Although dynamic light scattering is usually

Although dynamic light scattering is usually click here applied to determine the diameter distribution of spherical particles, it also facilitates the understanding of size distribution of dispersed carbon nanotubes [35–38]. Prior to centrifugation, the average particle size of 5 μg/ml PEI-NH-SWNTs and PEI-NH-MWNTs was the highest among the concentrations tested, due possibly to the inhomogeneous nature of the suspension. After centrifugation, the average particle

size of 5 to 100 μg/ml PEI-NH-SWNTs and PEI-NH-MWNTs in the supernatant was 229 ± 8 to 291 ± 34 and 287 ± 8 to 433 ± 102 nm, which were significantly lower than those before centrifugation (Figure 6). In addition, when the particle size of different concentrations of PEI-NH-SWNTs or PEI-NH-MWNTs was compared, no significant difference was observed. These results indicate that the centrifugation procedure effectively Wnt inhibitor reduced the particle size and increased the homogeneity of PEI-NH-CNTs. Figure 6 Average particle size of PEI-NH-SWNTs and PEI-NH-MWNTs before and after centrifugation. The average

particle diameters of 5, 50, and 100 μg/ml of PEI-NH-SWNTs (A) or PEI-NH-MWNTs (B) before and after removal of large aggregates through centrifugation was analyzed by dynamic light scattering. Before centrifugation, PEI-NH-SWNTs or PEI-NH-MWNTs were solubilized in ddH2O at a concentration of 1 mg/ml and sonicated for 15 min; after centrifugation, PEI-NH-SWNTs or PEI-NH-MWNTs were centrifuged at 3,000 rpm for aminophylline 30 min to remove large aggregates. Error bars represent standard deviations (n ≥ 3). *p < 0.05 and **p < 0.01 compared to PEI-NH-SWNTs or

PEI-NH-MWNTs of the same concentration before centrifugation. Zeta potential of PEI-NH-CNTs The zeta potential of 1 mg/ml pristine or PEI-grafted carbon nanotubes at 25°C and neutral pH was determined through dynamic light scattering. The zeta potential of pristine SWNTs and MWNTs was negative (Figure 7), similar to those reported in the literature [39, 40]. As expected, PEI functionalization increases the positive charge on the surface of PEI-NH-CNTs, resulting in positive zeta potentials, which were higher in PEI-NH-MWNTs compared to PEI-NH-SWNTs (Figure 7). The stability of PEI-NH-CNT suspension may therefore be maintained by electrostatic repulsion contributed by the cationic PEI. Figure 7 Zeta potential of pristine and PEI-functionalized carbon nanotubes. The zeta potential of 1 mg/ml pristine or PEI-grafted carbon nanotubes at 25°C and neutral pH was determined by dynamic light scattering. Error bars represent standard deviations (n ≥ 3). **p < 0.01 compared to PEI-NH-SWNTs.

The resulting plasmids were conjugated into S meliloti via E co

The resulting plasmids were conjugated into S. meliloti via E. coli S17-1 to introduce deletions by allelic exchange. Production of mutant strains was confirmed by PCR reactions designed to amplify DNA fragments spanning the gene of interest. CAS siderophore assay Chrome azurol S (CAS) assay mixtures for siderophore detection were prepared as described by Schwyn and Neilands Selleckchem FK506 [33]. Supernatants of S. meliloti cultures grown in VMM were mixed 1:1 with a CAS assay solution. After equilibrium was reached, the absorbance at 630 nanometers was measured. The relative siderophore activity was determined by measuring optical density ratios of different cultures. Procedures for continuous

pH and pH shift growth experiments S. meliloti strains were grown in Vincent minimal medium (VMM) [57] at 30°C at either pH 7.0 or pH 5.75 for growth tests at continuous pH values. VMM medium was composed of 14.7 mM K2HPO4, 11.5 mM KH2PO4, 0.46 mM CaCl2, 0.037 mM FeCl3, 1 mM MgSO4, 15.7 mM NH4Cl, 10 mM sodium succinate, 4.1 μM biotin, 48.5 μM H3BO3, 10 μM MnSO4, 1 μM ZnSO4, 0.5 μM CuSO4, 0.27 μM CoCl2, and 0.5 μM NaMoO4. Triplicate samples were measured for optical density at 580 nm, twice a day, for 7 days. For pH shift experiments cells of three independent cultures were grown in 30 ml of VMM with pH 7.0 to an O.D.580 of 0.8. Cell cultures of each flask were then centrifuged (10,000 × g, 2 min, 30°C)

and the supernatant was discarded. The cell pellets were resuspended in 30 ml VMM with pH 5.75 or 30 ml VMM with pH 7.0 (control) and incubated at 30°C. At six time points cell suspension samples of 5 ml CP-690550 in vitro were harvested from each flask and immediately centrifuged (10000 × g, 1 min, 4°C). The resulting pellets were instantly frozen in liquid nitrogen for later RNA preparation. Cell suspension samples were harvested at 0, 5, 10, 15, 30, and 60 minutes following the pH shift. To determine the

number of viable cells, dilutions of S. meliloti cultures grown 30 minutes after pH shift were plated on TY agar and incubated overnight at 30°C. RNA isolation RNA was isolated according to the protocol published by Rüberg et al. [59]. Total RNA was prepared using the RNeasy mini kit (QIAGEN, Hildesheim, Germany). By ribolysation (30 s; speed, 6.5; Hybaid, Heidelberg, Germany) cells were disrupted in the RLT buffer Nintedanib (BIBF 1120) provided with the kit in Fast Protein Tubes (Qbiogene, Carlsbad, CA). Transcriptional profiling using the SM14kOligo whole genome microarray For microarray hybridization, three independent bacterial cultures from each condition were prepared as biological replicates for RNA isolation. Accordingly, for each time point, dual-fluorescence-labeled cDNA probes were prepared to hybridize with three slides, respectively. For each preparation of Cy3 and Cy5 labeled cDNAs, 10 μg of total RNA were used [60]. To each microarray, the cDNA of the pH 7.0 and pH 5.75 grown cultures were mixed and hybridized.

We tested the difference between

pairs using distance bas

We tested the difference between

pairs using distance based NP-MANOVA, which yielded p = 0.085 for unweighted UniFrac and p = 0.197 for weighted UniFrac. Thus the two gold standards were not significantly different. Figure 3A shows the unweighted UniFrac analysis colored to distinguish communities from the 10 individuals studied. Figure 3B shows the same scatter plot colored by storage method, and Figure 3C shows the plot colored by extraction method. The data emphasizes that individuals differ substantially from KU-60019 chemical structure each other, and that storage and extraction methods have less pronounced effects. Also present in each individual cluster are the two replicates from 1 cm apart, emphasizing the reproducibility of

the method. Statistical analysis was carried out by asking whether unweighted UniFrac distances were greater within groups than between groups, then 10,000 label permutations were used to generate an empirical P-value. Clustering by subject was highly significant (P < 0.0001). No significance was seen for clustering by extraction SCH 900776 in vivo method (P = 0.16) or storage method (P = 0.98). We conclude that overall clustering, when analyzed for presence or absence of different bacterial groups, is dominated by differences between individuals. Figure 4 shows the weighted UniFrac analysis, which takes into account information on relative abundance, comparing the influence of individual of origin (Figure 4A), extraction method (Figure 4B), or storage method (Figure 4C). Again the differences among subjects were highly significant (P < 0.0001), but now the differences due to extraction methods were also significant (P = 0.001). Differences due to storage method were not significant. Thus when the proportional

representation of different taxa is taken in to account, both Fossariinae the subject of origin and the extraction method exert significant effects. We next investigated whether significant clustering could be detected when each extraction method was compared individually to the collection of other extraction methods. Again UniFrac distances were analyzed for within group and between group comparisons, and an empirical P-value generated from 10,000 permutations. No significant clustering was seen in the unweighted analysis. However, using weighted UniFrac significant clustering was seen for the phenol-bead beating method (P = 0.041) and the Qiagen method (P = 0.0014). The strong effect of the Qiagen method was driven in part by the fact that the most samples were analyzed using the Qiagen method, so the sample size was relatively large. Comparison of each method to the two gold standards using NP-MANOVA showed that the phenol bead beating and PSP methods both achieved p = 0.001.

2014) The cross-linking data indicate that Asp440 of CP47 (numbe

2014). The cross-linking data indicate that Asp440 of CP47 (numbering according to Liu et al. 2014) is in van der Waal’s contact with

Lys102 of Synechocystis CyanoQ, and that Lys120 of Synechocystis CyanoQ is within 12 Å of both Lys59 and Lys180 of PsbO. Although Asp440 of CP47 is conserved in both Synechocystis and T. elongatus, Lys102 and Lys120 of Synechocystis CyanoQ are replaced by Thr105 and Asp123, respectively, in T. elongatus CyanoQ (3ZSU numbering) (Fig. S8). These cross-linked residues in CyanoQ are found in a region containing helices selleck α2a, α2b and α3 and the H2-H3 cavity (Jackson et al. 2010) (Fig. 4). Highly conserved residues Arg79 and Asp119 found in the H2–H3 cavity highlighted in Fig. 4d are therefore good candidates for interacting with PsbO, whereas residue Gln101 might interact with CP47 (Fig. S8). In contrast, a recent structural analysis of the isolated PSII complex from the red alga Cyanidioschyzon merolae suggests that PsbQ’ binds near to CP43 (Krupnik et al. 2013) rather than CP47. Given the significant structural differences between PsbQ and CyanoQ with regard the N-terminus and surface charge, we do not yet 17-AAG chemical structure exclude the possibility that PsbQ and CyanoQ bind at different locations in PSII. Summary We have provided evidence

that CyanoQ binds to PSII

complexes isolated from the thermophilic cyanobacterium T. elongatus, although the degree of association is dependent on the purification method. The crystal structures of CyanoQ and spinach PsbQ are very similar despite limited sequence identity with a four-helix bundle the common structural feature. This robust fold is likely to be conserved in the other members of the PsbQ family. Changes in the surface properties through mutation would explain how binding specificity could be altered to allow PsbQ-like proteins to bind outside PSII. Acknowledgements We thank the staff of Diamond Light Source for their assistance, and the BBSRC (BB/E006388/1 and BB/I00937X/1) and EPSRC (EP/F00270X/1) for financial support. Flucloronide We are grateful to Dr Miwa Sugiura for providing the His-tagged CP43 strain of T. elongatus, and Dr Diana Kirilovsky for sending the His-tagged CP47 strain. Special thanks to Dr Michael Hippler for mass spectrometry analysis. Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Electronic supplementary material Below is the link to the electronic supplementary material.

However this trial do not assess the efficacy of oxaliplatin rein

However this trial do not assess the efficacy of oxaliplatin reintroduction

after additional lines of therapy (ie, irinotecan and anti-EGFR or anti-VEGF therapy) and do not analyze the role of a real treatment holiday. The OPTIMOX 2 phase II trial randomised 216 patients to receive fluorouracil maintenance between FOLFOX administration versus a treatment holiday. The primary objective was the duration of disease control (DDC), calculated as the sum of the duration of PFS induced with the initial FOLFOX therapy and with the subsequent reintroduction of FOLFOX. But most importantly, after induction of a response, metastases were allowed to progress back to baseline levels before FOLFOX was reintroduced. It was observed that continuing treatment with a maintenance chemotherapy led to a longer PFS, compared with pausing treatment (8.7 months vs LDE225 datasheet 6.9 months, P = 0.009) but overall survival data were

not available [39, 40]. DDC was almost identical in both arms (12.9 months vs 11.7 months, P not significant and duration of CFI seemed to depend on different clinical prognostic factors including Eastern Cooperative Oncology Group performance status, lactate dehydrogenase and alkaline phosphatase levels, number of metastatic sites. These data showed the possibility of identifying a favourable prognosis group which could benefit from an intermittent strategy. The COIN phase III study randomized 1630 patients with untreated metastatic colorectal cancer to receive either continuous oxaliplatin and fluoropyrimidine combination (arm A), continuous selleck chemicals llc chemotherapy plus cetuximab (arm B), or intermittent (arm C) chemotherapy. In arms A and B, treatment continued until development of progressive disease, cumulative toxic effects, or the patient chose to stop. In arm C, patients who had not progressed after six cycles of chemotherapy started a treatment holiday until evidence of disease progression, when the same treatment was restarted. Median survival was 15.8 months in arm A vs 14.4 months in arm C (hazard ratio 1.084, 80% CI 1.008–1.165). In the per-protocol population, more patients on continuous Protein kinase N1 than on intermittent treatment

had grade 3 or worse haematological toxic effects (15% vs 12%), whereas nausea and vomiting were more common on intermittent treatment (2% vs 8%). Other grade 3 or worse toxicities (such as peripheral neuropathy and hand–foot syndrome) were more frequent on continuous than on intermittent treatment [41]. Studies evaluating efficacy and feasibility of biological therapy administered during chemotherapy-free interval The NORDIC VII multicenter phase III trial randomly assigned 571 previously untreated patients to receive the standard Nordic FLOX, cetuximab and FLOX, or cetuximab combined with intermittent FLOX. Median PFS was 7.9, 8.3, and 7.3 months for the three arms, respectively (not significantly different). But OS was almost identical for the three groups (20.4, 19.7, 20.

Typhimurium (SB300; 200 CFU) harboring ampicillin resistant plasm

Typhimurium (SB300; 200 CFU) harboring ampicillin resistant plasmid pM973. The colonization SAHA HDAC in vivo efficiency of the challenged strain was evaluated at various host sites at day 3 post challenge (p.c.). Evaluation of serum and gut antibody response To measure the mucosal immune response, serum

IgG and secretory gut IgA responses were quantified by Western blot as described previously [34, 48]. Serum and gut washes were collected at day 30 p.v from MT5 and MT4 immunized mice and the PBS treated control mice. The protein fractions of lysates from the overnight-grown S. Typhimurium wild-type strain (SB300), ssaV mutant (MT5), ssaV and mig-14 double mutant (MT4) and S. Enteritidis P125109 (M1525) wild-type strain were separated on polyacrylamide gels and transferred to nitrocellulose membrane. The membrane was treated with suitably diluted serum sample or gut washes followed by incubation with conjugated α-mouse IgG (for serum; Santa cruz) and α-mouse IgA (for gut wash; Santa cruz). The blots were developed by ECL BTK inhibitor kit (Thermo Scientific). Statistical analysis Statistical analyses were performed

using the two-way ANOVA (GraphPad Prism 5). p < 0.05 was considered statistically significant. Results and discussion Additional mig-14 mutation in S. Typhimurium ssaV mutant shows significant attenuation in immunocompromised mice The attenuation of MT5 and MT4 strains in various immunocompromised mice was analyzed by normal infection experiment at day 4 p.i. In our initial observations, equivalent loads of MT5 and MT4 strains were detected in the cecal content of Nos2 −/−, Il-10 −/− mice (Figure 1A) whereas, MT4 showed reduced colonization in spleen and liver (Figure 1B, C and D) as compared to MT5. Similar experiment

was carried out to assess the performance of MT4 in wt C57BL/6 and CD40L −/− mice. It was observed that neither MT4 nor MT5 colonized spleen and liver of CD40L −/− and wild-type C57BL/6 mice (Figure 1C-D). Branched chain aminotransferase However, MT4 (ssaV, mig-14 mutant) colonized the mLN of wild-type mice as efficiently as MT5 (ssaV mutant) (Figure 1B). We also tested the attenuation profile in terms of competitive index of mig14::aphT single mutant against wild-type S. Typhimurium strain; it was appreciable that the mig14::aphT single mutant has reduced ability to colonize to systemic sites (Additional file 1: Figure S1 and Additional file 1: Figure S2); however, this reduced colonization in liver and spleen was not as sharp as in case of C57BL/6 mice infected with ssaV mutant MT5 (compare Additional file 1: Figure S2 with Figure 1C,D). Overall the data demonstrates that the deletion of mig-14 in the ssaV knockout background does not allow S. Typhimurium to colonize the systemic sites like liver and spleen in severely immunocompromised mice (Figure 1C and D). Figure 1 Analysis of MT4 attenuation in comparison to MT5 in Nos2 −/− , Il-10 −/− , CD40L −/− and wild-type C57BL/6 mice.

4a–f) was highly reproducible Fig  2 3D-landscapes of selected g

4a–f) was highly reproducible. Fig. 2 3D-landscapes of selected gel areas. Relative spot intensities from controls (a, c, e) and RF-EME exposed cells (b, d, f) are depicted as spot heights to demonstrate the specific induction of some proteins relative to the local spot environment. The indicated proteins are also listed in Table 1 Fig. 3 Identification details of isolated 2D gel spots. After tryptic digestion and peptide find more separation by nano-flow

liquid chromatography, isolated peptides were fragmented in an ion trap mass spectrometer. a–c Peptides identified in the spot identified as ubiquitin carboxyl-terminal hydrolase 14 (z, peptide charge; Score, Spectrum Mill peptide score; SPI, scored peak intensity). b Assignment of identified peptides to protein sequence. c MS2 spectrum of the peptide AQLFALTGVQPAR. d–f Peptides identified in the spot identified as 26S protease

regulatory subunit 6B, e assignment of identified peptides to protein sequence, f MS2 spectrum of the peptide ENAPAIIFIDEIDAIATK Fig. 4 The RF-EME induced increase of 35S incorporation rates was reproducibly observed Selleck PLX4032 in different cell types. a b and c, d show two independent experiments with Jurkat cells. e, f is a representative example for cultured human fibroblasts showing the highest induction of 35S incorporation rates by RF-EME, g, h shows a representative example of quiescent (metabolically inactive) primary human white blood cells (WBC). Here, RF-EME hardly induced ID-8 detectable increases in 35S incorporation rates; compared to untreated controls (g), activated WBC (i) displayed higher 35S incorporation rates, RF-EME induced a further increase in 35S incorporation rates (j), which indicates that activity renders cells sensitive to RF-EME Fibroblasts Cultured human fibroblasts showed the highest level of responsiveness to RF-EME (Fig. 4e, f; Table 2) with an average protein synthesis increase of 128 ± 22% (three independent experiments). Thirteen of the fourteen proteins whose rate of de novo synthesis was increased in Jurkat cells were also synthesized at a higher rate in fibroblasts. As well as these, the rates of synthesis of annexin

A1 and A5 were found to be significantly increased (Table 2). This finding suggests that the proteome alterations in responsive cells induced by RF-EME exposure are characteristic for this kind of cell stress. White blood cells Primary mononuclear cells isolated from peripheral blood (white blood cells, WBC) responded only marginally to RF-EME (Fig. 4g, h; Table 3). The apparent increase in 35S incorporation was less than 10%, which is within the margin of error of the applied methodology. Inflammatory stimulation of WBCs by treatment with lipopolysaccharide and phytohaemagglutinin increased the level of protein synthesis by these cells (compare Fig. 4g–i), which is consistent with the induction of cell proliferation as previously described in more detail (Traxler et al. 2004).

Photosynth Res 46(1–2):93–113 Arnold WA (1991) Experiments Photo

Photosynth Res 46(1–2):93–113 Arnold WA (1991) Experiments. Photosynth Res 27(2):73–82 Barber J (2004) Engine of life and big bang of evolution: a personal perspective. Photosynth Res 80(1–3):137–155 Benson AA (2002) Paving the path. Annu Rev Plant Biol 53:1–25 Calvin M (1989) Forty years of photosynthesis and related activities.

Photosynth Res 21(1):1–16 Chance B (1991) Optical method. Annu Rev Biophys Biophys Chem 20:1–28 Clayton RK (1988) Memories of many lives. Photosynth Res 19(3):205–224 Devault D (1989) Tunneling enters biology. Photosynth Res 22(1):5–10 Drews G (1996) Forty-five years of developmental biology of photosynthetic bacteria. KPT-330 Photosynth Res 48:325–352 Duysens LNM (1989) The discovery of the two photosynthetic systems: a personal account. Photosynth Res 21(2):61–79 Feher G (1998) Three decades of research in bacterial photosynthesis and the road leading to it: a personal account. selleck chemicals llc Photosynth Res 55(1):1–40 Feher G (2002) My road to biophysics: picking flowers

on the path to photosynthesis. Annu Rev Biophys Biomol Struct 31:1–44 Forti G (1999) Personal recollections of 40 years in photosynthesis research. Photosynth Res 60(2–3):99–110 French CS (1979) Fifty years of photosynthesis. Annu Rev Plant Physiol 30:1–26 Frenkel AW (1993) Recollections. Photosynth Res 35(2):103–116 Fujita Y (1997) A study on the dynamic features of photosystem stoichiometry: accomplishments and problems for future studies. Photosynth Res 53(2–3):83–93 Fuller RC (1999) Forty years of microbial photosynthesis research: where it came from and what it led to. Photosynth Res 62(1):1–29 Gaffron H (1969) Resistance to knowledge. Annu Rev Plant Physiol 20:1–40 Gerhart D (1996) Forty-five years of developmental biology of photosynthetic bacteria. Photosynth Res 48(3):325–352 Gest H (1994) A microbiologist’s odyssey: bacterial viruses to photosynthetic bacteria. Photosynth Res 40(2):129–146 Gest H (1994) Discovery of the heliobacteria. Photosynth Res 41(1):17–21 Gest H (1999) Memoir of a 1949 railway journey with photosynthetic bacteria. Photosynth Res 61(1):91–96 Gibbs M (1999) Educator and editor. Annu Rev Plant

Physiol Plant Mol Biol 50:1–25 Good NE (1986) Confessions of a habitual selleck chemical skeptic. Annu Rev Plant Physiol 37:1–22 Gunsalus IC (1984) Learning. Annu Rev Microbiol 38:1–26 Hatch MD (Hal) (1992) I can’t believe my luck. Photosynth Res 33(1):1–14 Hill R (1975) Days of visual spectroscopy. Annu Rev Plant Physiol 26:1–11 Jagendorf AT (1998) Chance, luck and photosynthesis research: an inside story. Photosynth Res 57(3):215–229 Joliot P (1993) Earlier researches on the mechanism of oxygen evolution: a personal account. Photosynth Res 38(3):214–223 Kamen MD (1986) A cupful of luck, a pinch of sagacity. Annu Rev Biochem 55:1–34 Kamen MD (1989) Onward into a fabulous half-century. Photosynth Res 21(3):137–144 [Also see Kauffman GB (2002) Martin D.

hydrophila subsp

hydrophila subsp. Selleckchem Daporinad hydrophila CECT 839T 130 – - 120 102 114 108 79 81 22 Environment, Tin of milk with a fishy odor – NA, NA, NA   A. hydrophila subsp. ranae CIP 107985 131 – - 121 103 115 109 80 82 101 Non-human, Frog I NA, Thaïland, NA   A. hydrophila CECT 5734 163 – - 150 132 144 137 104 12 127 Non-human, Fish I Valencia, Spain, 1987   A. hydrophila subsp. hydrophila CCM 2280 171 – - 69 139 152 145 111 115 134 Non-human, Snake – NA, NA, 1963   A. hydrophila subsp. hydrophila CCM 2282 172 – - 158 140 153 146 47 116 135 Non-human,

Nile Monitor ND NA, NA, 1963   A. hydrophila subsp. hydrophila CCM 4528 174 – - 160 15 17 148 13 118 137 Human, Stool ND NA, Czech Republic, 1993 A. veronii (n=71) BVH22 13 – - 13 11 12 4 8 11 12 Human, Wound I Alès, Fr, 2006   BVH23 13 – - 13 11 12 4 8 11 12 Human, Wound I Saint-Brieux, Fr,2006   BVH25b 13 – - 13 11 12 4 8 11 12 Human, Respiratory tract I Saint-Brieux, Fr,2006   BVH26a 13 – - 13 11 12 4 8 11 12 Human, Wound I Saint-Brieux, Fr,2006   BVH27a 13 – - 13 11 12 4 8 11 12 Human, Wound I Reunion Island, Fr,2006   BVH28a 13 Epigenetics activator – - 13 11 12 4 8 11 12 Human, Wound

I Reunion Island, Fr,2006   BVH61 46 5 D 46 29 31 31 34 34 40 Human, Stool I Antibes, Fr,2006   BVH71 54 5 D 46 29 31 31 26 34 40 Human, Stool ND Martinique Island, Fr, ND   BVH47 33 – D 33 29 31 31 26 16 31 Human, Blood I Roubaix, Fr,2006   ADV102 33 – D 33 29 31 31 26 16 31 Human, Stool ND Montpellier, Fr, 2008   BVH18 10 – - 10 9 10 10 7 9 9 Human, Wound I Villeneuve sur Lot, Fr, 2006   AK249 Vitamin B12 10 – - 10 9 10 10 7 9 9 Environment, Water lake   Annecy, Fr, 1998   ADV129 85 8 H 78 64 74 69 56 56 67 Human, Stool ND Montpellier, Fr, 2009   ADV133 89 8 H 82 64 74 69 56 56 67

Human, Wound I Montpellier, Fr, 2010   BVH 90 66 7 G 61 6 58 55 45 43 53 Human, Stool I Dunkerque, Fr, 2006   AK236 106 7 G 61 6 58 55 45 68 53 Environment, Water lake – Annecy, Fr, 1998   BVH37 25 – - 25 21 23 24 20 19 23 Human, Blood I La Roche sur Yon, Fr, 2006   BVH46 25 – - 25 21 23 24 20 19 23 Human, Blood I Roubaix, Fr, 2006   BVH56 42 4 E 42 36 40 24 32 6 23 Human, Blood I Versailles, Fr, 2006   ADV101 74 4 E 42 57 40 24 32 19 23 Human, Stool ND Montpellier, Fr, 2008   A. veronii bv. veronii CECT 4257T 143 – - 131 114 125 120 11 19 110 Human, Respiratory tract I Michigan, USA, NA   A.

**AmpR: Ampicillin resistance, KanR: Kanamycin resistance, TetR:

**AmpR: Ampicillin resistance, KanR: Kanamycin resistance, TetR: Tetracycline resistance. Figure 1 Construction of mutant strains. ORFs are indicated by boxed arrows (not drawn to scale). The locations Palbociclib clinical trial of the primers used to amplify the fragments and generate the deletions are indicated by solid arrows. The dash line box indicated

the location of the deletion of chromosomal sequence and insertion of an antibiotic resistant cassette (cat or aphA3). (a), (b), (c), and (d) are diagrams for operons cj0309c-cj0310c, cj0423-cj0425, cj1169c-cj1170c and cj1173-cj1174, respectively. The involvement of the PSMR efflux systems in aerobic and oxidative stress survival in C. jejuni was tested next. In this experiment,

the ability of bacterial cells to grow on MH agar was assessed under different oxygen levels (5% O2 or 18.5% O2). The PSMR mutants and their wild-type strain grew comparably under microaerobic environment (5% O2) (Figure 2A). However, under aerobic conditions (18.5% O2), all mutants showed declined growth compared with the wild-type strain (Figure 2A) and the decline was more prominent with KO73Q and DKO01Q (~100 fold difference). Kinase Inhibitor Library To confirm the phenotype associated with the mutant strains, a partial complementation of the double knock-out mutant with the wild-type copy of cj1173-cj1174 was constructed as described in material and methods. As shown in Figure 2B, the complementation partly restored the mutant’s ability to grow under high oxygen tension. These results indicated that the two PMSR systems facilitate C. jejuni adaptation to aerobic environment. Additionally, we performed disk diffusion assay using hydrogen peroxide, cumene, and menadione, which did not show any significant differences (p > 0.05) in bacterial growth inhibition between the wild-type and PSMR mutant strains (result not shown), suggesting that the two putative efflux systems are not directly involved in the resistance to the examined

oxidants. Figure 2 Comparison of oxygen tolerance of C. jejuni wild-type NCTC 11168 and its mutant strains. For (A) and (C), 5 μl of serial dilutions (from left to right: 107-101 CFU/ml) of overnight cultures were spotted onto MH agar plates and incubated at Sodium butyrate either 18.5% or 5% O2. For (B), 5 μl of serial dilutions (from left to right: 105-101 CFU/ml) of overnight cultures were spotted onto MH agar plates and incubated at either 18.5% or 5% O2. Results are representative of three independent experiments. Since the PSMR mutants demonstrated enhanced susceptibility to the high-level oxygen concentration, we further examined their contribution to colonization of chickens. Both the wild-type and the mutant strains were equally motile as determined by swarming on semi-solid agar.