Partial characterization of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> bacteriophage F4

Large Enterococcus faecalis F4 bacteriophage (described earlier) consisting of double-stranded linear DNA of approximately 60 kb was characterized. Library was prepared of its random DNA fragments and selected recombinants were sequenced. Three phage essential genes were characterized: DNA polymerase, replicative DNA helicase and a minor capsid protein, showing only limited homology to other known phage encoded genes. The occurrence of these genes among enterococci was determined by PCR method. Only two out of 40 tested isolates possessed all three genes, another three isolates contained at least one of the genes, demonstrating low frequency F4 lysogens among natural enterococcal isolates.     

Transfer of the chromosomally encoded tetracycline resistance gene <Emphasis Type="Italic">tet</Emphasis>(M) from marine bacteria to <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>

The transferability of the tetracycline (TC) resistance gene tet(M) from marine bacteria to human enteric bacteria was examined by a filter-mating method. Vibrio spp., Lactococcus garvieae, Bacillus spp., Lactobacillus sp., and Paenibacillus sp. were used as donors, and Escherichia coli JM109 and Enterococcus faecalis JH2-2 were used as recipients. The combination of Vibrio spp. and E. coli resulted in 5/68 positive transconjugants with a transfer rate of 10⁻⁷ to 10⁻³; however, no transfer was observed with E. faecalis. In case of L. garvieae and E. faecalis, 6/6 positive transconjugants were obtained with a transfer rate of 10⁻⁶ to 10⁻⁵; however, no transfer was observed with E. coli. The tet(M) gene of Bacillus, Lactobacillus, and Paenibacillus were not transferred to either E. coli or E. faecalis. tet(M) transfer was confirmed in positive E. coli and E. faecalis transconjugants by polymerase chain reaction (PCR) and Southern hybridization. All the donor strains did not harbor plasmids, while they all harbored transposon Tn916. In the transconjugants, the transposon was not detected by PCR, suggesting the possible transfer of tet(M) from the marine bacterial chromosome to the recipient chromosome. This is the first report to show that tet(M) can be transferred from marine bacteria to human enteric bacteria in a species-specific manner.     

Identification of <Emphasis Type="Italic">O</Emphasis>-GlcNAcylated proteins in <Emphasis Type="Italic">Plasmodium falciparum</Emphasis>

Background

Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been discussed as potential anti-malarial drug targets due to their involvement in many cell processes. O-GlcNAcylation is a widespread PTM found in different organisms including Plasmodium falciparum. The aim of this study was to identify O-GlcNAcylated proteins of P. falciparum, to learn more about the modification process and to understand its eventual functions in the Apicomplexans.

Methods

The P. falciparum strain 3D7 was amplified in erythrocytes and purified. The proteome was checked for O-GlcNAcylation using different methods. The level of UDP-GlcNAc, the donor of the sugar moiety for O-GlcNAcylation processes, was measured using high-pH anion exchange chromatography. O-GlcNAcylated proteins were enriched and purified utilizing either click chemistry labelling or adsorption on succinyl-wheat germ agglutinin beads. Proteins were then identified by mass-spectrometry (nano-LC MS/MS).

Results

While low when compared to MRC5 control cells, P. falciparum disposes of its own pool of UDP-GlcNAc. By using proteomics methods, 13 O-GlcNAcylated proteins were unambiguously identified (11 by click-chemistry and 6 by sWGA-beads enrichment; 4 being identified by the 2 approaches) in late trophozoites. These proteins are all part of pathways, functions and structures important for the parasite survival. By probing clicked-proteins with specific antibodies, Hsp70 and α-tubulin were identified as P. falciparum O-GlcNAc-bearing proteins.

Conclusions

This study is the first report on the identity of P. falciparum O-GlcNAcylated proteins. While the parasite O-GlcNAcome seems close to those of other species, the structural differences exhibited by the proteomes provides a glimpse of innovative therapeutic paths to fight malaria. Blocking biosynthesis of UDP-GlcNAc in the parasites is another promising option to reduce Plasmodium life cycle.

     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Horizontal Transfer of the Tetracycline Resistance Gene <Emphasis Type="Italic">tetM</Emphasis> Mediated by pCF10 Among <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> in the House Fly (<Emphasis Type="Italic">Musca domestica</Emphasis> L.) Alimentary Canal

The house fly (Musca domestica L.) alimentary canal was evaluated for the potential of horizontal transfer of tetM on plasmid pCF10 among Enterococcus faecalis. Two sets of experiments were conducted: (1) house flies without surface sterilization and (2) surface-sterilized flies. Both sets of flies were exposed to E. faecalis OG1RF:pCF10 as donor for 12 h and then E. faecalis OG1SSp as recipient for 1 h. Another group of flies received the recipient first for 12 h followed by exposure to the donor strain for 1 h. House flies were screened daily to determine the donor, recipient, and transconjugant bacterial load for up to 5 days. In addition, the sponge-like mouth parts used for food uptake (labellum) of surface-sterilized house flies were removed and analyzed for donors, recipients, and transconjugants, separately. In both groups of flies (n = 90 flies/group), transfer occurred within 24 h after exposure with a transconjugant/donor rate from 8.6 × 10⁻⁵ to 4.5 × 10¹. Transconjugants were also isolated from the house fly labellum. Our data suggest that the house fly digestive tract provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among enterococci. Our results emphasize the importance of this insect as a potential vector of antibiotic-resistant bacterial strains.     

<Emphasis Type="Italic">Tolypothrix tenuis</Emphasis> stress response to nickel

Summary Metal ions are both essential and potentially toxic. The aim of this work was to demonstrate that diazotrophic cyanobacterium Tolypothrix tenuis N° 54 can tolerate toxic concentrations of Ni²⁺ in order to use the biomass in biofilters or as biofertilizer. For this purpose, growth, pigment and protein contents and catalase activity of T. tenuis growing in increasing concentrations of Ni²⁺ ranging from 10⁻¹⁰ to 10⁻⁴ M were assesed. The strain did not grow at Ni²⁺ concentration of 10⁻⁴ M, but at lower concentrations there were no significant differences with the control; it was tolerant at 10⁻¹⁰ and 10⁻⁸ M. Nickel concentration of 10⁻⁶ M is toxic for this cyanobacterial strain, because dry weight decreased by 30%; allophycocyanin and phycoerythrin decreased by 92% and 98%, respectively and protein content increased by 42%. Chlorophyll a concentration was more than double the control value in 10⁻¹⁰ and 10⁻⁸ M, but in 10⁻⁶ M it decreased by 19%. Catalase (E.C. 1.11.1.6) activity doubled the control value in the lowest nickel concentration whereas in 10⁻⁸ M there was no significant difference with the control and in 10⁻⁶, it decreased by 78%. The living biomass of this strain could be used as a step in the bioremediation process in waters contaminated with concentrations of nickel lower than 10⁻⁶ M and eventually as a biofertilizer.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Cytolysin gene expression in<Emphasis Type="Italic"> Enterococcus faecalis</Emphasis> is regulated in response to aerobiosis conditions

Here we investigate the expression of cylL(L)and cylL(S), the genes that encode the structural subunits of the cytolysin/haemolysin of Enterococcus faecalis, in response to aerobiosis conditions. Haemolysis assays of E. faecalis strains cultured under aerobic and anaerobic conditions revealed three different haemolytic phenotypes, one of which exhibited greater haemolysis under anaerobic conditions than under aerobic conditions, and was shown to be associated with the presence of the cyl genes. Reporter gene studies revealed that cylL(L) L(S) promoter activity was significantly greater (up to 8.6-fold) under anaerobic compared to aerobic conditions throughout batch growth, demonstrating that these genes are regulated in response to the degree of aerobiosis. Band shift assays confirmed the binding of a protein factor to the region between 202 and 37 bp upstream of the cylL(L)start codon, and a higher level of binding was observed with anaerobically derived cell-free extracts than with extracts of aerobically grown cells. This is the first report of an oxygen-regulated virulence factor in E. faecalis (that is distinct from the quorum-sensing regulatory system reported previously), and may be of in vivo relevance for the bacterium in biofilms and other environments characterised by oxygen gradients.     

A novel family of mitochondrial proteins is represented by the <Emphasis Type="Italic">Drosophila</Emphasis> genes <Emphasis Type="Italic">slmo</Emphasis>, <Emphasis Type="Italic">preli-like</Emphasis> and <Emphasis Type="Italic">real-time</Emphasis>

Mitochondria play essential roles in development and disease. The characterisation of mitochondrial proteins is therefore of particular importance. The slowmo (slmo) gene of Drosophila melanogaster has been shown to encode a novel type of mitochondrial protein, and is essential in the developing central nervous system. The Slmo protein contains a conserved PRELI/MSF1p domain, found in proteins from a wide variety of eukaryotic organisms. However, the function of the proteins of this family is currently unknown. In this study, the evolutionary relationships between members of the PRELI/MSF1p family are described, and we present the first analysis of two novel Drosophila genes predicted to encode proteins of this type. The first of these, preli-like (prel), is expressed ubiquitously during embryonic development, whilst the second, real-time (retm), is expressed dynamically in the developing gut and central nervous system. retm encodes a member of a novel conserved subclass of larger PRELI/MSF1p domain proteins, which also contain the CRAL-TRIO motif thought to mediate the transport of small hydrophobic ligands. Here we provide evidence that, like Slmo, both the Prel and Retm proteins are localised to the mitochondria, indicating that the function of the PRELI/MSF1p domain is specific to this organelle.Edited by P. Simpson     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

<Emphasis Type="Italic">Wolinella succinogenes</Emphasis> response to ox-bile stress

The bacterium Wolinella succinogenes is the only known species of its genus. It was first isolated from cow ruminal fluid, and in cattle, it dwells in the reticulum and rumen compartments of the stomach. The global protein response of W. succinogenes to ox-bile was investigated with the aim to understand bile-tolerance mechanisms of the bacterium. Bacteria were grown in liquid media supplemented with different bile concentrations to determine its effects on growth and morphology. Proteomic analyses served to identify 14 proteins whose expression was modulated by the presence of 0.2% bile. Quantitative real-time PCR analyses of the expression of selected genes were employed to obtain independent confirmation of the proteomics data. Proteins differentially expressed revealed metabolic pathways involved in the adaptation of W. succinogenes to bile. The data suggested that bile stress elicited complex physiological responses rather than just specific pathways, and identified proteins previously unknown to be involved in the adaptation of bacteria to bile.