Interaction of bacteriophage P1 with an epiphytic Pantoea agglomerans strain-the role of the interplay between various mobilome elements.

P1 is a model, temperate bacteriophage of the 94 kb genome. It can lysogenize representatives of the Enterobacterales order. In lysogens, it is maintained as a plasmid. We tested P1 interactions with the biocontrol P. agglomerans L15 strain to explore the utility of P1 in P. agglomerans genome engineering. A P1 derivative carrying the Tn9 (cmR) transposon could transfer a plasmid from Escherichia coli to the L15 cells. The L15 cells infected with this derivative formed chloramphenicol-resistant colonies. They could grow in a liquid medium with chloramphenicol after adaptation and did not contain prophage P1 but the chromosomally inserted cmR marker of P1 Tn9 (cat). The insertions were accompanied by various rearrangements upstream of the Tn9 cat gene promoter and the loss of IS1 (IS1L) from the corresponding region. Sequence analysis of the L15 strain genome revealed a chromosome and three plasmids of 0.58, 0.18, and 0.07 Mb. The largest and the smallest plasmid appeared to encode partition and replication incompatibility determinants similar to those of prophage P1, respectively. In the L15 derivatives cured of the largest plasmid, P1 with Tn9 could not replace the smallest plasmid even if selected. However, it could replace the smallest and the largest plasmid of L15 if its Tn9 IS1L sequence driving the Tn9 mobility was inactivated or if it was enriched with an immobile kanamycin resistance marker. Moreover, it could develop lytically in the L15 derivatives cured of both these plasmids. Clearly, under conditions of selection for P1, the mobility of the P1 selective marker determines whether or not the incoming P1 can outcompete the incompatible L15 resident plasmids. Our results demonstrate that P. agglomerans can serve as a host for bacteriophage P1 and can be engineered with the help of this phage. They also provide an example of how antibiotics can modify the outcome of horizontal gene transfer in natural environments. Numerous plasmids of Pantoea strains appear to contain determinants of replication or partition incompatibility with P1. Therefore, P1 with an immobile selective marker may be a tool of choice in curing these strains from the respective plasmids to facilitate their functional analysis.

Symbiotic efficiency of Rhizobium leguminosarum sv. trifolii strains originating from the subpolar and temperate climate regions.

Red clover (Trifolium pratense L.) is a forage legume cultivated worldwide. This plant is capable of establishing a nitrogen-fixing symbiosis with Rhizobium leguminosarum symbiovar trifolii strains. To date, no comparative analysis of the symbiotic properties and heterogeneity of T. pratense microsymbionts derived from two distinct geographic regions has been performed. In this study, the symbiotic properties of strains originating from the subpolar and temperate climate zones in a wide range of temperatures (10-25 °C) have been characterized. Our results indicate that all the studied T. pratense microsymbionts from two geographic regions were highly efficient in host plant nodulation and nitrogen fixation in a wide range of temperatures. However, some differences between the populations and between the strains within the individual population examined were observed. Based on the nodC and nifH sequences, the symbiotic diversity of the strains was estimated. In general, 13 alleles for nodC and for nifH were identified. Moreover, 21 and 61 polymorphic sites in the nodC and nifH sequences were found, respectively, indicating that the latter gene shows higher heterogeneity than the former one. Among the nodC and nifH alleles, three genotypes (I-III) were the most frequent, whereas the other alleles (IV-XIII) proved to be unique for the individual strains. Based on the nodC and nifH allele types, 20 nodC-nifH genotypes were identified. Among them, the most frequent were three genotypes marked as A (6 strains), B (5 strains), and C (3 strains). Type A was exclusively found in the temperate strains, whereas types B and C were identified in the subpolar strains. The remaining 17 genotypes were found in single strains. In conclusion, our data indicate that R. leguminosarum sv. trifolii strains derived from two climatic zones show a high diversity with respect to the symbiotic efficiency and heterogeneity. However, some of the R. leguminosarum sv. trifolii strains exhibit very good symbiotic potential in the wide range of the temperatures tested; hence, they may be used in the future for improvement of legume crop production.

Efficient traceless modification of the P1 bacteriophage genome through homologous recombination with enrichment in double recombinants: A new perspective on the functional annotation of uncharacterized phage genes.

The advent of high-throughput omic technologies has caused unprecedented progress in research on bacteriophages, the most abundant and still the least explored entities on earth. Despite the growing number of phage genomes sequenced and the rejuvenation of interest in phage therapy, the progress in the functional analysis of phage genes is slow. Simple and efficient techniques of phage genome targeted mutagenesis that would allow one to knock out particular genes precisely without polar effects in order to study the effect of these knock-outs on phage functions are lacking. Even in the case of model phages, the functions of approximately half of their genes are unknown. P1 is an enterobacterial temperate myophage of clinical significance, which lysogenizes cells as a plasmid. It has a long history of studies, serves as a model in basic research, is a gene transfer vector, and is a source of genetic tools. Its gene products have structural homologs in several other phages. In this perspective article, we describe a simple and efficient procedure of traceless P1 genome modification that could also serve to acquire targeted mutations in the genomes of certain other temperate phages and speed up functional annotations of phage genes.

Effect of amide protoporphyrin derivatives on immune response in Apis mellifera.

The intracellular microsporidian parasite Nosema ceranae is known to compromise bee health by induction of energetic stress and downregulation of the immune system. Porphyrins are candidate therapeutic agents for controlling Nosema infection without adverse effects on honeybees. In the present work, the impact of two protoporphyrin IX derivatives, i.e. PP[Asp]2 and PP[Lys]2, on Apis mellifera humoral immune response has been investigated in laboratory conditions in non-infected and N. ceranae-infected honeybees. Fluorescence spectroscopy analysis of hemolymph showed for the first time that porphyrin molecules penetrate into the hemocoel of honeybees. Phenoloxidase (PO) activity and the expression of genes encoding antimicrobial peptides (AMPs: abaecin, defensin, and hymenoptaecin) were assessed. Porphyrins significantly increased the phenoloxidase activity in healthy honeybees but did not increase the expression of AMP genes. Compared with the control bees, the hemolymph of non-infected bees treated with porphyrins had an 11.3- and 6.1-fold higher level of PO activity after the 24- and 48-h porphyrin administration, respectively. Notably, there was a significant inverse correlation between the PO activity and the AMP gene expression level (r = - 0.61696, p = 0.0143). The PO activity profile in the infected bees was completely opposite to that in the healthy bees (r = - 0.5118, p = 0.000), which was related to the changing load of N. ceranae spores in the porphyrin treated-bees. On day 12 post-infection, the spore loads in the infected porphyrin-fed individuals significantly decreased by 74%, compared with the control bees. Our findings show involvement of the honeybee immune system in the porphyrin-based control of Nosema infection. This allows the infected bees to improve their lifespan considerably by choosing an optimal PO activity/AMP expression variant to cope with the varying level of N. ceranae infection.

Impact of Protoporphyrin Lysine Derivatives on the Ability of Nosema ceranae Spores to Infect Honeybees.

The effect of two protoporphyrin IX derivatives conjugated with single (PP[Lys(TFA)-OH)]2) or double (PP[Lys(TFA)-Lys(TFA)-OH]2) lysine moieties on the infectious capacity of Nosema ceranae spores was examined, and their efficacies were compared with those of a cationic porphyrin (H2TTMePP). Honeybees were inoculated with spores preincubated with porphyrins or with untreated spores (control). A significantly lower level of infection was observed in the bees infected with the porphyrin-treated spores than in the infected control. Porphyrins 1 and 2 reduced the infectious capability of microsporidia more efficiently than porphyrin 3, with bee mortality declining to almost 50%. Confocal analysis of the midguts of infected bees revealed distinct differences in the number of spores between the control group and the group infected with PP[Lys(TFA)-Lys(TFA)-OH]2-treated spores. Notably, bees with a reduced level of infection consumed less sucrose syrup than the control bees, indicating a reduction in digestive disorders and an improvement in food absorption.

Bioactivity studies of porphyrinoids against microsporidia isolated from honeybees.

Microsporidian infections are dangerous to honeybees due to the absence of an efficient treatment for nosemosis. In the present work, the abilities of several porphyrins to directly inactivate microsporidia derived from Nosema-infected honeybees were studied in vitro. Amide derivatives of protoporphyrin IX (PPIX) conjugated with one and two amino acid moieties were synthesized, and their activities were compared with those of two cationic porphyrins, TMePyP and TTMePP. The most active porphyrins, PP[Lys-Asp]2, PP[Lys-TFA]2, PP[Asp(ONa)2]2 and PP[Lys-Lys]2 at concentrations as low as 10-50 µM exerted significant effects on microsporidia, reducing the number of spores by 67-80% compared to the control. Live-cell imaging of the spores treated with porphyrins showed that only 1.6% and 3.0% of spores remained alive after 24 h-incubation with 50 µM PP[Asp(ONa)2]2 and PP[Lys-Asp]2, respectively. The length of the amino acid side chains and their identity in the PPIX molecules affected the bioactivity of the porphyrin. Importantly, the irradiation of the porphyrins did not enhance their potency in destroying Nosema spores. We showed that the porphyrins accumulated inside the living spores but not inside dead spores, thus the destruction of the microsporidia by non-metallated porphyrins is not dependent on photosensitization, but is associated with their active transport into the spore cell. When administered to honeybees in vivo, PPIX[Lys-TFA]2 and PPIX[Lys-Lys]2 reduced spore loads by 69-76% in infected individuals. They both had no toxic effect on honeybees, in contrast to zinc-coordinated porphyrin.

Porphyrins inactivate Nosema spp. microsporidia.

The study of organic/inorganic molecules with activity against intracellular fungi of the phylum Microsporidia is of critical importance. Here, for the first time, the inactivation of these parasitic fungi by porphyrins is reported. The biological effects of porphyrins (10 µM and 100 µM) on the microsporidian Nosema ceranae was investigated in honeybee hosts using cage experiments. A significant reduction in the number of spores (from 2.6 to 5 fold) was observed in Nosema-infected honeybees with a sucrose-protoporphyrin amide [PP(Asp)2] syrup diet compared to the control honeybees. PP(Asp)2 and the other porphyrin examined in vitro, TMePyP, had a direct impact on the microsporidia. Notably, neither porphyrin requires light excitation to be active against microsporidia. Moreover, microsporidia preincubated with these porphyrins exhibited decreased ability to infect honeybees. In particular, PP(Asp)2, possessing amphiphilic characteristics, exhibited significant inactivation of microsporidia, preventing the development of the microsporidia and diminishing the mortality of infected honeybees. In addition, the porphyrin-treated spores examined by scanning electron microscopy (SEM) showed morphological changes in their exosporium layers, which were distinctly deformed. Thus, we postulate that the mechanism of action of porphyrins on microsporidia is not based on photodynamic inactivation but on the destruction of the cell walls of the spores.

[Gastroenterological manifestations of von Hippel-Lindau disease]. / Objawy zespolu von Hippel-Lindau zwiazane z przewodem pokarmowym.

Von Hippel-Lindau disease is rare autosomal dominant disorder that results from mutation of VHL gene. Typical manifestations of this syndrome include haemangioblastomas of retina, cerebellum and spinal cord, endolymphatic sac tumors, clear cell cancer and kidney cysts, pheochromocytoma, pancreatic cysts and neuroendocrine tumors. The differential diagnosis of pancreatic lesions in patients with von Hippel Lindau syndrome plays an important role. The pancreas in VHL disease is not only site of benign lesions (cysts, serous systic adenomas) but also of potentially malignant (neuroendocrine) and malignant tumors(metastases).The gastroenterological manifestations can be the first symptoms of von Hippel-Lindau disease.

[Gastroenterological manifestations of von Hippel-Lindau disease - a case report]. / Objawy zespolu von Hippel-Lindau zwiazane z przewodem pokarmowym ­ opis przypadku.

Gastrointestinal organs are involved in the course of von Hippel Lindau disease. Typically pancreas in von Hippel Lindau syndrome is a site of cystic and solid tumors. Differential diagnosis of pancreatic lesions includes benign lesions (cysts, serous cystic adenomas), potentially malignant (neuroendocrine) and malignant tumors(metastases).In this work we present a patient with VHL syndrome with pancreatic cysts and neuroendocrine tumor.