CIp20, which is a derivative of CIp10 [76], contains the URA3 and HIS1 markers. CIp20-GUP1 was linearized with StuI, transformed into C. albicans gup1Δ/gup1Δ to create the GUP1-reintegrant strain CF-Ca001. The integration of CIp20-GUP1 at the RPS1 locus was confirmed by PCR with primers TTGTATCACAACCCTCCC and GTGGTTGGAGCTTTGATG. The control strains were generated by transforming the parental strain (BWP17) and the homozygous C. albicans gup1Δ/gup1Δ with the empty CIp20 plasmid

linearized with StuI. Sensitivity to lipid biosynthesis inhibitors (i) Drop tests Drop tests were performed from YPD cellular young cultures suspensions, containing approximately 1 × 106 cells/ml. Ten-fold serial dilutions were made, and 5 μl of each suspension was applied on the selective media. Sapanisertib in vivo Results were scored after 3-5 days of incubation at 30°C. Selective conditions were as follow: clotrimazole (68.8 and 172 μg/ml), ketoconazole selleck inhibitor (106.3 and 265.7 μg/ml), fluconazole (30.6, 91.8 and 153 μg/ml) and fenpropimorph (60, 120 and 240 μg/ml), amphotericin B (25 μg/ml) and nystatin (2.5 μg/ml). All chemicals were obtained at the highest available grade from Sigma Aldrich. (ii) Methyl-blue diffusion test Alternatively, we assayed the sensitivity to lipid biosynthesis inhibitors with a methyl-blue-diffusion

plate test. Sterile filter disks (BBL) of 6 mm diameter were placed on top of YPD methyl-blue plates seeded with 5 ml of a wt or Cagup1Δ mutant strain young cultures. The filter disks were impregnated with 5 to 10 μl of the following drugs: clotrimazole (137.6 μg/ml), ketoconazole (212.6 μg/ml), fluconazole (91.8 μg/ml), fenpropimorph (80 μg/ml), amphotericin B (25 μg/ml) and nystatin (2.5 μg/ml). The plates were incubated at 30°C, and halos of inhibition were scored after 3 days. Again, all chemicals were obtained at the highest available grade

(Sigma-Aldrich). Filipin/Sterol fluorescence microscopy Sterol-lipid distribution was assessed in vivo using filipin. This was performed basically as described before [19, 40]. For fluorescence microscopy, cells were mounted directly on slides with a 10 μl drop of anti-fading agent Vectashield (Vector Laboratories) to Avelestat (AZD9668) overcome the instability of filipin, and immediately observed by light microscopy (LM). Colony morphology and differentiation To observe different colony morphology/differentiation, equal volumes of young cultures of each strain were diluted and spotted onto non-inducing (YPD at 30°C) and hyphal-inducing (Spider medium and on 10% FBS at 37°C) conditions, and also in YPD at 37°C. Cultures were allowed to grow for 3-5 days. Colonies on agar surface were observed under magnifying lens (10 times) and photographed. Spider medium colonies were also thoroughly observed by light microscopy.

J Bacteriol 2005,187(5):1591–603.PubMedCrossRef 11. Patten CL, Kirchhof MG, Schertzberg MR, Morton RA, Schellhorn HE: Microarray analysis of RpoS-mediated gene expression in Escherichia coli K-12. Mol Genet Genomics 2004,272(5):580–591.PubMedCrossRef 12. Mandel MJ, Silhavy RG7112 chemical structure TJ: Starvation for different nutrients in Escherichiacoli

results in differential modulation of RpoS levels and stability. J Bacteriol 2005,187(2):434–442.PubMedCrossRef 13. Farewell A, Kvint K, Nyström T: Negative regulation by RpoS: a case of sigma factor competition. Mol Microbiol 1998,29(4):1039–1051.PubMedCrossRef 14. Ferenci T: Maintaining a healthy SPANC balance through regulatory and mutational adaptation. Mol Microbiol 2005, 57:1–8.PubMedCrossRef 15. Dong T, Chiang SM, Joyce C, Yu R, Schellhorn HE: Polymorphism and selection of rpoS in pathogenic Escherichiacoli . BMC Microbiol 2009, 9:118.PubMedCrossRef 16. Atlung T, Nielsen HV, Hansen FG: Characterisation of the allelic variation in the rpoS gene in thirteen K12 and six other non-pathogenic Escherichiacoli strains. Mol Genet Genomics

2002,266(5):873–81.PubMedCrossRef 17. King T, Ishihama A, Kori A, Ferenci T: A regulatory SCH727965 in vitro trade-off as a source of strain variation in the species Escherichiacoli . J Bacteriol 2004,186(17):5614–20.PubMedCrossRef 18. Spira B, Ferenci T: Alkaline phosphatase as a reporter of σ S levels and rpoS polymorphisms in different E.coli strains. Arch Microbiol 2008, 189:43–47.PubMedCrossRef 19. Ferenci T, Zhou Z, Betteridge T, Ren Y, Liu Y, Feng L, Reeves PR, Wang L: Genomic sequencing reveals regulatory mutations Sitaxentan and recombinational events in the widely used MC4100 lineage of Escherichiacoli K-12. J Bacteriol

2009,191(12):4025–9.PubMedCrossRef 20. Spira B, Hu X, Ferenci T: Strain variation in ppGpp concentration and RpoS levels in laboratory strains of Escherichiacoli K-12. Microbiology 2008,154(Pt 9):2887–2895.PubMedCrossRef 21. Gentry DR, Hernandez VJ, Nguyen LH, Jensen DB, Cashel M: Synthesis of the stationary-phase sigma factor sigma s is positively regulated by ppGpp. J Bacteriol 1993,175(24):7982–9.PubMed 22. Becker G, Klauck E, Hengge-Aronis R: Regulation of RpoS proteolysis in Escherichiacoli : the response regulator RssB is a recognition factor that interacts with the turnover element in RpoS. Proc Natl Acad Sci USA 1999,96(11):6439–44.PubMedCrossRef 23. Hengge-Aronis R, Fischer D: Identification and molecular analysis of glgS , a novel growth-phase-regulated and rpoS -dependent gene involved in glycogen synthesis in Escherichia coli . Mol Microbiol 1992,6(14):1877–1886.PubMedCrossRef 24. Gruber TM, Gross CA: Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol 2003, 57:441–466.PubMedCrossRef 25. Taschner NP, Yagil E, Spira B: A differential effect of σ S on the expression of the PHO regulon genes of Escherichiacoli .

002), 27 (p = 0.001), and 48 (p = 0.003) (Table 3). For fat-free mass, there was no significant difference between groups (p = 0.137). However, a significant difference was observed among the four testing sessions indicating that fat-free mass significantly

increased Selleck KU55933 at days 6 (p = 0.001), 27 (p = 0.001), and 48 (p = 0.001). There was also a significant increase at day 48 compared to days 6 (p = 0.012) and 27 (p = 0.022) (Table 3). There was no significant difference between groups for thigh muscle mass (p = 0.236); however, a significant difference was observed among the four testing sessions which revealed thigh muscle mass to be significantly increased at days 27 (p = 0.017) and 48 (p = 0.016). Increases were also seen at day 27 (p = 0.012) and 48 (p = 0.041) compared to day 6 (Table 3). Table 3 Body Composition Variables Variables Day 0 Day 6 Day 27 Day 48 Body Weight (kg)   * (p = 0.015) * (p = 0.006) * (p = 0.027) PLA 77.91 (18.36) 77.94 (17.76) 78.52 (18.64) Ilomastat supplier 78.80 (18.50) CRT 89.42 (22.08) 90.76 (22.60) 90.55 (22.54) 90.09 (22.86) CEE 73.69 (14.94) 74.49 (14.48) 74.91 (15.19) 75.32 (15.21) Fat-Free Mass (kg)   * (p = 0.001) * (p = 0.001) * (p = 0.001) PLA

54.55 (10.05) 55.10 (9.60) 56.05 (10.19) 56.25 (10.22) CRT 63.27 (10.79) 64.68 (11.18) 65.54 (11.68) 65.12 (11.39) CEE 59.06 (8.46) 59.74 (8.16) 60.01 (8.52) 60.11 (8.11) Fat Mass (kg)   * (p = 0.002) * (p = 0.001) * (p = 0.003) PLA 14.34 (8.92) 13.80 (8.65) 13.66 (8.89) 13.68 (8.94) CRT 21.55 (12.63) 21.09 (12.40) 20.20 (12.06) 20.08 (12.15) CEE † (p = 0.043) 10.44 (7.28)

10.41 Calpain (7.49) 10.50 (7.59) 10.88 (7.88) Thigh Mass (kg)     * (p = 0.017) * (p = 0.016) PLA 8.07 (1.77) 8.17 (1.73) 8.31 (1.73) 8.36 (1.71) CRT 8.93 (1.78) 9.17 (1.79) 9.28 (1.84) 9.34 (1.93) CEE 7.58 (.81) 8.06 (1.35) 8.22 (1.31) 8.21 (1.36) Data are expressed as mean (± SD). * indicates a significant difference at the respective testing session. † indicates a significant difference between groups (p < 0.05). Body water There was no significant difference between groups for total body water (p = 0.276). However, a significant difference existed among the four testing sessions indicating that total body water was significantly increased at days 27 (p = 0.022) and 48 (p = 0.001). There was also a significant increase at day 48 compared to day 6 (p = 0.002) (Table 4). No significant difference between groups existed for intracellular body water (p = 0.198). A significant difference was observed among the four testing sessions indicating there to be increases in intracellular body water at days 27 (p = 0.023) and 48 (p = 0.001).

The triangles are theoretical lines obtained by Equation 5. The insets are ESR of the samples with oblique sputtering angle of 0° and 60°. Here the saturation magnetization 4πM s was obtained by static VSM measurement; the perpendicular selleck chemical magnetic anisotropy constant could be acquired by fitting the experimental data with Equation 5. The fitted result showed that K⊥ of 60° was 16.3 × 103 erg/cm3 larger than the 12.9 × 103 erg/cm3 of 0°, which indicated increase with increasing oblique sputtering angle. Generally, the K⊥ of continuous film was almost zero due to strong demagnetization energy. In our case, the decrease of demagnetization energy was caused by shape anisotropy of nanostructure

films, which induced the increase of K⊥. Therefore, the increase of K⊥ induced inhomogeneities of magnetic anisotropy, which resulted in the increase of linewidth and/or damping factor. Conclusions The static and dynamic magnetic

properties of CoZr/AAO films with different oblique sputtering angles have been investigated. All the properties and parameters were found to be dependent on magnetic anisotropy field which was induced by the shape of the AAO template and oblique sputtering. The competition between the two factors resulted in the trend of dependence on anisotropy field H k and remanence ratio M r /M s, with various oblique sputtering angles. The resonance frequency change of CoZr/AAO films was also attributed to the effect of properties and oblique selleck chemicals sputtering. Enhanced microwave absorption was confirmed by complex permeability measurement comparing with continuous film on a Si all substrate. Acknowledgments This work is supported

by the National Basic Research Program of China (grant no. 2012CB933101), the National Science Fund for Distinguished Young Scholars (grant no. 50925103), and the National Natural Science Foundation of China (grant no. 11034004 and 50902064). References 1. Encinas-Oropesa A, Demand M, Piraux L, Ebels U, Huynen I: Effect of dipolar interactions on the ferromagnetic resonance properties in arrays of magnetic nanowires. J Appl Phys 2001, 89:6704.CrossRef 2. Fish GE: Soft magnetic materials. Proc IEEE 1990, 78:947–972.CrossRef 3. Yamaguchi M, Suezawa K, Arai KI, Takahashi Y, Kikuchi S, Shimada Y, Li WD, Tanabe S, Ito K: Microfabrication and characteristics of magnetic thin-film inductors in the ultrahigh frequency region. J Appl Phys 1999, 85:7919.CrossRef 4. Che RC, Peng LM, Duan XF, Chen Q, Liang XL: Microwave absorption enhancement and complex permittivity and permeability of Fe encapsulated within carbon nanotubes. Adv Mater 2004, 16:401–405.CrossRef 5. Gilbert TL: Classics in magnetics a phenomenological theory of damping in ferromagnetic materials. IEEE Trans Magn 2004, 40:3443–3449.CrossRef 6. Kittel C: On the gyromagnetic ratio and spectroscopic splitting factor of ferromagnetic substances. Phys Rev 1949, 76:743–748.CrossRef 7.

J Immunol Methods 1998,221(1–2):35–41.PubMedCrossRef Conflicts of interests Patents for the in vitro and in vivo use of EndoS have been applied for by Genovis AB and Hansa Medical AB, respectively. MC is listed as inventor on these applications that are pending.

Hansa Medical AB in part funded this study, but had no influence on the design of study, interpretation of data, or the final form of the manuscript. MC is a part time scientific consultant for Hansa Medical AB. Authors’ contributions JS participated in the CP-690550 solubility dmso design of the study, performed experiments and drafted the manuscript. MC and VN conceived of the study. CO performed experiments. AH designed the study and performed experiments. All authors read and approved the final manuscript.”
“Background Genes that are highly conserved between different types of organisms code for important biological functions and are therefore usually well studied and described. One group of conserved genes whose function has remained enigmatic until recently is the Kae1(OSGEP)/YgjD

family. Genes from this family occur in almost all bacterial, RG7112 order archaeal and eukaryotic genomes. The gene family consists of two groups: one group, GCP1/OSGEPL/Qri7, is of bacterial origin, the other, GCP2/OSGEP/Kae, is supposed to originate from archaea [1]. In Escherichia coli, Kae1/YgjD is essential for viability [2, 3]; in Arabidopsis thaliana and Saccharomyces cerevisia, deletion mutants exhibit deleterious phenotypes [4–6]. A biochemical activity for YgjD has recently been described: as already suggested by [7], Srinivasan and colleagues [8] showed that Kae1/YgjD protein (of Saccharomyces cerevisiae and Escherichia Mannose-binding protein-associated serine protease coli, respectively) is required to add a threonyl carbamoyl adenosine (t6A) modification to a subset of tranfer-RNAs that recognize codons with an adenin at the first position. Transfer-RNAs undergo complex modifications and maturation steps [9] required for translational fidelity [10–12]. Mutations in these modification pathways can be lethal or cause severe defects [13–15], and the involved genes are highly conserved in different organisms [14–16]. Because ygjD is

essential, it is not possible to delete the gene and study the phenotypic consequences. As an alternative, one can put the gene under control of an inducible promoter, and investigate the consequence of turning off its expression, and thereby depleting the YgjD protein. Our aim here is to get insights into the morphological changes that come about when the YgjD protein is depleted from growing Escherichia coli cells. In two studies ([3] and [17]), the authors have noticed an effect on cell size in YgjD depletion strains, suggesting a role of YgjD for cell division and/or cellular elongation. However, while Katz et al. observed shorter cells under YgjD depletion conditions, Handford et al. observed a mixed population of elongated and short cells.

Based on

these observations, further work should now concentrate on understanding the molecular mechanisms responsible so that the underlying process are understood and used to help develop better treatment and prevention and www.selleckchem.com/products/gant61.html control strategies. Methods Bacterial strains and plasmids E. coli 345-2RifC, E. coli 345-8 and 343-9 are all commensal isolates of porcine origin. E. coli 345-2RifC is marked with a no-cost rifampicin-resistance mutation in RpoB (H526Y). Strains 99-24 and 99-40 are human urinary isolates, whilst E. coli K12 JM109 is a laboratory strain. Study strains were chosen on the basis that they did not carry acquired antibiotic resistance genes and that they exhibited good growth characteristics in laboratory media, with doubling ranging between 21 and 27 minutes in nutrient broth. Their phylogenetic group was determined as described previously [27]. The relatedness of the isolates was investigated by randomly amplified polymorphic DNA (RAPD) PCR [37]. The broad-host range plasmids

RP1, pUB307, BIX 1294 mouse R46, pVE46 and N3 were introduced into host strains by conjugation using the agar mating method [26]. The 345-2RifC(pVE46) strain used was a variant passaged in the laboratory, the same from which silent isolates arose [26]. Derivatives of 345-2RifC(pVE46) and 345-2RifC(RP1), carrying silent antibiotic resistance genes were as described previously [26]. The characteristics of strains and plasmids used in this study are listed in Table 3. DNA sequencing and analysis DNA of IncN plasmid N3 was prepared

by alkaline SDS maxiprep and CsCl/EtBr density gradient centrifugation [38]. The E. coli N3 plasmid was sequenced to approximately CYTH4 37-fold shotgun sequence, totalling 1711 end sequences, from pUC19 (with insert sizes of 2-4 kb; 4-6 kb) genomic shotgun libraries that were sequenced using big-dye terminator chemistry on ABI3730 automated sequencers. The assembly was generated using phrap2gap. All repeat regions and gaps were bridged by read-pairs or end-sequenced polymerase chain reaction (PCR) products again sequenced with big dye terminator chemistry on ABI3730 capillary sequencers. The sequence was manipulated to the ‘Finished’ standard [39]. Competition experiments to assay in vitro fitness To assess the fitness impact of the plasmids upon E. coli host strains growth competition between plasmid-carrying and plasmid-free isogenic strain pairs was carried out as described previously in Davis minimal medium with 25 mg/ml glucose (DM25) [24]. To estimate bacterial counts, competition cultures were diluted as appropriate and spread in triplicate onto IsoSensitest agar (Oxoid) and onto IsoSensitest agar containing the relevant antibiotic.

As mentioned above, this emphasizes the need for a standardized preparation

procedure to exclude any influence of the sample preparation procedure on the quality of the protein spectra. Other studies also showed that bacterial protein profiles may be altered by varying growing conditions and extraction solvents. For example, triflouroacetic acid can be used instead of formic acid or different matrix solutions can be applied [23, 38, 39]. To overcome this problem, all leptospiral samples included in this study were cultured and extracted under standardized conditions. Furthermore, as proposed by Welker et al. [40] to ensure the quality of an established protein reference spectra database, each genomospecies was represented by several strains. Beyond this, MSP creation was performed twice, in two self-contained laboratories. Gamma-secretase inhibitor The quality of the established database was confirmed by defined measurements. To exclude any influence of the preparation method sample protein extracts of the reference strains were spotted and measured four times in each laboratory. Reliable species identification for all used strains was successful. Only one field isolate, L. kirschneri serovar Grippotyphosa, did match with the same score value for L. kirschneri and L. interrogans. This indicates that the differentiation of closely related species

by MALDI Biotyper™ is difficult. In this Ralimetinib case, 16S rRNA sequencing revealed the correct species to be L. kirschneri. The close phylogenetic relationship of the two species was confirmed in former sequencing projects [41–43]. Nevertheless, a clear separation of the species L. borgpetersenii and L. interrogans was possible. Studies showed that the genome of the two species L. interrogans and L. borgpetersenii differ in their chromosome size and gene numbers. In comparison to the other two pathogenic species, L. borgpetersenii contains the smallest genome size with 3,931 kb. This pathogenic Etomidate species is not adapted

for the existence in the outer environment [1, 44], which may be due to the loss of genes in the evolutionary process. Differences in the bacterial genome structure followed by the transcription of different proteins in the host and under laboratory conditions can result in the loss of protein peaks in MALDI-TOF MS spectra leading to differences in the proteome profiles. This observation is well-described for other microorganisms such as Brucella spp. [37, 45]. Considering these known leptospiral genomic variations, we hypothesize that it is possible to distinguish lepotspiral strains on the basis of discriminating peaks in their protein profiles. The most critical point for successful subtyping of gram-positive and gram-negative bacteria is the rigorous control of the extraction procedure, as described for Salmonella enterica[46].

Curr Osteoporos Rep 4:57–63CrossRefPubMed 36. Rauch F, Schoenau E (2001) Changes in bone density during childhood and adolescence: an approach based on bone’s biological organization. J Bone Miner Res 16:597–604CrossRefPubMed 37. Kyttälä P, Ovaskainen M, Kronberg-Kippilä C et al (2008) Lapsen ruokavalio ennen kouluikää, The Diet of Finnish Preschoolers. Kansanterveyslaitoksen julkaisuja B 32/2008 38. Houghton

LA, Vieth R (2006) The case against ergocalciferol (vitamin D2) as a vitamin supplement. Am J Clin Nutr 84:694–697PubMed 39. Heaney RP, Davies KM, Chen TC, Holick STA-9090 mouse MF, Barger-Lux MJ (2003) Human serum 25-hydroxy-cholecalciferol response to extended oral dosing with cholecalciferol. Am J Clin Nutr 77:204–210PubMed 40. Viljakainen HT, Palssa A, Kärkkäinen M, Jakobsen J, Lamberg-Allardt C (2006) How much vitamin D3 do the elderly need? J Am Coll Nutr 25:429–435PubMed 41. Millen AE, Bodnar LM (2008) Vitamin D assessment KU-57788 mw in population based studies: a review of

the issues. Am J Clin Nutr 87:1102S–1105SPubMed 42. Gurlek A, Pittelkow MR, Kumar R (2002) Modulation of growth factor/cytokine synthesis and signaling by 1, 25-dihydroxyvitamin D(3): implications in cell growth and differentiation. Endocr Rev 23:763–786CrossRefPubMed 43. Litonjua AA, Weiss ST (2007) Is vitamin D deficiency to blame for the asthma epidemic? J Allergy Clin Immunol 120:1031–1035CrossRefPubMed 44. Lapillonne A (2010) Fenbendazole Vitamin D deficiency during pregnancy may impair maternal and fetal outcomes. Med Hypotheses 74:71–75CrossRefPubMed”
“Introduction Treatment with bisphosphonates significantly reduces the risk of fractures in men and women with osteoporosis. The evidence is based on high-quality phase III randomized controlled trials (RCTs) with fracture as an endpoint [1–10]. The benefits of bisphosphonates also extend to other disorders of bone metabolism such as glucocorticoid-induced osteoporosis [11], Paget’s disease [12] and bone metastases [13, 14]. Treatment with bisphosphonates is

not without adverse effects, but they are generally minor and occur in a minority of patients. The most common adverse effect is gastrointestinal upset with the oral formulations, the frequency of which decreases with intermittent treatment such as once-weekly or monthly regimens. Intravenous (IV) administration of nitrogen-containing bisphosphonates may induce an acute phase reaction which manifests as fever, myalgia and arthralgia, although these side effects usually resolve within a few days of onset [3, 7, 15]. High doses of bisphosphonates given intravenously may impair renal function, and the kidney is a major route of elimination of the bisphosphonates. For this reason, bisphosphonates are not recommended for use in patients with severe renal impairment [16–18].

The plasmids were transformed into the wildtype and strain ALSM3 to generate strains ALSM20, ALSM13, ALSM33, and ALSM34. Luciferase assay Luciferase assays were performed by withdrawing 1 ml culture. The OD600 was

measured and samples were held on ice until the start of the assay. 100 μl of each sample were mixed find more with 3× assay buffer (75 mM tricine, 15 mM MgSO4, 1.5 mM EDTA, 1.5 mM DTT, 900 μM ATP, 3 mg/ml (w/v) BSA, and 3% (w/v) D-Glucose, pH = 7.8) and incubated 10 min prior to injection of 100 μl D-luciferin (120 μM final concentration) solved in 20 mM tricine (pH 7.8). D-Luciferin (Carl-Roth, Karlsruhe, Germany) was resuspended in 20 mM tricine (pH = 7.8, 1 mg/ml), aliquoted and stored at -70°C until use. Luminescence was recorded for 35 s (POLARstar OPTIMA luminometer, BMG LABTECH) and normalized against the OD600 to calculate the relative light units (RLU). For calculation of the fold change, the RLU were normalized against the RLU of time zero. All measurements were done in triplicate. RNA extraction and quantitative real-time RT PCR S. mutans wildtype was incubated anaerobically in BM medium containing 0.5% (w/v) sucrose until early-log phase. A sample was withdrawn for time zero, transferred into the double volume of RNA-protect (Qiagen, Hilden, Germany)

Apoptosis inhibitor and centrifuged according to the manufacturer’s instructions. The cultures were split in two halves and free malic acid was added to one of them (final concentration 25 mM). After two hours samples for RNA extraction were withdrawn and treated as described above. For lysis, cells were incubated with lysozyme (2.5 mg/ml culture pellet) and mutanolysin (50 U/ml culture pellet) at room temperature for 45 min. The mixture was transferred into RLT buffer containing sterile, acid washed glass beads (diameter 106 μm) and vortexed for 3 min. Subsequent RNA extraction was carried out using the

RNeasy mini kit (Qiagen). Genomic DNA was removed using the DNAse I (Qiagen) in-solution digestion protocol. The quality of the total RNA was controlled on a denaturating formaldehyde agarose Fossariinae gel. Synthesis of cDNA was carried out using random hexamers and SuperScript II reverse transcriptase (Invitrogen, Karlsruhe, Germany), followed by purification using the PCR Purification kit (Qiagen). All reactions included a control without SuperScript II to assess genomic DNA contamination. Real-time PCR was performed using the LightCycler 480 system (Roche, Mannheim, Germany) and the reaction mixtures were prepared using the Quantitect SYBR Green PCR Kit (Qiagen). Changes in the level of gene expression were calculated automatically by the LightCylcer 480 software using the ΔΔC T method. The gyrase A gene (Smu.1114) was used as the housekeeping reference gene. All steps were performed according to the manufacturer’s protocols. All measurements were done in duplicate. Acid killing and hydrogen peroxide killing The ability of S.

coli. The restriction endonucleases, T4 DNA ligase and Pfu polymerase were provided by Promega (Promega Corporation, Madison, WI). Complementation of the gpsX mutant For complementation of the gpsX mutant 223 G4 (gpsX-), a 2,299-bp DNA fragment containing the intact open reading frame (ORF) of gpsX and 230 bp upstream of the 5′ end to 21 bp downstream of the 3′ end of the ORF, was amplified from the genomic DNA of Xac strain 306 using the primers C10-F (5′ -tcgaggtaccgttggtgtcgtcctcgaaat-3′) and C10-R (5′ – tcgtaagcttctcaccccgcaataaacaac-3′),

respectively Adriamycin nmr containing KpnI and HindIII restriction enzyme sites (underlined). The PCR product was digested with KpnI and HindIII and cloned into the complementary vector pUFR053 [33] to construct the recombinant plasmid pJU3110 (Table 2). The recombinant plasmid was transferred into the gpsX mutant 223 G4 (gpsX-) by triparental conjugation as described elsewhere [57], resulting in strain C233G4 (gpsX+) (Table 2). Quantitative determination of EPS production To estimate EPS production, strains were cultured in 100 ml NB or XVM2 liquid

medium containing 2% (wt/vol) various sugars (fructose, galactose, glucose, maltose, mannose, Selleckchem PU-H71 sucrose, and xylose) at 28°C with shaking at 200 rpm for 24 hours (in NB) or 48 hours (in XVM2). EPS was precipitated from the culture supernatant at different time point post inoculation with ethanol, dried, and weighed as described elsewhere [35]. Lipopolysaccharides (LPS) analysis Bacterial strains were cultured at 28°C in NB or XVM2 liquid medium with shaking (200 rpm). Five-milliliter samples acetylcholine of cultures at the exponential stage were collected and the LPS samples were isolated as described previously [23]. LPS was separated

by SDS-PAGE and visualized using silver staining following the manufacturer’s instructions (Bio-Rad Laboratories, Inc., Hercules, CA). Standard LPS from Salmonella entenica serovar Typhimurium was obtained from Sigma. The test was performed three times independently. Capsule assays Bacterial capsules were stained using a capsule-staining kit (Eng Scientific Inc., Clifton, NJ, USA) following the manufacturer’s instructions. The samples were photographed using a light microscope (Leica DMLB2; Leica Microsystems GmbH, Wetzlar, Germany) with a digital camera. The experiment was repeated three times. Biofilm formation assays Biofilm formation on polystyrene and glass surfaces were examined as described previously [23] with modifications. The average of four replicates was used for quantitative measurement. Assays of biofilm formation on leaf surfaces were conducted as described previously [58] with modifications. Briefly, 20 μl of each bacterial suspension (108 cfu/ml) was incubated on the abaxial surface of citrus leaves, and the leaves were kept at 28°C in a humidified chamber. After 24 h of incubation, biofilm formation on the leaf surface was visualized using crystal violet staining.