE, PemI, and their homologs have a swapped
hairpin β-barrel fold shared by AbrB and SpoVT-type of transition state regulators
(<xref ref-type="bibr" rid="evt175-B15">Coles et al. 2005</xref>). Each monomer of AbrB
contains two β-hairpins that interweave with that of the dimer to form two layers of
β sheets connected by a short α-helix. Residues from the β1 loop extend into
the major groove to make base-specific interactions. Residues in the β2 loop and the
α-helix are also critical for DNA-binding ability of AbrB (<xref ref-type="bibr" rid="evt175-B79">Sullivan et al. 2008</xref>). A bioinformatics approach showed that HTH
domain containing antitoxins are found to co-occur with RelE/ParE, Zeta, HipA, GinD, and a
large number of other uncharacterized toxins. The RHH domain-containing antitoxins are found
together with ParE/RelE and CcdB/MazF type toxins, while the AbrB-type antitoxins are found
with Doc, CcdB/MazF, and VapC-type toxins (<xref ref-type="bibr" rid="evt175-B49">Leplae et
al. 2011</xref>).</p><p>The exploitation of TA modules presents an effective strategy for the development of novel
antibacterials as they are present in most bacterial pathogens, but have no human homolog.
Disruption of the preformed TA complex or prevention of formation of the TA complex could
thus release the toxin to exert its lethal effect. In case of MazF-type toxins, it may be
possible to achieve partial disruption of the TA complex in two ways: 1) disruption of TA
interactions at the active site, thus allowing the toxin to cleave free mRNA and 2) causing
allosteric activation of the toxin. Also, MazF toxins are proposed to be more impervious to
the development of resistance in comparison to CcdB-like toxins. Previous reports have
established that a targeted peptide can disrupt the TA interaction (<xref ref-type="bibr" rid="evt175-B52">Lioy et al. 2010</xref>; <xref ref-type="bibr" rid="evt175-B14">Chopra et
al. 2011</xref>) and the utility of TA disruption for antimicrobial development (<xref ref-type="bibr" rid="evt175-B52">Lioy et al. 2010</xref>). There is a scarcity of
structural information and data on the amino acids and residues that define the binding
hotspots between toxin and antitoxin (<xref ref-type="bibr" rid="evt175-B86">Williams and
Hergenrother 2012</xref>). MoxXT from <italic>Bacillus anthracis</italic> (<xref ref-type="bibr" rid="evt175-B3">Agarwal et al. 2010</xref>; <xref ref-type="bibr" rid="evt175-B14">Chopra et al. 2011</xref>) and YdcDE from <italic>B</italic>acillus
<italic>subtilis</italic> (<xref ref-type="bibr" rid="evt175-B69">Pellegrini et al.
2005</xref>) are TA systems consisting of a toxin with a SH3-like barrel domain and an
antitoxin with a RHH motif at the N-terminus. In an initial Position-Specific Iterative
(PSI)-Blast search, a large number of homologs could be identified for MoxT in the nr
(nonredundant) sequence database, including MazF, Kid, and YdcE. MoxXT and the previously
reported YdcDE are TA systems in which the antitoxin belongs to the CopG family as distinct
from the SpovT/AbrB-type antitoxin found in other members of the MazF/PemK family. In the
present work, we carried out exhaustive search and filtering to identify MazF family TA
modules with simultaneous identification of the different folds of their cognate antitoxin.
The various pathogenic bacteria in which they could act as drug targets were identified.
Further, we carried out the sequence analysis of the MazF family toxins in order to detect
the presence of conserved residues at the binding sites for the antitoxin.</p><p>Evolutionary studies on operons have shown that they may either be transferred by
horizontal gene transfer or by direct descent. They may be transmitted either as a whole, as
a single gene, or as a subset of genes (<xref ref-type="bibr" rid="evt175-B67">Omelchenko et
al. 2003</xref>). In view of the unique addiction properties of TA operons, their transfer
as operons is necessitated. However, diversity in the TA system is evident in the presence
of different types of antitoxins paired to the same toxin. Therefore, gene neighborhood
analysis was carried out to probe the evolution of the toxins with different types of
cognate antitoxins. Another complexity to be considered during evolution of TA modules is
their plasmid or chromosomal location. Therefore, we also analyzed the phylogenetic tree
obtained in context of chromosomal/plasmid location. Network modeling was carried out to
identify the hubs of gene transfer in the set of MazF family toxins. Earlier studies have
proposed that the toxin forms a distinct evolutionary module with the C-terminal residues of
the antitoxin, while the upstream promoter region of the DNA and the N-terminal DNA-binding
domain forms another distinct evolutionary module (<xref ref-type="bibr" rid="evt175-B78">Smith and Magnuson 2004</xref>). In view of this, we searched for different types of
regulatory elements present in the noncoding genomic region upstream of the TA operon in
each of the different subfamilies. Our analysis yields insights into the distribution of
MazF family TA modules in pathogenic organisms, their multibinding sites, mode of evolution,
and conserved upstream motifs of the MazF family TA modules.</p></sec><sec sec-type="materials|methods"><title>Materials and Methods</title><sec><title>Identification of MazF/PemK/Kid Toxin Family Homologs</title><sec><title>Search for Homologous Sequences</title><p>The sequence of MoxT from <italic>B. anthracis</italic> was used for a sequence
similarity search against the nr public database using PSI-Blast (<xref ref-type="bibr" rid="evt175-B5">Altschul et al. 1997</xref>). A cut off e-value threshold of 0.005 was
used for PSI-Blast, and the run was iterated till convergence. Distinct representatives
from the hits were obtained, and the database was searched with these sequences as
queries to perform an exhaustive search for additional distant homologs of MazF protein
using the generalized methodology as described earlier (<xref ref-type="bibr" rid="evt175-B6">Anantharaman and Aravind 2003</xref>; <xref ref-type="bibr" rid="evt175-B35">Guglielmini et al. 2008</xref>). The putative toxins without a
cognate antitoxin partner were discarded. In order to qualify the hits as putative TA
systems, sequences were filtered according to the following criteria: 1) the length of
the toxin should be 60–150 amino acids; 2) an open reading frame (ORF) of length
40–90 amino acids transcribed in the same direction as the toxin must be present
upstream of the putative toxin; 3) the separation or overlap between the putative
antitoxin and toxin ORFs must not exceed 30 bases; 4) sequences should have a complete
PemK fold. Fold prediction of the obtained toxin sequences was carried out using Batch
CD search (<xref ref-type="bibr" rid="evt175-B58">Marchler-Bauer et al. 2011</xref>) in
order to confirm that the fold of the toxin was SH3 barrel as found in MazF. 5) Finally,
the toxin should be a member of a two-gene operon. The number of genes present in each
operon was predicted using the Database of prOkaryotic OpeRons (DOOR) database (<xref ref-type="bibr" rid="evt175-B57">Mao et al. 2009</xref>). Entries not found in DOOR
were searched for in the microbes online (<xref ref-type="bibr" rid="evt175-B18">Dehal
et al. 2010</xref>) and PropDB databases (<xref ref-type="bibr" rid="evt175-B81">Taboada et al. 2012</xref>).</p><p>Clusters of the toxin sequences were made using Blastclust (L 0.75; S 1.0) using the
resources available at <ext-link ext-link-type="uri" xlink:href="http://toolkit.tuebingen.mpg.de/">http://toolkit.tuebingen.mpg.de/</ext-link> (last accessed November 24, 2013). The
clusters with a single type of cognate antitoxin were combined. The sequence alignment
in each individual cluster and combination of clusters was carried out using MUltiple
Sequence Comparison by Log-Expectation (MUSCLE) (<xref ref-type="bibr" rid="evt175-B21">Edgar 2004</xref>) and manual sequence editing/analysis of individual clusters was
done using Bioedit version 7.0.5.3. The secondary structure assignment of the sequences
was carried out using STRuctural Alignments of Proteins (STRAP) (<xref ref-type="bibr" rid="evt175-B29">Gille and Frommel 2001</xref>). The pattern of secondary structures
and the placement of gaps in the alignment were inspected manually. Fold prediction of
the cognate antitoxin protein was also carried out using a Conserved Domain Database
(CDD) or Protein Homology/analogY Recognition Engine V 2.0 (PHYRE2) (<xref ref-type="bibr" rid="evt175-B46">Kelley and Sternberg 2009</xref>) to identify the
DNA-binding motif. Intergenic sequences upstream of the two gene TA operons were
collected for motif analysis.</p></sec><sec><title>Sequence Coding</title><p>For further analysis, the selected sequences were coded as follows: the genus
constitutes the first two letters and species name constitutes the next two letters
followed by the GenBank ID (GI) number. Plasmid and chromosomal location was indicated
by characters “p” and “c,” respectively, after the GI number.
Toxins with different cognate partners were encoded separately as follows: ab:
AbrB/SpovT, co: MetJ/Arc/CopG, du: Duf(3018), du1: Duf(104), du2: Duf(2281), wi: winged
HTH, re: relE, ph: Phd/YefM, and hyp: No conserved domain.</p></sec><sec><title>Presence of TA Modules in Strains of Pathogenic Organisms</title><p>The diseases caused by the organisms in which the sequences of the clustered TA modules
were present were determined with the help of the Kyoto Encyclopedia of Genes and
Genomes (KEGG) Disease resources. The human pathological strains were listed using the
PathoSystems Resource Integration Center (PATRIC) database (<xref ref-type="bibr" rid="evt175-B30">Gillespie et al. 2011</xref>). Using Blast search, it was confirmed
that the toxin homologs were present in all the pathogenic strains of the bacteria.</p></sec><sec><title>Tree Building</title><p>Bayesian and maximum likelihood methods were used to construct the phylogenetic tree
using the final alignment for the identified MazF family TA modules. ProtTest v2.4
(<xref ref-type="bibr" rid="evt175-B1">Abascal et al. 2005</xref>) was run to
determine that the Le and Gasquel, 2008 (LG) model was the most appropriate model to
evaluate trees. As MrBayes 3.1.2 (<xref ref-type="bibr" rid="evt175-B4">Altekar et al.
2004</xref>) does not support the LG model of evolution, the parameter was set to nst
= mixed with gamma distribution of rates and invariant site categories used for
the analysis. The MrBayes tree was built using the Cyberinfrastructure for Phylogenetic
Research (CIPRES) science gateway (<xref ref-type="bibr" rid="evt175-B89">Miller et al.
2010</xref>). Two parallel runs, each consisting of four chains, were run
simultaneously for 1.0 × 10<sup>7</sup> generations. Every 1,000th tree was
sampled, and the first 25% of the generations were omitted from topology and
probability reconstruction. In all analyses, convergence of the two parallel runs was
observed. Maximum likelihood tree was computed with PhyML (<xref ref-type="bibr" rid="evt175-B20">Dereeper et al. 2008</xref>) using the Whelan and Goldman, 2001 (WAG)
amino acid substitution model. The two methods produced similar trees.</p></sec><sec><title>Network Modeling</title><p>The split network was created using the Neighbor-Net algorithm (<xref ref-type="bibr" rid="evt175-B12">Bryant and Moulton 2004</xref>) and uncorrected <italic>P</italic>distances with EqualAngle split transformation settings as implemented in
Splitstree4.12.8 (<xref ref-type="bibr" rid="evt175-B40">Huson and Bryant 2006</xref>).
The graph was then visualized using the EqualAngle layout.</p></sec><sec><title>Search for Conserved Motifs in the Operator Region</title><p>A data set of upstream noncoding DNA sequences in representative members of each TA
cluster of the tree was constructed. The presence of motifs in each set was probed using
the Multiple Em for Motif Elicitation (MEME) suite (<xref ref-type="bibr" rid="evt175-B8">Bailey et al. 2009</xref>). Where a number of similar motifs were
detected using the MEME suite, an attempt to combine the motifs was made using Glam2,
which helps in determining the conserved motif from the DNA or protein sequences (<xref ref-type="bibr" rid="evt175-B23">Frith et al. 2008</xref>). MEME usually finds the
most statistically significant (low <italic>P</italic> value) motifs first. The e-value
of a motif is based on its log-likelihood ratio, width, sites, the background letter
frequencies, and the size of the training set. Glam2 was run on the data set of upstream
sequences to detect the presence of conserved motifs. The sequences were scanned for the
presence of 10 shared motifs of length 6–50 bases with any number of repetitions
in the data set. For determination of statistically significant <italic>P</italic>values, the MEME run was also performed with the “shuffle sequences” option.
The occurrence of the upstream motifs found was also confirmed using Bioprospector
(<xref ref-type="bibr" rid="evt175-B53">Liu et al. 2001</xref>).</p></sec></sec></sec><sec sec-type="discussion"><title>Results</title><sec><title>TA Module Search and Filtering</title><p>We carried out exhaustive search and filtering to detect MazF family TA modules in
prokaryotic genomes. Over 400 toxin sequences were identified with an exhaustive PSI-Blast
search. Of these, singlet toxins with no upstream gene (overlapping or within 30 bases)
were discarded. Toxins that could not be annotated with a complete PemK fold were
discarded from our data set. Further elimination of TA modules on the basis of their size
as well the direction of transcription of the ORF was done in accordance with the method
followed in <xref ref-type="bibr" rid="evt175-B35">Guglielmini et al. (2008)</xref>. After
extensive operon prediction using the resources as described in the Materials and Methods
section, the TA operons present in operons having more than two members were discarded to
yield a final data set of 263 TA operons. Two toxins annotated in Genbank as CcdB-type
toxins were identified at the 2nd iteration of the PSI-Blast search (GI: 304312769,
58616201). Sequence alignment of MazF family toxins with these identified CcdB toxins
showed that they do not share the same secondary structure pattern. As a result, they did
not align well with other sequences. Therefore, these CcdB-type toxins were not grouped
with the MazF family toxins in the present study.</p></sec><sec><title>Phyletic Distribution of the MazF/PemK Family TA Modules</title><p>After filtering out the sequences that did not fulfill the outlined criteria for the
MazF-type toxins belonging to a TA operon, the final set of 263 toxin sequences of TA
operons with two predicted gene members. The GI numbers, sequence codes, and source
organism of the sequences used in this analysis are listed in <ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">supplementary table S1</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">Supplementary Material</ext-link> online). Of the two-membered TA operons, 95%
were found in bacteria, one sequence was from the <italic>Lactobacillus johnsonii</italic>prophage Lj771, while the rest were distributed in Archaea (Euryarchaeotes). Within the
bacterial subgroup, 110 sequences (42%) were concentrated in the family
Proteobacteria, 67 (25%) were in Firmicutes, while the rest were found to be
distributed in Actinobacteria, green sulfur bacteria, Spirochetes,
Cytophaga-Flavobacteria-Bacteroides (CFB) group bacteria, green nonsulfur group bacteria,
Deinococcales, and others. In the present work, we detected the occurrence of five
AbrB-linked two-membered TA modules in Archaea and one in virus. The distribution analysis
based on genomic location was also carried out. A total of 215 (80%) of the MazF TA
systems were located on the chromosomes, with only one fifth of the sequences being
plasmid encoded.</p></sec><sec><title>Clustering of Toxins by Sequence Similarity</title><p>The data set of 263 toxin sequences from two gene operons segregated into five clusters
having 20 or more members using Blastclust, with parameters as described in the Materials
and Methods section. In subsequent discussions, these clusters will be referred to as
Clusters I through V. The gene neighborhood analysis of the selected toxin sequences was
carried out to identify their cognate antitoxin partner. Four main different classes of
antitoxins were found upstream to the MazF family toxins, namely: AbrB/SpoVT, CopG,
Duf(3018), and winged HTH. Duf(3018) is an uncharacterized family of bacterial proteins
with unknown function. The TA systems with AbrB/SpovT-type of antitoxin were relatively
large in number as compared to antitoxins of other types. The AbrB-linked and Duf(3018) TA
systems were found to be highest in number in Proteobacteria, while CopG-linked TA systems
were predominantly found in Firmicutes. The single Duf(104) family protein containing TA
module was from <italic>Archaeglobus profundus</italic>, while the single Duf(2281)
originates from the Cyanobacteria <italic>Trichodesmium erythraeum IMS101</italic>. All
the five winged HTH antitoxin containing TA modules were from Archaea. Two putative hybrid
TA systems mixing MazF-type toxins with RelB-type antitoxins were also identified (Toxin
GI: 83592312 and 213692408) and provide a putative evolutionary link between the MazF and
the RelE superfamilies. All the 48 Cluster I toxins showed high within-group sequence
conservation, were paired with either CopG family or uncharacterized antitoxins, and had a
chromosomal location. Other chromosomal toxins in clusters also shared high sequence
similarity. However, the same is not true of the plasmid-borne toxins.</p></sec><sec><title>Sequence Analysis and Structure Comparison</title><p>The crystal structure of the MazEF complex shows the presence of two intimate binding
sites of interaction between the C-terminal residues of MazE and MazF. The sites have been
described previously as Site 1 and Site 2 (<xref ref-type="bibr" rid="evt175-B43">Kamada
et al. 2003</xref>) and are shown in the biological dimer in <xref ref-type="fig" rid="evt175-F1">figure 1</xref> (inset). Site 1 constitutes an interface between the
disordered C-terminal region of the antitoxin and one of the toxin monomers. Site 1 is the
most intimate site of interaction between antitoxin and toxin and thus has the maximum
contribution to binding. Specific interaction at Site 1 occurs by the occupation of a
pocket in Chain A of MazF by Trp 73 of MazE. The pocket at Site 1 becomes exposed to
solvent upon disordering of the S1–S2 loop. Sequence/structural analysis has shown
that the hydrophobic pocket at Site 1 as well as a disordered S1–S2 loop is also
conserved in CcdB and Kis. Site 2 consists of a crevice constituted by the dimeric
interface of the two MazF monomers overlaid by α-helix H2 of MazE. Together, Site 1,
disordering of the S1–S2 loop and Site 2 have been proposed as conserved features of
TA interaction in the MazEF family (<xref ref-type="bibr" rid="evt175-B43">Kamada et al.
2003</xref>). Interactions of a small molecule or peptide at either Site 1 or Site 2 can
help to disrupt the TA interaction, serving as a potential antibacterial strategy (<xref ref-type="bibr" rid="evt175-B86">Williams and Hergenrother 2012</xref>). Residues
occurring with high probability (>0.9) in binding hotspots between MazE and MazF were
predicted by hotspot analysis of the crystal structure of the MazEF using PCRPi (<xref ref-type="bibr" rid="evt175-B42">Jubb et al. 2012</xref>). The hotspot residues
identified coincide with both Site 1 and Site 2. Further analysis was done to identify the
evolutionary conservation of sequence and structure of the binding sites suitable for
targeting by small molecules. <fig id="evt175-F1" position="float"><label>F<sc>ig</sc>.
1.—</label><caption><p>Superposition of residues at Site 1 of <italic>E.
coli</italic> MazF (red) with structurally equivalent residues in <italic>B.
subtilis</italic> YdcE (yellow). The inset shows the full length superposition of
Chain A of the two toxin proteins with Site 1 residues in red and Site 2 residues in
blue.</p></caption><graphic xlink:href="evt175f1p"></graphic></fig></p><p>While YdcE, a toxin obtained from <italic>B. subtilis</italic>, has only 24%
sequence identity with MazF, the fold is common (<xref ref-type="fig" rid="evt175-F1">fig.
1</xref>, inset). Superposition of the structure of dimer of YdcE (PDB ID:1NE8) (<xref ref-type="bibr" rid="evt175-B32">Gogos et al. 2003</xref>) with MazF was possible with
root mean square deviation (rmsd) of 1.54 Å among 166 residues. As shown in <xref ref-type="fig" rid="evt175-F1">figure 1</xref>, the residues of the pocket at Site 1 in
MazF are conserved in YdcE. Thus, the pocket at Site 1 is conserved structurally in the
toxins of MazF family even in the absence of high sequence identity. Further, the pocket
at Site 1 is conserved in both AbrB-linked toxins MazF and Kid (<xref ref-type="bibr" rid="evt175-B43">Kamada et al. 2003</xref>) as well as CopG-linked toxins YdcE and MoxT.
Sequence analysis of the Cluster I–V toxins was carried out to determine the extent
of conservation at the various sites. The sequence alignment of MazF toxins as obtained in
CopG-linked Cluster I is shown in <ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">supplementary figure S1</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">Supplementary Material</ext-link> online) with the residues involved in interactions at
the two sites marked as 1 and 2, respectively. As shown in this figure, within this
cluster (Cluster I), there is a high degree of conservation in the S1–S2 loop as
well as at Sites 1 and 2. A consurf rendering showing conservation of the residues of the
binding pocket at Site 1 is shown in <xref ref-type="fig" rid="evt175-F2">figure 2</xref>.
The residues of the pocket (highlighted) in Site 1 were found to be highly conserved and
are overlaid by the conserved residues constituting the loop (not highlighted). <fig id="evt175-F2" position="float"><label>F<sc>ig</sc>. 2.—</label><caption><p>Consurf rendering of
1NE8 pocket showing the cartoon of the entire protein. The highly conserved residues
are shown as purple balls. The pocket residues (11, 87, 83, 81, 44, 46, 25, 27) are
highlighted by a yellow halo. The residues overlying the highlighted pocket are
constituted by the loop S1–S2 (residues 12–27 in
1NE8).</p></caption><graphic xlink:href="evt175f2p"></graphic></fig></p><p>All members of Cluster II, Cluster III, and Cluster V, including <italic>Escherichia
coli</italic> MazF (PDB ID: 1UB4) and Kid (PDB ID: 2C06), were paired with
AbrB/Spovt-type of antitoxin. Multiple sequence alignment of all 3 clusters together with
the secondary structure notation derived from experimentally derived structures and
catalytic residue information is shown in <ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">supplementary figure S2</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">Supplementary Material</ext-link> online). The alignment in the three different panels
<italic>A</italic>, <italic>B</italic>, and <italic>C</italic> of the <ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">supplementary figure S2</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">Supplementary Material</ext-link> online) corresponds to the three clusters and shows
the distinct pattern of conservation in the three sets. There are some minor deviations in
the side chains lining the hydrophobic pocket at Site 1 in CopG as compared to the
AbrB-linked toxins, which may lead to small changes in the size and shape of the pocket.
However, it is expected that the compounds that bind to Site 1 in AbrB-linked toxins will
be able to bind to the same site in CopG-linked toxins also. At Site 2, the sequence in
this cluster is not well conserved either within this cluster or with respect to Cluster
1. Thus, on the basis of sequence conservation, Site 1 is better suited for design of
compounds to act by disruption of the TA complex in both groups. Cluster I toxins show
high conservation throughout the sequence, Cluster II + III + V toxins show high
sequence conservation in the N-terminal domain and at Site 1 in the C-terminal domain. In
comparison, though the structure at Site 2 is conserved, the sequence is not conserved in
the Clusters II, IV, and V.</p><p>All sequences in Cluster IV were paired with Duf(3018)-type of antitoxin partners and had
predominantly chromosomal location with few plasmid borne modules. These have not been
validated experimentally, and no information about the mode of action of these putative
toxins is available at present. However, the toxins may be ribonucleases in view of the
high sequence similarity to toxins in the other clusters identified. The sequence
alignment of Cluster IV toxins is shown in <ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">supplementary figure S3</ext-link> (<ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">Supplementary Material</ext-link> online). In Cluster IV sequences, sequence
conservation of the N-terminal domain is similar to that of the AbrB-linked toxins. The
sequence conservation in the C-terminal region differs markedly, showing highly conserved
residues interspersed throughout the C-terminal domain of the toxin. Thus, there is a
clear difference between the pattern of conservation in the toxin sequences in the three
clusters.</p><p>In Cluster I, the catalytic residues of <italic>B. anthracis</italic> have been
established as H59 and E78 (<xref ref-type="bibr" rid="evt175-B3">Agarwal et al.
2010</xref>). As the catalytic residues of Kid are known to be R73 and D75 (<xref ref-type="bibr" rid="evt175-B44">Kamphuis, Monti, van den Heuvel, Lopez-Villarejo, et
al. 2007</xref>), the E78 residue could be inferred by sequence similarity D75. However,
the R73 is replaced by a nonreactive L in MoxT, therefore H59 was inferred by structural
proximity to D75 in the modeled MoxT. Therefore, other charged residues in a structurally
favorable position can act as an acid–base pair to mediate catalysis. In accordance
with previous studies, sequence analysis (<ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">supplementary fig. S1</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">Supplementary Material</ext-link> online) shows that the catalytic residues E78 and H59
are conserved in majority of the sequences in the CopG-linked subfamily of toxins
(catalytic residues marked by asterisk). In Clusters II, III, and V (<ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">supplementary fig. S2</ext-link>, <ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">Supplementary Material</ext-link> online), the residue at D75 of Kid is found to be
identical in MazF and replaced by a polar residue (E or N) in other sequences. However,
R73 of Kid is replaced by a nonpolar residue in a majority of the sequences. The
difference in the catalytic residues of Kid and MazF has been noted previously. H28 has
been proposed as a general base to stabilize the catalytic intermediate in MazF (<xref ref-type="bibr" rid="evt175-B50">Li et al. 2006</xref>). Accordingly, the residues at
analogous to H28 position of the alignment are found to be polar/charged in most of the
sequences of Clusters II, III, and V. In Cluster IV Duf-linked toxins, the position
analogous D75 of Kid is occupied by D in all the sequences. However, the position
analogous to R73 of Kid is replaced by M. The position analogous to H59 of the CopG-linked
toxins is also occupied by a nonpolar residue in most of the sequences. Therefore, the
catalytic acid of the Cluster IV toxin remains to be determined.</p></sec><sec><title>TA Modules of the MazF/PemK Family: Presence in Pathological Organisms</title><p>It is well known that MazF family toxins are present in pathogenic bacteria (<xref ref-type="bibr" rid="evt175-B27">Gerdes et al. 2005</xref>). However, due to their
occurrence in plasmids, as well as their linkage with mobile genetic elements, their
presence within a bacterial species is heterogenous, and their occurrence in clinical
isolates is required to be confirmed (<xref ref-type="bibr" rid="evt175-B86">Williams and
Hergenrother 2012</xref>). In this work, we have identified MazF family TA modules with
conserved sequence features. Further, we screened the KEGG Disease resources and PATRIC
database to confirm the presence of these TA modules in all the known pathogenic strains
of these bacteria. TA modules linked to both CopG-type antitoxins and AbrB-type antitoxins
were found to be present in some important disease-causing bacteria. <xref ref-type="table" rid="evt175-T1">Table 1</xref> shows the species distribution of the
different MazF-type toxins of the CopG-linked Cluster (I) and the AbrB-linked clusters
(II, III, and V) in pathogenic bacteria as well as the disease caused. The presence of
homologs of the CopG-linked toxins was detected in all the pathogenic strains of
<italic>Clostridia</italic> spp. and <italic>Listeria monocytogenes</italic>.
AbrB-linked toxins are found in all the pathological strains of
<italic>Enterobacter</italic> spp., <italic>Klebsiella pneumoniae, Salmonella
enterica</italic> and <italic>Neisseria meningitidis.</italic> A set of toxins with no
identifiable N-terminal domains were found in <italic>Staphylococcus aureus</italic> in
Cluster I. <table-wrap id="evt175-T1" position="float"><label>T<sc>able</sc>1</label><caption><p>Pathogenic Organisms Containing MazF Type TA Modules and the
Diseases Caused by Them </p></caption><table frame="hsides" rules="groups"><thead align="left"><tr><th rowspan="1" colspan="1"></th><th rowspan="1" colspan="1">Organism</th><th rowspan="1" colspan="1">Disease</th></tr></thead><tbody align="left"><tr><td rowspan="4" colspan="1"><bold>Cluster I TA modules</bold></td><td align="left" rowspan="1" colspan="1"><italic>C. perfringens</italic></td><td align="left" rowspan="1" colspan="1">Food poisoning and gas gangrene</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Li. monocytogenes</italic></td><td align="left" rowspan="1" colspan="1">Listeriosis</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>St. aureus</italic></td><td align="left" rowspan="1" colspan="1">Nosocomial infections</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>C. botulinum</italic></td><td align="left" rowspan="1" colspan="1">Botulism</td></tr><tr><td rowspan="4" colspan="1"><bold>Clusters II, III, and V TA modules</bold></td><td align="left" rowspan="1" colspan="1"><italic>Enterobacter sp.</italic></td><td align="left" rowspan="1" colspan="1">Shigellosis</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>K. pneumoniae</italic></td><td align="left" rowspan="1" colspan="1">Kliebsella pneumonia</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>S. enterica</italic></td><td align="left" rowspan="1" colspan="1">Enteric fever</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>N. meningitidis</italic></td><td align="left" rowspan="1" colspan="1">Meningitis</td></tr></tbody></table></table-wrap></p></sec><sec><title>Phylogenetic Analysis</title><p>Molecular phylogenetic analysis is now a well-established computational technique for
studying evolutionary relationships between gene families and genomes. Traditionally,
phylogenetic analysis has been used to infer shared ancestry and divergence as well as
gene speciation or duplication events. It has also been used to predict functions of novel
proteins. However, in view of the large number of bacterial genomes now available, it is
now possible to organize different protein families into trees that enable us to analyze
these groups in the context of important biological information like interaction partners,
function, etc. This will also help to elucidate evolutionary phenomenon like coevolution,
gene/domain shuffling, linkage, etc.</p><p>Here, we carried out the phylogenetic analysis of the entire set of MazF family of toxins
and analyzed the tree in context of their cognate antitoxins and the genomic location. The
MrBayes tree depicting the interrelationships between the members of the MazF toxin family
is shown in <xref ref-type="fig" rid="evt175-F3">figure 3</xref>. As shown in <xref ref-type="fig" rid="evt175-F3">figure 3</xref>, the toxins linked to the three dominant
types of antitoxins, namely AbrB, CopG, and Duf(3018), cluster together on distinct
branches of the tree. Blastclust results show that chromosomal TA modules have better
conservation among the toxins in comparison to plasmid borne modules. However, the plasmid
borne TA modules are interspersed between the chromosomal sequences on the tree,
indicating a possible gene transfer between plasmid and chromosomal TA modules from
divergent species. In the AbrB group, a larger number of plasmid borne TAs are
interspersed between the chromosomal toxins. The single reported TA module from the
<italic>L</italic>. <italic>johnsonii</italic> prophage LJ771 may be integrated into the
prophage genome from the bacterial host. Though another toxin linked to AbrB antitoxin is
present in <italic>L. johnsonii</italic>, the closest branch neighbor for the prophage
toxin on the inferred tree is a TA module occurring in the <italic>L. salivarius</italic>plasmid. <fig id="evt175-F3" position="float"><label>F<sc>ig</sc>. 3.—</label><caption><p>MrBayes
computed tree of the MazF family toxins in two gene operons. All the branches had
bootstrap values > 50%. The Abrb, CopG, Duf(3018), and winged HTH-linked
toxin clusters are highlighted in red, blue, green, and yellow, respectively. In
each highlighted cluster, branches and taxon labels are colored by type of antitoxin
as described. However, taxon labels corresponding to plasmid borne TAs in each
cluster are colored gray. Taxon labels of toxins for which the antitoxins could not
be annotated are highlighted in pink.</p></caption><graphic xlink:href="evt175f3p"></graphic></fig></p><p>The two toxins coupled with RelB-type antitoxins were present as a long branch in the
midst of the AbrB-linked toxins. However, another RelB-linked toxin clustered closely with
CopG-linked toxins. Thus, evolutionary links are indicated between RelBE family with both
these subgroups of MazF toxins. The Duf(2281), Duf(104), with Phd antitoxin linked toxins
did not occur in any known clusters. The winged HTH-linked toxins occurring in Archaea
make a small separate cluster close to the root, having both plasmid and chromosomal
members. The toxins linked to antitoxins for which the toxin fold could not be determined
were found predominantly interspersed with CopG family toxins. These may represent similar
antitoxin N-terminal fold as the CopG cluster in which they occur, but could not be
detected by the fold prediction methods used.</p></sec><sec><title>Evolutionary Network of MazF Family TA Modules</title><p>Network modeling of the MazF family toxins was carried out as described. The resulting
network is shown in <xref ref-type="fig" rid="evt175-F4">figure 4</xref>. The Neighbor-Net
analysis results support the clustering of different subfamilies of MazF subfamilies, as
previously observed in the phylogenetic tree. In cases of higher frequencies of internode
gene transfer, the calculated splits give rise to a reticulate network of nodes that helps
to detect conflicting phylogenies suggestive of horizontal gene transfer. The figure shows
that reticulations are dense within each subfamily. A comparatively lower number of
reticulations join distinct subfamilies. The MazF toxins linked to AbrB-type antitoxins
form one large, segmented, reticulated cluster with chromosomal toxins (red squares)
frequently interspersed by plasmid-borne toxins (gray circles). The chromosomal
CopG-linked and Duf-linked toxin subfamilies also occur in clearly demarcated and densely
reticulated zones. It was observed that the AbrB-linked toxins occur in several
reticulated clusters near the origin as well as further away from it, indicating several
ancestral as well as recent instances of divergence. In contrast, the CopG-linked and the
Duf-linked toxin branches clustered and were linked by edges near the end of the branch.
This suggests that there was more recent divergence in these two groups as compared to the
AbrB-linked toxins. <fig id="evt175-F4" position="float"><label>F<sc>ig</sc>.
4.—</label><caption><p>Split network of MazF-type toxins. Red square:
AbrB-linked chromosomal toxins; gray circles: AbrB-linked plasmid-borne toxins; dark
blue squares: CopG-linked chromosomal toxins; dark green squares: Duf-linked
chromosomal toxins; light green circles: Duf-linked plasmid-borne toxins; dark pink:
Chromosomal toxin sequences with hypothetical antitoxins; light pink: Plasmid-borne
toxin sequences with hypothetical antitoxins; orange squares: Winged HTH-linked
chromosomal toxins; yellow circles: Winged HTH-linked plasmid-borne
toxins.</p></caption><graphic xlink:href="evt175f4p"></graphic></fig></p></sec><sec><title>Motif Discovery</title><p>The representative sequence clusters as described before were analyzed for the presence
of conserved motifs upstream of their operon. The significance of the motif was confirmed
by the fact that it was not found upon using the shuffled upstream sequences. A gapped two
block gapped motif consisting of a twelve base inexact repeat separated was found in the
AbrB set and is shown in <xref ref-type="fig" rid="evt175-F5">figure
5</xref><italic>a</italic>. The motif was found with high significance in upstream
elements of 76 out of the 79 representative AbrB antitoxin-containing TA modules. Four
consecutive bases of the ten base repeat form a symmetrical palindrome. In a majority of
the sequences, the detected motif was present directly upstream of the start site, either
on the same or the complementary strand. Detection of the palindrome on the complementary
strand is equivalent to an inversion of the recognition sequence in the region upstream of
the TA module and may correspond to varying modes of structural recognition by the
regulatory proteins. <fig id="evt175-F5" position="float"><label>F<sc>ig</sc>.
5.—</label><caption><p>Graphical representation of sequence motifs detected in
the upstream DNA sequence of TA operons of each subfamily. (<italic>a</italic>) Logo
of motif detected upstream of AbrB-linked TA modules. An inexact ten base repeat is
underlined in black. The purple lines depict the presence of a four-base palindrome
in the motif. (<italic>b</italic>) Logo of motif upstream of CopG-linked TA modules
(underlined in red). (<italic>c</italic>) Logo of motif upstream of Duf(3018)-linked
TA modules. The 12 base motif is underlined in red and the six base palindromic
sequence is shown by purple lines.</p></caption><graphic xlink:href="evt175f5p"></graphic></fig></p><p>The significant motif found in the upstream sequences of the CopG-linked toxins is shown
in <xref ref-type="fig" rid="evt175-F5">figure 5</xref><italic>b</italic>. A single-block
ungapped motif was also identified with high significance in 21 out of 25 of the Duf(3018)
antitoxin-linked sequences. This motif consists of a six base palindrome on the same
strand as shown in <xref ref-type="fig" rid="evt175-F5">figure 5</xref><italic>c</italic>(underlined bases). In most instances, the six base palindromic motif occurs directly
upstream of the TA operon, and is present either on the same or the complementary strand.
Thus, TA modules with distinct type of antitoxins share unique motifs in their upstream
sequences. These may represent regulatory motifs.</p></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>In the present work, we have identified 263 TA modules after discarding the singlet or
“bad” toxins (<xref ref-type="bibr" rid="evt175-B35">Guglielmini et al.
2008</xref>). It has been reasoned that these TA modules lack an antitoxin and thus, may
also lack the toxic ribonuclease activity. However, the possibility that the antitoxin gene
may be present elsewhere in the genome or on extrachromosomal elements and that its proteic
product may be supplied “from outside” exists (<xref ref-type="bibr" rid="evt175-B64">Nariya and Inouye 2008</xref>) and needs to be explored. The presence of
67 predicted TA operons having more than two genes further raises interesting questions
about the functionality of the additional genes in the operon and the mechanism by which
these mosaic operons are formed. According to earlier reports, the MazF family toxins were
thought to be confined to the bacterial domain (<xref ref-type="bibr" rid="evt175-B68">Pandey 2005</xref>). However, our results are in line with a later study reporting the
presence of potential MazF toxins in Archaea (<xref ref-type="bibr" rid="evt175-B35">Guglielmini et al. 2008</xref>).</p><p>The CcdB-type toxins are grouped in the same superfamily as MazF as they share the same
fold, even though the biological function/cellular targets are different (<xref ref-type="bibr" rid="evt175-B6">Anantharaman and Aravind 2003</xref>). While CcdB targets
DNA gyrase and causes inhibition of DNA replication, MazF inhibits protein synthesis due to
its ribonuclease activity. Previous sequence analysis studies have also not reported any
significant sequence homology between MazF and CcdB-type toxins (<xref ref-type="bibr" rid="evt175-B35">Guglielmini et al. 2008</xref>). The two CcdB-like sequences identified
in our database search did not share sufficient sequence similarity with the MazF family
toxins and were thus discarded from the data set. However, an approach based on structural
similarity may be able to unify the MazF and CcdB families.</p><sec><title>MazF Family Toxins Occur with Four Main Types of Antitoxins. Individual TAs Link MazF
Evolutionarily to Other Families</title><p>Four main types of antitoxins were identified to occur with the MazF family toxins in the
present study namely, AbrB/SpoVT, CopG, Duf(3018), and winged-HTH. The single antitoxin
proteins from the Members of Duf(104) (COG2880) have previously been reported as putative
antitoxins (<xref ref-type="bibr" rid="evt175-B56">Makarova et al. 2009</xref>). Structure
of one of the members of Duf(104) family AF2212 from <italic>Archaeglobus
fulgidus</italic> is known (PDB ID: 2NWT). The Structural Classification of Proteins
(SCOP) classification of 2NWT shows that it shares the double split β-barrel fold
with AbrB/SpovT antitoxins. Another antitoxin belonged to Duf(2281) (COG 3553), a domain
family of bacterial proteins. The nuclear magnetic resonance structure of one member with
Duf(2281) domain from <italic>Pseudomonas aeruginosa</italic> has been determined (PDB
ID:2JPI). Comparison of 2JPI structure with representative structures in PDB shows that it
has high similarity with the structure of transcription initiation factor, Transcription
Factor IID (PDB ID: 1QNA).</p><p>A TA module having a RelE toxin combined with a MazE-type antitoxin has been identified
previously (<xref ref-type="bibr" rid="evt175-B73">Schmidt et al. 2007</xref>). Similarly,
in our data set, a single TA module having a Phd/YefM-type antitoxin (Toxin GI: 256827703)
links the MazF family with the FIC domain Doc family (<xref ref-type="bibr" rid="evt175-B25">Garcia-Pino et al. 2008</xref>). These results are in agreement with
previous studies on putative antitoxin partners for MazF-type toxins (<xref ref-type="bibr" rid="evt175-B56">Makarova et al. 2009</xref>). A recent comprehensive
study catalogs Toxin-Antitoxin (TA) partners by sequence similarity as well as
“Guilt by association” (GA) technique in prokaryotic genomes. In this study,
MazF and CcdB was treated as one superfamily, and five categories of antitoxin partners
were identified, of which the GA category constitutes a large group (<xref ref-type="bibr" rid="evt175-B49">Leplae et al. 2011</xref>). Thus, while MazE and CcdA-type antitoxins
are well established as partners for the MazF family, the Duf domain linked TAs as found
in the present study may have formed part of the GA category. They have not been reported
or validated previously. Validation of the proposed TA function of the Duf-linked proteins
is required to establish them as members of the MazEF family.</p><p>The observed high sequence conservation of the chromosomal toxins within a subfamily
agrees with previous works that view the integration of plasmid TA operons into
chromosomal locations as further continuation of their “selfish” behavior
(<xref ref-type="bibr" rid="evt175-B49">Leplae et al. 2011</xref>). Chromosomal
integration of the TA modules limits their genetic mobility by horizontal gene transfer
and may underlie the observation that chromosomal TA modules, which are mostly inherited
by vertical descent, and have higher sequence conservation as compared to plasmid-borne
modules.</p></sec><sec><title>A Conserved Pocket in a Multibinding Site of MazF Family Toxins Is a Potential
Drug-Binding Hotspot</title><p>Targeting protein–protein interactions (PPIs) for development of therapeutic
molecules poses the problem of tethering a small molecule to a large, featureless protein
surface. However, studies have shown that the regions involved in protein–protein
interface formation are inherently flexible or intrinsically disordered, which may allow
small molecules to diffuse in and disrupt the complex (<xref ref-type="bibr" rid="evt175-B82">Thangudu et al. 2012</xref>). Successful targeting of PPIs is also made
possible due to the ability of the small molecules to bind to a small fraction of the
residues contributing to the interface, also known as binding hotspots, which contribute
the most to the free energy of interaction. Further direction to the success of the
approach is promised by the recognition that, while one of the protein surfaces is
disordered, flat, and featureless, the other consists of one or a series of well-defined
pockets. These pockets form multibinding sites and are highly conserved (<xref ref-type="bibr" rid="evt175-B42">Jubb et al. 2012</xref>).</p><p>The MazF family TA systems are proposed to have a conserved mode of binding to the
C-terminal of their antitoxin through Site 1 and Site 2 (<xref ref-type="bibr" rid="evt175-B43">Kamada et al. 2003</xref>). YdcE of <italic>B. subtilis</italic> and
<italic>B. anthracis</italic> toxin MoxT have high sequence and structural similarity
with MazF. Various peptides based on the sequence of MoxX and MazE were capable of
disrupting the MoxXT interaction (<xref ref-type="bibr" rid="evt175-B3">Agarwal et al.
2010</xref>). A rationally designed peptide could disrupt the MoxXT interaction by
42% at micromolar concentration (<xref ref-type="bibr" rid="evt175-B14">Chopra et
al. 2011</xref>). Thus, it has been shown that some molecules are able to disrupt the
interactions of the MazF family TA systems.</p><p>At Site 1 of the MazEF complex, the disordered, extended and essentially featureless
C-terminal half of the antitoxin binds via a hydrophobic group to a pocket on the
corresponding surface of the toxin. Sequence analysis has shown that Site 1 is also highly
conserved among the MazF family TA modules. Also, disruption of Site 2 TA interactions by
a small molecule or peptide is expected to cause a decrease in the toxic ribonuclease
activity due to proximity of Site 2 to the catalytic site (<xref ref-type="bibr" rid="evt175-B3">Agarwal et al. 2010</xref>). In view of the contribution of Site 1 to
binding, nature of the interface with the disordered antitoxin C-terminus (<xref ref-type="bibr" rid="evt175-B43">Kamada et al. 2003</xref>), predicted
sequence/structure conservation, and possible modulation by a variety of peptides, it
represents a multibinding site for further drug design and discovery efforts.</p><p>As proposed on the basis of modeling studies, Site 1 may also be targeted for disruption
by the bacteria’s own quorum sensing mechanisms, resulting in enhanced ribonuclease
activity of the toxin (<xref ref-type="bibr" rid="evt175-B9">Belitsky et al. 2011</xref>).
The action of MazEF mediated quorum sensing Extracellular Death Factor (EDF) is yet to be
confirmed in independent experiments from other laboratories (<xref ref-type="bibr" rid="evt175-B83">Van Melderen 2010</xref>; <xref ref-type="bibr" rid="evt175-B36">Guglielmini and Van Melderen 2011</xref>; <xref ref-type="bibr" rid="evt175-B39">Hayes
and Van Melderen 2011</xref>). However, a recent report in respect of EDF-like peptides
is that MazEF mediated cell death in <italic>E. coli</italic> can also be caused by quorum
sensing peptides obtained from the supernatant of <italic>B. subtilis</italic> and
<italic>P. aeruginosa</italic> (<xref ref-type="bibr" rid="evt175-B48">Kumar et al.
2013</xref>). While the mediation of programmed cell death by TA modules remains
contested, support for the increase in ribonuclease activity of MazF is gained from
cross-neutralization experiments between Kis toxin and the antitoxin of CcdAB, which has a
similar structural and functional organization as found in the MazEF system. The
C-terminal domain of CcdA is found to be sufficient for CcdB inhibition, as has been
established for the MazEF family toxins. It was shown that CcdA binding to Kis was
accompanied by an increase in ribonuclease activity (<xref ref-type="bibr" rid="evt175-B77">Smith et al. 2012</xref>). CcdB and Kid share a similar fold even in
the absence of apparent sequence similarity. However, Site 1, disordered loop and mode of
toxin binding are similar to that of MazF and Kid. Therefore, Site 1 is indicated as the
possible site mediating the interaction of CcdA with Kid. This is also in agreement with
the observation that the physical interaction of CcdA with Kid did not cause a decrease in
ribonuclease activity. The ribonuclease catalytic site (near to Site 2) could function
unhindered by the presence of antitoxin at this site.</p><p>The structures of CcdB and Kid show the presence of a hydrophobic tyrosine that
stabilizes their closed-loop conformation. However, in the antitoxin-bound disordered-loop
conformation of toxin, the F17 that is structurally analogous to the CcdB and Kid tyrosine
residues is partially disordered. Therefore, the invariant hydrophobic W73 of MazE may
compete with the MazF F17 for interactions with the tryptophan-binding pocket of MazF,
causing the closed loop to open up during TA complex formation. Specific occupation of the
tryptophan-binding pocket would then serve as a mechanism of antidote recognition. The
structure of the MazEF complex shows that the MazF-bound MazE residues at Site 1 (residues
67–73) adopt an extended β-strand like structure. Thus, the presence of the
binding hotspot at Site 1 also suggests the mechanism of specific recognition of the
extended, disordered, and essentially featureless C-terminal residues 67–73 of MazE
at Site 1 of MazF. The recognition of the C-terminal residues of MazE by MazF at Site 1 is
additionally aided by the occupation of another small hydrophobic pocket on the surface of
MazF by His68 of MazE. Also, Q77 of the A chain of MazF makes a bidentate interaction with
the backbone of residues E69 and I71 of MazE (<xref ref-type="bibr" rid="evt175-B43">Kamada et al. 2003</xref>).</p></sec><sec><title>Roaming of Catalytic Residues in the MazF Family Toxins</title><p>Sequence analysis of the catalytic site residues showed that these differ in Clusters
I–V. Combined sequence and structure analysis has been used to propose and validate
putative toxin active site residues on the basis of spatial proximity to the conserved
active site residue (<xref ref-type="bibr" rid="evt175-B3">Agarwal et al. 2010</xref>).
The phenomenon of occurrence of distributive active site residues that are placed in
different positions in the binding pocket has been termed as the roaming of catalytic
residues and is also seen in the RelB family of toxins (<xref ref-type="bibr" rid="evt175-B10">Blower et al. 2011</xref>).</p></sec><sec><title>MazF Family TA Modules with Conserved Sequence Features in Pathological Strains of
Bacteria</title><p>It has been noted that the occurrence of TA modules is variable in different strains of
the same organism (<xref ref-type="bibr" rid="evt175-B86">Williams and Hergenrother
2012</xref>). However, in our study, the presence of homologs of the toxins was detected
in all pathogenic strains of the bacteria. Specifically, the presence and conservation of
toxins linked to short antitoxins lacking an identifiable N-terminal domain in
<italic>S</italic><italic>t</italic>. <italic>aureus</italic> is of interest in view of
the role of this organism in nosocomial infections and the prevalence of multiple drug
resistance in these bacteria (<xref ref-type="bibr" rid="evt175-B76">Shopsin and
Kreiswirth 2001</xref>; <xref ref-type="bibr" rid="evt175-B37">Haddadin et al.
2002</xref>). One short TA module has been validated as a functional TA pair in
<italic>S</italic><italic>t</italic><italic>. aureus</italic> (<xref ref-type="bibr" rid="evt175-B24">Fu et al. 2007</xref>). Novel antibiotic strategies are also important
in <italic>S</italic><italic>t</italic><italic>. aureus</italic> as the community
associated nature of the drug-resistant strains raises the possibility of its rapid and
widespread colonization in the milieu outside the hospital (<xref ref-type="bibr" rid="evt175-B26">Gardam 2000</xref>; <xref ref-type="bibr" rid="evt175-B61">Miller and
Kaplan 2009</xref>).</p></sec><sec><title>Linkage and Shuffling in the MazF Family TA Modules</title><p>The clustering of the different subfamilies on the branches of the tree indicates the
tight evolutionary linkage of each subfamily of TA modules. Thus, the two-gene TA operon
is predominantly transferred as a whole, a situation that is perhaps intensified by the
addictive nature of the TA interaction. However, as toxin singlets and TA operons
consisting of more than two genes were not included in the current analysis, the
possibility of their forming a dynamic continuum with the two-gene member TA operons
remains to be explored.</p><p>The presence of different branches of MazF-type toxins linked to distinct antitoxins
indicates that such prokaryotic TA systems may have arisen by gene shuffling wherein; the
toxin has acquired a different antitoxin partner during evolution. A new antitoxin gene is
incorporated from a distant source accompanied by the loss of the previous antitoxin gene
by xenologous displacement (<xref ref-type="bibr" rid="evt175-B67">Omelchenko et al.
2003</xref>). Such xenologous displacement events are few in the two-membered MazEF
family TA operons, and can occur either in plasmid or chromosomal TA modules as observed
at the interface of each distinct antitoxin-linked subfamily. The probable mechanism for
xenologous displacement is the slow accumulation of mutations at the TA interface. Some of
the mutations may render the toxin dysfunctional, leading to decay of the TA system as
previously studied for the ccd<sub>O157</sub> system. Inactivation of the toxin is
considered the first and safer (nonlethal) step of TA systems degradation. Subsequent
inactivation of the antitoxin may also occur. Study of the ccd<sub>O157</sub> homologs
revealed that 61% of the TA systems possessed both active components, 21%
had an inactive toxin, and 2% had both inactive components. Additionally, the
plasmid-borne TA modules were found to be under higher selection pressure to retain their
function (<xref ref-type="bibr" rid="evt175-B62">Mine et al. 2009</xref>).</p><p>The ability of toxin to successfully pair with different types of antitoxin can be
attributed to the modular structure of antitoxin. The N-terminus of the antitoxin, which
comprises diverse DNA-binding structural motifs, is mainly involved in mediating
transcriptional regulation of the operon by binding to its own upstream DNA. The antitoxin
binds to the toxin via its C-terminus, which is unstructured or partially unstructured in
toxin-free state (<xref ref-type="bibr" rid="evt175-B65">Nieto et al. 2007</xref>) but
adopts an extended structure on binding to the toxin. Especially, the C-terminal stretch
of the antitoxin comprising Site 1 and Site 2 can be modulated at distinct sequence
positions by selected mutations at the TA interface. Thus, the toxin can exist in
combination with different cognate antitoxin partners. The two main sites of interaction
between TA, namely Sites 1 and Site 2, show a unique pattern of conservation of residues
in the three different subfamilies in the present study. Therefore, gene shuffling is
accompanied by simultaneous changes in the TA interaction sites in order that the
biological complex formation is maintained. Across the entire MazF family, Site 1 shows
higher conservation across species and antitoxin types as compared to Site 2. Site 2
coincides with the catalytic site and the interface of the biologically required toxin
dimer in MazF family toxins. Yet, Site 2 mutations are preferred by the TA pair in spite
of the possible loss of function incurred by making changes at this site. There may be two
mechanisms to ensure antitoxin recognition at Site 1 of the toxin. Firstly, as apparent
from sequence analysis, large changes at Site 1 of toxin are resisted as they would lead
to loss of antitoxin recognition and cause unchained action of the toxin. Secondly, the
sequence variability of the antitoxin at the C-terminal end suggests that a large number
of peptides with varying sequence and structure can be recognized by the toxin at the
invariant Site 1 of MazF family toxins.</p><p>Though it has often been shown that cognate antitoxins are unique to their antitoxins
even when the antitoxins are from the same family, instances of cross neutralization
between TA pairs have been reported (<xref ref-type="bibr" rid="evt175-B34">Grady and
Hayes 2003</xref>; <xref ref-type="bibr" rid="evt175-B65">Nieto et al. 2007</xref>;
<xref ref-type="bibr" rid="evt175-B70">Ramage et al. 2009</xref>; <xref ref-type="bibr" rid="evt175-B22">Fiebig et al. 2010</xref>). Instances of cross-neutralization of toxins
by homologous antitoxins have been recorded within the CcdB family, which shares
structural similarity with MazF (<xref ref-type="bibr" rid="evt175-B71">Santos-Sierra et
al. 2002</xref>; <xref ref-type="bibr" rid="evt175-B85">Wilbaux et al. 2007</xref>).
Point mutations did not adversely affect the ability of a CcdA antitoxin to neutralize its
toxin. However, a frameshift mutation in CcdA yielded an inactive antitoxin (<xref ref-type="bibr" rid="evt175-B62">Mine et al. 2009</xref>). This indicates that mutations
at the TA interface are carefully modulated out of sheer necessity of maintaining the TA
interface. Functional interactions between ChpB and ParD, two homologous systems in
<italic>E. coli</italic>, have also been reported (<xref ref-type="bibr" rid="evt175-B72">Santos Sierra et al. 1998</xref>). In the MazF family TA modules,
functional interactions of MazE and Kid system caused the neutralization of the toxic
activity of Kid with an altered stoichiometry of interaction as compared to the MazEF
complex (<xref ref-type="bibr" rid="evt175-B45">Kamphuis, Monti, van den Heuvel,
Santos-Sierra, et al. 2007</xref>). Variation in TA stoichiometry may be another
mechanism for accommodating changes at the TA interface. Complex formation has also been
demonstrated for Kis with CcdB, though with a lower efficiency of neutralization as
compared to CcdA,and may suggest their common evolutionary origin (<xref ref-type="bibr" rid="evt175-B77">Smith et al. 2012</xref>). Functional and physical interactions of the
MazEF and the VapBC TA systems have also been reported (<xref ref-type="bibr" rid="evt175-B88">Zhu et al. 2010</xref>), though the structural basis of such
interactions is yet unknown.</p></sec><sec><title>Location and Mobility of the Three Toxin Subfamilies</title><p>Our results indicate the passage of TA modules between plasmid and chromosomal DNA of
different bacteria akin to that established for antibiotic resistance genes (<xref ref-type="bibr" rid="evt175-B66">Ochman et al. 2000</xref>) and secondary metabolites.
As the AbrB-type toxins are often accompanied by transposon-like elements flanking the
AbrB-type modules (<xref ref-type="bibr" rid="evt175-B27">Gerdes et al. 2005</xref>), this
may account for the widespread occurrence of these TA modules on both plasmids as well as
chromosomes. Plasmid-chromosomal transfer may also occur by means of conjugative plasmids,
that are known to mediate transfer of large fragments of bacterial chromosomes (<xref ref-type="bibr" rid="evt175-B87">Wollman et al. 1956</xref>).The TA modules are often
passengers of the bacterial mobilome, which enjoys horizontal mobility by means of
bacteriophages, plasmids, and transposable elements. Horizontal gene transfer between the
mobilome and chromosome may be mediated by specialized Gene Transfer Agents. Phylogenetic
studies have also shown that replicon fusion is a common event during prokaryotic
evolution, further blurring the boundaries between the plasmid and the chromosome (<xref ref-type="bibr" rid="evt175-B47">Koonin and Wolf 2008</xref>). TA modules have
previously been included within the resistome domain of the bacterial mobilome (<xref ref-type="bibr" rid="evt175-B56">Makarova et al. 2009</xref>), where the term resistome
was expanded to include genes responsible for resistance to stress caused by factors other
than antibiotics. Indeed, the role of the TA systems in making their host cells resistant
to stress by and inducing persister cell formation has been well documented (<xref ref-type="bibr" rid="evt175-B41">Jayaraman 2008</xref>; <xref ref-type="bibr" rid="evt175-B28">Gerdes and Maisonneuve 2012</xref>). The occurrence of antibiotic
tolerance and persistence is also found to be high in cells overproducing MazF family
ribonuclease toxins (<xref ref-type="bibr" rid="evt175-B55">Maisonneuve et al.
2011</xref>). The frequent transfer of MazF family TA modules between plasmid and
chromosomal locations as shown in the present work establishes these selfish elements to
be part of the resistome. Also, though the TA module may be “domesticated”
into performing the normal cellular functions of the cell by integration into the
chromosome, it may also revert to a purely plasmid-borne selfish nature.</p><p>The phylogenetic tree of the MazF family TA modules shows that the single RelB
antitoxin-containing module is closely evolutionarily linked to the AbrB-linked toxins,
while the archaeal-winged HTH-containing modules form a separate cluster near the root of
the tree. Due to their notable structural similarity, the winged HTH motif is also an
elaboration of the structural scaffold present in HTH. The HTH domain is thought to be an
ancient conserved domain found in the last universal common ancestor of all extant life
forms (<xref ref-type="bibr" rid="evt175-B7">Aravind et al. 2005</xref>). Therefore, the
winged HTH-linked archaeal MazF Toxins may either represent the putative ancestral
sequences for the MazF family toxins or can be attributed to archaeobacterial gene
transfer (<xref ref-type="bibr" rid="evt175-B47">Koonin and Wolf 2008</xref>).</p></sec><sec><title>Bacterial Genome Stabilization by Transfer of a TA Module via a Bacteriophage</title><p>The bacterial prophage is an important agent of change during prokaryotic evolution.
Bacteriophages and plasmids often integrate into chromosomes reversibly or irreversibly.
Reversible integration can mobilize chromosomal genes, while irreversible integration
serves to domesticate the mobile elements into resident genes (<xref ref-type="bibr" rid="evt175-B66">Ochman et al. 2000</xref>; <xref ref-type="bibr" rid="evt175-B80">Sundin 2007</xref>). The LJ771-borne TA module helps in the stabilization of the
prophage (<xref ref-type="bibr" rid="evt175-B19">Denou et al. 2008</xref>). Thus, both the
TA module and the prophage become incurably integrated in the chromosome of <italic>L.
johnsonii.</italic> Once the TA operon is incorporated in the prophage, the selfish and
addictive nature of the TA module ensures its own propagation.</p></sec><sec><title>AbrB-Linked Toxins Form a Large Hub of Gene Transfer in an Evolutionary Network of
MazF Family Toxins</title><p>In view of the pervasive horizontal gene transfer in prokaryotic genomes, the validity of
tree thinking is questionable. A more accurate representation can be achieved with the
help of directed network graphs, with weighted edges corresponding to the intensity of the
flow of genes. The MazF family TA evolution can be conceptualized as a network graph with
the chromosomal modules at the center, plasmid borne modules at the periphery, and
distinct zones of toxins, representing the different antitoxin-linked subfamilies. The
concept of distinct horizontal gene transfer highways and hubs in such a graph has already
been introduced (<xref ref-type="bibr" rid="evt175-B31">Gogarten and Townsend
2005</xref>). Thus, the highly mobile AbrB-linked toxins occurring in superintegrons
(<xref ref-type="bibr" rid="evt175-B27">Gerdes et al. 2005</xref>) form a hub of gene
transfer between replicons as reflected in the widespread occurrence of the AbrB-linked TA
subfamily in divergent bacteria. The presence of specific mechanisms that permit such
mobility in the chromosomal CopG-linked and Duf-linked toxins remains to be
investigated.</p></sec><sec><title>Unique DNA Motifs Are Present in the Upstream Region of Each Toxin Subfamily</title><p>Many of the known TA modules are negatively autoregulated at the level of transcription.
The autoregulation is mediated by binding of the antitoxin to the promoter region and is
enhanced in presence of the toxin. Several antitoxins have been shown to bind to
characteristic palindromic repeats of DNA in their upstream ORF (<xref ref-type="bibr" rid="evt175-B54">Madl et al. 2006</xref>; <xref ref-type="bibr" rid="evt175-B60">Mattison et al. 2006</xref>; <xref ref-type="bibr" rid="evt175-B74">Schreiter et al.
2006</xref>; <xref ref-type="bibr" rid="evt175-B51">Li et al. 2008</xref>). Further, the
number, length, sequence and distance between the palindromes can modulate the strength of
the protein–DNA interaction. We found that unique motifs were present in the
upstream region of the subfamilies identified.</p><p>As the promoter region has been identified in both the MazEF and the Kis Kid TA modules,
these sequences were scanned for the presence of the pattern found. Though the binding
site identified by footprinting analysis is much larger, the MazEF module can be expressed
by two promoters, namely P2 and P3 occurring at a distance of 13 base pairs (<xref ref-type="bibr" rid="evt175-B59">Marianovsky et al. 2001</xref>). Of these, P2, the
upstream promoter is active in exponentially growing cells and has ten times the activity
of the P3 promoter. Autoregulation by MazEF is attributed to the presence of an unusual
alternating palindrome in its upstream DNA. The alternating promoter consists of three
parts, namely a, b, and c. The middle a part is complementary to both the upstream c and
downstream b parts of the palindrome. The MazEF complex may bind to either the c or the b
arms of the palindrome, leading to inhibition of its own expression. The downstream b arm
also overlaps with the strong P2 promoter (<xref ref-type="bibr" rid="evt175-B59">Marianovsky et al. 2001</xref>). Examination of the sequence of the MazEF promoter
showed that the 10 base repeat containing a 4 base palindrome found by Glam2 is present
both in the c and the b arms of the MazEF palindrome. However, the region identified in
the motif finding by Glam2 corresponds to the downstream b arm of the palindrome. The Kis
Kid promoter also contains two repeats, I and II. Motif I contains a perfect palindrome,
whereas Motif II contains an imperfect palindrome. The binding of the Kid and Kis Kid
complex is tighter to promoter I as compared to promoter II (<xref ref-type="bibr" rid="evt175-B63">Monti et al. 2007</xref>). The region containing the motif shown in
<xref ref-type="fig" rid="evt175-F5">figure 5</xref><italic>a</italic> coincides with
the promoter I of the Kis Kid upstream DNA. Thus, in both MazEF and Kis Kid systems, the
promoter sequence established to be important for tight binding to the TA complex has been
recognized. The significance of such regulatory motifs has been probed in conjunction with
the varying stoichiometry of interaction in the Kis Kid module. It has been proposed that
the expression of the Kis Kid TA system is tightly controlled by the molar ratio of the
toxin and antitoxin, with additional complexity being provided by cooperative interactions
between the two promoter regions (<xref ref-type="bibr" rid="evt175-B63">Monti et al.
2007</xref>). A palindromic pattern or repeat is not obvious in the motif found upstream
of the CopG-linked toxins (<xref ref-type="fig" rid="evt175-F5">fig.
5</xref><italic>b</italic>). However, two regions of the upstream sequence consisting of
a long and a short palindrome have been established to be important for MoxXT binding in
<italic>B. anthracis</italic> (<xref ref-type="bibr" rid="evt175-B14">Chopra et al.
2011</xref>). The region recognized in MoxXT promoter by Glam2 contains the long
palindrome. Thus, the motifs conserved in the upstream region of the different subfamilies
of MazF family TA modules may correspond to regulatory sequences recognized by the
distinct N-terminal domain of the antitoxin in each subfamily. Conservation of specific
motifs in the upstream region of each TA subfamily is indicative of the fact that distinct
upstream regulatory elements of DNA form an evolutionary module along with the N-terminal
DNA-binding domain of the antitoxin. Though there is a strong correlation of the unique
motifs with each subfamily of MazF marked by unique N-terminal domain of the antitoxin,
there remains the possibility remains that the motifs identified may represent binding
sites for the TA complex, transcription factors or other cis-regulatory factors that
modulate the action of the TA modules.</p></sec></sec><sec sec-type="conclusions"><title>Conclusion</title><p>Toxin proteins of the MazF/PemK/Kid family are found to occur in a majority with three
types of cognate antitoxin partners, namely AbrB, CopG, and Duf(3018). The modular nature of
antitoxin protein wherein the N-terminus has the DNA-binding motif, whereas the extended
C-terminus binds to the toxin groove, facilitates such multiple antitoxin partners. AbrB was
found to be the most prevalent type of antitoxin in the MazF family. Five populated clusters
of MazF toxins were obtained and sequence analysis was used to identify an evolutionarily
conserved toxin-binding hotspot for antitoxin/peptides. As disruption of TA modules is a
potential drug target, a search for homologous two-member TA modules was made in all the
strains of the pathogenic organisms. MazF family TA modules were present in the pathological
strains of <italic>C. perfringens, L</italic><italic>i</italic><italic>. monocytogenes,
S</italic><italic>t</italic><italic>. aureus,</italic> and <italic>C. botulinum</italic>.
The specific effect of disruption of TA interaction in these organisms requires further
investigation.</p><p>Phylogenetic analysis showed that the toxin sequences with distinct cognate antitoxin
partners clustered together on the branches of the Bayes tree, indicating that the operons
are usually inherited as a whole. However, fewer gene shuffling events mediate the
acquisition of new antitoxins. Acquisition of a new antitoxin needs to be accompanied by
accumulation of mutations at the TA interface. The requirement for specificity in the
C-terminal domain is perhaps aided by the disordered, extended nature of the C-terminal
residues of the antitoxin sequence and structure plasticity. The plasmid borne TA modules
were found to be interspersed with the chromosomal TA modules of the same subfamily
presenting evidence of the exchange between TA modules of the plasmid and chromosomes of
different bacteria. Especially, the plasmid–chromosomal exchange is frequent in the
AbrB family of toxins, and may be attributed to the presence of upstream transposon-like
elements in AbrB family TA operons. Interchange of genes between Archaea, divergent
bacteria, closely related bacteria, the bacterial mobilome and the chromosome lends to the
view that these exist in dynamic and stable equilibrium with each other. Network modeling of
evolution in the MazF family TA modules shows that the chromosomal AbrB-linked TA modules
are present as hubs of gene transfer. We found the conservation of specific upstream motifs
in the AbrB and CopG family. The motifs identified could be correlated with the known
operator regions of AbrB and CopG family TA modules. Taken together, phylogenetic analysis
of MazF family TA modules unifies many observed phenomenon and presents a snapshot into the
mechanisms of bacterial evolution. Further, while the presence of a possible multibinding
hotspot outlines an opportunity for inducing TA disruption, the emergent role of TA modules
in persister cell formation indicate that caution is required while pursuing this path.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><p><ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">Supplementary tables S1</ext-link> and <ext-link ext-link-type="uri" xlink:href="http://gbe.oxfordjournals.org/lookup/suppl/doi:10.1093/gbe/evt175/-/DC1">figures S1–S3</ext-link> are available at <italic>Genome Biology and
Evolution</italic> online (<ext-link ext-link-type="uri" xlink:href="http://www.gbe.oxfordjournals.org/">http://www.gbe.oxfordjournals.org/</ext-link>).</p><supplementary-material id="PMC_1" content-type="local-data"><caption><title>Supplementary Data</title></caption><media mimetype="text" mime-subtype="html" xlink:href="supp_5_12_2268__index.html"></media><media xlink:role="associated-file" mimetype="application" mime-subtype="pdf" xlink:href="supp_evt175_supp_fig_and_table.pdf"></media></supplementary-material></sec></body><back><ack><title>Acknowledgments</title><p>This work was supported by <funding-source>Indian Council of Medical
Research</funding-source> grant number <award-id>64/5/2011-BMS</award-id>. S.B.
acknowledges Dr Aparup Das, NIMR, New Delhi, and Pooja Khurana, NSIT, for illuminating
discussions about the methodology and interpretation of the tree. N.C. acknowledges
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