Temperate phages, maintained as prophages in their lysogens, have been the subject of speculation concerning their benefit to the host: selective advantage, increased virulence, and other traits with varying degrees of direct and/or indirect impact on the host have been identified [11
]. The challenge in this area has been how to identify phage-encoded genes that directly affect their lysogen, because many/most phage genes are annotated as encoding hypothetical proteins. In addition, there will always be a small background population undergoing spontaneous induction in the absence of discernible stimuli [19
], potentially confounding the identification of lysogen-restricted prophage gene expression. In a specific E. coli
lysogen of Stx2-phage 933W, a phage very closely related to Φ24B
, the spontaneous induction rate was calculated as 0.014% [28
], which means that in a lysogen culture fourteen cells per 100,000 are undergoing prophage induction. Other recent work was demonstrated that various induction agents and growth conditions differentially effects induction in a prophage-dependent manner [29
]. Assuming a burst size similar to that of bacteriophage Lambda (170 ± 10 virions cell-1
], a significant amount of phage structural protein production can occur in an uninduced lysogen culture.
In order to mitigate this effect, the growth phase at which the ratio of lysogens to free phage was high (two to three hours post inoculation) was targeted. However, the cell density at this point was very low and 5-6 hours was chosen as the standardised incubation time as a compromise. In this study, 26 genes from the bacteriophage Φ24B were identified by either CMAT or 2D-PAGE as being expressed in E. coli lysogen culture. No genes were identified by both CMAT and 2D-PAGE methods, perhaps due in part to the low absolute number of Φ24B genes identified by the latter approach. However, the level of redundancy in the genes identified by the CMAT clones was lower than expected, given the number of clones screened and the calculated phage genome coverage; however, putative positive clones were selected conservatively in an attempt to limit the number of false positives. Additionally, CMAT-based identification may also introduce bias into library screening due to differences in protein immunogenicity and antigenicity. It is important to note that the best characterised lysogen-restricted gene, cI (encoding lambdoid phage repressor), was not identified using either CMAT or 2D-PAGE, indicating that this study was not exhaustive. Nevertheless, the paucity of information on lysogen-restricted gene expression is such that these data represent a significant step forward in our understanding of phage/host interactions and lysogen biology.
Of the 26 phage genes identified in this study, Tsp, encoding the characterised tail spike protein of Φ24B
] was a known structural protein and therefore not expected to be expressed by a stable lysogen (Tables &), while the expression profiles of the other 25 proteins were unknown. Therefore the resulting challenge was to identify the fraction of the culture (lysogens or cells undergoing lysis) that were responsible for expression of these 26 phage genes as well as determining testable hypotheses to assign function to the identified gene products. Five genes identified during the CMAT screening were chosen for gene expression profiling due to their genome location, potential function or degree of conservation across a range of phages (Table ). The CDS CM18 encodes a Lom orthologue, which was expected to be expressed in the lysogen as the lambda lom
gene is associated with the alteration of the lysogen's pathogenic profile after location of Lom in the outer membrane [32
]. However, expression of lom
in the Φ24B
lysogen unexpectedly appears to be uncoupled from the phage regulatory pathways, because it is expressed at similar levels in an infected cell regardless of whether that cell exists as a stable lysogen or is undergoing prophage induction. The CDS CM2 encodes a putative Dam methyltransferase. Bacterial-encoded Dam methyltransferase has been shown to be essential for maintenance of lysogeny in E. coli
infected with Stx-phage 933 W [35
]. The expression pattern of the Φ24B
-encoded Dam methyltransferase could indicate that it is fulfilling a similar role, or supplementing the function of the host-encoded Dam methylase in lysogens infected with this phage. The functions of CM5 and CM7 are unknown. CM7 is an ORF of 8 kb, and as the amount of DNA that can be packaged by a phage is limited, such a large gene is likely to be conserved only if it confers an advantage to the phage or its lysogen; it may be significant that this large gene is associated with several other phages (Table ). CM5 is a small CDS located on the complementary strand to the one encoding CM7, in a region with few other CDS, though it is directly upstream of another CMAT-identified CDS, CM6. The data (Figure ) indicate that the expression of these 3 genes is linked to prophage induction, a surprising outcome as CM7 does not appear to be a phage structural gene, has been indicated by bioinformatic analyses (data not shown) to be a probable outer membrane protein, and is downstream of CM18, whose regulation is uncoupled from expression of the late genes.
Distribution of the proteins identified by CMAT and 2D-PAGE across phage genomes
The qPCR expression profile for the phage genes identified as being expressed in the lysogen by 2D-PAGE, P1, P2, P3, P4, P5 and P6, indicated that only the expression of P2 and P3 were restricted to lysogen cultures with a stable prophage. The genes for both P2 and P3 lie downstream of the cI
gene. However, their expression levels are one and five orders of magnitude greater, respectively, than the expression levels of cI
, the lambdoid phage repressor gene. It is known that in Lambda phage, the cI
gene transcript is leaderless, possessing no ribosome binding site for initiation of translation, with transcription and translation beginning at the AUG start codon [36
]. If this causes the 5' end of the transcript to be less stable and more easily subject to degradation, the higher level of P3 transcript could simply be due to possession of a longer half life than those genes at the 5' end of the transcript.
The genes encoding P2 and P3 are conserved in many other phages (Table ). They have no bioinformatically identifiable promoters of their own, so are likely to be driven by pRM
] for a cogent review of the related lambda phage), but differences in the levels of transcription between these 3 genes implies that there is still more to discover about the right operator region of this phage. The proteins P1, P4, P5 and P6 all exhibit gene expression profiles that suggest they are expressed following prophage induction. These genes are scattered across the phage genome (Figure ) and are shared by various phages (Table ). The protein P4 appears to be part of the lambda Red recombinase system [38
] and the data presented here suggest that this is most active upon prophage induction. This could be relevant to the mechanisms that underpin diversification, evolution and production of new phages by lysogens carrying an inducible prophage along with one or more inducible or remnant prophages [11
]. The proteins P1, P5 and P6 are scattered across the genome on the strand typically associated with expression of genes linked to lysogenic infection (e.g. cIII, N, cI
). Two genes encoding proteins P1, P5 and P6 are found in other phages, but have no known function.
In summary, genome sequencing of prophages and bacteriophages has identified that these viral elements encode higher numbers of hypothetical genes than those to which we can currently assign a function. These genes are often conserved across many bacteriophages, but do not appear to encode structural proteins. For these genes to remain present in the phage genome, especially considering the fluidity of the genetic composition of lambdoid phages [43
], they must surely provide an important function in either the phage life cycle or that of the lysogen itself. In attempting to identify prophage genes whose expression was restricted to the stable prophage state, our goal was to identify prophage genes that were candidates for influencing the fitness of the bacterial host. However, the study was hampered by the fact that lysogen-restricted gene expression can be at very low levels, and phage genes associated with phage replication are expressed at very high levels.