Functional Dissection of P1 Bacteriophage Holin-like Proteins Reveals the Biological Sense of P1 Lytic System Complexity

P1 is a model temperate myovirus. It infects different Enterobacteriaceae and can develop lytically or form lysogens. Only some P1 adaptation strategies to propagate in different hosts are known. An atypical feature of P1 is the number and organization of cell lysis-associated genes. In addition to SAR-endolysin Lyz, holin LydA, and antiholin LydB, P1 encodes other predicted holins, LydC and LydD. LydD is encoded by the same operon as Lyz, LydA and LydB are encoded by an unlinked operon, and LydC is encoded by an operon preceding the lydA gene. By analyzing the phenotypes of P1 mutants in known or predicted holin genes, we show that all the products of these genes cooperate with the P1 SAR-endolysin in cell lysis and that LydD is a pinholin. The contributions of holins/pinholins to cell lysis by P1 appear to vary depending on the host of P1 and the bacterial growth conditions. The pattern of morphological transitions characteristic of SAR-endolysin–pinholin action dominates during lysis by wild-type P1, but in the case of lydC lydD mutant it changes to that characteristic of classical endolysin-pinholin action. We postulate that the complex lytic system facilitates P1 adaptation to various hosts and their growth conditions.     

Unraveling Protein Interactions between the Temperate Virus Bam35 and Its Bacillus Host Using an Integrative Yeast Two Hybrid–High Throughput Sequencing Approach

Bacillus virus Bam35 is the model Betatectivirus and member of the family Tectiviridae, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. Interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, which are known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein–protein interactions (PPIs) network for a tectivirus–host system by studying the Bam35–Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and high-throughput sequencing (Y2H-HTS). We generated and thoroughly analyzed a genomic library of Bam35′s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait–prey couples. Initial analysis of the raw data enabled the identification of over 4000 candidate interactions, which were sequentially filtered to produce 182 high-confidence interactions that were defined as part of the core virus–host interactome. Overall, host metabolism proteins and peptidases were particularly enriched within the detected interactions, distinguishing this host–phage system from the other reported host–phage PPIs. Our approach also suggested biological roles for several Bam35 proteins of unknown function, including the membrane structural protein P25, which may be a viral hub with a role in host membrane modification during viral particle morphogenesis. This work resulted in a better understanding of the Bam35–B. thuringiensis interaction at the molecular level and holds great potential for the generalization of the Y2H-HTS approach for other virus–host models.     

Genome Study of a Novel Virulent Phage vB_SspS_KASIA and Mu-like Prophages of Shewanella sp. M16 Provides Insights into the Genetic Diversity of the Shewanella Virome

Shewanella is a ubiquitous bacterial genus of aquatic ecosystems, and its bacteriophages are also isolated from aquatic environments (oceans, lakes, ice, and wastewater). In this study, the isolation and characterization of a novel virulent Shewanella phage vB_SspS_KASIA and the identification of three prophages of its host, Shewanella sp. M16, including a mitomycin-inducible Mu-like siphovirus, vB_SspS_MuM16-1, became the starting point for comparative analyses of phages infecting Shewanella spp. and the determination of their position among the known bacterial viruses. A similarity networking analysis revealed the high diversity of Shewanella phages in general, with vB_SspS_KASIA clustering exclusively with Colwellia phage 9A, with which it forms a single viral cluster composed of two separate viral subclusters. Furthermore, vB_SspS_MuM16-1 presented itself as being significantly different from the phages deposited in public databases, expanding the diversity of the known Mu-like phages and giving potential molecular markers for the identification of Mu-like prophages in bacterial genomes. Moreover, the functional analysis performed for vB_SspS_KASIA suggested that, despite the KASIA host, the M16 strain grows better in a rich medium and at 30 °C the phage replication cycle seems to be optimal in restrictive culture conditions mimicking their natural environment, the Zloty Stok gold and arsenic mine.     

Pantoea Bacteriophage vB_PagS_MED16—A Siphovirus Containing a 2′-Deoxy-7-amido-7-deazaguanosine-Modified DNA

A novel siphovirus, vB_PagS_MED16 (MED16) was isolated in Lithuania using Pantoea agglomerans strain BSL for the phage propagation. The double-stranded DNA genome of MED16 (46,103 bp) contains 73 predicted open reading frames (ORFs) encoding proteins, but no tRNA. Our comparative sequence analysis revealed that 26 of these ORFs code for unique proteins that have no reliable identity when compared to database entries. Based on phylogenetic analysis, MED16 represents a new genus with siphovirus morphology. In total, 35 MED16 ORFs were given a putative functional annotation, including those coding for the proteins responsible for virion morphogenesis, phage–host interactions, and DNA metabolism. In addition, a gene encoding a preQ₀ DNA deoxyribosyltransferase (DpdA) is present in the genome of MED16 and the LC–MS/MS analysis indicates 2′-deoxy-7-amido-7-deazaguanosine (dADG)-modified phage DNA, which, to our knowledge, has never been experimentally validated in genomes of Pantoea phages. Thus, the data presented in this study provide new information on Pantoea-infecting viruses and offer novel insights into the diversity of DNA modifications in bacteriophages.     

APTC-C-SA01: A Novel Bacteriophage Cocktail Targeting Staphylococcus aureus and MRSA Biofilms

The high infection and mortality rate of methicillin-resistant Staphylococcus aureus (MRSA) necessitates the urgent development of new treatment strategies. Bacteriophages (phages) have several advantages compared to antibiotics for the treatment of multi-drug-resistant bacterial infections, and thus provide a promising alternative to antibiotics. Here, S. aureus phages were isolated from patients and environmental sources. Phages were characterized for stability, morphology and genomic sequence and their bactericidal activity against the biofilm form of methicillin-susceptible Staphylococcus aureus (MSSA) and MRSA was investigated. Four S. aureus phages were isolated and tested against 51 MSSA and MRSA clinical isolates and reference strains. The phages had a broad host range of 82–94% individually and of >98% when combined and could significantly reduce the viability of S. aureus biofilms. The phages had a latent period of ≤20 min and burst size of >11 plaque forming units (PFU)/infected cell. Transmission electron microscopy (TEM) identified phages belonging to the family of Myoviridae. Genomic sequencing indicated the lytic nature of all four phages, with no identified resistance or virulence genes. The 4 phages showed a high complementarity with 49/51 strains (96%) sensitive to at least 2/4 phages tested. Furthermore, the frequency of bacteriophage insensitive mutant (BIM) generation was lower when the phages were combined into the phage cocktail APTC-C-SA01 than for bacteria exposed to each of the phages alone. In conclusion, APTC-C-SA01, containing four lytic S. aureus phages has the potential for further development as a treatment against MSSA and MRSA infections.     

CRISPR/Cas9–Mediated Genome Editing for Pseudomonas fulva,a Novel Pseudomonas Species with Clinical,Animal, and Plant–Associated Isolates

As one of the most widespread groups of Gram–negative bacteria, Pseudomonas bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. Pseudomonas fulva is a novel species of Pseudomonas with clinical, animal, and plant–associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time–consuming and laborious endeavor. Here, by using the CRISPR–Cas system, a versatile gene–editing tool with a two–plasmid strategy was developed for a native P. fulva strain isolated from the model organism silkworm (Bombyx mori) gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic–related gene, oqxB, was knocked out, resulting in the slow growth of the P. fulva deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked–in gfp exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel Pseudomonas species.     

Identification of Genes Involved in Fe–S Cluster Biosynthesis of Nitrogenase in Paenibacillus polymyxa WLY78

NifS and NifU (encoded by nifS and nifU) are generally dedicated to biogenesis of the nitrogenase Fe–S cluster in diazotrophs. However, nifS and nifU are not found in N₂-fixing Paenibacillus strains, and the mechanisms involved in Fe–S cluster biosynthesis of nitrogenase is not clear. Here, we found that the genome of Paenibacillus polymyxa WLY78 contains the complete sufCDSUB operon, a partial sufC2D2B2 operon, a nifS-like gene, two nifU-like genes (nfuA-like and yutI), and two iscS genes. Deletion and complementation studies showed that the sufC, sufD, and sufB genes of the sufCDSUB operon, and nifS-like and yutI genes were involved in the Fe–S cluster biosynthesis of nitrogenase. Heterologous complementation studies demonstrated that the nifS-like gene of P. polymyxa WLY78 is interchangeable with Klebsiella oxytoca nifS, but P. polymyxa WLY78 SufCDB cannot be functionally replaced by K. oxytoca NifU. In addition, K. oxytoca nifU and Escherichia coli nfuA are able to complement the P. polymyxa WLY78 yutI mutant. Our findings thus indicate that the NifS-like and SufCDB proteins are the specific sulfur donor and the molecular scaffold, respectively, for the Fe–S cluster formation of nitrogenase in P. polymyxa WLY78. YutI can be an Fe–S cluster carrier involved in nitrogenase maturation in P. polymyxa WLY78.     

Characterization of Phages YuuY,KaiHaiDragon, and OneinaGillian Isolated from Microbacterium foliorum

Microbacterium foliorum is a Gram-positive bacteria found in organic matter. Three lytic bacteriophages, KaiHaiDragon, OneinaGillian, and YuuY, were isolated from M. foliorum strain NRRL B-24224. Phage YuuY in particular expresses a broad host range as it possesses the ability to infect closely related bacterial species Microbacterium aerolatum at a high plating efficiency. Characterization tests were performed on all three Microbacterium phage to assess morphology, genomic characteristics, pH and thermal stabilities, life cycle, and the type of receptor used for infection. All three phages showed similar pH stability, ranging from pH 5–11, except for KaiHaiDragon, which had a reduced infection effectiveness at a pH of 11. YuuY possessed a significantly higher temperature tolerance compared to the other Microbacterium phages as some phage particles remained viable after incubation temperatures of up to 80 °C. Based on the one-step growth curve assay, all three Microbacterium phages possessed a relatively short latent period of 90 min and an approximately two-fold burst size factor. Moreover, all three phages utilize a carbohydrate receptor to initiate infection. Based on bioinformatics analysis, YuuY, KaiHaiDragon and OneinaGillian were assigned to clusters EA10, EC, and EG, respectively.     

Contribution of Invariant Natural Killer T Cells to the Clearance of Pseudomonas aeruginosa from Skin Wounds

Chronic infections are considered one of the most severe problems in skin wounds, and bacteria are present in over 90% of chronic wounds. Pseudomonas aeruginosa is frequently isolated from chronic wounds and is thought to be a cause of delayed wound healing. Invariant natural killer T (iNKT) cells, unique lymphocytes with a potent regulatory ability in various inflammatory responses, accelerate the wound healing process. In the present study, we investigated the contribution of iNKT cells in the host defense against P. aeruginosa inoculation at the wound sites. We analyzed the re-epithelialization, bacterial load, accumulation of leukocytes, and production of cytokines and antimicrobial peptides. In iNKT cell–deficient (Jα18KO) mice, re-epithelialization was significantly decreased, and the number of live colonies was significantly increased, when compared with those in wild-type (WT) mice on day 7. IL-17A, and IL-22 production was significantly lower in Jα18KO mice than in WT mice on day 5. Furthermore, the administration of α-galactosylceramide (α-GalCer), a specific activator of iNKT cells, led to enhanced host protection, as shown by reduced bacterial load, and to increased production of IL-22, IL-23, and S100A9 compared that of with WT mice. These results suggest that iNKT cells promote P. aeruginosa clearance during skin wound healing.