Time-resolved, phage, and host genome-wide transcriptomics (RNA-Seq) and proteomics (tandem mass spectrometry) data were collected and analyzed from population-wide infections and compared to samples from uninfected controls, in biological triplicates (Supplementary Dataset S1 Tab. 1, Supplementary Table S1 and S2), and analyzed using available genomes and genome annotations (Supplementary Dataset S2). The infections included (Fig. 1; Supplementary Figure S1): (1) generalist phi38:1 (72.5 Kb dsDNA podophage) on its original host of isolation, host38 [44], (2) generalist phi38:1 on host18, and (3) specialist phi18:3 (71.4 Kb dsDNA podophage) on its original host of isolation, host18 [44]. Phages phi18:3 and phi38:1 do not share any homologous gene (Supplementary Figure S2) and belong to different genera, Cba183likevirus and Cba401likevirus, respectively, based on their genome similarity [8]. Phage phi18:3 does not infect host38 [21]. Critical new analyses from the previously reported phi38:1 transcriptomes [30] are used to both compare with significant new data—phi18:3 transcriptomes, and proteomes of all three infections—and address the hypotheses generated from prior work.

Fig. 1 The C. baltica—phage model system investigated. Generalist phage phi38:1 infects more efficiently its original host of isolation (strain NN016038; “host38”) than alternative host strain #18 (“host18”). Specialist phage phi18:3 efficiently infects its original host of isolation, “host18,” and does not infect host38 [21] Full size image

Phage genomes and transcriptomes suggest drivers of host-range and infection efficiency

On host18, specialist phi18:3 had an efficient, 75-min infection (Fig. 2a) and a similar temporal transcriptome to that of phi38:1 infecting less efficiently this same host (Fig. 2b). Namely, both phages expressed all their genes by 60 min, and with early, middle, and late expression categories where transcript abundances peaked at 0, 15–20, and ≥40 min post infection, respectively (Fig. 2c, d; Supplementary Tables S3-S4 and Supplementary Dataset S1 tabs 9-10). Additionally, genes in each temporal category were functionally similar for both phages. Namely, expressed early were host takeover (i.e., those that begin phage gene expression and control host functions) genes, including a ribosyltransferase, serine/threonine phosphatase, or a lysozyme. Expressed middle were the DNA metabolism genes, and expressed late were the structural and lysis genes (Supplementary Figures 2, 3). Such transcriptional patterns are common among phages across diverse bacterial hosts and environments [30, 32, 33, 37, 59,60,61,62,63] and suggests that, regardless of their efficiency, phages that lack an RNA polymerase (RNAP) may successfully utilize the host’s RNAP for transcription, similar to well-known Enterophages (e.g., lambda or T4) [64,65,66]. Thus, the data at this point suggest that these universalities in phage transcription can be predicted via RNA-seq and are independent of host type, host range, and infection efficiency.

Fig. 2 Infection dynamics and expression profile of phi18:3 and phi38:1 infecting C. baltica #18 (“host18”). One-step growth curve of the a efficient phi18:3 and b inefficient phi38:1 infections on host18. Represented are the average values of three biological replicates and their standard error, as well as the latent period (LP) and burst size (BS). Time 0 represents the dilution of the infection after a 15-min adsorption. Both phages express their genes in early, middle, and late categories, with highest expression at c 0, 15, and ≥30 min for phi18:3, and d 0, 20, and ≥40 min for phi38:1. The average log 2 RPKM values of the genes in each category and standard deviation are represented in the graphs. Numbers of genes in each temporal category are represented in parentheses Full size image

Beyond these overall similarities in phage transcriptomes, some expressed genes (Supplementary Tables S3-S4, Supplementary Dataset S2; Supplementary Figures S3, S4) suggested unique generalist versus specialist strategies from their predicted functions. For example, unique to generalist phi38:1 are 16 tRNAs (two transcribed early and 14 late) and DNA replication genes (e.g., DNA polymerase and primase, middle expressed, and late translated). Such genes might help a generalist phage utilize host translation and replicate phage DNA, respectively, across diverse hosts [67] given that phage genomes are known to code for genes that reprogram host metabolism [68, 69] and that they are not identified in a specialist phage (phi18:3). In contrast, expressed genes that were unique to phi18:3 included an anti-restriction gene and methylases (“anti-R/M” genes; transcribed and translated early), and the metabolic regulator MazG (middle transcribed and translated late). In Escherichiacoli, phages overcome host R/M defenses by, for example, expressing a suite of anti-restriction genes (e.g., T3, T7), or injecting such proteins into the cell (e.g., P1, T4) [70], and/or by self-methylating their DNA via methylases (e.g., T2, T4) [62]. Additionally, E. coli’s maz system is induced to prevent translation from stalling in response to diverse stresses, including phage infection [70, 71]. A variety of environmental phages encode for MazG, including cyanophages that transcribe it as a middle gene during their efficient infection [33]. Thus, these genes might enable phi18:3 to efficiently infect host18 by overcoming host R/M defenses through direct interaction with host’s endonucleases (anti restriction) [72] and self-DNA methylation (methylases) [70], and by preventing host metabolic shutdown during infection (MazG) [70, 71]. Other phages that lack such genes and still have efficient infections [28, 30,31,32] either protect their DNA through alternative modifications (e.g., glycosylation [70]) or, as in the case for phi38:1 infecting host38, do not trigger the host’s R/M system and might have adapted to utilize the host’s functions without the need for MazG [30].

Absence of transcriptional–translational synchronization in inefficient infections

The presence of early proteins in phi18:3 (Fig. 3a, b, Supplementary Figure S3) suggested that this phage was able to rapidly utilize the host’s translational machinery to translate very early (i.e., within 15 min of infection). Notably, among these early proteins was the anti-restriction protein, which would be one of the proteins needed immediately upon infection to evade host R/M systems. To test the hypothesis that phi18:3 utilizes host translation early we measured phage protein dynamics (Fig. 3) and observed that, indeed, most (88%) of phi18:3’s early transcripts produced early proteins (Supplementary Figure S3) and that overall protein abundances paralleled those of the transcripts, with early, middle and late classifications readily apparent (Fig. 3a–c). One exception was a DNA replication protein that peaked early despite having a middle transcript (Fig. 3b). Presumably this protein was detected prior to its transcript because it was contained in the virion and introduced upon infection, even though it was not previously detected in a preliminary virion screen with other phages [8]. Other phages, including cyanophage Syn9 [33], T4 [73], and other Enterophages [70] also carry proteins in the virion that function in host takeover and early phage expression as T4’s Alt protein [73]. Notably, the observed 45-min peak in protein abundance (Fig. 3a–c) was not an artifact as interpreted from multiple proteomics controls, and yet is difficult to ascribe to particular biology. One possibility would be that while our population-wide averaged measurements captured the overall signal in these phage–host interactions, the 45-min anomaly might result from cell-to-cell variability in phage–host interaction initiation and infection dynamics. This could perhaps result from some asynchronous infections that are even coupled with variation caused by phenotypic heterogeneity in the underlying clonal population of cells [74]—phenomena that might be evaluated with single-cell ‘omics technologies as they emerge for prokaryotic phage–host systems.

Fig. 3 Temporal transcriptional–translational dynamics of phi18:3 and phi38:1. For each infection, three vertical graphs represent the early, middle, or late phage transcripts. In those, averaged transcript expression is graphed with the thickest line, with “n” representing how many genes were averaged. The standardized, relative protein abundances of those transcripts are individually graphed in thinner lines, and they are early, middle, late, or a variation of those. The number in each category is also represented by an “n.” Time 0 represents the dilution of the infection after a 15-min adsorption. a–c Proteins are early (35%), middle (2%), middle late (5%), or late (58%) and they largely appear after their respective a early, b middle, and c late transcripts when phi18:3 infects host18. d–f Some (14%) phage proteins are early but most (86%) are late regardless of d early, e middle, or f late transcription of their genes when phi38:1 infects host18. g–i Phage proteins are early (4%), middle (4%), middle late (51%), late (39%), or constant (1%; protein abundance does not significantly change over time; see Supplementary Dataset S1, Tab. 10) and closely follow the g early, h middle, or i late transcription of their genes when phi38:1 infects host38. Calculations are in Supplementary Dataset S1 Full size image

In contrast, the abundances of 86% of phi38:1’s proteins peaked later than the “late” transcriptional group (Supplementary Figure S4a-b) and largely plateaued after 60 min (Fig. 3d–f). These findings suggest either a mixed protein dynamics signal deriving from new rounds of infection happening after 150 min (when 4% of the cells lyse based on phageFISH data [15])—which could be resolved with better single-cell technologies—or slower protein dynamics and delayed translation signal deriving from population-wide data. If the latter, such biological signal could explain the previously observed delayed particle production [30] and even lysis, as follows. Namely, among phi38:1’s genes with non-delayed translation was a peptidase (gp021) that was transcribed and translated early (Supplementary Table S3; Supplementary Figure S4a-b). Given that a variety of peptidases function as endolysins and accumulate in the cell throughout the infection until holins are made [75, 76], one hypothesis is that the holin (which is unknown in phi38:1’s genome) is delayed or not produced in the inefficient infection. Regardless, these expression data together suggest that only specialist phi18:3 synchronizes host-derived translation with transcription to synthesize proteins in a timed and regulated manner.

To determine whether phi38:1 could have better translation in another host, we measured its protein dynamics while infecting its original host, host38, and compared them to our prior transcriptome dynamics [30]. This revealed that phage proteins were no longer delayed as when infecting host18 and instead were produced in early, middle, and late categories, with over half of them (55%) being middle/middle late (Fig. 3g–i; Supplementary Figure S4c-d). Notably, upregulation of host38’s translation genes was also middle/middle late [30], thus suggesting that phage phi38:1 translation in this host is mainly timed with the activation of host translation as occurs with phi18:3 on host18. Given that phage protein translation is expected to closely follow transcription based on lessons from phages infecting (efficiently) well-known systems (e.g., Bacillus phage phi29 synthesizes >60% of its proteins before middle transcription [77]), the inability of phi38:1 to synchronize translation with transcription in host18 may contribute to this inefficient infection.

Hosts’ transcriptomes and proteomes reveal causes for inefficient phage infections

Given that phage proteome analyses suggested differences in utilizing host functions, we next examined host18’s transcriptome and proteome during phi18:3 and phi38:1 infections and compared them to host38’s responses. Globally, host18’s transcription was repressed throughout infection by phi18:3, but not by phi38:1 (Fig. 4a), as evidenced by the relative decrease in host transcript abundance, which was not derived from a sequencing skew toward phage transcripts (Supplementary Figure S5). This suggested that, like phi38:1 on its original host38 [30], phi18:3 successfully shutdown transcription in its own original host (Fig. 4a), whereas phi38:1 did not in an alternative host [30]. Phage induced, host-transcriptional repression as a sign of host takeover has been described across phage–host model systems [28,29,30, 33, 35,36,37]. Mirroring the transcriptional response, the host’s proteome was differentially impacted by phage infection. Namely, host18’s protein abundances significantly decreased with phi18:3 (t-test, p ≤ 0.05; Fig. 4b) and increased with phi38:1 (Fig. 4c). In contrast, when phi38:1 infected host38 host protein abundances no longer significantly increased (t-test, p > 0.05; Supplementary Figure S6b). Thus, these proteomic data suggest that variably efficient infections differentially impact the host proteome such that only the former stalls host protein synthesis, which is likely phage mediated, whereas the latter does not impact the host proteome. Here, the increase in host18 protein abundances at 4 h during the infection with phi38:1 (Fig. 4c) suggests that this host continues translating, perhaps as a defense mechanism against the phage.

Fig. 4 Global transcriptome and proteome during C. baltica #18 (“host18”) infection with phi18:3 and phi38:1. OE over expressed, UE under expressed. a Host genes are globally UE throughout the infection with phi18:3, and OE throughout the infection with phi38:1. Represented is the fold change of gene expression between infected and uninfected cells, normalized by the fraction of infected cells. b, c Host protein abundances b significantly (t-test between 0 and 60 min, p ≤ 0.05) decrease by the end of the infection with phi18:3, and c significantly (t-test against 0 min, p ≤ 0.05) increase throughout the infection (≥120 min) with phi38:1. None of the uninfected host protein abundances significantly increase (t-test against 0 min, p > 0.05) over time. Represented in b and c is the average values of three biological replicates and standard error. Statistical analyses can be found in Supplementary Dataset S1 (Tab. 12). Time 0 represents the dilution of the infection after a 15-min adsorption Full size image

With global host responses suggesting differences in host takeover in the variably efficient infections, we next examined phage impacts on specific host functions. First, we evaluated the response of host18’s R/M system for three reasons: (1) both C. baltica hosts lack the common bacterial CRISPR-Cas defenses [78], (2) bacteria commonly restrict incoming phage DNA [70, 79, 80], and (3) early expression of 22 early proteins by phi18:3 suggested a phage counter defense [70]. Those early proteins included both the anti-R/M genes and potentially the 14 proteins with unknown functions that surrounded those anti-R/M genes and which are predicted to be transcribed under the same promoter (Supplementary Table S3; Supplementary Figure S3). Indeed, host18 R/M genes were differentially over expressed during infection with phi18:3, but not with phi38:1 (Fig. 5a, Supplementary Table S5). Proteomics suggested that overall protein abundances significantly (t-test, p ≤ 0.05) decreased during phi18:3 infection, but increased with phi38:1 (Supplementary Figure S7). Notably, our annotation of these genes as R/M derives from in silico predictions of restriction endonucleases and methyltransferases (see Methods section), and awaits experimental evaluation. Additionally, as R/M genes are known to have ubiquitous roles [81], beyond defense to phage the genes shown here might also function in SOS response or DNA replication. Should they be involved in defense against phage DNA, data here presented suggest that host18 transcriptionally activated its R/M system against phi18:3, but its proteins likely encountered phi18:3’s early translated genes involved in anti-R/M counter defenses and were inactivated, as occurs in other phage–host systems [70]. Such transcriptional activation, is not commonly reported in phage–host interactions as it is in, for example, chlorella virus [81]. While this remains an open question, the observed over-expression of R/M genes (Fig. 5a) could be an example of inducible R/M systems against not just phage phi18:3 DNA entry, but also replication. In contrast, the lack of these genes in phi38:1 would prevent such inactivation and likely allow host18 R/M proteins to accumulate from the genes’ basal expression and contribute to the previously observed host targeting of phi38:1 DNA [15, 30].

Fig. 5 Expression of host18’s restriction/modification (R/M) and translation genes in response to phi18:3 and phi38:1. OE over expressed; UE under expressed. Represented in a stacked bar graph is the fold change of expression of infected relative to uninfected cells, normalized by the fraction of infected cells. Solid bars represent the differential expression values whereas dotted bars represent the non-differentially expressed genes. a The R/M system is clearly differentially expressed in response to phi18:3; mostly OE throughout the infection; and UE occurs during middle infection with phi38:1. Host18 displays basal expression (non-differentially expressed genes) of the R/M system regardless of phage infection. b Host18 genes involved in protein translation are OE 4.5-fold (early), 1.7-fold (middle), and 1.4-fold (late) higher in response to phi18:3 than to phi38:1. The values of expression in host18 infected with phi38:1 do not significantly increase over time (t-test, p-value > 0.05; Supplementary Dataset S1 Tab. 7). Host18 displays basal expression (non-differentially expressed genes) of the translation genes regardless of phage infection. Time 0 represents the dilution of the infection after a 15-min adsorption Full size image

Second, we examined host translation genes because phi18:3 synthesized early proteins (Fig. 3a–c) and because previous transcriptome data suggested that differential transcription of host translation genes in host38 versus host18 contributed to phi38:1’s virion formation delay [30]. Here, host18 transcriptomes (Fig. 5b) and proteomes (Supplementary Figure S8) suggested that phi18:3 activated the host’s translational machinery for its reproduction better than phi38:1 did. Namely, from the beginning of the infection (early) host18’s genes had higher differential expression with phi18:3 (e.g., 6.5-fold) than with phi38:1 (1.4-fold; Fig. 5b). Additionally, such difference in translation activation at the early stage was reflected in the proteome: host18 infected with phi18:3 had produced 93% of those translation proteins, whereas only 2% when infected with phi38:1 (Supplementary Figure S8), thus suggesting that phi18:3 was better poised to utilize host18’s translation than phi38:1. The difference between this efficient translation in phi18:3-infected host18 and that observed in phi38:1 infected host38—the other, more efficient, original host infection—was that phi18:3-infected host18 cells expressed their translation genes early—4.5-fold higher than when infected with phi38:1 (Fig. 5b)—whereas over-expression began during mid infection in host38 (Fig. 5c in ref. [30]). We posit that host translational activation enabled better phage transcriptional–translational synchronization and thus efficient infections on the original hosts, and that early activation of translation in host18 enabled phi18:3 to translate early its host-takeover proteins.

Third, although the majority (86%) of phi38:1’s proteins were produced late when infecting host18, some (14%) were produced early (Fig. 3d–f; Supplementary Table S4), which suggests that phi38:1 proteins may be encountering host18’s proteases and contributing to disrupting transcriptional–translational synchronization. Notably, alternative explanations might exist for the delay in phi38:1 protein abundances as previously mentioned, but here we considered the highly conserved protease ClpP because it can target several phage (e.g., lambda, P1) proteins and also be inhibited by phage counter defenses [82, 83]. This host18 gene was differentially under expressed (−0.67-fold) in response to phi18:3, but not to phi38:1 (Fig. 6a), where the gene was not even differentially expressed. Protein abundances significantly (t-test, p ≤ 0.05) decreased (~3×) from start to end of the phi18:3 infection (Fig. 6b, c). These findings suggest that ClpP was inactivated only by phi18:3 and not phi38:1, which would in turn lead to ClpP accumulation to possibly target degradation of phi38:1 proteins. Such dynamics would be consistent with delays both in virion synthesis previously observed [30] and protein synthesis observed here (Fig. 3d–f). While ClpP effects are well-studied in phage lambda [84], this and other conserved proteases might constitute underexplored bacterial strategies that modulate phage infection efficiency.

Fig. 6 Gene expression and protein abundance of “host18” protease ClpP during infection with phi18:3 and phi38:1. a The gene is only differentially expressed, and under expressed (UE), upon phi18:3 infection. Protein abundance over time during b phi18:3 infection decreases significantly (t-test, p ≤ 0.05), but during c phi38:1 infection increases significantly (t-test, p ≤ 0.05). The abundances in the controls b, c are not statistically significantly different from time 0 (t-test, p > 0.05), unlike in the infected treatment. Represented in b, c are the average of three biological replicates and their standard error. The t-tests can be found in Supplementary Dataset S1 (Tab. 7). Time 0 represents the dilution of the infection after a 15-min adsorption Full size image

A mechanistic model for drivers of variable efficient phage–host interactions

These data provide support for recent mechanistic work in environmental phage–host model systems, while also bringing new perspective on the mechanisms driving infection efficiency as follows. First, host takeover in efficient infections seems to involve global repression of host transcription across systems [28,29,30, 33, 35,36,37]. Our findings with a specialist phage are consistent with these studies. Additionally, they show that host takeover in efficient infections might also stall the host proteome. Second, here efficient infections also seem to activate the host’s translational machinery to translate phage genes to proteins. Unlike giant phages that can code for translation genes [85] and complement the host’s translation machinery, phages are host-dependent for synthesizing proteins. Third, we also show that the timing of such activation either occurs early, if the phage requires producing proteins to counteract host defenses against phage DNA (e.g., in specialist phi18:3 to fight host18 R/M), or later, perhaps reflecting that phages have alternative DNA protection strategies (e.g., glycosylation [70]), can localize to the cell poles [81], or the hosts lack, or fail to activate R/M defenses (e.g., in generalist phi38:1 infecting host38 [30]). In contrast, phage protein dynamics during inefficient infections are not only delayed but they likely lose temporal synchronization with their transcriptional counterparts. We suggest that host proteases, for example, ClpP, likely reduce phage protein abundances in inefficient infections. As these proteases are highly conserved [86] and phages can have inhibitors for them [84], proteases might be a widespread, underexplored, and critical mechanism driving phage infection efficiency. Finally, here we have provided insight into only some of the possibly additional mechanisms contributing to variation in infection efficiency. Specifically, additional mechanisms could be found in the ~260 unique bacterial genes (published in Supplementary Dataset S1 in ref. [30]), likely beyond the best described ones that are not present in these hosts, such as CRISPR-Cas, and the conserved toxin/antitoxin and abortive systems. While mechanistic knowledge of phage–host interactions in nature is arguably in its infancy, this and other work [28,29,30, 33, 35,36,37] establish a baseline with which to investigate infection efficiency and phage–host interactions in more phylogenetically diverse lytic phage–host systems. While such work is largely lytic cycle focused, recent ‘omics-enabled measurements in lysogenic infections are revealing both the complexity of these systems ([59, 87,88,89, 103]) and more fully helping establish paradigms for naturally occurring phage–host interaction biology.

Defining and better characterizing infection efficiency—a discussion just beginning

The work presented here invites discussing infection efficiency, as efficiency is a term with relative uses in the literature. Briefly, in phage studies, the term “efficient” is used to describe either infections or phages that lyse the most cells the fastest [28, 29], have the shortest LP/best growth rate [104], display better intracellular capabilities (e.g., in DNA replication [105] or gene transcription [37], or are proficient at hijacking host RNAP [37]). In contrast, the eukaryotic viral literature evaluates whether each step of the complex viral infection, from attachment to dispersion, is efficient [106,106,107,108,109,110,111,112,113].

Against this vast eukaryotic virus literature, we sought to classify each of the three infections presented here as efficient or inefficient. The infection of phi18:3 on host18 has both the shortest LP and largest burst size (BS), thus it can safely be classified as the most efficient infection of the three. However, phi38:1’s infections on host18 and host38 are more difficult to classify since the former has a LP of > 2.5 h and a BS of ~39, and the latter has a LP of 75 min and BS of nearly 10. To better clarify the longer term implications of such infection dynamics, we performed new experiments where we infected host18 and host38 separately with phi38:1 for 28 h and measured cell density and phage production (Supplementary Figure S9). The results showed that (A) infected host18 cells maintained lower OD than infected host38 cells, and that (B) more phage progeny was produced, and earlier, in host38 than in host18.

Given all knowledge of phi38:1’s infection, we assessed the relative efficiencies for each stage of infection as is often evaluated in the eukaryotic virus literature. This revealed that phi38:1 on host18 is, compared to the infection of either phi38:1 on host38 or phi18:3 on host18: (1) less efficient at forming plaques [21]; (2) less efficient at infecting cells [15, 21, 30]; (3) less efficient at overcoming host defenses (this work); (4) equally efficient at transcribing phage genes [30]; (5) less efficient at repressing host transcription [30]; (6) less efficient at repressing and taking over host translation [30]; (7) less efficient at replicating DNA [15, 30]; (8) less efficient at producing and/or assembling phage particles [30]; (9) less efficient at lysing cells [15]; and (10) less efficient at producing progeny throughout 28 h (this work). Given that phi38:1 on host18 is less efficient for nine out of ten steps, such infection is the least efficient of the three studied here.

Pragmatically, such inefficient infections are challenging to study using population-wide measurements. Specifically, synchronizing infections is not simple, as even our best efforts were still prey to a low fraction of cells initially infected and cell lysis spanning long time periods (4% of cells initially lysed at 2.5 h as compared to the bulk by 11 h [15]). To compensate for this, we assumed that the transcriptome and proteome of uninfected cells would be similar to uninfected control cells. While we see this as a reasonable assumption due to the lack of evidence for uninfected to infected cell communication mechanisms and the strong signal expected for infected cells (driven by the here observed higher RNA in infected versus uninfected samples; Supplementary Dataset S1 Tab. 1), the ability to apply single-cell ‘omics technologies to these systems would be invaluable for experimentally evaluating such assumption.