Of Terms in Biology: Phage–Plasmid (2|2)

by Christoph
 

In the first part of this piece on phage–plasmids (P–Ps), I focused on examples that are well known today, and on the fact that they are re­pre­sentatives of larger families with dozens of members, rather than peculiar solitary cases. As a reminder, phage–plasmids transfer horizontally between cells as viruses and vertically within cellular lineages as plasmids. Here now, in the second part, I will introduce some lesser-known examples of phage–plasmids.
 

Cosmids  Barbara Hohn and John Collins introduced the most widely used lab-constructed cosmid, pHC79, in 1980: "Cosmids are plasmids containing (i) the λ sequence which is required for packaging into a λ capsid (cos, for cohesive end site), (ii) antibiotic resistance gene(s), and (iii) single restriction sites useful for cloning. Upon restriction and insertion of large fragments of DNA into these cosmids, long concatenated DNA structures are formed which mimic the natural packaging substrate. Packaging, cleavage of the cos sequence to yield cohesive ends, and sub­sequent transduction impose a size selection: only those hybrids are obtained that fill a phage head with approx. 37 to 50 kb long DNA. In this way only hybrids carrying a large insert are recovered at the expense of small hybrids and parent vector molecules. After transduction into a λ-sensitive bacterium the hybrid cosmids replicate as plasmids and are selected for by using the antibiotic resistance carried by them." (Figure 4)
 

Figure 4. Photographs and densitometer tracings of 0.4% agarose gels of a hybrid-cosmid pool of E. coli DNA, cloned into the BamHl site of pHC79. Lane A: undigested hybrid-cosmid DNA, with the positions in kb of three supercoiled marker plasmids and extra­po­lations (numbers in parentheses). Lane B: Com­plete BamHI digest of the E. coli hybrid pool. Lane C: Com­plete BamHI digest of original uncloned E. coli DNA. Source. Frontispiece: Detail from the Tabula Peutin­geriana 1–4th century CE. Facsimile Ed. by Conradi Mil­lieri, 1887/1888. Source

Every lab that worked with cosmids at that time when it came to cloning larger genome fragments also used two strains, BHB2688 and BHB2690, and the protocol provided by Barbara Hohn together with the strains: "λ and cosmid DNA, as well as in vitro recombinants thereof, are conveniently packaged using a combination of two lysates each of which is defective in another step of λ morphogenesis." Today, it takes some courage to read (and understand!) the genotypes of the two strains, and many prefer to resort to commercial sources for "packaging extracts." 
 

Fosmids  Shizuya et al. (1992) showed that it is possible to clone, and stably maintain, up to ~300 kb segments of human DNA in E. coli. Their pBAC108L vector was a fosmid whose rep­lication was based on the E. coli F plasmid: "The regulatory genes include oriS, repE, parA, and parB. The oriS and repE genes mediate the unidirectional replication of the F factor while parA and parB maintain copy number at a level of one or two per E. coli genome." They introduced the now common term BAC (bacterial artificial chromosome) for this type of vector and the Human Genome Project relied heavily on such BACs for sequencing H. sapiens. Fosmids or BACs contain a λ cos site, but only for mapping reasons (with the λ terminase employed as "rare cutter" restriction enzyme) as fosmids with lengths exceeding ~50 kb cannot be pack­aged anymore in λ heads. Therefore, fosmids should better not be considered phage–plasmids.
 

Phasmids  I wrote about phasmids (also called phagemids) earlier in STC: "Among hard-core entomologists – the folks studying insects – the 'walking sticks' are known as Phasmatodea, or Phasmids for short. Apparently, there had been an incidence of "Ho­ri­zon­tal Term Transfer" three decades ago that went largely undetected. We molecular biologists also knew phasmids: cloning vectors that carried, in addition to the usual (dsDNA) plasmid rep­li­ca­tion origin, a secondary origin derived from phages like f1 that, given a helper phage, enabled pro­duction of single-stranded DNA perfect for Sanger sequencing. Yet, as soon as we learned (while still in the pre-PCR days) to sequence the inserts in double-stranded plasmid vectors using cus­tom-made primers – in both directions at once, without fumbling with phasmids or tedious sub­cloning in M13 vectors, yeah! – phasmids rapidly fell into oblivion. Thus endeth the confusion of terms." Not quite though, phasmids are still popular among people who work with phage display: translatable full or partial gene sequences are inserted into the coat protein gene of the phasmid genome so that the phage particle produced during propagation exposes the inserted gene as an epitope of its coat protein. Depending on the purpose of the test, you then need a "fishing rod", for example an antibody, to fish the desired phages out of the supernatant. That's a pretty stringent coupling of genotype to phenotype, isn't it?
 

Figure 5. Electron microscopic analysis of phasyl DNA and phages. Electron micrographs (negative staining, magnification x50,000) of DNA prepared from M13/phasyl phages (A) or from cells infected with this phage mixture (B) are shown (M13, large circles; phasyl, small circles). Source. C) Linear maps. Plus and minus strand origins and the arp gene with its promoter are in­dicated. In pKH1D7 and pKH1R9, the plus strand origin is inactivated by insertion of a Km^r fragment. In pAR9 and pKH1R9, the postulated origin for autonomous replication is removed by deletion of 241 bp. The arp gene is inactivated by a 36-bp deletion in plasmids pAD7 and pKH1D7 which does not affect the putative origin for autonomous replication. The plasmids were tested for replication in strain H411 (gene II positive; replication in the phage modus) or in strain C600 with plasmid pAN12-4 (Arp⁺, autonomous repl.). Source

Phasyl  The genome size and gene content of naturally occurring phage–plasmids differ wi­de­ly. Decreasing in size, we have the S. enterica Typhimurium LT2 phage SSU5 with 103,229 bp (130 orfs), E. coli phage P1 with 93,601 bp (117 orfs), E. coli phage N15 with 46,363 bp (28 orfs) and satellite phage P4 with 11,624 bp (13 orfs). Can (satellite) phage–plasmids get any smaller? Yes, they can. E. coli phasyl has a length of 1,282 bp and two thirds of its genome encodes only one protein, Arp (autonomous replication of phasyl) with 285 residues. In 1988, Wolfgang Seu­fert from Walter Messer's lab at the MPI MolGen, Berlin found during routine cloning experi­ments with M13mp18 phages (RF form 7,249 b) and host strain E. coli JM103 unexpectedly a "mini-phage" that was not one of the known, infrequently occurring M13 deletion derivatives because it did not hybridize with M13 (Figure 5AB). Sequencing revealed two motifs for (+) and (–) strand replication of ssDNA phages but in rerverse order as compared to M13 or fd. Gielow et al. (1990) could show that phasyl replicates in the phage mode when geneII of phage fd was supplied in trans, and in the plasmid mode when Arp was supplied in trans but only when the arp promoter region was intact (Figure 5C). The arp promoter region lacks significant simila­rities with other plasmid replication origins but is functional. As was found later by del Solar et al. (1998), Arp belongs to a large heterogeneous family of plasmid and phage Rep proteins for θ-replication (see here). Like the satellite phage–plasmid P4, phasyl needs a helper phage for propagation as phage, but it is still the smallest known naturally occurring two-origin replicon. (Disclosure: I joined Walter Messer's lab as a postdoc shortly after their fun adventure with phasyl and still saw a faint, special smirk on their faces when someone mentioned it.)
 

Looking back  Although it has long been obvious to all experts that there are overlaps be­tween phages and plasmids on all levels of mosaicism, they have been reluctant to abandon the clear distinctions they held dear. In 1979, the first mention of a "'phage–plasmid' chimera" was made in a publication, but not without quotation marks (air quotes) and with the quali­fication that it was a lab artifact (I could not find any earlier PubMed entries). Seven years later, Lagos et al. (1986) still make some verbal contortions when they speak "...of "phasmid" (phage-plasmid) P4" in the abstract of their paper, and Seufert et al. (1988) still coyly refer to phasyl as "a phage-plasmid hybrid". Only from 1988 on, the term "phage‑plasmid" became normalized in the literature mainly due to the work of the group led by Gianni Dehò and Daniela Ghisotti, who spent years investigating the life cycle of the peculiar satellite phage P4 (that satellite phages are not so peculiar after all was recently highlighted by Roberto here in STC). Sorry for this lengthy excursion into the history of a technical term. But it shows very nicely how arduous it is to overcome established concepts and classifications, not least when it comes to dealing with editors and reviewers of scientific journals.
 

Do you want to comment on this post? We would be happy about it! Please comment on Mastodon, Bluesky, or on 𝕏 (formerly Twitter).