Vol. 187, No. 23
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
A Second Case of
Ϫ1 Ribosomal Frameshifting Affecting a Major
Virion Protein of the Lactobacillus Bacteriophage A2
Isabel Rodrı´guez, Pilar Garcı´a, and Juan E. Sua
Received 23 June 2005/Accepted 12 September 2005
؊1 translational frameshift to generate two
of the gene. The major head gene presents a similar recoding ability. A2 is the only phage described with two
Ϫ1 frameshifting is generated through a back-
ward displacement of the translating ribosome to the position
Ϫ1, which results in the synthesis of two polypeptides, one that
arises from translation of the gene in the 0 frame and another
that is identical to it until the frameshifting position but whose
sequence corresponds to the message encoded in the
from that point onwards (1). Two cis-acting elements are com-
monly necessary for
Ϫ1 frameshifting. The ﬁrst is a hep-
tanucleotide sequence that allows tandem slippage of the
tRNAs located at the functional A and P sites of the ribosome
and repairing of their respective anticodon triplets with the
codons that result in the
Ϫ1 frame. The second is a structure
that promotes ribosome pausing at the slippery sequence, such
as a stem loop or a pseudoknot that starts in its 3
Ј vicinity (3,
5, 11) or a Shine-Dalgarno-like sequence located about 10
Ј of the slippery sequence (10, 15). The functional
Ϫ1 frameshifting may be to produce two proteins
in a deﬁned ratio. In the case of viral genes the “frameshifted”
product is usually essential for viability, both in retroviruses,
where synthesis of a ﬁxed proportion of gpGag-Pol is required
for efﬁcient packaging of reverse transcriptase (21), and in
bacteriophages, for example, in lambda the fusion protein
gpG-T is essential for tail assembly, even though it does not
become part of the mature virion (12). However, in the case of
cellular genes the frameshifted products may be dispensable,
(15) although, as in the gamma subunit of DNA polymerase III
of Escherichia coli (2), it may be important for the activity of
the enzyme (19).
A2 is a temperate bacteriophage that belongs to the family
Siphoviridae (8). The two major proteins of the capsid share
their amino termini, which matched an internal sequence of
rise to two polypeptides of different sizes, the smaller (gp5A)
resulting from canonical translation of orf5 and the larger
(gp5B) being generated by a
Ϫ1 ribosomal frameshift at the
penultimate codon of orf5 mRNA, resulting in a product that
is 85 amino acids longer than gp5A. Frameshifting is depen-
dent on a slippery region with the sequence CCCAAAA and
on a stem loop that begins 9 nucleotides after the end of the
slippery sequence. Both gp5A and gp5B appear to be essential
for phage viability, because although lysogens harboring
prophages that produce only one or the other protein become
lysed upon induction with mitomycin C, no viable phage prog-
eny are observed (7).
In this work, data are presented which indicate that
frameshifting during the phage A2 lytic cycle is not restricted
the transcript for the major tail proteins. In addition, some
requirements for this recoding event are studied.
Bacteriophage A2 was propagated and assayed on Lactoba-
different versions of orf10 cloned into plasmid pET11a (22).
Plasmid constructions, site-directed mutagenesis, and protein
labeling and detection were performed as described previously
Puriﬁcation of gp10A and gp10B was achieved after overex-
ϫ g for 90 min) and successively loaded
onto Q-Sepharose, Mono Q, and Superdex 75 columns (Phar-
macia). The puriﬁed proteins were digested with porcine tryp-
sin (Promega), and the resulting peptides were analyzed by
matrix-assisted laser desorption ionization–time of ﬂight mass
spectrometry, essentially as previously described (7).
To achieve overproduction of gp10A and gp10B in L. casei,
orf10 with its 3
Ј-adjacent region was placed under the nisin
) using plasmids pEM117 and pEM110 and the
procedure described previously was followed (14). The result-
ing plasmid, pEM110-orf10, was electroporated into an L. casei
derivative that expressed the quorum-sensing system that pro-
induction (9). Overexpression was obtained by ad-
dition of nisin to exponential cultures of this L. casei strain.
gp10-speciﬁc antibodies were obtained from rabbits, injected
at 2-week intervals with 1 mg of pure gp10A emulsiﬁed 1:1 in
incomplete Freund’s adjuvant. For Western blotting of L. casei
extracts, proteins were separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and electrotransferred to
an Immobilon-P membrane (Millipore). The membrane was
incubated with anti-gp10 rabbit immunoglobulin G (IgG) (1:
* Corresponding author. Mailing address: Area de Microbiologı´a,
Facultad de Medicina, Universidad de Oviedo, Julia
´n Claverı´a 6,
33006 Oviedo, Spain. Phone: 34-985103559. Fax: 34-985103148. E-mail:
IgG (1:5,000) (Jackson ImmunoResearch Laboratories). The
Western blots were developed using the BM chemilumines-
cence blotting substrate (POD) (Roche Diagnostics).
؊1 frameshifting. In silico analysis of the
A2 genome revealed a heptanucleotide (CCCAAAA) at the
end of the major tail protein gene (orf10) which was identical
to the slippery sequence previously encountered in orf5 (7).
This heptanucleotide was immediately followed by a putative
H-type pseudoknot (
⌬G ϭ Ϫ31.9 kcal/mol), thus suggesting
Ϫ1 frameshift might occur at the end of the orf10
residues polypeptide, to a second one of 283 amino acids (Fig.
1a and b).
To determine whether these structures were functional, a
DNA segment that comprises orf10 plus its 400-bp downstream
stretch was cloned into pET11a and overexpressed. Two
polypeptides of sizes compatible with that of the expected gp10
and with the product that would result from a
Ϫ1 frameshift in
the predicted position were observed (Fig. 2a, lane 1). Mass
spectrometry of the tryptic peptides of these two polypeptides
revealed that all those present in the smaller one (gp10A) were
also present in the bigger one (gp10B), including one that
would end at the lysine encoded by the second codon of the
slippery sequence. In addition, gp10B generated two peptides
of 2,279 and 1,792 Da, matching the expected masses of two
tryptic peptides that would result from translation of the mes-
sage downstream of orf10 when read in the
Ϫ1 frame (Fig. 1a).
To conﬁrm the role of the slippery sequence, point muta-
tions were introduced in different positions of the heptanucle-
otide and their effect on frameshifting was tested. Any muta-
tions that induced a change in the dipeptide Pro-Lys encoded
by the slippery sequence in both the 0 and
Ϫ1 frames abolished
gp10B (Fig. 2a, lanes 2 to 7), suggesting the need for compat-
ibility of the anticodons of the tRNAs located at the A and P
ribosomal sites with their complementary codons in both
To test the functionality of the putative pseudoknot, a 48-bp
the intermediate loop (nomenclature used is as described in
reference 16) while preserving the slippery sequence and the
reading frame after the deleted DNA stretch (Fig. 1a). The
clone harboring the mutant gene would be expected to gener-
ate gp10A (21.8 kDa) and a shorter form of gp10B (28.0 kDa
instead of 29.6 kDa of the wild-type form), if the pseudoknot
did not play a role in frameshift occurrence. However, only
gp10A was visualized in the gels (Fig. 2b, lane 2), thus con-
ﬁrming the role of this secondary structure in frameshift oc-
To test whether frameshifting occurred in L. casei as well as
in E. coli, orf10 with its adjacent 3
Ј sequence was cloned in a
plasmid under the control of the nisin promoter. After induc-
tion of the system, two polypeptides were observed that re-
acted with gp10-speciﬁc antibodies and had masses compatible
with the expected sizes of gp10A and gp10B (Fig. 2c, lane 2),
indicating that the
Ϫ1 frameshift signals were being recognized
in L. casei.
Both gp10A and gp10B are late proteins that become incor-
porated into mature A2 virions.
To determine the production
kinetics of both proteins, L. casei lysogenic exponential cul-
tures were induced with mitomycin C, and samples collected
during the lytic development of the phage were analyzed for
their protein content by Western blotting (Fig. 2d). Expression
of orf10 became evident at 90 min postinduction, but initially
only gp10A was observed. Detection of gp10B was delayed
until 240 min postinduction (under those conditions the cul-
tures start to lyse at about 300 min postinduction). This might
reﬂect a very different rate of biosynthesis for these proteins,
the canonical form being very favored over the frameshifted
product, which would reach a concentration detectable by the
antibodies only after a long period of accumulation. Both
FIG. 1. a) DNA and deduced protein sequences of the A2 genome region surrounding the 3
Ј end of the major tail protein gene (orf10); in the
DNA, the slippery sequence is indicated in boldface and italics, and the ﬁrst stem-loop is indicated with converging arrows. The 48-bp segment
deleted from it is boxed. Relevant polypeptides obtained by tryptic digestion of gp10A and/or gp10B are underlined. b) Proposed secondary
structure of the mRNA frameshift stimulatory element located downstream of the slippery sequence.
on July 28, 2018 by guest
A2), indicating that they contribute to the ﬁnal structure of the
Ϫ1 frameshifting appears to be quite common
among genes involved in virion morphogenesis of tailed bac-
teriophages. It was ﬁrst described for the gene that encodes the
major head proteins of T3 and T7 (4) and later for generation
of the G-T fusion proteins of lambda (12) and many other
siphoviruses and myoviruses infecting both Eubacteria and Ar-
chaea (23). What makes A2 peculiar is that
occurs upon translation of both the major head and tail genes
and that the four resulting polypeptides are part of the virion
particle. In addition, A2 does not have a system similar to the
one that promotes formation of the G-T fusion protein in
other phages: the orf located in a position similar to that of the
lambda gene g is immediately followed by several stop codons
in all three possible frames, thus precluding formation of any
No amino acid sequence similarities were found between the
canonical products of the genes that encode the major head
and tail proteins of A2, i.e., gp5A and gp10A; however, their
frameshifted counterparts, gp5B and gp10B, showed extensive
homology in their extra carboxy-terminal ends. These stretches, of
85 and 81 amino acids, respectively, are 42% identical (63%
similar) (Fig. 3). The predicted structures of these two protein
segments are related to bacterial immunoglobulin-like folding
domains deﬁned by pfam02368 (Big 2) and COG5492 (13).
Similar domains are found in tail components of phages such
as N15 and K (18, 20), bacterial surface proteins involved in
FIG. 2. a) Effect on frameshifting of different point mutations (underlined) at the slippery sequence of orf10 (in boldface). b) Outcome of the
deletion of the central part of the stem loop located downstream of the slippery sequence (lane 2); lane 1, control gene. orf10 (and its variants)
were placed under the control of the P10 promoter of T7 and overexpressed in vivo in E. coli in the presence of a
S-labeled amino acid mix and
d) Time course of accumulation of gp10A and gp10B in exponential cultures of an A2 lysogenic culture of L. casei induced with mitomycin C (0.5
g/ml); the ﬁgures above the lanes indicate time postinduction in minutes, L. casei stands for an uninduced culture, and A2 corresponds to puriﬁed
virions (the two bands below gp10A in this last lane are degradation products of this protein). The proteins were separated by sodium dodecyl
sulfate–12% polyacrylamide gel electrophoresis and autoradiographed (E. coli) or detected by Western blotting (L. casei).
FIG. 3. Alignment of the amino acid sequences encoded by the
stretches located in the 3
Ј vicinity of the major head (MHP) and tail
(MTP) A2 genes after
Ϫ1 frameshifting with the COG5492 and
PFAM02368 (bacterial and phage surface proteins containing immu-
noglobulin-like domains) consensus motifs. The sequences of the por-
tions of three individual proteins with different functions (a
the text for references) that contain these motifs are also included for
substrates, such as an agarase from Microscilla spp. or a
3-endoglucanase from Bacillus circulans (24, 25) (Fig. 3). The
in bacterial products mainly involved in carbohydrate recogni-
tion might indicate that the phage proteins function in the
stabilization of the phage particle on the polysaccharide-based
cell envelope once the speciﬁc binding by the tail tip has oc-
curred (A. Davidson, personal communication). The phage A2
has an extraordinarily long tail of about 280 nm, which would
justify the need of these “molecular hooks” at the level of both
the head and tail of the infecting virion to retain its infectivity,
thus explaining why gp5B is essential for phage propagation
This work was supported by CICYT grants BMC2002-0638 and
and the FEDER Plan. P.G. and I.R. are holders, respectively, of a
fellowship and a scholarship associated with these grants.
We thank K. F. Chater for critical reading of the manuscript. The
proteomics service of the National Biotechnology Centre (CSIC) is
1. Baranov, P. V., R. F. Gesteland, and J. F. Atkins. 2002. Recoding: transla-
tional bifurcations in gene expression. Gene 286:187–201.
2. Blinkova, A., C. Hervas, P. T. Stukenberg, R. Onrust, M. E. O’Donnell, and
1993. The Escherichia coli DNA polymerase III holoenzyme
contains both products of the dnaX gene, tau and gamma, but only tau is
essential. J. Bacteriol. 175:6018–6027.
3. Brierley, I. 1995. Ribosomal frameshifting viral RNAs. J. Gen. Virol. 76:
4. Condron, B. G., J. F. Atkins, and R. F. Gesteland. 1991. Frameshifting in
gene 10 of bacteriophage T7. J. Bacteriol. 173:6998–7003.
5. Condron, B. G., R. F. Gesteland, and J. F. Atkins. 1991. An analysis of
sequences stimulating frameshifting in the decoding of gene 10 of bacterio-
phage T7. Nucleic Acids Res. 19:5607–5612.
6. Garcia, P., V. Ladero, and J. E. Suarez. 2003. Analysis of the morphogenetic
cluster and genome of the temperate Lactobacillus casei bacteriophage A2.
Arch. Virol. 148:1051–1070.
7. Garcia, P., I. Rodriguez, and J. E. Suarez. 2004. A
Ϫ1 ribosomal frameshift
in the transcript that encodes the major head protein of bacteriophage A2
mediates biosynthesis of a second essential component of the capsid. J.
8. Herrero, M., C. G. de los Reyes-Gavila
393-A2, a bacteriophage that infects Lactobacillus ca-
9. Kuipers, O. P., P. G. G. A. De Ruyter, M. Kleerebezem, and W. M. De Vos.
1998. Quorum sensing-controlled gene expression in lactic acid bacteria.
J. Biotechnol. 64:15–21.
10. Larsen, B., N. M. Wills, R. F. Gesteland, and J. F. Atkins. 1994. rRNA-
mRNA base pairing stimulates a programmed
Ϫ1 ribosomal frameshift. J.
11. Larsen, B., R. F. Gesteland, and J. F. Atkins. 1997. Structural probing and
mutagenic analysis of the stem-loop required for Escherichia coli dnaX ri-
bosomal frameshifting: programmed efﬁciency of 50%. J. Mol. Biol. 271:47–
translational frameshift is required for the synthesis of a bacteriophage
lambda tail assembly protein. J. Mol. Biol. 234:124–139.
13. Marchler-Bauer, A., and S. H. Bryant. 2004. CD-Search: protein domain
annotations on the ﬂy. Nucleic Acids Res. 32:W327–W331. [Online.]
14. Martin, M. C., M. Fernandez, J. M. Martin-Alonso, F. Parra, J. A. Boga, and
2004. Nisin-controlled expression of Norwalk virus VP60
protein in Lactobacillus casei. FEMS Microbiol. Lett. 237:385–391.
15. Mejlhede, N., J. F. Atkins, and J. Neuhard. 1999. Ribosomal
ing during decoding of Bacillus subtilis cdd occurs at the sequence CGA
AAG. J. Bacteriol. 181:2930–2937.
16. Moon, S., Y. Byun, H. J. Kim, S. Jeong, and K. Han. 2004. Predicting genes
Ϫ1 and ϩ1 frameshifts. Nucleic Acids Res. 32:4884–4892.
17. Nolling, J., G. Breton, M. V. Omelchenko, K. S. Markarova, Q. Zeng, R.
Gibson, H. M. Lee, J. Dubois, D. Qiu, J. Hitti, Y. I. Wolf, R. L. Tatusov, F.
Sabathe, L. Doucette-Stamm, P. Soucaille, M. J. Daly, G. N. Bennett, E. V.
Koonin, and D. R. Smith.
2001. Genome sequence and comparative analysis
of the solvent-producing bacterium Clostridium acetobutylicum. J. Bacteriol.
18. O’Flaherty, S., A. Coffey, R. Edwards, W. Meaney, G. F. Fitzgerald, and R. P.
2004. Genome of staphylococcal phage K: a new lineage of Myoviridae
infecting gram-positive bacteria with a low G
ϩC content. J. Bacteriol. 186:
19. Pritchard, A. E., H. G. Dallmann, B. P. Glover, and C. S. McHenry. 2000. A
novel assembly mechanism for the DNA polymerase III holoenzyme DnaX
complex: association of deltadelta
Ј with DnaX(4) forms DnaX(3)deltadeltaЈ.
EMBO J. 19:6536–6545.
20. Ravin, V., N. Ravin, S. Casjens, M. E. Ford, G. F. Hatfull, and R. W.
2000. Genomic sequence and analysis of the atypical temperate
bacteriophage N15. J. Mol. Biol. 299:53–73.
21. Ribas, J. C., and R. B. Wickner. 1998. The Gag domain of the Gag-Pol fusion
protein directs incorporation into the L-A double-stranded RNA viral par-
ticles in Saccharomyces cerevisiae. J. Biol. Chem. 273:9306–9311.
22. Studier, F. W., A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff. 1990. Use
of T7 RNA polymerase to direct expression of cloned genes. Methods En-
23. Xu, J., R. W. Hendrix, and R. L. Duda. 2004. Conserved translational frame-
shift in dsDNA bacteriophage tail assembly genes. Mol. Cell 16:11–21.
24. Yamamoto, M., R. Aono, and K. Horikoshi. 1993. Structure of the 87-kDa
beta-1,3-glucanase gene of Bacillus circulans IAM1165 and properties of the
enzyme accumulated in the periplasm of Escherichia coli carrying the gene.
Biosci. Biotechnol. Biochem. 57:1518–1525.
25. Zhong, Z., A. Toukdarian, D. Helinski, V. Knauf, S. Sykes, J. E. Wilkinson,
2001. Sequence analysis
of a 101-kilobase plasmid required for agar degradation by a Microscilla
isolate. Appl. Environ. Microbiol. 67:5771–5779.