Bacterial group II introns: not just splicing
Nicolás Toro, José Ignacio Jiménez-Zurdo & Fernando Manuel Garcı́a-Rodrı́guez
Grupo de Ecologı́a Genética de la Rizosfera, Estación Experimental del Zaidı́n, Consejo Superior de Investigaciones Cientı́ficas, Granada, Spain
Correspondence: Nicolás Toro, Grupo de
Ecologı́a Genética de la Rizosfera, Estación
Experimental del Zaidı́n, Consejo Superior de
Investigaciones Cientı́ficas, Profesor Albareda
1, 18008 Granada, Spain. Tel.:
134 958181600; fax: 134 958129600;
e-mail: [email protected]
Received 30 November 2006; revised 12
January 2007; accepted 12 January 2007.
First published online March 2007.
DOI:10.1111/j.1574-6976.2007.00068.x
Abstract
Group II introns are both catalytic RNAs (ribozymes) and mobile retroelements
that were discovered almost 14 years ago. It has been suggested that eukaryotic
mRNA introns might have originated from the group II introns present in the
alphaproteobacterial progenitor of the mitochondria. Bacterial group II introns are
of considerable interest not only because of their evolutionary significance, but
also because they could potentially be used as tools for genetic manipulation in
biotechnology and for gene therapy. This review summarizes what is known about
the splicing mechanisms and mobility of bacterial group II introns, and describes
the recent development of group II intron-based gene-targetting methods.
Bacterial group II intron diversity, evolutionary relationships, and behaviour in
bacteria are also discussed.
Editor: Rafael Giraldo
Keywords
catalytic RNA; homing; splicing; retroelements;
Lactococcus lactis ; Sinorhizobium meliloti .
Introduction: group II introns
Group II introns are large catalytic RNAs (ribozymes) and
mobile retroelements [reviewed by Pyle & Lambowitz
(2006)] that splice by means of a lariat intermediate, in a
mechanism similar to that of spliceosomal introns. The
transfer of these introns either to intronless alleles (retrohoming) or to ectopic sites (retrotransposition) is mediated
by ribonucleoprotein (RNP) complexes consisting of the
intron-encoded protein (IEP) and the excised intron RNA
lariat. Group II introns were initially identified in the
mitochondrial and chloroplast genomes of lower eukaryotes
and plants (Michel et al., 1989). They were later identified in
bacteria (Ferat & Michel, 1993), and have recently been
found in the archaeal genus Methanosarcinales (Toro, 2003;
Dai & Zimmerly, 2003; Rest & Mindell, 2003). It is thought
that both nuclear spliceosomal introns and non-Long Terminal Repeat (LTR) retrotransposons evolved from mobile
group II introns [Sharp, 1985; Cech, 1986; Cavalier-Smith,
1991; Eickbush, 1994; Zimmerly et al., 1995; recently
reviewed by Koonin (2006)]. According to this hypothesis,
group II introns originated in bacteria and invaded the
nucleus of a primitive eukaryote, possibly from the alphaproteobacterial progenitor of the mitochondria; they were
then fragmented to form the spliceosome (Cavalier-Smith,
1991; Stoltzfus, 1999). It has recently been suggested that the
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spread of group II introns and their decay into spliceosomal
introns created a strong selective pressure, necessitating
compartmentalization of the nucleus and cytoplasm (Martin & Koonin, 2006). In addition to their possible evolutionary significance in eukaryogenesis, mobile group II
introns are of interest because they have been used to
develop a new type of gene-targetting vector (Lambowitz &
Zimmerly, 2004; Lambowitz et al., 2005), with potential
applications in biotechnology and medicine.
A typical group II intron (Fig. 1) consists of a highly
structured RNA with six distinct double-helical domains (DI
to DVI) and an internally encoded (ORF within DIV)
reverse transcriptase (RT) maturase. The IEP is required for
in vivo folding of the intron RNA into a catalytically active
structure (Michel & Ferat, 1995). Unlike organellar introns,
most bacterial group II introns have an IEP (Lambowtiz &
Zimmerly, 2004). The IEPs of these introns (Fig. 1) have
four conserved domains: an N-terminal RT domain; domain X – a putative RNA-binding domain associated with
RNA splicing or maturase activity-, a C-terminal DNAbinding domain (D); and a DNA-endonuclease domain
(En). More than half of the bacterial group II IEPs annotated
to date lack the En domain, and many also lack the D domain
(Martı́nez-Abarca & Toro, 2000b; Zimmerly et al., 2001;
Belfort et al., 2002; Toro, 2003; Lambowitz & Zimmerly,
2004). It has been suggested that all current group II introns
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Bacterial group II introns
Fig. 1. Generic secondary structure of group II intron RNA and domain structure of the intron-encoded proteins (IEP). The model shows the ribozyme
domains (DI–DVI), the EBS/IBS elements, the bulged adenosine within DVI, and the sequences involved in tertiary interactions (Greek letters) operating in
splicing as described in the text. Major differences between IIA and IIB introns are indicated in brackets. Note that IIC introns lack the EBS2 element.
Boxed are the schematics (drawn to scale) of the Ll.LtrB (IIA), RmInt1 (IIB) and GBSi1 (IIC) IEPs encoded within intron DIV, with indication of the predicted
reverse transcriptase (RT), maturase (X), variable DNA-binding region (D) and conserved DNA endonuclease (En) domains. Numbers above the RT
domain identify conserved amino acid sequence blocks characteristic of RTs. The question mark denotes a functionally uncharacterized C-terminal
extension of 20 amino acid residues in the RmInt1 IEP.
are descended from an RT-encoding group II intron in
bacteria (Toor et al., 2001). Three main phylogenetic subclasses (IIA, IIB and IIC) of group II introns have been
described on the basis of IEP and conserved intron RNA
structures (Michel et al., 1989; Toor et al., 2001; Zimmerly
et al., 2001; Ferat et al., 2003; Toro, 2003), with the IIC
subclass unique to bacteria. Bacterial group II introns differ
markedly from organellar introns in not generally being
located in conserved genes and in being mostly associated
with mobile DNAs or located within intergenic regions (Dai
& Zimmerly, 2002; Toro, 2003).
Group II introns, which may predate prokaryotes
(Koonin, 2006), are still present in many bacteria. So, what
role do group II introns play in the bacterial genome?
Although they are generally seen as ‘selfish’ elements,
studies of group II introns are continually discovering
FEMS Microbiol Rev 31 (2007) 342–358
surprising features such as the association of some lineages
with specific genetic signals or with the host replication
machinery for mobility.
Bacterial group II intron splicing
Group II introns splice by means of a lariat intermediate,
in a mechanism resembling that of spliceosomal introns.
Intron excision from eukaryotic mRNA requires a
set of host-cell snRNA molecules and proteins, whereas
the catalytic functions involved in group II intron splicing
reside within the RNA molecule itself. Nevertheless,
the folding of the intron RNA into the catalytically
active structure in vivo depends on the maturase activity
of the IEP encoded within intron DIV, outside the catalytic
core of the ribozyme.
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344
Self-splicing mechanisms and products
Several bacterial group II introns have been found to selfsplice in the absence of the IEP when incubated under
nonphysiological high-salt conditions in vitro: Cal.x1 from
Calothrix (Ferat & Michel, 1993); IntB from Escherichia coli
(Ferat et al., 1994); Ll.LtrB from Lactococcus (Matsuura
et al., 1997); AV1 (Avi.groEL) and AV2 from Azotobacter
(Adamidi et al., 2003; Ferat et al., 2003; Kosaraju et al.,
2005); GBSi1 from Streptococcus (Granlund et al., 2001);
B.a.I1, B.a.I2 and B.hI1 from Bacillus (Robart et al., 2004;
Tourasse et al., 2005; Toor et al., 2006); Gk.Int1 from
Geobacillus (Chee & Takami, 2005); and RmInt1 from
Sinorhizobium (Costa et al., 2006a). However, bacterial
group II ribozymes are generally only moderately reactive
and display slow kinetics in vitro. The models chosen for
investigations of group II intron catalysis and splicing
mechanisms have therefore tended to be the more efficient
large mitochondrial group II ribozymes of the two major
group II intron subclasses, IIA and IIB, from eukaryotic
organisms (Costa et al., 1997, 2000; Su et al., 2001).
The results of these studies suggest that the pathway
generally used for group II intron splicing involves two
sequential transesterification reactions (Fig. 2a). The first
involves a nucleophilic attack on the 5 0 splice junction by the
2 0 -OH of a bulged nucleotide, typically an adenosine residue
(bulging A) located in DVI near the 3 0 end of the intron,
releasing the 5 0 exon and generating an intron-3 0 exon
branched intermediate. The second reaction involves a
nucleophilic attack on the 3 0 splice site by the free 3 0 -OH of
the last nucleotide of the 5 0 -exon, yielding the ligated exons
and the intron RNA lariat with a 2 0 –5 0 phosphodiester bond
as the major splicing products. Alternatively, the splicing
reaction may be initiated by hydrolysis (Fig. 2b), followed by
the second step, resulting in the excision of the intron as a
linear molecule rather than a lariat. Most of the self-splicing
Nicolás Toro et al.
bacterial group II introns identified to date display both
excision pathways in vitro. The exceptions are some bacterial
class C introns such as GBSi1, identified in some Streptococcus B isolates, which splice solely via a linear intermediate
molecule (Granlund et al., 2001). In addition, it has been
reported that yeast intron aI5g can be also excised as a true
circular form in vitro (Fig. 2c). The circle seems to result
from the formation of a 2 0 –5 0 phosphodiester bond between
the last and first residues of the intron, and it has been
proposed that this reaction requires the 3 0 exon to have been
released first from precursor molecules by a trans-splicing
reaction triggered by 5 0 exon molecules previously generated
by the so-called spliced exon reopening (SER) reaction.
Subsequent to trans splicing, the 2 0 -OH of the terminal
intron residue will attack the 5 0 splice site, releasing the
5 0 exon and an intron circle (Murray et al., 2001).
Group II ribozyme positioning on the precursor mRNA
substrate and cleavage site fidelity are determined principally by base-pairing interactions between short-sequence
elements located in loop structures of intron DI (exonbinding sites, EBS) and sequence stretches flanking the
splice site (intron-binding sites, IBS). EBS1 and EBS2, each
typically five to seven nucleotides long, recognize their
partner sequences – IBS1 and IBS2 – at the 3 0 end of the 5 0
exon. Interestingly, IIC introns are self-splicing-competent,
despite lacking the EBS2–IBS2 pairing (Granlund et al.,
2001; Toor et al., 2006). The recognition of the 3 0 intron–
exon junction involves two additional base-pair interactions. The first is a single tertiary base-pair interaction
between the last position of the intron (g 0 ) and another
intron nucleotide (g) between DII and DIII. The second
involves the first one or several specific exon and intron
nucleotides. The identity of this IBS for the 3 0 exon can be
used to differentiate between the two major subclasses of
group II introns. In IIA introns, the sequence immediately
upstream from EBS1 (d sequence) base-pairs with one to
Fig. 2. Mechanisms of group II intron splicing through (a) branching, (b) hydrolysis and (c) circles formation. 5 0 and 3 0 exons are indicated with black
and grey boxes, respectively, whereas the line represents the intron RNA. Double arrows indicate the reversibility of some reactions.
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345
Bacterial group II introns
three nucleotide residues (d 0 position) of the 3 0 exon for
intron splicing (3 nt in the Lactococcus lactis Ll.LtrB intron).
In contrast, most IIB introns display no significant signs of
d–d 0 complementarity. These group II ribozymes recognize
the first nucleotide in the 3 0 exon, referred to as IBS3 rather
than d 0 , by canonical base-pairing with an intron nucleotide,
referred to as EBS3 rather than d, located in the so-called
‘coordination loop’ of DI (Costa et al., 2000; De Lencastre
et al., 2005). The d position is involved in a new tertiary
interaction with another residue (denoted d 0 ), also located
in the coordination loop (Costa et al. 2000). These nucleotides in the loop are involved in aligning the two exons for
the second step of splicing (d–d 0 and IBS3–EBS3 interactions). The three-dimensional arrangement of the active site
of group II ribozymes can be further modelled by a complex
network of tertiary interactions involving the various RNA
domains (Michel & Ferat, 1995; Costa et al., 2000; Swisher
et al., 2001 and references therein).
Thus, 5 0 exon binding through DI interactions and the
conserved DV are involved in the formation of the minimal
catalytic core of the ribozyme, with DII and DIII also
contributing to RNA folding and catalytic efficiency (Fedorova et al., 2003; for a review see Pyle & Lambowitz, 2006).
The branch site of DVI, both exons, the essential catalytic
regions of DV (bulge and AGC triad regions), conserved
nucleotides in the joining region (J) between DII and DIII
(J2/3), and the e–e 0 substructure are all in close proximity
before the first splicing step (De Lencastre et al., 2005). It has
recently been shown that the branch site binds, with specific
polarity, to the coordination loop substructure, thereby
placing the nucleophilic adenosine in a position in which it
can react with the 5 0 splice site (Hamill & Pyle, 2006). The
docking site for DV, a tetraloop receptor motif composed of
the z–z and k–k 0 interactions, is separated from the coordination loop by a short helical stem (reviewed by Lehmann &
Schmidt, 2003). The choice of branch site is strictly controlled
by a molecular caliper, which measures the distance between
the base of DV and the nucleophilic 2 0 -hydroxyl in DVI (Chu
et al., 2001). Thus, DV and the branch-site receptors function
together to position the nucleophile correctly, so as to
promote catalysis (Hamill & Pyle, 2006).
Alternative EBS-IBS interactions, shifted from the
expected EBS-IBS pairings, have also been described in the
bacterial group IIB introns B.a.I2 and RmInt1 (Robart et al.,
2004; Costa et al., 2006a, b). As shown in vitro, the 3 0 splice
site of B.a.I2 is flexible but depends on potential g–g 0 and
IBS3–EBS3 pairings downstream from the wild-type site.
Alternative splice-site usage either creates frame shifts within the exon-encoded ORF or joins the two exonic ORFs in
frame, ultimately affecting the output of host mRNA
translation in vivo (Robart et al., 2004). On the other hand,
it has been suggested that an RmInt1 ribozyme reaction at a
surrogate IBS sequence located 3 0 to the intron might be
FEMS Microbiol Rev 31 (2007) 342–358
responsible for the generation of unconventional self-splicing products along with the ligated exons and the excised
intron. These unconventional self-splicing products include
a 3 0 exon truncated at its 5 0 end, truncated variants of the
linear and lariat forms of the intron –3 0 exon intermediate,
and putative circular molecules derived from this intermediate (Costa et al., 2006a). The self-splicing profiles of
B.a.I2 and RmInt1 may therefore highlight mechanisms
underlying the control of host-gene expression by bacterial
group II introns.
Protein-assisted splicing in vivo
Only a minority of bacterial group II introns have been
unequivocally shown to be functional or to undergo splicing
in vivo: the L. lactis Ll.LtrB (Mills et al., 1996; Shearman
et al., 1996); the Sinorhizobium meliloti RmInt1 (Martı́nezAbarca et al., 1998); a Clostridium difficile intron (Roberts
et al., 2001); the Bacillus anthracis B.a.I2; and the Trichodesmium erythraeum RIR-3 (Robart et al., 2004; Meng et al.,
2005) introns. The best characterized of these introns to date
are Ll.LtrB and RmInt1, as representatives of subclasses IIA
and IIB, respectively. Initial sequencing and genetic analysis
have shown that the splicing of these group II introns in vivo
requires the IEP encoded within intron DIV (Mills et al.,
1996; Shearman et al., 1996; Martı́nez-Abarca et al., 1998;
Muñoz-Adelantado et al., 2003). Bacterial group II intronencoded maturases function more efficiently when
expressed in cis, within the same precursor transcript
encoding the ribozyme core. However, trans-encoded IEPs
can promote the splicing of genomic copies of nonfunctional ORF-less introns, a property that has evolutionary
and biotechnological implications (Cui et al., 2004; Meng
et al., 2005; Nisa-Martı́nez et al., 2007).
Conserved RT and X domains are common to both
Ll.LtrB and RmInt1 IEPs, being the X domains associated
with the splicing-promoting (maturase) activity of the
proteins. However, most of the available information on
the mechanisms involved in IEP-assisted bacterial group II
intron splicing is based on the lactoccocal intron (Matsuura
et al., 1997, 2001; Saldanha et al., 1999; Wank et al., 1999;
Singh et al., 2002; Cui et al., 2004). Ll.LtrB and its encoded
protein, LtrA, are efficiently expressed and functional in
E. coli, facilitating the detailed biochemical characterization
of both the intron RNA and the IEP (Matsuura et al., 1997).
The purified LtrA protein binds to the unspliced precursor
RNA via a rapid bimolecular reaction, followed by a slower
unimolecular step involving a change in RNA conformation
that ultimately promotes Ll.LtrB splicing when assayed
in vitro under low salt concentrations shown to inhibit the
self-splicing reaction (Saldanha et al., 1999). In certain
specific conditions, LtrA also binds nonspecifically to other
group II introns and RNA molecules. However, this
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346
nonspecific binding is inefficient for RNA splicing, and LtrA
is therefore considered to be an intron-specific splicing
factor (Saldanha et al., 1999). This specificity is based on
protein recognition of a unique structural feature in the
intron subdomain DIVa as a primary high-affinity binding
site; this site contains the Shine–Dalgarno sequence and the
initiation codon of the LtrA ORF (Wank et al., 1999; Singh
et al., 2002). This binding, which also autoregulates LtrA
translation, requires domain X and parts of the RT domain
of the IEP, and extends further into the 5 0 and 3 0 conserved
regions of the catalytic core and DIV through weaker
secondary contacts that are sufficient to support residual,
but inefficient, splicing in vivo when the high-affinity
binding site in DIVa is deleted (Wank et al., 1999; Matsuura
et al., 2001; Singh et al., 2002; Cui et al., 2004). Further
chemical probing and RNA footprinting experiments have
demonstrated that this array of protein–RNA contacts
promotes the generation of a novel set of conserved longrange tertiary interactions in the Ll.LtrB RNA, which are not
stably formed in the absence of the maturase under low-salt
conditions, but which are required to model the active
conformation of the ribozyme core in vivo (Matsuura et al.,
2001). The major protein-assisted splicing products of Ll.LtrB
are the conventional linear and lariat forms of the intron,
along with exons ligated in frame (Saldanha et al., 1999).
The maturase activity of the RmInt1 IEP has not been
biochemically characterized. However, recent studies have
demonstrated that RmInt1 splices in vivo leading to the
formation of the intron lariat form, along with circular
molecules in which the first and last intron residues are
ligated (Molina-Sánchez et al., 2006). Genetic analysis of the
conserved X domain of the RmInt1 IEP not only confirmed
the requirement of the maturase for intron splicing in vivo
but also supported an additional role for the IEP in
controlling the balance between the two intron excision
pathways (Molina-Sánchez et al., 2006). Thus, in vivo the
presence of the IEP seems to ensure cleavage at the correct 3 0
splice site or at position 11 in the 3 0 exon before intron
RNA circle formation. Circular RNAs had previously only
been described as splicing products of group II introns in
eukaryotic cells, in which they were thought to be linked to
cellular senescence (Murray et al., 2001 and references
therein). These novel findings therefore open up new perspectives for investigation of the functional diversity of
bacterial group II ribozymes and their biological significance.
As described above, analyses of the splicing reaction,
using the purified Ll.LtrB-encoded maturase and RNA,
predict a remarkable self-sufficiency of bacterial group II
introns in the promotion of their own splicing. However, it
has also been shown that the efficiency of the in vivo splicing
reaction of the group II intron RmInt1 decreases when the
intron is expressed in a genetic background other than that
of its natural bacterial host, S. meliloti (Martı́nez-Abarca
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Nicolás Toro et al.
et al., 1998, 1999). Several nuclear gene products have been
identified as RNA chaperones involved in the splicing of
group II introns from yeast, algae or higher plants (Lambowitz & Perlman, 1990; Lambowitz et al., 1999; Lehmann
& Schmidt, 2003; Ostheimer et al., 2003). It remains unclear
whether this is also the case for bacterial group II introns,
and to what extent splicing efficiency depends on host
functions.
Bacterial group II intron mobility
mechanisms
Group II introns act both as large catalytic RNAs and as sitespecific retroelements (more recently reviewed by Lambowitz & Zimmerly, 2004; Pyle & Lambowitz, 2006). After
splicing, the intron RNA lariat and the IEP remain associated, forming a ribonucleoprotein particle (RNP) promoting intron insertion into DNA target sites identical to the
splice site through retrohoming, which is the principal and
most efficient group II intron RNA-based mobility pathway
(Fig. 3). Retrohoming has been unequivocally demonstrated
for the lactococcal Ll.LtrB and rhizobial RmInt1 introns,
using engineered ‘twintron’ shuttle constructs in which the
bacterial group II intron was interrupted by a DNA sequence
encoding a splicing-competent group I intron (tdI). Consistent with a mobility pathway involving an RNA rather
than a DNA intermediate, the group I intron was found to
be absent from the homing products generated in plasmidborne target sites as a result of tdI splicing in the precursor
twintron RNA (Cousineau et al., 1998; Martı́nez-Abarca
et al., 2004). Group II introns can also insert, at a lower
frequency, into noncognate sequences resembling the homing site – a process known as ectopic transposition (retrotransposition) – which contributes to group II intron
dispersal in nature (Cousineau et al., 2000; Martı́nez-Abarca
& Toro, 2000a; Muñoz et al., 2001; Dai & Zimmerly, 2002;
Ichiyanagi et al., 2002).
Of all the bacterial group II introns characterized as
splicing-competent in vivo, only the L. lactis Ll.LtrB and
S. meliloti RmInt1 introns have been shown to be efficient
mobile genetic elements, leading to the use of these elements
as model experimental systems for deciphering the bacterial
group II intron mobility pathways and mechanisms (Lambowitz & Zimmerly, 2004 and references therein).
Retrohoming through target DNA-primed
reverse transcription (TPRT)
The retrohoming pathway of bacterial group II introns was
first investigated for Ll.LtrB, by plasmid-based genetic assays
in vivo, in E. coli or L. lactis. It was then further dissected by
characterization of the biochemical activities of the intronencoded RNPs reconstituted with a purified LtrA expressed
in E. coli and an Ll.LtrB RNA lariat excised from a precursor
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Bacterial group II introns
Fig. 3. Retrohoming pathways of bacterial group II introns. RNP complexes recognize the target site on double- or single-stranded DNA primarily by the
EBS-IBS (d–d 0 in subgroup IIA introns) pairings, whereas the IEP also binds specifically to key nucleotide residues at distal 5 0 - and 3 0 -exon regions
indicated by dotted lines in the diagram (a). The En-dependent retrohoming mechanism (TPRT) used by the L. lactis Ll.LtrB intron involves DNA
unwinding assisted by the domain D of the IEP, reverse splicing of the intron RNA into the top strand of the target DNA, and antisense strand cleavage
catalyzed by the En domain. The cleaved antisense strand is then used by the IEP (RT activity) as primer to reverse transcribe the reverse-spliced intron
RNA (b). The major pathway used for retrohoming by the Sinorhizobium meliloti RmInt1 intron (c) is linked to DNA replication involving invasion of the
DNA strand that serves as a template for lagging strand synthesis. The reverse transcription of the inserted intron RNA could be primed by RNA primers
synthesised by the primase or partial polymerization of the Okazaki fragments by DNA polymerase III. A second possible minor retrohoming pathway (c)
may occur independently of the target-site strand, and involve alternative priming mechanisms. A cDNA copy of the intron is completely integrated in
the new location by the host repair functions.
transcript in vitro (Matsuura et al., 1997; Cousineau et al.,
1998; Saldanha et al., 1999; Aizawa et al., 2003). Neither the
Ll.LtrB RNA itself nor the purified LtrA protein displays
reverse splicing or endonuclease activities of the RNP
particle (Saldanha et al., 1999). Group II intron RNPs
initially bind DNA nonspecifically and then search for
DNA target sites (Aizawa et al., 2003). Mutagenesis experiments, DNA footprinting, and modification interference
FEMS Microbiol Rev 31 (2007) 342–358
mapping have shown that Ll.LtrB-encoded RNPs recognize
a relatively long DNA target site (Fig. 3a) extending from
position 25 to 19 relative to the intron insertion site
(Guo et al., 2000; Mohr et al., 2000; Karberg et al., 2001;
Singh & Lambowitz, 2001). Retrohoming then occurs by a
target DNA-primed reverse transcription mechanism
(TPRT) involving several sequential reactions (Fig. 3b).
The RNA component of the RNP cleaves the sense-strand
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348
precisely at the exon junction in the double-stranded DNA
recipient allele, by a reverse splicing reaction that integrates
the intron RNA at the target site, whereas LtrA uses its
endonuclease domain (En) to cleave the antisense-strand at
position 19 relative to the insertion site. The 3 0 end of the
cleaved antisense strand is then targetted to the active site of
the reverse transcriptase, where it is used as a primer for
reverse transcription of the inserted intron RNA by the RT
domain of LtrA, generating a cDNA copy of the intron that
is subsequently integrated into its new location by homologous recombination-independent repair mechanisms
(Cousineau et al., 1998).
The target sequence is recognized principally by the RNA
component of the RNP complex, through EBS2-IBS1, EBS1EBS2 and d-d 0 base-pairing interactions, which, for Ll.LtrB,
involve positions 12 to 8, 6 to 1 and 11 to 13,
respectively. The nucleotide residues within the IBS and d 0
regions contribute to DNA target-site recognition to different extents, but mutations in these nucleotides have an
overall inhibitory effect on the reverse splicing activity of the
reconstituted RNPs and Ll.LtrB homing frequencies in vivo;
nonetheless, both can be restored to wild-type levels by
introducing the corresponding complementary mutations
into the intron RNA (Guo et al., 2000; Mohr et al., 2000).
The variable DNA-binding domain (D) is involved in the
recognition of a subset of key nucleotide residues (positions
T-23, G-21 and A-20) in the distal 5 0 exon region via major
groove interactions bolstered by phosphate-backbone contacts (Singh & Lambowitz, 2001). LtrA may also be involved
in the recognition of distal nucleotides of IBS2, probably on
the complementary strand of the target site, as the restoration of EBS2-IBS2 base-pairing at these positions does not
result in the recovery of wild-type reverse splicing levels or
homing frequencies. Mutations at these key positions in the
distal 5 0 exon region block reverse splicing of the intron
RNA into double-stranded, but not into single-stranded
DNA, substrates. This suggests that the interaction of these
positions with LtrA triggers local DNA unwinding, thereby
placing the intron RNA in a position to base-pair with the
IBS and d 0 sequences for reverse splicing into doublestranded DNA target sites (Mohr et al., 2000). Consistent
with LtrA interaction and base-pairing of the intron RNA
being concerted rather than sequential to promote the
efficient unwinding of DNA at cognate sites, KMnO4
modification experiments revealed that RNPs reconstituted
with intron RNAs with EBS/d mutations preventing basepairing with the DNA target site do not trigger DNA
unwinding. Recent data indicate that before the reverse
splicing of intron RNA, RNPs bend the target DNA by
maintaining initial contacts with the 5 0 exon while engaging
in 3 0 exon interactions, gradually bringing the scissile
phosphate into position for bottom-strand cleavage (Noah
et al., 2006). Second-strand cleavage, catalyzed by the
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Nicolás Toro et al.
conserved En domain of LtrA, occurs after a time lag, and
requires additional interactions of the protein with fixed
positions in the 3 0 exon, the most critical of which being
position T15, which lies within a single-stranded region
after initial DNA unwinding (Mohr et al., 2000; Singh &
Lambowitz, 2001).
En-independent retrohoming is linked to DNA
replication in bacteria
TPRT-based retrohoming requires the generation of a
primer for reverse transcription of the intron RNA via
second-strand cleavage, catalyzed by the conserved En
domain of the IEP. Phylogenetic analysis based on the
sequence and domain structure of group II intron IEPs, of
both bacterial and eukaryotic origin, assigned a putative
mitochondrial lineage to the Ll.LtrB intron, with most
bacterial introns located elsewhere in the tree. Furthermore,
the C-terminal En domain is found in only c. 40% of the
bacterial group II IEPs currently annotated in databases
(Martı́nez-Abarca & Toro, 2000b; Zimmerly et al., 2001; Dai
& Zimmerly, 2002). The S. meliloti RmInt1 intron is the best
characterized of the group II introns clustering on the
bacterial branch. RmInt1 encodes a protein with conserved
RT and maturase domains and a functionally uncharacterized C-terminal extension of 20 amino acid residues that
appears to be unrelated to the DNA-binding (D) domain of
LtrA (Martı́nez-Abarca et al., 2000; San Filippo & Lambowitz, 2002). Despite the absence of recognizable D and En
domains, RmInt1 retrohomes very efficiently, with 100% of
the intron-containing strains undergoing insertion events at
plasmid-borne target sites (homing frequency), and 20–45%
of target copies within a single cell invaded by the intron
(homing efficiency) (Martı́nez-Abarca et al., 2000; JiménezZurdo et al., 2003). However, experiments with engineered
En mutant Ll.LtrB derivatives and target sites have shown
that Ll.LtrB can retrohome, albeit much less efficiently,
without second-strand cleavage (D’Souza & Zhong, 2002;
Zhong & Lambowitz, 2003). These findings suggest that
there are alternative pathways operating in the mobility of
En bacterial group II introns linked, as described below, to
DNA replication (Muñoz-Adelantado et al., 2003; Zhong &
Lambowitz, 2003; Martı́nez-Abarca et al., 2004).
RmInt1 mobility (Fig. 3a and c) depends on the intron
RNA and the IEP, with the RmInt1-encoded RNP complex
recognizing a DNA target site extending 20 nt into the 5 0
exon and 5 nt into the 3 0 exon, as inferred from genetic
characterization of the intron and target site coupled to
homing assays in vivo (Jiménez-Zurdo et al., 2003; MuñozAdelantado et al., 2003). Subsequent mutational analysis
demonstrated that this site is recognized primarily by the
intron RNA, through the characteristic pairing pattern of
IIB intron splicing: EBS2-IBS2, EBS1-IBS1 and EBS3-IBS3,
FEMS Microbiol Rev 31 (2007) 342–358
349
Bacterial group II introns
involving positions 13 to 9, 7 to 1 and 11 in the
RmInt1 target site, respectively (Jiménez-Zurdo et al., 2003).
Unlike Ll.LtrB, RmInt1 has less stringent requirements for
recognition of the distal 5 0 and 3 0 exon regions, with only
single nucleotide positions (T-15 and G14) required for
wild-type retrohoming (Jiménez-Zurdo et al., 2003). These
positions were initially thought to be recognized by the
RmInt1 IEP, but recent findings suggest that there may be an
alternative intron–exon pairing between an intron segment
overlapping with the EBS2 exon-binding site and a 5 0 exon
site located just distal of IBS2 relative to the splice junction
(IBS2; Costa et al., 2006a, b). Thus, pairing of the first A of
EBS2 with the last T of IBS2 at position 15 of the intron
target site might render the last T of IBS2 critical for
homing. The removal of nucleotides 20 to 16 from the
RmInt1 target site dramatically decreases homing efficiency
(Jiménez-Zurdo et al., 2003). The presumably small number
of IEP–target-site interactions raises questions about the
identity of the IEP domains involved and mechanistic
implications for DNA target-site recognition.
RmInt1-derived RNP particles have RT activity and
promote the reverse splicing of intron RNA into both
single-stranded and double-stranded DNA substrates,
through RNA-mediated cleavage at the intron insertion site.
However, they cannot carry out the En-dependent secondstrand cleavage in double-stranded DNA substrates
(Muñoz-Adelantado et al., 2003). The efficiency of the
reverse splicing reaction is markedly lower with long double-stranded DNA substrates. RmInt1-RNPs have higher
levels of RT activity on exogenous synthetic substrates than
on the endogenous intron RNA template, the reverse
transcription of which in vitro has no obvious priming bias,
suggesting that positioning of the reverse transcriptase on
the precursor RNA facilitates initiation of the RT reaction
various distances downstream from the intron (MuñozAdelantado et al., 2003). According to this biochemical
analysis, the RmInt1-encoded RNP complex should recognize the key nucleotide residues in both single-stranded and
double-stranded DNA target sites. However, the limited
contact with the IEP is insufficient for the promotion of
local DNA unwinding, suggesting that the preferred retrohoming pathway of RmInt1 involves reverse splicing into a
transiently single-stranded DNA target site and that there
may be alternative, uncharacterized priming mechanisms
for reverse transcription of the intron RNA (MuñozAdelantado et al., 2003).
Homing assays in vivo, using recipient plasmids in which
the RmInt1 target site was inserted in both orientations
relative to the origin of replication of the plasmid, have
provided evidence for two distinct retrohoming pathways
for RmInt1 (Fig. 3c). The main mechanism of mobility is
favoured by cell division and involves the reverse splicing of
the intron RNA into single-stranded DNA at DNA replicaFEMS Microbiol Rev 31 (2007) 342–358
tion forks, with a bias towards the DNA strand that serves as
a template for lagging strand synthesis (Martı́nez-Abarca
et al., 2004). This preference suggests that the intron should
be inserted once the replication fork has passed the DNA
target site, avoiding the potentially disruptive passage of the
DNA polymerase complex through the region occupied by
the RNP. Reverse transcription of the intron RNA is then
primed by the nascent lagging strand, using either the RNA
primer synthesized by the primase or partially polymerized
Okazaki fragments. There is a second possible but minor
retrohoming pathway (Fig. 3c), independent of DNA replication, that may involve reverse splicing into either doublestranded or transiently single-stranded DNA target sites and
alternative priming mechanisms, such as random nonspecific DNA nicks (Schäfer et al., 2003), nascent leading strand
(Zhong & Lambowitz, 2003) or de novo initiation priming
(Wang & Lambowitz, 1993). The blocking of Ll.LtrBmediated second-strand cleavage by mutations in the En
LtrA domain or substitutions of key nucleotide residues in
the 3 0 exon revealed a minor retrohoming pathway for the
lactococcal intron, dependent on DNA replication, with
preferential use of the nascent leading strand to prime
reverse transcription – a bias opposite to that for RmInt1
retrohoming (Zhong & Lambowitz, 2003).
Other naturally occurring En group II introns include
those belonging to the IIC subgroup of bacterial introns.
These introns are often found inserted at target sites with
recognizable IBS1 but not IBS2 sequences. The IBS2
sequence is replaced by a palindromic Rho-independent
transcription terminator motif or similar structures (Granlund et al., 2001; Centron & Roy, 2002; Dai & Zimmerly,
2002). Recent data suggest that exon recognition by IIC
introns rely on IBS1 and IBS3 pairings, and putatively on a
new nonpairing interaction with a stem-loop (Toor et al.,
2006).
Dependence of retrohoming on host functions
Group II introns are genetic elements with molecular
features allowing them to maintain themselves and to spread
in a genome. However, their mobility depends, to a variable
extent, on host genetic background, suggesting that retrohoming requires the recruitment of host functions for its
successful completion (Karberg et al., 2001; Toro et al.,
2003). In addition to requiring the replication machinery
during key initial stages, the retrohoming pathway of En
bacterial group II introns requires, at later stages, repair
functions poorly characterized for both endonucleasedependent and -independent retrohoming. A recent study
of Ll.LtrB homing efficiency in various E. coli mutants with
defective DNA/RNA repair functions led to the construction
of a retrohoming completion model including exonucleases
(Recj, MutD and PolI) and RNAses (RNase H) for DNA
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350
resection and removal of the intron RNA template after
first-strand cDNA synthesis, together with complexes of
DNA polymerases and repair polymerases (PolII, PolIII,
PolIV and PolV) for synthesis and proofreading of the
second strand of the intron cDNA (Smith et al., 2005).
However, further functional characterization is required to
determine unambiguously the role of these genes and
proteins in the retrohoming pathway.
Ectopic transposition
Bacterial group II introns can also retrotranspose, at low
frequency (typically 104 to 105), to nonallelic sites by
means of an RNA intermediate (Cousineau et al., 2000;
Martı́nez-Abarca & Toro, 2000a; Ichiyanagi et al., 2002).
However, retrotransposition events were first demonstrated
for eukaryotic mitochondrial group II introns, the RNPs of
which were shown to promote reverse splicing of the intron
RNA into noncognate DNA target sites (Mueller et al., 1993;
Yang et al., 1998; Dickson et al., 2001).
RNA-based retrotransposition was first investigated in
bacteria using an Ll.LtrB twintron variant carrying the selfsplicing group I td intron and a kanamycin resistance gene
for the selection of mobility events (Cousineau et al., 2000).
In contrast to the situation described for mitochondrial
group II introns, these experiments suggested that there may
be a major En-independent and RecA-dependent Ll.LtrB
retrotransposition pathway in L. lactis, probably involving
reverse splicing of the intron RNA into cellular RNA rather
than into DNA targets (Cousineau et al., 2000). However,
the results of other studies are not consistent with this RNAtargetted mechanism as the preferred general pathway for
the retrotransposition of bacterial group II introns: (1) the
S. meliloti RmInt1 intron displays RecA-independent insertion into an ectopic site within the oxi1 gene (Martı́nezAbarca & Toro, 2000a); (2) many bacterial group II introns
are located in nontranscribed intergenic regions (Martı́nezAbarca & Toro, 2000a; Dai & Zimmerly, 2002); (3) for the
group II introns analysed to date, the IEP has been shown to
have an affinity for DNA, rather than for RNA.
Ll.LtrB retrotransposition was analysed further, using
improved twintron donor constructs incorporating the
kanamycin resistance gene as a retrotransposition indicator
gene (RIG) inserted into intron DIV and interrupted by the
td intron. Retrotransposition events can thus be reliably
detected on the basis of kanamycin resistance once the td
intron has been spliced out of the precursor RNA mobility
intermediate (Ichiyanagi et al., 2002). The pattern of Ll.LtrB
spread within the L. lactis genome, as shown by RIG
analysis, is consistent with intron retrotransposition into
double-stranded or single-stranded DNA targets through a
homologous recombination-independent mechanism, as
described for the mitochondrial and bacterial RmInt1
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Nicolás Toro et al.
introns. Insertion events were biased toward the use of the
lagging strand as a template. This suggests a possible major
En-independent retrotransposition pathway involving
reverse splicing of the intron RNA into transiently singlestranded DNA target sites at replication forks, and the use of
the nascent lagging strand to prime reverse transcription of
the inserted intron RNA, a mechanism similar to the retrohoming mechanism of En bacterial group II introns
(Ichiyanagi et al., 2002). An analysis of ectopic insertion
sites for bacterial group II introns revealed that bona fide
IBS1 sequences were conserved, whereas the IBS2 element
and key nucleotide positions in the 5 0 and 3 0 exon sequences
recognized by the protein component of the RNP in TPRTbased retrohoming events were not (Cousineau et al., 2000;
Martı́nez-Abarca & Toro, 2000a, b; Dai & Zimmerly, 2002;
Ichiyanagi et al., 2002). However, these target sites retain
essential recognition elements for insertion events independent of second-strand cleavage. Nevertheless, recent studies
have reported a preferential En-dependent Ll.LtrB retrotransposition mechanism in E. coli, suggesting that the
retrotransposition strategies of bacterial group II introns
may be largely influenced by the host (Coros et al., 2005).
During the retrotransposition of the lactoccocal group II
intron Ll.LtrB in E. coli, insertion occurs preferentially
within the Ori and Ter macrodomains of the chromosome.
This bipolar distribution of retrotransposition events results
from the presence of the IEP (LtrA) at the poles of the cell
(Zhao & Lambowitz, 2005; Beauregard et al., 2006).
Bacterial group II intron retrotransposition has been
shown to occur in natural bacterial populations and is
favoured by targets in transmissible genetic elements, such
as plasmids, providing further support for this mobility
mechanism as the major dissemination strategy of bacterial
group II introns in nature (Muñoz et al., 2001; Ichiyanagi
et al., 2003).
Group II introns as biotechnological tools
Derivatives of group II intron Ll.LtrB have been used for
gene disruption in various gram-negative (E. coli, Shigella
flexneri and Salmonella typhimurium; Karberg et al., 2001)
and gram-positive (i.e. Lactococcus lactis, Clostridium
perfringens and Staphylococcus aureus; Chen et al., 2005;
Yao et al., 2006) bacteria.
A number of characteristics render group II introns
suitable for use as biotechnological tools: (1) they are mobile
elements, integrating with high efficiency into their DNA
targets by a homologous recombination-independent process; (2) they recognize the target DNA mainly by base
pairing, so target specificity can be changed by modifying
the EBS sequences within the intron RNA; (3) they can
mobilize foreign genetic information inserted within the
intron; and (4) minimal host functions are required to
FEMS Microbiol Rev 31 (2007) 342–358
351
Bacterial group II introns
support intron mobility and are probably provided by
conserved housekeeping proteins.
Group II introns were first engineered to increase the
frequency and efficiency of mobility (Guo et al., 2000;
Nisa-Martı́nez et al., 2007; Plante & Cousineau, 2006).
Initial modifications to Ll.LtrB included a deletion in the
domain IV loop removing most of the LtrA ORF (DORF
intron), with the intact LtrA ORF cloned and expressed
downstream from the 3 0 exon. This configuration increased
the frequency of mobility to almost 100% (Guo et al., 2000).
Smaller DORF Ll.LtrB intron derivatives were recently
constructed in which LtrA was inserted upstream from the
5 0 exon; these derivatives also had a higher homing efficiency (Plante & Cousineau, 2006). Similar engineering has
been applied to the RmInt1 intron, resulting in a higher
retrohoming efficiency than for the wild-type intron, with
constructs in which the IEP has been deleted from domain
IV and expressed either upstream from the 5 0 exon or
downstream from the 3 0 exon. The retrohoming efficiency
obtained was also higher (reaching 90%) if the constructs
carried short stretches of wild-type 5 0 and 3 0 exons (20/
15) flanking the DORF intron (Nisa-Martı́nez et al., 2007).
The greater mobility of the engineered introns seems to be
the result of greater stability of the intron RNA, probably
owing to a decrease in susceptibility to degradation by host
nucleases (Guo et al., 2000). In these constructs, the IEP
promotes splicing of the DORF intron. However, after
insertion into a new location, splicing of the DORF
intron cannot occur in the absence of the IEP, resulting in
disruption of the target gene. Introns targetted to the
antisense strand lead to unconditional disruption of
the gene. However, if DORF intron derivatives are retargetted to the sense strand, insertion of the intron generates
a conditional disruption, as splicing can take place if the IEP
is expressed in trans (Frazier et al., 2003; Nisa-Martı́nez
et al., 2007).
Another type of conditional gene disruption has also been
achieved with Ll.LtrB. This conditional mutagenesis is based
on the temperature-sensitive splicing displayed by this
group II intron. The essential gene hsa of S. aureus was
disrupted by an Ll.LtrB-DORF derivative in the sense
orientation with respect to gene transcription. It could
therefore be removed by RNA splicing, permitting the
translation of Hsa. As IEP-assisted splicing is temperaturesensitive, hsa mutants can grow at 32 1C, a temperature at
which splicing can take place, but cannot grow at 43 1C, a
temperature at which splicing cannot occur (Yao et al.,
2006).
Thus, it is currently possible to reprogram group II
introns to insert into any desired target sequence. Guo et al.
(2000) have developed a method based on a random intron
library, making it possible to select introns inserting into the
selected target DNA. Two plasmids are used in this system.
FEMS Microbiol Rev 31 (2007) 342–358
The donor plasmid expresses a set of intron molecules from
a DORF-IEP configuration with randomized EBS sequences
that also contain a T7 promoter, replacing the ORF within
DIV. The second plasmid (the recipient) harbours the
selected target sequence upstream from a promoterless tetR
gene. A sequence encoding an E. coli rrnB T1 transcription
terminator capable of terminating transcription by both the
E. coli and T7 RNA polymerases is inserted upstream from
the target site, and an rrnB T2 terminator, which terminates
transcription by the E. coli RNA polymerase, but not by the
T7 RNA polymerase, is inserted between the target site and
the tetR gene. A phage T7 TF terminator is inserted downstream from the tetR gene to act as a terminator for the T7
RNA polymerase. Insertion of the intron carrying the T7
promoter into the target site activates expression of the tetR
gene. This system can be used to select modified introns
from the randomized library that can insert into the chosen
gene. These introns can be further modified to improve
base-pairing with the target DNA. This system was designed
for use in E. coli, but genes from other organisms could be
cloned and used in this system to select the intron disrupting
the cloned gene most efficiently. This technique has been
used to select an intron disrupting the human CCR5 gene
(coreceptor for human HIV-1 virus; Guo et al., 2000). The
CCR5 gene, carried by a plasmid within embryonic kidney
cells, was then disrupted with the selected intron, which was
introduced into the human cells as RNPs.
The retargetting of bacterial group II introns can be
optimized with a computer algorithm, which scans the
target sequence, selects the best position for IEP recognition,
and then designs primers to modify the intron EBS1, EBS2
and d sequences accordingly (Perutka et al., 2004). The IBS1
and IBS2 sequences in the 5 0 exon of the donor plasmid are
also modified to make them complementary to the retargetted EBS sequences for efficient RNA splicing. The IEP
recognizes few enough positions with a sufficient flexibility
for the identification of several target sites in any gene.
Introns integrated into the target DNA can be selected by
colony PCR, or using a selectable marker, such as an
antibiotic resistance gene. The selectable marker strategy is
possible because group II introns can carry foreign DNA
sequences within their RNA core. This heterologous DNA is
carried in DIV of the group II intron, after removal of the
IEP sequence. The presence of this foreign DNA generally
has a negative effect on homing, but the modified introns
nonetheless display detectable homing (Guo et al., 2000;
Ichiyanagi et al., 2002; Frazier et al., 2003; Zhong et al.,
2003). The main concern with conventional antibiotic
resistance genes is that they are expressed from the donor
plasmid independently of homing, which can hinder the
selection of true homing events. The development of the
retrotransposition-activated selectable marker (RAM) system has overcome this problem. The RAM system is based
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352
on RIG markers, interrupted by a splicing-competent tdI
group I intron (Zhong et al., 2003). The selectable marker is
thus expressed only if retrohoming occurs, facilitating the
monitoring of group II intron dispersal through retrohoming. Almost all resistant, RAM-containing colonies display
disruption of a single target. Subsequent modifications, in
which the TpR gene was flanked by Flp Recombinase Target
sequence (FRT), have adapted the system for multiple gene
disruptions (Broach et al., 1982). The inserted FRT-flanked
RAM marker can thus be excised by expressing the Flp
recombinase, leading to 100% excision. Once the marker has
been excised, a second mutagenesis with the same RAM is
possible. Engineered group II introns carrying both the
RAM-Tp marker and randomized EBSs have been used to
generate a library of E. coli mutants (Zhong et al., 2003).
Most of the intron insertions were found to be located close
to the origin of replication of the chromosome, indicating a
bias favouring this region. However, the library was complex
enough for the detection by PCR of insertions in less
favoured regions. The integrated introns can be isolated
and used to obtain single knockouts in any gene (Yao et al.,
2005).
Bacterial group II intron diversity and
evolution
Group II introns have been found in c. 25% of sequenced
bacterial genomes (Lambowitz & Zimmerly, 2004) and they
are present in the Proteobacteria, Cyanobacteria, Acidobacteria, Actinobacteria, Bacteroides and Firmicutes (gram-positive bacteria) groups (Zimmerly group II-intron database,
http://www.fp.ucalgary.ca/group2introns). Group II introns
were discovered more recently in the Archaea, in which they
Nicolás Toro et al.
have been found only in two closely related methanogenic
species, Methanosarcina mazei and Methanosarcina acetivorans, probably as a result of horizontal transmission from
Eubacteria (Dai & Zimmerly, 2003; Rest & Mindell, 2003;
Toro, 2003). Group II introns are currently classified into
three main phylogenetic subclasses: IIA, IIB and IIC (Michel
et al., 1989; Toor et al., 2001, 2002; Zimmerly et al., 2001;
Ferat et al., 2003; Toro, 2003). Organellar introns from
mitochondria and chloroplasts are most closely related to
bacterial IIA and IIB (IIB1, IIB2) introns, whereas IIC
introns are missing from Cyanobacteria and there are no
close relatives of these introns among organellar introns
(Fig. 4).
The RNA structures of the group II intron ribozyme and
the IEP seem to have coevolved, leading to the ‘retroelement
ancestor hypothesis’, which suggests that all extant group II
introns descended from an RT-encoding group II intron in
bacteria with RNA structural features not conforming to
canonical IIA or IIB structures (Robart & Zimmerly, 2005).
Two different lineages, the canonical IIA and IIB, then
became associated with organelles and the ORF was
repeatedly lost, resulting in the formation of numerous
ORF-less organellar introns (Toor et al., 2001; Lambowitz &
Zimmerly, 2004; Robart & Zimmerly, 2005). ORF-less
introns have also been found in Cyanobacteria (Nakamura
et al., 2002), Bacillus (van der Auwera et al., 2005; Tourasse
et al., 2006) and Archaea (Dai & Zimmerly, 2003),
but they seem to be derived from intron-carrying ORFs.
Interestingly, truncated group II introns are also present in
bacteria (Dai & Zimmerly, 2002; Fernández-López et al.,
2005). Some have a 5 0 truncation that could be accounted
for by incomplete reverse transcription during TPRT.
However, 5 0 truncations account for only a minority of
fragmented introns (Robart & Zimmerly, 2005). Introns
Fig. 4. Phylogenetic relationships between group II intron ORFs and correspondence with RNA structural class. The consensus phylogenetic unrooted
tree shown was obtained using the maximum-parsimony method, and values are for 1000 bootstrap replicates (shown as percentages). The IEP RTs used
for the alignment were the same as previously described (Zimmerly et al., 2001; Toro, 2003). The various mitochondrial group II intron-encoded ORFs
and their accession numbers are as follows: M.p. atpA,1, M.p. atpA,2, M.p. cob,3, M.p. coxI,1, M.p. ORF732, M.p. coxI,2 and M.p. 18S rRNA geneM68929 are from the liverwort Marchantia polymorpha; O.b.nad1,4-M63034 is from the green plant Oenothera berteriana; S.p. cob,1-X54421, K.l.
coxI,1-X57546, S.c. aI1 and S.c. aI2-AJ011856 are from the yeasts Schizosaccharomyces pombe, Kluyveromyces lactis and Saccharomyces cerevisiae,
respectively; N.c. coI,1-S07649 and P.a. coI,1-X55026 are from the fungi Neurospora crassa and Podospora anserina, respectively; P.l.LSU,2.-S58504
and P.l.LSU,1-S58503 are from the brown alga Pylaiella littoralis; P.p. ORF544 and P.p. ORF546-AF114794 are from the red alga Porphyra purpurea. The
chloroplast group II intron-encoded ORFs are S.o. petD,1-P19593 from the green alga Scenedesmus obliquus and B.m. rbcl,1-X55877 from Bryopsis
maxima. The bacterial IEPs are in bold and underlined and named according to Dai & Zimmerly (2002), except for those from the uncultured marine
bacterium (UMB), for which we used its accession number (AAL78688). M.a.I1 (gene name MA4626, AE011185) and M.a.I2 (gene name MA3645,
AE011073) are from Methanosarcina acetivorans str.C2A, and M.m.I1 is from Methanosarcina mazei Goe1 (gene name MM2683, AE013515). RmInt1
(S.me.I1) is from Sinorhizobium meliloti (Y11597), as are S.me.I3 (AL0603646) and S.me.I4 (AJ496462); E.c.I2 is from Escherichia coli (X77508), as are
E.c.I4 (AB024946) and E.c.I5 (AF074613); B.j.I2 (elsewhere B.j.F1) is from Bradyrhizobium japonicum (AF322013), as are B.j.F4/B.j.F5 (AF322012) and
B.j.I1 (AF322013); S.ma.I1 is from Serriata marcescens (AF027768); S.p.I1 is from Streptococcus pneumoniae (AF030367); B.h.I1 is from Bacillus
halodurans (AP01507); P.p.I1 and P.p.I2 are from Pseudomonas putida (AF101076 and Y18999, respectively); P.a.I1 is from Pseudomonas alcaligenes
(AF323437); R.e.I1 is from Ralstonia eutropha (AF261712); C.d.I1 is from Clostridium difficile (X98606); B.me.I1 is from Bacillus megaterium
(AB022308); B.a.I1 and B.a.I2 are from Bacillus anthracis (AF065404) and S.f.I1 is from Shigella flexneri (U97489); C.sp.I1 is from Calothrix sp.
(X71404); L.l.LtrB (L.l.I1) is from Lactococcus lactis; N.a.I1 and N.a.I2 are from Novosphingobium aromaticivorans (AF079317).
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FEMS Microbiol Rev 31 (2007) 342–358
353
Bacterial group II introns
like RmInt1 tend to be inactivated by fragmentation, with
loss of the 3 0 terminus. In some cases, such inactivation
arises from the insertion of other mobile elements and
further genetic rearrangements (Dai & Zimmerly, 2002;
Fernández-López et al., 2005). The fragmentation of
group II introns, together with the accumulation of mutations and natural selection may well be responsible for
the current status of self-splicing group II introns in
bacteria, with the intrinsically large population size and
genome simplification (genomic streamlining) also playing
important roles (Lynch, 2006).
FEMS Microbiol Rev 31 (2007) 342–358
The IEP of full-length bacterial group II introns generally
contains both the RT and maturase domains, but different
groups of introns can be distinguished on the basis of the
presence or absence of the D and/or En domains. The
absence of the D and En domains may result from their
specific loss in some introns. However, whole intron groups
within the IIB class (Fig. 4), such as the IIB3 (also known as
the D class, Zimmerly et al., 2001) and IIB5 lineages (also
known as the E class, Toro et al., 2002; Lambowitz &
Zimmerly, 2004; Robart & Zimmerly, 2005), seem to lack
the D and En domains entirely (Toro, 2003). The IIC class of
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354
Nicolás Toro et al.
require further investigation, it is also plausible that the
ancestor of these introns was actually a catalytic RNA intron
functioning in the absence of the RT protein (Fig. 5),
perhaps in extreme environments capable of supporting
such catalytic activity (Lambowitz & Zimmerly, 2004).
The acquisition of the RT-maturase protein may have
enabled group II introns to extend into bacterial populations, in which they survived and spread by inserting into
nonessential genes, such as other mobile elements.
Their mobility would have been linked to the replication
machinery of the host cell, with insertion into singlestranded DNA target sites at a replication fork, using
a nascent lagging DNA strand as the primer for reverse
transcription. Subsequent acquisition of the D and En
domains would then have enabled group II introns to insert
efficiently by reverse splicing into double-stranded DNA
target sites (Lambowitz & Zimmerly, 2004). This acquisition
may have provided group II introns present in the alphaproteobacterium ancestor of the mitochondria, which
invaded an archaeal host during eukaryogenesis (Fig. 5),
with the features required for the massive invasion of the
host-cell genome, leading to the creation of spliceosomal
introns (Koonin, 2006).
Conclusions and perspectives
Fig. 5. Hypothesis for the evolution of group II introns. The model
predicts a catalytic group II intron RNA functioning in the absence of an
RT and its evolution in bacteria by acquisition of the RT-maturase IEP
leading to the ancestor of the three current intron lineages IIA, IIB and IIC
(a). IIA and IIB introns present in the alphaproteobacterium ancestor of
mitochondria would lead to massive invasion of the archaeal host
genome during eukaryogenesis (Koonin, 2006), thus giving rise to
spliceosomal introns (b).
introns (Fig. 4), characterized by the absence of an EBS2
sequence and a shorter ribozyme DV (Toor et al., 2001;
Toro, 2003), also lacks the En domain, but still has a putative
D domain (San Filippo & Lambowitz, 2002). It is widely
accepted that the ancestral RT probably lacked the D and En
domains (Fig. 5) and that their acquisition and loss in some
cases may have contributed to the evolution of the various
bacterial group II intron lineages.
Phylogenetic analyses of IEP and the intron RNA structures (Martı́nez-Abarca & Toro, 2000b; Toor et al., 2001;
Zimmerly et al., 2001; Toro, 2003) have shown the IIB and
IIA classes to be sister lineages and the IIC class to be the
most divergent lineage (Fig. 4). So which of these lineages of
group II introns is the oldest? The data currently available
provide no final answer to this question, but, according to
the retroelement hypothesis, the earliest branching class of
introns is most likely to be class IIC.
Regardless of the evolutionary relationships between the
current subclasses of group II introns in bacteria, which
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
Since the discovery 14 years ago of group II introns in
bacteria, information has accumulated about the architectural and functional organization of group II introns as
catalytic RNAs, the mechanism of interaction between the
IEP and the intron RNA catalytic core leading to RNP
assembly, the mechanisms of intron mobility and DNA
target site recognition, and the development of these retroelements as new gene-targetting vectors. These advances
have provided new insight into the fundamental nature and
applications of these ribozymes.
Improvements in our understanding of the course of
evolution of group II introns in bacteria should also provide
clues to the origin of the spliceosome and the possible role of
these introns in eukaryogenesis. What role do group II
introns play in bacterial genomes? Although some group II
introns are inserted into nonessential regions of the genome,
others are found in essential genes. The current situation
may result from bacterial genome streamlining and
population size thus decreasing intron numbers or leading
to such genetic elements being entirely absent in some
bacterial species. There may also be unknown mechanisms
in bacteria controlling intron expression and mobility.
Despite extensive debate concerning the way in which
introns have played a critical role in eukaryote evolution
by inserting into protein-coding genes, very little is
known about group II intron evolution in bacteria. Progress
in bacterial genome sequencing programs may shed light on
FEMS Microbiol Rev 31 (2007) 342–358
355
Bacterial group II introns
the evolution of bacterial group II introns, revealing lineages
other than the current known subclasses IIA, IIB and IIC,
and possibly on group II intron RNAs functioning in the
absence of the RT protein. This work may involve sequencing metagenomes from extreme environments. Studies on
natural bacterial populations may also improve our understanding of the current contribution of group II introns to
bacterial evolution and the possible functional role of these
introns. The current view of group II introns as selfish
elements maintained throughout prokaryotic evolution
may turn out to be a simplistic assumption based on our
current lack of knowledge of bacterial group II intron
evolution.
Future studies on group II introns will focus not only on
fundamental evolutionary aspects, but also on the development of gene-targetting vectors. The development of such
tools based on other bacterial introns, with specific mechanistic intron mobility features different from those of Ll.LtrB,
should make this technology more broadly applicable. The
mobility mechanism of these bacterial introns does not
depend on recombination. This feature makes these
retroelements potential tools for genetic manipulation in
higher eukaryotes with low levels of recombination, such
as plants and animals. Like the splicing factors identified
for group II introns in organelles, bacterial group II
introns probably use certain host factors that might
contribute to intron RNA folding and mobility. In the next
few years, efforts should be made to identify such factors,
which might increase the functionality of these bacterial
group II introns in various prokaryotic and eukaryotic
hosts.
Studies on the architectural and functional organization
of group II introns will continue to expand our knowledge
about the splicing mechanism of these ribozymes, and will
also contribute to parallel investigations on related elements,
such as spliceosomes and retrotransposons. The excision of
group II introns as circles has been demonstrated to occur
in vivo for yeast introns such as aI2 and for some plant
mitochondrial introns. The recent detection of circular
intron molecules in bacteria indicates that this particular
mode of excision may be more widespread in nature than
initially thought. The splicing mechanism involved in group
II intron circle formation, and the biological role of such
circles in intron spread remain key issues that we can expect
to see resolved in the next few years.
Acknowledgements
Work at the authors’ laboratory was supported by the
Spanish Ministerio de Educación y Ciencia (BIO200502312) and Junta de Andalucı́a (CVI-01522). F.M.G.-R.
holds an I3P postdoctoral fellowship (CSIC), and J.I.J.-Z. is
a Ramón & Cajal hired scientist.
FEMS Microbiol Rev 31 (2007) 342–358
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