BACKGROUND
bacteriophage Φ <dig> was the first member of the cystoviridae to be isolated  <cit> .
in  <dig> we isolated a number of phages that were members of the cystoviridae  <cit> .
some were close relatives of Φ <dig> while others were rather distantly related in that they shared little or no base sequence similarity although their gene order was similar and they all contained genomes of three segments of dsrna enclosed in a polyhedral shell that was, in turn, encased in a lipid-containing membrane.
all members of this family have an inner core composed of  <dig> molecules of the major structural protein p <dig>   <dig> hexamers of the packaging ntpase p <dig>   <dig> molecules of polymerase p <dig> and about  <dig> molecules of auxilliary protein p <dig>  the core is encased in a shell of protein p <dig> in all members except the Φ <dig> group.
this is designated as the nucleocapsid.
the nucleocapsid is covered by a lipid-containing membrane which has protein p <dig> as its major component and proteins p <dig> and p <dig> which determine host specificity.
we proposed four groups represented by phages Φ <dig>  Φ <dig>  Φ <dig> and Φ <dig>  the phages in the last three groups attached to host cells through rough lps while the Φ <dig> group attached to type iv pili.
we have recently isolated a new collection of phages and they seem to fit into the previously proposed groups with some important distinctions.
in this paper we describe bacteriophage Φ <dig> which has similarity to Φ <dig>  <cit>  in the amino acid composition of several of its proteins but whereas Φ <dig> attaches to rough lps, Φ <dig> attaches to type iv pili.
Φ <dig> is of special interest in that it is dependent upon a host protein, glutaredoxin <dig> to activate its transcription upon initiating infection .
it appears that different members of the cystoviridae use different host proteins to activate or to regulate transcription  <cit> .
the control of transcription in Φ <dig> involves the nature of the first base of the segment l transcript while that of Φ <dig> and its close relatives involves the nature of the second base.
RESULTS
twenty five new isolates of members of the cystoviridae were obtained from the leaves of radish, carrot and onion plants.
five of the isolates showed similarity to previously isolated Φ <dig>  <cit>  although their host ranges differed from that of Φ <dig>  radish leaves were incubated with lb broth.
the liquid was mixed with a culture of pseudomonas syringae lm <dig> which is a rough lps derivative of the original host strain for the cystoviruses  <cit> .
plaques were tested for sensitivity to chloroform.
an isolate named Φ <dig> was found to contain three segments of dsrna.
the sizes of the rna segments differed from those of the known cystoviruses.
the host range of the phage was similar to that of Φ <dig> in that it did not propagate on strains missing type iv pili but did propagate on strain hb10y which has type iv pili and smooth lps.
phage was purified by sedimentation and equilibrium banding in sucrose or renocal  gradients.
purified phage was analyzed by polyacrylamide gel electrophoresis .
the migration of the proteins was similar to that seen for most of the cystoviridae and that of protein p <dig> was similar to that of Φ <dig> in that it appeared to have a molecular weight of  <dig> kd rather than that of  <dig> kd shown by most of the cystoviridae.
cdna was prepared from the genomic dsrna of the phage or from transcripts produced in vitro by nucleocapsids of the virus.
cdna was prepared using random hexamers or polya tailing in conjunction with oligodt priming.
the sequences were compiled into the maps shown in figure  <dig>  the sizes of the genomic segments were found to be  <dig> bp,  <dig> bp and  <dig> bp respectively for segments s, m and l. blast searches showed no significant nucleotide similarity with other phages but searches of amino acid sequence showed significant similarity to many of the gene products of bacteriophage Φ <dig>   <cit> .
in particular, the amino acid sequence of the viral rna polymerase, p <dig>  was closely related to that of Φ <dig>  several of the differences shown by Φ <dig> relative to Φ <dig> were present in p <dig> of Φ <dig>  this was true of the regions in p <dig> of Φ <dig> at nucleotide positions k <dig> and r225; r <dig> and r 270; and s <dig> that deal with triphosphate binding and catalytic sites  <cit> .
moreover, the 5' terminal sequences of the segment transcripts resembled, but were not identical to those of the Φ <dig> genomic segments .
Φ <dig> differs from other members of the cystoviridae in the base sequences at the 5' termini of plus strand copies of the genome.
whereas most examples have gg at the 5' termini of segments s and m and gu at the 5' end of l, Φ <dig> has gaa at the termini of segments s and m and acaa at the 5' of segment l. Φ <dig> has the sequence of gc at the 5' termini of segments s and m and acaa at the 5' terminus of l. bacteriophage Φ <dig> and its close relatives have identical sequences, gaaauuu, at the 5' termini of all three transcripts  <cit> .
the 3' sequences of the three plus strands contained a  <dig> base near identity at the terminus.
this sequence produced a structure with two hairpin stem loops that differ in sequence from those of phi <dig> and other members of the cystoviridae but probably function as protection against host exonucleases   <cit> .
amino acid similarity to some of the proteins of the Φ <dig> l segment was also found, but at a lower level than found for Φ <dig> .
an exception was the finding that protein p <dig> had striking similarity to p <dig> of Φ <dig>  a phage that otherwise had little similarity to Φ <dig> .
a strong relationship was found between the product of gene  <dig> and protein flgj  of the host organism p. syringae.
protein p <dig> is a muramidase in all the cystoviridae while flgj is a host flagellar protein that has peptidoglycan hydrolase activity.
the similarity of Φ <dig> p <dig> to flgj is greater than that of Φ <dig> to that of p <dig> protein of any of the other cystoviruses, even Φ <dig>  it seems clear that gene <dig> was derived from the host muramidase gene.
the cystoviridae are capable of acquisition of genetic material from the host.
although acquisition is much more likely if pac sequences are on the introduced rna, we have shown acquisition in cases where pac sequences are not present  <cit> .
a needleman-wunsch alignment
b p. syringae flgj glycosidase
c no significant similarity
d relationship to Φ13
the arrangement of the genes is similar to that of most of the cystoviridae  <cit> .
polar relationships are evident for genes  <dig> and 12; and  <dig> and  <dig>  the arrangement of genes  <dig> and  <dig> is consistant with a polar arrangement but gene  <dig> does seem to have a proper shine-dalgarno sequence  <cit> .
in these cases the orfs for genes  <dig> and  <dig> do not have proper shine-dalgarno sequences associated with them .
reverse genetics
the cdna copies of the genomic segments were inserted into a derivative of plasmid pt7t319u  that had the t <dig> rna polymerase promoter replaced by the promoter of sp <dig> rna polymerase so that transcription would start efficiently at the terminal g of segments s and m. the fidelity of the cdna constructs was tested by their activity in the production of live virus resulting from their electroporation into strain lm <dig>  lm <dig> is a derivative of lm <dig> carrying plasmid plm <dig> that expresses the sp <dig> rna polymerase.
three different constructs of the l segment were utilized; they contained 5' sequences beginning with the normal acaa start, or with gacaa or gcaa.
segments m and s were normal .
the cdna plasmids are cole <dig> derivatives and unable to replicate in pseudomonads.
the constructs with acaa or gacaa produced about six thousand plaques from  <dig> cells while the construct of gcaa produced about one hundred plaques.
the amount of transcript with acaa would be expected to be much lower than that for gacaa, suggesting that its efficiency in plaque production would be greater than that for gacaa on a per molecule basis.
while the phage resulting from the l segment with the normal 5' sequence of acaa behaved identically to that of wild type Φ <dig>  the phage with the gcaa sequence showed novel transcription behavior.
whereas wild type Φ <dig> nucleocapsids transcribe only genomic segments s and m in vitro; the mutant with gcaa instead of acaa at the 5' terminus transcribes all three genomic segments in vitro .
the differences in template activity for genomic segment l as opposed to s and m is used by most members of the cystoviridae to effect temporal regulation of transcription.
in the case of Φ <dig> the l segment has the sequence gu at the 5' end of the plus strand while s and m have gg.
the polymerase favors g over u as the second nucleotide and it is proposed that the second base is the first to be paired during transcription  <cit> .
a host protein, yajq, is able to alter this selection so as to enable active transcription of the l segment upon entry into the host cell  <cit> .
a change of gu to gg in segment l of Φ <dig> results in in vitro transcription of segment l  <cit> .
although the structure of the polymerase of Φ <dig> has not been studied, it seems likely that in this case the terminal nucleotide would be paired first and that g is preferred to a.
the host specificity of Φ <dig> is different from that of its close relative Φ12; however it was possible to construct viable phage with a middle segment containing the pac sequence of Φ <dig> and the genes  <dig> and  <dig> of Φ <dig>  gene  <dig> codes for the host attachment protein while gene  <dig> codes for its membrane bound anchor  <cit> .
the plasmid plm <dig> has the 5' region of Φ2954m up to the sphi site at position  <dig> and the sequence of Φ13m from sacii at nucleotide  <dig>  the resulting phage, Φ <dig> does not plate on the normal host of Φ <dig>  hb10y but does plate on strains that have rough lps such as lm <dig> or lm <dig>
we have also constructed a plasmid with the pac sequence of Φ2954m and the genes  <dig> and  <dig> of Φ <dig>  the resulting phage has the same plating properties as Φ <dig> with respect to pilus attachment.
another test of the functionality of the cdna copy of segment m was to determine whether bacteriophage Φ <dig> could acquire the transcript of this plasmid in order to change its host range.
plasmid plm <dig>  which carries the cdna copy of Φ <dig> genomic segment m, was electroporated into strain lm <dig> before infection with Φ <dig>  these cells were plated along with those of hb10y and plaques were obtained.
these plaques plated on hb10y but not on a strain missing the type iv pili.
the genomic segments of these phages were consistent with the segments l and s of Φ <dig> and m of Φ <dig> .
the finding that Φ <dig> is able to acquire segment m of Φ <dig> is intriguing in that the pac sequences in m are very different for both phages.
this is reminiscent of the case of bacteriophage Φ <dig> acquiring segment m of Φ <dig> in which case there is again very little sequence similarity in the pac sites  <cit> .
this is so despite the observation that small changes in the pac sequences of Φ <dig> m or s drastically reduce the ability of Φ <dig> to acquire these segments  <cit> .
the finding that it is possible to change the host attachment proteins is of special interest in that it shows that the proteins p <dig> and p <dig> are able to recognize viral membrane that contains the major membrane protein p <dig> of distantly related phages of the same family.
another test of genomic packaging was the production of a genomic segment containing segments s and m joined together.
the apai to xbai segment of m was joined to the psti site that is present in the vector following the 3' end of the cdna copy of segment s. a plasmid plm <dig> containing this construct was able to donate its transcript to phage Φ <dig> that contains segments s and l of Φ <dig> and an m segment that has the pac sequence of Φ2954m but the genes of segment m of Φ <dig>  a phage that plates on host cells containing rough lps.
electroporating plasmid plm <dig> into strain lm <dig> produced a phage with the entire genome contained in a single segment.
this plasmid contained the cdna copies of the complete segment s with the sequence of segment m beginning with the apai site at position  <dig> to the xbai site following its c terminus with segment l beginning with an mfei site at position  <dig> that was converted to xbai.
the observation that phage were produced in high yield from this plasmid is consistent with the previous observations of the preparation of single segment genomes in Φ <dig> and Φ <dig>  it also suggests that the open reading frames of genes  <dig> and  <dig>  starting at  <dig> and  <dig>  are not necessary for phage production.
CONCLUSIONS
Φ <dig> has a number of properties similar to other members of the cystoviridae; however, it shows some interesting differences.
in particular, it regulates transcription by altering the first nucleotide of the segment l transcript relative to those of segments s and m while most other cystoviruses regulate by altering the second nucleotide.
the cdna copies of the genome have been shown to be accurate and they allow manipulation of the structure of the genome.
Φ <dig> will be an important component in the investigation of the temporal control of transcription in the cystoviridae.

 bacteriophage Φ was the first member of the cystoviridae to be isolated. in we isolated a number of phages that were members of the cystoviridae. some were close relatives of Φ while others were rather distantly related in that they shared little or no base sequence similarity although their gene order was similar and they all contained genomes of three segments of dsrna enclosed in a polyhedral shell that was, in turn, encased in a lipid-containing membrane. all members of this family have an inner core composed of molecules of the major structural protein p hexamers of the packaging ntpase p molecules of polymerase p and about molecules of auxilliary protein p the core is encased in a shell of protein p in all members except the Φ group. this is designated as the nucleocapsid. the nucleocapsid is covered by a lipid-containing membrane which has protein p as its major component and proteins p and p which determine host specificity. we proposed four groups represented by phages Φ Φ Φ and Φ the phages in the last three groups attached to host cells through rough lps while the Φ group attached to type iv pili. we have recently isolated a new collection of phages and they seem to fit into the previously proposed groups with some important distinctions. in this paper we describe bacteriophage Φ which has similarity to Φ in the amino acid composition of several of its proteins but whereas Φ attaches to rough lps, Φ attaches to type iv pili. Φ is of special interest in that it is dependent upon a host protein, glutaredoxin to activate its transcription upon initiating infection. it appears that different members of the cystoviridae use different host proteins to activate or to regulate transcription. the control of transcription in Φ involves the nature of the first base of the segment l transcript while that of Φ and its close relatives involves the nature of the second base.  twenty five new isolates of members of the cystoviridae were obtained from the leaves of radish, carrot and onion plants. five of the isolates showed similarity to previously isolated Φ although their host ranges differed from that of Φ radish leaves were incubated with lb broth. the liquid was mixed with a culture of pseudomonas syringae lm which is a rough lps derivative of the original host strain for the cystoviruses. plaques were tested for sensitivity to chloroform. an isolate named Φ was found to contain three segments of dsrna. the sizes of the rna segments differed from those of the known cystoviruses. the host range of the phage was similar to that of Φ in that it did not propagate on strains missing type iv pili but did propagate on strain hb10y which has type iv pili and smooth lps. phage was purified by sedimentation and equilibrium banding in sucrose or renocal gradients. purified phage was analyzed by polyacrylamide gel electrophoresis. the migration of the proteins was similar to that seen for most of the cystoviridae and that of protein p was similar to that of Φ in that it appeared to have a molecular weight of kd rather than that of kd shown by most of the cystoviridae. cdna was prepared from the genomic dsrna of the phage or from transcripts produced in vitro by nucleocapsids of the virus. cdna was prepared using random hexamers or polya tailing in conjunction with oligodt priming. the sequences were compiled into the maps shown in figure the sizes of the genomic segments were found to be bp, bp and bp respectively for segments s, m and l. blast searches showed no significant nucleotide similarity with other phages but searches of amino acid sequence showed significant similarity to many of the gene products of bacteriophage Φ. in particular, the amino acid sequence of the viral rna polymerase, p was closely related to that of Φ several of the differences shown by Φ relative to Φ were present in p of Φ this was true of the regions in p of Φ at nucleotide positions k and r225; r and r 270; and s that deal with triphosphate binding and catalytic sites. moreover, the 5' terminal sequences of the segment transcripts resembled, but were not identical to those of the Φ genomic segments. Φ differs from other members of the cystoviridae in the base sequences at the 5' termini of plus strand copies of the genome. whereas most examples have gg at the 5' termini of segments s and m and gu at the 5' end of l, Φ has gaa at the termini of segments s and m and acaa at the 5' of segment l. Φ has the sequence of gc at the 5' termini of segments s and m and acaa at the 5' terminus of l. bacteriophage Φ and its close relatives have identical sequences, gaaauuu, at the 5' termini of all three transcripts. the 3' sequences of the three plus strands contained a base near identity at the terminus. this sequence produced a structure with two hairpin stem loops that differ in sequence from those of phi and other members of the cystoviridae but probably function as protection against host exonucleases. amino acid similarity to some of the proteins of the Φ l segment was also found, but at a lower level than found for Φ. an exception was the finding that protein p had striking similarity to p of Φ a phage that otherwise had little similarity to Φ. a strong relationship was found between the product of gene and protein flgj of the host organism p. syringae. protein p is a muramidase in all the cystoviridae while flgj is a host flagellar protein that has peptidoglycan hydrolase activity. the similarity of Φ p to flgj is greater than that of Φ to that of p protein of any of the other cystoviruses, even Φ it seems clear that gene was derived from the host muramidase gene. the cystoviridae are capable of acquisition of genetic material from the host. although acquisition is much more likely if pac sequences are on the introduced rna, we have shown acquisition in cases where pac sequences are not present. a needleman-wunsch alignment b p. syringae flgj glycosidase c no significant similarity d relationship to Φ13 the arrangement of the genes is similar to that of most of the cystoviridae. polar relationships are evident for genes and 12; and and the arrangement of genes and is consistant with a polar arrangement but gene does seem to have a proper shine-dalgarno sequence. in these cases the orfs for genes and do not have proper shine-dalgarno sequences associated with them. reverse genetics the cdna copies of the genomic segments were inserted into a derivative of plasmid pt7t319u that had the t rna polymerase promoter replaced by the promoter of sp rna polymerase so that transcription would start efficiently at the terminal g of segments s and m. the fidelity of the cdna constructs was tested by their activity in the production of live virus resulting from their electroporation into strain lm lm is a derivative of lm carrying plasmid plm that expresses the sp rna polymerase. three different constructs of the l segment were utilized; they contained 5' sequences beginning with the normal acaa start, or with gacaa or gcaa. segments m and s were normal. the cdna plasmids are cole derivatives and unable to replicate in pseudomonads. the constructs with acaa or gacaa produced about six thousand plaques from cells while the construct of gcaa produced about one hundred plaques. the amount of transcript with acaa would be expected to be much lower than that for gacaa, suggesting that its efficiency in plaque production would be greater than that for gacaa on a per molecule basis. while the phage resulting from the l segment with the normal 5' sequence of acaa behaved identically to that of wild type Φ the phage with the gcaa sequence showed novel transcription behavior. whereas wild type Φ nucleocapsids transcribe only genomic segments s and m in vitro; the mutant with gcaa instead of acaa at the 5' terminus transcribes all three genomic segments in vitro. the differences in template activity for genomic segment l as opposed to s and m is used by most members of the cystoviridae to effect temporal regulation of transcription. in the case of Φ the l segment has the sequence gu at the 5' end of the plus strand while s and m have gg. the polymerase favors g over u as the second nucleotide and it is proposed that the second base is the first to be paired during transcription. a host protein, yajq, is able to alter this selection so as to enable active transcription of the l segment upon entry into the host cell. a change of gu to gg in segment l of Φ results in in vitro transcription of segment l. although the structure of the polymerase of Φ has not been studied, it seems likely that in this case the terminal nucleotide would be paired first and that g is preferred to a. the host specificity of Φ is different from that of its close relative Φ12; however it was possible to construct viable phage with a middle segment containing the pac sequence of Φ and the genes and of Φ gene codes for the host attachment protein while gene codes for its membrane bound anchor. the plasmid plm has the 5' region of Φ2954m up to the sphi site at position and the sequence of Φ13m from sacii at nucleotide the resulting phage, Φ does not plate on the normal host of Φ hb10y but does plate on strains that have rough lps such as lm or lm we have also constructed a plasmid with the pac sequence of Φ2954m and the genes and of Φ the resulting phage has the same plating properties as Φ with respect to pilus attachment. another test of the functionality of the cdna copy of segment m was to determine whether bacteriophage Φ could acquire the transcript of this plasmid in order to change its host range. plasmid plm which carries the cdna copy of Φ genomic segment m, was electroporated into strain lm before infection with Φ these cells were plated along with those of hb10y and plaques were obtained. these plaques plated on hb10y but not on a strain missing the type iv pili. the genomic segments of these phages were consistent with the segments l and s of Φ and m of Φ. the finding that Φ is able to acquire segment m of Φ is intriguing in that the pac sequences in m are very different for both phages. this is reminiscent of the case of bacteriophage Φ acquiring segment m of Φ in which case there is again very little sequence similarity in the pac sites. this is so despite the observation that small changes in the pac sequences of Φ m or s drastically reduce the ability of Φ to acquire these segments. the finding that it is possible to change the host attachment proteins is of special interest in that it shows that the proteins p and p are able to recognize viral membrane that contains the major membrane protein p of distantly related phages of the same family. another test of genomic packaging was the production of a genomic segment containing segments s and m joined together. the apai to xbai segment of m was joined to the psti site that is present in the vector following the 3' end of the cdna copy of segment s. a plasmid plm containing this construct was able to donate its transcript to phage Φ that contains segments s and l of Φ and an m segment that has the pac sequence of Φ2954m but the genes of segment m of Φ a phage that plates on host cells containing rough lps. electroporating plasmid plm into strain lm produced a phage with the entire genome contained in a single segment. this plasmid contained the cdna copies of the complete segment s with the sequence of segment m beginning with the apai site at position to the xbai site following its c terminus with segment l beginning with an mfei site at position that was converted to xbai. the observation that phage were produced in high yield from this plasmid is consistent with the previous observations of the preparation of single segment genomes in Φ and Φ it also suggests that the open reading frames of genes and starting at and are not necessary for phage production.  Φ has a number of properties similar to other members of the cystoviridae; however, it shows some interesting differences. in particular, it regulates transcription by altering the first nucleotide of the segment l transcript relative to those of segments s and m while most other cystoviruses regulate by altering the second nucleotide. the cdna copies of the genome have been shown to be accurate and they allow manipulation of the structure of the genome. Φ will be an important component in the investigation of the temporal control of transcription in the cystoviridae. 
although the pac sequences of Φ show no similarity to those of Φ segment m of Φ could be acquired by Φ resulting in a change of host specificity. phage were constructed with one, two or three genomic segments. the base sequences for many of the genes and for the segment termini were similar but not identical to those of bacteriophage Φ however, the host specificity was for the type iv pili of pseudomonas syringae hb10y rather than for the rough lps to which Φ attaches. reverse genetics techniques enabled the production of infectious phage from cdna copies of the genome.
 bacteriophage Φ is a member of the cystoviridae and is distinct from Φ the first member of that family. we have recently isolated a number of related phages and five showed high similarity to Φ in the amino acid sequences of several proteins. bacteriophage Φ is a member of this group.  Φ was isolated from radish leaves and was found to have a genome of three segments of double-stranded rna, placing it in the cystoviridae. the base sequences for many of the genes and for the segment termini were similar but not identical to those of bacteriophage Φ however, the host specificity was for the type iv pili of pseudomonas syringae hb10y rather than for the rough lps to which Φ attaches. reverse genetics techniques enabled the production of infectious phage from cdna copies of the genome. phage were constructed with one, two or three genomic segments. phage were also produced with altered transcriptional regulation. although the pac sequences of Φ show no similarity to those of Φ segment m of Φ could be acquired by Φ resulting in a change of host specificity.  we have isolated a new member of the bacteriophage family cystoviridae and find that although it shows similarity to other members of the family, it has unique properties that help to elucidate viral strategies for genomic packaging and gene expression. 