There are a large number of prophages in wheat phyllosphere bacteria whose activity, diversity and functions are unknown
Widespread and largely unknown prophage activity, diversity, and function in two genera of wheat phyllosphere bacteria
Article，2023-11-2，The ISME Journal， [IF 11]
DOI：https://doi.org/10.1038/s41396-023-01547-1
Original link:https://doi.org/10.1038/s41396-023-01547-1
First author:Peter Erdmann Dougherty
Conversation author:Lars Hestbjerg Hansen
Main units:
1.Department of Plant and Environmental Sciences, University of Copenhagen
2. Department of Microbial Ecology, Netherlands Institute of Ecology
- Summary -
Environmental bacteria harbor a large number of prophages, but their diversity and natural functions remain largely elusive. This paper studied the prophage activity and diversity in 63 strains of Erwinia and Pseudomonas isolated from wheat flag leaves. By introducing and validating virus-induced sequencing (VIP-Seq), the researchers identified and quantified the activity of 12 spontaneously induced prophages and found that some phyllosphere bacteria produced more than 10 viruses in overnight culture. 1>8 cells/ml, significant induction was also observed in plants. Sequencing results and plaque assays reveal that Erwinia aphid prophages account for most of the intraspecific genetic diversity and are extensively involved in microbial warfare by splitting bacterial hosts into distinct factions, revealing prophage-mediated microbial warfare The importance of diversity. When comparing spontaneously active prophages to predicted prophages, we also found that the insertion sequence was closely related to inactive prophages. In conclusion, we discovered the presence of a wide range of prophages of largely unknown function and diversity among plant phyllosphere bacteria.
- Introduction -
Although it has become an oft-cited statement that phages are the most abundant entities on Earth, many phages do not exist as the free-floating virus particles we usually imagine, but rather as prophages integrated into the genomes of most bacteria middle. From this perspective, "most" microbiologists work with phages, albeit indirectly and often unconsciously. Therefore, a better understanding of the activity and function of prophages may provide insights into the behavior of their bacterial hosts.
Prophages represent the dormant phase of a temperate phage infection and are phage genomes that replicate perpendicularly to their host bacteria. Prophages can remain in this state indefinitely until they are induced to produce phage virus particles and burst from the cell, or until they are tamed by mutations that render them inducible. Although phages are nearly ubiquitous among bacterial taxa, their distribution is not uniform. A recent study of 10,370 bacterial and archaeal genomes found that predicted prophages were present in 75% of the genomes, with an average of 3.24 prophages per genome. In contrast to the strictly predatory nature of virulent (non-integrating) phages, these temperate phages are a double-edged sword for their bacterial hosts. The vast majority of temperate phage infections result in immediate viral replication and bacterial death, but when integrated, prophages may conditionally enhance host fitness through virulence factors. Beneficially, prophages can also confer resistance to infection by related phages to their hosts, although the breadth of this resistance varies. Modeling of both microbial systems also suggests that prophage induction can serve as an efficient self-replicating weapon for the host. However, in contrast to virulent phages, temperate phages are generally prohibited as biocontrol agents due to the potential transmission of virulence factors.
There are many possible triggers for prophage induction, including DNA damage, bacterial toxins, and phage-encoded communication systems. Many prophages also exhibit so-called spontaneous induction, whereby lysogens produce detectable levels of free phage in regular bacterial cultures, although there is evidence that "spontaneous" induction is a response to some trigger, e.g. DNA damage observed in a subset. To further complicate matters, prophage induction rates are affected by culture medium choice, and indirect effects such as quorum sensing may also affect induction rates in unpredictable ways. Despite these interesting dynamics, little is known about the role of prophages in microbial ecology, particularly in the phyllosphere. Prophage induction can be widely observed in intestinal bacteria, and there is evidence that they may play an important role in regulating microbial communities. Despite our increasing understanding of their importance, phages have not been commonly studied in the field of plant-beneficial synthetic bacterial consortia (SynComs). However, there are indications that prophages are important players in the phyllosphere. Although prophages were not studied, phage depletion has been shown to alter bacterial composition. A recent metagenomic study of the wheat phyllosphere also found that an estimated 24% of phages were temperate, with the most abundant phage being the temperate phage Hamidonella virus APSE, which protects aphids from parasitic wasps .
Identifying prophages can be difficult. Although bioinformatics tools such as PHASTER and VIBRANT enable in silico prophage prediction from bacterial genome assemblies, sequence-based prophage identification has limitations, such as determining whether a prophage is viable and the inability to predict the relative induction rate of prophages or whether they are Under what conditions it is induced. To quantify induced prophages, traditional plaque assays are widely used. However, since this requires a susceptible host, many studies have used culture-free techniques (TEM, epifluorescence microscopy, qPCR). Recently, several tools have been developed to identify prophage activity by sequence alignment of whole-genome shotgun (WGS) reads to bacterial assemblies. Both PropagAtE and hafeZ directly search for regions with high read coverage (calculating prophage/host read coverage), while Prophage Tracer searches for inconsistent reads to estimate prophage excision rates (although it cannot detect prophage excision rates with multiple active prophages). Accurate quantification of excision in the phage host). However, high levels of background chromosome coverage limit the detection limit of WGS data. To address this issue, the Tranductomics pipeline sequences only the encapsulated DNA of the inducing phage, thereby increasing detection limits and enabling the study of transduction patterns (without phage quantification). Based on the advantages of these tools, weintroduced and validated Virion Induction Analysis Sequencing (VIP-Seq). By quantifying the DNA concentration in the encapsulated DNA, combined with read alignment and inconsistent read checking, VIP-Seq enables the identification and absolute quantification of all induced prophage titers with high sensitivity.
We applied VIP-Seq and other techniques to a collection of strains isolated from a single environment, providing the first study of prophages in the phyllosphere. Among 63 newly sequenced strains of Erwinia and Pseudomonas isolated from single-field wheat flag leaves, we identified 120 spontaneously induced prophages from 23 new genera, quantified their titers, and found Many are highly induced in overnight cultures. We also used wheat seedlings to further demonstrate that widespread phage induction can also occur in plants. By investigating the importance of microdiversity undetected by conventional metagenomic studies, we find that prophages are major promoters of intraspecific diversity and warfare, fragmenting their hosts into incompatible "phagocytic" species. Finally, comparison with bioinformatic prophage predictions also revealed major differences and suggestions for IS-mediated prophage inactivation. Our results reveal for the first time that prophages are ubiquitous, active, genetically diverse, and influential among wheat phyllosphere bacteria.
- method -
Identification and quantification of active prophages using virion induction assay sequencing (VIP-Seq)
Identification and quantification of active prophages using
Virion Induction Profiling Sequencing (VIP-Seq)
We used VIP-Seq to identify and quantify prophages in overnight bacterial culture supernatants. Briefly, bacterial supernatants are concentrated and subjected to DNase digestion, then encapsulated DNA is quantified and reads mapped back to the bacterial host (Figure 1).
Figure 1 VIP-Seq workflow for identification and quantification of active prophages in bacterial cultures.
Created by BioRender.com.
- result -
Erwinia and Pseudomonas strains from the phyllosphere contain high titers of spontaneously induced multiple prophages
Erwinia and Pseudomonas strains from the phyllosphere
harbour diverse prophages spontaneously induced at
high titres
Many spontaneously induced prophages (SIPs) are highly induced in overnight culture, with titers as high as 3*108/ml of virions (Figure 2A-B). In some strains, virion titers were even comparable to colony-forming unit (CFU) counts; in aphid strain Z9_1, the aggregated virion/CFU ratio was 0.32 (Fig. 2C).
There was a significant difference in SIP content between Erwinia and Pseudomonas (Fig. 2, A-C). In the median, strains contained 2 SIPs, while aphidicola strain Z9_3 had the most, containing 4 SIPs. In contrast, six Pseudomonas strains appeared to lack SIPs in overnight cultures. Erwinia virion aggregation titer (median (IQR) 6.5*107(2.4-12)*10< a i=3>7 viral particles/ml) was higher than Pseudomonas (median (IQR) 5.0*105 (2.9-12)*105 virus particles/ml). Although these differences were reduced after adjusting for CFU concentration (Fig. 2C), they were still significant (p=8.9*10-4, Kruskal - Wallis rank sum test)
Studying the SIP genome revealed many shared genome segments interspersed with unaligned regions (Figure 2D).
Figure 2 Overview of bacterial strains and their SIP content.
A Cladogram of 45 Erwinia and 18 Pseudomonas strains based on genome-wide similarity (UPGMA clustering). Also shown are active phage clusters at the species level in each strain. Finally, aggregate virion titers in overnight cultures of each strain are shown as bar graphs.
B Violin plot of estimated virus counts/ml for all 120 Erwinia and Pseudomonas active prophages.
C Violin plot of estimated virus number/CFU for all 120 Erwinia and Pseudomonas active prophages.
D Representative from 28 identified prophage species-level clusters, labeled with the name of each cluster. The Kalvehoegda cluster is marked "*" and annotation indicates that this region may be a phage satellite. Next, clinker plots show the amino acid arrangement of coding regions between species representatives.
VIP-Seq validation
Validation of VIP-Seq
Except for W02_4 supernatant, VIP-Seq titers were in good agreement with EPI counts, and the EPI counts of T4, B01_5, and W01_1 accounted for 54%, 65%, and 19% of the EPI counts, respectively (Figure 3). In contrast, PFU is more variable; when T4's plaque count exceeds EPI count, B01_5 and W01_1 have PFU/EPI ratios of 4*10-2, respectively. It is shown that PFU can only be used conditionally to quantify induced prophage titers. -4. Since both strains contained multiple SIPs that could potentially form PFU, we sequenced plaques on the two indicator strains to determine which SIP was forming plaques (Figure 3B,C). After adjusting for the relative titers of plaques in overnight cultures, the adjusted PFU/EPI ratios were 1.05 and 1.3*10-5 and 2.8*10
In particular, the very long prophage genome length from Vibrant is due in part to the merging of several pairs of closely spaced prophages (i.e., B01.5 prophages Glittertind_A and Skarstind_A, Figure 3B) into a single prophage prediction.
In W02_4 supernatant, both VIP-Seq and plaque assay were negative, while EPI count was 2.5*104VLP /mL (Figure 3A,D).
Figure 3 Comparison of VIP-Seq quantification with plaque and fluorescence microscopy counts.
A The phage titer determination method was used to compare the phage titers of four strains: T4 virulent phage, B01_5 phage, W01_1 phage and W02_4 phage. Phage titers of sensitive strains B01.10 (B01.5 SIPs) and B01.5 (W01.1 SIPs) were determined using VIP-Seq, fluorescence microscopy and plaque counting. W02_4 supernatant showed no plaque against any of the strains tested. For VIP-Seq, the supernatants of B01_5, W01_1, and W02_4 were concentrated with Amcon filters before DNA extraction, unlike the high-titer T4. Each error bar represents the standard deviation of three technical replicates.
B−D read coverage of VIP-seq libraries constructed on untreated overnight cultures of B01_5, W01_1 and W02_4 respectively. Phage regions of B01_5 and W01_1 are amplified and organized (maintaining relative proportions), while the full genome is shown in W02_4.
Erwinia host phage capable of bacterial warfare
Erwinia host prophages capable of bacterial warfare
While many SIPs in Erwinia supernatants showed broad host range on competing strains isolated from the same environment (Fig. 4A), Pseudomonas supernatants failed to produce a single visible plaque, despite Some clearing areas of turbidity were observed. There were significant differences in host range, with Erwinia supernatants imprinting on 0 (aphid B07.5) and 27 (aphids Z9.1 and Z9.3) competing strains (Fig. 4B). Prophage susceptibility varied similarly, from 0 (multiple strains) to 30 (aphid E. N2.3). There are also at least two examples of widespread infection, both with E. billingiae W05.1 and new E. sp strains leaving patches on aphid strains. Some supernatants also showed multiple plaque morphologies on the same host (the three leftmost points in Figure 4B correspond to the supernatants of W11.1, Z9.1, and Z9.3, respectively, containing 2, 3 and 4 identified SIPs), indicating that multiple SIPs may form plaques.
Figure 4 Erwinia supernatant plaque test
A Heat map showing the results of a 45 × 45 Erwinia supernatant plaque assay, with supernatant host shown vertically and host sensitivity shown horizontally. Bar graphs showing cumulative host range/sensitivity adjacent to the respective axis.
B Plaque analysis of 45 Erwinia supernatants on aphid N2.3 plates showed a wide range of susceptibility and multiple plaque morphologies. The image is cropped sharply.
High levels of prophage induction observed in plants
High levels of prophage induction observed in planta
Next, CFU and PFU (utilizing B01_5 SIP Glittertind_A plaques on B01.10) were monitored for five days in the experimental and control groups (Figure 5, Supplementary Table S7). While the PFU of the cell-free control dropped below the lower limit of detection on the second day, the PFU of the B01-5 treatment remained relatively stable for all five days (Figure 5B). In fact, PFU even increased statistically significantly between days 0 and 3 (P=0.04, Kruskal-Wallis rank and test), dropped on day 5. This relative stability of PFU contrasts with CFU counts of B01_5 counts, which decreased by nearly three orders of magnitude between days 0 and 5. Due to these different trends, the PFU/CFU ratio of the B01_5 treatment varied greatly over the course of the time series. Although PFU/CFU was initially much lower than the PFU/CFU ratio of overnight in vitro culture (4.7×10-3), by the third day it has climbed to more than 500 times the in vitro ratio (Fig. 5C), showing a very high plant induction rate.
Figure 5 E. aphidicola strain produces prophage in plants.
A The first leaf at 12 days old is marked as FL.
B CFU and PFU wheat seedlings were inoculated on the first leaves of 12-day-old Heerup seedlings. The first treatment (B01_5) was washing aphid strain B01_5 twice in PBS buffer, while the second treatment (control) was washing the cell-free supernatant of B01_5. Leaves were washed in SM buffer and colonies were counted on Pseudomonas selection agar, while plaques were counted using a soft agar overlay with sensitive strain B01_10 as host. Each data point is the mean of four biological replicates, and error bars represent maximum/minimum values. The detection limit for the average of four biological replicates is 1.5 PFU or CFU/leaf (indicated by the black dashed line), and data points below this value are artificially set to 1.3 for clarity.
C PFU/CFU ratios treated with B01_5 cells, and PFU/CFU ratios recorded in vitro indicated by the blue dashed line. The orange line connects the mean of each data point, while all data points are shown as black dots.
Comparison of active prophage with bioinformatics predictions suggests possible IS-mediated inactivation of prophage
Comparing active prophages to bioinformatic predictions suggests possible IS-mediated prophage inactivation
Checking that the tools correctly predicted the SIPs identified by VIP-Seq, we found that Vibrant predicted 117/120 (0.98) and PHASTER predicted 109/120 (0.91) (Figure 6A). Neither PHASTER nor VERIFIANT predicted the possible phage satellite Kalvehoegda_A. Both tools also have relatively low nucleotide precision because they often add extraneous host DNA within VIP-Seq validated precursors. These differences are evident even when only high-confidence prophage predictions are considered, as the distribution of genome size and relative GC content differs from experimentally active prophages (Fig. 6B,C). In addition to predicting >90% of SIPs, PHASTER and Vibrant also predicted many prophages for which no SIPs were observed (Fig. 6D).
Surprisingly, half (53%) of the PHASTER predictions completely lacked structural gene annotation and were therefore unlikely to be credible prophages; indeed, none of these predictions were SIPs (Fig. 6E, Supplementary Table S9).
Figure 6 Bioinformatics analysis of SIPs
A Performance of PHASTER and VIBRANT phage predictions compared to 120 SIPs discovered using VIP-Seq. Performance metrics are detailed in Section 2.6.
B Comparative distribution of prophage region sizes for high-quality prophage predictions with all SIPs.
C is the same as (B), but the GC content of the prophage region is relative to that of the host bacteria.
D Bar chart showing the number of spontaneously active phage predictions observed at each confidence level for PHASTER and VIBRANT (labeled "high", "medium", and "low" by prediction confidence).
E Sankey diagram illustrating the predicted composition of PHASTER prophages from 40 aphid genomes.
- discuss -
The impact of prophage activity in agriculture could be substantial. Although temperate phages are generally considered unsuitable for biocontrol because of their tendency to integrate into prophages, we found that many phyllosphere prophages have been involved in bacterial warfare. These results also highlight the importance of prophages in engineering beneficial microbial-synthetic consortia. Although rarely considered in this context, it is likely that many members of the synthetic microbiota contain active prophages. Some of these may kill disease-causing strains and promote the establishment of beneficial communities, while others may eliminate bacteria that benefit the plant or spread harmful genes. In any case, they should not be ignored. Prophages combine traditional phage-based biocontrol with traditional bacterial-based synthetic flora, providing a potential toolbox for next-generation plant microbiome engineering and sustainable agriculture.
references
Dougherty, P.E., Nielsen, T.K., Riber, L. et al. Widespread and largely unknown prophage activity, diversity, and function in two genera of wheat phyllosphere bacteria. ISME J 17, 2415–2425 (2023). https://doi.org/10.1038/s41396-023-01547-1
- About the Author -
Corresponding Author
University of Copenhagen
Lars Hestbjerg Hansen
professor
Lars Hestbjerg Hansens' Environmental Microbial Genomics Group has extensive expertise in performing highly customized genomic and metagenomic studies of prokaryotes and eukaryotes using in-house high-throughput and whole-genome sequencing (Illumina and Nanopore sequencers). The group uses information based on sequence analysis to characterize the genomes of key species and their prevalence in natural and artificial microbial communities and metagenomes, including how phage information and other mobile genetic factors influence microbial evolution and traits. Group activities cover microorganisms in natural, agricultural and health-related systems.
research direction:
Leaf hub species and bacteriophages as agricultural biological control agents
Use of altered bases in phage genomes
Manipulating the gut microbiome through FMT or phage therapy
Genetic origins of antibiotic resistance
Mobility of genetic parasites and their role in bacterial evolution
Microbiomics, Mobilityomics and Viromics
Epigenetics of Bacteria and Phages
Laboratory official website: https://plen.ku.dk/forskning/mikrobiel-oekologi-og-bioteknologi/environmental-microbial-genomics/
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