They are commonly

used to manufacture fermented milk products and some species are considered probiotics. Many health benefits are associated with their use, including the ability to modulate the immune system (Gill, Midostaurin supplier 1998; Salminen et al., 1998) as well as antitumor, antimetastatic properties (Tomita et al., 1994; Matsuzaki et al., 1996). Intraperitoneal administration of Lactobacillus casei induced the production of cytokines such as interferon γ (IFNγ), interleukin-1 (IL-1) and tumor necrosis factor α (TNFα), which could contribute to the inhibition of tumor growth and increased survival of tumor-bearing mice (Matsuzaki, 1998). Several Lactobacillus species stimulate cells of the innate immune system in vitro, namely natural killer cells (Kato find more et al., 1984; Haller et al., 1999) and macrophages. Stimulation of these cells can induce proinflammatory cytokines such

as TNFα (Haller et al., 1999), IFNγ and IL-12 (Miettinen et al., 1998; Hessle et al., 1999; Kato et al., 1999; Morita et al., 2002), and regulatory cytokines such as IL-10 (Christensen et al., 2002). TNFα directly induces tumor apoptosis and enhances the tumoricidal activity of macrophages (Wang et al., 1996), while IL-12 has potent antitumor and antimetastatic effects against tumors by the stimulation of cytotoxic CD8+ T cells and natural killer cells. IL-12 also enhances the production of Th1 cytokines such as IFNγ. IL-10 plays a regulatory role in allergy (Akbari et al., 2001) and anti-inflammatory responses

(Kuhn et al., 1993). Toll-like Avelestat (AZD9668) receptors (TLRs) are pattern recognition receptors that recognize molecules that are common to pathogens, but absent in the host. TLR4 is essential for the recognition of lipopolysaccharide, while lipoproteins from gram-positive bacteria are recognized by TLR2 (Takeuchi et al., 1999). Major cell wall components of gram-positive bacteria, such as peptidoglycan and lipoteichoic acid, signal through TLR2 (Schwandner et al., 1999; Matsuguchi et al., 2003) and stimulate cytokine production. The mannose and Fcγ receptors and CD14 are associated with bacterial phagocytosis, which can also result in cytokine production. Unmethylated CpG dinucleotides in the bacterial DNA have stimulatory effects on mammalian immune cells (Lipford et al., 1998). Hemmi et al. (2000) showed that the cellular response to CpG DNA is mediated by TLR9 as TLR9 knockout mice did not respond to CpG DNA and the immune cells from these mice did not produce inflammatory cytokines upon stimulation with CpG DNA. This study aims to evaluate the immunostimulatory properties of three commonly consumed lactobacilli species: L. casei, Lactobacillus rhamnosus and Lactobacillus bulgaricus. Analysis of splenocyte TNFα, IL-12p40 and IL-10 production after stimulation with ‘live’ and lyophilized lactobacilli was performed. The role of TLRs and phagocytosis in the stimulation of cytokine production was also examined.

It has been shown that serologic diagnosis is very sensitive in confirming the diagnosis. It has 100% sensitivity on the first serum specimen tested at a reference laboratory, BAY 73-4506 order if drawn within 3 months of onset of lymphadenopathy.9 This can eliminate the need for further invasive workup. Our series has a male to female ratio of 6.5 : 1, which is likely due to the high proportion of returned male missionary travelers being seen at our center. Travelers occasionally acquire two or more infections concurrently. Comorbidities, likely also resulting from travel, were noted in 50% of the patients and included chronic diarrhea (three), suspected dengue fever (one),

latent tuberculosis acquisition (one), culture positive Salmonella typhi (one), serologic evidence of Chagas disease (one), and carbon monoxide

Wnt inhibitor poisoning during travel (one). While life-threatening toxoplasmosis is generally associated with the immunosuppressed populations, there have been a number of case reports in immunocompetent individuals. Documented complications include disseminated disease,10 bronchiolitis, pneumonitis,11 pneumonia in a pregnant woman,12 fatal myocarditis, pericarditis, simultaneous myocarditis and polymyositis,13 hepatosplenomegaly and hepatitis, diffuse encephalitis,14 encephalitis with quadriparesis and chorioretinitis, and Guillain-Barré syndrome.1,15 Several of these complications were noted in relationship with an atypical strain of Toxoplasma, for example in the well-described community outbreak of multivisceral toxoplasmosis in Patam, a Surinamese village near the French Guianan border. In our series, 2 of 14 (14%) patients required hospital admission—one for febrile illness with concern for endocarditis, and one for unexplained fever and lymphadenopathy. Both were discharged home once the diagnosis of toxoplasmosis was established. Atypical lymphocytes are often seen in patients with acute toxoplasmosis. Atypical lymphocytosis

Endonuclease was noted in 3 of 14 (21%) patients in our series, all of whom presented during the acute phase of symptoms. Clinicians should recognize atypical lymphocytes as a sign of acute toxoplasmosis and if the symptomatology is appropriate, order toxoplasma serologies. In all of our patients where toxoplasmosis was clinically suspected, diagnosis was established by a positive IgM and a positive IgG titer. Ideally, repeat serologic testing with fourfold rise in IgG titers is recommended, but the self-limiting nature of the illness and this retrospective study design precluded this confirmatory testing. Tests for IgM and IgG antibodies should be used for initial evaluation of suspected toxoplasmosis. Acute infection is supported by documented seroconversion of IgM and IgG antibodies or a greater than fourfold rise in IgG antibody titer in sera run in parallel.

There are four conserved aspartate residues within the amino acid sequence of AroR (Fig. 2b), with aspartate 58 X-396 research buy residue predicted to be the most likely site of transphosphorylation. The receiver

domain is linked to an AAA+ATPase domain that precedes the DNA-binding domain at the C-terminus. The presence of an AAA+ATPase domain is indicative of a transcription factor activity associated with the activation of σ54 promoters. In analogous response regulators from the NtrC/DctD family, ATPase activity is coupled to a hexameric or a heptameric ring assembly that is required for the formation of an open RNA polymerase complex at the initiation of transcription (Gao & Stock, 2009). Furthermore, AroR sequence analysis shows the presence of a highly conserved ESELFGHEKGAFTGA

sequence motif that is essential for binding to the σ-factor of the σ54-RNAP (Yan & Kustu, 1999; Xu & Hoover, 2001; Bordes et al., 2003). We have previously detected a putative σ54-like promoter region upstream of aroB, and in a recent study of H. arsenicoxydans, it was shown, through transposon insertions, that alternative N sigma factor (σ54) of RNA polymerase is involved in the control of the arsenite oxidase gene expression (Koechler et al., 2010). aroR- and aroS-like genes appear to be conserved within gene clusters associated with arsenite oxidation (Fig. 1a). However, Akt inhibitor in members of the Alphaproteobacteria that include NT-26, the aroR and aroS genes are in the same Resveratrol orientation as the arsenite oxidase genes,

whereas in members of the Betaproteobacteria, they are in the opposite orientation with a gene involved in oxyanion binding or phosphate/phosphonate transport in between them (Fig. 1a). Both AroS and AroR share high sequence similarity (∼80% identity) to analogous proteins from A. tumefaciens and O. tritici, with sequence similarities declining significantly to the next closest sequence homologues from Xanthobacter autotrophicus exhibiting sequence identities of 43% and 56% for AroS and AroR, respectively. In all the other identified organisms, which have homologous proteins, sequence identities range from approximately 38% to 23% for AroS-like proteins and 43% to 38% for AroR-like proteins, with significantly higher sequence conservation of AroR compared with that of AroS, possibly reflecting differences between various stimuli activating these sensors. The arsenite oxidase gene cluster consisting of aroB, aroA, cytC and moeA1 encodes two transcripts, one transcript that is constitutive and only contains cytC and moeA1 and another transcript that is inducible with arsenite and that contains all the genes and that is most likely regulated through an involvement of a putative σ54-like promoter upstream of aroB (Santini et al., 2007).

There are four conserved aspartate residues within the amino acid sequence of AroR (Fig. 2b), with aspartate 58 selleck compound residue predicted to be the most likely site of transphosphorylation. The receiver

domain is linked to an AAA+ATPase domain that precedes the DNA-binding domain at the C-terminus. The presence of an AAA+ATPase domain is indicative of a transcription factor activity associated with the activation of σ54 promoters. In analogous response regulators from the NtrC/DctD family, ATPase activity is coupled to a hexameric or a heptameric ring assembly that is required for the formation of an open RNA polymerase complex at the initiation of transcription (Gao & Stock, 2009). Furthermore, AroR sequence analysis shows the presence of a highly conserved ESELFGHEKGAFTGA

sequence motif that is essential for binding to the σ-factor of the σ54-RNAP (Yan & Kustu, 1999; Xu & Hoover, 2001; Bordes et al., 2003). We have previously detected a putative σ54-like promoter region upstream of aroB, and in a recent study of H. arsenicoxydans, it was shown, through transposon insertions, that alternative N sigma factor (σ54) of RNA polymerase is involved in the control of the arsenite oxidase gene expression (Koechler et al., 2010). aroR- and aroS-like genes appear to be conserved within gene clusters associated with arsenite oxidation (Fig. 1a). However, Akt inhibitor in members of the Alphaproteobacteria that include NT-26, the aroR and aroS genes are in the same Dichloromethane dehalogenase orientation as the arsenite oxidase genes,

whereas in members of the Betaproteobacteria, they are in the opposite orientation with a gene involved in oxyanion binding or phosphate/phosphonate transport in between them (Fig. 1a). Both AroS and AroR share high sequence similarity (∼80% identity) to analogous proteins from A. tumefaciens and O. tritici, with sequence similarities declining significantly to the next closest sequence homologues from Xanthobacter autotrophicus exhibiting sequence identities of 43% and 56% for AroS and AroR, respectively. In all the other identified organisms, which have homologous proteins, sequence identities range from approximately 38% to 23% for AroS-like proteins and 43% to 38% for AroR-like proteins, with significantly higher sequence conservation of AroR compared with that of AroS, possibly reflecting differences between various stimuli activating these sensors. The arsenite oxidase gene cluster consisting of aroB, aroA, cytC and moeA1 encodes two transcripts, one transcript that is constitutive and only contains cytC and moeA1 and another transcript that is inducible with arsenite and that contains all the genes and that is most likely regulated through an involvement of a putative σ54-like promoter upstream of aroB (Santini et al., 2007).

“
“Recordings of large

neuronal ensembles and neural stimulation of high spatial and temporal precision are important requisites for studying the real-time dynamics of neural networks. Multiple-shank silicon probes enable large-scale monitoring of individual Selleckchem Small molecule library neurons. Optical stimulation of genetically targeted neurons expressing light-sensitive channels or other fast (milliseconds) actuators offers the means for controlled perturbation of local circuits. Here we describe a method to equip the shanks of silicon probes with micron-scale light guides for allowing the simultaneous use of the two approaches. We then show illustrative examples of how these compact hybrid electrodes can be used in probing local circuits in behaving rats and mice. A key advantage of these devices is the enhanced spatial precision of stimulation that is achieved by delivering light close to the recording sites of the probe. When paired with the expression of light-sensitive actuators within genetically specified neuronal populations, these devices allow the relatively straightforward and interpretable manipulation of network activity. One of the important challenges in neuroscience is to identify Sotrastaurin cell line the causal links between the collective activity of neurons and behavior. While the study of correlations between ensemble neuronal activity and behavior has produced unprecedented progress in the past decade (Buzsaki et al., 1992;

Wilson & McNaughton, 1993; Harris et al., 2003; Gelbard-Sagiv et al., 2008; Yamamoto & Wilson, 2008; Battaglia et al., 2009; Rizk et al., 2009), the correlational Sclareol nature of these measurements leaves ambiguous the cause-and-effect relationship. A more thorough understanding requires at least two additional steps. The first one is the identification of the multiple neuronal cell types that uniquely contribute to the assembly behavior, rather like members of an orchestra. There are at least two dozen

excitatory and inhibitory neuron types in the cortex, with diverse targets, inputs and uniquely tuned biophysical properties, and existing methods have serious limitations for identifying and segregating these neuron types (Freund & Buzsaki, 1996; Klausberger et al., 2003; Markram et al., 2004; Klausberger & Somogyi, 2008). The second step is a principled manipulation of the spiking activity of these identified cell groups. The recently developed molecular optogenetic tools provide a means to achieve each of the above experimental goals (Deisseroth et al., 2006; Zhang et al., 2007a; O’ Connor et al., 2009). Optical stimulation of genetically targeted neurons expressing light-sensitive channelrhodopsin-2 (Chr2 has recently been reported to be a rapid activator of neuronal firing with potential cell-type selectivity (Nagel et al., 2003; Boyden et al., 2005; Li et al., 2005; Ishizuka et al., 2006; Han & Boyden, 2007; Zhang et al., 2007b).

C at position 98 and T at position 253 were common characters in all the strains of P. coccineus (including MUCL 38420) and in

the Chinese strains of P. sanguineus (including CIRM-BRFM 542). C/G substitution at positions 152 and 206 was specific to the East Asian strains of Pycnoporus, and T/C substitution (at position 56) was specific to the Australian strains of Pycnoporus. The phylogenetic trees inferred from ITS1-5.8S-ITS2 and β-tubulin gene sequences (Figs 1 and 2) clearly differentiated the group of P. cinnabarinus strains from the group of P. puniceus strains (100% bootstrap support). The group of the P. coccineus strains from Australia (including strain MUCL 38420), the P. sanguineus strains from China (including CIRM-BRFM 542 of unknown origin) with the Japanese strain of P. coccineus, selleck chemicals and the strain of P. coccineus Palbociclib from the Solomon Islands (positioned alone), formed a well supported clade (84% bootstrap value with ITS). Due to the high similarity of their ITS sequences, the strains of P. sanguineus from Madagascar, Vietnam, New Caledonia, French Guiana and Venezuela could not be distinguished phylogenetically. β-Tubulin molecular data might be of slightly more help than ITS data to disclose genetic polymorphism within these P. sanguineus strains with two groups, although weakly supported (Fig. 2). In

this study, the functional lac3-1 gene, which protein products showed high variability in enzymatic activity between the species of Pycnoporus (Uzan et al., 2010), was targeted to infer the phylogenetic relationships within the genus Pycnoporus, out and especially within the P. sanguineus and P. coccineus species. PCR amplification resulted in laccase F2-R8 products of about 1640 bp. Comparison

between gene and predicted cDNA fragment sequences showed that the corresponding partial coding regions were interrupted by eight introns. A positional homology among these introns could be observed. It is noteworthy that the eight intron lengths were strictly similar for the East Asian strains of Pycnoporus on the one hand, and for the Australian strains on the other (data not shown). The nine exons corresponded to sequences of 1182 nucleotides. The 36 deduced partial proteins (corresponding to about 75–80% of the full length protein) displayed sequence similarity ranging from 87.6% to 99.7%. The 36 laccase sequences from Pycnoporus strains were aligned in 1185 nucleotide positions after hand-refining (see File S3). These regions of the laccase gene had 33% variable positions among the strains of Pycnoporus studied. Informative nucleotide site variations were localized in the conserved copper-binding domains, especially domains II and III with T/C substitution specific to the East Asian strains of Pycnoporus. Phylogenetic construction of our worldwide sample of Pycnoporus lac3-1 sequences led to distinct groups that were correlated with the geographic origin of the strains (Fig. 3).

Once encapsulated, the trapped bacteria subsequently die (Silva et al., 2002). Cytotoxic factors targeting immunocytes are good candidates for the mediators of immunosuppression (Clarke, 2008). Plu1961/Plu1962 caused death of CF-203 cells via necrosis. Further studies on the necrotic and apoptotic activities of Plu1961/Plu1962 against insect hemocytes will be necessary to elucidate its role in immunosuppression. Confocal

microscopy revealed that Plu1961/Plu1962 caused a notable decrease in cellular tubulin of CF-203 cells. Microtubule, one of the principal components of cytoskeleton, is critical to cell shape, cell movement, intracellular transport of organelles, and the separation of chromosomes during mitosis (Archuleta

et al., 2011). As a result, microtubule is a prime target for pathogens and their virulence factors. Mouse macrophages treated with Bacillus anthracis lethal toxin (LT) induced INCB024360 datasheet a notable decrease in the level of cellular tubulin and altered stability of the microtubule network (Chandra et al., 2005). Treatment of human colonocytes with Clostridium difficile toxin A resulted in tubulin deacetylation and subsequent microtubule depolymerization (Nam et al., 2010). Assembly of the two components www.selleckchem.com/products/torin-1.html is essential for binary toxins to exhibit their cytotoxicity (Schleberger et al., 2006). However, the stage at which the assembly of the binary toxin components occurs is debatable. Previous study suggested that intoxication by binary toxins initially involved specific, receptor-mediated binding of ‘B’ component to a targeted cell as monomers that form homoheptamers on the cell surface. The ‘B’ heptamer–receptor complex then acts as a docking platform that subsequently translocates the enzymatic ‘A’ component into the cytosol. Once inside the cytosol, ‘A’ component can inhibit normal cell functions (Barth et al., 2004).

It was reported that at low toxin concentrations, complex formation might enhance the efficiency of the binary toxin (Kaiser et al., 2006). Our data demonstrated that ID-8 when co-expressed in the same cytoplasm, Plu1961 and Plu1962 could interact with each other and form a complex. This could in part explain the observation that Plu1961 and Plu1962 mixed in vitro did not affect the growth of mammalian cells, but while co-expressed in the same cytoplasm, Plu1961/Plu1962 exhibited cytotoxic effect against B16, 4T1, and HeLa cells. In conclusion, we have identified XaxAB-like binary toxin from P. luminescens TT01, which exhibits highly injectable toxicity against insect larvae. Plu1961/Plu1962 mixture could cause rapid cell necrosis when applied to insect midgut CF-203 cells. However, co-expression in the same cytoplasm is essential for Plu1961/Plu1962 to exhibit cytotoxic activity against mammalian cells. The biological role of Plu1961/Plu1962 in the infection process needs further study.

, 2000; Mallick et al.,

2007). The metabolism of 2-hydroxy-1-naphthoic acid by the cell-free extract of strain PWTJD grown on phenanthrene was evidenced by the change in color of the reaction mixture to slightly yellowish and an increase in absorbance at 297 and 334 nm with time (Fig. 3a). An almost similar spectrum was obtained in the HPLC analysis for peak VI (Fig. 2), indicating the possible presence of a ring cleavage product of Selumetinib 2-hydroxy-1-naphthoic acid in the resting cell transformation analysis. However, no change was observed in the spectral pattern when 1,2-dihydroxynaphthalene was incubated with the cell-free extract of phenanthrene-grown cells. All this information indicated the direct ring cleavage of 2-hydroxy-1-naphthoic acid by a ring cleavage dioxygenase present in the strain PWTJD similar to the earlier report from Gram-positive Staphylococcus sp. (Mallick et al., 2007). Like the previous report on the meta-cleavage of 2-hydroxy-1-naphthoic acid (Mallick et al., 2007), it was also observed that the ring-cleavage dioxygenase possessed dissociable ferric iron at the catalytic center because

an increase in the ring-cleavage activity was noticed when the cell-free extract was supplemented with 1 mM FeCl3. In addition, on treatment of the cell-free extract with deferroxamine mesylate, a ferric chelating reagent, the resultant cell-free extract preparation did not show 2-hydroxy-1-naphthoic DOK2 acid ring-cleavage activity. However, the ring-cleavage activity selleck products could be restored on further treatment with FeCl3 solution, verifying the role of ferric iron in catalysis. On the other hand, EDTA, a ferrous chelating reagent, had no impact on the enzyme activity. In the lower pathway of the

degradation of phenanthrene, the metabolism of salicylaldehyde to salicylic acid has been demonstrated in the spectrophotometric analyses (Fig. 3b) by the cell-free extract of both phenanthrene and 2-hydroxy-1-naphthoic acid-grown cells of strain PWTJD. An increase in the absorbance at 296 nm and a simultaneous decrease in absorbance at 254 and 330 nm was observed, indicating the formation of salicylic acid (Fig. 3b) when salicylaldehyde was incubated with a crude cell-free extract (Eaton & Chapman, 1992). Because salicylaldehyde itself has absorbance around 340 nm, the formation of NADH (λmax at 340 nm) from NAD+ during this transformation could not be observed during the early stage of transformation, but became apparent in the later stages of incubation (Fig. 3b). On the other hand, catechol was found to be metabolized by the cell-free extracts of either phenanthrene, 2-hydroxy-1-naphthoic acid or salicylic acid-grown cells of strain PWTJD with the formation of a yellow-colored product, 2-hydroxymuconaldehyde acid (Kojima et al., 1961; Nozaki, 1970), having a λmax at 374 nm (Fig.

Escherichia coli BW25113 (ΔaraBD) (Datsenko & Wanner, 2000) and BL21 (DE3) were grown in M9 medium supplemented with 0.2% casamino acids and 0.5% glycerol at 37 °C. The primers used in this study are summarized in Table 1. The coding sequences of ygfX alone or ygfYX were PCR-amplified using primers YGFX-F and YGFX-R1, or YGFY-F

and YGFX-R1, respectively. The fragments were cloned into pBAD24 vector (Guzman et al., 1995) and designated as pBAD24-ygfX and pBAD24-ygfYX, respectively. The coding sequence of YgfX in a fusion with His6-tag at the C-terminal (YgfX−His) was also cloned into pBAD24 using YGFX-F and YGFX-R2. A truncated protein of YgfX (YgfX(C); cloned from V49 to Z135) was cloned into buy Nutlin-3 pCold-Km (unpublished results, Inouye laboratory) using YGFXs-F and YGFX-R1. His6-tagged FtsZ and MreB were constructed previously (Tan et al., 2011). FLAG-tagged FtsZ and MreB

were also previously constructed in pET17b, having a tag at the C-terminal end (H. Masuda and M. Inouye, unpublished results). For examining the growth rate, 0.2% arabinose was added to the cultures during the early exponential phase. His6-tagged YgfX(C), FtsZ, and MreB were expressed in E. coli BL21(DE3). Protein expression was induced for 2 h by adding 1 mM IPTG when the OD600 nm reached 0.8. The cells were collected by brief centrifugation at 8000 g and lysed by French pressure press (Thermo Fisher Scientific, MA). FtsZ and MreB were purified as described before (Tan et al., 2011). YgfX(C)−HIS was purified from the insoluble materials after being dissolved learn more in 8 M urea (pH 8.0). Proteins were purified

using Ni-NTA agarose according to the manufacturer’s instructions (Qiagen, CA). Inner and outer membrane proteins were isolated following the method described previously (Hobb et al., 2009). Briefly, the total membrane proteins were collected from the lysate by ultracentrifugation at 100 000 g for 1 h. The pellet was washed, then resuspended in 1% (w/v) N-lauroylsarcosine in 10 mM HEPES, pH 7.4, and incubated at 25 °C for 30 min with gentle agitation. The inner and outer membrane fractions were further separated by ultracentrifugation. His6-tag pulldown assays were carried out by incubating the cell lysate containing YgfX−HIS and the cell lysate containing FsZ−FLAG or MreB−FLAG (lysis buffer: 50 mM HEPES-KOH, pH 7.5, 10 mM MgCl2, 200 mM KCl, 0.1 mM EDTA, and 10% ID-8 glycerol) overnight at 4 °C. Ni-NTA agarose (0.5 mL) was added to the lysate, and the mixture was incubated at room temperature for 1 h. The beads were washed three times with 20 mL of the same lysis buffer containing 20 mM imidazole. Protein complexes were then separated by 17.5% SDS-PAGE and visualized by Western blot using monoclonal anti-FLAG antibody conjugated with horseradish peroxidase (Sigma-Aldrich, MO). The effect of YgfX on FtsZ and MreB polymerization was determined by a sedimentation method as previously described (Anand et al., 2004) with a few modifications.

Escherichia coli BW25113 (ΔaraBD) (Datsenko & Wanner, 2000) and BL21 (DE3) were grown in M9 medium supplemented with 0.2% casamino acids and 0.5% glycerol at 37 °C. The primers used in this study are summarized in Table 1. The coding sequences of ygfX alone or ygfYX were PCR-amplified using primers YGFX-F and YGFX-R1, or YGFY-F

and YGFX-R1, respectively. The fragments were cloned into pBAD24 vector (Guzman et al., 1995) and designated as pBAD24-ygfX and pBAD24-ygfYX, respectively. The coding sequence of YgfX in a fusion with His6-tag at the C-terminal (YgfX−His) was also cloned into pBAD24 using YGFX-F and YGFX-R2. A truncated protein of YgfX (YgfX(C); cloned from V49 to Z135) was cloned into Epigenetics inhibitor pCold-Km (unpublished results, Inouye laboratory) using YGFXs-F and YGFX-R1. His6-tagged FtsZ and MreB were constructed previously (Tan et al., 2011). FLAG-tagged FtsZ and MreB

were also previously constructed in pET17b, having a tag at the C-terminal end (H. Masuda and M. Inouye, unpublished results). For examining the growth rate, 0.2% arabinose was added to the cultures during the early exponential phase. His6-tagged YgfX(C), FtsZ, and MreB were expressed in E. coli BL21(DE3). Protein expression was induced for 2 h by adding 1 mM IPTG when the OD600 nm reached 0.8. The cells were collected by brief centrifugation at 8000 g and lysed by French pressure press (Thermo Fisher Scientific, MA). FtsZ and MreB were purified as described before (Tan et al., 2011). YgfX(C)−HIS was purified from the insoluble materials after being dissolved RXDX-106 mouse in 8 M urea (pH 8.0). Proteins were purified

using Ni-NTA agarose according to the manufacturer’s instructions (Qiagen, CA). Inner and outer membrane proteins were isolated following the method described previously (Hobb et al., 2009). Briefly, the total membrane proteins were collected from the lysate by ultracentrifugation at 100 000 g for 1 h. The pellet was washed, then resuspended in 1% (w/v) N-lauroylsarcosine in 10 mM HEPES, pH 7.4, and incubated at 25 °C for 30 min with gentle agitation. The inner and outer membrane fractions were further separated by ultracentrifugation. His6-tag pulldown assays were carried out by incubating the cell lysate containing YgfX−HIS and the cell lysate containing FsZ−FLAG or MreB−FLAG (lysis buffer: 50 mM HEPES-KOH, pH 7.5, 10 mM MgCl2, 200 mM KCl, 0.1 mM EDTA, and 10% (-)-p-Bromotetramisole Oxalate glycerol) overnight at 4 °C. Ni-NTA agarose (0.5 mL) was added to the lysate, and the mixture was incubated at room temperature for 1 h. The beads were washed three times with 20 mL of the same lysis buffer containing 20 mM imidazole. Protein complexes were then separated by 17.5% SDS-PAGE and visualized by Western blot using monoclonal anti-FLAG antibody conjugated with horseradish peroxidase (Sigma-Aldrich, MO). The effect of YgfX on FtsZ and MreB polymerization was determined by a sedimentation method as previously described (Anand et al., 2004) with a few modifications.