The impact of the neisserial DNA uptake sequences on genome evolution and stability

Background  

Efficient natural transformation in Neisseria requires the presence of short DNA uptake sequences (DUSs). Doubts remain whether DUSs propagate by pure selfish molecular drive or are selected for 'safe sex' among conspecifics.     

An Arabidopsis GRIP domain protein locates to the trans-Golgi and binds the small GTPase ARL1

GRIP domain proteins are a class of golgins that have been described in yeast and animals. They locate to the trans-Golgi network and are thought to play a role in endosome-to-Golgi trafficking. The Arabidopsis GRIP domain protein, AtGRIP, fused to the green fluorescent protein (GFP), locates to Golgi stacks but does not exactly co-locate with the Golgi marker sialyl transferase (ST)-mRFP, nor with the t-SNAREs Memb11, SYP31 and BS14a. We conclude that the location of AtGRIP is further to the trans side of the stack than STtmd-mRFP. The 185-aa C-terminus of AtGRIP containing the GRIP domain targeted GFP to the Golgi, although a proportion of the fusion protein was still found in the cytosol. Mutation of a conserved tyrosine (Y717) to alanine in the GRIP domain disrupted Golgi localization. ARL1 is a small GTPase required for Golgi targeting of GRIP domain proteins in other systems. An Arabidopsis ARL1 homologue was isolated and shown to target to Golgi stacks. The GDP-restricted mutant of ARL1, AtARL1-T31N, was observed to locate partially to the cytosol, whereas the GTP-restricted mutant AtARL1-Q71L labelled the Golgi and a population of small structures. Increasing the levels of AtARL1 in epidermal cells increased the proportion of GRIP-GFP fusion protein on Golgi stacks. We show, moreover, that AtARL1 interacted with the GRIP domain in a GTP-dependent manner in vitro in affinity chromatography and in the yeast two-hybrid system. This indicates that AtGRIP and AtARL1 interact directly. We conclude that the pathway involving ARL1 and GRIP domain golgins is conserved in plants.     

ER quality control can lead to retrograde transport from the ER lumen to the cytosol and the nucleoplasm in plants

Quality control in the secretory pathway is a fundamental step in preventing deleterious effects that may arise by the release of malfolded proteins into the cell or apoplast. Our aims were to visualise and analyse the disposal route followed by aberrant proteins within a plant cell in vivo using fluorescent protein technology. A green fluorescent protein (GFP) fusion was detected in the cytosol and the nucleoplasm in spite of the presence of an N-terminal secretory signal peptide. In contrast to secreted GFP, the fusion protein was retained in the cells where it was degraded slowly, albeit at a rate much higher than that of the endoplasmic reticulum (ER)-retained derivative GFP-HDEL. The fusion protein could not be stabilised by inhibitors of transport or the cytosolic proteasome. However, the protein is a strong lumenal binding protein (BiP) ligand. Complete signal peptide processing even after long-term expression in virus-infected leaves rules out the possibility that the documented accumulation in the cytosol and nucleoplasm is because of the bypassing of the translocation pores. The data are consistent with the hypothesis that the fusion protein is disposed off from the ER via a retrograde translocation back to the cytosol. Moreover, accumulation in the nucleoplasm was shown to be microtubule dependent unlike the well-documented diffusion of cytosolically expressed GFP into the nucleoplasm. The apparent active transport of the GFP fusion into the nucleoplasm may indicate an as yet undiscovered feature of the ER-associated degradation (ERAD) pathway and explain the insensitivity to degradation by proteasome inhibitors.     

Dynamic changes in the frequency and architecture of plasmodesmata during the sink-source transition in tobacco leaves

Summary The sink-source transition in tobacco leaves was studied noninvasively using transgenic plants expressing the green-fluorescent protein (GFP) under control of theArabidopsis thaliana SUC2 promoter, and also by imaging transgenic plants that constitutively expressed a tobacco mosaic virus movement protein (MP) fused to GFP (MP-GFP). The sink-source transition was measured on intact leaves and progressed basipetally at rates of up to 600 m/h. The transition was most rapid on the largest sink leaves. However, leaf size was a poor indicator of the current position of the sink-source transition. A quantitative study of plasmodesmatal frequencies revealed the loss of enormous numbers of simple plasmodemata during the sink-source transition. In contrast, branched plasmodesmata increased in frequency during the sink-source transition, particularly between periclinal cell walls of the spongy mesophyll. The progression of plasmodesmal branching, as mapped by the labelling of plasmodesmata with MP-GFP fusion, occurred asynchronously in different cell layers, commencing in trichomes and appearing lastly in periclinal cell walls of the palisade layer. It appears that dividing cells retain simple plasmodesmata for longer periods than nondividing cells. The rapid conversion of simple to branched plasmodesmata is discussed in relation to the capacity for macromolecular trafficking in developing leaf tissues.     

Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.     

Viunalikeviruses are environmentally common agents of horizontal gene transfer in pathogens and biocontrol bacteria

Bacteriophages have been used as natural biocontrol and therapeutic agents, but also as biotechnological tools for bacterial engineering. We showed recently that the transducing bacteriophage ϕMAM1 is a ViI-like phage and a member of the new genus, ‘Viunalikevirus''. Here, we show that four additional ViI-like phages and three new environmentally isolated viunalikeviruses, all infecting plant and human pathogens, are very efficient generalised transducers capable of transducing chromosomal markers at frequencies of up to 10⁻⁴ transductants per plaque-forming unit. We also demonstrate the interstrain transduction of plasmids and chromosomal markers, including genes involved in anabolism, genes for virulence and genes encoding secondary metabolites involved in biocontrol. We propose that all viunalikeviruses are likely to perform efficient horizontal gene transfer. Viunalikeviruses therefore represent useful agents for functional genomics and bacterial engineering, and for chemical and synthetic biology studies, but could be viewed as inappropriate choices for phage therapy.Combined morphological, genomic and phylogenetic analyses have recently led to the proposed creation of a new phage genus, ‘Viunalikevirus'', within the Myoviridae family (Adriaenssens et al., 2012a). The first member of this proposed genus, Salmonella phage ViI, was isolated in the 1930s (Craigie and Yen, 1938) and multiple viunalikeviruses have been sequenced and characterised since 2010 (Pickard et al., 2010; Anany et al., 2011; Hooton et al., 2011; Kutter et al., 2011; Matilla and Salmond, 2012; Park et al., 2012; Adriaenssens et al., 2012a, 2012b; Hsu et al., 2013; Luna et al., 2013; Shahrbabak et al., 2013). Viunalikeviruses are characterised as virulent (lytic) phages showing similar genome size, extensive DNA homology, strong gene synteny and a complex adsorption apparatus, which uses tail spike proteins as host-recognition determinants (Adriaenssens et al., 2012a).We recently isolated the ViI-like phage, ϕMAM1, that infects several environmental and clinical isolates belonging to Serratia and Kluyvera genera (Matilla and Salmond, 2012). During the characterisation of ϕMAM1, we showed that it mediates highly efficient generalised transduction (Matilla and Salmond, submitted for publication). These observations were consistent with a previous report, that the Salmonella phage ViI was also capable of transduction (Cerquetti and Hooke, 1993) and we have confirmed that phage ViI can transduce chromosomal markers and plasmids at frequencies of up to 4.6 × 10⁻⁵ transductants per plaque-forming unit (p.f.u.; Figure 1a; Supplementary Table 1).Open in a separate windowFigure 1Transduction capabilities of viunalikeviruses. (a) Transduction frequencies of LIMEstone1, LIMEstone2, ViI and CBA120 phages. The graph also shows transduction efficiencies of LIMEstone phages within and between Dickeya solani strains. Transduction efficiency was defined as the number of transductants obtained per p.f.u. In all cases, error bars represent the standard deviations (n=3). (b) Skimmed milk agar plates showing protease production in the wild-type (wt) Dickeya solani strains MK10, MK16 and IPO 2222. LIMEstone1- (LS1) and LIMEstone2- (LS2) mediated transduction of the spp::Km marker from the protease negative mutant strain MK10P1 to the wild-type strains MK10, MK16 and IPO 2222 result in a protease-negative phenotype. (c–e) LIMEstone-mediated transduction of the oocN::Km marker from the oocydin A-negative mutant strain MK10oocN to the wild-type strains MK10 (c), MK16 (d) and IPO 2222 (e) results in an oocydin A-negative phenotype and, consequently, in the generation of strains defective in their antimicrobial activity against the plant pathogenic oomycete, Pythium ultimum. The anti-oomycete assays were performed as described previously (Matilla et al., 2012).Most generalised transducers utilise a headful packing strategy where phage terminases recognise specific sequences (pac sites) in the DNA and perform cycles of packing that result in mature phage particles (Fineran et al., 2009a). Indeed, phage terminases with reduced specificity for pac sequences may lead to the evolution of efficient transducing phages (Schmeiger, 1972). Based on the high similarity between the terminases of ϕMAM1, ViI and those of other previously sequenced viunalikeviruses, we hypothesised that all of these ViI-like phages should be capable of transduction in their respective bacterial hosts. To test this hypothesis, we investigated three additional viunalikeviruses, Escherichia coli phage CBA120 (Kutter et al., 2011), and Dickeya phages LIMEstone1 and LIMEstone2 (Adriaenssens et al., 2012b). All the bacteriophages, bacterial strains, plasmids and primers used in this study are listed in the Supplementary Tables 2 and 3. Experimental procedures are presented as Supplementary Material.The LIMEstone phages specifically infect some strains of the emerging plant pathogen, Dickeya solani (Adriaenssens et al., 2012b), and here we showed that they also infect the recently sequenced D. solani strains MK10, MK16 and IPO 2222. As predicted, we confirmed that the LIMEstone phages effected efficient transduction of various auxotrophic markers between Dickeya solani strains (Figure 1a; Supplementary Table 4). To our knowledge, only one Dickeya transducing phage, ϕEC2, has been isolated previously (Resibois et al., 1984). Additional mutant strains were constructed and the generalised nature of the transduction was confirmed by transfer of multiple chromosomal markers, including mutations in the gene cluster encoding biosynthesis of the anti-oomycete haterumalide, oocydin A (Matilla et al., 2012) and in the locus for synthesis and secretion of protease virulence factors. Transduction frequency was higher at an multiplicity of infection (m.o.i.) of 0.1 and 0.01 with efficiencies of up to 10⁻⁴ transductants per p.f.u. (Figure 1a; Supplementary Tables 4 and 5).We also demonstrated transduction of a kanamycin resistance-marked plasmid pECA1039-Km3 between strains MK10, MK16 and IPO 2222 at frequencies of up to 8.6 × 10⁻⁵ (Supplementary Table 4). Plasmid pECA1039 (originally isolated from the phytopathogen, Pectobacterium atrosepticum) encodes a bifunctional type III Toxin-Antitoxin (TA) system, ToxIN, with abortive infection capacity. Although ToxIN aborts infection of various enterobacteria by diverse phages (Fineran et al., 2009b) it did not protect against infection by the tested viunalikeviruses, ϕMAM1, ViI, CBA120, LIMEstone1 or LIMEstone2 (not shown). Furthermore, another type III TA system, TenpIN, from the insect pathogen, Photorhabdus luminescens (Blower et al., 2012), failed to protect against any of the five ViI-like phages (not shown).In addition, we also tested the transduction capacity of the E. coli phage, CBA120, and confirmed transduction of plasmid-borne antibiotic resistances at a frequency of up to 10⁻⁴ transductants per p.f.u. (Figure 1a; Supplementary Table 6).We decided to test our hypothesis that the viunalikeviruses may all be generalised transducers by first isolating new viunalikeviruses from the environment. From treated sewage effluent, we isolated three new bacteriophages infecting Dickeya solani, ϕXF1, ϕXF3 and ϕXF4, as defined initially by their very characteristic ViI-like morphology in electron microscopy (Figures 2a–c). As predicted, all of these new phages were able to transduce chromosomal markers and plasmids at frequencies of up to 3 × 10⁻⁶ transductants per p.f.u. (Figure 2e; Supplementary Table 7). Sequencing of structural and non-structural protein-encoding genes of ϕXF1, ϕXF3 and ϕXF4 showed high nucleotide homology (between 80% and 100%) with the corresponding orthologs in LIMEstone1 (Supplementary Figure 1), indicating that these virgin environmental isolates also clade within the Viunalikevirus genus.Open in a separate windowFigure 2Environmental isolation and characterisation of new viunalikeviruses with generalised transduction functionality. Transmission electron micrographs of phages ϕXF1 (a), ϕXF3 (b), ϕXF4 (c) and ϕXF28 (d) are shown. As an internal control, ϕXF28 was an example of a new lytic phage isolated from the same environment but showing no transduction capabilities. Bars, 50 nm. (e) Transduction frequencies of the new viunalikeviruses ϕXF1, ϕXF3 and ϕXF4. Transduction experiments were performed using 10⁹ cells with ϕXF1, ϕXF3, ϕXF4 at an m.o.i. of 0.01. Transduction efficiency was defined as the number of transductants obtained per p.f.u. Error bars represent the standard deviations (n=3).Although we did not have access to other ViI-like Salmonella phages SFP10 (Park et al., 2012), ϕSH19 (Hooton et al., 2011) and Marshall (Luna et al., 2013), Escherichia phage PhaxI (Shahrbabak et al., 2013), Shigella phage ϕSboM-AG3 (Anany et al., 2011) and Klebsiella phage 0507-KN2-1 (Hsu et al., 2013), our results allow us to predict that all of these phages will mediate generalised transduction. Importantly, these phages would be expected to contribute to the horizontal gene transfer of virulence factors and antimicrobial-resistance determinants in diverse environments.Viunalikeviruses do not seem to be limited to the enterobacteria as bacteriophages showing ViI-like morphology have been isolated in Acinetobacter (Ackermann et al., 1994), Bordetella (Adriaenssens et al., 2012b) and Sinorhizobium (Werquin et al., 1988). Furthermore, another ViI-like morphotype phage (ϕM12 of Sinorhizobium meliloti) has also been shown to be an efficient transducer (Finan et al., 1984). Taken together, these results suggest that, even in the absence of strongly predictive comparative genomic detail, a characteristically discrete ViI-like morphology in electron microscopy may be sufficient to identify new phages as strong candidates for possession of generalised transduction capacity.The emergence and dissemination of antibiotic-resistant pathogens coupled with low discovery rates for new antimicrobials, plus increasing legal constraints on the use of chemical pesticides, have (re)focussed attention on the potential use of bacteriophages for ‘natural biocontrol'' of human, animal and plant pathogens. Several viunalikeviruses have been proposed as candidate therapeutic agents for the control of bacterial infections (Anany et al., 2011; Hooton et al., 2011; Park et al., 2012; Hsu et al., 2013; Shahrbabak et al., 2013) and the LIMEstone phages have been used in successful field trials for biocontrol of D. solani infections (Adriaenssens et al., 2012b). However, their efficient transduction capacities could provide a route for dissemination of virulence factors, such as proteases (Marits et al., 1999). In fact, we have demonstrated the interstrain transduction of plasmids and oocydin A, auxotrophy and protease markers between three different D. solani strains, at high frequencies (Figures 1 and ​and2;2; Supplementary Tables 4 and 7). Also, the irregular distribution of the oocydin A gene cluster within the Dickeya genus and the fact that its genomic context varies between strains raises the possibility of phage-mediated horizontal gene transfer between bacterial strains. These results emphasize strongly that when considering the genomics of phages for ‘phage therapy'' the absence of genes readily defined as playing roles in lysogeny or bacterial virulence may be insufficient to inspire confidence that use of a particular therapeutic phage presents no risk–particularly among the high efficiency-transducing viunalikeviruses.     

Development of pan-specific antibody against trimethyllysine for protein research

Background  

Trimethylation of the Nε-lysine residues in a protein is one of the most important events of posttranslational modifications. Simple methods for rapid detection and isolation of the Nε-trimethylated protein species are needed. This report introduces a novel method to prepare the affinity purified antibody specific for the Nε-trimethylated lysine (tMeK). The applications of the purified antibody are also reported in this paper.     

The effect of cumulative endurance exercise on leptin and adiponectin and their role as markers to monitor training load

Leptin and adiponectin play an essential role in energy metabolism. Leptin has also been proposed as a marker for monitoring training load. So far, no studies have investigated the variability of these hormones in athletes and how they are regulated during cumulative exercise. This study monitored leptin and adiponectin in 15 endurance athletes twice daily in the days before, during and after a 9-day simulated cycling stage race. Adiponectin significantly increased during the race (p = 0.001) and recovery periods (p = 0.002) when compared to the baseline, while leptin decreased significantly during the race (p < 0.0001) and returned to baseline levels during the recovery period. Intra-individual variability was substantially lower than inter-individual variability for both hormones (leptin 34.1 vs. 53.5%, adiponectin 19% vs. 37.2%). With regards to exercise, this study demonstrated that with sufficient, sustained energy expenditure, leptin concentrations can decrease within the first 24 hours. Under the investigated conditions there also appears to be an optimal leptin concentration which ensures stable energy homeostasis, as there was no significant decrease over the subsequent race days. In healthy endurance athletes the recovery of leptin takes 48-72 hours and may even show a supercompensation-like effect. For adiponectin, significant increases were observed within 5 days of commencing racing, with these elevated values failing to return to baseline levels after 3 days of recovery. Additionally, when using leptin and adiponectin to monitor training loads, establishing individual threshold values improves their sensitivity.