Kohsuke Furuse

Grouping of RNA coliphages based on analysis of the sizes of their RNAs and proteins.

Abstract In order to elucidate the intergroup relationships among four groups of RNA coliphages (RNA phages), we studied the sizes of their RNAs by measuring the sedimentation velocity of the RNA in a sucrose density gradient and the electrophoretic mobility of the RNA and those of proteins in polyacrylamide gel. The RNAs of group I, II, III, and IV phages (including serological intermediates) were found to have sedimentation coefficients of 24, 23, 27, and 28 S by sucrose density gradient centrifugation analysis and to have average molecular weights of 1.21, 1.20, 1.39, and 1.42 × 106 daltons by gel electrophoretic analysis, respectively. In the virions of group I and II phages, there were two kinds of protein (maturation protein and coat protein). In those of group III and IV phages, an additional protein, read-through (IIb or Al) protein (average molecular weights: 3.85 × 104 for group III, and 3.90 × 104 for group IV phages) was detected. The average molecular weights of coat protein from groups I, II, III, and IV were 1.40, 1.29, 1.69, and 1.73 × 104, respectively. Those of maturation protein were 4.48, 4.45, 4.50, and 4.8 × 104, respectively. Read-through protein was synthesized not only in cells infected with group III and IV phages, but also in a cell-free protein synthesizing system directed by groups III and IV phage RNAs. These results indicate that a distinct difference (about 20%) in molecular size of RNA exists between groups I and II and groups III and IV, which reflects the presence of readthrough protein in groups III and IV. The above results suggest that the molecular sizes of RNAs and virion proteins may offer a useful means for grouping RNA phages, because the present results were in good agreement with those of grouping of RNA phages based on serological property. In this respect, the serologically intermediate phages, JP34 and MX1, were classified into groups II and IV, respectively.

Relationships among four groups of RNA coliphages based on the template specificity of GA replicase

Abstract In order to elucidate the intergroup relationships among four groups of RNA coliphages, we investigated the template activity of various phage RNAs with GA replicase in an in vitro complementary (minus strand) RNA synthesizing system. Phage RNAs of groups I and II showed almost equal template activities with GA replicase, those of group III revealed no template activity with the same replicase, while those of group IV showed lower but distinct template activity with the same replicase. On this basis, some affinity between group II and IV phage RNAs may exist.

Distribution of RNA Coliphages in Senegal, Ghana, and Madagascar

The distribution patterns of RNA coliphages (phages) in Senegal, Ghana, and Madagascar were investigated by collecting sewage samples from domestic drainage in November, 1980. In Senegal, among 65 sewage samples collected mainly from Dakar and its vicinity, 14 (22%) contained RNA phages (16 strains). By serological analysis, 13 of the 16 strains were found to belong to group III. This is consistent with the distribution pattern of RNA coliphages in tropical and subtropical regions of Asia. In Ghana, however, among 106 samples collected from Accra, Suhum, and their vicinities, only seven (7%) contained RNA phages (seven strains) (groups I, II, and III [1:3: 3]). In Madagascar, among 124 samples collected from Antananarivo, Moramanga, and their vicinities, seven (6%) contained RNA phages (seven strains) (groups I, II, III, and IV [1:1:1:4]). In spite of the low isolation frequency, it can be said that Madagascar appears to have a unique distribution pattern (abundance of group IV phages) which differs from that of any other countries we have examined. The generality of the distribution pattern of RNA phages in the tropical region (abundance of group III phages) was thus verified at least in Senegal.

Characterization of an Intergroup Serological Mutant from Group II RNA Phage GA

Starting from the group II RNA phage GA which has an amber mutation in the maturation protein cistron, a spontaneous mutant of group II phage GA, whose serological and electrophoretic properties became similar to those of group I phage MS2, was isolated and analyzed. The mutant has now become sensitive to anti‐MS2 serum and resistant to anti‐GA serum. Analysis of the nucleotide sequence of the coat protein gene revealed that G↔A transition was the main change. The deduced amino acid sequence showed that five amino acids were substituted in the mutant, and three of the five became identical to MS2, resulting in increased molecular weight of the coat protein. However, it did not complement MS2. These results suggested that the serological change from group II phage GA type to group I phage MS2 type is induced spontaneously at high frequency by minor nucleotide changes in coat protein gene, and confirmed the previous results at the RNA level that MS2 and GA were related although the closeness between them seems somewhat remoter than that of groups III and IV (18, Inokuchi et al, unpublished data for the nucleotide sequence of group IV phage SP).

Three complementation subgroups in group IV RNA phage SP

Abstract We isolated twelve suppressor sensitive ( sus ) mutants from the RNA phage SP which belongs to group IV and classified them into three cistrons (genes 1, 2, and 3) by complementation tests. Using representative mutants of each cistron, synthesis of infectious RNA, phage antigen (serum blocking power) and defective particles under nonpermissive conditions were examined. Gene 1 mutants had a defect in maturation protein synthesis, gene 2 mutants in coat protein synthesis, and gene 3 mutants in RNA synthesis, respectively. Thus, group IV RNA phage SP has (at least) three genes corresponding to RNA replicase, coat protein, and maturation protein, as already shown in group I (f2, R17) and group III (Qβ) RNA phages. From intergroup complementation tests between several sus mutants of groups I, III, and IV, Qβ(III) had a fairly close relationship to SP(IV), while f2(I) had no such close relationships to Qβ(III) or SP(IV). These are consistent with the results of grouping by several biological and physicochemical criteria and support the idea that groups I and II may be assembled into one group and groups III and IV into another.

Continuous Survey of the Distribution of RNA Coliphages in Japan

In order to demonstrate the stability and continuity of RNA coliphages (phages) in their natural habitats, we investigated the amount and group types of RNA phages in sewage samples collected continuously from domestic drainage in Japan proper and islands in the seas adjacent to Japan (abbreviated simply as islands, hereafter) over a 5‐yr period from 1973 to 1977. It was found that the frequencies of isolation of RNA phages were fairly high and constant. The group types of RNA phages isolated were also stable in the three cities, Choshi, Niigata, and Toyama in Japan proper. The average for the three cities was group II: III = 3: 1. The investigation in islands revealed that the frequencies of isolation of RNA phages were fairly high as in the case of the above three cities in Japan proper and the group types of RNA phages isolated were also stable. That is to say, group II phages were predominant on Rishiri Island, Rebun I., Iki I., and Tsushima I., which are located relatively near to mainland Japan, while group III phages were predominant on Amamiohshima I., mainland Okinawa, Ishigakijima I., and Iriomotejima I., which are located south of Kyushu. It can thus be said that the RNA phages in the domestic drainage of Japan proper and islands remained more or less stable over at least the 5‐yr period, and an apparent difference in the geographical distribution of RNA phages in Japan exists between Kyushu and Amamiohshima I.

Electrophoretic properties of RNA coliphages.

Miyake et al (9, 10, 12) previously reported that RNA coliphages showed group specific electrophoretic profiles on cellulose acetate membranes. Based on this characteristic, we extensively studied the interand intra-group relationships of RNA coliphages classified into four groups (I to IV) according to serological and certain physicochemical properties (5, 9, 13). All RNA phages used here, except for MS2 (1), f2 (7), and R17 (11), were originally isolated in our laboratory and were classified into four groups (2-5,9, 12, 13). Growth of the phages was carried out as described previously (8). Electrophoresis of the phages on cellulose acetate membranes (Separax, jookoo Sangyo Co., japan) was performed as described by Miyake et al (10) with slight modifications (see legend to Fig. 1). As shown in Fig. 1, among the four groups, the electrophoretic profile of group I phage was more group specific than that of the other groups. For example, all the group I phages tested here migrated to the anode (+) side (Fig. 1(A)). Among these phages, MS2 and FRI migrated to the position of fraction 7. Phages f2, R17, ZR, and BOI moved slightly faster than MS2, and jP50l to the furthest (+) side. These results suggest that jP50l is apparently distinct from other group I phages [subgroup I(a)] in this character and should be assigned to a subgroup of I(b). This is supported by the fact that jP50l is serologically different from other group I phages (Furuse et al, unpublished data). Most of the group II phages such as GA, SD, THI, BZ13, and KUl migrated to the position of fraction 3 in a direction opposite to the group I phages (Fig. 1 (B)). It should be noticed in this panel that phages jP34 and jP500, which are serologically close to the group I phage MS2 (2) but classified into group II on the basis of other characters (5,6), moved to a position adjacent to that of MS2. On this basis, group II phages can be divided into two subgroups: (a) GA, SD, THl, BZ13, and KUl, and (b) jP34 and jP500. The electrophoretic profiles of group III phages were slightly complicated. As shown in Fig. 1(C), phages Qf3 and VK migrated to the cathode (-) side, whereas TW18 and ST migrated to the (+) side. Although the mobility of Qf3 was almost the same as that of VK, the mobility of ST was distinctly different from that of TW18. Group III phages can thus be separated into three subgroups on the basis of this character as follows: (a) Qf3 and VK, (b) TW18, and (c) ST.

Pseudolysogenization by RNA Phage Qβ

We isolated fairly stable lysogenic‐like bacteria from a lysogenic state established between an amber mutant for the maturation protein gene of RNA phage Qβ (Qβam205) and its nonpermissive host BE110. These bacteria contained few mature phages intracellularly (less than 10−3 plaque forming unit per cell), continued to grow with a potentiality to produce Qβam205 spontaneously, and showed an immunity‐like response against homologous phage infection. These characteristics were maintained by growth in liquid medium containing anti‐Qβ serum. We designated these cells as pseudolysogenic bacteria. The relative amounts of RNA genomes in these pseudolysogenic cells (about 102 infectious RNA strands per cell) indicated that the RNA genomes could replicate in nonpermissive cells and be distributed in daughter cells synchronizing well with cell division.

The Host-Dependent Restriction of Growth of an RNA Coliphage FI

Phage FIC is a spontaneous host‐dependent mutant of phage FI which is classified into the fourth group of RNA Escherichia coli phages (RNA coliphages). The mutant phage (FIC) grows normally in E. coli strain Q13 (permissive host), but poorly in strain A/λ (non‐permissive host) (9). Attempts to elucidate the regulatory mechanism of growth of the mutant phage in the non‐permissive host revealed the following: (a) growth of the mutant phage was specifically restricted in E. coli strains that have certain suppressor genes for amber mutation; (b) the mutant phage RNA (FIC‐RNA) could not produce progeny in the spheroplasts of the non‐permissive host; (c) adsorption of the mutant phage to, and penetration of the mutant phage RNA into, the non‐permissive host were normal; and (d) biosynthesis of the phage‐specific late protein and RNA did not occur in the non‐permissive host. Based on these results we conclude that phage FIC is a spontaneous azure‐type mutant of the fourth group of RNA coliphage FI.

Isolation and characterization of RNA phages for Caulobacter crescentus.

Abstract We have isolated six RNA-containing bacteriophages for Caulobacter crescentus from sewage and river water collected in Japan. These phages were different serologically from phage ∅Cb5 which is an RNA-containing Caulobacter phage reported previously. Two of these six isolates, named ∅Cp2 and ∅Cp18, were different from each other serologically, in plaque morphology, and in stability of infectivity. Phages ∅Cp2 and ∅Cp18, however, were similar in virion size (29.2 and 28.5 nm, respectively), sedimentation coefficient of the viron (79.5 and 80.7 S, respectively), sedimentation coefficient of the phage RNA (30.7 and 29.6 S, respectively), and RNA base composition (both poor in adenine and rich in uracil). These two RNA phages are discussed in comparison with RNA phages reported previously for Caulobacter .