Nucleotide sequence of a ssRNA phage from Acinetobacter: kinship to coliphages

J. Klovins1,2, G. P. Overbeek1, S. H. E. van den Worm1, H.-W. Ackermann3 and J. van Duin1

Department of Biochemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands1
Biomedical Research Centre, University of Latvia, Riga, Latvia2
Félix d'Hérelle Reference Centre for Bacterial Viruses, Department of Microbiology, Medical Faculty, Laval University, Québec, Canada G1K 7P43

Author for correspondence: J. van Duin. Fax +31 71 527 4340. e-mail j.duin{at}chem.leidenuniv.nl


   Abstract
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Abstract
Introduction
Methods
Results and Discussion
References
 
The complete nucleotide sequence of ssRNA phage AP205 propagating in Acinetobacter species is reported. The RNA has three large ORFs, which code for the following homologues of the RNA coliphage proteins: the maturation, coat and replicase proteins. Their gene order is the same as that in coliphages. RNA coliphages or Leviviridae fall into two genera: the alloleviviruses, like Q{beta}, which have a coat read-through protein, and the leviviruses, like MS2, which do not have this coat protein extension. AP205 has no read-through protein and may therefore be classified as a levivirus. A major digression from the known leviviruses is the apparent absence of a lysis gene in AP205 at the usual position, overlapping the coat and replicase proteins. Instead, two small ORFs are present at the 5' terminus, preceding the maturation gene. One of these might encode a lysis protein. The other is of unknown function. Other new features concern the 3'-terminal sequence. In all ssRNA coliphages, there are always three cytosine residues at the 3' end, but in AP205, there is only a single terminal cytosine. Distantly related viruses, like AP205 and the coliphages, do not have significant sequence identity; yet, important secondary structural features of the RNA are conserved. This is shown here for the 3' UTR and the replicase-operator hairpin. Interestingly, although AP205 has the genetic map of a levivirus, its 3' UTR has the length and RNA secondary structure of an allolevivirus. Sharing features with both MS2 and Q{beta} suggests that, in an evolutionary sense, AP205 should be placed between Q{beta} and MS2. A phylogenetic tree for the ssRNA phages is presented.


   Introduction
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Abstract
Introduction
Methods
Results and Discussion
References
 
ssRNA bacteriophages or Leviviridae infect a wide variety of Gram-negative bacteria, provided these express the proper pili on their surface to facilitate entrance of the viral RNA into the cytoplasm. Pili-specific phages were first discovered in Escherichia coli by T. Loeb and N. D. Zinder, but soon after, similar creatures were found in Caulobacter and Pseudomonas (Zinder, 1975 ). ssRNA phages cause lytic infection; this takes a few hours under laboratory conditions. On average, each infected cell produces some 104 new phage particles and titres of about 1013 p.f.u./ml can easily be reached.

Based on different physical and serological properties and on their genetic map, ssRNA coliphages have been divided in two genera: Levivirus and Allolevivirus. Each genus falls into two species, previously called groups. MS2 (species I) and GA (species II) are leviviruses, while Q{beta} (species III) and SP (species IV) are alloleviviruses (Furuse, 1987 ; van Duin, 2000 ). The best-studied levivirus is MS2, while Q{beta} is the prototype species of the alloleviviruses. Their typical genetic maps are shown in Fig. 1(a). The major difference between MS2 and Q{beta} is the read-through protein, a minor capsid constituent that is absent in members of the genus Levivirus. On the other hand, the lysis gene is absent from members of the genus Allolevivirus. In the latter, the maturation protein has a second function: lysing the host (Karnik & Billeter, 1983 ; Winter & Gold, 1983 ).



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Fig. 1. (a) Genetic map of representative RNA coliphages and non-coliphages. Maturation (A) is a minor capsid component necessary for attachment to host pili. Read-through is a coat-protein extension present in the capsid in small amounts and, like the A-protein, necessary for infectivity. (b) Relevant part of the bacterial phylogenetic tree showing the relative positions of Acinetobacter, Pseudomonas and Escherichia. The tree was derived by maximum likelihood analysis of small subunit rRNA sequences. The distance scale indicates the expected number of changes per sequence position, for those positions changing at the medium rate. Taken from Olsen et al. (1994) .

 
The molecular biology of the RNA coliphages has been extensively studied and many aspects of regulation of translation and replication have been clarified (Biebricher, 1999 ; van Duin, 1988 , 1999 ; Weber, 1999 ). In addition, the virions of several phages have been crystallized and the capsid structure determined (Valegrd et al., 1990 ; Tars et al. 2000 ).

Recent sequence analysis of a large number of RNA coliphages (Inokuchi et al., 1986 , 1988 ; Adhin, 1989 ; Beekwilder, 1996 ; Olsthoorn, 1996 ; Groeneveld, 1997 ) has revealed a considerable uniformity. Species belonging to the same genus, such as Q{beta} and SP, show no variation in genetic map, some difference in RNA secondary structure and about 70% sequence identity. Between strains, this lack of diversity is even more pronounced and phages sampled from different continents (Furuse, 1987 ) show, in general, sequences that are very similar to their type species. For example, the strains MS2, R17, f2, M12 and JP501, which all belong to species I, are more than 95% identical in their RNA sequence. There can be several reasons for this lack of diversity. It may reflect a recent global expansion due to their close association with humans and domesticated livestock. Also, there may be functional constraints on the genome that limit polymorphism to extremely low levels, even at the high mutation rate of these phages. Another, and possibly major, reason for this lack of diversity is that competition among these coliphages is strong and worldwide, ensuring that only the very best sequences survive. Therefore, to assess whether nature has discovered other ways to construct RNA bacteriophages, one should either examine coliphages that have evolved in complete genetic isolation from the global pool, which may be difficult, or turn to phages infecting bacteria other than E. coli.

In a previous study, we examined PP7, a ssRNA phage infecting Pseudomonas aeruginosa via polar pili (Bradley, 1966 ; Olsthoorn et al., 1995 ). The map of PP7, as derived from its sequence, showed this phage to be a regular member of the genus Levivirus (Fig. 1a). As PP7 could be readily classified by the co-linearity of its map with MS2 and GA, it appeared possible that no other types of RNA phages would exist but the two genera found in E. coli.

We report here the sequence of Acinetobacter phage 205. Acinetobacter, Pseudomonas and Escherichia are all members of the gamma subdivision of the proteobacteria. In evolutionary terms, Acinetobacter and Pseudomonas are relatively close to each other and both are about equally distant from E. coli (Fig. 1b) (Olsen et al., 1994 ). Members of the genus Acinetobacter are ubiquitous, free-living Gram-negative bacilli that can be found in soil, water, food and sewage. More recently, they have been recognized as an important nosocomial pathogen in hospital patients (reviewed by Towner, 1997 ).

AP205 is the first example of a ssRNA phage with a different genetic map. Although the three major genes occupy positions identical to those in PP7- and MS2-type coliphages, there is no lysis gene at the usual position. Furthermore, two small ORFs are found ahead of the maturation gene.


   Methods
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Abstract
Introduction
Methods
Results and Discussion
References
 
{blacksquare} Phage and phage host.
AP205 was isolated from Quebec sewage by the enrichment technique (Coffi, 1995 ). The host bacterium, a type strain of Acinetobacter genospecies 16 (Bouvet & Jeanjean, 1989 ), had been isolated originally from urine and was obtained through P. J. M. Bouvet (Pasteur Institute, Paris, France). Phage and host were grown at 28 °C in brain–heart infusion broth or on trypticase soy agar (Difco). High-titre lysates were prepared from plates with confluent lysis. Phage and host are held in the Félix d’Hérelle Centre under the accession numbers HER424 and HER1424, respectively.

{blacksquare} Electron microscopy.
Phage AP205 was sedimented at 25000 g for 60 min in a JA-18.1 rotor using a Beckman J2-21 (Palo Alto) centrifuge and was washed twice in 0·1 M ammonium acetate (pH 7·0). Particles were deposited onto a copper grid with carbon-coated Formvar films, stained with 2% potassium phosphotungstate (pH 7·2) and examined under a Philips EM 300 electron microscope. Magnification was controlled with catalase crystals (Luftig, 1967 ).

{blacksquare} Plasmids and E. coli strains.
Plasmids containing fragments of AP205 cDNA were propagated in E. coli strains JM109 [thi {Delta}(lac-proAB) traD36 proAB laclqZ{Delta}M15] and TOP10 (Invitrogen). Expression of AP205 cDNA was studied in E. coli K12 strain M5219 (M72 lacZam trpAam Smr/{lambda}bio252 cI857 DH1), which encodes the thermosensitive {lambda} repressor and the transcription anti-termination factor N (Remaut et al., 1981 ).

Plasmids used for cloning AP205 cDNA were pUC18 and pCR4-TOPO (Invitrogen). cDNA fragments used for protein expression were cloned into pPLa2311 under the control of the PL promoter.

{blacksquare} Phage RNA isolation, cDNA synthesis and sequencing.
Acinetobacter HER1424 was infected with a lysate of AP205 at OD650=0·2 and cells were grown at 28 °C for several hours. After cell lysis (which did not always happen), cell debris was removed by centrifugation. Phages were precipitated with ammonium sulphate and purified by CsCl centrifugation, as described for MS2 (Voorma et al., 1971 ).

RNA was isolated using RNA Insta-Pure buffer (Eurogentec), according to the recommendation of the supplier. In a denaturing agarose gel, the RNA migrated as a single band slightly slower than Q{beta} RNA. Phage RNA was tailed with ATP at the 3' end using poly(A) polymerase (Pharmacia) at the following conditions: 50 mM Tris–HCl (pH 7·5), 10 mM MgCl2, 2·5 mM MnCl2, 250 mM NaCl, 0·5 mg/ml BSA, 0·1 mM ATP and 2 U poly(A) polymerase. The reaction was carried out at 37 °C for 30 min and the RNA was recovered by phenol extraction and alcohol precipitation.

Information on the N-terminal amino acid sequence of the coat protein was used to design degenerate primers for PCR. Reverse transcription (RT) was carried out on polyadenylated phage RNA using a poly(dT) oligonucleotides and AMV reverse transcriptase (Sigma), as recommended by the supplier.

The RT product was used directly in PCR with the following primers: 5' GGGAATTC(T)25, corresponding to the 3' poly(A) region, and 5' CCCAAGCTTAAYAARCCNATGCARCC, the degenerate primer corresponding to amino acids three to eight of the coat protein and extended with an HindIII site and the triplet CCC. The RT–PCR mixture contained many fragments of different lengths that were all shorter than expected. PCR products were purified and, after digestion with EcoRI/HindIII, ligated into pUC18. A total of 20 clones were sequenced, 13 of which contained sequences corresponding to the start of the coat protein gene. Seven clones contained cDNA fragments from various other regions (some upstream of the coat protein start), which, most probably, was the result of inaccurate priming during PCR. We identified these sequences by their similarity in amino acid sequence to replicases and maturation proteins from other ssRNA phages. Only one of the 20 clones actually contained the 3' end.

Based on the sequences determined, a set of new PCR primers was designed to obtain cDNA from unknown regions. These primers were used for PCR and the resulting products were cloned directly into pCR4-TOPO. After sequencing this set of clones, we obtained the almost complete phage sequence, missing only the 5' end.

To determine the 5'-terminal sequence, an oligonucleotide, Mat9R, complementary to nt 387–407 was used in the RT reaction under the conditions described above. The product obtained was purified by phenol extraction and precipitation. The cDNA was tailed with dATP by terminal transferase (Gibco) and, subsequently, PCR was carried out using the primers Mat9R and 5' GG(T)25. The PCR product was cloned into pCR4-TOPO and sequenced. On this sequence gel, we also ran the RT product obtained with primer Mat9R. The RT product migrated exactly at the position of the first G of the sequence gel, indicating that the real 5' end of the phage RNA has been sequenced.

Three independent polyadenylations and subsequent RT–PCRs were performed with forward primers complementary to regions around 3091, 3691 and 4083, respectively. In all cases, the same 3' terminus was found and this terminus is therefore likely to represent the true end of the phage RNA.

Sequencing was carried out with the T7 DNA Polymerase kit. All sequences were determined on at least two independent clones in both directions.

{blacksquare} Detection of lysis gene expression in E. coli.
AP205 nt 66–401, embedded between two EcoRI sites, was obtained by PCR and inserted in two orientations into the EcoRI site present behind the thermoinducible PL promoter of pPLa2311 (Remaut et al., 1981 ). The sense insertion is called pRBS-lysis, the opposite orientation is called pControl. As a further control, pPL-C-R was made; this plasmid contains AP205 nt 2047–2631 as an EcoRI fragment.

E. coli M5219 cells harbouring these three plasmids were grown in rich medium at 28 °C until A650=0·25. The PL promoter was then induced by shifting the culture to 42 °C. A650 was further recorded.

{blacksquare} Software.
Computer programs used for sequence analysis and amino acid alignments were from the DNASTAR software. Secondary structure predictions were made by eye and by RNA MFOLD software, version 3.1 (Zuker et al., 1999 ; Mathews et al., 1999 ).


   Results and Discussion
Top
Abstract
Introduction
Methods
Results and Discussion
References
 
Discovery and general features of AP205
Sewage from the city of Québec, Canada, was surveyed for the presence of bacteriophages able to grow on various species of Acinetobacter. One such phage, AP205, was tentatively classified as belonging to the family Leviviridae because of its appearance under electron microscopy. It had the size (27–30 nm) and spherical or hexagonal shape of the ssRNA coliphages. In addition, it was seen attached to the sides of bacterial pili of about 6 nm in diameter (Fig. 2). AP205 was therefore considered to be the first ssRNA phage found in Acinetobacter (Coffi, 1995 ). We have sequenced the RNA of this phage and our analysis confirms this conclusion.



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Fig. 2. Phage particles adsorbed to the sides of a pilus in Acinetobacter. Phosphotungstate, final magnification 148500x. Bar, 100 nm.

 
Assigning the genes
Fig. 1(a) shows the genetic map of AP205 in relation to those of Q{beta}, MS2 and PP7, a phage that propagates in P. aeruginosa. The AP205 replicase gene could be assigned by the presence of several amino acid motifs that are conserved and are typical for these phage-coded RNA-dependent RNA polymerases. Fig. 3(a) shows the amino acid sequence alignment of six different replicases. The central part of these enzymes is best conserved. Note the fully conserved (Y)GGD motif present in all polymerases.



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Fig. 3. (a) Amino acid alignment of the replicase proteins in four ssRNA coliphages and two non-coliphages. Fully conserved residues are in bold. Alignment is by CLUSTAL (Thompson et al., 1994 ). SP (species IV) and Q{beta} (species III) are alloleviviruses, MS2 (species I) and GA (species II) are leviviruses. (b) Amino-acid alignment of the conserved parts of maturation proteins in six different RNA phages. Fully conserved residues are in bold.

 
The maturation gene was assigned in a similar way. Maturation proteins of ssRNA phages are known to be very variable. Not surprisingly, therefore, the similarity between AP205 and the other phages is not high. Fig. 3(b) shows the three regions where sequences in maturation proteins are conserved.

The N-terminal 21 aa of the coat protein were sequenced (R. Schmitt, unpublished results) and they correspond to our RNA sequence. Although all phage coat proteins analysed so far have a similar secondary and tertiary structure, there are only five universally conserved amino acids, even in alignments based on protein structure (Tars et al., 1997 , 2000 ). Not unexpectedly then, a meaningful alignment of the phage coat proteins, including AP205, could not yet be constructed (K. Tars, personal communication).

Genome and protein length
Judged by its map, AP205 belongs to the RNA phages with short genomes, such as MS2 (species I) and GA (species II). However, its genome is longer than that of Q{beta} and is only 9 nt shorter than that of SP (species IV), the longest ssRNA phage known today. This is due to longer intercistronic regions, a longer maturation protein (longer by about 50 aa) and, in particular, the two extra reading frames preceding the maturation sequence. The length of the coat protein, 130–133 aa, is remarkably constant in all RNA phages.

Extra reading frames
(a) ORF1 may encode a lysis protein.
ORF1 (nt 134–238) is only 35 aa long but it contains a pronounced Shine–Dalgarno (SD) sequence at the optimal distance upstream of the AUG start codon (Table 1). It seems therefore that the ORF is functional. Comparing its amino acid sequence with those present in NCBI did not produce anything similar. However, ORF1 may encode a lysis protein. There is a clustering of positively charged residues at the N terminus, while hydrophobic amino acids are concentrated near the C terminus. This distribution is generally found in RNA phage lysis proteins (Young, 1992 ). A length of 35 aa is sufficient for a lysis function, since expression of the C-terminal 32 aa of the MS2 lysis protein suffices to cause bacteriolysis (Berkhout et al., 1985 ).


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Table 1. Nucleotide sequences around the start of the five ORFs

 
When ORF1 was cloned into an expression vector behind the strong inducible PL promoter, its induction in E. coli, performed here at two different cell densities, halted cell growth (Fig. 4, pRBS-lysis). Expression of AP205 cDNA covering the nt 2000–2700, where the coliphage L protein is located, did not show any effect on cell growth (pPL-C-R). Neither did we find any recognizable ORF in this region, apart from the coat and replicase proteins. Also, when we cloned the ORF1 gene in the reverse orientation, behind the PL promoter, the culture grew undisturbed upon induction (pControl). These results suggest that ORF1 translates into a peptide that has a lysis function.



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Fig. 4. Growth curves of E. coli containing the indicated plasmids after the induction of the {lambda} PL promoter controlling transcription of various sections of AP205 cDNA. pRBS-lysis contains the complete ORF1 (nt 66–401). pControl contains the same DNA but in the reverse orientation. pPL-C-R contains the sequence 2047–2631. For pRBS-lysis, the promoter was induced at two different cell densities (x and {diamondsuit}).

 
The MS2 lysis protein and its relatives are not, by themselves, mureine-degrading enzymes. Rather, they short-circuit the cytoplasmic membrane, thereby presumably activating the bacterial autolysins (Goessens et al., 1988 ; Walderich et al., 1988 ). Lysis proteins are remarkably variable in size and, even among the four closely related species I and II coliphages (MS2, fr, GA and KU1), amino acid sequence identity of this protein is no more than 20%. When we include PP7 in the comparison, sequence identity becomes insignificant; the same is true for the comparison with the AP205 lysis protein. There are apparently no precise amino acid requirements for these proteins that simply dissipate the proton-motive force by inserting in the inner membrane. These relaxed requirements would make it not very difficult for a phage to evolve a lysis gene, either by exploiting a second reading frame in an already dedicated sequence (PP7 and the coliphage leviviruses) or by using a vacant sequence (AP205). It is conceivable that the lysis gene has arisen more than once in the ssRNA phages and that in fact the lysis proteins in MS2 (PP7) and AP205 are functional analogues rather than homologues.

(b) ORF2.
This reading frame is even more enigmatic. As the AUG start codon is preceded by an extensive SD sequence (AGGAG, Table 1), its translation is likely. However, there is no role for another small protein in ssRNA phages and a search through the database did not produce credible homologues.

Two features in the sequence suggested that ORF2 might play a role in the translation of the maturation gene. First, the maturation gene, although containing an AUG start codon, does not have a significant SD sequence (Table 1). Second, this start codon is part of an extremely strong hairpin (Fig. 5a). Both features seem to be aimed at suppressing independent translation from this start site (de Smit & van Duin, 1990 ). Production of the A-protein may therefore be coupled somehow to reading ORF2. Accordingly, we isolated the A-protein from CsCl-purified phage particles by gel electrophoresis and determined the sequence of the ten N-terminal amino acids. To our surprise the sequence corresponds to that predicted by ORF2. This result implies that there is either a translational rephasing from ORF2 to that of the A-protein gene or a sequencing error such that ORF2 and A-protein is, in fact, one reading frame. This last possibility is unlikely because single nucleotide substitutions in the hairpin, which break up the basepairing, only produce the ORF2 protein, as identified by antibodies raised against ORF2-derived synthetic peptides. The simplest interpretation of such results is that the strong hairpin (Fig. 5a) causes a shift from ORF2 to the A-protein frame. The precise nature of this shift and its dependence on RNA structure elements are the focus of our present investigations.



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Fig. 5. (a) Very stable hairpin predicted to form at the beginning of the maturation gene. Start codon and a minimal SD sequence are marked. (b) Proposed RNA secondary structures at the 5' terminus of several ssRNA phages. More examples have been presented elsewhere (Beekwilder et al., 1996 ). For the complete 5' UTR structure of MS2, see Groeneveld et al. (1995) . Structures shown are predicted by RNA MFOLD, except for the two long-distance base pairs at the 5' terminus of AP205. There is strong comparative evidence for the presence of a 5'-terminal hairpin in all ssRNA phages. (c) Replicase-operator hairpins in several RNA phages. Bases shown to have sequence-specific interaction with the coat protein are encircled. Start codons are boxed. MX1 and M11 are RNA coliphages belonging to species III. The operator structure for group IV coliphages SP and NL95 is the same as in Q{beta} except for one or two basepair substitutions. All structures shown are predicted by RNA MFOLD. Those of the coliphages are confirmed by comparative analysis and structure probing.

 
5' and 3' termini lack the consensus sequence
All coliphages sequenced begin their RNA with 5' GGG and end with CCCAOH 3', where the A is added during termination of replication. These termini were also found in the small RNAs (6S RNAs) that can be multiplied by Q{beta} replicase. The removal of one C from the terminus of Q{beta} RNA had strong negative effects on replication (Weber, 1999 ). Exceptions to the three-C rule have only lately been found in some Q{beta} RNA revertants, where only two terminal Cs were present, while the third internal one had reverted to U (Schuppli et al., 2000 ). A similar deviation from the rule was noticed in the Pseudomonas phage PP7. Here, the beginning and end of the RNA contained only two Gs and two Cs, respectively (Olsthoorn et al., 1995 ).

The rule is even further relaxed in AP205. The chain does begin with 5' GG but ends only in a single C. Initially, it was thought that double or triple Cs were selected by nature because initiation would require the strong stacking energies between two GTP molecules. It seems now that initiation of replication in the RNA phages is not strictly dependent on double terminal C residues.

Conserved RNA structures
Phage RNA contains two kinds of information. Besides encoding the viral proteins, it assumes a specific higher order structure that is responsible for regulation of replication and translation and many other functions. It can easily be shown that compromising this structure by introducing amino acid-neutral mutations in codon wobble positions decreases the fitness of the phage by many orders of magnitude (Klovins et al., 1997 ). Therefore, the shape of the RNA in these phages must be maintained even if the sequence changes.

(a) The 5'-terminal hairpin.
A first example is the 5'-terminal hairpin, which is believed to be necessary to initiate strand separation during replication (Zamora et al., 1995 ; Beekwilder et al., 1996 ). For AP205, this hairpin is shown in Fig. 5(b), next to its equivalents in PP7 and MS2. All of them have the extremely stable upper part containing four to seven consecutive C{bullet}2G pairs. However, there is one difference. Unlike what is found in all other RNA phages, the 5' AP205 hairpin does not seem to include the two terminal Gs. These are probably paired to a more downstream sequence to form a four-way junction, very similar to how the 3' UTR is folded (see below).

(b) The operator hairpin.
All ssRNA coliphages share the same special way to control translation of their replicase gene. The start region of this gene can fold into a phage-specific hairpin structure that has affinity for its own coat protein. When the concentration of coat protein during infection becomes high enough, a dimer binds this stem–loop structure and precludes further ribosome binding. This complex is also considered to be the nucleation point for capsid formation. In Fig. 5(c), we show this so-called operator hairpin for AP205, PP7, two representatives of the leviviruses (MS2 and GA) and three representatives of the alloleviviruses (Q{beta}, MX1 and M11). Replicase start codons are boxed and nucleotides that interact in a base-specific way with the coat protein are encircled (Witherell et al., 1991 ; Valegrd et al., 1994 ).

There are pronounced differences between the operators of the two coliphage genera. The leviviruses have a 4 nt loop with As at the first and fourth position. Furthermore, there is the typical bulged A 2 bp from the loop. All these three As have base-specific interactions with the coat protein dimer. The alloleviviruses on the other hand have a 3 nt loop and of the whole hairpin structure, only the A in the third position of this loop shows base-specific interactions with the coat protein dimer. This Q{beta}-type operator also lacks the bulged A 2 bp from the loop. Instead, there is a bulge further down the stem. Its function, if any, is not known but it was shown not to contribute to coat-protein binding (Witherell & Uhlenbeck, 1989 ). This may explain why its position and identity vary. For Q{beta}, it is an A on the 5' side of the stem but for MX1 and M11 it is a C on the 3' side of the stem at 4 and 5 bp, respectively, from the loop.

The AP205 operator has features from both leviviruses and alloleviviruses. The 4 nt loop with A residues in the first and last position is typical for the leviviruses. On the other hand some features resemble the Q{beta}-type hairpin; for instance, the absence of the bulged A 2 bp from the loop. Instead, AP205 has the bulge further down on the 3' side of the stem, very similar to what is found for MX1 and M11. (Note that the bulged A in PP7 also resembles that of Q{beta}.) Finding that the AP205 operator has features of both leviviruses and alloleviviruses suggests that AP205 is more related to the alloleviviruses than MS2 is.

(c) Phylogenetic relationship between AP205 and other RNA.
In coliphages, the 3' UTR forms a separate domain in the RNA secondary structure model. This domain is defined and delimited by long-distance pairing of the five or six terminal nucleotides to a sequence just downstream of the replicase stop codon, shown as ld IX in Fig. 6 for a levivirus (MS2) and for an allolevivirus (SP). Justification of the structure has been given elsewhere (Beekwilder et al., 1995 , Adhin et al., 1990 ). There are substantial differences between the two genera. In MS2, the domain is much larger and contains five more stem–loop structures: V1, U4, U5, U6 and U3, which is located between ld IX and the replicase-terminator hairpin R1. Although we do not yet know the precise function of these hairpins, they are characteristic features for the two phage genera and we can use their absence or presence as a marker in a phylogenetic comparison. It is clear that AP205 is very similar to PP7 and that both resemble SP much more than MS2. In fact, both non-coliphages represent an even simpler form of SP by folding into a four-way junction without any nucleotides between the stems U1 and V. It is interesting then that, although the genetic map of AP205 and PP7 is that of a levivirus, their 3' end structure is typical for an allolevivirus. Sharing properties with both genera indicate that AP205 and PP7 hold intermediary positions between MS2 and Q{beta} and in fact supports the evolutionary tree shown in Fig. 7.



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Fig. 6. Comparison of RNA secondary structures in the 3' UTR in four different RNA phages. SP is an allolevivirus (species IV). The difference between Q{beta} and SP is that Q{beta} has a small stem–loop of 12 nt between hairpins V and U1, whereas SP only has 3 nt here (CGC). SP is therefore even more similar to AP205 and PP7 than Q{beta} is. The boxed sequence at the top of hairpin U1 is conserved in all RNA phages. In Q{beta} this sequence was shown to form a pseudoknot with its complement about 1200 nt upstream. The stop codon of the replicase is boxed. ld is long-distance interaction. Structures for the coliphages were derived by phylogenetic comparison (Adhin et al., 1990 ; Beekwilder et al., 1995 ; Olsthoorn et al., 1995 ) and are usually predicted by computer programs. End structures for PP7 and AP205 are predicted by RNA MFOLD, except ld IX. Comparison with the folding of the coliphages, especially species IV, strongly argues for its presence, however. The shadowed areas in MS2 are the structure elements absent from SP.

 


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Fig. 7. Tentative phylogenetic tree of ssRNA bacteriophages based on genetic maps and RNA secondary structure features. The tree is intended to show the main lines of descent. A, Maturation; C, coat; R, replicase; L, lysis; RT, read-through.

 
This tentative, qualitative tree is based on features of the phage RNA that are more stably inherited than the nucleotide sequence, namely the genetic map and the specific folding of the RNA. Applying this principle, it is clear that AP205, PP7 and MS2 are more related to each other than to Q{beta} because they share the same map. From the 3' end structure and, to a lesser degree, from the replicase-operator hairpin, we cannot but conclude that AP205 and PP7 are closer to Q{beta} (SP) than MS2 is. Finally, our tree places PP7 between MS2 and AP205 because PP7 shares its map with MS2 but not its 3' end. On the other hand PP7 shares its 3' end but not its map with AP205.

This unexpectedly places Q{beta} and MS2 at opposite ends of the tree, even though these phages colonize the same host. The ancestor to the RNA phages in this scheme has a genome containing only the three major genes and the simple 3' end (as in PP7, AP205 and SP). Possibly in this prototype, the A-protein acted as a lysis protein as it still does today in Q{beta}. The branching off to the alloleviviruses now needs nothing more than an insertion or a duplication between coat and replicase that potentially produces the read-through protein. Duplications and deletions in phage RNA genomes have been observed (Olsthoorn & van Duin, 1996a , b ; Licis et al., 2000 ). Evolution of the leviviruses from the ancestor only requires the ‘discovery' that the lysis function can also be carried out by a small peptide. This peptide can be translated either from an already dedicated section of the RNA genome by using a different reading frame (MS2 and PP7) or from a vacant part (AP205). The advantage of this functional shift from A-protein to lysis protein is not evident but it is conceivable that the separation of functions may improve both the infection process and cell lysis, as suggested by Bollback & Huelsenbeck (2001) . We believe that the variable position of the lysis gene supports the idea that the gene was added to a pre-existing genome, here, the ancestor. The alternative, jumping, cannot be excluded but seems unlikely. It requires a double cross-over of the replicase.

A further step in the evolution of the coliphages is the enormous expansion of the 3' UTR in GA and even more so in MS2, where five more hairpins are present than in AP205 and PP7. Interestingly the same tendency is seen in the alloleviviruses of E. coli. Whereas SP has expanded the simple four-way junction with a 3 nt bulge between V and U1, in Q{beta}, this bulge has grown to a regular hairpin (data not shown) and, as far as this feature is concerned, we could consider that Q{beta} has evolved further than SP and MS2 has evolved further than GA. The functional significance of this 3' UTR expansion is not clear, but deletion of any of these hairpins from Q{beta} leads to complete, or almost complete, loss of viability (H. Weber, personal communication).

The above evolutionary scheme is different from our previous view based only on PP7 as a non-coliphage (Olsthoorn et al., 1995 ). The present phylogenetic tree is also different from a recent evolutionary reconstruction based on protein sequence comparison (Bollback & Huelsenbeck, 2001 ). Although speculative, our descendence tree is attractive because only small, simple steps are needed to proceed from the ancestor to the two existing genera.

(d) Conservation of a pentanucleotide sequence in the 3'-terminal hairpin loop.
Another most intriguing feature of the 3' UTR is the conservation of a primary sequence in the loop of the 3'-terminal hairpin (U1). In all RNA coliphages and in AP205, this sequence is UGCUU (the variant CGCUC is found in PP7). Its precise position in the loop varies. Considering that the sequence is part of the loop of a non-coding hairpin, its preservation can hardly be accidental. For Q{beta} RNA, it was found that the UGCUU motif pairs to a single-stranded sequence positioned 1200 nt upstream that connects two large RNA structural domains in the replicase gene (Klovins & van Duin, 1999 ). The resulting long-range pseudoknot has the effect of bringing the 3' terminus of the RNA chain near to the major Q{beta} replicase-binding site. Indeed, Q{beta} mutants in which the pseudoknot was disrupted by mismatches showed complete loss of replication, while double mutants in which basepairing was restored had almost wild-type levels of replication, as measured in an in vitro assay. However, these double mutants, though showing wild-type replication in vitro, were not viable even when only a single G{bullet}2C pair in the pseudoknot was reversed. Therefore, these experiments suggested that not only the pseudoknot per se was important but also that one or both constituent sequences were required in cis. The conservation of the UGCUU sequence in AP205 supports this idea. It may be significant that, despite the variable lengths of ld IX and hairpin U1 in various phages, the distance between the conserved sequence and the 3' end is remarkably constant: namely 17 nt in Q{beta} and AP205, 16 nt in MS2 and 15 nt in PP7.

Concluding remarks
Based on its genetic map, in particular on the absence of a read-through protein, we suggest that AP205 is classified as a member of the genus Levivirus. Unlike those in other leviviruses, the lysis protein of AP205 is encoded at the 5' end of the genome in a non-overlapping fashion. The variable position of this small gene raises the possibility that it arose more than once and it could be a late addition to the phage genome, as originally suggested by Zinder (1980) , albeit on different grounds.

Several features in the structure of AP205 RNA resemble the allolevivirus Q{beta} much more than the levivirus MS2. Based both on these features and on the genetic map, we propose an evolutionary tree for the ssRNA phages in which the coliphages Q{beta} and MS2 are the least related of all phages. The ancestor to all RNA phages is supposed to be a levivirus not yet encoding a separate lysis protein, but using its maturation protein for cell lysis.


   Acknowledgments
 
We like to thank Ruediger Schmitt, Regensburg University, and Reinout Amons, Leiden University, for performing the N-terminal sequence analysis of the AP205 coat protein and the AP205 maturation protein, respectively. Maarten de Smit is acknowledged for evolutonaary discussions. This work was supported by NATO grant LST.CLG974896.


   Footnotes
 
The GenBank accession number of the sequence reported in this paper is AF 334111.


   References
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Abstract
Introduction
Methods
Results and Discussion
References
 
Adhin, M. R. (1989). A comparative analysis of RNA coliphages. PhD thesis, Leiden University, Leiden, The Netherlands.

Adhin, M. R. & van Duin, J. (1990). Scanning model for translational reinitiation in eubacteria. Journal of Molecular Biology 213, 811-818.[Medline]

Beekwilder, M. J. (1996). Secondary structure of the RNA genome of bacteriophage Q{beta}. PhD thesis, Leiden University, Leiden, The Netherlands.

Beekwilder, M. J., Nieuwenhuizen, R. & van Duin, J. (1995). Secondary structure model for the last two domains of single-stranded RNA phage Q{beta}. Journal of Molecular Biology 247, 903-917.[Medline]

Beekwilder, M. J., Nieuwenhuizen, R., Poot, R. & van Duin, J. (1996). Secondary structure model for the first three domains of Q{beta} RNA. Control of A-protein synthesis. Journal of Molecular Biology 256, 8-19.[Medline]

Berkhout, B., de Smit, M. H., Spanjaard, R., Blom, T. & van Duin, J. (1985). The amino terminal half of the MS2-coded lysis protein is dispensable for function: implications for our understanding of coding region overlaps. EMBO Journal 4, 3315-3320.[Abstract]

Biebricher, C. K. (1999). Mutation, competition and selection as measured with small RNA molecules. In Origin and Evolution of Viruses , pp. 65-85. Edited by E. Domingo, R. G. Webster & J. Holland. London:Academic Press.

Bollback, J. P. & Huelsenbeck, J. P. (2001). Phylogeny, genome evolution, and host specificity of single-stranded RNA bacteriophages (family Leviviridae). Journal of Molecular Evolution 52, 117-128.[Medline]

Bouvet, P. J. M. & Jeanjean, S. (1989). Delineation of new proteolytic genomic species in the genus Acinetobacter. Research in Microbiology 140, 291-299.[Medline]

Bradley, D. E. (1966). Structure and infective process of a P. aeruginosa phage containing RNA. Journal of General Microbiology 45, 83-96.

Coffi, H. (1995). Lysotypie des Acinetobacter. Thèse de Maîtrise dès Sciences, Laval University, Québec, Canada.

de Smit, M. H. & van Duin, J. (1990). Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. Proceedings of the National Academy of Sciences, USA 87, 7668-7672.[Abstract]

Furuse, K. (1987). Distribution of the coliphages in the environment: general considerations. In Phage Ecology , pp. 87-124. Edited by S. M. Goya, C. P. Gerba & G. Bitton. New York:Wiley Interscience.

Goessens, W. H. F., Driessen, J. M., Wilschut, J. & van Duin, J. (1988). A synthetic peptide corresponding to the C-terminal 25 residues of phage MS2 coded lysis protein dissipates the protonmotive force in Escherichia coli membrane vesicles by generating hydophilic pores. EMBO Journal 7, 867-873.[Abstract]

Groeneveld, H. (1997). Secondary structure of bacteriophage MS2 RNA. Translational control by kinetics of RNA folding. PhD thesis, Leiden University, Leiden, The Netherlands.

Groeneveld, H., Thimon, H. & van Duin, J. (1995). Translational control of maturation-protein synthesis in phage MS2: a role for the kinetics of RNA folding? RNA 1, 79-88.[Abstract]

Inokuchi, Y., Takahashi, R., Hirose, T., Inayama, S., Jacobson, A. B. & Hirashima, A. (1986). The complete nucleotide sequence of group II RNA coliphage GA. Journal of Biochemistry 99, 1169-1180.[Abstract]

Inokuchi, Y., Jacobson, A. B., Hirose, T., Inayama, S. & Hirashima, A. (1988). Analysis of the complete nucleotide sequence of the group IV RNA coliphage SP. Nucleic Acids Research 16, 6205-6221.[Abstract]

Karnik, S. & Billeter, M. (1983). The lysis function of RNA bacteriophage Q{beta} is mediated by the maturation protein. EMBO Journal 2, 1521-1526.[Medline]

Klovins, J. & van Duin, J. (1999). A long-range pseudoknot in Q{beta} RNA is essential for replication. Journal of Molecular Biology 294, 875-884.[Medline]

Klovins, J., Tsareva, N. A., de Smit, M. H., Berzins, V. & van Duin, J. (1997). Rapid evolution of translational control mechanisms in RNA genomes. Journal of Molecular Biology 265, 372-384.[Medline]

Licis, N., Balklava, Z. & van Duin, J. (2000). Forced retroevolution of an RNA bacteriophage. Virology 271, 298-306.[Medline]

Luftig, R. B. (1967). An accurate measurement of the catalase crystal period and its use as an internal marker for electron microscopy. Journal of Ultrastructure Research 20, 91-102.[Medline]

Mathews, D. H., Sabina, J., Zuker, M. & Turner, D. H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. Journal of Molecular Biology 288, 911-940.[Medline]

Olsen, G. J., Woese, C. R. & Overbeek, R. (1994). The winds of (evolutionary) change: breathing new life into microbiology. Journal of Bacteriology 176, 1-6.[Medline]

Olsthoorn, R. C. L. (1996). Structure and evolution of RNA phages. PhD thesis, Leiden University, Leiden, The Netherlands.

Olsthoorn, R. C. L. & van Duin, J. (1996a). Evolutionary reconstruction of a hairpin deleted from the genome of an RNA virus. Proceedings of the National Academy of Sciences, USA 93, 12256-12261.[Abstract/Free Full Text]

Olsthoorn, R. C. L. & van Duin, J. (1996b). Random removal of inserts from an RNA genome: selection against single-stranded RNA. Journal of Virology 70, 729-736.[Abstract]

Olsthoorn, R. C. L., Garde, G., Dayhuff, T., Atkins, J. F. & van Duin, J. (1995). Nucleotide sequence of a single-stranded RNA phage from Pseudomonas aeruginosa: kinship to coliphages and conservation of regulatory RNA structures. Virology 206, 611-625.[Medline]

Remaut, E., Stanssens, P. & Fiers, W. (1981). Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage {lambda}. Gene 15, 81-93.[Medline]

Schuppli, D., Georgijevic, J. & Weber, H. (2000). Synergism of mutations in Q{beta} RNA affecting host factor dependence of Q{beta} replicase. Journal of Molecular Biology 295, 149-154.[Medline]

Tars, K., Bundule, M., Fridborg, K. & Liljas, L. (1997). The crystal structure of bacteriophage GA and a comparison of bacteriophages belonging to the major groups of Escherichia coli leviviruses. Journal of Molecular Biology 271, 759-773.[Medline]

Tars, K., Fridborg, K., Bundule, M. & Liljas, L. (2000). The three-dimensional structure of bacteriophage PP7 from Pseudomonas aeruginosa at 3·7 resolution. Virology 272, 331-337.[Medline]

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673-4680.[Abstract]

Towner, K. J. (1997). Clinical importance and antibiotic resistance of Acinetobacter spp. Journal of Medical Microbiology 46, 721-746.[Abstract]

Valegrd, K., Liljas, L., Fridborg, K. & Unge, T. (1990). The three-dimensional structure of the bacterial virus MS2. Nature 345, 36-41.[Medline]

Valegrd, K., Murray, J. B., Stockley, P. G., Stonehouse, N. J. & Liljas, L. (1994). Crystal structure of an RNA bacteriophage coat protein-operator complex. Nature 371, 623-626.[Medline]

van Duin, J. (1988). The single-stranded RNA bacteriophages. In The Viruses , pp. 117-167. Edited by H. Fraenkel-Conrat & R. Wagner. New York:Plenum.

van Duin, J. (1999). Single-stranded RNA phages. In Encyclopedia of Virology, pp. 1663–1668. London: Academic Press.

van Duin, J. (2000). Leviviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses , pp. 645-646. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego:Academic Press.

Voorma, H. O., Benne, R. & den Hertog, T. J. A. (1971). Binding of aminoacyl-tRNA to ribosome programmed with bacteriophage MS2-RNA. European Journal of Biochemistry 18, 451-462.[Medline]

Walderich, B., Ursinus-Wossner, A., van Duin, J. & Höltje, J.-V. (1988). Induction of the autolytic system of Escherichia coli by specific insertion of bacteriophage MS2 lysis protein into the bacterial cell envelope. Journal of Bacteriology 170, 5027-5033.[Medline]

Weber, H. (1999). Q{beta} replicase. In Encyclopaedia of Molecular Biology , pp. 2085-2087. Edited by T. E. Creighton. New York:John Wiley.

Winter, R. G. & Gold, L. (1983). Overproduction of bacteriophage Q{beta} maturation (A2) protein leads to cell lysis. Cell 33, 877-885.[Medline]

Witherell, G. W. & Uhlenbeck, O. C. (1989). Specific RNA binding by Q{beta} coat protein. Biochemistry 28, 71-76.[Medline]

Witherell, G. W., Gott, J. M. & Uhlenbeck, O. C. (1991). Specific interaction between RNA phage coat proteins and RNA. Progress in Nucleic Acid Research and Molecular Biology 40, 185-220.[Medline]

Young, R. (1992). Bacteriophage lysis: mechanism and regulation. Microbiology Reviews 56, 430-481.

Zamora, H., Luce, R. & Biebricher, C. K. (1995). Design of artificial short-chained RNA species that are replicated by Q{beta} replicase. Biochemistry 34, 1261-1266.[Medline]

Zinder, N. D. (1975). RNA Phages. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Zinder, N. D. (1980). Portraits of viruses: RNA phage. Intervirology 13, 257-270.[Medline]

Zuker, M., Mathews, D. H. & Turner, D. H. (1999). Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. In RNA Biochemistry and Biotechnology , pp. 11-43. Edited by J. Barciszewski & B. F. C. Clark. Amsterdam:Kluwer.

Received 26 September 2001; accepted 20 February 2002.