Inhibition of growth of bacteriophage BF23 by the ColIb plasmid: Identification of the ibfA and ibfB genes of the ColIb plasmid

Summary A 3.7 Mdal DNA fragment of plasmid ColIb which carried all the genetic information determining the growth inhibition of bacteriophage BF23 (Ibf phenotype) but not for production of colicin Ib protein (Cib) and immunity to colicin Ib (Imm) was cloned in the pBR322 vector. We also cloned the 8.7 Mdal DNA fragment that was responsible for both Cib and Imm but not for Ibf phenotype. Thus, these results clearly showed that the gene(s) determining Ibf are different from those for Cib or Imm. Dissection of the Ibf-DNA revealed that at least two genes, ibfA and ibfB, were involved in the Ibf phenotype. The ibfA gene was mapped within a 1.45 Mdal EcoRI DNA fragment (E-13); mutation of this gene by deletion or by insertion of Tn5, a kanamycin resistance transposon, resulted in the complete loss of Ibf phenotype. The ibfB gene, mapped around a 0.32 Mdal HindIII DNA fragment (H-7), was found to be active in trans to ibfA, and its function seemed to promote ibfA activity. The genetic map of the ibf genes in relation to other ColIb markers was determined as ibfB-ibfA-imm-cib. Attempts to identify the ibfA gene product in the minicell system, however, did not succeed.Preliminary results were presented in the 1980 Annual Meeting of the Japan Molecular Biology Society. (Uemura and Mizobuchi Abst Ann Mol Meet 1980, p 35)     

ColIb plasmid genes that inhibit the replication of T5 and T7 bacteriophage

The colicin Ib (ColIb) plasmid genes that inhibit the replication of the T5-like and T7 bacteriophage have been cloned on an approximately 7200-bp ClaI fragment and their sites relative to each other and to the colicin immunity (imm) gene have been mapped. The inhibition of wild-type T7 by the clone is shown to be caused by the same gene or genes (pic) that cause the inhibition of T7 kinase-negative mutants and is a different gene than the one that causes inhibition of T5 (ibf or abi). The pic gene does not hybridize to the pif genes of the F plasmid that also cause the replication of T7 to be inhibited. The abi gene and the pic gene map very closely together but are under the control of different promoters. The abi gene has a maximum size of 900 bp and lies approximately 3000 bp away from the immunity gene, distal to the colicin gene. A site which maps in or near the gene binds very tightly to Escherichia coli RNA polymerase. The pic gene or genes lie between the abi gene and the imm gene and are contiguous with abi. Promoters for pic have been mapped and hypotheses to explain the inhibition of T7 by a cloned gene but not the whole ColIb plasmid are presented.     

Relationships among genes and gene products of bacteriophage BF23

Twenty-five gene products of bacteriophage BF23 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functions were studied in relation to type I and II genes classified by means of genetic complementation tests. All the type I mutants were defective in the synthesis of a tail protein, L3. In addition, 4 type I gene products, L5 (gp21), L7 (gp20), L8 (gp29), and L9 (gp25), were identified as constituents of tails (gp21 denotes that a protein is a product of gene 21). Three type IIb mutants in genes 10, 14, and 19 diminished substantially the production of late proteins, including tail and head proteins, and the two other type IIb mutants in genes 1 and 2 were defective in the synthesis of both early and late proteins. Of 14 type IIa mutants, at least 6 were defective in phage DNA synthesis and 2 were defective in the synthesis of head proteins. The defect in the head donor activities of type IIa mutants in extract complementation tests was due to the failure of the formation of mature heads containing DNA. The above results support directly the results of the genetic characterization of BF23 genes.     

Terminally redundant deletion mutants of bacteriophage BF23.

Deletion mutants of bacteriophage BF23 were isolated and the positions of the deletions were determined. Two different deletable regions were detected: one in the same region as previously reported for bacteriophage T5, which is closely related to BF23; and the other within both terminal repetitions. The former deletable region lay between positions 0.31 and 0.36, which represented the fractional lengths of the BF23 ( + ) DNA as measured from its left end. The latter deletion was evenly divided between the two terminal repetitions. The deletion in the left terminal repetition lay between positions 0.044 and 0.078 and was repeated in the corresponding region of the right terminal repetition between positions 0.966 and 1.0. The size of the DNA transferred to host cells during the first step of DNA transfer by BF23 carrying deletions in the terminal repetitions of its DNA was less than the size of DNA transferred during the first step by wild-type BF23 by an amount equal to the size of the deletion in each terminal repetition. This finding suggests the existence of a specific mechanism for delineating the position at which the first step of DNA transfer is stopped.     

Isolation and genetic characterizaion of Escherichia coli K-12 mutations affecting bacteriophage T5 restriction by the ColIb plasmid.

A mutant derivative of Escherichia coli K-12 has been isolated which is permissive for bacteriophage T5 infection even when harboring a wild-type ColIb plasmid. The fully permissive phenotype was the result of two mutations that are located near the rpsL-rpsE region on the E. coli chromosome and are recessive to the wild-type alleles. These mutations had little or no effect on induction of colicin synthesis and did not affect the expression of antibiotic resistance by the resistance plasmids R64drd11 or R1drd19. Cells harboring the mutant alleles grew more slowly than isogenic wild-type derivatives in either minimal or complete media.     

Physical map of bacteriophage BF23 DNA: restriction enzyme analysis.

Cleavage maps of bacteriophage BF23 DNA have been constructed for the restriction endonucleases SalI (3 fragments), BamHI (5 fragments), EcoRI, (8 fragments), BalI (13 fragments), and HpaI (49 fragments, 32 of which have been ordered). The maps were determined by (i) analysis of deletion mutants, (ii) digestion with two endonucleases, (iii) digestion of isolated fragments with a second enzyme, (iv) analysis of partial digests, and (v) digestion after treatment with lambda exonuclease.     

Cloning, sequencing, and expression of bacteriophage BF23 late genes 24 and 25 encoding tail proteins.

Two bacteriophage BF23 late genes, genes 24 and 25, were isolated on a 7.4-kb PstI fragment from the phage DNA, and their nucleotide sequences were determined. Gene 24 encodes a minor tail protein with the expected M(r) of 34,309, and gene 25 located 4 bp upstream of gene 24 encodes a major tail protein with the expected M(r) of 50,329. When total cellular RNA isolated from either phage-infected cells or cells bearing the cloned genes was analyzed by the primer extension method using the primers specific to either gene 25 or gene 24, we identified a possible late gene promoter, designated P25, in the 5'-flanking region of gene 25. This promoter was similar in structure to Escherichia coli promoters for sigma 70. Studies of the translational gene 25- and gene 24-lacZ fusions in the cloned gene system revealed that the promoter P25 was responsible for the expression of both genes 25 and 24 even in the absence of the regulatory genes which were absolutely required for late gene expression in the normal phage-infected cells. These results indicate that the two genes constitute an operon under the control of P25 and that the regulatory gene products of BF23 do not participate directly in specifying the late gene promoter.     

The relationship of the delta transfer factor and KColIb to the ColIb plasmid

The structures of the colicin Ib plasmid (ColIb), the delta transfer factor and a plasmid determining kanamycin resistance and colicin Ib production called KColIb, were compared. Radiolabelled mini-ColIb plasmids and isolated DNA fragments of ColIb were used as probes for nitrocellulose blots of digests of the other two large plasmids. The structure of delta was consistent with it having one large deletion of about 10 MDa in the SB fragment and two insertions of approximately 6 MDa and 12 MDa in the SB and SA fragments of the ColIb plasmid. It was hypothesized that KColIb had six small insertions in SA, SB, SE and near the junction of the SB and SD fragments. However, ColIb, KColIb and delta were homologous for at least 70% of their lengths. The highly conserved regions in the three plasmids were the regions that corresponded to fragments SA, SC and SD of ColIb. In addition, delta and KColIb differed from ColIb at similar sites. The possible evolution of these plasmids is discussed.     

Physical map of bacteriophage BF23 DNA: terminal redundancy and localization of single-chain interruptions.

The DNA of bacteriophage BF23 possesses two structural features, localized single-chain interruptions and a large terminal repetition, previously described for T5, a closely related virus. As is the case for T5, single-chain interruptions occur with variable frequencies at a small number of fixed sites within one strand of the double-stranded BF23 genome. The sites where interruptions occur with the highest frequencies were napped by an electrophoretic analysis of the single-stranded fragments produced by denaturation of BF23 DNA. The positions of these fragments were determined by degrading BF23 DNA to various extents with lambda exonuclease and observing the relative order with which they were (i) degraded or (ii) released intact from the undenatured duplex. The exact locations of the interruptions were determined from analysis of analogous duplex fragments produced by degrading exonuclease III-treated BF23 DNA with a single-strand-specific endonuclease. BF23 has five principal sites (located at 7.9, 18.7, 32.4, 65.8, and 99.6% from the left end of the DNA) where interruptions occur in most molecules. The principal interruptions in T5 DNA occur at similar positions. The locations of eight secondary interruptions in BF23 DNA were also determined. In general, BF23 DNA has fewer secondary interruptions than t5 dna, although there is at least one location where an interruption occurs with a greater frequency in BF23. The presence of a terminal repetition in BF23 DNA was demonstrated by annealing ligase-repaired molecules that had been partially digested with lambda exonuclease. If the complementary sequences at both ends of the DNA were exposed by exonuclease treatment, the duplex segment that resulted from annealing could be released by digestion with a single-strand-specific endonuclease. This segment was analyzed by agarose gel electrophoresis and found to represent 8.4% of BF23 DNA.     

Inhibition of bacterial growth and malolactic fermentation in wine by bacteriophage

Bacteriophage present in wine can attack bacterial starter cultures and inhibit the malolactic fermentation. The possibility of starter culture failure due to phage attack was studied in a commercial dry red wine of pH 3·23, inoculated with a multiple strain starter culture. During two stages of malolactic fermentation, bacterial growth and malate degradation in the wine were inhibited. A phage capable of lysing isolates of Leuconostoc oenos was isolated from the wine. The isolated phage had an icosahedral head of 42–45 nm diameter and a flexible, regularly cross-striated tail 197–207 nm long with a small baseplate. The results confirm that phage can attack bacterial starter cultures in wine at low pH.     

Genetic and physical characterization of the ColIb plasmid using ColIb-R222 hybrids

Summary The presence of the ColIb plasmid in Escherichia coli cells inhibits the growth of bacteriophages BF23 and T5 (Ibf phenotype; inhibition of BF23 and T5 growth). To understand this abortive infection, we devised a method of isolating mutants that were defective in some ColIb phenotypes including Ibf. This method consisted of transduction of the tet (Tc^r; tetracycline resistance) or cml (Cm^r; chloramphenicol resistance) gene of plasmid R222 with phage P22 into ColIb, construction of Tc^rCm^rIbf⁺ Imm⁺ (immunity to colicin Ib) Cib^- (no production of colicin Ib) recombinants by crossing between the transductants, and isolation of deletion mutants from the recombinants by phage P1 transduction. By this procedure, pKM25-2 (Tc^rCm^sIbf^-Imm^-Cib^-) and pKM25-1 (Tc^rCm^sIbf⁺Imm⁺Cib^-) were isolated. Construction of the cleavage map of the ColIb plasmid by restriction endonucleases and comparative analyses of the DNA fragments produced from the mutant plasmids revealed that the genes determining Ibf and Imm mapped on a 4.60 Mdal HindIII fragment (H-3) and the gene determining Cib on a 1.71 Mdal EcoRI fragment (E-12).These results together with other observations (Wilkins et al. 1981; Hama personal communication) also show the approximate positions of the genes for Rep (replication), Inc (incompatibility), and Sog (suppression of dnaG) as well as Ibf, Imm, and Cib phenotypes on the cleavage map of the ColIb plasmid.Preliminary data were reported in the 1979 Annual Meeting of the Japan Molecular Biology Society (Uemura and Mizobuchi, Abst Ann Mol Biol Meet 1979, p 36)     

Physical map of the dispensable region of the genome of E. coli bacteriophage BF23

Using 13 deletion mutants of bacteriophage BF23, physical as well as genetic structures of that portion of the genome which is dispensable for phage growth were investigated. The dispensable region covers at least 15% of the genome of wild type BF23, extending from about 0.2 to 0.35 map unit. Restriction endonuclease (EcoRI and HindIII) cleavage sites and the sites of single-strand interruptions in this dispensable region were localized. It was found that the dispensable region contains an interruption site, which is missing in the mutant BF23st(0) used by Okada and Shimura (1980). Wild-type phage DNA is heterogeneous in the presence or absence of specific single-strand interruptions in this or in a neighboring region of the genome.     

Abortive infection by bacteriophage BF23 due to the colicin Ib factor. II. Involvement of pre-early proteins

The mechanism by which the growth of phage BF23 is arrested in cells carrying colicinogenic factor Ib involves certain phage-specific pre-early proteins. BF23 induces the extensive formation of proteins lc and ld, but very little formation of protein le, whereas BF23h^? (a mutant that is not arrested in cells carrying colicinogenic factor Ib) induces very small amounts of proteins lc and ld, but extensive amounts of protein le. Proteins lc and ld may be oligomers of protein le, and it is the presence of these putative oligomers that is necessary for the arrest of phage growth.     

Cloning, sequencing, and recombinational analysis with bacteriophage BF23 of the bacteriophage T5 oad gene encoding the receptor-binding protein.

Binding of bacteriophage T5 to its receptor, the Escherichia coli FhuA protein, is mediated by tail protein pb5. In this article we confirm that pb5 is encoded by the T5 oad gene and describe the isolation, expression, and sequencing of this gene. In order to locate oad precisely, we analyzed recombinants between BF23, a T5-related phage with a different host range, and plasmid clones containing segments of the T5 chromosome. This analysis also showed that oad has little or no homology with hrs, the analogous BF23 gene. We were able to overproduce a protein that comigrates with pb5 after fusing a 2-kb segment containing oad to a phage T7 promoter. This segment contains an open reading frame that can encode a protein of the appropriate size. Its deduced amino acid sequence does not closely resemble that of any other protein in the database. The sequence upstream of the open reading frame shows typical characteristics of a promoter region with two overlapping, divergently orientated promoters.