The mkaC virulence gene of the Salmonella serovar Typhimurium 96 kb plasmid encodes a transcriptional activator

The intracellular growth and virulence of Salmonella serovar Typhimurium for mice is dependent on a plasmid-borne gene cluster termed mka. We studied the regulatory interactions of the genes mkaA, mkaB, mkaC and mkaD using lacZ gene fusions. Complementation experiments with cloned DNA fragments encoding each of the four MKa proteins indicated that mkaC enhances the expression of beta-galactosidase from the mkaA-, mkaB- and mkaC-lacZ gene fusions in trans. An mkaD-lacZ fusion or mkaA-lacZ fusion that did not contain DNA proximal to mkaB was not inducible with MkaC, indicating that at least mkaB and mkaA are induced together as an operon. MkaC is thus the first virulence protein whose function has been resolved.     

Experimental manipulation of food availability leads to short‐term intra‐clutch adjustment in egg mass but not in yolk androgen or thyroid hormones

In birds, mothers can affect their offspring's phenotype and thereby survival via egg composition. It is not well known to what extent and time‐scales environmental variation in resource availability, either via resource constrains or adaptive adjustment to predicted rearing conditions, influences maternal effects. We experimentally studied whether egg and yolk mass and yolk hormone levels respond to short‐term changes in food availability during laying in wild great tits Parus major. Our treatment groups were: 1) food supplementation (mealworms) from the 1st until the last egg; 2) food supplementation from the 1st until the 5th egg, where the effect of cessation of the supplementary food treatment could also be studied; 3) no food supplementation (controls). We analysed both nutritional resources (egg, yolk and albumen mass), and the important developmental signals, yolk androgens (testosterone and androstenedione), and for the first time in a wild population, yolk thyroid hormones (thyroxine and 3,5,3′‐triiodothyronine). Egg mass is a costly resource for females, androgens most likely non‐costly signals, whereas thyroid hormones may be costly signals, requiring environmental iodine. In the food supplemented group egg, yolk and albumen mass increased rapidly relative to controls and when food supplementation was halted, egg and albumen mass decreased, indicating rapid responses to resource availability. Yolk androgen and thyroid hormone levels were not affected by food supplementation during laying. Thyroxine showed an increase over the laying sequence and its biological meaning needs further study. The rapid changes in egg mass to variation in within‐clutch food availability suggest energetic/protein/nutrient constrains on egg formation. The lack of a response in yolk hormones suggest that perhaps in this species the short‐term changes in resource availability during egg laying do not predict offspring rearing conditions, or (for thyroid hormones) do not cause systemic changes in circulating hormones, and hence do not affect maternal signaling.     

Sympatric occurrence of three leaf beetle species of Macroplea Samouelle, 1819 (Coleoptera,Chrysomelidae, Donaciinae) in Finland with a key to species in Northern Europe

Macroplea Samouelle, 1819 is the only known fully aquatic leaf beetle genus with three European species that have earlier been classified by their assumed water salinity preferences. We studied the inter- and intraspecific variation of the specimens living in Northern Europe using both molecular (cytochrome c oxidase subunit I, COI) and morphological evidence. The variation in the COI sequences between M. mutica (Fabricius, 1792) and M. pubipennis (Reuter, 1875) was 8.4%–9%, M. mutica and M. appendiculata (Panzer, 1794) – 3.9%–4.9%, and M. appendiculata and M. pubipennis – 8.8%–9.2%. All three species were sampled together in the Bothnian Sea on the same water plants, showing that neither salinity nor plant species bear a decisive importance in their occurrence in the region. Phylogenetic results suggest the existence of two currently unknown Macroplea species that are evolutionarily close to M. appendiculata. A key to the Nordic species is provided.     

Comparative proteome profiling of bovine and human Staphylococcus epidermidis strains for screening specifically expressed virulence and adaptation proteins

The present study reports a comparative proteome cataloging of a bovine mastitis and a human‐associated Staphylococcus epidermidis strain with a specific focus on surfome (cell‐wall bound and extracellular) proteins. Protein identification by 1DE coupled with LC‐MS/MS analyses resulted in 1400 and 1287 proteins from the bovine (PM221) and human (ATCC12228) strains, respectively, covering over 50% of all predicted and more than 30% of all predicted surfome proteins in both strains. Comparison of the identification results suggests elevated levels of proteins involved in adherence, biofilm formation, signal transduction, house‐keeping functions, and immune evasion in PM221, whereas ATCC12228 was more effective in expressing host defense evasion proteases, skin adaptation lipases, hemagglutination, and heavy‐metal resistance proteins. Phenotypic analyses showed that only PM221 displays protein‐ and DNA‐mediated adherent growth, and that PM221 was more efficient in cleaving tributyrin, a natural compound of milk fat under low CO₂ conditions. These findings are in line with the identification data and suggest that distinct expression of lipases and adhesive surfome proteins could lead to the observed phenotypes. This study is the first extensive survey of S. epidermidis proteomes to date, providing several protein candidates to be examined for their roles in adaptation and virulence in vivo. All MS data have been deposited in the ProteomeXchange with identifier PXD000404 ( http://proteomecentral.proteomexchange.org/dataset/PXD000404 ).     

Novel Vascular Endothelial Growth Factor D Variants with Increased Biological Activity

Members of the vascular endothelial growth factor (VEGF) family play a pivotal role in angiogenesis and lymphangiogenesis. They are potential therapeutics to induce blood vessel formation in myocardium and skeletal muscle, when normal blood flow is compromised. Most members of the VEGF/platelet derived growth factor protein superfamily exist as covalently bound antiparallel dimers. However, the mature form of VEGF-D (VEGF-D^ΔNΔC) is predominantly a non-covalent dimer even though the cysteine residues (Cys-44 and Cys-53) forming the intersubunit disulfide bridges in the other members of the VEGF family are also conserved in VEGF-D. Moreover, VEGF-D bears an additional cysteine residue (Cys-25) at the subunit interface. Guided by our model of VEGF-D^ΔNΔC, the cysteines at the subunit interface were mutated to study the effect of these residues on the structural and functional properties of VEGF-D^ΔNΔC. The conserved cysteines Cys-44 and Cys-53 were found to be essential for the function of VEGF-D^ΔNΔC. More importantly, the substitution of the Cys-25 at the dimer interface by various amino acids improved the activity of the recombinant VEGF-D^ΔNΔC and increased the dimer to monomer ratio. Specifically, substitutions to hydrophobic amino acids Ile, Leu, and Val, equivalent to those found in other VEGFs, most favorably affected the activity of the recombinant VEGF-D^ΔNΔC. The increased activity of these mutants was mainly due to stabilization of the protein. This study enables us to better understand the structural determinants controlling the biological activity of VEGF-D. The novel variants of VEGF-D^ΔNΔC described here are potential agents for therapeutic applications, where induction of vascular formation is required.Vascular endothelial growth factors (VEGFs)3 are considered as key growth factors inducing angiogenesis and lymphangiogenesis during embryogenesis as well as maintaining vasculature during adulthood. Their abnormal expression is found in pathological conditions such as cancer and retinopathies (1). Five mammalian VEGFs, VEGF-A, -B, -C, -D and placenta growth factor (PlGF), are known (2) as well as Orf virus-derived VEGF-E proteins (3) and multiple homologues from snake venoms (VEGF-Fs) (4). Several members of the VEGF family exist as different isoforms, either as a result of the alternative splicing of their mRNAs or due to proteolytic processing. These forms vary in their specificities and affinities to three main VEGF receptors, co-receptors such as neuropilins and heparan sulfate proteoglycans and other components of the extracellular matrix, translating into different biological effects (5).VEGFR-2 is an important receptor regulating vasculogenesis and angiogenesis. It is mainly expressed on endothelial cells, but expression is also found in several other cell types (6). Mammalian VEGFR-2 ligands include VEGF-A (7), VEGF-C (8), and VEGF-D (9). In addition to VEGFR-2, VEGF-C and VEGF-D are ligands for VEGFR-3, which mainly mediates lymphangiogenesis in adults but also participates in the formation of blood vessels during embryogenesis (2).Because of their importance in angiogenesis, VEGFs have been suggested as potential therapeutic agents in different pathological conditions to improve compromised blood flow. Studies aiming at inducing angiogenesis in vivo have been performed by introducing VEGFs to tissues either directly as recombinant proteins (10) or using gene therapy vectors (11). Findings from several laboratories have shown that VEGFs have strong angiogenic activity in vivo, and they could be used for the treatment of conditions like lower limb ischemia and ischemic coronary artery disease. The short in vivo half-life of these growth factors and the requirement for sustained angiogenic stimulus makes gene therapy a preferred option. Of the VEGFs, VEGF-A and the mature form of VEGF-D (VEGF-D^ΔNΔC, see below) are the strongest agents to induce vascular formation (12, 13).VEGFs share structural similarity with platelet-derived growth factors. Together they are classified as the VEGF/platelet-derived growth factor family, belonging to a larger family of cystine knot growth factors. The members of this family share a cystine knot motif, which is found in many extracellular proteins and is conserved among numerous species (14). Characteristic of the cystine knot proteins is that they contain a conserved structure of antiparallel β-sheets connected by three disulfide bonds. Typically cystine knot growth factors form dimers, which within the VEGF/platelet-derived growth factor family are often linked by intersubunit disulfide bonds. The crystal structures have been solved for VEGF-A (15), PlGF (16), VEGF-B (17), VEGF-E (18), and two snake venom VEGF-Fs, vammin and VR-1 (19).There are currently no published structures of VEGF-C or VEGF-D. They can be divided into their own subfamily based on sequence similarity and several characteristic features; 1) they are the only VEGFs that bind to VEGFR-3, 2) they are expressed as long precursor forms having poor receptor binding affinities, and 3) they require proteolytic processing at their N-terminal and C-terminal ends to become more active. In contrast to other members of the VEGF family, the mature, proteolytically processed ΔNΔC-forms of VEGF-C and VEGF-D exist predominantly as non-covalently bound dimers, even though they have the conserved cysteine residues that form the intersubunit disulfide bonds in other VEGFs (8, 20). However, both VEGF-C and VEGF-D also have an additional cysteine residue located at the dimer interface (8, 20). Mutation of this residue in VEGF-C only minimally altered the receptor binding affinity (21), but it stabilized the dimer structure (56).In the current study we investigated the importance of residues at the subunit interface for the function of VEGF-D^ΔNΔC. We built homology models of VEGF-D^ΔNΔC and used alanine scanning and site-specific mutagenesis as well as tested the biological activity of various mutated forms of VEGF-D^ΔNΔC. Our study revealed that the conserved cysteine residues (Cys-44 and Cys-53), which are known to form intersubunit disulfide bridges in other VEGFs, were essential for the activity of the recombinant VEGF-D^ΔNΔC. Furthermore, the monomer to dimer ratio of VEGF-D^ΔNΔC could be regulated by mutagenesis. In addition, it was found that replacement of the “extra” cysteine (Cys-25) by various amino acids, preferably Ile, Leu, or Val, actually enhanced the activity of VEGF-D^ΔNΔC. This was at least partially due to increased stability of the protein.     

Real-time monitoring of antimicrobial activity with the multiparameter microplate assay

Kinetic measurements of the bacteriostatic, bactericidal and bacteriolytic activities of six model antibiotics (ampicillin, erythromycin, nalidixic acid, polymyxin B, tetracycline, and trimethoprim) against Escherichia coli as target bacteria were performed by bioluminescence, fluorescence, and optical density based real-time assay. Additionally, plate counting was used as a control measurement. The gfp and insect luciferase (lucFF) genes were cloned into cells used for measurements to enable fluoro-luminometric detection. Bacteria were exposed to antibiotics for 10 h, and the effects of antimicrobial agents were established. Inhibitory concentration of 50% (IC(50)), minimum bactericidal concentration (MBC), and bactericidal concentration of 50% (BC(50)) of each antibiotic were calculated from these procedures. Bacteriostatic, bactericidal or bacteriolytic actions of each antibiotic, as well the time interval from exposure to visible effect, were readily observed from kinetic data. No significant differences were observed between data obtained with the different methods employed. Ampicillin and polymyxin B were clearly bacteriolytic, nalidixic acid and tetracycline showed bactericidal effects, and erythromycin and trimethoprim were bacteriostatic drugs. The assay has the advantage of speed and accurately discerns between lytic, cidal and static compounds. Thus, this reliable and fully automated novel kinetic assay with high sample capacity offers new possibilities for real-time detection, making it suitable for diverse high throughput screening (HTS) applications.     

Hcp2, a Secreted Protein of the Phytopathogen Pseudomonas syringae pv. Tomato DC3000, Is Required for Fitness for Competition against Bacteria and Yeasts

When analyzing the secretome of the plant pathogen Pseudomonas syringae pv. tomato DC3000, we identified hemolysin-coregulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the P. syringae pv. tomato DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of the hcp genes and tested the fitness of hcp knockout mutants in host plant colonization and in intermicrobial competition. We found that the hcp2 gene is expressed most actively at the stationary growth phase and that the Hcp2 protein is secreted via the T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and does not contribute to virulence in or colonization of tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with the suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition with yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive under conditions where it has to compete with other microorganisms for resources.