To construct plasmid pENA9, a 025-kb DNA fragment containing the

To construct plasmid pENA9, a 0.25-kb DNA fragment containing the phaR promoter region was amplified by PCR. Cyclopamine After digestion with EcoRI and HindIII, the DNA fragment was inserted into pLC4 (Ray et al., 1985), which carries the xylE reporter gene. To construct plasmid pENA10, a 2-bp mutation was introduced into the −35 region of the putative σA-like promoter of phaR using a two-step PCR method (Higuchi et al., 1988). The resulting DNA fragment was digested with EcoRI plus HindIII and cloned into pLC4. Disruptions of the chromosomal phaC, sigB, sigH, spo0A, spo0F, or sigF gene of B. thuringiensis by integrations of plasmid pRN5101-derived pENA1, pENA2, pENA3, pENA4, pENA5, or pENA6, respectively,

through a Campbell-like single-crossover recombination were performed as described previously (Fedhila et al., 2002). The correct integrations were verified by PCR. Construction of the abrB deletion mutant BNA7, in which the abrB gene was replaced with the kanamycin resistance gene kan via a two-step gene replacement event, was performed according to the method described previously (Arnaud et al., 2004). The correct replacement of the chromosomal abrB sequence was verified by PCR. To construct the abrB spo0A double mutant BNA8, the pRN5101-derived plasmid pENA4 was introduced into

the abrB deletion mutant BNA7. Chromosomal integration of pENA4 through a single-crossover recombination was carried out as described above. The correct integration was verified click here by PCR. The PHB contents of B. thuringiensis cells were determined by GC as described Celastrol previously (Tseng et al., 2006) as well as using the method involving spectrophotometric determination of crotonic acid generated from digestion of PHB with concentrated sulfuric acid (Law & Slepecky, 1961). The sample preparation was carried out according to the method described in the literature (Tian et al., 2005), but with a slight modification as described previously (Chen et

al., 2009). Thin sections were examined on a JEM 2000EXII TEM (JEDL Inc.). Transformation of B. thuringiensis cells by electroporation was carried out as described previously(Bone & Ellar, 1989). Total RNA was isolated from B. thuringiensis according to the previously described method (Zuber & Losick, 1983). Northern blotting and primer extension analysis were performed using standard methods (Sambrook & Russell, 2001). An established method was used for spectrophotometric measurement of XylE (catechol 2,3-dioxygenase) activity (Ray et al., 1985). Protein concentrations were determined using the BCA protein assay method according to the instructions of the manufacturer (Pierce Biotechnology Inc.). Measurement of the PHB contents of B. thuringiensis revealed that this bacterium gradually accumulated PHB in the stationary phase after growth in LB medium (Fig. 1a). To demonstrate that the B. thuringiensis homolog of the B.

, 2005) These include two ATP-binding cassette (ABC) transporter

, 2005). These include two ATP-binding cassette (ABC) transporters, TcyABC and TcyJKLMN, and a symporter TcyP (Burguiere et al., 2005). The TcyJKLMN and TcyP uptake systems are high-affinity transporters

while TcyABC is a low-affinity l-cystine transporter (Burguiere et al., 2005). The TcyJKLMN transporter, encoded within a large operon called the ytmI operon, was found to be the most sensitive to l-cystine starvation compared with other transporters in that its expression was repressed more than 200-fold in the presence of sulfate or l-cystine (Carlsson, 1970). In addition, the expression of the ytmI operon was induced during disulfide stress by the thiol oxidant diamide (Chapot-Chartier et al., 1993). TcyP and TcyABC l-cystine transporters have also been identified in Staphylococcus aureus and were shown to be negatively regulated by the CymRSA regulator, a global regulator that controls cysteine metabolism Selleck Olaparib in response to its availability (Coppee et al., 2001). Cysteine metabolism has not been extensively studied in S. mutans. However, Sperandio et al. 2010 recently characterized two LysR-type transcriptional regulators, CysR and HomR, which activate transcription of genes involved in cysteine metabolism and transport. These authors also identified two l-cystine importers, TcyABC and TcyDEFGH, whose expression was activated by CysR and HomR, respectively (Sperandio et al.,

2010). We sought to characterize the tcyABC tri-cistronic operon encoding the TcyABC transporter in S. mutans. Mutagenesis of tcyABC severely impaired the ability of

S. mutans to transport l-cystine and survive under cystine starvation conditions. BAY 80-6946 solubility dmso We also identified a novel Lys-type regulator of TcyABC which we termed TcyR. Unlike most Lys-type regulators, TcyR was found to repress transcription of the tcyABC operon. Streptococcus mutans strain UA159 was used to construct mutants. Unless otherwise specified, strains were routinely cultured in Todd-Hewitt yeast extract (THYE) medium (BD Biosciences) at 37 °C in air with 5% CO2 without agitation. Mutant strains were propagated in THYE agar plates supplemented with erythromycin at 10 μg mL−1. Optical density (OD) was measured using an Ultrospec 3000 UV/Visible Spectrophotometer (Fisher Scientific). Streptococcus mutans UA159 was Chlormezanone used as the wild-type strain. The S. mutans ΔtcyA (SmTcyA), ΔtcyB (SmTcyB), ΔtcyC (SmTcyC), ΔtcyABC (SmTcyABC), and ΔtcyR (SmTcyR) mutants were constructed in UA159 by a PCR ligation-based deletion strategy as described previously (Cvitkovitch et al., 1997). Briefly, an erythromycin resistance cassette was used to disrupt the tcyC, tcyABC, and tcyR coding regions in the S. mutans UA159 wild-type chromosome using the primer pairs listed below. To confirm successful integration of the erythromycin gene into these coding regions, chromosomal DNA was isolated from erythromycin-resistant transformants and subjected to validation using PCR and nucleotide sequence analysis.

Other exclusion criteria were a history of central nervous system

Other exclusion criteria were a history of central nervous system (CNS) infection, stroke, serious head injury, or other neurologic event likely to affect cognition. Many patients were exposed to low doses of psychoactive drugs, either on a prescription or recreational basis. Given that this is a clinical reality in this population, we excluded only those in whom drug effects might be expected to substantially affect cognition. Of the patients originally referred selleck screening library for the study, only three

met one of these exclusion criteria (one with MoCA <20, one with a history of another CNS process, and one with intoxication at the time of testing). The protocol was approved by the ethics board of the McGill University Health Centre, and all participants provided informed consent. All tests Pirfenidone price were administered

in the same session by a trained research technician, in a quiet room, in the patient’s choice of either French or English. Clinical information was collected using a semi-structured interview at the time of testing, supplemented by clinic chart review. Patient age, sex, educational level, and mother tongue were recorded and evaluated for their impact on cognitive test performance. Age was coded into 5-year bins and educational level was coded as some vs. no education at the university level. Mother tongue was coded as English, French or other. Clinical characteristics deemed relevant to cognitive test performance and HIV-infection-related

variables were also recorded, including the presence of self-reported cognitive complaints (no/yes), and Selleckchem Sunitinib the presence of depressive symptoms as evaluated with the Beck Depression Inventory II (BDI-II; minimal, mild, moderate or severe). The MoCA was administered and scored according to the published instructions for this test ( Individual items of the MoCA test were coded dichotomously as failed or passed for each patient, with the exception of Serial 7s subtraction. For this item we used a polytomous scoring system of 0 to 5 based on the sum of correct responses over five consecutive subtractions. Participants performed seven tasks examining different aspects of frontal lobe function. Reversal learning. Participants learned to make response selections based on feedback. The score was the total number of correct selections [28]. Emotion recognition. Participants rated the degree of emotional expression in a series of faces and were scored based on the difference between ratings of emotional and neutral faces [29]. Letter 2-back task. In this working memory test, a series of letters was presented and participants were scored on their ability to detect letters matching the one presented two trials previously [30]. Stop-signal task.

The genes required for PGA synthesis in B anthracis


The genes required for PGA synthesis in B. anthracis

are part of the cap operon (capBCADE) located on the pX02 plasmid (Makino et al., 1989; Uchida et al., 1993; Candela et al., 2005). The capD gene encodes a γ-glutamyltranspeptidase, or PGA depolymerase, which was found to be required for the covalent anchoring of PGA to the peptidoglycan in B. anthracis (Candela & Fouet, 2005). PGA producers that lack capD produce a loose slime layer instead of a covalently linked capsule (Candela & Fouet, 2005). While all Group I and II isolates in this study tested negative for the four cap genes tested (including capD), they were still able to produce a covalently linked PGA capsule. Homologs to several of the cap genes AC220 have been found in other bacteria, such as Bacillus subtilis, Bacillus licheniformis, and Staphylococcus epidermidis (Ashiuchi & Misono, 2002; Urushibata et al., 2002; Candela & Fouet, 2005, 2006; Candela et al., 2005; Kocianova Selleckchem Verteporfin et al., 2005). It is possible that the Group I and II isolates contain PGA synthesis genes that more closely resemble these homologs than those in the cap operon of B. anthracis and are thus too divergent to amplify using the cap PCR primers used in this study. A clear difference in colony morphology on bicarbonate

agar was evident for about half of the isolates. These isolates, as well as the B. cereus G9241-positive control, produced dull or dry colonies, whereas virulent strains of B. anthracis produce the shiny or the mucoid morphology. Thus, although commonly used for capsule expression and observation in B. anthracis, the colony morphology of other species on bicarbonate agar alone is not an accurate indicator of capsule production. The d-PGA capsule of B. anthracis is required for virulence, in addition to Phospholipase D1 the tripartite toxin encoded by the pX01 plasmid. The role in virulence, if any, of the B. anthracis-like PGA capsules produced by

Group I and II isolates, however, is unknown. We cannot be certain that these isolates were responsible for the infections, as Bacillus spp. are frequent contaminates of clinical samples, and no further epidemiological data were available (Farrar & Reboli, 2006). However, during the course of this study, 20 clinical isolates from CDC’s Special Bacteriology Reference Laboratory’s (SBRL) culture collection were identified as having a high degree of 16S rRNA gene sequence similarity (99.87–100%) to one or more of the Group I or II isolates, and were subsequently tested for capsule production. These isolates had been sent by various states or territories (AK, CA, CO, MO, OH, PA, PR, TN, VA, and VT) for identification from 2001 to 2008, and were ultimately identified by SBRL as either Bacillus spp. or Brevibacterium spp. Nineteen of the 20 isolates tested positive for capsule production, detected by both methods described above.

suis 2 of 110 kDa (Fig 2b), confirming that HtpS is a cell surfa

suis 2 of 110 kDa (Fig. 2b), confirming that HtpS is a cell surface-associated protein of S. suis 2. To determine whether rHtpS-elicited antibodies could affect C3 deposition on the surface of S. suis 2, 05ZYH33 binding of C3 was detected by FCM after incubation with different concentrations of rabbit anti-HtpS sera. C3 deposition on S. suis 2 was low (41.9±2.01%) in the absence of rabbit anti-HtpS sera. When S. suis 2 was incubated with increasing concentrations of rabbit anti-HtpS sera, the C3 deposition on the bacterial surface was increased significantly in a rabbit anti-HtpS sera concentration-dependent manner, with up to 62.9±4.20% bacteria positive

with 50% rabbit anti-HtpS PD-0332991 datasheet sera (Fig. 4). Normal human sera and preimmune rabbit sera

were used as controls and induced only weak changes in C3 deposition compared with rabbit anti-HtpS sera (data not shown). The bactericidal experiment was adopted to evaluate the bactericidal activity of the rabbit anti-HtpS antibody. As shown in Fig. 5, 72.9±3.88% of S. suis 2 bacteria could survive after incubation with whole blood not containing rabbit anti-HtpS antibody. In the presence of 5% rabbit anti-HtpS antibody, the survival of the bacteria in whole blood was significantly reduced to 51±7.74%. To determine BIBF 1120 datasheet whether rHtpS can protect mice against S. suis 2 infection, mice were immunized with rHtpS and challenged with a lethal dose of S. suis 2 05ZYH33. ELISA test results revealed that titers of rHtpS-specific antibody of the group immunized with rHtpS ranged from 204 800 to 819 200 before challenge with S. suis 2. After the challenge, all 10 mice of the negative control group died

within 24 h postinoculation, while only two out of 10 mice immunized with rHtpS died in the same period. The remaining eight mice only exhibited rough hair in the first 24 h postinoculation, and then recovered and survived (Fig. 6). The mortality rate was significantly reduced in mice immunized with rHtpS (P<0.001), indicating that rHtpS confers protection in mice. So far, histidine triad family proteins have been documented in group A, B, C, G streptococcal species, S. pneumoniae, as well as S. suis 2 (Adamou et al., 2001; Kunitomo et al., 2008; Aranda et al., 2009). tuclazepam Although the function of this family is not clear, histidine triad protein family members, Pht proteins of S. pneumoniae and HtpA of S. pyogenes, have proved to be good candidates for subunit vaccines due to their strong ability to protect mice against bacterial infection (Adamou et al., 2001; Zhang et al., 2001; Kunitomo et al., 2008). Crystal structure analysis of PhtA revealed that the histidine triad domain of histidine triad protein family members was a zinc-binding fold (Riboldi-Tunnicliffe et al., 2004, 2005). Recently, Ogunniyi et al.

baumannii BM4547 and P aeruginosa PU21

as recipients and

baumannii BM4547 and P. aeruginosa PU21

as recipients and the five NDM-1-positive E. coli J53 transconjugants as donors. Mixes of donor and recipients cells were incubated for 18 h at 37 °C for S. typhimurium LT2, A. baumannii BM4547, P. aeruginosa PU21 and P. mirabilis CIP103181 and for 3 h at 37 °C for K. pneumoniae CIP15153. In addition, E. coli J53 transconjugant carrying a c. 70-kb IncF-type blaCTX-M-15-positive plasmid was included for comparison, as IncF-type plasmids conjugate efficiently among Enterobacteriaceae (personal data). Transfer frequencies were calculated by dividing the number of transconjugants by the number of donor cells. Statistical analysis was performed Trametinib in vitro using the Student’s t-test; a P-value of ≤ 0.05 was considered significant. Transformation experiments were performed as described previously by electroporation CH5424802 order of a plasmid DNA suspension from the five NDM-1-positive E. coli J53 into

rifampicin-resistant P. aeruginosa and A. baumannii reference strains (Potron et al., 2009). pAT-RTG-4 (shuttle vector) and pInt-Veb plasmids were used as positive control for electroporation in A. baumannii and P. aeruginosa (Aubert et al., 2003; Potron et al., 2009). Selection was performed on agar plates supplemented with ticarcillin (50 μg mL−1). MICs of carbapenems and cefotaxime were determined using the E-test strips (bioMérieux, Marcy l’Etoile, France). The five blaNDM-1-carrying plasmids of Enterobacteriaceae studied here belonged to various incompatibility groups (L/M, FII, A/C and two untypeable plasmids). IncL/M, IncA/C and IncFII plasmid types have been frequently described in Enterobacteriaceae carrying other β-lactam resistance determinants (Carattoli, 2009). IncL/M- and IncA/C-type plasmids

are broad-host range plasmids, whereas IncF-type plasmids are narrow-host range plasmids (Novais et al., 2007). Vasopressin Receptor The five NDM-1-positive plasmids were self-conjugative using E. coli J53 as recipient at frequencies ranging from 10−4 to 10−8 transconjugants/donor (Table 1). The blaNDM-1 gene was the single carbapenem resistance marker located on those plasmids. Using blaNDM-1-positive E. coli J53 transconjugants as donors, second-step transconjugants were obtained using E. coli, K. pneumoniae, S. typhimurium and P. mirabilis as recipient species. In E. coli JM109, transfer frequencies ranged from 10−4 to 10−8 transconjugants per donor depending on plasmid type (Table 2). The lowest transfer frequencies were obtained with the untypeable plasmid p419 and IncA/C-type plasmid pKp7. No difference of transfer rate was observed using E. coli Tc601 and E. coli Tc271 as donors when different temperatures were used during the mating-out assays (Table 2). Using Tc419 and TcKp7 as donors, the transfer rate was significantly higher at 30 °C compared with that observed at 25 °C and 37 °C (P < 0.05), as reported for other blaNDM-1-positive plasmids (Walsh et al., 2011). For E.

Anaemia was defined as a haemoglobin level ≤12 or ≤14 mg/dL for w

Anaemia was defined as a haemoglobin level ≤12 or ≤14 mg/dL for women and men, respectively [17]. Patients could develop anaemia or, for those with anaemia, worsening anaemia was defined as a haemoglobin level ≤8 mg/dL. For the liver function tests, 40 IU/L was taken as the ULN (for GSI-IX both ALT and AST) [18]. Patients were followed until they experienced an event or to the date of their last measurement for each clinical or laboratory marker in EuroSIDA. It should be noted that not all patients in all groups had information on these markers available for all analyses; therefore, the number of patients included in each analysis

differed according to the availability of data. Patients with the event at baseline were excluded from analyses. Any factor that was significant at the 10% level in univariate analyses (P<0.1) was included in multivariate analyses. In multivariate analyses, statistical significance was attained

if P<0.05. All analyses were performed using sas 9.1 (SAS Institute, Cary, NC, USA). A total of 6634 patients started a nevirapine- (1600; 24%), efavirenz- (3109; 47%) or lopinavir- (1925; 29%) based cART regimen after 1 January 2000. A total of 1750 patients (26%) were excluded from the analysis because they had no CD4 cell count or viral load measurement prior to starting treatment: 410 (26%) on nevirapine, 888 (29%) on efavirenz, and 452 (23%) on lopinavir. A total of 1039 patients (21%) were excluded because of previous exposure to any of the three Lepirudin drugs: 339 on nevirapine (28%), 297 on efavirenz (13%) and 403 on lopinavir (27%). Nine hundred and fifty-nine

patients (25%) did not achieve suppression, had stopped treatment within the first 3 months or did not have sufficient follow-up and were therefore excluded: 248 (29%) on nevirapine, 459 (24%) on efavirenz, and 252 (24%) on lopinavir. Thus, a total of 2886 patients were included in the analysis; 603 of these patients (21%) were on a nevirapine-based cART regimen, 1465 (51%) on an efavirenz-based cART regimen, and 818 (28%) on a lopinavir-based cART regimen. Patients excluded from the analysis had similar characteristics to those included, but were more likely to have previous cART exposure (64%vs. 57%, respectively; P<0.0001) and to have a prior AIDS diagnosis (32%vs. 26%, respectively; P<0.0001). Table 1 compares the characteristics of the patients in each group at the time of starting their new regimen. A lower proportion of patients starting nevirapine were treatment naïve: 28%, compared with 38% of patients starting efavirenz and 38% of patients starting lopinavir. Patients on nevirapine had a higher median CD4 count [359 cells/μL; interquartile range (IQR) 230–583 cells/μL] and a lower median viral load (2.70 log10 copies/mL; IQR 1.70–4.56 log10 copies/mL) compared with those on efavirenz [median CD4 count 323 cells/μL (IQR 190–535 cells/μL) and median viral load 3.59 log10 copies/mL (IQR 1.70–4.

The fundamental process in JIA is chronic inflammation, in which

The fundamental process in JIA is chronic inflammation, in which the immune system understandably plays a critical role.[1] Both innate and adaptive immune systems have been implicated in the pathogenesis of various subtypes of JIA. Over the last two decades our understanding of the pathophysiology of this condition has

improved a great deal and several new genetic associations have been PCI-32765 order recognized.[1, 3, 4] Family studies have provided firm evidence for genetic susceptibility in JIA. Although many candidate genes have been tentatively identified, most of these lack validation studies on different populations and appropriate sample sizes.[1, 3, 4] Human leukocyte antigen (HLA) linkages have been noted in oligoarticular and polyarticular forms of

JIA. Oligoarticular JIA has been shown to be associated with HLA-A2, DR5 and DR8, whereas DRB1*04, DRB1*07 and DQA1*03 are said to be protective.[1, 3, 4] HLA-A2, DRB1*08, DQA1*04 and DPB1*03 are associated with RF-negative polyarticular JIA and DRB1*04, DQA1*03 and DQB1*03 with RF-positive polyarticular JIA. RF-positive polyarthritis is also associated with HLA-DR4, DR1 and DR14, whereas DQA1*02 is protective.[1, 3, 4] HLA associations for oligoarticular JIA and RF-negative polyarticular JIA overlap, LEE011 suggesting that these are genetically related. However, RF-positive polyarticular JIA appears to be a genetically distinct disorder and has HLA linkages similar to adult rheumatoid arthritis. Quite understandably, the clinical course, response to treatment and complications associated with RF-positive polyarticular JIA are also similar to adult rheumatoid arthritis. Several non-HLA genes

have now been discovered to be linked with subtypes of JIA and the list of putative markers has been expanding over the years. Although many such associations have been previously suggested, these have not been subsequently replicated in follow-up studies in different populations. Coproporphyrinogen III oxidase Independent confirmations could be obtained for only a few candidate genes like, such as ‘Protein tyrosine phosphatase, non-receptor type 22 (PTPN22)’, ‘Migration Inhibitory Factor (MIF)’, ‘Solute carrier 11 member 1 (SLC11A1)’ encoding for the natural resistance-associated macrophage protein 1, ‘WNT1 inducible signaling pathway protein 3 (WISP3)’ and ‘Tumour necrosis factor α gene (TNFA)’.[1, 3, 4] Thompson et al.[5] in a landmark study, examined a cohort of 809 JIA cases of non-Hispanic European ancestry and reported that ‘PTPN2’, ‘COG6’ and ‘ANGPT1’ were associated with oligoarticular and RF-negative polyarticular JIA. These are also known to be associated with type 1 diabetes mellitus, Crohn’s disease and multiple sclerosis, thus emphasizing the fact that common genetic mechanisms may underlie many autoimmune diseases and could influence therapeutic interventions.[5] In a subsequent study published in 2012, Thompson et al.

Importantly, the assay can be performed at bedside and in rural a

Importantly, the assay can be performed at bedside and in rural areas

using only Selleck AZD0530 a water bath (Tomita et al., 2008). Several LAMP assays have been developed to detect common causative pathogens of BM such as Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Escherichia coli and Staphylococcus aureus (Seki et al., 2005; Yamazaki et al., 2008; Hanaki et al., 2011; Kim et al. 2011; McKenna et al. 2011). However, no LAMP assay has been reported to detect Streptococcus agalactiae and Streptococcus suis, which are two of the most common pathogens of BM in some countries (Mai et al., 2008; Chiba et al., 2009). Moreover, it is laborious and expensive by performing multiple reactions for each sample to detect bacterial pathogen. Thus, we aimed to design and develop a LAMP assay capable of detecting multiple bacterial species based on the nucleotide sequences of the 16S rRNA genes of the bacteria. The broad range LAMP primers were designed to be specific for eubacterial 16S rRNA-specific gene. This gene was chosen because

of its highly conserved regions among species and has been widely used as a target for broad range PCR method (Gray et al., 1984; Lane et al., 1985). The partial nucleotide sequences of the 16S rRNA genes of S. aureus (GenBank FJ907240.1), S. pneumoniae (Z22807), S. suis (Z22776.1), S. agalactiae (Z22808), N. meningitidis (Z22806), H. influenzae (Z22809.1) and E. coli (AY513502.1) were PD0332991 chemical structure retrieved from the GenBank database and were aligned to identify potential target regions using multialin software (Corpet, 1988). Several conserved regions were chosen for designing of LAMP primer set using the LAMP primer design software Primer Explorer

version 4 (Eiken Chemical Co., Ltd, Tokyo, Japan). A set of four primers including two outer primers (forward primer F3 and backward primer B3) and two inner primers [forward inner primer (FIP) and backward inner primer (BIP)] that identified six distinct regions on the potential target sequence was designed. This study Aldol condensation was approved by the institutional ethical review committees of the Institute of Tropical Medicine, Nagasaki University. Serotypes 3 and 10 of S. pneumoniae were isolated from upper respiratory tract in Vietnamese patients. Two strains (8-01 and 8-02) of S. suis serotype 2, E. coli, S. aureus and S. agalactiae were also isolated from Vietnamese patients. In addition, H. influenzae and N. meningitidis were isolated from Japanese patients. The S. pneumoniae was cultured on rabbit blood Muller Hinton agar, while other bacteria were grown on rabbit blood brain heart infusion agar. Grown bacteria were harvested and suspended in normal saline. The cells were pelleted, suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.

Ten thousand events for each sample were collected using facsdiva

Ten thousand events for each sample were collected using facsdiva™ software and the data were stored and calculated after mathematical modeling using modfit lt™ software version 3.0 (Verity Software House, Topsham, ME). Cells treated with 100 μM H2O2 for 2 min were used as positive controls. Cell lysate preparation was performed as described previously (Chauvatcharin et al., 2005). Briefly, bacterial cells in 20 mL cultures were harvested and washed once with 50 mM sodium phosphate buffer pH 7.0 (PB). Cell pellets were resuspended in PB containing 1.0 mM

phenylmethylsulfonyl fluoride, a protease inhibitor, and lysed by intermittent sonication. Cleared lysates, separated by centrifugation at 10 000 g for 10 min, were used for the catalase activity assay (Beers & Sizer, 1952) and total protein determination (Bradford, 1976). One unit of catalase was defined as the amount of enzyme Selleckchem EPZ015666 capable of catalyzing the turnover of 1 μmol substrate min−1 under an assay condition. In order to test whether catalases were required for heat shock tolerance in X. campestris

pv. campestris, a series of mutants lacking catalases, that is, katA, katG, and katA-katG mutants (Jittawuttipoka et al., 2009), were assessed for their ability to survive the heat treatment by exposing the exponential-phase cultures of the mutant strains to a high temperature of 45 °C for 10 and 15 min. The results are illustrated in Fig. 1. Inactivation of katA reduced Megestrol Acetate the bacterial viability by 100-fold, while the katG mutant showed roughly a 10-fold selleck screening library reduction in the survival after the heat treatment at 45 °C for either 10 or 15 min of treatment compared with a parental strain.

The katA-katG double mutant was over 1000-fold more sensitive to the heat treatment than a parental strain. In X. campestris pv. campestris, KatA is the major catalase responsible for 80% of the total catalase activity in the exponential-phase cells, while the remaining 20% of the activity could be accounted for by KatG (Jittawuttipoka et al., 2009). When the total catalase activity in the kat mutant strains was taken into consideration, a correlation between the ability to survive the heat treatment and the total catalase activity emerged (Table 1). Among the X. campestris pv. campestris kat mutants, the katG mutant had the highest total catalase activity (4.7 ± 0.5 U mg−1 protein) and also the highest heat-treatment survival rate among the kat mutants. The katA mutant had intermediate levels for both the survival of heat treatment and the total catalase activity (Table 1). The katA katG double mutant, whose catalase activity was not detectable, also showed the lowest heat-treatment survival (Fig. 1 and Table 1). The ectopic expression of katG from pKatG (pBBR1MCS containing a full-length katG) (Jittawuttipoka et al., 2009) could complement the reduced heat resistance of the katG mutant as well as the katG katA double mutant (Fig. 1).