INTRODUCTION

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Bacteriophage 6 was isolated from bean straw infested with Pseudomonas syringae pv. phaseolicola (25). It contains a genome of three segments of double-stranded RNA (22) packaged inside a procapsid which is covered by a shell of protein P8 and a lipid-containing membrane containing additional viral proteins (25). The genome of 6 has been cloned and sequenced, and the life cycle and structure of the phage have been subjects of considerable investigation (1, 8).

We have developed a model for the mechanism of genomic packaging in 6 and accumulated evidence to support it (7, 15, 17). The model involves the binding of segment S to sites on the outside of the empty procapsid so as to position the 5' end at an entry portal. Upon packaging of segment S, the binding sites are lost and new sites for segment M appear on the outside of the particle. The process repeats, and the sites for segment M disappear and those for segment L appear. We had shown previously that each of the segments contains a packaging sequence of about 200 nucleotides near the 5' ends of the plus strands (5). We embarked upon the search for relatives of 6 in the hope that a comparison of the sequences in the packaging regions of the segments would suggest important structural or sequence motifs. Up until now, 6 has been alone in the family Cystoviridae and alone in the genus Cystovirus (11). It is now clear that this group is composed of many more phages, some very similar to 6 and some rather distantly related.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial strains, phage, and plasmids. P. syringae pv. phaseolicola HB10Y (HB) (25) was used as the primary host for phage plating. LM2333 was selected as a mutant of HB resistant to several DNA phages. LM2489 is a derivative of LM2333. MPO.16 is a derivative of HB that lacks type IV pili and is resistant to 6 (19, 20). MR is a derivative of P. aeruginosa that has the type IV pilus of P. syringae (19). LM2509 is a derivative of LM2489 that lacks pili and is resistant to 6. Strain ERA is an isolate of P. pseudoalcaligenes. 6 infects ERA when it has a mutation in gene 3 called h1 (9). S4 is a derivative of ERA that contains a nonsense suppressor mutation. S4M is a derivative of S4 that has mutated so as to be infected by 6 without the h1 mutation. P. syringae pv. phaseolicola Ro49dRa1 is a rough derivative (21). SL1102 and SL3789 are strains of Salmonella typhimurium LT2 from the Salmonella Genetic Stock Centre, and they have lipopolysaccharide (LPS) defects due to mutations rfaE543 and rfaF511, respectively (18).

Media. The media used were LC and MB (23). Ampicillin plates contained 200 µg of ampicillin per ml in LC agar.

Enzymes and chemicals. All restriction enzymes, T4 DNA ligase, T4 DNA polymerase, T4 polynucleotide kinase, Klenow enzyme, and exonuclease BAL 31 were purchased from Promega Corp. (Madison, Wis.), New England Biolabs (Boston, Mass.), and Boehringer GmbH (Mannheim, Germany).

Reverse transcriptase reaction. About 3 µg of RNA in 20 µl of water was mixed with 200 ng of oligonucleotide primer OLM297, which is essentially identical to the first 16 nucleotides of the three genomic segments of 6 and also carries a PstI site. Its sequence is GGGGGGCTGCAGAAAAAAACTTTATATA. The mixture was heated at 100°C for 4 min and quickly cooled to 42°C, and 12 µl was mixed with 4 µl of RT buffer (5×; Promega), 2 µl of deoxynucleoside triphosphates (2 mM each), and 2 µl of avian myeloblastosis virus reverse transcriptase (5 U). The mixture was incubated at 42°C for 1 h.

PCR. A 10-µl volume of the RT mixture was supplemented with 0.2 mM deoxynucleoside triphosphates; 1 µg of OLM297; 1 µg of OLM15, OLM298, or OLM296; 2.5 U of Pwo DNA polymerase; and PCR buffer components (Boehringer GmbH) in 50 µl. The mixture was taken through 30 cycles of 94°C for 1 min, 45°C for 30 s, and 72°C for 2 min. The final product was extracted with phenol, ethanol precipitated, and dissolved in 20 µl of DNA buffer. Oligonucleotides OLM15, OLM298, and OLM296 are complementary to the plus strands of 6 segments S (nucleotides 1158 to 1141), M (nucleotides 464 to 445), and L (nucleotides 656 to 634).

Cloning and sequencing. The PCR products were cut with PstI and either SacI, MunI, or XhoI and ligated to vector pLM1454 that had been cut with PstI and SacI, EcoRI, or SalI, respectively. The ligates were then introduced into Escherichia coli JM109 by transformation, and white colonies were picked from Luria-Bertani plates containing ampicillin and 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal). Small DNA preparations were made, and the plasmids were analyzed to determine their restriction site patterns. Sequencing was done at the New York University Medical Center Sequencing Facility. In this way, we determined the sequences of the 5' regions of the RNAs of 7, 9, 10, and 11.

LPS analysis. A qualitative test for LPS structure is the gel electrophoresis procedure of de Kievit and Lam (5). Samples were prepared as previously described and stained with silver after periodate treatment.

Nucleotide sequence accession numbers. Sequences have been deposited in GenBank with the following accession numbers: 7 S, AF125682; 7 M, AF125681; 7 L, AF125680; 9 S, AF125679; 9 M, AF125678; 9 L, AF125677; 10 S, AF125676; 10 M, AF125675; 10 L, AF125674.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Leaves of the various plants were incubated for several hours in Luria-Bertani broth at room temperature. The liquid was passed through a 0.2 µm filter, and aliquots of about 100 µl were plated on lawns of P. syringae pv. phaseolicola HB. The numbers of plaques appearing ranged from zero to several thousand. Most plaques contained virus that appeared to have DNA genomes. In order to screen out these phages, we isolated a mutant of HB that was resistant to several of the dominant plaque types. This strain was designated LM2333. 6 infected LM2333 with the same efficiency as HB. The original strategy for the isolation of new phages was to plate the extracts on LM2333, test infection of HB, and then test for plaque formation on MPO.16, which is a derivative of HB that lacks type IV pili and cannot be infected by 6. Phages that infected the first two strains and not MPO.16 would be tested for chloroform sensitivity and, if sensitive, would be then analyzed for the presence of a double-stranded RNA (dsRNA) genome.

Close relatives of 6. In this way, we isolated 7, 9, 10, and 11, all of which infected LM2333 and HB and not MPO.16. These phages contained genomes of three pieces of dsRNA. The RNA segments were of approximately the same sizes as those of 6 (Table 1). RT-PCR analysis of this group, with primers derived from 6 sequences, proved difficult but possible. When the resulting cDNA clones were sequenced, it was found that the nucleotide sequences differed from that of 6 by about 15 to 20%. Within open reading frames (ORFs), the base sequence changes were concentrated in the third base of codon triplets so that the amino acid sequence remained highly conserved. The 5' ends of the genomic plus strands contain the pac sequences, which are about 200 nucleotides in length and unique and specific for the packaging of each segment (6). The sequences of the pac regions in this group of phages were about 90% identical; however, the sequence changes were often complementary in regions where presumed stem-loop structures are present (27). Changes in one strand of the stems were often compensated by complementary changes in the other strand (7).

View this table: [in this window] [in a new window]   TABLE 1.   Properties of the new bacteriophages relative to 6

View larger version (59K): [in this window] [in a new window]   FIG. 1.   Comparison of the nucleotide sequences of the 5' regions of segment L RNA plus strands in viruses closely related to 6. Note that the pac sequence ends at about nucleotide 205. Gene 14 begins at nucleotide 270 in 6 and 10 and at nucleotide 473 in 7 and 9. ORF E is not present in 6 or 10 and starts at nucleotide 268 in 7 and 9.

Distant relatives of 6. It was subsequently found that additional phages (8, 12, and 13) could be isolated that infect P. pseudoalcaligenes ERA, which is an alternative host for 6 (9). These phages infected LM2333 but not HB, the normal host of 6. The plating characteristics of the various isolates are shown in Table 1. These phages were able to infect a derivative of LM2333, designated LM2509, that is resistant to 6 due to the loss of the specific type IV pilus. Various strains of P. syringae were tested for the ability to be infected by 8, 12, and 13. The only positive candidate was a rough strain, Ro49dRa1. These results suggested that phages, 8, 12, and 13 were attaching directly to rough LPS or some element exposed in this type of outer membrane. It also seemed reasonable that strain LM2333 and its derivatives were rough mutants of HB and that this was the reason for their general resistance to many DNA phages. E. coli JM109 is known to be somewhat rough in its LPS composition (24). Plating of concentrated suspensions of these phages on lawns of JM109 showed killing but not plaque formation. The phages did not show this killing of strain HB. A gel analysis of LPS of strains that plated this group of phages indicated that the strains on which they grew had truncated LPS (Fig. 2). A collection of S. typhimurium strains with various mutations causing changes in LPS was obtained from the Salmonella Genetic Stock Centre. The phages were plated on these strains. Phages 12 and 13 were able to kill strains that had lost O antigen, but they did not form plaques. Phage 8 was able to kill the strains that had lost O antigen but was also able to form plaques on strains that harbored an rfaC, rfaE, or rfaF mutation (18). These strains have either no heptose or only one of the two normal heptose residues in their LPS. The efficiency of plating of 8 on the heptoseless strains was similar to that on the pseudomonads, although the plaques were small and turbid.

View larger version (87K): [in this window] [in a new window]   FIG. 2.   Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel of LPSs from various cultures. The samples are as follows: HB, smooth; RO, P. syringae rough; 2489, rough derivative of HB; JM, E. coli JM109; ERA, P. pseudoalcaligenes; PA, P. aeruginosa smooth.

View larger version (43K): [in this window] [in a new window]   FIG. 3.   dsRNA extracted from cells resistant to kanamycin due to carrier state infections. Lanes: 6, dsRNA isolated from 6 virions; 13/2509, RNA isolated from kanamycin-resistant cells of LM2509 (P. syringae without type IV pili); 13/3789, RNA isolated from kanamycin-resistant S. typhimurium SL3789. 13 contains a segment M with kan inserted in the 3' noncoding region.