Pantoea comprises species found in a variety of different environmental sources. Pantoea spp. are often recovered from plant material and are capable of both benefitting the plants and acting like phytopathogens. Some species of Pantoea (including P. agglomerans) are considered opportunistic human pathogens capable of causing various infections in immunocompromised subjects. In this study, a strain of P. agglomerans (identified by 16S rRNA gene sequencing) was isolated from a dead specimen of an unidentified Latvian grasshopper species. The retrieved strain of P. agglomerans was then used as a host for the potential retrieval of phages from the same source material. After rounds of plaque purification and propagation, three high-titer lysates corresponding to putatively distinct phages were acquired. Transmission electron microscopy revealed that one of the phages was a myophage with an unusual morphology, while the two others were typical podophages. Whole-genome sequencing (WGS) was performed for each of these isolated phages. Genome de novo assembly and subsequent functional annotation confirmed that three different strictly lytic phages were isolated. Elaborate genomic characterization of the acquired phages was performed to elucidate their place within the so-far-uncovered phage diversity.The bacterial genus  Pantoea includes multiple Gram-negative, yellow-pigmented rods that are frequently isolated from a variety of environmental and higher-organism-associated sources [The bacterial genus  sources , hence tPantoea spp. are still, arguably, mainly thought of as bacteria associated with plants, capable of both having positive effects on plants  a,4,5 aPans e.g., ,7,8,9). ,8,9. Pans —are omnipresent in every environment where bacteria thrive. As natural predators of bacteria that have the ability to rapidly propagate themselves upon invading a susceptible host bacteria population, phages can be harnessed as biocontrol agents to eliminate unwanted bacteria. However, phages generally have a very limited host range, which might be as narrow as several strains of a particular bacterial species. Thus, isolation and characterization of new bacteriophages capable of infecting representatives of the bacterial genera or species even of modest economic or healthcare importance are of particular interest, especially in a case such as when a host has none or just a few bacteriophages yet known to infect it. In addition to the expansion of the knowledge on the uncovered phage diversity, which is of fundamental importance, such studies might potentially provide a good lead for further practical application evaluation of a given novel phage or its proteins.Pantoea-infecting phages that have their complete genomes publicly available. These phages represent all the tailed phage morphotypes , and their genomes range from 36,790 to 149,913 base pairs, showing a percentage GC content of 39–55.35%. Based on the complete genome-associated metadata, strains of Pantoea agglomerans are indicated as the specific host of these phages most commonly (n = 12) followed by P. deleyi (n = 3) and P. dispersa (n = 2). Eleven of the so far sequenced Pantoea phages have been isolated from plant sources . Additionally, five Pantoea phages were previously retrieved from water-associated sources and another three from soil samples. This makes the phages Nifs112, Nufs112, and Nafs113 described in this study the first three Pantoea phages to be isolated directly from an insect-associated source , isolate LS5-2, the isolation host of phages Nifs112, Nufs112, and Nafs113, formed beige colonies at all of the three incubation temperatures tested . The fastest growth was observed at +30 °C, and the slowest was observed at RT. After their appearance, the LS5-2 colonies were initially beige but turned yellow-orange during the subsequent incubation.Pantoea. More specifically, as LS5-2 shared a well-supported most recent common ancestor with P. agglomerans DSM 3493 in the phylogeny, and both sequences had only a few differences, the bacterial isolate LS5-2 was considered to be a strain of P. agglomerans  than that of Nufs112 (54.4 ± 1.8 nm). With the opposite holding true for tail lengths, the tail of Nifs112 was measured to be around 11.2 ± 1.6 nm in length, whereas Nufs112 had a tail of 12.8 ± 2 nm. However, a more detailed TEM examination involving at least several tens of virions, additionally taking the planes at which the virions were seen into account, would be necessary when drawing conclusions on whether these capsid size and tail length differences were meaningful. What stood out in the micrographs, however, were the tail appearances, with Nifs112 having seemingly slimmer tails in contrast to the “chunky stubs” of Nufs112  that has been observed on numerous occasions for a plethora of different podophages . CombineAlthough having an unremarkable plaque appearance, Nafs113 virions were determined to have a less-common myovirus morphotype—an elongated head with an approximately 3:1 length (121.4 ± 6.8 nm)-to-width (43.4 ± 3.8 nm) ratio, to which a 88.5 ± 3.2 nm long contractile tail with a width of 15.4 ± 0.6 nm (uncontracted state) was attached , Nufs112 , and Nafs113  complete genomes, respectively  were found to belong to the family lymerase ). HoweveAutographiviridae podophages. No protein, however, was found to be present in more than a single studied phage at the 95% identity threshold. Lowering the threshold to 50% only identified five proteins that were shared between Nifs112 and Nufs112—a terminase large subunit, phosphatase, a HTH domain-containing protein, and two hypothetical proteins. Setting an identity threshold to 30% revealed that phosphatase was the only protein that could be considered to be shared by all three of the phages. As expected, the lowest threshold used (30%) also revealed that Nifs112 and Nufs112 might have up to 20 homologous proteins, i.e., the ones involved in either virion morphogenesis  or those responsible for nucleic acid metabolism, modification, and repair , and several other, mostly hypothetical, proteins. At this threshold, a homolog of HNH endonuclease from Nafs113 (ORF115 product) was also identified in the Nifs112 proteome (ORF30 product).Roary  pangenoAfter functional annotation of the studied phage genomes, termini types were also additionally verified using the TerL phylogeny reconstruction approach based on a dataset of Merrill and colleagues . The TerHamiltonella phage APSE-1 representing the same TerL sequence clade also has a terminally redundant and circularly permuted genome, with the sequence and electron microscopic analysis carried out indicating that “most likely the permutations are randomly distributed over the genome” [Shigella phage Sf6 noted “little evidence for specific termini”, but they also found out that Sf6 DNA appeared to be cleaved at many sites within a large region of about 1800 bp, including a possible pac site for the initiation of a packaging series from a DNA concatemer [Pantoea phage Nafs113’s genome’s circular permutation , however, was not further looked at experimentally at the time being.Yet, the TerL phylogeny reconstruction showed that the TerL of Nafs113 clustered together with Sf6-type headful-packaging-employing phages. This further supported our assumptions regarding the Nafs113 headful genome packaging, as, for example, the  genome” . Researcncatemer . The extP. agglomerans . The genomes of Nifs112 (50.2%) and Nufs112 (47.7%) deviated way more in that regard, although a Pantoea-infecting phage with a GC% content as low as 39% was previously documented  similar to that of its host species, 70119.1; ).Pantoea agglomerans phages uncovered so far is noticeably lower than that of their host  possibly allows us to propose two of the studied phages (Nifs112 and Nafs113) as novel species within the already-recognized phage genera, whereas Nufs112 might even be a sole representative for a novel phage genus based on the ICTV adopted genome nucleotide sequence similarity phage genus and species demarcation criteria .Pantoea phage Nifs112 (OK570184.1), the highest-total-scoring hit was documented to a complete genome of the Erwinia phage vB_EamP-S2, representing an Eracentumvirus S2 species within the genus Eracentumvirus . This hit to Erwinia phage vB_EamP-S2 was followed by hits to other Erwinia phages either officially or tentatively belonging to the Eracentumvirus genus  among the top five highest-scoring hits.When querying the Pantoea phage Nufs112 (OK570185.1), the top hit to a cultured phage complete genome was noted to the Klebsiella phage 6939  unclassified at the genus and species level. This hit to Klebsiella phage 6939 was followed by hits to the phage Reminis, showing a similar intergenomic distance, and recognized or putative representatives of the Gajwadongvirus genus , showing a >60% intergenomic distance to Nufs112.In the case of the Pantoea phage Nafs113 (OK570186.1) had only a single meaningful hit—to Pantoea phage vB_PagM_LIET2  representing a sole Lietduovirus LIET2 species within the genus Lietduovirus, with hits to other phage genomes having a ≤1 percent query coverage.Interestingly, the genome of the When the studied phage genomes were queried against the bacterial sequences found in GenBank (taxid: 2) using megablast, hits to bacterial genomes were limited to up to a few hundred nucleotides (less than a few percent query coverages) for each of the three phages, indicating a lack of highly similar prophages or the remnants of thereof in the so-far-sequenced bacterial isolates.Having gained a glimpse at the genome nucleic acid sequence-wise similarity to other phages, a reconstruction of the selected analogous protein phylogenies was performed within the context of their most closely related counterparts encoded by other phage genomes . For thiEracentumvirus. Although the Eracentumvirus clade with Nifs112 had a well-supported MRCA in three of the trees (except the endolysin tree), its closest neighbors differed across all four of the trees with regard to taxonomical placement.In all four of the trees, proteins from Nifs112 were most like their counterparts from the representatives of Pantoea phage vB_PagM_LIET2 belonging to the genus Lietduovirus, and in all of the trees save for an endolysin tree, there was a rather long branch leading to their two-sequence clade, indicating distinctness of their selected homologous proteins from the homologs found in other phages.Selected proteins from Nafs113 shared an MRCA with the Klebsiella phage 6939 and the Ralstonia phage Reminis. These two phages, however, shared a well-supported MRCA in all four of the trees, whereas Nufs112 reliably clustered with them only in the case of MCP and TerL. Interestingly, the phage Reminis and the Pantoea phage MR4 seemed to be classified in GenBank as tentative representatives of the genus Gajwadongvirus, but from the generated trees, only MR4, and not Reminis, seemed to form monophyletic clades together with the officially recognized Gajwadongviruses PP99 and ECBP5, which together boasted very short within-clade evolutionary distances. Thus, the designation of Reminis as a Gajwadongvirus representative did not really seem justified based on the selected protein phylogenies. While an evolutionary link to the recognized Gajwadongvirus proteins could also be established for Nufs112, their counterparts seemed to have diverged in a more distant past and were very unlikely to share an immediate ancestry.The situation with Nufs112 was revealed to be more ambiguous, as it did not reliably fall within the same monophyletic group, and the tree topologies surrounding the corresponding sequences of Nufs112 proteins differed to an extent. Based on the phylogenies, the selected Nufs112 proteins were most similar to the homologs from the To get a better proteome-based overview of the place of the studied phages within the context of cultured and sequenced phage diversity, 18,553 complete phage genomes  available at INPHARED  were suEscherichia (35 phages), and other bacterial genera were represented by only up to eleven different phages infecting them. Apart from each other, there were no other first neighbors designated as Pantoea phages identified for either Nifs112 or Nufs112. However, three Pantoea agglomerans phages  were among the first neighbors of Nafs113, with the phage LIET2, expectedly from the previously determined BLASTN search, being the most similar one. Notably, VC_437_0, to which Nifs112 was clustered based on its proteome, otherwise comprised exclusively Erwinia-infecting phages, either officially or tentatively representing the genus Eracentumvirus.From the vConTACT2-generated network, only the first neighbors of the studied phages were of interest, excluding the studied phages themselves—six unique neighbors were identified for Nafs113, nine for Nifs112, and 22 for Nufs112, whereas sixty-three of the phages were the first neighbors for both Nifs112 and Nufs112 . The stuAutographiviridae, with sixty-seven being further classified into the Molineuxvirinae subfamily comprising the clusters VC_436_0 (at least six genera representatives) and VC_437_0 (Eracentumvirus), and nineteen into the Colwellvirinae subfamily . Interestingly, while fifty-eight Molineuxvirinae phages were the first neighbors of both Nifs112 and Nufs112 and only nine representatives of this subfamily were uniquely identified as the first neighbors of Nifs112, all nineteen Colwellvirinae representatives were neighbors of Nufs112 exclusively . However, some of the (mostly tentative) designations in the taxonomy associated with the phage genome submissions were not supported at the chosen intergenomic distance criteria—Vectrevirus representatives were split into two VIRIDIC clades; Uliginvirus were split into five VIRIDIC clades at the given intergenomic similarity threshold; the bacteriophage Reminis was tentatively classified as Gajwadongvirus (likely incorrectly); the tentative Tuodvirus Escherichia phage vB_EcolP_P433.1 seemed way more similar to two clades, in which recognized or tentative Vectrevirus representatives are found, than to the officially recognized Tuodvirus phD2B.Most of the genus-level designations in the retrieved complete-genome sequence-associated taxonomy coincided with the VIRIDIC clusterization at a 70% intergenomic similarity threshold, and such clades were monophyletic in the generated intergenomic distance NJ tree  to be so similar to a plethora of Autographiviridae podophages. A look at the ICTV virus metadata resource (VMR_20-190822_MSL37.3) revealed that this stemmed from the misattribution of a taxonomy, as the Vibrio phage Vc1  is a completely different phage to the Vibrio phage VC1 , but the taxonomy associated with the latter was mistakenly expanded to the former in the GenBank.In addition, it seemed very unbelievable to find a single alleged Eracentumvirus, interestingly, so far comprising only Erwinia phages;Nifs112 was a representative of the genus Gajwadongvirus representatives but did not look like a representative of that genus itself;Nufs112 was related to Lietduovirus, but the question of whether it represents a novel species therein remains.Nafs113 could firmly be considered a representative of the genus Nevertheless, combining the results of the performed “overview” analyses for our studied phage place within the so-far-uncovered phage diversity, it was revealed that:Pantoea phage Nifs112 is a linear 46,202 bp long dsDNA molecule  with a GC% content of ~50.2%. The genome of Nifs112 (OK570184.1) was found to contain 59 open reading frames, all of which were located on a direct strand; no tRNA genes were found. ATG was predicted to serve as a start codon for fifty-one of the ORFs; GTG—four, CTG—two, and TTG–four ORFs. The majority of the predicted ORFs had a well-identifiable Shine–Dalgarno motif in the sequence span 20 bp upstream of the selected start codons  of the phages . Moreover, twenty of them were found in all three of the phages even at the 90% identity threshold set for Roary. All three of the phages shared a conserved genome architecture, with the main differences being in the presence or absence of short ORFs encoding different hypothetical proteins without an annotation. Pairwise homologous protein identity percentages clearly indicated that Nifs112 was more closely related to the phage vB_EamP-S2 than to Era103 .Acinetobacter bacteriophages identifiable using CD search, and Acinetobacter podophages are known to have a host-capsular-polysaccharide-degrading ability coupled to their tailspikes [Most of the ORF products of Nifs112 and vB_EamP-S2 were highly similar, showing exceptional conservation within the most functionally annotated products. The difference of interest, however, lay in the tailspike protein that was thought to contain an EPS depolymerase activity and showed a conservation only of about 50% between the two phages, with the sequence encoding the second half of the respective proteins being particularly different . Respectilspikes . Previouilspikes  and was ilspikes . Thus, wErwinia phage vB_EamP-S2 (YP_009797612.1) seemed to be a fusion of two hypothetical proteins encoding ORFs found in both Nifs112 (UJH95776.1 from ORF4 (+2 frame) and UJH95777.1 from ORF5 (+3 frame)) and Era103 (YP_001039634.1 and YP_001039635.1) that were in close proximity of each other in both cases. The corresponding product/products so far seemed to be unique to Eracentumvirus representatives and remained without even a hint at the possible function. The ORF encoding nucleotidyl transferase in the genome of vB_EamP-S2 was also split in two in the genome of Nifs112, and it looked like it might now be translated with a −1 frameshift. Although these two ORFs overlapped, no evident slippery signal was found in the region of the overlap of ORF21 (+1 frame) and ORF22 (+3 frame) or ~60 bases upstream until the in-frame stop codon preceding the start codon of the ORF22, and the raw reads supported this sequence unambiguously. Moreover, both ORF21 and ORF22 had Shine–Dalgarno motifs that could reasonably well support the translation of their products . However, given that most BLASTP-found homologs approximately correspond to their concatenated length, the question of whether such products would be functional is open.Additionally, a region encoding the second hypothetical protein in the genome of the Pantoea phage Nifs112 might not show a sufficient amount of similarity to representatives of either of the Eracentumvirus S2  might not be an optimal host for Nifs112 could further be reasoned by the fact that, for example, the closely related Erwinia phage VyarbaL formed plaques of 3–3.5 mm in diameter and a well-marked halo on its host strain of E. amylovora  [Erwinia phage S2, representing the same genus Eracentumvirus species as VyarbaL, was able to lyse not only multiple strains of E. amylovora but also at least a single strain of both P. agglomerans (strain Em283) and P. ananatis (strain 351 Lys) [Summing up the analyses, it seemed that the isolated and characterized _EamP-S2  and Vyar_EamP-S2 ) or EracphiEa100 ) speciesublished , which, VyarbaL) . Additio351 Lys) .Pantoea phage Nufs112 is a linear 45,951 bp long dsDNA molecule  with a GC% content of ~47.7%. The genome of Nufs112 (OK570185.1) was predicted to have 67 open reading frames and no tRNA genes; every ORF was detected on a direct strand. ATG was predicted to be a start codon for fifty-eight of the ORFs, TTG for 6, GTG for 3, and no ORFs were presumably starting with a CTG. Only several of the identified ORFs did not have a highly preserved Shine–Dalgarno motif complementary to the 16S rRNA tail of P. agglomerans in the sequence span 20 bp upstream of the selected start codons . Nearly all of the functionally annotated ORF products of Nufs112 were revealed to perform either analogous or similar functions to those identified for Nifs112 products and are typically expected to be present in an ntatives ), and sePantoea phage Nufs112 was most closely related to the Klebsiella phage 6939 and the phage Reminis . From the taxonomically officially recognized phages, Nufs112, indeed, was so far most similar to the representatives of the Gajwadongvirus phage genus (Escherichia phage ECBP5 and Pectobacterium phage PP99).The data presented previously indicated that the Pantoea phage Nufs112 only shared a hypothetical product of ORF67 with Reminis under such a strict threshold. Notably, it was shown that both the recognized Gajwadongvirus species (represented by ECBP5 and PP99) shared up to 32 of the proteins, having a >90% pairwise identity, whereas Reminis had only a single hypothetical protein shared with a recognized representative of Gajwadongvirus at such a threshold  comprised one hundred and thirty-five products, with twenty-nine of them being encoded by the genome of each phage in the comparison. At this threshold, Nufs112 encoded up to twenty-one proteins not found in the proteomes of its closest relatives, nineteen of which remained without any functional annotation , whereas at the 90% threshold there was no protein shared between the five phages, which was not very surprising given their large pairwise intergenomic distances. The hreshold . In linePantoea phage Nufs112 (OK570185) and closely related phages, genomes of the Ralstonia phage Reminis (MN478376) and the Klebsiella phage 6939 (OL362271) were rearranged to ensure collinearity with Nufs112, for which the exact genome termini were identified. In addition, it was revealed that all of the five genomes should be colinear, as seen within the virions of the respective phages. Consistent with the expectations based on the results described previously, nearly all of the proteins found in Nufs112 that were also present in the Klebsiella phage 6939 and the Ralstonia phage Reminis, and the Gajwadongvirus representatives  and theeminis MN8376 and Eracentumvirus representatives, the genomes of Nufs112 and its closest relatives also had a well-defined modular structure . The region from 7.5 kbp up to ~23 kbp contained ORFs encoding proteins responsible for DNA replication, modification, and repair, as well as several additional functions not reliably falling within any of the defined protein functional groups. Functionally annotated proteins encoding ORFs in this part of the genome were interspaced by up to another 16 hypothetical proteins encoding ORFs. The remainder of the genome (~23 kbp to ~46 kbp) was mostly comprised of ORFs encoding virion morphogenesis and putative host lysis proteins that did not form a lysis cassette with sequential ORFs encoding up to four of the lysis proteins  but were interspaced by other morphogenesis proteins coding ORFs.As in the case of the Nifs112 comparison with tructure . The genKlebsiella phage 6939 (URY99237.1) and pairwise similarities. Considering that tail fiber/fibers (their sequence) is one of the factors influencing the host range of phages, it is not uncommon for phages of different hosts to have these analogous proteins be diverged. However, it is worth noting that the Gajwadongviruses ECBP5, PP99, and MR4  were all isolated on different hosts , despite having very similar putative tail fiber proteins (pairwise identity >90% over 306 aa length of the respective proteins).The main difference that stood out when comparing the proteome contents of these phages was, however, a putative tail fiber encoding ORF .These ORF products all had an identifiable T7 tail-fiber-protein conserved domain (pfam03906) at their N termini but differed greatly in lengths (242 aa in Reminis (QGH45085.1); 306 aa in Gajwadongviruses PP99 (YP_009788795.1) and ECBP5 (AID17699.1); 506 aa in Nufs112 (UJH95883.1); 769 aa in The predicted tailspike protein of Nufs112  had an identifiable pectate lyase superfamily protein domain (pfam12708), which was previously also identified in other phages . The actPantoea phage Nufs112 was sufficiently divergent from any of the so-far-uncovered phages, which allows us to propose it to be the founding member of a novel phage genus within the family Autographiviridae.Consolidating the results, the Ralstonia solanacearum was indicated as the host in the metadata of MN478376, the genome of Reminis (MN478376) was not organized taking the SDTRs of 377 bp mentioned by the authors into account, and no SDTRs were present and/or annotated in MN478376, proteome contents and evolutionary analyses using MN478376 indicated that it is a podophage, and not a siphophage, among others).Of note, during the preparation of the genome architecture comparison figure between the phages related to Nufs112, and other analyses performed within this study, several discrepancies between the results presented in the article describing the phage Reminis  and refePantoea phage Nafs113 is a linear 75,899 bp long dsDNA molecule with a GC% content of ~54.1%. No defined termini could be predicted as an implication of the presumed headful packaging strategy that results in the progeny virions each having terminally redundant genomes that are circularly permuted when compared to each other. The scaffold representing the genome of Nafs113 was opened in a way that ensured collinearity with the closest relative, which in this particular case also followed the convention of cutting the circularized scaffold before ORFs encoding terminase subunits. Although, frankly, in the case of both phages, at the time there seemed to be no reason to assume that the ORF preceding TerL-encoding ORF encodes TerS, which is a protein known not to show such a high degree of evolutionary conservation as its larger counterpart, other than empirical phage genomics observations of the common TerS- and TerL-encoding ORF appearance in tandem. However, it is worth noting that with the growth of the number of phage genome sequences in the public biological sequence repositories, more and more exceptions to most “phage genomics rules” begin to appear.The complete genome of the The genome of Nafs113 (OK570186.1) was found to contain 130 open reading frames, 43 of which were located on a direct strand, whereas 87 were located on a reverse strand. No tRNA genes were found in the genome of Nafs113. ATG was predicted to serve as a start codon for one hundred and sixteen of the ORFs, GTG for nine, TTG for four ORFs, and a single ORF was predicted to have CTG as a start codon.P. agglomerans antiSD sequence)) of Nafs113 was only −4.57 ± 2.36 kcal/mol (average ± SD) as calculated by free_align.pl script [Interestingly, despite the fact that most of the predicted ORFs had a well-identifiable Shine–Dalgarno motif in the sequence span 20 bp upstream of the selected start codons , the onel script , whereasPantoea phage Nafs113 was demonstrated to be rather unique among the so-far publicly available sequenced phages (bar the Pantoea phage vB_PagM_LIET2) based on its complete-genome nucleotide sequence, it was not unexpected that only 30% of the predicted Nafs113 ORF products could be assigned a putative function. Nevertheless, a number of the reasonably well-conserved proteins responsible for virion morphogenesis/structural features expected for a tailed dsDNA phage were identified in the genome of Nafs113 despite its genomic nucleotide sequence being very uncommon among the so-far-uncovered phage diversity. Both tail-tape-measure-protein- and tail-sheath-protein-encoding ORFs were identified, which would be sufficient to identify Nafs113 as a myophage had the TEM virion analysis not yet been made. Upon closer inspection, holin, endolysin, and putative inner and outer spanins, representing the lysis proteins expected for a phage of a Gram-negative host bacterium, could be identified. In addition, a number of nucleases and other proteins involved in DNA replication, modification, and repair were annotated. The genome of Nafs113 contained several additional functions performing proteins as well; however, their annotations did not allow us to reliably categorize them into any of the phage product groups we categorized proteins into according to their function , representing a Pantoea stewartii WceF—a glycan biofilm-modifying enzyme with a bacteriophage tailspike-like parallel beta-helix fold. It was recently shown that WceF is a glycosidase active on stewartan, which acts as the main P. stewartii EPS biofilm component, and that WceF is very similar to bacteriophage tailspike proteins [Another marked difference between the two genomes was that one of the two ORFs that were either fused into one (LIET2) or split into two (Nafs113) encoded what was annotated as a 1218 aa long EPS depolymerase-domain-containing protein in LIET2 (YP_009843765.1) or two putative tail-spike (UJH95983.1 and UJH95984.1)-encoding ORFs in Nafs113. While a CD search failed to identify any conserved domains that would imply EPS depolymerase activity for either of these three proteins, the top HHpred hit for each of the three of them was to a PDB structure , whereas the same search using the amino acid sequence of the putative ORF108 product (UJH96005.1) as a query revealed only a match to the hypothetical protein HWC07_gp107 from the Pantoea phage vB_PagM_LIET2.A similar fusion/split was observed for two Nafs113 hypothetical proteins (UJH96005.1 and UJH96006.1) encoded, this time, from frames −1 (ORF107) and −3 (ORF108) in Nafs113, respectively, whose products appeared to be fused in LIET2 (YP_009843788.1). Again, in this case, too, no apparent +1 frameshift signal, was observed, and the presumed Shine–Dalgarno motif for translation of the ORF107 product appeared “stronger” than that of ORF108. However, in this case, the ORF107 product (UJH96005.1) had a lot of BLASTP hits to hypothetical proteins of similar length (~208 aa) from the bacteria  collected geographically  close and temporally proximal  to each other. Speculatively, it can be proposed that the consequences of differences in horizontal gene exchange events by their ancestral phage populations  might explain most of the differences observed between genome architectures and proteome contents as, we believe, the ecological niches and environmental factors naturally influencing both LIET2 and Nafs113, as well as their respective host species strains, might naturally be very similar.All things considered, there is undeniably a very close evolutionary relationship between the Pantoea phages LIET2 and Nafs113 were higher than the same phage species level threshold of <5% differences over complete genome lengths, and some genomic features were different, there is no sufficient evidence in favor of creating a novel phage species within the genus Lietduovirus for Nafs113 instead of considering it another isolate representing the Lietduovirus LIET2 species. Additional research on both of these phages might, however, shed light on more of their functional differences that may warrant their possible delineation at the species level in the future.Given their great difference from any other phage sequenced so far, at this time, we are rather convinced that, for practical reasons, even though intergenomic distances between Orthoptera) specimen, as described previously for the phage Nocturne116 [Studied phages were isolated alongside their host bacteria from a dead unidentified Latvian grasshopper , and subjected to a double agar overlay assay . Phage-nSeveral rounds of individual plaque purifications were carried out, and three different lines of plaques consistently giving the same negative colony appearances were established and assumed to represent three distinct phages.g, 30 min), and the resulting supernatant was decanted and filtered through a 0.45 μm pore size syringe filter. For all three of the investigated phages, such propagation yielded filtrate with a titer of up to 1–2 × 1010 PFU/mL.For phage propagation, soft agar layers from 20 double agar overlay plates  showing confluent lysis of the bacterial lawn were collected and homogenized by vortexing with the addition of 5 mL LB liquid medium per plate. Next, the homogenized soft agar layer mixture was subjected to centrifugation . The obtained phage pellets were dissolved in ~4 mL of TE buffer . Acquired phage suspensions were next layered on top of a CsCl solution (CsCl—0.6 g per mL of the TE buffer). For each phage, a single Ultra-Clear centrifuge tube  was filled with 11.5 mL of the CsCl solution, and 2 mL of a concentrated phage sample was loaded on top of the CsCl solution. Solutions were then spun at +4 °C for 20 h  in an SW 40 Ti rotor (Beckman Coulter)-equipped Beckman Optima L-100XP ultracentrifuge. Distinct horizontal phage bands were formed for each of the phages, and these were collected by pipetting. Collected phage-containing bands were desalted using NAP-25/Sephadex G-25 columns  and PBS as an exchange buffer.Phages were then concentrated by centrifugation of the filtrates in a Beckman Optima L-100XP ultracentrifuge with a 70 Ti rotor  for 5 min, rinsed with 1 mM EDTA solution, and negatively stained using 0.5% uranyl acetate before being left to dry for 2 h. After drying, the stained phage-sample-containing grids were examined using a JEM-1230 transmission electron microscope , and virions were pictured with a Morada 11 MegaPixel TEM CCD microscope-mounted camera using iTEM imaging software .Phage particle dimensions were determined with the help of ImageJ software  utilitiTo release the phage genomic DNA, the purified and concentrated phage sample was incubated at +56 °C for 1 h with the addition of proteinase K and SDS . DNA extraction was performed with the Genomic DNA Clean & Concentrator-10  kit as per the manufacturer’s protocol. Quality and approximate concentration of the obtained phage genomic DNA were evaluated on a NanoDrop ND—1000 spectrophotometer  and then diluted accordingly to verify the specific dsDNA amount using a Qubit fluorometer  dsDNA high-sensitivity quantification assay (Invitrogen).The TruSeq DNA Nano Low Throughput Library Prep Kit  protocol, compatible with the TruSeq DNA Single Indices Set A (Illumina), was followed to prepare the Illumina MiSeq-compatible DNA libraries . To prepare the appropriate input for the library preparation step, 200 ng of the dsDNA from each of the studied phages was randomly fragmented with a target fragment length of 550 bp in mind using a Covaris S220 focused ultrasonicator .Resultant library quality control was performed using an Agilent 2100 bioanalyzer  with a high-sensitivity DNA kit (Agilent) and a Qubit fluorometer (Invitrogen) dsDNA high-sensitivity quantification assay (Invitrogen). Libraries were then sequenced on the Illumina MiSeq system (Illumina) using a 500-cycle MiSeq Reagent Kit v2 nano (Illumina) for three of the twelve uniquely indexed libraries comprising the run.Overnight culture of the bacterial host isolate LS5-2 was used for genomic DNA isolation as described for the phage genomic DNA extraction  recommendations , 5 min at +70 °C, followed by a hold at +4 °C). The ABI PRISM 3130xl system (Thermo Fisher Scientific) was used as a sequencer. Raw read chromatograms were manually inspected and trimmed in GeneStudio (v. 2.2.0.0.). After, the trimmed reads were joined into a contig and used for consensus sequence calling  in the same software.Sanger-based sequencing of the extracted amplified partial 16S rRNA sequence of the host was carried out in two reactions (27F primer for forward and 1492R primer for reverse read) according to the BigDyePectobacterium carotovorum strain NCPPB 312 (JQHJ01000001) representing an outgroup. Multiple sequence alignment (MSA) was performed using MAFFT  with further manual visual refinement in Inkscape .The resulting partial 16S rRNA gene sequence of the host was queried against the bacterial 16S rRNA gene sequences publicly available at EzBioCloud . All val. 7.453; ), and th. 7.453;  reconstr. 7.453; . The NJ . 7.453; . The resPrior to de novo assembly, demultiplexed read datasets were inspected with the help of FastQC , and coDe novo assembled “circularized” contigs were next used alongside the corresponding library untrimmed reads for possible genome termini identification using PhageTerm . Next, https://phagesdb.org/DNAMaster/ accessed on 12 April 2021) and were followed by putative ORF product functional annotations using publicly available biological sequence repositories and web services as described previously [P. agglomerans 16S rRNA tail . Longer possible coding sequence overlaps were also evaluated when annotating phages within this study. Additionally, TMHMM [The open reading frame (ORF) and tRNA gene predictions were performed with the corresponding tool implementations in DNA master  were usCUAG-5′;  when evay, TMHMM  and Phoby, TMHMM  web servBacillus phage BJ4 (AOZ61694) and Bacillus phage SBP8a (AOZ62321), as well as the TerL sequences of the studied Pantoea phages Nufs112 (UJH95886.1), Nifs112 (UJH95823.1), and Nafs113 (UJH95900.1)). Multiple TerL sequence alignment was performed using MAFFT .To further verify the packaging strategies/termini types the isolated phages employed, the terminase large subunit (TerL) amino acid sequence ML tree was reconstructed using an extended dataset of Merrill and colleagues . Extensi. 7.453; ), and ML. 7.453; ), select. 7.453;  inferenc. 7.453; ) replicaFirst, a BLASTN search against all the viral sequences (taxid:10239) publicly available at the non-redundant nucleotide collection was performed for complete-genome sequences of studied phages to evaluate their novelty and get a hint at the most appropriate further analyses for the task of elucidating the place of isolated phages within the so-far-uncovered phage diversity.Pantoea phages Nifs112 (OK570184.1), Nufs112 (OK570185.1), and Nafs113 (OK570186.1) were already publicly available in GenBank in late January 2022, we took advantage of the INPHARED [As complete annotated genomes of INPHARED  databaseINPHARED ) proteomINPHARED ). Nodes http://tree.bio.ed.ac.uk/software/figtree/ accessed on 10 May 2021) and Inkscape .The vConTACT2-identified first-neighbor genomes were next subjected to VIRIDIC  analysiIndividual protein phylogenies were reconstructed for the major capsid protein , terminase large subunit , endolysin , and phosphatase  of the studied phages .First, these sequences were subjected to a BLASTP search against the non-redundant protein sequences of viral origin (taxid:10239) under other default settings. For a given protein from the studied phages, the ten highest-scoring hits encoded by other cultured phage genomes were selected and downloaded regardless of the annotations, combining hits into multifastas comprising proteins of presumably analogous functions.Next, respective analogous protein amino-acid-sequence-containing datasets  were aligned using MAFFT  and subhttp://tree.bio.ed.ac.uk/software/figtree/ accessed on 10 May 2021). Inkscape  was then used to combine, as well as annotate, the trees based on the phage complete-genome-associated metadata .The resulting tree was midpoint rooted and visualized in FigTree  and Cliv.2.2.2; ) were sePantoea phage Nifs112 (OK570184) was compared with two representatives of the genus Eracentumvirus—Erwinia phages vB_EamP-S2 (NC_047917) and Era103 (NC_009014).That way, the complete genome of the Pantoea phage Nufs112 (OK570185) was compared with the genomes of the Klebsiella phage 6939  and the Ralstonia phage Reminis , both of which had to be rearranged taking the identified SDTR location in the genome of Nufs112 into account. Additionally, genus Gajwadongvirus representatives Escherichia phage ECBP5 (KJ749827) and Pectobacterium phage PP99 (NC_047802) were also used for Nufs112-related comparisons.The Pantoea phage Nafs113 was compared with the sole representative of the genus Lietduovirus, Pantoea phage vB_PagM_LIET2 (NC_048751).The https://inkscape.org accessed on 10 May 2021).Generated figures were further visually refined in Inkscape  was empPantoea agglomerans LS5-2. The isolated phages were very different from each other and, to our knowledge, were so far the first three Pantoea sp.-infecting phages isolated from an insect source.Three bacteriophages infecting the same bacterial strain were isolated from an unidentified Latvian grasshopper species alongside its host Pantoea podophage Nifs112 (OK570184) was proposed to represent a novel species within the phage family Autographiviridae, subfamily Molineuxvirinae, genus Eracentumvirus (comprising so far exclusively phages infecting Erwinia).The Pantoea podophage Nufs112 (OK570185) was evolutionary moderately related to two of the phages yet without a standing in the official phage taxonomy  and, to a lower extent, to representatives of the phage genus Gajwadongvirus. The sufficient amount of difference Nufs112 showed in comparison to either of the so-far-sequenced phages, however, allowed us to propose it as an exemplar isolate for the creation of a new phage species that would represent a novel phage genus.The Pantoea myophage Nafs113 (OK570186) had quite an unusual capsid with an approximately 3:1 length-to-width ratio. Nafs113 was very distantly related to any of the so-far completely sequenced phages that are publicly available, except for the Pantoea phage vB_PagM_LIET2, with whom Nafs113 shared a very close evolutionary relationship. For practicality reasons, at this time we propose to consider Nafs113 as an isolate of the phage species Lietduovirus LIET2, the so-far single phage species within the genus Lietduovirus, currently represented by the Pantoea phage vB_PagM_LIET2 alone.The Pantoea spp. but also of closely related bacteria .Additional microbiological characterization of these three lytic phages will next be undertaken to determine their possible host ranges, lifecycle characteristics, and virion stability, ultimately to evaluate their potential to be used for biocontrol of not only


Phages are efficient in diagnosing, treating, and preventing various diseases, and as sensing elements in biosensors. Phage display alone has gained attention over the past decade, especially in pharmaceuticals. Bacteriophages have also found importance in research aiming to fight viruses and in the consequent formulation of antiviral agents and vaccines. All these applications require control over the stability of virions. Phages are considered resistant to various harsh conditions. However, stability-determining parameters are usually the only additional factors in phage-related applications. Phages face instability and activity loss when preserved for extended periods. Sudden environmental changes, including exposure to UV light, temperature, pH, and salt concentration, also lead to a phage titer fall. This review describes various formulations that impart stability to phage stocks, mainly focusing on polymer-based stabilization, encapsulation, lyophilization, and nano-assisted solutions. Bacteriophages are viruses infecting bacteria. The name is derived from “bacteria” and the Greek φαγεῖν (phagein), meaning “to devour”. Bacteriophages are obligate parasites that annex the host’s molecular machinery to complete their life cycle. Hundreds of new virions are folded inside a single bacterial cell. In most cases, bacteria are disrupted, and phages are released (lytic cycle). Chronic phages, e.g., filamentous phages f1, fd, or M13, do not lyse their host cells, but progeny virions are secreted continuously ,3. In thStraboviridae , the Caudoviricetes class , or the family Autographiviridae . Tail fibers are attached to the tail and have a substantial positive charge. The head has a significant negative charge [Caudoviricetes class phages are filamentous  or nearly spherical (isometric) phages .The average size of a virion (single phage) is around 50 nm to 200 nm. However, the largest bacteriophages are more than 800 nm . In oceae charge . Such asCaudoviricetes, initial recognition is based on electrostatic interactions—positively charged tail fibers are attracted to the negatively charged surface of bacteria. The following steps of host recognition utilize specific receptor-binding proteins, which discriminate the appropriate host. This selectivity is the basis for the utilization of phages in sensing.Only recognizing a proper and viable host assures the multiplication of virions and the completion of the phage life cycle. Thus, very often, a multi-step “identification” process is used. In the case of The phage display method boosts the importance of bacteriophages. The possibility of directly studying the link between genotype and phenotype has allowed for numerous applications. Phage display is quite similar to the immunodetection assay but, instead of relying on antibodies, it relies on phages as recognition elements. Briefly, a library of genetically modified phages expressing different peptides on their capsids is prepared. Then, the phage library is exposed to the molecule (embedded on the surface) to determine specifically binding peptides .Confidence in phage therapy is re-emerging in the pharmaceutical world. The FDA approved the use of phages in critically ill COVID patients with secondary bacterial infections in 2020. Several phage-based products emerged shortly after, for example, GangaGen, ContraFect, Phagelux, and Phagomed, to fight S. aureus in India, the USA, China, and Austria, respectively . In addiApproximately 13% of deaths are related to bacterial diseases . BacteriFoodborne illnesses remain a significant cause of worldwide death despite many advances in pathogen surveillance and food sanitation methods. According to the WHO, 600 million foodborne infections occurred in 2010, resulting in over 400,000 deaths. Besides being a huge social burden, it is also a massive drain on the economy of nations. The average incident is estimated to cost around USD ~1500 per person . MoreoveBeing natural antibacterial agents, bacteriophages are considered an alternative to antibiotics, especially at the dawn of the antibiotic-resistance era. There are several advantages to using phages to fight bacteria.Phages can be produced easily and cheaply in large quantities and easily purified. By only infecting a bacteria solution, one can obtain a large number of progeny phages.E. coli phage PK1A2 in vitro [Bacteriophages are considered non-toxic to eukaryotes because structural elements of the virion cannot bind to eukaryotic cells . There ain vitro . The virThe lysis of bacteria resulting from phage infection supports the inflammatory response against bacteria . TherefoPhages undergo evolution, and thus, they remain effective against bacteria . DespitePhagoburn project) [Bacteriophages have been proposed for medical use since the early 1900s ,30. Due project) . The firproject) .de facto makes phages incapable of infecting bacterial cells [Phage administration without any stabilizing additives triggers an inflammatory response as a result of releasing pathogen-associated molecular patterns (PAMPs) from lysed bacteria  . Howeveral cells .Phages are also tested for biocontrol applications, e.g., in the food industry and agriculture. Phage biocontrol is increasingly accepted as green and natural technology for the specific targeting of pathogens . Phages Among the many bacteriophages, some examples are considered good surrogates for studies on eukaryotic, often dangerous, viruses. The most common examples are MS2 , Phi6 55,56, PhiAs such, methods developed for phage stabilization might be applicable to the stabilization of other viruses. This is still important, as bacteriophages can be used to formulate vaccines. Depending on the particular type of vaccine, phages can either carry the genomes of other viruses, affinity bind eukaryotic virus antigens on the capsid’s surface ,62, or pThe major drawback of using phages as antimicrobials is the stability of virions. The primary criteria are virulence, selectivity, host range, ease of manipulation, and modification. Even though phages are capable of retaining their activity after exposure to stress factors , about 5 log below the required rate of infection (ROI) [The matter of phage suspension stability is not to be underestimated. Improper preparation of such suspension can affect the effectiveness of the phage therapy approach. For instance, in the on (ROI) .With the shift in attention towards phage-based pharmaceutics, the precise nature and uniformity of bacteriophages are gaining increased appreciation. The preliminary testing of phage therapy did not concern the stability of phages; however, commercial circulation would require stable concentrations and known dosages. Phages often lose activity during formulation and storage. The most common factors affecting the viability of phages (and phage proteins) include exposure to organic solvents, high temperatures, pH, and ionic strength .Encyclopedia Britannica). Because of a large number of subunits, removing one of them does not change the properties of the entire molecule. Polymers can be divided into natural  and synthetic  [A polymer is a substance or material of large molecular mass composed of repeating subunits (opylene) . AlthougP. aeruginosa. Such hydrogels were effective in the elimination of bacteria embedded on the surfaces of gel beads. The authors, however, did not provide quantitative values of these antibacterial properties. [Listeria monocytogenes. While pullulan itself was not effective for phage stabilization after drying, the combination of pullulan and trehalose as a stabilizing matrix allowed the maintenance of about 7 log active phages after 60 days . In comparison, in the trehalose matrix, only 3 log phages remained active after the same amount of time [The effect of sugars as stabilizers against protein unfolding is known ,76,77,78perties. . Leung e of time .Polyacrylamide can be used to stabilize phage particles for strictly research purposes, such as protein nuclear magnetic resonance spectroscopy. Trempe et al. reported the stabilization of a filamentous Pf1 phage using 5% polyacrylamide as a polymer-stabilized liquid crystal (PSLC). This approach allowed for measurements of dipolar couplings with a single sample and, therefore, a more accurate analysis of protein structure based on the comparison of theoretical and experimental tensor parameters .PEGylation is the attachment of polyethylene glycol (PEG) groups to the target molecule, commonly used in food and drug formulation to increase molecules’ stability. PEG is biocompatible and reduces the immunogenicity of the molecule, but it is non-degradable . PEG is Polymers might also have an indirect effect on phages. Richter et al. explained the phenomenon of phage titer loss when phages are stored in plastic (mainly polypropylene) labware . The autPolymers are also used as protective matrices in which phages are embedded. Encapsulation allows for better stability and modulates the long-term release of active phage particles . VariousSalmonella was encapsulated within alginate microparticles associated with calcium carbonate to prolong their gut residence time [The applications of encapsulation also include phage cocktails. For example, a cocktail of three phages against nce time ,102. Pacnce time .Malik et al. listed the types of polymers used for phage encapsulation as agarose, alginate, chitosan, pectin, whey protein, gelled milk protein, hyaluronic acid methacrylate, hydroxypropyl methylcellulose poly(N-isopropyl acrylamide), poly(DL-lactide: glycolide), polyesteramide, polyvinyl pyrrolidone, polyethylene oxide/polyvinyl alcohol, cellulose diacetate, and polymethyl methacrylate . AlginatSalmonella [Salmonella phages have been produced to maintain elevated levels of phage particles in the gut [Salmonella phages, protecting them against the acidic conditions of the intestine [Liposomes are biocompatible nanoparticles composed of a lipid bilayer surrounding therapeutic cargo. This approach is promising in achieving directed delivery while surviving the extreme conditions of the stomach and intestine when administered orally . Liposomlmonella . Pharmac the gut . Nanoparntestine . This tentestine . Howeverntestine .While aiming for perfection, modifications in the approach to encapsulate phages are gaining popularity. For example, microfluidic devices are used to produce calcium alginate capsules containing bacteriophages . These cLyophilization (dehydration process), first used for food storage, has become a very commonly used method for phage stabilization and long-term storage. At first, lyophilization involved freezing the phage stock, lowering the pressure, and removing the water  . Now, thFor phage storage, freeze-drying and spray-drying methods are the most important. Freeze-drying is, relatively, the cheapest method for preparing phage powders. One of the first attempts was the freeze-drying of mycobacteriophages; when stored in the dark, phage lyophilizates were suitable for over two years . The aveStaphylococcus aureus ISP phage to prolong the storage time up to 37 months, with a titer loss of about 1 log [6000) in the M13 phage model. After seven days, the phage titer was 2 log higher in the sucrose and trehalose solution compared to PEG [The storage time can be extended by adding some cryoprotectants. Merabishvili et al. proposed adding 0.5 M sucrose and trehalose solutions to the ut 1 log . This obut 1 log . Additiod to PEG . A simild to PEG . Recentld to PEG . Other sd to PEG .Pseudomonas phages—PEV2 and PEV40. They showed that phage powder prepared in the mixture containing 70% trehalose and 30% leucine could be stored for 12 months with a titer decrease of about 0.5 log [Spray-drying allows for a reduction in the decrease in the phage titer during the procedure . Still,  0.5 log . Chang e 0.5 log . In addi 0.5 log . The sen 0.5 log . Moreove 0.5 log . Phage s 0.5 log ) contain 0.5 log .Pseudomonas aeruginosa [Acinetobacter baumannii [Burkholderia cenocepacia [Salmonella enterica [Campylobacter jejuni [Mycobacterium tuberculosis [Freeze-drying and spray-drying were combined into a novel atmospheric spray-freeze-drying (ASFD) ,135. Botruginosa ,143,144,aumannii , Burkholocepacia , Salmoneenterica , Campylor jejuni , or Mycorculosis . Howeverrculosis .Another long-term method of storing and stabilizing bacteriophages is freezing mature virions within bacterial cells. In this approach, bacteriophages at a proper multiplicity of infection (MOI) are mixed with their host bacterium and incubated for a short time. Next, infected cells are frozen and stored at −80 °C. After reviving and washing, phages are released and can actively infect bacterial cells. Golec et al. proved that this method allows for phage storage with minor or no losses in phage titer for about 10 months, depending on a particular phage .The implementation of nanotechnology in medicine has the potential to solve the stability issue. Nanoparticles can be viewed as vectors for drug solubilization while overcoming sequential biological barriers in the body . Phages E. coli cells [B. anthracis can be targeted using phage probes modified and stabilized with gold nanoparticles [Nanoscience plays an essential role in the immobilization of phages; bacteriophages can be chemically or genetically modified to bind strongly to nanomaterials . Gold nali cells . Additioarticles . For morarticles . The onearticles .In some experiments on vaccine formulations, nanolayers of aluminum oxide were used to stabilize the λ bacteriophage, which ensured the controlled release of antigens in vivo . VaccineApart from imparting stability, nanoparticles have been shown to enhance the functioning of phage-based biosensors by exploring rapid and sensitive approaches . Carbon-P. aeruginosa planktonic and biofilm states, with high stability under a broad range of temperatures, pHs, and salt concentrations [Salmonella enterica by causing the deformation of biofilms [The synergistic effect of phages and nanoparticles has been widely used for targeting biofilms and eliminating pathogenic infections ,167. Fortrations . In othetrations . Phage vtrations . Metallibiofilms . Alternabiofilms .Vaccination against infectious diseases saves over three million lives yearly. However, some estimations suggest this number could be doubled if all the problems related to proper storage were solved . AccordiDespite the advantages, research on phages is troublesome due to their instability in phage cocktails and varying phage titers . Table 12), but only for up to 5 min [Phage delivery is also often compromised due to its degradation and clearance by the body’s defense mechanisms . Many teto 5 min . Due to This review presents an update on the state of the art of bacteriophage stabilization, including the research published only last year (since May 2021). The review is mostly focused on polymer-based stabilization, encapsulation, lyophilization, and nano-assisted solutions; the problems and future perspectives of such approaches are also highlighted.