Bacterial group II introns: not just splicing

Bacterial group II introns: not just splicingNicolas Toro, Jose Ignacio Jimenez-Zurdo & Fernando Manuel Garcıa-Rodrıguez

Grupo de Ecologıa Genetica de la Rizosfera, Estacion Experimental del Zaidın, Consejo Superior de Investigaciones Cientıficas, Granada, Spain

Correspondence: Nicolas Toro, Grupo de

Ecologıa Genetica de la Rizosfera, Estacion

Experimental del Zaidın, Consejo Superior de

Investigaciones Cientıficas, 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

Editor: Rafael Giraldo

Keywords

catalytic RNA; homing; splicing; retroelements;

Lactococcus lactis ; Sinorhizobium meliloti .

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.

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 (retro-

homing) 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 Term-

inal 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 alpha-

proteobacterial progenitor of the mitochondria; they were

then fragmented to form the spliceosome (Cavalier-Smith,

1991; Stoltzfus, 1999). It has recently been suggested that the

spread of group II introns and their decay into spliceosomal

introns created a strong selective pressure, necessitating

compartmentalization of the nucleus and cytoplasm (Mar-

tin & Koonin, 2006). In addition to their possible evolu-

tionary 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; do-

main X – a putative RNA-binding domain associated with

RNA splicing or maturase activity-, a C-terminal DNA-

binding 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

(Martınez-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

FEMS Microbiol Rev 31 (2007) 342–358c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

are descended from an RT-encoding group II intron in

bacteria (Toor et al., 2001). Three main phylogenetic sub-

classes (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

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.

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.

FEMS Microbiol Rev 31 (2007) 342–358 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

343Bacterial group II introns

Self-splicing mechanisms and products

Several bacterial group II introns have been found to self-

splice 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 50 splice junction by the

20-OH of a bulged nucleotide, typically an adenosine residue

(bulging A) located in DVI near the 30 end of the intron,

releasing the 50 exon and generating an intron-30 exon

branched intermediate. The second reaction involves a

nucleophilic attack on the 30 splice site by the free 30-OH of

the last nucleotide of the 50-exon, yielding the ligated exons

and the intron RNA lariat with a 20–50 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

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 Strepto-

coccus 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 20–50 phosphodiester bond between

the last and first residues of the intron, and it has been

proposed that this reaction requires the 30 exon to have been

released first from precursor molecules by a trans-splicing

reaction triggered by 50 exon molecules previously generated

by the so-called spliced exon reopening (SER) reaction.

Subsequent to trans splicing, the 20-OH of the terminal

intron residue will attack the 50 splice site, releasing the

50 exon and an intron circle (Murray et al., 2001).

Group II ribozyme positioning on the precursor mRNA

substrate and cleavage site fidelity are determined princi-

pally by base-pairing interactions between short-sequence

elements located in loop structures of intron DI (exon-

binding 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 30 end of the 50

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 30 intron–

exon junction involves two additional base-pair interac-

tions. The first is a single tertiary base-pair interaction

between the last position of the intron (g0) 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 30 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 30 exons are indicated with black

and grey boxes, respectively, whereas the line represents the intron RNA. Double arrows indicate the reversibility of some reactions.


344 Nicolas Toro et al.

three nucleotide residues (d0 position) of the 30 exon for

intron splicing (3 nt in the Lactococcus lactis Ll.LtrB intron).

In contrast, most IIB introns display no significant signs of

d–d0 complementarity. These group II ribozymes recognize

the first nucleotide in the 30 exon, referred to as IBS3 rather

than d0, 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 d0), also located

in the coordination loop (Costa et al. 2000). These nucleo-

tides in the loop are involved in aligning the two exons for

the second step of splicing (d–d0 and IBS3–EBS3 interac-

tions). 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, 50 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 (Fedor-

ova 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–e0 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 50 splice site (Hamill & Pyle, 2006). The

docking site for DV, a tetraloop receptor motif composed of

the z–z and k–k0 interactions, is separated from the coordi-

nation 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 20-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 30 splice

site of B.a.I2 is flexible but depends on potential g–g0 and

IBS3–EBS3 pairings downstream from the wild-type site.

Alternative splice-site usage either creates frame shifts with-

in 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 30 to the intron might be

responsible for the generation of unconventional self-spli-

cing products along with the ligated exons and the excised

intron. These unconventional self-splicing products include

a 30 exon truncated at its 50 end, truncated variants of the

linear and lariat forms of the intron –30 exon intermediate,

and putative circular molecules derived from this inter-

mediate (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 (Martınez-

Abarca et al., 1998); a Clostridium difficile intron (Roberts

et al., 2001); the Bacillus anthracis B.a.I2; and the Trichodes-

mium 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; Martınez-Abarca et al., 1998;

Munoz-Adelantado et al., 2003). Bacterial group II intron-

encoded 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 nonfunc-

tional ORF-less introns, a property that has evolutionary

and biotechnological implications (Cui et al., 2004; Meng

et al., 2005; Nisa-Martınez 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



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 50 and 30 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 long-

range 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-Sanchez 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-Sanchez et al., 2006). Thus, in vivo the

presence of the IEP seems to ensure cleavage at the correct 30

splice site or at position 11 in the 30 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 per-

spectives 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 (Martınez-Abarca

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 (Lam-

bowitz & 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 mobilitymechanisms

Group II introns act both as large catalytic RNAs and as site-

specific retroelements (more recently reviewed by Lambow-

itz & Zimmerly, 2004; Pyle & Lambowitz, 2006). After

splicing, the intron RNA lariat and the IEP remain asso-

ciated, forming a ribonucleoprotein particle (RNP) promot-

ing 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). Con-

sistent 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 plasmid-

borne target sites as a result of tdI splicing in the precursor

twintron RNA (Cousineau et al., 1998; Martınez-Abarca

et al., 2004). Group II introns can also insert, at a lower

frequency, into noncognate sequences resembling the hom-

ing site – a process known as ectopic transposition (retro-

transposition) – which contributes to group II intron

dispersal in nature (Cousineau et al., 2000; Martınez-Abarca

& Toro, 2000a; Munoz 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 (Lam-

bowitz & Zimmerly, 2004 and references therein).

Retrohoming through target DNA-primedreverse 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 intron-

encoded RNPs reconstituted with a purified LtrA expressed

in E. coli and an Ll.LtrB RNA lariat excised from a precursor



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 experi-

ments, DNA footprinting, and modification interference

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

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–d0 in subgroup IIA introns) pairings, whereas the IEP also binds specifically to key nucleotide residues at distal 5 0- and 30-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.



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 30 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 homo-

logous 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, EBS1-

EBS2 and d-d0 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 d0

regions contribute to DNA target-site recognition to differ-

ent 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 50 exon region via major

groove interactions bolstered by phosphate-backbone con-

tacts (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 restora-

tion 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 50 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 d0 sequences for reverse splicing into double-

stranded 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 base-

pairing 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 50 exon while engaging

in 30 exon interactions, gradually bringing the scissile

phosphate into position for bottom-strand cleavage (Noah

et al., 2006). Second-strand cleavage, catalyzed by the

conserved En domain of LtrA, occurs after a time lag, and

requires additional interactions of the protein with fixed

positions in the 30 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 DNAreplication 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

(Martınez-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 uncharacter-

ized C-terminal extension of 20 amino acid residues that

appears to be unrelated to the DNA-binding (D) domain of

LtrA (Martınez-Abarca et al., 2000; San Filippo & Lambow-

itz, 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) (Martınez-Abarca et al., 2000; Jimenez-

Zurdo 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 (Munoz-Adelantado et al., 2003; Zhong &

Lambowitz, 2003; Martınez-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 50

exon and 5 nt into the 30 exon, as inferred from genetic

characterization of the intron and target site coupled to

homing assays in vivo (Jimenez-Zurdo et al., 2003; Munoz-

Adelantado 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,



involving positions �13 to �9, �7 to �1 and 11 in the

RmInt1 target site, respectively (Jimenez-Zurdo et al., 2003).

Unlike Ll.LtrB, RmInt1 has less stringent requirements for

recognition of the distal 50 and 30 exon regions, with only

single nucleotide positions (T-15 and G14) required for

wild-type retrohoming (Jimenez-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 50 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

(Jimenez-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 second-

strand cleavage in double-stranded DNA substrates

(Munoz-Adelantado et al., 2003). The efficiency of the

reverse splicing reaction is markedly lower with long dou-

ble-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 (Munoz-

Adelantado et al., 2003). According to this biochemical

analysis, the RmInt1-encoded RNP complex should recog-

nize 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 retro-

homing 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 (Munoz-

Adelantado 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 replica-

tion forks, with a bias towards the DNA strand that serves as

a template for lagging strand synthesis (Martınez-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 repli-

cation, that may involve reverse splicing into either double-

stranded or transiently single-stranded DNA target sites and

alternative priming mechanisms, such as random nonspe-

cific DNA nicks (Schafer et al., 2003), nascent leading strand

(Zhong & Lambowitz, 2003) or de novo initiation priming

(Wang & Lambowitz, 1993). The blocking of Ll.LtrB-

mediated second-strand cleavage by mutations in the En

LtrA domain or substitutions of key nucleotide residues in

the 30 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 (Gran-

lund 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 retro-

homing 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 endonuclease-

dependent 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



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 10�4 to 10�5), to nonallelic sites by

means of an RNA intermediate (Cousineau et al., 2000;

Martınez-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 self-

splicing 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 RNA-

targetted mechanism as the preferred general pathway for

the retrotransposition of bacterial group II introns: (1) the

S. meliloti RmInt1 intron displays RecA-independent inser-

tion into an ectopic site within the oxi1 gene (Martınez-

Abarca & Toro, 2000a); (2) many bacterial group II introns

are located in nontranscribed intergenic regions (Martınez-

Abarca & 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

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 single-

stranded 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 retro-

homing 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 50 and 30 exon sequences

recognized by the protein component of the RNP in TPRT-

based retrohoming events were not (Cousineau et al., 2000;

Martınez-Abarca & Toro, 2000a, b; Dai & Zimmerly, 2002;

Ichiyanagi et al., 2002). However, these target sites retain

essential recognition elements for insertion events indepen-

dent of second-strand cleavage. Nevertheless, recent studies

have reported a preferential En-dependent Ll.LtrB retro-

transposition 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 (Munoz 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 pro-

cess; (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



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-Martınez 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 30 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

50 exon; these derivatives also had a higher homing effi-

ciency (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 50 exon or

downstream from the 30 exon. The retrohoming efficiency

obtained was also higher (reaching 90%) if the constructs

carried short stretches of wild-type 50 and 30 exons (�20/

15) flanking the DORF intron (Nisa-Martınez 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 retar-

getted 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-Martınez

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 temperature-

sensitive, 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.

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 down-

stream 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 50 exon of the donor plasmid are

also modified to make them complementary to the retar-

getted 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) sys-

tem has overcome this problem. The RAM system is based



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 retrohom-

ing. 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 andevolution

Group II introns have been found in c. 25% of sequenced

bacterial genomes (Lambowitz & Zimmerly, 2004) and they

are present in the Proteobacteria, Cyanobacteria, Acidobac-

teria, Actinobacteria, Bacteroides and Firmicutes (gram-posi-

tive 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

have been found only in two closely related methanogenic

species, Methanosarcina mazei and Methanosarcina acetivor-

ans, 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; Fernandez-Lopez et al.,

2005). Some have a 50 truncation that could be accounted

for by incomplete reverse transcription during TPRT.

However, 50 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 gene-

M68929 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).



like RmInt1 tend to be inactivated by fragmentation, with

loss of the 30 terminus. In some cases, such inactivation

arises from the insertion of other mobile elements and

further genetic rearrangements (Dai & Zimmerly, 2002;

Fernandez-Lopez et al., 2005). The fragmentation of

group II introns, together with the accumulation of muta-

tions 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).

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



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 struc-

tures (Martınez-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

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 popula-

tions, 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 single-

stranded 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 alpha-

proteobacterium 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

Since the discovery 14 years ago of group II introns in

bacteria, information has accumulated about the architec-

tural 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 retro-

elements 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

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).



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 sequen-

cing metagenomes from extreme environments. Studies on

natural bacterial populations may also improve our under-

standing 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 develop-

ment of gene-targetting vectors. The development of such

tools based on other bacterial introns, with specific mechan-

istic 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 Educacion y Ciencia (BIO2005-

02312) and Junta de Andalucıa (CVI-01522). F.M.G.-R.

holds an I3P postdoctoral fellowship (CSIC), and J.I.J.-Z. is

a Ramon & Cajal hired scientist.

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Bacterial group II introns: not just splicing

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