Other immobilization techniques that take advantage of the ability of RNA to form base-pairs could also serve to slow RNA exchange. Although dextran/PEG ATPS and ATP/pLys coacervate systems do not provide suitably stable compartmentalization of reactants for long periods

of time, such systems SB525334 do enable transient localization and concentration of RNA molecules. Focusing on the potential usefulness of these systems for sub-compartmentalization within protocells may be a productive direction for future research (Hyman and Brangwynne 2011). Fatty acid and phospholipid vesicle systems compatible with dextran/PEG ATPSs have been developed (Helfrich et al. 2002; Long www.selleckchem.com/products/Cyclosporin-A(Cyclosporine-A).html et al. 2005; Dominak et al. 2010; this study), and it may be possible to develop similar vesicle systems that are compatible with the ATP/pLys coacervate system. This might be achieved by using net-neutral zwitterionic phospholipids or non-ionic amphiphiles as membrane forming molecules, as they would not interact strongly with the coacervate CP-868596 clinical trial components, thus avoiding precipitation. Such a system would be similar to cellular organelle-based compartmentalization. In a prebiotic setting, a lipid-based membrane could encapsulate all components, and selective chemical

partitioning into the two phases could provide an early protocell with the ability to partition compounds internally and accelerate reactions within the protocell, including for example the assembly of RNA complexes and ribozyme catalysis (Strulson et al. 2012). Megestrol Acetate Thus, understanding

how ATPSs and coacervates interact and combine with fatty acid and phospholipid vesicles may lead to a greater understanding of the possibilities for the development of early cells in an RNA world. Methods Chemicals Tris(hydroxymethyl) aminomethane (Tris), sodium chloride, magnesium chloride hexahydrate, D-(+)-glucose, 2-mercaptoethanol, adenosine 5′-triphosphate (ATP) disodium salt hydrate, adenosine 5′-diphosphate (ADP) disodium salt, adenosine 5′-monophosphate (AMP) disodium salt, guanosine 5′-triphosphate (GTP) sodium salt hydrate, guanosine 5′-diphosphate (GDP) sodium salt, guanosine 5′-monophosphate (GMP) disodium salt hydrate, uridine 5′-triphosphate (UTP) trisodium salt hydrate, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) trisodium salt, enzyme catalase from bovine liver, polyethylene glycol (PEG) 8 kDa, dextran 9–11 kDa from Leuconostoc mesenteroides, dextran sulfate sodium salt 9–20 kDa from Leuconostoc spp., diethylaminoethyl-dextran (DEAE-dextran) hydrochloride >500 kDa, poly-L-lysine (pLys) hydrobromide 1–5 kDa, poly-L-lysine hydrobromide 4–15 kDa, poly-L-lysine hydrobromide 15–30 kDa, and Sepharose 4B (45–165 μm bead diameter) beads were purchased from Sigma-Aldrich Corporation (St. Louis, MO).

Sequence analysis of several M. synoviae strains suggested that MSPA was more antigenically variable than MSPB [6, 10, 11]. Consistently, in isogenically derived M. synoviae clones that have lost their haemadsorbing and/or haemagglutinating

activity, MSPA was no more detectable by polyclonal antisera or monoclonal antibodies, suggesting extensive antigenic variation [12]. The molecular basis underlying the generation of antigenic variants of M. synoviae vlhA genes has been selleck compound elegantly demonstrated in a study conducted by Noormohammedi et al. 2000 [17]. It resides in the ability of a single strain to undergo high frequency site-specific recombination, owing to the availability in the genome sequence of a significant pool of pseudogenes (vlhA-related partial sequences). Recombination between the single complete vlhA gene and one of the multiple pseudogene copies ensures the creation of a new vlhA gene variant. To date, three expressed vlhA gene variants (vlhA1, vlhA4, and vlhA5) find more have been characterized in M. synoviae strain WVU 1853 [17]. These genes are equally sized and show extensive sequence variability in a 400-bp DNA BI2536 segment in the middle of the vlhA sequence, suggesting that the recombination event, though introduced

sequence variations, tended to preserve the overall sequence length and composition. Although it has been concluded that the potential of vlhA genes to vary is considerable, there is no indication as to which extent a vlhA gene could diverge without losing its properties. Previous studies from our laboratory have identified in M. synoviae strain WVU 1853, an immunodominant MEK inhibitor vlhA variant (termed MS2/28.1) [18] whose haemagglutinin region displayed a dramatic sequence shift and was considerably reduced in size, relative to the previously characterized expressed vlhA genes (vlhA1, vlhA4, and vlhA5) [17]. To better evaluate the extent of antigenic variation that could be tolerated by the M. synoviae haemagglutinin, we sought to know whether this highly divergent vlhA member was properly processed and

whether it remained functionally competent. Our results provide evidence that the antigenic repertoire of M. synoviae vlhA genes might be wider than previously perceived. Results Isolation of the MS2/28.1 fragment The complete nucleotide sequence of MS2/28, the λ phage-derived DNA fragment (GenBank accession number MSU66315) harbouring the immuno-reactive MS2/28.1 sequence, has been previously described [18]. It is 2657 bp long and contained two partial ORFs, referred herein to as, MS2/28.1 (5′ end) and MS2/28.2 (3′ end) (GenBank accession numbers ORF G2149016 and ORF G2149017, respectively). MS2/28.1 lacked its N-terminal sequence, whereas the C-terminal region of MS2/28.2 was incomplete. The two partial ORFs shared 71% and 61.

Forward and reverse oligonucleotide primers for each upstream intergenic region of the cluster were used to PCR amplify regions from the first strand cDNA to create second strand cDNA. In some instances, sequencing was used to confirm the correct amplification of second strand cDNA, either by direct sequencing of PCR products or by sequencing of TOPO TA cloning vectors (Invitrogen) containing the intergenic region. All RG7112 cell line cDNA PCR products were visualized on agarose gels. Use of promoter prediction and β-galactosidase reporter gene assays to search for promoter

activity Each intergenic region upstream of the genes in the jamaicamide pathway was examined for conserved binding regions (in comparison to the σ70 E. coli consensus promoter) using the BPROM predictor (http://​www.​softberry.​com;

Table 1). The upstream (up-) regions of genes predicted to contain a promoter (jamA, jamB, jamC, jamD, jamG, jamI, jamN, and jamQ) were amplified with specific primers Vistusertib in vivo (Additional file 1: Table S1) from fosmids produced previously [6]. Each upstream section was individually cloned into the pBLUE TOPO vector (Invitrogen) and transformed into TOP-10 E. coli. Plasmid purification (Qiagen) and sequencing (Seqxcel, Inc., La Jolla, CA) were used to confirm the sequence and direction of the inserts. Verified clones were used to measure NVP-BSK805 research buy relative promoter activity using the β-galactosidase reporter gene assay (Invitrogen), standardized against total soluble protein content as measured by BCA assay (Pierce). For this protocol, colonies from freshly streaked selective (100 μg ml-1 ampicillin) plates were grown overnight in LB media (5 ml). Cultures were pelleted at 4000 RPM, and the pellets were washed once with 3 ml of chilled PBS. After an additional centrifugation step, the pellets were resuspended in 1 ml PBS. The tube contents were centrifuged at 14,000 RPM for 5 min, and subsequently resuspended in 110 μl lysis buffer (0.25 mM Tris, pH 8.0). The cells were lysed Isoconazole by freeze-thawing the suspensions 4 times with dry ice and a 37°C water bath, followed by another centrifugation

period. The supernatant was removed from each tube for use in the β-galactosidase and BCA assays in a 96 well plate. O-nitrophenol measurements from ONPG cleavage in the assay were taken at 420 nm, while BCA readings were taken at 570 nm (Thermo-Electron Multiskan Ascent plate reader). Serial dilutions of lysis buffer from each culture were made to find an optimal range for colorimetric detection of o-nitrophenol and for a comparison of relative activity (identified as a 3-fold dilution). Relative activity calculations were made by determining the average nmol ONPG hydrolyzed min-1 mg soluble protein-1 value for each vector insert and dividing each value by the highest overall average value to obtain a percentage.

J Parenter Enteral Nutr 1990, 14:137S-146S.CrossRef 22. Rhoads JM, Wu G: Glutamine, arginine and leucine signaling in the intestine. Amino Acids 2009, 37:111–122.CrossRef 23. Rogero MM, Tirapegui J, Pedrosa RG, de Castro IA, de Olveira Pires IS, Chem B: Effect of alanyl-glutamine supplementation on plasma and tissue glutamine concentrations in rats submitted to exhaustive

exercise. Nutrition 2006, 22:564–571.CrossRefPubMed 24. McKenna MJ, Bangsbo J, Renaud JM: Muscle K+, Na+, and Cl disturbances and Na+-K+ pump inactivation: implications for fatigue. J Appl Physiol 2008, 104:288–295.CrossRefPubMed 25. Mourtzakis M, Saltin B, Graham T, Pilegaard H: Carbohydrate metabolism during prolonged selleck compound exercise and recovery: interactions between pyruvate dehydrogenase, fatty acids, and amino acids. see more J Appl Physiol 2006, 100:1822–1830.CrossRefPubMed 26. Sumida KD, Donovan CM: Enhanced hepatic gluconeogenic capacity for selected precursors after endurance training. J Appl Physiol 1995, 79:1883–1888.PubMed 27. Francesconi RP, Sawka MN, Pandolf KB, Hubbard RW, Young AJ, Muza S: Plasma renin and aldosterone responses at graded hypohydration levels during exercise-heat MK-0457 in vitro stress. J Appl Physiol 1985, 59:1855–1860.PubMed 28. Montain SJ, Laird JE, Latzka WA, Sawka MN: Aldosterone and vasopressin responses in the heat: hydration level and exercise intensity effects. Med Sci

Sports Exerc 1997, 29:661–668.PubMed 29. Kenefick RW, Maresh CM, Armstrong LE, Riebe D, Echegaray ME, Castellani JW: Rehydration with fluid of varying tonicities: effects on fluid regulatory hormones and exercise performance in the heat. J Appl Physiol 2007, 102:1899–1905.CrossRefPubMed 30. Koshimizu TA, Nasa Y, Tanoue A, Oikawa R, Kawahara Y, Kiyono Y, Adachi T, Tanaka T, Kuwaki T, Mori T, Takeo S, Okamura H, Tsujimoto G: V1a vasopressin receptors maintain normal blood pressure by regulating circulating blood volume and baroreflex sensitivity.

Proc Natl Acad Sci 2006, 103:7807–7812.CrossRefPubMed 31. Brandenberger G, Candas V, Follenius M, Libert JP, Kahn JM: Vascular fluid shifts and endocrine response to exercise in the heat. Eur J Appl Physiol 1986, 55:123–129.CrossRef 32. Convertino VA, Keil LC, Greenleaf JE: Plasma DCLK1 volume, renin, and vasopressin responses to graded exercise after training. J Appl Physiol 1983, 54:508–514.PubMed 33. Castell LM, Poortmans JR, LeClercq R, Brassuer M, Duchateau J, Newsholme EA: Some aspects of the acute phase response after a marathon race, and the effects of glutamine supplementation. Eur J Appl Physiol 1997, 75:47–53.CrossRef 34. Miles MP, Andring JM, Pearson SD, Gordon LK, Kasper C, Depner CM, Kidd JR: Diurnal variation, response to eccentric exercise, and association of inflammatory mediators with muscle damage variables. J Appl Physiol 2008, 104:451–458.CrossRefPubMed 35.

Analysis of CF isolates show increased expression Selleckchem LY294002 of QS, bacteriophage and other genes that are indicative of iron limited, stationary phase, and oxygen-limited growth

[23, 24] and many of these correlate with in vivo transcriptome analysis [25]. Despite the accumulation of evidence regarding gene expression during infection, the molecular basis for transmissibility is almost completely unknown. In this study, we employed a complementary proteomic approach involving two-dimensional gel electrophoresis (2-DE) and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2-DLC-MS/MS) with isobaric tags for relative and absolute quantitation (iTRAQ) to determine protein abundance differences between the reference strain P. selleck inhibitor aeruginosa PAO1, the virulent burn/wound isolate UCBPP-PA14 (PA14) and the early, transmissible CF-associated P. aeruginosa AES-1R. We identified over 1700 proteins of which 183 were present at statistically significant altered abundance between strains. This study identified 3 previously hypothetical proteins only expressed in strain

AES-1R, of which AES_7139 was the most abundant protein AZD1152 purchase detected on 2-DE gels. Other proteins present at elevated abundance in AES-1R compared to PA14 and PAO1 included several secreted and iron acquisition proteins, such as those associated with pyochelin synthesis and binding. AES-1R displayed an absence or decreased abundance of a number of porins including OprE, OprG and OprD, but elevated abundance of the multi-drug efflux protein MexX, part of the MexXY-OprM tripartite efflux pump. AES-1R also displayed differential abundance of proteins involved in lipopolysaccharide Chorioepithelioma and fatty acid biosynthesis. These data suggest that AES-1R expresses specific proteins and regulates the abundance of proteins shared with other P. aeruginosa strains to influence transmissibility and colonization of the CF lung. Methods Bacterial strains

and growth conditions P. aeruginosa PAO1 is a laboratory reference strain originally isolated from an infected burn/wound of a patient in Melbourne, Australia (American Type Culture Collection ATCC 15692), strain PA14 (UBPPC-PA14) was obtained from Dr. Laurence Rahme, Harvard Medical School, Cambridge, MA [26] and AES-1R was obtained from Prof. David Armstrong, Monash Medical Centre, Australia [7]. Strains were cultured in six replicates of 50 mL of salt modified Luria-Bertani broth (5 g/L NaCl) and grown to stationary phase (OD600 nm ~ 1.0) with incubation at 37°C and shaking at 250 × rpm (Additional file 1). Cultures were harvested, washed three times with phosphate-buffered saline and cells collected by centrifugation at 6,000 × g for 10 mins at 4°C. The resulting bacterial cell pellets were frozen, lyophilized and stored at -80°C. Phenotypic assays Phenotypic assays on P.

21-22 nts) are produced selleck screening library by a Dicer-1/Loquacious/Ago1 dependent mechanism [8, 9]. Intriguingly, components from these two pathways do not function exclusively from one another. Dicer-2 and an alternate spliceform of Loquacious interact to produce endogenous siRNAs (endo-siRNAs) [10, 11]. This alternate pathway is also an important regulator of

host gene expression and selfish genetic elements [12]. PIWI pathway products, piRNAs, 24-30 nts in length, are produced in a Dicer-independent manner [13]. Moreover, an additional sRNA size class has been described in the anti-Ago2 antibody immunoprecipitation of unusually small RNAs (usRNAs) (ca. 13-19 nts) [14]. Triggers for SRRPs are only partially understood. The

anti-viral and endo-siRNA pathways have a double-stranded RNA trigger which activates processing and loading of an 20-23 nt siRNA guide strand [15]. Once loaded, the RISC may be recycled. The miRNA pathway relies on microRNA-encoding genes that are processed in a DGCR8/Drosha-dependent manner [16]. In contrast to siRNAs, miRNAs, also 20-23 nts, bind to target transcripts with imperfect complementarity. PIWI pathway sRNA biogenesis is less understood but likely involves a selleck kinase inhibitor single-stranded RNA trigger (reviewed in [7]). Mosquito-borne dengue virus is a human health threat in tropical urban areas and causes sporadic outbreaks in the www.selleckchem.com/products/pd-1-pd-l1-inhibitor-2.html southern US [17, 18]. It is transmitted to humans by aedine mosquitoes and has bypassed the requirement for an enzootic amplification cycle, thus increasing the threat to public health. Arboviruses must successfully replicate in mosquitoes, escape anti-viral defense, and then invade salivary glands in order to be transmitted during blood feeding to subsequent hosts. Using radioisotopic detection, newly replicated Dengue virus serotype 2 (DENV2) genomes can be detected in Ae. aegypti Higg’s White Eye (HWE) midguts, the initial site of infection,

as early as 4 days post infection (dpi), and v iral i nterfering sRNAs (viRNAs) at 8 dpi [6, 19]. The best described anti-viral RNAi pathway relies on a Dicer-2 dependent mechanism whereby the Ago2 endonuclease cleaves target RNAs [20]. Silencing of RNAi component transcripts Ago2, R2D2 and Dicer-2 in Ae. aegypti Resminostat increases DENV2 titers; therefore these components play an important role in controlling arbovirus replication [3, 6, 21]. Another component of the RNA-induced Silencing Complex (RISC) is Tudor-SN (TSN), a transcriptional co-factor [3, 22]. Given the presence of a functional RNAi pathway, it remains a mystery as to how arboviruses overcome anti-viral defense to establish persistent infections and perpetuate the arbovirus disease cycle. sRNAs represent the product of host mRNA or viral RNA cleavage in an RNAi-specific manner.

3). CcmL and CsoS4A have been structurally characterized (Tanaka et al. 2008); both form pentamers and have a pronounced concave/convex sidedness similar to the hexamers. In contrast to the hexameric shell proteins, the electrostatic potential of these proteins is predominantly

positive (Fig. 6). The structures of CcmL and CsoS4A can be superimposed with an RMSD of 0.74 Å over 58 C-α atoms. The largest difference between the primary structures of these two proteins is in the region corresponding to an 8–10 amino acid loop on the concave face of the pentamer that seems to influence the charge of the concave face. A similar difference is seen between the paralogs CsoS4A and CsoS4B. In this region CsoS4B has more positively see more NVP-LDE225 charged residues than CsoS4A. The pores Based on the current models of carboxysome function and structure, pores in the shell protein hexamers provide conduits for the flux of metabolites; bicarbonate ions and RuBP diffuse in and 3PGA to diffuses out, while preventing the Proteasome inhibitor leakage of CO2 from the interior (Dou et al. 2008). The shell also prevents oxygen from diffusing in, reducing unwanted photorespiration by RuBisCO (Marcus et al. 1992). As the shell localizes CA and RuBisCO together, the overall rate of CO2 fixation by RuBisCO is enhanced; effectively, the carboxysome provides a focal point for the carbon concentrating mechanism (CCM) (Fig. 2). A key characteristic of carboxysome shell proteins is a narrow (~4–7 Å

diameter; Kerfeld et al. 2005) central pore that is formed at the 5- and 6-fold axis of symmetry by a loop in the hexamers and pentamers, respectively. Residues forming this loop tend to be conserved

among paralogs; for example, these residues are K-I-G-S and R-(A/V)-G-S in CcmK2 and CcmK4, respectively (Table 1). Such differences in residues flanking the pore likely Non-specific serine/threonine protein kinase influence the flux of metabolites into or out of the carboxysome by influencing the size and charge of the pore. All of the pores of structurally characterized carboxysome shell proteins are positively charged at the narrowest point (Fig. 9); presumably this provides a favorable attractive force for negatively charged metabolites such as bicarbonate. At the same time, a charged pore would not attract molecules lacking a dipole moment, such as CO2 and oxygen (Fig. 9). Table 1 List of structurally characterized BMC-domain proteins from the carboxysome and their dimensions Pfam00936 protein Carboxysome type Hexamer diameterb (Å) Hexamer edge lengthc (Å) Pore residues Pore diameter (Å) CsoS1A [2G13] α 72 36 FVGG 4 CsoS1C [3H8Y] α 72 36 FVGG 4 CcmK1 [3BN4] β 75 37 KIGS 4.8 (5.5) CcmK2 [2A1B] β 75 35 KIGS 5.5 (7) CcmK4 [2A10] β 75 37 RAGS 4 CsoS1Da [3F56] α 72 36 ERAF 12.5 (14) PDB IDs of the listed structures are in brackets. aCsoS1D is a tandem BMC-domain protein; values for the dimensions of the pseudohexamer are reported. b Hexamer diameter was measured from one vertex to its opposite vertex.

All authors read and approved the final manuscript.”
“Background The emergence of antimicrobial resistance is severely limiting treatment options for many important infectious diseases [1, 2]. Traditionally the problem of antimicrobial resistance has been approached buy DAPT by developing new compounds having increased potency. Unfortunately, development of new compounds is not keeping pace with the emergence of antibiotic-resistant pathogens. Consequently, new strategies are needed to preserve existing agents. One approach is to seek compounds that will enhance

the activity of distinct antimicrobial classes by blocking resistance mechanisms. For example, β-lactamase inhibitors extended the utility of β-lactams when delivered as combinations such as Augmentin (amoxicillin-clavulanic acid) [3], and inhibitors of efflux

pumps produced synergistic inhibition of growth against tetracycline-resistant Escherichia coli when used in combination with doxycycline [4]. The conventional strategy has been to identify genes whose inactivation increases the ability of compounds to block bacterial growth (decreases in minimal inhibitory concentration, MIC) [5]. Since some compounds kill bacteria by processes that are distinct from bacteriostatic p53 activator action [6, 7] and since deficiencies in repair of lethal damage may not affect bacterial growth, the possibility EX 527 in vivo exists that genes involved in bacterial survival are distinct from those that protect from growth inhibition. Finding genes whose products protect from the lethal effects of stress requires screening procedures that differ from those used for bacteriostatic effects. In the present work, we used the prototype quinolone, nalidixic acid, as

a probe for screening genes whose products protect E. coli from lethal effects of stress. Nalidixic acid was chosen as the initial screening agent because bacteriostatic and lethal action are distinct events that are sensitive to different drug concentrations (for review see [8]). Mutants of E. coli, obtained by Tn5-mediated insertional mutagenesis, were screened for those that had the same bacteriostatic susceptibility to nalidixic acid as the wild-type strain out while exhibiting greater sensitivity to the lethal action of the drug. We call this new phenotype hyperlethality. With this phenotype we could eliminate from consideration mutants with altered drug uptake, efflux, and target interactions, since these properties affect bacteriostatic activity. The decreased survival of the mutants was expected in some cases to arise from disruption of genes involved in protecting from lethal stress. The hyperlethal mutants were then examined by measuring the lethal action of several other antimicrobial and environmental stresses. This work defined a novel bactericidal phenotype and identified a diverse set of poorly characterized bacterial stress-response genes as a new source of potential targets for antimicrobial enhancement.

Conclusions EV71 and CA16 were highly diverse in the nucleotide sequences of vp1s and vp4s. The severity of illness of EV71 infected was not associated with the sequence variation of vp1s or vp4s. The sera positive rates of VP1 and VP4 of EV71 were lower than that of CA16, suggesting less exposure rate to EV71 than CA16 in Beijing population. The detection of serum antibodies by Western blot using VP1s and VP4s as antigens indicated that the immunological reaction to VP1 and VP4 of both EV71 and CA16 was different. IgM against VP1 but not VP4 was

generated in children after acute infections, which needs to be clarified further. Methods Clinical specimens and isolation of viruses Throat swabs and vesicle fluids were collected from infants and children with clinical diagnosis of HFMD or suspected

GSK690693 EV infection who visited the Affiliated Children’s Hospital to Capital Institute of Paediatrics during the HFMD seasons PF-6463922 mw of year 2007 to 2009. The specimens were inoculated in Vero cells after being delivered to the Laboratory of Virology, and CPE were observed by microscopy everyday. When the CPE reached ++++, the isolates were harvested and stored at-80°C until use. Serum specimens Serum specimens for the detection of IgM antibodies against the expressed VP1s and VP4s were collected from infants and children with acute EV infection, including 14 from children with acute EV71 infection and 12 from children with acute CA16 infection identified by RT-PCR, virus isolation from throat swabs and vesicle fluids, and immnofluorescence staining of IgM against

EV71 or CA16 in sera (data not shown). Another batch of 189 sera were collected for the detection of IgG antibodies against the expressed proteins, including 141 from adults for regular health check up and 48 children without acute EV infections. The study was performed according to the Declaration of Helsinki II and approved by Ethics Committee of Capital Institute of Paediatrics and written informed consent was obtained from IMP dehydrogenase all patients or from their caretakers. Identification of EV71 and CA16 from clinical specimens and isolated viruses by RT-PCR RNAs were extracted from clinical specimens and isolated virus strains using selleck screening library Trizol (Invitrogen, USA) following the instructions provided by manufacture. RT-PCR was carried out to identify EV71 and CA16 in the specimens and virus isolates. Viral cDNAs were generated using random primer (Invitrogen, USA) and M-MLV (Invitrogen, USA) by reverse transcription. EV consensus primers, EV71 and CA16 specific primers were synthesized according to Perara D’s [33] and Singh S’ [35], and used to detect EV71 and CA16 by PCR as described by our group previously [29]. The PCR products were analyzed by electrophoresis in a 2% agarose (GibcoBRL, US) gel and visualized by staining the gels with ethedium bromide.

The optimal reaction SB-715992 nmr pH of the endolysin was examined by adding dialyzed endolysins to cells of B. thuringiensis strain HD-73 resuspended in a Entinostat ic50 series of buffers (20 mM Tris) at various pH levels

(pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0), and the OD600 was monitored as described above. The optimal reaction temperature of the endolysin was tested in 20 mM Tris-HCl (pH 8.0) at temperatures of 10–80°C in 10°C increments, and the OD600 was again monitored. To analyze the endolysin thermostability, endolysins in 20 mM Tris-HCl (pH 8.0) were first treated at different temperatures (4°C, 30°C, 40°C, 50°C, and 60°C) for 1 h and the lytic activity was tested as described above. All experiments were carried out in triplicate. Labeling and binding activity assay of PlyBt33-IC To test binding activity, purified PlyBt33-IC was labeled with FITC (Sigma-Aldrich, Saint Louis, PFT�� in vivo MO) according to the manufacturer’s instructions. Following purification, PlyBt33-IC protein was dialyzed four times against FITC reaction buffer (7.56 g NaHCO3, 1.06 g Na2CO3, 7.36 g NaCl, with MilliQ water added to 1 l and the pH adjusted to 9.0). FITC was dissolved in dimethyl sulfoxide to a concentration of 1 mg/ml and added into the PlyBt33-IC suspension at a ratio of 150 μg FITC

to 1 mg PlyBt33-IC. Following 8 h incubation at 4°C in the dark, the reaction was stopped with NH4Cl at a final concentration of 50 mM for 2 h at 4°C in the dark. The labeled protein was dialyzed against PBS (8 g NaCl, 0.2 g KCl, 3.49 g Na2HPO4.12H2O, 0.24 g KH2PO4, with MilliQ water added to 1 l and the pH adjusted to 7.4) several times until the dialysis liquid was colorless, and stored at −20°C until required. BSA was also labeled as above and used as a control. FITC-labeled proteins were named FITC-PlyBt33-IC and FITC-BSA. The specific binding activity of PlyBt33-IC to the cell wall was assayed as described previously with some modifications [12, 46]. B. thuringiensis strain HD-73 was grown to mid-exponential phase in LB broth (OD600 = 0.8), and the cells were harvested by centrifugation (10,000 × g for 1 min) and Carbohydrate resuspended

in a one-tenth volume of PBS-T (pH 7.4, 0.01% Tween 20). FITC-labeled PlyBt33-IC was added to a 100 μl cell suspension to a final concentration of 0.0125 mg/ml and incubated at 30°C for 5 min. For fluorescence microscopy observation, the cells were harvested by centrifugation and washed twice with 500 μl PBS-T buffer. The pellet was then resuspended in 50 μl PBS-T. FITC-labeled BSA was used as a control. All cells were observed using an Olympus BX51 microscope. Acknowledgments This study was supported by the National Natural Science Foundation of China (No.31170123), the National Project (2009ZX08009-056B), and the projects of the Chinese Academy of Sciences (KSCX2-EW-G-16).