Newly isolated Nodularia phage influences cyanobacterial community dynamics.

Cyanophages, that is, viruses infecting cyanobacteria, are a key component driving cyanobacterial community dynamics both ecologically and evolutionarily. In addition to reducing biomass and influencing the genetic diversity of their host populations, they can also have a wider community-level impact due to the release of nutrients by phage-induced cell lysis. In this study, we isolated and characterized a new cyanophage, a siphophage designated as vB_NpeS-2AV2, capable of infecting the filamentous nitrogen fixing cyanobacterium Nodularia sp. AV2 with a lytic cycle between 12 and 18 hours. The role of the phage in the ecology of its host Nodularia and competitor Synechococcus was investigated in a set of microcosm experiments. Initially, phage-induced cell lysis decreased the number of Nodularia cells in the cultures. However, around 18%-27% of the population was resistant against the phage infection. Nitrogen was released from the Nodularia cells as a consequence of phage activity, resulting in a seven-fold increase in Synechococcus cell density. In conclusion, the presence of the cyanophage vB_NpeS-2AV2 altered the ecological dynamics in the cyanobacterial community and induced evolutionary changes in the Nodularia population, causing the evolution from a population dominated by susceptible cells to a population dominated by resistant ones.

Lipid fingerprints of intact viruses by MALDI-TOF/mass spectrometry.

A number of viruses contain lipid membranes, which are in close contact with capsid proteins and/or nucleic acids and have an important role in the viral infection process. In this study membrane lipids of intact viruses have been analysed by MALDI-TOF/MS with a novel methodology avoiding lipid extraction and separation steps. To validate the novel method, a wide screening of viral lipids has been performed analysing highly purified intact bacterial and archaeal viruses displaying different virion architectures. Lipid profiles reported here contain all lipids previously detected by mass spectrometry analyses of virus lipid extracts. Novel details on the membrane lipid composition of selected viruses have also been obtained. In addition we show that this technique allows the study of lipid distribution easily in subviral particles during virus fractionation. The possibility to reliably analyse minute amounts of intact viruses by mass spectrometry opens new perspectives in analytical and functional lipid studies on a wider range of viruses including pathogenic human ones, which are difficult to purify in large amounts.

Targeting risk factors for impaired wound healing and wound complications after kidney transplantation.

BACKGROUND: Because of potent immunosuppression, impaired wound healing and complications are frequent features after kidney transplantation (KTx). OBJECTIVE: To investigate the incidence and nature of impaired wound healing and complications at a single transplantation center in Norway. PATIENTS: Of 226 patients who underwent KTx, 199 (87%) were followed up prospectively for 1 year (2005) via close and meticulous wound inspection. RESULTS: The study revealed a high rate of wound complications (200-250/y) in a high-volume center. Fifty-four patients (27%) experienced prolonged wound healing, defined as gaps, secretions, or wound complications, at 3 to 5 weeks posttransplantation, and 41 patients (21%) had impaired wound healing, defined as gaps, secretions, or wound complications after 5 weeks posttransplantation. In total, 50 patients (25%) required surgical or radiologic reintervention. Complications included lymphocele in 29 patients (14.6%), wound dehiscence in 16 (8.0%), bleeding or hematoma in 10 (5.0%), and infection in 9 (4.5%). Risk factors associated with wound complications included recipient older than 60 years, body mass index greater than 30, hemoglobin concentration less than 10 g/dL, albumin concentration less than 36 g/dL, duration of surgery more than 200 minutes, no subcutaneous sutures, and sirolimus or everolimus therapy. At nominal and logistic regression analysis, recipient older than 60 years, body mass index greater than 30, and no subcutaneous sutures were independent risk factors. CONCLUSION: Risk factor analysis and previous documentation suggest that wound complications might be counteracted using the following measures: subcutaneous sutures, predialysis transplantation, sealing or ligation of lymphatic trunks, prophylactic fenestration, reduction of corticosteroid load, and avoiding sirolimus/everolimus therapy.

mRNA stability and the secretion signal of HrpA, a pilin secreted by the type III system in Pseudomonas syringae.

Gram-negative bacteria that are pathogenic for animals or plants utilise a specialised Type III secretion system to inject effector proteins into their eukaryotic target cells. The basis for selection of the proteins to be translocated via type III systems is still enigmatic. No clearly defined consensus amino acid sequence that could serve as a specific secretion signal has been identified, and the hypothesis that an mRNA secondary structure acts as the signal has several shortcomings. We have localised a secretion signal that is sufficient to ensure the secretion of the pilin HrpA, a substrate and an indispensable extracellular component of the type III secretion machinery of Pseudomonas syringae pv. tomato DC3000, to the first 15 codons. Transcription of hrpA starts at a single site 42 bp upstream of the first codon. Gene swapping experiments revealed that altering the continuity of the 5' non-translated leader with the region including the secretion signal radically decreased accumulation of the hrpA transcript. These results indicate that an mRNA secondary structure, possibly formed in this region, is important for efficient expression of the gene. The proposed secondary structure is not, however, indispensable for the secretion of HrpA and it does not couple secretion and translation.

Immunocytochemical localization of HrpA and HrpZ supports a role for the Hrp pilus in the transfer of effector proteins from Pseudomonas syringae pv. tomato across the host plant cell wall.

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 microm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

Mutational analysis of the Pseudomonas syringae pv. tomato hrpA gene encoding Hrp pilus subunit.

Plant pathogenic Pseudomonas syringae strains harbour a type III secretion pathway suggested to be involved in the delivery of effector proteins from the bacteria into plant cells. During plant interaction, the bacteria apparently produce surface appendages, termed Hrp pili, that are indispensable for the secretion process. We have created an insertion mutation library, as well as deletion mutations to hrpA, the structural gene encoding Hrp pilin. Analysis of the mutants revealed gene regions important for hrpA expression, pilus assembly and pilus-dependent autoagglutination of the bacteria. The majority of insertions in the amino-terminal half of the pilin were tolerated without bacterial interaction with plants being affected, while the carboxy-terminus appeared to be needed for pilus assembly. Insertions in the 5' non-translated region and the first codons within the open reading frame affected mRNA production or stability and abolished protein production.

Characterization of type IV pilus genes in Pseudomonas syringae pv. tomato DC3000.

Many strains of Pseudomonas syringae produce retractile pili that act as receptors for lytic bacteriophage phi 6. As these are also characteristics of type IV pili, it was postulated that P. syringae may possess genes for type IV pilus biogenesis. A cosmid clone bank of P. syringae pv. tomato DC3000 genomic DNA was used to complement a mutant of Pseudomonas aeruginosa defective in the PilD (XcpA) prepilin peptidase gene by selection for restoration of extracellular protein secretion, a function also known to require PilD. A cosmid able to complement this mutant was also able to complement mutations in the pilB and pilC genes, suggesting that, if the organization of these genes is similar to that of P. aeruginosa, the cosmid may contain the P. syringae pilA. This was confirmed by sequencing a region from this plasmid that was shown to hybridize at low stringency to the P. aeruginosa pilA gene. The deduced P. syringae PilA polypeptide possesses the characteristic properties of the type IV pilins. Heterologous expression of the P. syringae pilA in P. aeruginosa was also shown, conferring not only phi 6 phage sensitivity to P. aeruginosa pilA mutants but also sensitivity to PO4, a lytic bacteriophage specific for the pilus of P. aeruginosa. This suggests that additional components might be present in the mature pilus of P. aeruginosa that are the true receptors for this phage. Chromosomal mutations in P. syringae pv. tomato DC3000 pilA and pilD genes were shown to abolish its sensitivity to bacteriophage phi 6. To determine the importance of P. syringae pilus in plant leaf interactions, these mutations were tested under laboratory and field conditions. Although little effect was seen on pathogenicity, culturable leaf-associated population sizes of the pilA mutant were significantly different from those of the wild-type parent. In addition, the expression of the DC3000 pilA gene appears to contribute to the UV tolerance of P. syringae and may play a role in survival on the plant leaf surface.

Purified HrpA of Pseudomonas syringae pv. tomato DC3000 reassembles into pili.

Pseudomonas syringae pv. tomato DC3000 produces Hrp pili under inducing in vitro conditions. A preparation of partially purified extracellular filaments contains HrpA, flagellin and some minor contaminants. HrpA was separated from the major contaminant, the flagellin, by gel filtration to a fraction containing HrpA as well as its three N-terminally truncated forms. These were further separated by two steps of reversed phase chromatography. HrpA and its degradation products were each shown to reassemble into filament structures after denaturation and renaturation showing that HrpA alone is sufficient for formation of filament structures.

Hrp pilus: an hrp-dependent bacterial surface appendage produced by Pseudomonas syringae pv. tomato DC3000.

Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants. A number of Hrp proteins share significant similarities with components of the type III secretion apparatus and flagellar assembly apparatus in animal pathogenic bacteria. Here we report that Pseudomonas syringae pv. tomato strain DC3000 (race 0) produces a filamentous surface appendage (Hrp pilus) of 6-8 nm in diameter in a solid minimal medium that induces hrp genes. Formation of the Hrp pilus is dependent on at least two hrp genes, hrpS and hrpH (recently renamed hrcC), which are involved in gene regulation and protein secretion, respectively. Our finding of the Hrp pilus, together with recent reports of Salmonella typhimurium surface appendages that are involved in bacterial invasion into the animal cell and of the Agrobacterium tumefaciens virB-dependent pilus that is involved in the transfer of T-DNA into plant cells, suggests that surface appendage formation is a common feature of animal and plant pathogenic bacteria in the infection of eukaryotic cells. Furthermore, we have identified HrpA as a major structural protein of the Hrp pilus. Finally, we show that a nonpolar hrpA mutant of P. syringae pv. tomato DC3000 is unable to form the Hrp pilus or to cause either an HR or disease in plants.

Characterization of genes required for pilus expression in Pseudomonas syringae pathovar phaseolicola.

Nonpiliated, phage phi 6-resistant mutants of Pseudomonas syringae pv. phaseolicola were generated by Tn5 transposon mutagenesis. A P. syringae pv. phaseolicola LR700 cosmid library was screened with Tn5-containing EcoRI fragments cloned from nonpiliated mutants. The cosmid clone pVK253 complemented the nonpiliated mutant strain HB2.5. A 3.8-kb sequenced region spanning the Tn5 insertion site contained four open reading frames. The transposon-inactivated gene, designated pilP, is 525 bp long, potentially encoding a 19.1-kDa protein precursor that contains a typical membrane lipoprotein leader sequence. Generation of single mutations in each of the three remaining complete open reading frames by marker exchange also resulted in a nonpiliated phenotype. Expression of this gene region by the T7 expression system in Escherichia coli resulted in four polypeptides of approximately 39, 26, 23, and 18 kDa, in agreement with the sizes of the open reading frames. The three genes upstream of pilP were designated pilM (39 kDa), pilN (23 kDa), and pilO (26 kDa). The processing of the PilP precursor into its mature form was shown to be inhibited by globomycin, a specific inhibitor of signal peptidase II. The gene region identified shows a high degree of homology to a gene region reported to be required for Pseudomonas aeruginosa type IV pilus production.