🔬 Phage T1: A Lambdoid Phage with Attitude?

Bacteriophage Ecology Group News (BEG News) was published mostly quarterly as an online newsletter for a total of 24 issues, July 1999 through April 2005. As follows is a reprint of an article from Volume 18. The newsletter’s successors are the ongoing Phage.org website, phage-therapy.org, and the Bacteriophage Ecology Group Facebook page.

Coliphage T1, one of the original seven T phages suggested by Max Delbrück (3, 4) for concentrated study by the bacteriophage community, has been sequenced. This phage, which the International Council for the Taxonomy of Viruses (ICTV) has treated as a species within the "T1-like viruses" genus (family Siphoviridae), possesses a polyhedral head approximately 60 nm in diameter with a characteristically long (150 nm) flexible noncontractile tail. Other phages which may be part of this genus are: UC-1 (11), D20, Hi, 102, 103, 150, 168, and 174 (7). To this we can now add the TolC-specific phage TLS - previously known as U3 in many laboratories (5).

Initially made famous by its use as the selective agent in the famous fluctuation test conducted by Salvador Luria and Max Delbrück (12)A, T1 gained notoriety because of its resistance to desiccation and its virulence. Unsubstantiated horror stories exist about its effect on industrial laboratories employing fermentations involving Escherichia coliB. Furthermore, while its potential impact was long appreciated by the phage community, the increase in molecular studies by biologists/biochemists unaware of its virulence has often resulted in unwanted infections. Today many biotech firms market T1-resistant competent cells (i.e. strains carrying a tonA marker; e.g. Cambio, Epicentre, Invitrogen).

Unfortunately research on this interesting and important virus largely languished after the mid 1980s, and prior to the current project the only T1 sequence data to be found in GenBank is for two genes one of which encodes a DNA N-6-adenine-methyltransferase (dam) (19). [We have found that this sequence (GenBank Accession No. BAA94133) contains an internal inframe deletion]. T1 sequence data has also inadvertently ended up in GenBank. A sequence reported to encode a European squid (Loligo forbesi) neurofilament-like protein (X66695) is, in fact, T1 sequence. The sequence of T1 has now been completed (18) revealing many of the secrets of this interesting virus. In addition, Drs. Gregory German and Rajeev Misra (Department of Microbiology, Arizona State University) have completed the sequence of phage TLS (6). In the following paragraphs I will briefly summarize some of the common properties of these two viruses. The Phage T1 Genome

Previous studies on T1 DNA indicated that the genome size was in the order of 48.5 kb with a terminal redundancy of approximately 2800 bp (13). Sequencing has actually shown that the T1 genome size is 50.7 kb with terminal repeats of 1.9 kb. Phage TLS is about the same size (50.9 kb) but possesses shorter (1 kb) terminal repeats. While these two phages differ somewhat in their overall base composition: T1 is 45.6 mol%G+C while TLS is 42.7 G+C their genomes show considerable overall sequence similarity as illustrated by the following Dotplot. Major differences in sequence and genes occur at the ends of the two genomes.

It has long been known that T1 DNA is insensitive to EcoBI [TGA(N8)TGCT] and EcoKI [AAC(N6)GTGC] type I restriction endonucleases. The reason for this has been revealed to be a complete lack of these site in the DNA. Phage TLS DNA has 13 EcoBI sites and a single EcoKI site. While it is unknown how this phage responds to these restriction endonucleases, German and Misra have evidence that TLS encodes a protein which inhibits type I restriction enzymes.

The T1 genome harbours 77 ORFs while that of TLS has 86. As suggested by the Dotplot results and confirmed by protein alignments many of the genes are similar. One significant difference is the finding that TLS encodes both a Dam and a Dcm (N-5-cytosine methyltransferase) methylase.

The work of Bourque and Christensen (2), employing host temperature-sensitive DNA replication mutants, showed that DNA polymerase III, DNA primase (DnaG) and clamp-loading protein (DnaX) were required for T1 replication, while replisome-organizer protein DnaA, helicase-loading protein DnaC and replicative DNA helicase DnaB were not. Sequencing has revealed the T1/TLS encode their own helicases, primases and single-stranded DNA-binding proteins. The origin for replication occurs, as it does in Salmonella phage P22, within the helicase gene. In addition, both phages contain RecE and Erf homologs which are part, in the case of T1, of a general recombination system termed "grn."

In coliphage early transcription involves host holo-RNA polymerase recognition of promoters which contain variants of the canonical hexamers (-35 TTGACA; -10 TATAAT) separated by 15-19 bp (15). While T1 contains many incidences of this type of promoter sequence its molecular approach to transcription is unusual, particularly within the morphogenesis genes. The late region is divided up into a series of transcriptional modules (transcriptons; Figure below) containing RpoD-dependent promoters�and perhaps enhancers�and is flanked by rho-independent terminators. The latter differ from those of coliphage T4 by lacking a UUCG or GNRA loop sequence (16).

Both T1 and TLS possess numerous 21 nt direct repeats located in the intergenic regions or overlapping the translational terminators of the preceding genes. While their high AT content is reminiscent of UP-elements in E. coli (10), their position suggests that they may function in a manner equivalent to eukaryotic enhancers. This transcriptional model differs fundamentally from that displayed by coliphage HK022 (Q-mediated transcriptional read-through) (8) or T7 (multiple phage RNA polymerases-specific promoters) and may account for the short latent period of 13 minutes observed with coliphage T1 (1, 3, 17).

Excluding the genes for the terminase subunits phage T1 has 23 genes which are most probably involved in morphogenesis. SDS-PAGE analysis has shown that the T1 virion is composed of 13-15 structural proteins (14, 20, 21) while TLS preparations contains fewer structural proteins. As part of the analysis of coliphage T1, Dr. Nancy Martin (Queen's University) analyzed the T1 proteome by two-dimensional gel electrophoresis/mass spectrometry. [She would be most interested in discussing potential collaborative phage proteomic projects with interested members of the phage community]. Packaging occurs in a headful manner from pac sites which have been localized in TLS to a 60 bp region which contains six tandem repeats of GATT(T/r) [G. German, personal communication (6)]. The analogous packaging site in T1 contains five adjacent repeats of ATATA.

With a couple of exceptions T1/TLS proteins display low sequence similarity to other phage proteins in the databases. The exceptions are the lysis proteins which possess 40% amino acid identity with lysozymes of Escherichia coli prophage CP-933K, and Salmonella typhimurium PS119 and PS34; and, the tail assembly genes. The latter, T1 genes 38 to 31, are homologous to N15 genes 16 to 23. In addition, both phages code for Cor homologs! Within this cluster are four proteins encoded by linked genes which have been implicated in tail cone assembly (9). The latter are related to similar genes in other members of the Siphoviridae infecting, or carried by, members of the class gamma-Proteobacteria including Burkholderia thailandensis phage phiE125 (22), and coliphages HK97, HK022, N15 and phi80. All of the latter phages are classified as lambda-like viruses at NCBI Taxonomy Browser (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/) suggesting that, at a higher phylogenetic level, phages T1 and TLS might be said to be part of the order lambda within the Siphoviridae.

While many of the mysteries of T1 have been revealed through analysis of its genome sequence there are still many unanswered questions. Research contemplated or in progress will analyze of the temporal expression of the T1 genome, its regulation, and the role of the 21 nt direct repeats. How the host genome is degraded remains a mystery, and in light of the number of proteins potentially involved in morphogenesis the latter deserves further experimentation. Lastly, we have the universal phage genome question: what is the function of the 53% of the ORFs which failed to result in a BLAST hit?

For those who would like a preview look at the annotated T1 sequence data please visit: http://microimm.queensu.ca/Phage/. References

Borchert, L. D. and H. Drexler. 1980. T1 genes which affect transduction. Journal of Virology 33:1122-1128.

Bourque, L. W. and J. R. Christensen. 1980. The synthesis of coliphage T1 DNA: requirement for host dna genes involved in elongation. Virology 102:310-316.

Delbrück, M. 1945. The burst size distribution in the growth of bacterial viruses. Journal of Bacteriology 50:131-135.

Delbrück, M. and S. E. Luria . 1942. Interference between bacterial viruses. I. Interference between two bacterial viruses acting upon the same host, and the mechanism of virus growth. Archives of Biochemistry 1:111-114.

German, G. J. and R. Misra. 2001. The TolC protein of Escherichia coli serves as a cell-surface receptor for the newly characterized TLS bacteriophage. Journal of Molecular Biology 308:579-585.

German, G. J. and R. Misra. 2003. The T1-like TolC- and lipopolysaccharide-specific (TLS) bacteriophage genome and the evolution of virulent phages. Journal of Molecular Biology (submitted).

Hug, H., R. Hausmann, J. Liebeschuetz, and D. A. Ritchie. 1986. In vitro packaging of foreign DNA into heads of bacteriophage T1. Journal of General Virology 67:333-343.

Juhala, R. J., M. E. Ford, R. L. Duda, A. Youlton, G. F. Hatfull, and R. W. Hendrix. 2000. Genetic sequences of bacteriophages HK97 and HK022: Pervasive genetic mosaicism in the lambdoid bacteriophages. Journal of Molecular Biology 299:27-51.

Katsura, I. 2003. Tail assembly and injection, p. 331-346. In R. W. Hendrix, J. W. Roberts, F. W. Stahl, and R. A. Weisberg (eds.), Lambda II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Kolasa, I. K., T. Lozinski, and K. L. Wierzchowski. 2002. Effect of An tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase. Acta Biochimica Polonica 49:659-669.

Lundrigan, M. D., J. H. Lancaster, and C. F. Earhart. 1983. UC-1, a new bacteriophage that uses the tonA polypeptide as its receptor. Journal of Virology 45:700-707.

Luria, S. and M. Delbrück. 1943. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 2:491-511.

MacHattie, L. A., M. Rhoades, and C. A. J. Thomas. 1972. Large repetition in the non-permuted nucleotide sequence of bacteriophage T1 DNA. Journal of Molecular Biology 72:645-656.

Martin, D. T., C. A. Adair, and D. A. Ritchie. 1976. Polypeptides specified by bacteriophage T1. Journal of General Virology 33:309-319.

McLean, B. W., S. L. Wiseman, and A. M. Kropinski. 1997. Functional analysis of sigma-70 consensus promoters in Pseudomonas aeruginosa and Escherichia coli. Canadian Journal of Microbiology 43 :981-985.

Miller, E. C., E. Kutter, G. Mosig, F. Arisaka, T. Kunisawa, and W. R�ger. 2003. Bacteriophage T4 genome. Microbiology and Molecular Biology Reviews 67:86-156.

Roberts, M. D. and H. Drexler. 1981. T1 mutants with increased transduction frequency are defective in host chromosome degradation. Virology 112:670-677.

Roberts, M. D., N. L. Martin, and A. M. Kropinski. 2003. The genome and proteome of coliphage T1. Virology (in press).

Schneider-Scherzer, E., B. Auer, E. J. de Groot, and M. Schweiger. 1990. Primary structure of a DNA (N6-adenine)-methyltransferase from Escherichia coli virus T1. DNA sequence, genomic organization, and comparative analysis. Journal of Biological Chemistry 265:6086-6091.

Toni, M., G. Conti, and G. C. Schito. 1976. Viral protein synthesis during replication of bacteriophage T1. Biochemical & Biophysical Research Communications 68:545-552.

Wagner, E. F., H. Ponta, and M. Schweiger. 1977. Development of E. coli virus T1: The pattern of gene expression. Molecular and General Genetics 150:21-28.

Woods, D. E., J. A. Jeddeloh, D. L. Fritz, and D. DeShazer. 2002. Burkholderia thailandensis E125 harbors a temperate bacteriophage specific for Burkholderia mallei. Journal of Bacteriology 184:4003-4017.

AAt the time known as phage a. See pp. 482 and 483 of Abedon (2000) for a brief history of the original T set of coliphages.

BKnowledge of phage T1�s desiccation resistance likely forms the basis of the famous "Phage in a Letter" urban legend, which apparently has since morphed into "Phage M13 in a letter." M13 is also a desiccation-resistant phage, but one which few have rejected from their laboratories perhaps because M13 is relatively avirulent and otherwise popular as a platform for protein display. See: http://www.panix.com/~iayork/phage.shtml or http://www.urbanlegends.com/science/phage.html for popular discussion of the "Phage in a Letter" urban legend.

Andrew M. Kropinski
Department of Microbiology, University of Guelph, Guelph, Ontario, Canada

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