Survey teams worked with as many arriving groups as possible, interviewing and swabbing as many pilgrims as possible in each group after they passed through immigration. In each survey, pilgrims were asked for their consent to participate. A nasopharyngeal and throat swab were obtained after the interview. The questionnaire in the arrival

survey included questions about pilgrims’ demographics (age, gender, occupation, and nationality), medical history (chronic disease and smoking), vaccination history (including Selleckchem Selumetinib separate questions about vaccination against pandemic influenza A(H1N1) and against seasonal influenza), knowledge about H1N1 influenza (symptoms, transmission, and ways to avoid), and compliance with wearing face masks. The questionnaire used in the departure survey included only questions about age, gender, and pandemic influenza A(H1N1) vaccination history. Respiratory specimens were placed in viral transport media (VTM) at the point of collection and transported to Jeddah Regional Laboratory where they were stored at −80°C before testing. Specimens selected for analysis were thawed and subjected to total nucleic acid extraction using Corbett X-tractor Gene (Qiagen, Hilden, Germany) and RNA DNA CorProtocol 25101 (Qiagen). Extracts were then tested using the xTAG Respiratory Viral Panel (RVP) FAST assay (Luminex Molecular Diagnostics Inc.,

Toronto, Canada) per manufacturer’s instructions. The xTAG RVP FAST is a qualitative selleck compound multiplex amplification assay allowing the simultaneous detection of multiple viral nucleic acid targets. In addition to influenza A and B, this test can detect respiratory syncytial virus, parainfluenza virus 1, 2, 3, and 4; rhino-enterovirus, adenovirus, and minor respiratory viruses: coronaviruses, metapneumovirus, and bocavirus. Amplification of specific matrix target was used to detect influenza A and B. Seasonal influenza H1 and H3 subtypes were detected after amplification with hemagglutinin-specific primers and probes. Specimens positive for influenza A but negative for seasonal H1 and H3 were subjected www.selleck.co.jp/products/Fludarabine(Fludara).html to additional PCR amplification to detect pandemic H1 and avian H5 (Qiagen

Artus Influenza/H1 RG/LC for H1N1 and TIB MOLBIOL, LightMix kit, Berlin, Germany for H5N1). Demographics, medical history, vaccination history, knowledge of H1N1 influenza, and compliance with infection control practices among arriving pilgrims were analyzed as frequency distributions. Differences in the prevalence of respiratory viruses between the arriving and departing pilgrims were examined using chi-square test or Fisher exact, as appropriate. Differences in the prevalence of respiratory viruses between potential confounding groups such as age groups and getting pandemic influenza A(H1N1) vaccine were examined using chi-square test or Fisher exact, as appropriate. All p values were two-tailed. p Value <0.05 was considered as significant. SPSS (release 17.

Because a well-structured nucleolus was not observed in the nuclear sections of a large number of cells (i.e. up to 30% of exponentially growing epimastigotes), only nucleoli present as a single granular body were considered in our morphometric analysis, based on previous work (López-Velázquez et al., 2005). Figure 2a depicts representative micrographs of exponential and stationary nuclei in which the nucleolus (No) may be noted. The peripheral heterochromatin

is also find more depicted (H). Figure 2b shows the box-plot distribution of the measured area of the nucleoli, indicating that the median nucleolar area calculated based on exponentially growing cells is significantly larger (>2-fold, P<0.0001) than that of cells at the stationary phase. The nucleoli of trypanosomatids are not structured into three different components as in mammalian cells, but rather ABT 888 only into granular and dense fibrillar components (Ogbadoyi et al., 2000; López-Velázquez et al., 2005). Here, the granular component is clearly dominant in the nucleoli of exponentially growing cells (Fig. 3a); its presence is less evident in nuclei from the stationary phase

(Fig. 3b). In agreement with these differences in nucleolar architecture, a higher density of granules (presumably ribosomes) in the cytoplasm (Cy) of the exponentially growing cells was also noted (Fig. 2a). Regarding the heterochromatin appearance, a closer examination of this nuclear structure is presented in Fig. 4 where a compact and relatively homogeneous material is indicated by arrows. So far we have considered the nucleolus as a fibrogranular structure independent from heterochromatin. Nevertheless, localized interactions between these two nuclear compartments can be observed. The blockade of protein synthesis, as with cycloheximide, results in early alteration of pre-rRNA processing

and ribosome formation (Hadjiolov, 1985). Moreover, this drug can profoundly affect nucleolar organization (Ghosh & Paweletz, 1994). To analyse Selleck Ribociclib the potential effect of cycloheximide on the nucleolar size of epimastigotes, an exponentially growing culture was diluted and divided into three parts. Cycloheximide was added to one part, the drug vehicle was added to the second part, and the rest of the culture was left untreated. Cellular samples were then processed 1 and 2 days later for nucleolar analysis, as described above. Figure 5a indicates that cells treated with cycloheximide do not grow and that their nucleoli appear slightly smaller than those of control cells (Fig. 5b and c). The growth rate and the nuclear architecture of the cells treated with the drug vehicle were similar to those observed in the untreated control cells. Finally, in terms of transcription, run-on assays showed a fivefold diminished UTP incorporation rate in nuclei isolated from cells treated with cycloheximide for 24 h, as compared with control-cell nuclei (data not shown). The nucleoli of T.

, 1983), the Gammaproteobacteria Escherichia coli (Javelle et al., 2005) and Azotobacter vinelandii (Kleinschmidt & Kleiner, 1978),

to which we can now add the Betaproteobacteria H. seropedicae. Thus membrane association of GS could be functionally relevant in bacteria. To determine whether the presence of ammonium in the culture medium would alter the content and dynamics of the membrane-associated proteins in H. seropedicae we used 2D-PAGE to analyze the membrane fraction of cells grown in 20 mM NH4Cl (nitrogen sufficiency, Alpelisib price +N), 5 mM glutamate (nitrogen limitation, −N) or 5 mM glutamate and collected 5 min after the addition 1 mM NH4Cl to the medium (ammonium shock, SH). Comparative analysis of the 2D-PAGE images indicated protein spots with reproducible different levels in the treatments (Table 2). Spot 151 in the SH treatment was over 10 times more abundant in conditions of ammonium shock and nitrogen limitation when compared with nitrogen sufficiency. The same spot did not show altered abundance when we compared SH AZD2281 with −N (Fig. 2). This suggests that the amount of this protein associated with the membrane is regulated by the availability of nitrogen during cell growth but its cellular localization is not affected

by an ammonium shock. Spot 151 was identified by MALDI-TOF analysis as the product of the orf1 gene in the orf1amtBglnK operon (Table 2). Previous bioinformatic analysis indicated that orf1 encodes a noncytoplasmic protein with unknown localization (Noindorf et al., 2006). A signal peptide (residues 1–21) was found using signalp 2.0, and the experimentally

determined pI (5.37) and molecular weight (MW; 28 kDa) of Orf1 are in good agreement with calculated values for the mature polypeptide (pI of 5.32 and MW of 26 kDa). Orf1 was not predicted to contain any transmembrane helices. A Pfam domain search indicated the presence of the Gcw-chp domain (E value=1.2e−48); this domain is present in a group of bacterial proteins of unknown function found predominantly in Proteobacteria. blastp analysis identified Orf1 homologues in members of the Alpha-, Gamma- and Epsilonproteobacteria. Fossariinae We propose to designate the gene located upstream of H. seropedicae glnK as nlmA and the gene product as NlmA. The expression of nlmA has been studied already (Noindorf et al., 2006). Studies of a lacZ gene fusion indicated that the gene is cotranscribed with glnK and amtB from a σ54-dependent promoter that is activated by the transcriptional regulator NtrC under nitrogen-limiting conditions. The proteomic data presented here support the proposed mechanism of transcription regulation. Quantitative differences were observed for spots 195 and 196 between the treatments (Table 2). Spot 195 was not detected when cells were grown in +N and was over six times more abundant after an ammonium shock when compared with the −N condition (Fig. 2).

2b). This suggested that, in addition to the previously identified promoter (P1), there may be a second promoter MK-2206 in vitro (P2) that was specifically activated in the WT strain in solid

culture. The transcription start sites controlled by these two promoters were identified by high-resolution S1 nuclease mapping, as shown in Fig. 2c (refer also to Fig. S4). The putative −35 and −10 sequences, which are similar to the consensus sequences of the Streptomyces spp. housekeeping gene promoters (TTGACW-N16−18-TAGWWT, where W=A or G), were located in P1, but not in P2. We identified an AdpA-binding site approximately 90 bp upstream of the transcription start site in P1 (Fig. 2c). We introduced a mutation (5′-ATCACTAGTG-3′) into the AdpA-binding sequence (5′-TGTCCGGATT-3′). By electrophoretic mobility shift assay (EMSA), we confirmed that AdpA could not bind to the 40-bp DNA fragment (position −113 to −74, relative to the transcription start site in P1) containing the mutated AdpA-binding sequence (Fig. 3a). We then examined the effect of this mutation on the generation of transcripts from the two promoters. To this end, we introduced this mutation into pTYMbldK-g, and thereby generated pTYMbldKmut. When pTYMbldKmut was integrated into the selleck products chromosome of the ΔbldKB-g strain, aerial mycelium formation was restored (Fig. 3b). Furthermore, the bldKB-g transcription profiles in the ΔbldKB-g SGR3787∷pTYMbldKmut strain, grown

in both SMM liquid (Fig. 3d) and on YMPD agar (Fig. 3e), were similar to those in the ΔbldKB-g SGR3787∷pTYMbldK-g strain. These results indicated that binding of AdpA to the sequence upstream Edoxaban of bldKB-g appeared not to influence the transcription of the bldK-g gene cluster. Thus, we concluded that reduced bldKB-g transcription in the ΔadpA strain grown in SMM liquid was an indirect consequence of AdpA being absent. The transcription

profile of bldKB-g in the ΔbldKB-g SGR3787∷pTYMbldK-g strain grown on YMPD agar was very different from that in the WT strain, as shown in Figs 2b and 3d. We speculate that this difference may be explained by the different chromosomal location of the operon: the pTYM vector was integrated into the coding sequence for SGR3787. Otherwise, the presence of two copies of bldKA-g, bldKC-g, bldKD-g, and bldKE-g in the complement strain may affect the transcription of bldKB-g by an unknown mechanism. It is worth noting that, unlike the entire bldK-g operon, the bldKB-g gene alone could not be introduced into either the ΔbldKB-g strain or the WT strain. These results suggested that regulation of the bldK-g operon was highly complex and that imbalanced expression of the bldK-g genes might cause a growth defect. The complex nature of bldK-g operon regulation was further implied by the remarkable differences between the transcription profiles of cells grown in SMM liquid and on YMPD agar. We have identified the BldK oligopeptide ABC transporter in S. griseus.

In multivariate regression analysis, treatment arm, baseline total body mass, CDC disease category, plasma HIV-1 RNA and HOMA index at baseline were independent significant predictors for change in body mass over 48 weeks. Patients in the ATV/r arm had a 2102 g [95% confidence interval (CI)

644, 3560 g; P=0.006] greater increase in total body mass compared with those on SQV/r. For the change in limb fat, treatment arm, baseline limb fat, age, CDC category, plasma HIV-1 RNA, LDL cholesterol and HOMA index were independent predictors. Patients in the ATV/r arm had a 614 g (95% CI 173, 1055 g; P=0.008) greater increase in limb fat compared with patients on SQV/r. Independent predictors for the change in SAT over 48 weeks were treatment arm, baseline SAT, age, ethnicity and CDC category. The increase in SAT was higher in the ATV/r arm (difference between arms 14 cm2; 95% CI 0.3, 28 cm2; P=0.048). The Selleckchem PS341 Framingham risk score could be calculated in 83 patients (SQV/r arm, n=40; ATV/r arm, n=43). The score was comparable between treatment arms at baseline [SQV/r arm, mean 3.6%, standard

deviation (SD) 3.5%; ATV/r arm, mean 3.5%, SD 5.6%], and remained stable after 48 weeks (data not shown). Plasma creatinine increased significantly (P<0.001) in both arms (SQV/r arm, +9 ± 1 μmol/L; HSP targets ATV/r arm, +6 ± 1 μmol/L) with no significant difference between arms (P=0.154). In the ITT analysis, eGFR Bay 11-7085 calculated using C&G, MDRD-4, MDRD-6 and CKD-EPI decreased significantly in the SQV/r arm. eGFR calculated using C&G and MDRD-6 remained stable in the ATV/r arm, but eGFR calculated using CKD-EPI and MDRD-4 decreased significantly. In contrast, eGFR calculated using cystatin C improved significantly in both arms. The difference in the change in eGFR between the arms was only significant using C&G (SQV/r vs. ATV/r, –9 ± 3 mL/min/1.73 m2 with a smaller change in the ATV/r arm; P=0.009). In the OT analysis, the same trend in the change in eGFR was observed in both arms, but none of these differences remained significant between the arms (Fig. 3). In the multivariate analysis, baseline eGFR calculated using C&G and

plasma HIV-1 RNA were independent significant predictors for the change in eGFR. Treatment arm was no longer a significant predictor of the change in eGFR. Minor nonsignificant decreases in plasma phosphate over 48 weeks were seen in both arms (SQV/r arm, −0.03 ± 0.04 mmol/L; ATV/r arm, –0.07 ± 0.04 mmol/L) with no significant difference between the arms (P=0.458). Severe hypophosphataemia [AIDS Clinical Trials Group (ACTG) grade 3/4] was observed in five patients (SQV/r arm, n=2; ATV/r arm, n=3). Glucosuria with normoglycaemia occurred in one patient (ATV/r) during follow-up. Fanconi’s syndrome was not observed. The mean (SD) CD4 count increase over 48 weeks was+190 (111) and+161 (124) cells/μL in the SQV/r and ATV/r arms, respectively (ITT).

18 mg, and the heme content

(mol mol−1 of protein) was estimated to 0.93, based on pyridine hemochrome analysis. The absorption maximum of the reduced-minus-oxidized difference spectrum of the pyridine hemochrome compound was 549.7 nm, supporting the notion of a c-type cytochrome. Figure 2 shows optical spectra of the purified protein in the oxidized and reduced states. The absorption maxima of reduced protein are 551 and 416 nm in the alpha and Soret bands, respectively, and 410 nm in the Soret band of the oxidized protein. To estimate the redox potential, optical spectra in the visible region were recorded from protein diluted into redox selleck kinase inhibitor buffer containing potassium hexacyanoferrate (II) and potassium hexacyanoferrate (III) in different proportions. Inset (b) in Fig. 2 shows the extent of reduction as a function of the redox potential of the buffer. A midpoint potential of 261 mV was obtained by curve fitting. The thermodynamics of chlorate reduction by the cytochrome depends on the difference between this potential and the potential of the chlorate/chlorite redox couple at pH=7. An estimate for the latter can be obtained from data given by Thompson (1986). The standard potential (pH=0) of the ClO3−/HClO2 redox couple is given as +1.16 V. From this, and a pKa value of 2 for the HClO2, a value of +0.708 V is obtained for the midpoint potential of the chlorate/chlorite couple at pH=7. This is considerably

higher than the potential found for GSK J4 the cytochrome, with the consequence that electron transfer from the cytochrome to chlorate is a thermodynamically favorable reaction. In a previous paper (Bäcklund et al., 2009), the chlorate-dependent reoxidation of reduced cytochrome c in periplasmic extract was demonstrated. In order to further investigate the reaction between the purified 9-kDa cytochrome

c-Id1 and chlorate reductase, the chlorate-dependent oxidation of the reduced cytochrome in the presence of purified chlorate reductase was studied. until Figure 3 shows spectra obtained in the visible region up to 12 min after the addition of chlorate. The time course of the reaction was obtained by plotting the A552 nm as a function of time and is shown in the inset. The solid line in the inset shows the fit of a single exponential function to the time course, demonstrating that the reaction is first-order. Similar first-order kinetics were observed at all concentrations of cytochrome c investigated. The effect of the concentration of cytochrome c-Id1 on the initial rates obtained from the curve fits are shown in Fig. 4. In the concentration range investigated, initial rates appear to increase linearly with the substrate concentration, indicating a KM value substantially higher than the highest substrate concentration investigated (4 μM) under present conditions. Using the estimated concentration of chlorate reductase, a kcat/KM of 7 × 102 M−1 s−1 was calculated from the slope of the line in Fig. 4.

Three phages φVh1, φVh2, and φVh4 had an icosahedral head of 60–115 nm size with a long, noncontractile tail of 130–329 × 1–17 nm, belonged Crenolanib to the family Siphoviridae. φVh3 had an icosahedral head (72 ± 5 nm) with a short tail (27 × 12 nm) and belonged to Podoviridae. REA with DraI and PFGE of genomic DNA digested with ScaI and XbaI and cluster analysis of their banding patterns indicated that φVh3 was distinct from the other three siphophages. PFGE-based genome mean size of the four bacteriophages φVh1, φVh2, φVh3, and φVh4 was estimated to be about 85, 58, 64, and 107 kb, respectively. These phages had the property of generalized transduction as demonstrated by transduction with plasmid pHSG 396 with

frequencies ranging from 4.1 × 10−7 to 2 × 10−9 per plaque-forming unit, suggesting a potential ecological role in gene transfer among aquatic vibrios. Vibrio harveyi, a gram-negative marine bacterium, has been described as a significant pathogen of marine vertebrates and invertebrates (Austin & Zhang, 2006). V. harveyi causes luminescent bacterial

disease (LBD) CDK activity in larval shrimp, resulting in considerable economic loss to shrimp hatcheries world over (Lavilla-Pitogo et al., 1990; Karunasagar et al., 1994). Pathogenicity mechanism of V. harveyi has been attributed to various virulence factors such as production of proteases (Liu & Lee, 1999), siderophores (Owens et al., 1996), and hemolysin (Zhang et al., 2001). Besides these virulence factors, the association of a V. harveyi myovirus-like (VHML) bacteriophage is reported to impart virulence Fossariinae to V. harveyi (Austin et al., 2003). Munro et al. (2003) also demonstrated that naïve

strains of V. harveyi could be converted into virulent strains by infecting them with bacteriophage VHML. It was almost three decades ago that the first description of bacteriophages infecting luminescent bacteria was reported (Keynan et al., 1974). After a long gap of 25 years, bacteriophage-mediated toxicity of V. harveyi in Penaeus monodon by the transfer of a gene controlling toxin production was reported (Ruangpan et al., 1999), followed by the description of VHML associated with toxin-producing strains (Oakey & Owens, 2000; Oakey et al., 2002). There are also some reports on the isolation and characterization of lytic bacteriophages of V. harveyi from coastal ecosystem and shrimp culture ponds (Shivu et al., 2007). A lytic bacteriophage was evaluated as a biocontrol agent of V. harveyi and was reported to provide encouraging results (Vinod et al., 2006; Karunasagar et al., 2007). In our earlier work, we reported isolation of bacteriophages of V. harveyi from shrimp hatchery (Chrisolite et al., 2008). Here, we present our work on the characterization of four selected bacteriophages with broad spectrum of infectivity against luminescent V. harveyi isolates, considering their potential as biocontrol agent of LBD in shrimp hatcheries.

Three phages φVh1, φVh2, and φVh4 had an icosahedral head of 60–115 nm size with a long, noncontractile tail of 130–329 × 1–17 nm, belonged find protocol to the family Siphoviridae. φVh3 had an icosahedral head (72 ± 5 nm) with a short tail (27 × 12 nm) and belonged to Podoviridae. REA with DraI and PFGE of genomic DNA digested with ScaI and XbaI and cluster analysis of their banding patterns indicated that φVh3 was distinct from the other three siphophages. PFGE-based genome mean size of the four bacteriophages φVh1, φVh2, φVh3, and φVh4 was estimated to be about 85, 58, 64, and 107 kb, respectively. These phages had the property of generalized transduction as demonstrated by transduction with plasmid pHSG 396 with

frequencies ranging from 4.1 × 10−7 to 2 × 10−9 per plaque-forming unit, suggesting a potential ecological role in gene transfer among aquatic vibrios. Vibrio harveyi, a gram-negative marine bacterium, has been described as a significant pathogen of marine vertebrates and invertebrates (Austin & Zhang, 2006). V. harveyi causes luminescent bacterial

disease (LBD) Afatinib in larval shrimp, resulting in considerable economic loss to shrimp hatcheries world over (Lavilla-Pitogo et al., 1990; Karunasagar et al., 1994). Pathogenicity mechanism of V. harveyi has been attributed to various virulence factors such as production of proteases (Liu & Lee, 1999), siderophores (Owens et al., 1996), and hemolysin (Zhang et al., 2001). Besides these virulence factors, the association of a V. harveyi myovirus-like (VHML) bacteriophage is reported to impart virulence Rucaparib to V. harveyi (Austin et al., 2003). Munro et al. (2003) also demonstrated that naïve

strains of V. harveyi could be converted into virulent strains by infecting them with bacteriophage VHML. It was almost three decades ago that the first description of bacteriophages infecting luminescent bacteria was reported (Keynan et al., 1974). After a long gap of 25 years, bacteriophage-mediated toxicity of V. harveyi in Penaeus monodon by the transfer of a gene controlling toxin production was reported (Ruangpan et al., 1999), followed by the description of VHML associated with toxin-producing strains (Oakey & Owens, 2000; Oakey et al., 2002). There are also some reports on the isolation and characterization of lytic bacteriophages of V. harveyi from coastal ecosystem and shrimp culture ponds (Shivu et al., 2007). A lytic bacteriophage was evaluated as a biocontrol agent of V. harveyi and was reported to provide encouraging results (Vinod et al., 2006; Karunasagar et al., 2007). In our earlier work, we reported isolation of bacteriophages of V. harveyi from shrimp hatchery (Chrisolite et al., 2008). Here, we present our work on the characterization of four selected bacteriophages with broad spectrum of infectivity against luminescent V. harveyi isolates, considering their potential as biocontrol agent of LBD in shrimp hatcheries.

, 2010). We used six different Pseudomonas strains, four of which produce well-characterized secondary metabolites that inhibit root-pathogenic fungi. Pseudomonas fluorescens DR54 produces viscosinamide: a membrane-bound cyclic lipopeptide with biosurfactant properties and broad antifungal activity (Nielsen et al., 1999; Thrane et al., Birinapant cost 2000). Pseudomonas fluorescens CHA0 produces various extracellular metabolites, two of them being DAPG (2,4-diacetylphloroglucinol), which causes membrane damage in fungi (Pythium) and inhibits zoospores, and pyoluteorin, which inhibits the fungal respiratory chain (Keel et al., 1992; Laville et al., 1992). Pseudomonas sp. DSS73 produces amphisin, an extracellular

cyclic lipopeptide with biosurfactant selleck kinase inhibitor properties and broad antifungal activity (Sørensen et al., 2001; Nielsen & Sørensen, 2003), and Pseudomonas chlororaphis MA342 produces DDR (2,3-de-epoxy-2,3-didehydro-rhizoxin), a membrane-bound compound that inhibits mitosis in eukaryotic cells (Hökeberg et al., 1997; Brendel et al., 2007). Two Pseudomonas strains, P. fluorescens type strain DSM50090T (Deutsche Sammlung von Mikroorganismen und Zellkulturen) and P. fluorescens ATCC43928 (American Type Culture Collection), produce no known antagonistic secondary metabolites. We further included the well-suited food bacterium Enterobacter aerogenes SC (Christensen

& Bonde, 1985) as a positive control, and a treatment only with phosphate buffer, but without bacteria, as a negative control. The bacteria for the protozoan growth experiments were pure cultures grown on tryptic soy broth (TSB) medium (3 g L−1, Difco Bacto, Detroit) at 22 °C for 24 h. Bacteria were then diluted 1/10 in weak phosphate buffer (‘Neff’s modified amoeba saline’; Page, 1988), which yields bacterial cultures with 5–10 × 107 cells mL−1. This approach yields

more reproducible results than if a fixed cell number (e.g. 5 × 107 cells mL−1) is used for standard comparison between cultures. This is because different bacterial cultures with similar cell numbers may vary considerably with regard to carbon content, because cell sizes differ (Lekfeldt & Rønn, 2008). Bacterial cell size depends on the growth medium. Here, all Gefitinib bacteria were cultivated on the same medium and microscopic evaluation demonstrated that differences between cell sizes were negligible. No biofilm formation was observed in the current set-up, even though both bacteria and protozoa settled at the bottom of the experimental units (data not shown). The protozoa used in the experiments belong to several very distantly related protozoan lineages (Adl et al., 2007). They included three amoeboid Rhizaria (Cercomonadida) Cercomonas longicauda (SCCAP C 1), Neocercomonas jutlandica (SCCAP C 161), and Heteromita globosa (SCCAP H 251), three non-amoeboid Excavata (Bodonidae) Bodo caudatus (SCCAP BC 330), Bodo designis (UJ), and B.

, 2010). We used six different Pseudomonas strains, four of which produce well-characterized secondary metabolites that inhibit root-pathogenic fungi. Pseudomonas fluorescens DR54 produces viscosinamide: a membrane-bound cyclic lipopeptide with biosurfactant properties and broad antifungal activity (Nielsen et al., 1999; Thrane et al., RG7204 datasheet 2000). Pseudomonas fluorescens CHA0 produces various extracellular metabolites, two of them being DAPG (2,4-diacetylphloroglucinol), which causes membrane damage in fungi (Pythium) and inhibits zoospores, and pyoluteorin, which inhibits the fungal respiratory chain (Keel et al., 1992; Laville et al., 1992). Pseudomonas sp. DSS73 produces amphisin, an extracellular

cyclic lipopeptide with biosurfactant selleck chemical properties and broad antifungal activity (Sørensen et al., 2001; Nielsen & Sørensen, 2003), and Pseudomonas chlororaphis MA342 produces DDR (2,3-de-epoxy-2,3-didehydro-rhizoxin), a membrane-bound compound that inhibits mitosis in eukaryotic cells (Hökeberg et al., 1997; Brendel et al., 2007). Two Pseudomonas strains, P. fluorescens type strain DSM50090T (Deutsche Sammlung von Mikroorganismen und Zellkulturen) and P. fluorescens ATCC43928 (American Type Culture Collection), produce no known antagonistic secondary metabolites. We further included the well-suited food bacterium Enterobacter aerogenes SC (Christensen

& Bonde, 1985) as a positive control, and a treatment only with phosphate buffer, but without bacteria, as a negative control. The bacteria for the protozoan growth experiments were pure cultures grown on tryptic soy broth (TSB) medium (3 g L−1, Difco Bacto, Detroit) at 22 °C for 24 h. Bacteria were then diluted 1/10 in weak phosphate buffer (‘Neff’s modified amoeba saline’; Page, 1988), which yields bacterial cultures with 5–10 × 107 cells mL−1. This approach yields

more reproducible results than if a fixed cell number (e.g. 5 × 107 cells mL−1) is used for standard comparison between cultures. This is because different bacterial cultures with similar cell numbers may vary considerably with regard to carbon content, because cell sizes differ (Lekfeldt & Rønn, 2008). Bacterial cell size depends on the growth medium. Here, all Phospholipase D1 bacteria were cultivated on the same medium and microscopic evaluation demonstrated that differences between cell sizes were negligible. No biofilm formation was observed in the current set-up, even though both bacteria and protozoa settled at the bottom of the experimental units (data not shown). The protozoa used in the experiments belong to several very distantly related protozoan lineages (Adl et al., 2007). They included three amoeboid Rhizaria (Cercomonadida) Cercomonas longicauda (SCCAP C 1), Neocercomonas jutlandica (SCCAP C 161), and Heteromita globosa (SCCAP H 251), three non-amoeboid Excavata (Bodonidae) Bodo caudatus (SCCAP BC 330), Bodo designis (UJ), and B.