Expansion of ribosomally produced natural products: A nitrile hydratase- and Nif11-related precursor family

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DOI: 10.1186/1741-7007-8-70 · Source: PubMed
Abstract
A new family of natural products has been described in which cysteine, serine and threonine from ribosomally-produced peptides are converted to thiazoles, oxazoles and methyloxazoles, respectively. These metabolites and their biosynthetic gene clusters are now referred to as thiazole/oxazole-modified microcins (TOMM). As exemplified by microcin B17 and streptolysin S, TOMM precursors contain an N-terminal leader sequence and C-terminal core peptide. The leader sequence contains binding sites for the posttranslational modifying enzymes which subsequently act upon the core peptide. TOMM peptides are small and highly variable, frequently missed by gene-finders and occasionally situated far from the thiazole/oxazole forming genes. Thus, locating a substrate for a particular TOMM pathway can be a challenging endeavor. Examination of candidate TOMM precursors has revealed a subclass with an uncharacteristically long leader sequence closely related to the enzyme nitrile hydratase. Members of this nitrile hydratase leader peptide (NHLP) family lack the metal-binding residues required for catalysis. Instead, NHLP sequences display the classic Gly-Gly cleavage motif and have C-terminal regions rich in heterocyclizable residues. The NHLP family exhibits a correlated species distribution and local clustering with an ABC transport system. This study also provides evidence that a separate family, annotated as Nif11 nitrogen-fixing proteins, can serve as natural product precursors (N11P), but not always of the TOMM variety. Indeed, a number of cyanobacterial genomes show extensive N11P paralogous expansion, such as Nostoc, Prochlorococcus and Cyanothece, which replace the TOMM cluster with lanthionine biosynthetic machinery. This study has united numerous TOMM gene clusters with their cognate substrates. These results suggest that two large protein families, the nitrile hydratases and Nif11, have been retailored for secondary metabolism. Precursors for TOMMs and lanthionine-containing peptides derived from larger proteins to which other functions are attributed, may be widespread. The functions of these natural products have yet to be elucidated, but it is probable that some will display valuable industrial or medical activities.
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RESEARCH ARTICLE
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Research article
Expansion of ribosomally produced natural
products: a nitrile hydratase- and Nif11-related
precursor family
Daniel H Haft*
1
, Malay Kumar Basu
1
and Douglas A Mitchell*
2,3,4
Abstract
Background: A new family of natural products has been described in which cysteine, serine and threonine from
ribosomally-produced peptides are converted to thiazoles, oxazoles and methyloxazoles, respectively. These
metabolites and their biosynthetic gene clusters are now referred to as thiazole/oxazole-modified microcins (TOMM).
As exemplified by microcin B17 and streptolysin S, TOMM precursors contain an N-terminal leader sequence and C-
terminal core peptide. The leader sequence contains binding sites for the posttranslational modifying enzymes which
subsequently act upon the core peptide. TOMM peptides are small and highly variable, frequently missed by gene-
finders and occasionally situated far from the thiazole/oxazole forming genes. Thus, locating a substrate for a particular
TOMM pathway can be a challenging endeavor.
Results: Examination of candidate TOMM precursors has revealed a subclass with an uncharacteristically long leader
sequence closely related to the enzyme nitrile hydratase. Members of this nitrile hydratase leader peptide (NHLP)
family lack the metal-binding residues required for catalysis. Instead, NHLP sequences display the classic Gly-Gly
cleavage motif and have C-terminal regions rich in heterocyclizable residues. The NHLP family exhibits a correlated
species distribution and local clustering with an ABC transport system. This study also provides evidence that a
separate family, annotated as Nif11 ni
trogen-fixing proteins, can serve as natural product precursors (N11P), but not
always of the TOMM variety. Indeed, a number of cyanobacterial genomes show extensive N11P paralogous
expansion, such as Nostoc, Prochlorococcus and Cyanothece, which replace the TOMM cluster with lanthionine
biosynthetic machinery.
Conclusions: This study has united numerous TOMM gene clusters with their cognate substrates. These results
suggest that two large protein families, the nitrile hydratases and Nif11, have been retailored for secondary
metabolism. Precursors for TOMMs and lanthionine-containing peptides derived from larger proteins to which other
functions are attributed, may be widespread. The functions of these natural products have yet to be elucidated, but it is
probable that some will display valuable industrial or medical activities.
Background
Bacteriocins are polypeptide-based natural products of
ribosomal origin, usually functioning as antibiotics toxic
to rival strains or species of bacteria [1]. Peptide products
resembling the bacteriocins in their size, precursor
sequence, posttranslational modifications and co-cluster-
ing with maturation enzymes occasionally prove to have a
signalling function or other non-antibiotic activity [2].
Collectively, these products represent a large reservoir of
molecules with vast potential. Bacteriocin production
and resistance mechanisms are, without question, major
contributors to microbial ecology dynamics. Despite
decades of research, including extensive work on low
molecular weight bacteriocins (microcins), these pro-
cesses are little understood. The small size and unusual
amino acid composition of microcin precursor peptides
hinder even the recognition of the open reading frame
(ORF) as the coding region of a real gene [3,4]. Further-
* Correspondence: DHaft@jcvi.org, douglasm@illinois.edu
1
The J Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850,
USA
2
Department of Chemistry, University of Illinois at Urbana-Champaign,
Urbana, IL 61801, USA
Full list of author information is available at the end of the article
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more, the low level of sequence similarity often found
even among microcins of the same general class impedes
identification of new microcins by sequence similarity.
These arguments represent possible explanations for the
reason why the study of ribosomally-produced peptide
natural products has lagged behind that of the well-
known non-ribosomal peptide synthetase and polyketide
synthase systems [5,6].
A subset of microcins has been recently described in
which the amino acid side chains of cysteine, serine and
threonine from a ribosomally produced precursor
undergo heterocyclization to generate a product with thi-
azole or (methyl)oxazole moieties. These include trich-
amide [7], the patellamides [8], goadsporin [9] and
microcin B17 [10], among others. Building on these ear-
lier studies, a research team led by Jack Dixon [4]
described three types of proteins that represent a con-
served biosynthetic machine for the formation of these
heterocycle-containing metabolites across numerous
microbial phyla. A zinc-tetrathiolate containing cyclode-
hydratase, flavin mononucleotide-dependent dehydroge-
nase and a docking scaffold protein are collectively
responsible for the installation of thiazole and (methyl/
oxazole modifications to a peptide precursor (Figure 1A).
In each case studied so far, the cyclodehydratase, dehy-
drogenase and docking scaffold proteins form a trimeric
complex (BCD) and serve to convert inactive, unstruc-
tured peptides into bioactive natural products [4,11,12].
The thiazole/oxazole heterocycles are biosynthesized
over two distinct chemical transformations. The first is
catalyzed by the cyclodehydratase (C), which converts
Cys and Ser/Thr residues into the corresponding thiazo-
line and (methyl)oxazoline with loss of water from the
amide backbone. In a second reaction, the dehydrogenase
(B) removes two electrons and two protons to afford the
aromatic thiazole and (methyl)oxazole [10,13]. The dock-
ing scaffold protein (D) appears to play a role in trimer
assembly and the regulation of enzymatic activity. For
each oxidized heterocycle formed, 20 Da is lost from the
parent peptide, which provides a convenient measure of
product formation by mass spectrometry (Figure 1)
[4,8,14]. This class of natural product has been termed
the thiazole/oxazole-modified microcins (TOMMs).
In a simplified view, the purpose of the TOMM biosyn-
thetic machinery is to recognize substrate and install
structural constraints that restrict peptide bond rotation,
thus endowing the modified peptide with a rigidified ter-
tiary structure. By restricting conformational flexibility at
the correct locations, the altered steric and electronic
properties of the molecule, in conjunction with the phys-
iochemical properties of the adjacent amino acids, lead to
a specific biological activity. This type of rationale could
also be extended to another family of post-translationally
modified peptides, the lantibiotics, with the only major
differences being the chemical composition (lanthionine
containing) and biosynthetic installation of the structural
constraints (Figure 1B) [15,16].
Again, similar to the lanthionine-containing peptides
(lantipeptides), TOMM precursor peptides are bipartite:
they contain an N-terminal leader sequence and a C-ter-
minal 'core' peptide. The leader sequence has been shown
in several cases to be critical to substrate recognition by
the modifying enzymes, while the core peptide serves as a
foundation upon which the active molecule is built
[11,17-19]. Outside of the leader region, TOMM precur-
sors tend to be rich in heterocyclizable residues (Cys, Ser,
Thr) and also in Gly, whose minimal side chain reduces
the energetic barrier required for cyclodehydration. Clues
that support the interpretation of an ORF as a TOMM
precursor include sequence similarity to previously iden-
tified TOMM precursors, a leader peptide cleavage motif,
and a hypervariable C-terminal core region rich in Gly,
Cys, Ser and Thr [4,11]. Also aiding the identification of a
TOMM cluster is the tendency of the modification
enzymes to cluster with other genes necessary for the
complete chemical maturation, export and immunity to
the natural product [4,20,21]. Identification of genes
encoding enzymes involved in lanthionine formation
[15,22,23], dehydroalanine production [9], peptide mac-
rocyclization [7,8,24,25] and thiazole/oxazole synthesis
Figure 1 The biosynthesis and defining chemical features of
TOMM and lanthionine-containing natural products. (A) Through
the action of a trimeric 'BCD' complex, consisting of a cyclodehy-
dratase (green), dehydrogenase (yellow) and docking/scaffolding pro-
tein (blue), thiazoles and (methyl/oxazoles are incorporated onto a
peptidic scaffold (black). These heterocycles are synthesized from ser-
ine/threonine (X = O) and cysteine (X = S) residues of an inactive pre-
cursor peptide and yield a bioactive natural product. The chemical
transformations carried out by the cyclodehydratase the dehydroge-
nase are shown, along with the corresponding mass change in Dal-
tons. (B) A bifunctional synthase, LanM, catalyzes both the dehydration
and Michael-type addition steps required to synthesize lanthionine
crosslinks.
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provide anchoring information for annotating post-trans-
lationally modified peptide biosynthetic clusters, such as
the TOMMs and lantipeptides. Identification of other
proteins (for example dehydrogenases, acetyltransferases,
methyltransferases, proteases and transporters) in the
local genomic region do not necessarily mark a biosyn-
thetic cluster on their own but instead, help to define the
extent and complexity of a proposed cluster [4].
Recent TOMM precursor identification by several
groups [3,8,24,26-29], including ours [4,30], provide a
growing number of short leader peptide sequences, a few
of which show a moderate level of similarity with one
another. However, many of the apparent TOMM biosyn-
thetic systems have remained orphan systems, in that the
thiazole/oxazole forming genes (encoding for the BCD
synthetase complex, Figure 1A) could be detected but the
TOMM precursors themselves could not be found. The
current availability of well over 1000 complete bacterial
and archaeal genomes permits the use of comparative
genomics methods to locate the substrates for orphan
TOMMs while simultaneously broadening the search for
previously unknown families of post-translationally mod-
ified peptides. Our results illustrate the power of applying
multiple informatics tools to the analysis of large num-
bers of fully sequenced genomes and suggest new oppor-
tunities to identifying secondary metabolite biosynthetic
systems.
Results and discussion
Using a combination of informatics tools against a large
number of sequenced genomes, we discovered several
protein families that appear to represent an entirely new
class of post-translationally modified peptide. The pre-
cursors have uncharacteristically long leader sequences
and large paralogous family counts per genome. Analysis
of the local genomic region predicts that these precursors
will have variable chemical fates, including thiazole/
oxazole and lanthionine formation. These families, sur-
prisingly, include one set of sequences with strong simi-
larity to the alpha subunit of the enzyme nitrile hydratase
(NHase) [31,32] while another set exhibits striking simi-
larity to nitrogen-fixing proteins from cyanobacteria
(Nif11) [33,34].
Description of NHase-related leader microcin family
One family of the newly discovered precursor peptides is
described by TIGRFAMs model TIGR03793 (Table 1)
and designated NHLP (nitrile hydratase-related leader
peptides - as described below). In five diverse bacterial
species, spanning several phyla, including firmicutes
(Syntrophomonas wolfei subsp. wolfei str. Goettingen,
Pelotomaculum thermopropionicum SI), proteobacteria
(Stigmatella aurantiaca DW4/3-1, Syntrophus aciditrop-
hicus SB [35]) and the chlorobi group (Chlorobium luteo-
lum DSM 273), NHLP precursors are found adjacent to a
cyclodehydratase-docking scaffold fusion protein
(TIGR03882), a required component of TOMM biosyn-
thesis [4]. The local genomic context of four of these bio-
synthetic clusters is illustrated in the upper portion of
Figure 2. Additional species provide further supporting
evidence for a link between NHLP and the cyclodehy-
dratase-docking scaffold by co-occurrence within the
same genome. Although not in close proximity to the
cognate NHLP substrate, the cyclodehydratase-docking
scaffold proteins from Microscilla marina ATCC 23134
(chlorobi group) and Methylobacterium sp. 4-46 (pro-
teobacteria) represent two examples of this genetic orga-
nization. Akin to the NHLP system, previous informatics
work has shown that the Bacillus anthracis and B. cereus
TOMM precursors are encoded more than one megabase
away from the modification cluster [30]. As with the
NHLPs from M. marina and Methylobacterium, recog-
nizing an orthologous cluster in B. licheniformis, in which
all components were clustered, accelerated the identifica-
tion of the precursors in B. anthracis and other members
of the B. cereus group.
An unmistakable feature of the NHLP family is its close
sequence similarity to the alpha subunit of NHase, which
is described by TIGR01323 (Table 1 and Figure 3). Previ-
ously characterized NHases (EC 4.2.1.84) are composed
of two subunits, alpha and beta, which together catalyze
the general reaction shown below [31,32].
For over 90% of genomes containing a member of the
NHase family (TIGR01323), that member occurs as the
highest scoring sequence in the genome to a search using
the fragment version (local-local scoring) Hidden Markov
Model (HMM) of TIGR03793. Fragment model searches
are preferred when match regions do not span the full
length of the seed alignment or the target sequence. This
is certainly the case when comparing sequences that have
either a large insertion or deletion (indel) relative to each
other. The median E-value for these HMM genome
search results is 1e-7, despite the short length (82 amino
acids) of the TIGR03793 model. As these sequences are
neither repetitive nor low in complexity in the regions
covered by the HMM, the consistently low E-values for
alignment between the two families predicts substantial
sequence similarity between NHases and NHLPs. Fur-
thermore, over three-quarters of the hits from
TIGR03793 (fragment model) to NHases found two
match segments, straddling a large indel region present in
the alpha subunit of NHase, but not in TIGR03793 family
sequences. The above described similarity and indel are
clearly evident in the alignment [36] shown in Figure 3A.
The sequences align convincingly over approximately 20
RCNHO RCONH
22
−≡ +
()
(1)
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residues N-terminal, and 50 residues C-terminal, to the
region deleted from the NHLP family.
The deleted region includes the NHase CxxCSC motif,
in which two of the three invariant cysteines are oxida-
tively modified - one to sulphenic acid, the other to sul-
phinic acid. Together, with a reduced cysteine thiol and
amide nitrogen of serine, these moieties serve as ligands
for the catalytic metal centre (Figure 3). NHase enzymes
use either a non-heme iron or a non-corrinoid cobalt
metal ion to activate water for hydrolyzing nitriles to
amides [31,32,37] (Equation 1). As all NHLPs lack the
entire active site region, they are suspected of being
devoid of NHase enzymatic activity. Supporting this is a
visual depiction of the segment of NHase missing in
NHLP, provided by the X-ray crystal structure of the
NHase from B. smithii (Figure 3B-3C) [38]. Another key
difference between the NHase and NHLP families is the
observation that the NHLPs harbour a classic leader pep-
tide cleavage site (Gly-Gly), which occurs at the extreme
C-terminal end of the region of similarity between the
TIGR01323 and TIGR03793 models (Figure 3A). This
motif also marks the end of sequence conservation
among members within TIGR03793. Following the Gly-
Gly motif is a hypervariable region, in which many
sequences are rich in residues that are targeted by post-
translational modifying enzymes (Cys, Ser, Thr, Figure 4).
This composition suggests that the hypervariable region
is the 'core peptide' and the homologous region com-
prises the leader sequence [18].
Phylogenetic profiling studies show connection to a
putative microcin export system
We computationally evaluated the candidacy of the
NHLP family as post-translationally modified peptide
precursors by the method of partial phylogenetic profil-
ing (PPP) [39], in which the profile serves as a query
against an entire genome. A phylogenetic profile was con-
structed on the basis of whether or not each sequenced
bacterial and archaeal genome carries a NHLP. Using
PPP, all sequences in the genome were evaluated to deter-
mine which best match the profile. In a collection of 1450
complete, or nearly complete, microbial genomes,
NHLPs occur in 14 species. Within these 14, each contain
between one and 12 copies of NHLP in their respective
genome (Figures 2, 3, 4). As shown in Table 2, the phylo-
genetic profile of these 14 NHLPs identified a three-gene
ABC transport cluster as the only high-scoring protein
family other than the NHLP precursor gene itself. The
top hits from our PPP search include families TIGR03794
(TransFuse), TIGR03796 (TransCleave), and TIGR03797
Table 1: Description of TIGR models
Accession Colour* Description Abbreviation
TIGR01323 N/A Nitrile hydratase, alpha subunit NHase
TIGR03603 N/A Cyclodehydratase (single ORF) C
TIGR03604 Blue Docking scaffold D
TIGR03882 Green Cyclodehydratase-docking (fused) CD
TIGR03605 Yellow Dehydrogenase B
TIGR03793 Black NHase-related leader peptide NHLP
TIGR03795 Dark gray Burkholderia NHLP NHLP-Burk
TIGR03798 Light gray Nif11-related peptide N11P
TIGR03794 Purple HlyD-like, type I secretion Trans-Fuse
TIGR03796 Red ABC transporter with peptidase Trans-Cleave
TIGR03797 Red ABC transporter Trans
*Colour coded in Figure 2.
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(TransABC), which are also described in Table 1. A nota-
ble difference between TIGR03796 and TIGR03797 is
that while both contain the adenosine triphosphate
(ATP)-binding cassette domain and permease domain,
the latter lacks the peptidase domain. TIGR03794 resem-
bles the HlyD membrane fusion protein of type I secre-
tion systems, suggesting a role in transport across the
outer membrane. Natural product export, including
unmodified and modified peptides, is often attributed to
a nearby ABC transport system that combines a protease
domain, permease domain and ATP-binding cassette,
either as multiple ORFs or as a single polypeptide
sequence [40,41]. The purpose of such a cassette is to
simply cleave the leader peptide and export the mature
product from the cell. NHLPs are adjacent to these trans-
porter cassettes in a diverse array of bacterial species,
including: Nostoc sp. PCC 7120* (cyanobacteria), Ana-
baena variabilis ATCC 29413 (cyanobacteria), M. marina
Figure 2 The genetic organization of TOMM and lanthionine biosynthetic clusters that utilize NHase- and Nif11-related precursor peptides.
Genomic regions are shown from selected organisms in which the precursor peptides are clustered with the cognate modification enzymes. In most
cases, a transport system is also visible in the local region. The TOMM precursors represented by Burkholeria cenocepacia (dark gray ORFs, NHLP-Burk)
are accompanied by a large, Ser/Thr kinase. Highly similar clusters have been identified in Acidovorax avenae and Delftia acidovorans. In several species,
precursors shown in black (NHLP) and light grey [Nif11-derived peptide (N11P)] may cluster with each other as well as with other modification and
transporter genes. Note: only those precursors closest to the cyclodehydratase-docking scaffold protein or LanM-like lanthionine synthase are shown.
For instance, Pelotomaculum thermopropionicum has 12 NHLPs (only two are shown) and seven N11P family precursors, while Cyanothece sp. PCC 7425
and P. marinus have 18 and 29 predicted N11P precursors, respectively (eight and seven are shown). Transport proteins, including those homologous
to HlyD (type I secretion, purple) and ABC transporters (red), correspond to the transport genes detected by PPP.
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ATCC 23134 (chlorobi group), C. luteolum DSM 273*
(chlorobi group), Victivallis vadensis ATCC str. BAA-548
(chlamydia group), and P. thermopropionicum SI* (firmic-
utes, * denotes a cluster shown in Figure 2). It is impor-
tant to note that not all of putative biosynthetic clusters
identified next to the ABC transporter genes are adjacent
to TOMM machinery. In the case of Nostoc sp. PCC 7120,
the NHLP and ABC transporter genes are adjacent to an
enzyme resembling LanM, which is involved in lanthion-
ine biosynthesis [16,42] (Figure 2). The findings from PPP
strongly support our interpretation of NHLPs as post-
translationally modified peptide precursors and further
argue that many, if not all, NHLP peptides will be sub-
jected to leader peptide cleavage upon export.
The fact that correlation to a transport cassette
emerges from PPP as a stronger relationship to the NHLP
family, rather than any posttranslational tailoring
enzyme, argues that the conservation in the leader pep-
tide reflects a common mechanism of handling by the
transport system (Table 2). The transport system appears
to be providing more evolutionary pressure in order to
maintain sequence similarity in this region than interac-
tion with modification enzymes, which are usually con-
sidered to be highly specific [11,18,19]. This finding
Figure 3 Alignment of nitrile hydratase (NHase) with nitrile hydratase leader peptide (NHLP) sequences. (A) Fourteen members of the NHase
alpha subunit protein family (TIGR01323), identified by locus tags, are shown aligned to the leader sequences of 28 members of the NHLP family
(TIGR03793). Along the top of the figure is a colour-coded region depicting the anticipated secondary structure for that region (red, alpha-helix; blue,
loop; green, beta-sheet). Relative to NHase, the NHLP sequences exhibit a 63-residue deletion that carries the residues required for iron/cobalt ligation,
the CxxCSC motif. Without the ability to bind the required catalytic metal, the truncation seen in NHLP is presumed to abolish NHase activity. Shown
in the truncated region is the canonical metal coordination architecture, with two of the three Cys thiol ligands being oxidized to sulphenic and sul-
phinic acids. Also shown is the putative leader sequence cleavage site for NHLP, which is not conserved with full-length NHase. (B) Crystal structure
of the NHase from Bacillus smithii, the most closely related NHase to the NHLP family with a known structure [38]. The N- and C-termini have been
labelled, the metal centre is shown as a cyan sphere and the beta subunit has been omitted for clarity. The colour coding is by secondary structure as
in panel A with the addition of the N- and C-terminal extensions (grey) that are not included in the alignment. The final residue of the NHase alignment,
Arg, is shown in blue stick format. (C) Same as in panel B, but with the insertion-deletion region omitted in NHLP's shown in wheat. This figure was
generated using a previously described, web-based program [36] and PyMOL.
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Figure 4 Sequence alignment of nitrile hydratase leader peptides (NHLPs) from natural combinatorial biosynthetic clusters. ClustalW align-
ment [44] of NHLPs from a putative thiazole/oxazole-modified microcin cluster from (A)Pelotomaculum thermopropionicum SI (12 sequences) and (B)
Azospirillum sp. B510 (eight sequences). Possible sites for thiazole formation are highlighted in cyan and sites of potential oxazole and methyloxazole
formation are yellow. The locus tag is given to the left of the sequence and the amino acid position is given on the right. An asterisk implies an invariant
residue, while the semicolon and period show positions that are highly and moderately related, respectively. Underlined red text indicates the puta-
tive leader peptide cleavage motif.
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Table 2: Partial phylogenetic profiling (PPP) results
Chlorobium luteolum
78186745 8 8 16 -16.124 NHLP
78186739 9 19 26 -13.212 Trans
78186738 9 22 29 -12.492 Trans-Cleave
78186736 8 16 17 12.045 Trans-Fuse
78187852 10 95 123 -7.475 (PAS domain)
Nostoc sp. PCC 7120
17229519 9 9 23 -18.140 NHLP
17229518 11 21 38 -16.662 NHLP
17229516 8 8 23 -16.124 NHLP
17233313 10 20 26 -14.927 Trans
17229512 10 20 26 -14.927 Trans
17229513 11 33 42 -13.969 Trans-Cleave
17233311 10 25 31 -13.698 Trans-Cleave
17229514 9 24 33 -12.080 Trans-Fuse
17233314 9 26 35 -11.709 Trans-Fuse
17228094 7 19 27 -9.451 (S-layer homol.)
Microscilla marina ATCC 23134
123986279 9 16 23 -14.108 Trans
123988060 9 21 28 -12.717 Trans-Cleave
123988059 7 10 14 -12.041 Trans-Fuse
123992175 8 29 54 -9.570 (HAMP domain)
Victivallis vadensis ATCC BAA-548
150259686 8 10 16 -14.479 Trans-Fuse
150259687 9 17 24 -13.784 Trans-Cleave
150259688 8 13 20 -13.033 Trans
150259679 6 6 15 -12.093 NHLP
150259681 5 6 8 -9.303 NHLP
150259680 4 4 6 -8.062 NHLP
150257768 10 88 119 -7.798 (GAF domain)
This table shows the results of PPP, where the profile contains 14 'YES' genomes having proteins recognized by TIGR03793, about 1% of
genomes and 1437 'NO' genomes. PPP scores each protein by selecting a BLAST score cutoff that gives the best possible fit between YES
genomes in the profile and the set of genomes in the BLAST hits list, then scoring the fit at that depth. Columns, from left to right, are gi
number, number of YES genomes encountered at the optimal depth, the total number of genomes at that depth, and the number of proteins
at that depth (which can differ from the total number of genomes because several proteins may come from the same genome), the PPP score
(a negative logarithm from the binomial distribution), and the protein family abbreviation. As PPP scores are not corrected for taxonomic
relationships between species, scores are for comparison within each genome only and are shown down to the first noise hit. Results are
shown in boldface except for noise hits. Note: Microscilla marina ATCC 23134 contains the nitrile hydratase leader peptide (NHLP) protein,
123988058, which is not detected by PPP. This gene is found co-clustered with the transport proteins identified by PPP, as shown.
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suggests a mix-and-match evolutionary pattern for post-
translationally modified peptide biosynthesis and export
systems, in which similarity in the leader peptide region
provides only indirect evidence of which class of modifi-
cation (thiazole/oxazole versus lanthionine) will occur.
The broader species distribution of the newly defined
putative export system, relative to the NHLP family
through which they were detected, provides a unique
opportunity to discover additional post-translationally
modified peptides families in emerging and existing
genomes.
Core peptide hypervariability and natural combinatorial
biosynthesis
The hypervariability observed in NHLPs after the Gly-
Gly motif is reminiscent of the variability in the core pep-
tides of experimentally validated antimicrobial peptides,
such as lichenicidin [42] and mersacidin [43]. An illustra-
tion of NHLP hypervariability is shown in Figure 4, where
members of TIGR03793 are aligned using ClustalW [44].
Intriguingly, all 12 substrates shown in panel A are from
the same organism, P. the rm op ro pio nic um SI, a thermo-
philic, clostridia class bacterium [45], while all eight
members shown in panel B are from Azospirillum sp.
B510, a proteobacterial rice endophyte [46]. Within the
local genomic context of the NHLPs from Azospirillum,
there are a LanM-like lanthionine-forming enzyme
(AZL_a09720) and an unfused docking scaffold protein
(AZL_a09740). While these two genes are plasmid-borne,
an additional copy of the unfused docking scaffold pro-
tein (AZL_022710) can be found on the chromosome,
along with a cyclodehydratase encoded 3 ORFs away
(AZL_022680). Therefore, it is not possible to determine
the chemical fate of the Azospirillum peptide precursors
at this time (Figure 4B). A more straightforward case is
demonstrated with P. thermopropionicum, which con-
tains one TOMM biosynthetic cassette and no discern-
able lanthionine-forming enzymes (Figure 2). This
implies that the single P. thermopropionicum cyclodehy-
dratase-docking scaffold fusion protein will process all of
the NHLPs into 12 distinct natural products (Figure 4A).
Supporting this is the observation that the leader peptide
region is highly conserved. The leader sequence of post-
translationally modified peptides typically contains spe-
cific binding motifs recognized by the modifying
enzymes [11,17,18]. This permits the selective modifica-
tion of the desired peptide in a complex environment,
such as the bacterial cytosol. Given that the P. thermopro-
pionicum genome is relatively small (3.0 megabase, 2930
coding sequences), if this organism is to produce an
extensive array of secondary metabolites, it must do so in
a highly genome-efficient manner. This is in contrast to
the much larger genome sizes of organisms renowned for
secondary metabolism, such as Streptomyces coelicolor
(8.7 megabase, 7825 coding sequences) [47]. Such exam-
ples of natural combinatorial biosynthesis are becoming
more frequent, as demonstrated with the cyanobactins by
Eric Schmidt's group [24]. It appears that natural combi-
natorial biosynthesis could be an underappreciated trait
of cyanobacteria, given that eight NHLPs were also iden-
tified in Nostoc punctiforme PCC 73102 (Figure 5 shows
an alignment of six of these).
NHLPs from Burkholderia
Members of a second putative microcin precursor family,
TIGR03795 (Table 1), occur near cyclodehydratase-dock-
ing scaffold fusion proteins in many proteobacteria of the
Burkholderia order, including Delftia acidovorans SPH-1,
two subspecies of Acidovorax avenae and multiple mem-
bers within the genus Burkholderia: B. cenocepacia, B.
ambifaria, B. pseudomallei, B. thailandensis, B. oklaho-
mensis and B. mallei [48-50]. TIGR03795 family
sequences occur exclusively as tandem gene pairs in the
Burkholderia genus, suggesting these may form a two-
peptide product, which are well-known (Figure 2) [41].
The tandem pairs in D. acidovorans and A. avenae are
fused to yield a single polypeptide, further suggesting that
the separate peptides from the Burkholderia genus func-
tion together. One member of each Burkholderia NHLP
(NHLP-Burk) pair contains either Cys-Cys, or a single
Cys, as the C-terminus (for example, Bcen_5137 and
Bcen_5138). Members of this family were discovered as
the top hits in their respective genomes to TIGR03793,
suggesting a sequence relationship to the NHLP sub-
strates described above. An alignment of NHase, NHLP
and NHLP-Burk reveals a moderate level of sequence
similarity. Relative to NHLP and NHase, NHLP-Burk
contains an insert of about 15 amino acids N-terminal to
a Pro-Xaa-Xaa-Pro motif conserved amongst the three
families. A major difference between NHLP and NHLP-
Burk lies in the leader peptide cleavage region (Table 3).
Non-thiazole/oxazole modified NHLPs
Besides the aforementioned case of Azospirillum, addi-
tional NHLP family members were found adjacent to a
LanM-like lanthionine synthase, instead of a cyclodehy-
dratase-docking fusion protein, in Nostoc sp. PCC 7120*
and N. punctiforme PCC 73102 (* shown in Figure 2,
lower panel). LanM is a bifunctional enzyme, responsible
for both the dehydration of Ser/Thr residues to dehydro-
alanine/butyrine and, subsequently, intramolecular
Michael-type addition of a Cys thiol to yield (methyl)lan-
thionines [15,23,51]. Aligning members of this family
revealed that sequence conservation is strong over nearly
90 amino acids, and ends with a typical leader sequence
cleavage motif, Gly-Gly (Figure 5) [18]. Reminiscent of
the TOMM-type NHLPs, the sequence C-terminal of the
Gly-Gly motif is short (average length 26) and highly vari-
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able. Although not depicted in Figure 5, over 60% of the
NHLPs adjacent to LanM-like proteins contain Cys in
their core peptide, meaning that these substrates are
capable of containing lanthionine crosslinks. Non-
TOMM NHLPs lacking Cys in the core peptide will pre-
sumably remain at the dehydrated state, unless new tai-
loring modifications are discovered that further process
these groups.
Post-translationally modified microcins derived from a
putative nitrogen-fixing protein
A third protein family, TIGR03798, reprises many of the
features of NHLP (Table 1) but are only found in bacteria
known to fix nitrogen, with most members also being
photosynthetic. TIGR03798 comprises a subset of the
Nif11 family (PF07862), which is heavily skewed to the
cyanobacteria. Nif11 proteins have no known function
[33]. TIGR03798 family members, such as NHLP, occur
in fairly large paralogous families. From this point on, we
will refer to TIGR03798 as Nif11-derived peptides
(N11P). N11P substrates are adjacent to the cyclodehy-
dratase-docking scaffold fusion protein in C. luteolum
(Figure 2) and nearby in P. t he r mo pr o pi on ic um . In many
cases, N11Ps are adjacent to ABC transport clusters (as
defined by TIGR03794, TIGR03796, and TIGR03797) in
Figure 5 Sequence alignment of nitrile hydratase leader peptides (NHLPs) from Nostdoc punctiforme PCC 73102. Shown is a ClustalW align-
ment of six selected NHLP substrates. N. punctiforme PCC 73102 has at least 16 total substrates, half of which are NHLP and the other half N11P. The
coloring scheme and notation are identical to Figure 4.
Table 3: Motif relationships in nitrile hydratase leader peptide (NHLP) and Nif11-related protein (N11P) leader sequences
to nitrile hydratase (NHase)
Accession Description Motif 1 Motif 2 Leader Suffix
TIGR01847 Gram-positive leader N/A ELSEKELAQIIGG 23 41
TIGR03898 lichenicidin leader IIRAWKDPEYRASLSSE ELSDEELESITGG 47 30
TIGR03798 N11P N/A ELSDEELEAVAGG 69 23
TIGR03793 NHLP IKKAWSDEEFKQALLNN ELSDEQLDAVAGG 87 27
TIGR03795 NHLP-Burk IALAWHDPEFRDELLAD N/A 108 11
TIGR01323 NHase VAKAWVDPEFKARLLKD GLSEEQLAALVTR 193 16
Sequence similarities and motif positions in NHase alpha subunit and putative leader sequences. Residues shared by at least 70% of the
calculated consensus sequences are shown in boldface. Leader peptide length is taken as the position of the C-terminal end of motif 2,
averaged after the shortest 10% and longest 10% removed of each family are removed as outliers and possible gene call errors. The similarity
of NHLP to N11P in motif 2 suggests intragenic recombination.
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C. luteolum, Synechococcus sp. WH 7803, C. phaeobacte-
roides, Desulfitobacterium hafniense and Eggerthella
lenta DSM 2243, among others. Additional N11P mem-
bers occur adjacent to LanM-like lanthionine-forming
enzymes in numerous species of cyanobacteria, including
N. punctiforme PCC 73102, Nostoc sp. PCC 7120,
Prochloroccocus marinus sp. MIT9313, and Cyanothece
sp. PCC 7425 (Figure 2) [52]. In the case of N. puncti-
forme PCC 73102, which also possess eight NHLP type
substrates (Figure 5), four LanM-like enzymes
(Npun_R3205, Npun_R3312, Npun_AF076, and
Npun_F5047) are expected to process an additional eight
N11P substrates for a total of 16 unique post-translation-
ally modified microcins.
Occurrence in the same genome with a LanM homolog,
although not necessarily clustered, is a feature of N11P
family proteins from Synechococcus sp. RS9916 and
Sinorhizobium medicae WSM419. Like NHLP and
NHLP-Burk, N11P sequences also have a classic leader
peptide cleavage motif, usually Gly-Gly, which marks the
end of family-wide similarity and the beginning of a low-
complexity region rich in Cys, Gly and Ser. As depicted by
a logo diagram (Figure 6) [53], the regions leading up to
the Gly-Gly motif in NHLPs and N11Ps are quite similar
to that of the leader peptides of family TIGR01847 (Table
3), which includes plantaricin A and lactococcin B
[41,54,55], two well-known, class II bacteriocins (unmod-
ified microcins).
Interfamily relationships of NHLP, NHLP-Burk and N11P
None of the three types of transport genes (Trans, Trans-
Cleave, Trans-Fuse) identified by PPP have a close
homolog in species with NHLP-Burk family members.
This implies that the export mechanism, if any, must dif-
fer. The occurrence of NHLP-Burk members in pairs,
fused in some genomes, suggests a two-chain structure. If
exported, these metabolites will likely require a different
transport mechanism. The NHLP and NHLP-Burk fami-
lies do exhibit extensive sequence similarity (motif 1,
Table 3), although not in the putative leader peptide
cleavage region (motif 2, Table 3). N11P does not show
clear evidence of direct similarity to the NHase alpha
subunit, as evidenced by extremely poor E-values (>1.0)
when querying all NHases against any N11P family mem-
ber. Nevertheless, N11P does exhibit regions of local
sequence similarity to NHLP (motif 2, Table 3). To vali-
date the similarity, TIGR03793 (NHLP) and TIGR03798
(N11P) were each searched against species that were
known to only contain members of the other family. For
instance, a TIGR03793 search against the draft genome of
Synechococcus sp. RS9916, which contains 31 N11P
sequences but no identifiable NHLP sequences, revealed
that 19 of the 24 nearest matches are actually members of
the N11P family. A similar search performed on Cyanoth-
ece sp. PCC 7425 returns 13 members of N11P as the top
scoring 15 sequences. Such searches also work with
members of the NHLP-Burk family. To illustrate, a search
with N11P against Burkholderia returns a member of
NHLP-Burk as the top hit. This cross-specificity,
although occurring at the 'noise' level, which is well below
the manually set trusted cutoff of each model, reflects
two regions of significant similarity between the three
precursor families. The more striking region, designated
motif 2 (Table 3), is the 13 amino acid stretch leading to
the Gly-Gly motif, similar to the leader peptide cleavage
region of model TIGR01847. In more classic lantibiotics,
such as lacticin 481, similarity of this region to class II
bacteriocins has been previously noted [56]. Another
region also shows strong sequence similarity between
NHLP, NHLP-Burk and N11P. This region, designated
motif 1, corresponds to the conserved sequence in the
NHase alpha subunit N-terminal to the active site Cys
residues (Figure 3). These results, in conjunction with the
noted paralogous duplication, are almost certainly the
result of intragenic recombination [57].
Conclusion
The proposed precursor families described in this report
dramatically expand the current repertoire of ribosomally
produced natural products. This revision includes hun-
dreds of peptides that exhibit (i) long leader peptide
regions, (ii) similarity to proteins and enzymes assigned
to other functions and (iii) locations distant to the
genomic regions used to encode their modification and
export genes. Microcins recognized by TIGR01847 have
Figure 6 Sequence logo comparison of classical Gram-positive
bacteriocin and nitrile hydratase leader peptides (NHLP)/Nif11-
derived peptides (N11Ps). (A) Logo depiction of TIGR01847, which is
representative of the sequence found near the peptide cleavage site of
Gram-positive bacteriocins. (B) Logo for the NHLP and N11P families
taken together. In addition to the classic Gly-Gly cleavage motif (posi-
tions 12-13), the two logos show an abundance of acidic residues at
positions 1, 4 and 6 and a significant preference for Leu at position 2
and 7, Ser/Thr at position 3, and either Val/Leu at position 10. This fig-
ure was generated online using a published algorithm [53].
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leader peptides predicted to end at an average length of
24 amino acids. However, the corresponding Gly-Gly
motifs in the new discovered families presented here end
at an average position of 83 and 70 for NHLP and N11P,
respectively. NHLPs demonstrate significant sequence
similarity to the alpha subunit of NHase, suggesting
strongly that they share a common ancestor. NHase is an
enzyme with a function unrelated to microcin production
and, thus, a broader implication of our findings is that a
small protein cannot be automatically excluded from
classification as a precursor to a natural product, even if it
is homologous to a protein with a known function.
The success of the approach employed here implies that
a parallel strategy could prove useful to unravelling other
natural product biosynthetic pathways. Possible applica-
tions are found in eukaryotic systems, such as in plants,
where complex natural product pathways exist, but the
requisite genes are not clustered. Clearly, the discovery of
new ribosomally produced natural products is far from
complete. Even within the families reported here, some
members of NHLP and N11P occur in species without
identified TOMM or lanthionine-forming enzymes. Fur-
thermore, numerous TOMM clusters remain orphans,
with candidate precursors yet to be identified. New tools
and concepts, such as those described here, will be of
importance in further defining the chemical genetic
scope of ribosomally produced natural products.
Note: While this manuscript was under review, an inde-
pendent report was published describing the in vitro
reconstitution and in vivo production of numerous N11P-
derived natural products from P. ma ri nu s sp. MIT9313
[58]. This finding strongly suggests that our informatics-
based predictions will hold up to further experimental
validation.
Methods
General
Multiple sequence alignments were generated using
MUSCLE [36] or ClustalW [44], inspected, and refined
manually. Refinements included trimming, removal of
truncated and other defective sequences, recruitment of
additional sequences, and realignment as necessary to
create representative seed alignments. Completed seed
alignments were used to construct HMMs. The resulting
new HMM-based protein family definitions, described in
this work, were deposited in the TIGRFAMs database
[59,60]. All HMM accessions refer to TIGRFAMs release
9.0 or Pfam release 22 [61].
In order to model regions of local sequence similarity
between different protein families, multiple alignments
were first generated, trimmed and used to train HMMs
for searches to gather additional candidate sequences
through an iterated, manual process. HMM construction
was performed with the Logical Depth 1.5.4 package soft-
ware-accelerated emulation of HMMER 2.3. The result-
ing motif models, of lengths 17 and 13, were searched
against the individual families TIGR01323, TIGR03793,
TIGR03795, TIGR03798 and the set of 20 proteins that
resulted from PSI-BLAST [62]. The PSI-BLAST itera-
tions were carried out to convergence, starting from the
predicted 49-residue leader peptide of a hypothetical lan-
thionine-containing peptide, gi|228993822 from B.
pseudomycoides SDM 12442), using composition-based
statistics and an E-value of 0.5. This search strategy pro-
vides a working definition for the set of lichenicidin-
related bacteriocins homologous in the leader peptide,
rather than the core peptide. All non-identical sequences
scoring above 0 bits to the respective motif HMMs were
aligned to the HMM, resulting in gapless alignments. For
each of these, a final HMM was built in order to emit a
consensus sequence.
Description of TIGR (The Institute for Genome Research)
models to locate biosynthetic genes
Previous work has identified many cyclodehydratase,
dehydrogenase and docking scaffold genes [4,24,27]. In
alpha/delta-proteobacteria, actinobacteria, cyanobacte-
ria, and chlorobi type bacteria, the cyclodehydratase and
docking scaffolds tend to be found encoded as a single
ORF, while other taxa usually produce separate protein
products [4]. TIGR03604 describes the docking protein
in both fused and unfused cases. TIGR03603 identifies
cyclodehydratases that occur as separate genes adjacent
to the docking scaffold gene, but a new model,
TIGR03882, had to be developed to reliably identify the
cyclodehydratase region of the enzymes fused to the
docking scaffold. All regions identified by TIGR03882 are
fused to a docking scaffold domain, and iteration by PSI-
BLAST demonstrates, as expected, weak similarity to a
set of known proteins: ThiF of thiamine biosynthesis
[63,64], MoeB of molybdopterin biosynthesis [65], ubiq-
uitin E1 conjugating enzymes and the cyclodehydratases
identified by TIGR03603. The sequence similarity
between post-translationally modified microcins and thi-
amine/molybdopterin biosynthetic proteins have been
previously documented [66]. MccB, an enzyme involved
in microcin C7 biosynthesis, also shares considerable
similarity to ThiF/MoeB/E1. The Walsh and Schulman
groups have recently characterized the MccB protein,
confirming the earlier report [67,68]. TIGR03882 recog-
nizes the cyclodehydratase domains of the TriA protein
for trichamide biosynthesis in Trichodesmium eryth-
raeum [7] and the PatD protein of patellamide biosynthe-
sis in Prochloron didemni [8]. The corresponding
cyanobactin-type TOMM precursors of these systems are
recognized by TIGR03678 [67,68]. Succinct descriptions
of all TIGR models of interest to this study are tabulated
in Tables 1 and 3.
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An examination of the genes in the vicinity of orphan
cyclodehydratase-docking scaffold fusion proteins
revealed no examples of short peptides qualitatively simi-
lar to those previously presented by Lee and Mitchell et
al. [4]. Previously identified peptides featured leader
sequences of approximately 25 amino acids, followed by
regions of very low complexity, often of a repetitive
nature, and highly enriched in cysteine, serine and threo-
nine. However, our latest survey identified somewhat
larger peptides nearby which warranted further investiga-
tion as potential TOMM precursors. For each family,
founding members were aligned in order to build HMMs
and search results were manually inspected in order to
set cutoffs for each family. The three families, now repre-
sented by TIGRFAMs models TIGR03793, TIGR03795
and TIGR03798 (Table 1) serve as the basis for this
report.
Partial phylogenetic profiling
Selected TIGRFAM models were searched against a col-
lection of 1450 complete or nearly complete bacterial and
archaeal genomes. All genomes with at least one protein
scoring above the trusted cutoff of the model were
assigned the value 1 ('YES') in the phylogenetic profile
built to represent that model, while all other genomes
were assigned the value 0 ('NO'). By PPP [39], the phylo-
genetic profile serves as a query to find which genes in a
genome may belong to protein families that can best
match that profile. PPP produces a score for each protein
in a genome by exploring increasing depths in the list of
best BLAST matches to that protein. PPP also records the
growing set of genomes from which those protein
matches originate. At each depth, PPP counts the num-
bers of genomes agreeing ('YES') and disagreeing ('NO')
with the query profile and uses the binomial distribution
to score the odds of obtaining at least that many agree-
ments. The overall score for each protein is based on a
depth for which the negative log
10
of the score is maxi-
mized, corresponding to an optimum for the working size
of a candidate protein family. Each phylogenetic profile
was used to query all genomes assigned as YES in the
profile. Top-scoring proteins were identified for further
analysis. In essence, PPP makes it possible to detect a
protein family that matches a query profile, even if that
family has never previously been defined.
List of abbreviations
ABC: ATP-binding cassette; ATP: adenosine triphos-
phate; HMM: hidden Markov model; indel: insertion-
deletion; N11P: Nif11-derived peptide; NHase: nitrile
hydratase; NHLP: nitrile hydratase leader peptide;
NHLP-Burk: Burkholderia type TOMM substrate family;
Nif11: nitrogen fixation protein of unknown function;
ORF: open reading frame; PPP: partial phylogenetic pro-
filing; TIGR: The Institute for Genomic Research;
TOMM: thiazole/oxazole-modified microcin.
Authors' contributions
DHH conceived the study and constructed the TIGRFAM models. DAM partici-
pated in the model validation and expanded the scope of the study. MKB
developed improved software tools for detecting protein family relationships.
DHH and DAM analysed and interpreted the data. DHH and DAM wrote the
paper. All authors read and approved the final manuscript.
Acknowledgements
The authors wish to thank Eric Eisenstadt and members of the Mitchell Labora-
tory for the critical review of the manuscript. This work was supported by
grants 1 R01 HGO04881, HHSN266200400038C and by institutional funds pro-
vided by the Department of Chemistry at the University of Illinois.
Author Details
1
The J Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850,
USA,
2
Department of Chemistry, University of Illinois at Urbana-Champaign,
Urbana, IL 61801, USA,
3
Department of Microbiology, University of Illinois at
Urbana-Champaign, Urbana, IL 61801, USA and
4
Institute for Genomic Biology,
University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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Cite this article as: Haft et al., Expansion of ribosomally produced natural
products: a nitrile hydratase- and Nif11-related precursor family BMC Biology
2010, 8:70
    • "All proteins with 100 % identity were removed and are represented as larger nodes on the network (size is dependent on the number of redundant proteins). Groups are number for reference within the manuscript from this bioinformatics study although several have been identified previously [32]. "
    [Show abstract] [Hide abstract] ABSTRACT: Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a burgeoning class of natural products with diverse activity that share a similar origin and common features in their biosynthetic pathways. The precursor peptides of these natural products are ribosomally produced, upon which a combination of modification enzymes installs diverse functional groups. This genetically encoded peptide-based strategy allows for rapid diversification of these natural products by mutation in the precursor genes merged with unique combinations of modification enzymes. Thiazole/oxazole-modified microcins (TOMMs) are a class of RiPPs defined by the presence of heterocycles derived from cysteine, serine, and threonine residues in the precursor peptide. TOMMs encompass a number of different families, including but not limited to the linear azol(in)e-containing peptides (streptolysin S, microcin B17, and plantazolicin), cyanobactins, thiopeptides, and bottromycins. Although many TOMMs have been explored, the increased availability of genome sequences has illuminated several unexplored TOMM producers. All YcaO domain-containing proteins (D protein) and the surrounding genomic regions were were obtained from the European Molecular Biology Laboratory (EMBL) and the European Bioinformatics Institute (EBI). MultiGeneBlast was used to group gene clusters contain a D protein. A number of techniques were used to identify TOMM biosynthetic gene clusters from the D protein containing gene clusters. Precursor peptides from these gene clusters were also identified. Both sequence similarity and phylogenetic analysis were used to classify the 20 diverse TOMM clusters identified. Given the remarkable structural and functional diversity displayed by known TOMMs, a comprehensive bioinformatic study to catalog and classify the entire RiPP class was undertaken. Here we report the bioinformatic characterization of nearly 1,500 TOMM gene clusters from genomes in the European Molecular Biology Laboratory (EMBL) and the European Bioinformatics Institute (EBI) sequence repository. Genome mining suggests a complex diversification of modification enzymes and precursor peptides to create more than 20 distinct families of TOMMs, nine of which have not heretofore been described. Many of the identified TOMM families have an abundance of diverse precursor peptide sequences as well as unfamiliar combinations of modification enzymes, signifying a potential wealth of novel natural products on known and unknown biosynthetic scaffolds. Phylogenetic analysis suggests a widespread distribution of TOMMs across multiple phyla; however, producers of similar TOMMs are generally found in the same phylum with few exceptions. The comprehensive genome mining study described herein has uncovered a myriad of unique TOMM biosynthetic clusters and provides an atlas to guide future discovery efforts. These biosynthetic gene clusters are predicted to produce diverse final products, and the identification of additional combinations of modification enzymes could expand the potential of combinatorial natural product biosynthesis.
    Full-text · Article · Dec 2015
    • "Only two of the nine cyanothecamide putative toxin genes have been experimentally implicated as precursors to identifiable mature patellamide-like com- pounds [30]. Yet, the capacity for biosynthetic machinery to modify substrate peptides with suitable N-terminal domains despite drastic variability in the C-terminal portion of the peptide has been demonstrated in other bacteriocins [31, 32]. The features of this particular gene block raise the possibility that bacteriocin loci encoding post-translationally modified peptides could, through elaboration of sequence diversity in multiple cognate peptide substrates, confer a greater breadth of functional diversity to producing organisms than previously appreciated [33]. "
    [Show abstract] [Hide abstract] ABSTRACT: Bacteriocins are peptide-derived molecules produced by bacteria, whose recently-discovered functions include virulence factors and signaling molecules as well as their better known roles as antibiotics. To date, close to five hundred bacteriocins have been identified and classified. Recent discoveries have shown that bacteriocins are highly diverse and widely distributed among bacterial species. Given the heterogeneity of bacteriocin compounds, many tools struggle with identifying novel bacteriocins due to their vast sequence and structural diversity. Many bacteriocins undergo post-translational processing or modifications necessary for the biosynthesis of the final mature form. Enzymatic modification of bacteriocins as well as their export is achieved by proteins whose genes are often located in a discrete gene cluster proximal to the bacteriocin precursor gene, referred to as context genes in this study. Although bacteriocins themselves are structurally diverse, context genes have been shown to be largely conserved across unrelated species. Using this knowledge, we set out to identify new candidates for context genes which may clarify how bacteriocins are synthesized, and identify new candidates for bacteriocins that bear no sequence similarity to known toxins. To achieve these goals, we have developed a software tool, Bacteriocin Operon and gene block Associator (BOA) that can identify homologous bacteriocin associated gene blocks and predict novel ones. BOA generates profile Hidden Markov Models from the clusters of bacteriocin context genes, and uses them to identify novel bacteriocin gene blocks and operons. Results and conclusions We provide a novel dataset of predicted bacteriocins and context genes. We also discover that several phyla have a strong preference for bacteriocin genes, suggesting distinct functions for this group of molecules. Software Availability
    Full-text · Article · Oct 2015
    • "PCC 9605. This putative precursor peptide was aligned with selected NHLP precursor peptides from N. punctiforme PCC 73102, and a conserved region near the peptide cleavage site (double glycine motif ) was identified [87] (Additional file 6). The wide distribution and range of bacteriocin gene clusters identified from the Subsection V cyanobacteria is consistent with previous reports from other cyanobacteria. "
    [Show abstract] [Hide abstract] ABSTRACT: Cyanobacteria are well known for the production of a range of secondary metabolites. Whilst recent genome sequencing projects has led to an increase in the number of publically available cyanobacterial genomes, the secondary metabolite potential of many of these organisms remains elusive. Our study focused on the 11 publically available Subsection V cyanobacterial genomes, together with the draft genomes of Westiella intricata UH strain HT-29-1 and Hapalosiphon welwitschii UH strain IC-52-3, for their genetic potential to produce secondary metabolites. The Subsection V cyanobacterial genomes analysed in this study are reported to produce a diverse range of natural products, including the hapalindole-family of compounds, microcystin, hapalosin, mycosporine-like amino acids and hydrocarbons. A putative gene cluster for the cyclic depsipeptide hapalosin, known to reverse P-glycoprotein multiple drug resistance, was identified within three Subsection V cyanobacterial genomes, including the producing cyanobacterium H. welwitschii UH strain IC-52-3. A number of orphan NRPS/PKS gene clusters and ribosomally-synthesised and post translationally-modified peptide gene clusters (including cyanobactin, microviridin and bacteriocin gene clusters) were identified. Furthermore, gene clusters encoding the biosynthesis of mycosporine-like amino acids, scytonemin, hydrocarbons and terpenes were also identified and compared. Genome mining has revealed the diversity, abundance and complex nature of the secondary metabolite potential of the Subsection V cyanobacteria. This bioinformatic study has identified novel biosynthetic enzymes which have not been associated with gene clusters of known classes of natural products, suggesting that these cyanobacteria potentially produce structurally novel secondary metabolites.
    Full-text · Article · Sep 2015
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