Genetic diversity among five T4-like bacteriophages

BACKGROUND: Bacteriophages are an important repository of genetic diversity. As one of the major constituents of terrestrial biomass, they exert profound effects on the earth's ecology and microbial evolution by mediating horizontal gene transfer between bacteria and controlling their growth. Only limited genomic sequence data are currently available for phages but even this reveals an overwhelming diversity in their gene sequences and genomes. The contribution of the T4-like phages to this overall phage diversity is difficult to assess, since only a few examples of complete genome sequence exist for these phages. Our analysis of five T4-like genomes represents half of the known T4-like genomes in GenBank. RESULTS: Here, we have examined in detail the genetic diversity of the genomes of five relatives of bacteriophage T4: the Escherichia coli phages RB43, RB49 and RB69, the Aeromonas salmonicida phage 44RR2.8t (or 44RR) and the Aeromonas hydrophila phage Aeh1. Our data define a core set of conserved genes common to these genomes as well as hundreds of additional open reading frames (ORFs) that are nonconserved. Although some of these ORFs resemble known genes from bacterial hosts or other phages, most show no significant similarity to any known sequence in the databases. The five genomes analyzed here all have similarities in gene regulation to T4. Sequence motifs resembling T4 early and late consensus promoters were observed in all five genomes. In contrast, only two of these genomes, RB69 and 44RR, showed similarities to T4 middle-mode promoter sequences and to the T4 motA gene product required for their recognition. In addition, we observed that each phage differed in the number and assortment of putative genes encoding host-like metabolic enzymes, tRNA species, and homing endonucleases. CONCLUSION: Our observations suggest that evolution of the T4-like phages has drawn on a highly diverged pool of genes in the microbial world. The T4-like phages harbour a wealth of genetic material that has not been identified previously. The mechanisms by which these genes may have arisen may differ from those previously proposed for the evolution of other bacteriophage genomes

Ω mutagenesis in Gram-negative bacteria: a selectable interposon which is strongly polar in a wide range of bacterial species

We have used the 2.0-kb DNA fragment Ω [Prentki and Krisch, Gene 29 (1984) 303–313] to mutagenize in vitro a broad-host-range plasmid carrying the entire meta-cleavage pathway of the Pseudomonas putida TOL plasmid pWW0. The mutant plasmids were subsequently introduced by conjugal mobilization into a variety of Gram-negative bacteria. The Ω fragment carries a selectable marker (aadA+; SpcR/SmR), which is expressed in all species tested, as well as flanking transcription and translation termination signals and synthetic Polylinkers. Expression of the plasmid-bome catechol 2,3-dioxygenase (C230) gene, situated downstream from the site of Ω insertion, was substantially reduced in all strains tested. The transcription terminators originally cloned from bacteriophage T4 gene 32, are apparently functional in a wide range of hosts. Insertional mutagenesis with the Ω ‘interposon' can thus be used in a wide variety of species, with the advantages of a positive selection for the presence of the fragment, the termination of RNA and protein synthesis beyond the site of insertion, and genetic stability of the resulting mutation

A plasmid expression vector that permits stabilization of both mRNAs and proteins encoded by the cloned genes

Two new expression vectors have been constructed to take advantage of several useful properties of bacteriophage T4-infected Escherichia coli. These plasmids, pRDB8 and pRDB9, contain the promoter region and start codon of T4 gene 32, a contiguous multiple cloning site (MCS), and translation and transcription termination signals. DNA fragments inserted into the MCS are transcribed and translated at a high level in both uninfected and phage T4-infected cells. Furthermore, the extreme stability of the hybrid mRNA after infection permits the specific biosynthetic labeling of the protein encoded by the cloned gene. In addition, the cloned gene product is stabilized, since the host-mediated degradation of foreign proteins is inhibited by phage infection. The properties of this expression system were demonstrated with the constant region of a rabbit immunoglobulin lambda light chain (C lambda) gene. Although proteolytic degradation of the C lambda fusion protein was rapid in uninfected cells, degradation was blocked in phage-infected cells and the protein accumulated in greater amounts

Gene 32 transcription and mRNA processing in T4-related bacteriophages

We have analysed transcription and mRNA processing for the gene 32 region of five phages related to T4. Two different organizations of gene 32 proximal promoters were found. In T4 and M1, middle‐ and late‐mode promoters are separated by 50 nucleotides and located within an upstream open reading frame. In T2, K3, Ac3, and Ox2, the 626 bp T4 sequence that includes these promoters is replaced by a 59 bp sequence containing overlapping middle and late promoters. The RNase E‐dependent processing of the g32 mRNAs is conserved in all of the phages. The processing site immediately upstream of g32 in T4 and M1 has been replaced in the other phages by a different sequence that is also cleaved by RNase E. The remarkable conservation of these regulatory features, despite the sequence divergences, suggests that they play an important role in the control of gene expression

A bacteriophage T4 expression cassette that functions efficiently in a wide range of Gram-negative bacteria

We have constructed a derivative of the broad-host-range vector RSF1010. This plasmid, pαΩ, contains an expression cassette derived from bacteriophage T4 gene 32, into which we have inserted the coding sequence for the xylE enzyme (C2,30) of the TOL plasmid pWWO. The composite plasmid, pαxylEΩ, was transferred by conjugal mobilisation into a variety of Gram-negative bacteria (Agrobacter, Paracoccus, Erwinia, Pseudomonas, Rhizobium and Xanthomonas). High levels of C2,30 activity were found in almost all of the extracts. Polyacrylamide gel electrophoresis of these extracts revealed a prominent protein band at 35 kDa whose identity as the C2,30 gene product was confirmed by immunoblotting. We have mapped the 5′ ends of the gene 32/xylE hybrid transcripts. In all of the Gram-negative bacteria, the proximal P2 promoter is the most efficient promoter in the cassette. In most of the strains a weaker and more distal promoter activity (P1) was also detected. In both uninfected and phage-infected Escherichia coli cells, the transcript produced from this promoter is processed at a specific site upstream from the gene 32 start codon. The same processing occurred in all the bacterial species examined. The decay of the hybrid xylE transcript has been analyzed in E. coli and Erwinia, and in both strains this mRNA was among the most stable

RNase E, an endoribonuclease, has a general role in the chemical decay of <i>Escherichia coli</i> mRNA: evidence that <i>rne</i> and <i>ams</i> are the same genetic locus

Escherichia coli RNase E is known to process RNA precursors at specific sites. We show that this endoribonuclease has a general role in E. coli mRNA turnover and affects the stability of specific transcripts. The effect of the rne mutation on functional stability of mRNA was much less pronounced than that on chemical stability, although the expression of some genes was affected. The E. coli ams (altered mRNA stability) mutation was found to have phenotypes indistinguishable from those of the rne mutation, affecting both 9S RNA and T4 gene 32 mRNA processing. The rne and ams mutations were both complemented by the same 3.7 kb fragment of E. coli DNA and are probably allelic. RNase E is the first endoribonuclease identified as having a general role in the chemical decay of E. coli mRNA

Specificity of <i>Escherichia coli</i> endoribonuclease RNase E: in vivo and in vitro analysis of mutants in a bacteriophage T4 mRNA processing site

Endoribonuclease RNase E has an important role in the processing and degradation of bacteriophage T4 and Escherichia coli mRNAs. We have undertaken a mutational analysis of the -71 RNase E processing site of T4 gene 32. A series of mutations were introduced into a synthetic T4 sequence cloned on a plasmid, and their effects on processing were analyzed in vivo. The same mutations were transferred into T4 by homologous recombination. In both the plasmid and the phage contexts the processing of the transcripts was similarly affected by the mutations. Partially purified RNase E has also been used to ascertain the effect of these mutations on RNase E processing in vitro. The hierarchy of the efficiency of processing of the various mutant transcripts was the same in vivo and in vitro. These results and an analysis of all of the known putative RNase E sites suggest a consensus sequence RAUUW (R = A or G; W = A or U) at the cleavage site. Modifications of the stem-loop structure downstream of the -71 site indicate that a secondary structure is required for RNase E processing. Processing by RNase E was apparently inhibited by sequences that sequester the site in secondary structure