Molecular genetic analysis of bacteriophage P22 gene 3 product, a protein involved in the initiation of headful DNA packaging.

Bacteriophage P22 DNA packaging events occur in processive series on concatemeric phage DNA molecules. At the point where such series initiate, the DNA is recognized at a site called pac, and most molecular left ends are generated within six short regions called end sites, which are present in a 120 base-pair region surrounding the pac site. The bacteriophage P22 genes 2 and 3 proteins are required for successful generation of these ends and DNA packaging during progeny virion assembly. Mutants lacking the 162-amino-acid gene 3 protein replicate DNA and assemble functional procapsids. In this report we describe the nucleotide changes and DNA packaging phenotypes of a number of missense mutations of gene 3, which give the phage a higher than normal frequency of generalized transduction. In cells infected by these mutants, more packaging events initiate on the host chromosome than in wild-type infections, so the mutations are thought to affect the specificity of packaging initiation. In addition to having this phenotype, these mutations affect the process of phage DNA packaging in detectable ways. They may: (1) alter the target site specificity for packaging; (2) make target site recognition more promiscuous; (3) affect end site utilization; (4) alter the pac site; and (5) cause apparent random DNA packaging series initiation on phage DNA.

Sequence comparison of the genes for immunity, DNA replication, and cell lysis of the P22-related Salmonella phages ES18 and L.

Complementation and hybridization experiments with the generalized transducing Salmonella phages P22, ES18 and L revealed strong similarity between the phages L and P22; the genome of ES18 shows a mosaic structure. About half of its genome, including the early genes, is similar or completely homologous to P22; the other half of the morphologically different ES18 does not show any similarity to P22 nor to E. coli phage lambda. Sequence comparison of the early genes has confirmed that the C-immunity region of ES18 is identical with that of P22, whereas the same region of phage L shows poor (repressor gene) or no similarity. The 5'-terminus of the DNA replication gene 12 of ES18, however, is homologous to the same section of gene O of phage lambda. The lysis genes of ES18 again are identical to those of P22; only gene 15 is mosaic-like and has more similarity to gene Rz of phage lambda. These results will be discussed in terms of the theory of modular genome organization.

Isolation of fragments with pac function for phage P22 from phage LP7 DNA and comparison of packaging gene 3 sequences.

Three PstI DNA fragments of the P22-related Salmonella phage, LP7, have been cloned. They contain sequences recognized as pac signals by the packaging apparatus of P22. One of these fragments corresponds to the P22 DNA fragment carrying gene 3 which comprises the pac signal of phage P22. The product of gene 3, Gp3, is involved in the recognition of pac and the packaging process. Gene 3 of LP7 and most of the adjacent gene 2 have been sequenced. The pac analogous segments of the other two PstI fragments have been narrowed down by subcloning and by transduction of the resulting hybrid plasmids under recombination-defective conditions.

Sequences of tRNA-encoding genes and associated open reading frames of Streptomyces lividans.

A gene library of Streptomyces lividans has been screened for tRNA-encoding genes with labeled Streptomyces tRNA as a probe. By sequence analysis of hybridizing fragments, two single genes have been identified which code for tRNA(Asp) and tRNA(Gly). Associated with the tRNA(Gly) gene, there are three open reading frames (ORFs) which might code for gene products possibly involved in active transport processes through the bacterial membrane. The transcriptional organization of tRNAGly and the following ORFs was examined by high-resolution S1 mapping. A third clone carried a cluster of genes which encode two tRNA(Gln) and three tRNA(Glu). This cluster corresponds to a similar cluster previously described for Streptomyces rimosus [Plohl and Gamulin, Mol. Gen. Genet. 222 (1990) 129-134].

Transduction of multiple drug resistance of Salmonella enterica serovar typhimurium DT104.

Epidemic strain Salmonella typhimurium DT104 is characterized by various multiresistance patterns. At least some of the resistance genes are organized as integrons. Resistance genes of DT104 isolates can be efficiently transduced by P22-like phage ES18 and by phage PDT17 which is released by all DT104 isolates so far analyzed. Cotransduction tests demonstrate that the resistance genes, although not organized in a unique integron, are tightly clustered on the Salmonella chromosome. The spread of resistance genes in this strain by generalized transduction is discussed.

Isolation of ribosomal RNA operons of Streptomyces lividans and sequence analysis of a 5S-rRNA gene.

Three clones containing rRNA genes have been isolated from a gene library of Streptomyces lividans. Two clones carried entire but different rRNA-operons. The third clone comprised the 3'-end of a 23S-rRNA gene, the entire 5S-rRNA gene and the spacer region between them. The nucleotide sequence starting within the 23S-rRNA gene beyond the putative transcription terminator downstream the 5S-rRNA gene has been determined (EMBL acc. no X58874).

Sequence analysis of cohesive ends of the actinophage RP3 genome and construction of a transducible shuttle vector.

The sequence of the cohesive ends of actinophage RP3 DNA has been determined. As with all other phages of Gram-positive bacteria that have been studied sofar, RP3 DNA has 3'-protruding ends. A shuttle cosmid has been constructed containing this cos area which can be efficiently transduced by phage RP3 to host cells of Streptomyces rimosus.

Bacillus cereus may produce two or more diarrheal enterotoxins.

Bacillus cereus strains were tested for production of diarrheal enterotoxin by the reverse passive latex agglutination test and for presence of B. cereus enterotoxin gene (bceT) by polymerase chain reaction. About 50% of 56 B. cereus strains reacted positive in broth culture in the reverse passive latex agglutination test, while the bceT gene was detected in 41.1%. Sixteen percent of the strains were positive for both diarrheal enterotoxin and bceT gene. A 741 bp probe prepared from the polymerase chain reaction product detected bceT gene in all strains that were positive with the polymerase chain reaction. This study indicates a likelihood of two or more enterotoxins being produced by B. cereus which may be involved in causing diarrheal type food poisoning.

Construction and transduction of a shuttle vector bearing the cos site of Streptomyces phage phi C31 and determination of its cohesive ends.

A plasmid has been constructed which is a bifunctional vector for Escherichia coli and for streptomycetes, containing the cos site of Streptomyces phage phi C31. It can be efficiently transduced into S. lividans 66 and into several other Streptomyces species. The nucleotide sequence of a 311-bp DNA fragment containing the cohesive ends has been determined. The cohesive ends consist of 10 bases protruding at the 3'-end of the phage genome.

Molecular characterization and module composition of P22-related Salmonella phage genomes.

Genomes of newly isolated Salmonella phages were analysed by comparison of their EcoRI restriction patterns and by hybridization. Characteristic hybridization probes from reference phages P22, ES18 and E. coli phage lambda were chosen. Four probes selected from the lysis region examined the dispersal of the lambdoid lysis genes. Other probes characterized were the replication genes and part of the structural genes. The complex immunity region was investigated by means of hybridization as well as biological tests. The results showed the relationship of the isolated phages to the P22 branch of the lambdoid phages and revealed their modular genome organization consisting of different proportions of P22-related sequences. DNA restriction patterns of phages released from Salmonella strains sampled in limited geographical areas were significantly less heterogeneous than those of phages released from the worldwide sampled SARA collection. The use of prophage restriction patterns as a tool for the typing of Salmonellae to support the epidemiologic classification of pathogenic strains is discussed.

pac sites are indispensable for in vivo packaging of DNA by phage P22.

F' pro+ plasmids were selected and used as donors to prepare P22 transducing phages. Two types of result were observed. pro+ from type I donors cannot be packaged by wild-type P22 to yield transducing particles unless a prophage pac site is introduced into the plasmid. Transposon Tn10 also allows initiation of packaging. pro+ from type II plasmids can be transduced with the same efficiency as pro+ DNA on the chromosome, indicating that a chromosomal pac site was included when the F' pro+ was excised from the Hfr strain. The usefulness of type I plasmids as a test substrate for pac signals is discussed.

Packaging signals for phage P22 on the chromosome of Salmonella typhimurium.

Numbers of transducing particles for a set of 28 different chromosomal markers were determined in a lysate of Salmonella phage P22. The results were plotted with regard to the map positions of the transduced loci. Maxima of the resulting curve are interpreted as positions of start signals ("pac-sites") for packaging series on the Salmonella chromosome. 5-6 pac-sites could be found as a minimum estimate.

Molecular survey of the Salmonella phage typing system of Anderson.

Typing phages for Salmonella and the prophages of their typical propagation strains were analyzed at the DNA level. Most of them belong to the P22 branch of the lambdoid phages. Acquisition of new plating properties of the typing phages by propagation in particular strains can be due to different host specific modifications of the DNA or to recombination events with residing prophages which are reflected by changes in the respective DNA restriction patterns. It is concluded that the actually available set of typing phages is a historically unique combination of strains.

In vitro packaging of plasmid DNA oligomers by Salmonella phage P22: independence of the pac site, and evidence for the termination cut in vitro.

In vitro packaging experiments with phage P22 using artificially ligated plasmid concatemers have shown that the pac site is not necessary for DNA packaging although in vivo this initiation signal is indispensable. This indicates that the phage-coded protein gp3 also executes other important functions during phage maturation in addition to the recognition of pac, or that its site specificity is lost in vitro. It has been shown previously that gp3 is necessary for in vitro packaging. Further, it was demonstrated that DNA which is only 74% of headful size cannot be packaged. Oversized DNA, however, is cut in vitro to unit length.

Selective transduction of recombinant plasmids with cloned pac sites by Salmonella phage P22.

Recombinant plasmids having PstI fragments of P22 DNA inserted in the vector pBR322 can be transduced efficiently by Salmonella phage P22, irrespective of the cloned phage sequences. When the rec function of the donor cells and the corresponding recombination system erf of the infecting phage are simultaneously inactivated, only plasmids containing the P22 pac site can be transduced. By this selective, generalized transduction an EcoRV DNA fragment of the P22 related phage L has been identified that carries a base sequence recognized by phage P22 as a packaging signal. Experiments in which only one of the two recombination systems was inactivated, showed that the bacterial rec system obviously promotes cointegrate formation between plasmid and phage DNA much more efficiently than the phage-coded erf system, allowing the specialized plasmid transduction observed by Orbach and Jackson (1982).

Spot-transformation with plasmids.

A method is described which allows transformation of bacterial cells on the surface of agar plates. It is suitable for transferring large numbers of different plasmids, such as gene libraries, into a new genetic background. One nanogram of plasmid DNA is sufficient for transformation.

Selection of bacterial pac sites recognized by Salmonella phage P22.

A gene library of chromosomal PstI fragments from Salmonella typhimurium strain DB5575 has been established. By means of phage P22 mediated transduction, ten different clones which contained inserts that promoted plasmid transduction were selected out of a total of about 7,000 clones. Seven of these clones carried inserts that stimulated transduction independently of general and int-promoted recombination and were interpreted as carrying pac analogous signals. The remaining three clones carried inserts that promoted transduction under recombination proficient conditions, whereas transduction occurred at reduced rate in the absence of recombination. These were believed to have short regions of homology with P22 DNA.

Appearance of transducing particles and the fate of host DNA after infection of Salmonella typhimurium with P22-mutants with increased transducing ability (HT-mutants).

The kinetics of production of transducing particles for different bacterial markers were followed by premature lysis of Salmonella typhimurium cells infected with P22 phages. The were compared for cells infected with wild type phage or with HT-mutants which show increased transduction frequencies. Measuring the sedimentation velocity of bacterial DNA of cells infected with wild type or HT-phages, it was shown that: a) there is no cutting of DNA at random; b) original fragments necessary for packaging host DNA into transducing particles cannot be smaller than 10 phage-genome lengths; c) cutting of transducing fragments leads immediately to the right length; d) there is no loss of precipitable DNA due to phage infection.

Altered cotransduction frequencies exhibited by HT-mutants of Salmonella-phage P22.

Phage P22-mutants with increased transduction ability (HT-mutants) show in comparison to wild type 22, different frequencies of contransduction for markers on two different transducing fragments of the Salmonella chromosome. The data are interpreted as indicating that host DNA to be packaged is cut by HT-mutants at sites different from those cut by wild-type phage, due to an altered specificity of the nuclease responsible for this step.

Bacterial DNA synthesized under phage control in a DNA-defective Salmonella-mutant and packaged into a special fraction of transducing particles of phage P22.

Lysates of P22 contain a small fraction of transducing particles with bacterial DNA replicated semiconservatively after the time of infection. It was demonstrated that the presence and relative amount of this class of transducing particles was unchanged, if infection of Salmonella occured under a condition nonpermissive for bacterial DNA replication. Analysis of particles with DNA fragments derived from different regions of the Salmonella chromosome indicated that the replication of the bacterial DNA carried by these transducing particles was not initiated specifically at the normal origin for bacterial chromosome replication.