Further research is required to explore the detailed mechanisms. “
“l-Asparaginase-producing microbes are conventionally screened on phenol red l-asparagine-containing plates. However, sometimes the contrast of the zone obtained (between yellow and pink) is not very sharp and distinct. In the present investigation, an improved method for screening of the microorganisms producing extracellular l-asparaginase is reported wherein bromothymol blue (BTB) is incorporated as pH indicator in l-asparagine-containing medium instead of phenol red. Plates containing BTB at acidic pH are yellow and turn dark blue at alkaline Alectinib pH. Thus, a dense dark blue zone is formed around microbial colonies producing l-asparaginase, differentiating between enzyme

producers and non-producers. The present method is more sensitive and accurate than the conventional method for screening of both fungi and bacteria producing extracellular l-asparaginase. Furthermore, BTB gives a transient green colour at neutral pH (7.0) and dark blue colour at higher pH 8.0–9.0, indicating the potency of the microorganism for l-asparaginase production. learn more “
“Studies of enterohemorrhagic Escherichia

coli (EHEC) infection mechanisms using mammals require large numbers of animals and are both costly and associated with ethical problems. Here, we evaluated the pathogenic mechanisms of EHEC in the silkworm model. Injection of a clinically isolated EHEC O157:H7 Sakai into either the silkworm hemolymph or intraperitoneal fluid of mice killed the host animals. EHEC O157:H7 Sakai deletion mutants of the rfbE gene, which encodes perosamine synthetase, a monosaccharide

component synthetase of the O-antigen, or deletion mutants of the waaL gene, which encodes O-antigen ligase against the lipid A-core region of lipopolysaccharide (LPS), had attenuated killing ability in both silkworms and mice. Introduction of the rfbE gene or the waaL gene into the respective mutants Etofibrate restored the killing ability in silkworms. Growth of both mutants was inhibited by a major antimicrobial peptide in the silkworm hemolymph, moricin. The viability of both mutants was decreased in swine serum. The bactericidal effect of swine serum against both mutants was inactivated by heat treatment. These findings suggest that the LPS O-antigen of EHEC O157:H7 plays an important defensive role against antimicrobial factors in the host body fluid and is thus essential to the lethal effects of EHEC in animals. Infectious diseases caused by enterohemorrhagic Escherichia coli (EHEC) O157:H7 are a serious clinical problem and are associated with encephalopathy and nephropathy (Tarr, 1995; Law, 2000). An understanding of the molecular mechanisms of EHEC O157:H7 virulence is important for establishing effective therapeutic strategies. Unlike other E. coli strains, EHEC produces Shiga toxins and hemolysins. Shiga toxins are encoded by the stx1 and stx2 genes on the phage DNA that is integrated into the EHEC genome (Sato et al.

Nevertheless, Ruxolitinib to further

stabilize expression, integration of the expression cassette into the Y. lipolytica genome was carried out. The integrative vector pMMR10 has a unique NotI site that allows linearization and integration of the vector into the YlLEU2 gene. NotI-digested vector was used to transform Y. lipolytica E150 strain and the transformants were selected for a Leu+ phenotype on YNB medium. Three transformants (T1, T2 and T3) were analyzed by Southern blot using a fragment of the YlLEU2 gene as a probe. The W29 strain, carrying the complete YlLEU2 gene, was used as a control. Two of the transformants (T2 and T3) showed a single copy of the vector correctly integrated into YlLEU2 gene (Fig. 2). Transformant T2 was used in subsequent experiments. Transformant GSK J4 order cells were grown in YNB media with and without copper sulfate. The time course of the Alt a 1 expression/secretion was examined by SDS-PAGE run under reducing conditions and Western blot (Fig. 3). No recombinant allergen was detected when transformants were grown without copper sulfate. The recombinant Alt a 1 was purified from the culture medium with a yield of

approximately 0.5 mg L−1 culture. Natural and rAlt a 1 were purified from A. alternata and Y. lipolytica supernatant culture media, respectively, by immunoaffinity chromatography. A high degree of purity was achieved with this single-step purification procedure, as was demonstrated by SDS-PAGE (Fig. 4a). Comparison of natural allergen from A. alternata and the recombinant form Benzatropine produced in Y. lipolytica was performed using Western blot, dot blot, and ELISA-inhibition experiments with sera from A. alternata-allergic patients. Natural and recombinant

Alt a 1 migrate as a 28-kDa protein under non-reducing conditions, but the protein breaks down into 16- and 15-kDa subunits under reducing conditions. Western blotting showed that both natural and recombinant allergen reacted with IgE from the pool of patients’ sera, in both reducing (data not shown) and non-reducing conditions (Fig. 4a). Reactivity of natural and recombinant Alt a 1 against rabbit anti-Alt a 1 serum, checked by Western blotting, showed similar results (data not shown). An IgE-dot blot was performed to confirm recognition by human IgE of non-denatured Alt a 1 proteins using sera from 42 A. alternata-allergic patients and 17 control patients. In our population of patients, 41 sera (97.6%) reacted with nAlt a 1 and 37 (88.1%) with its recombinant counterpart (Fig. 4b). None of the sera from control patients reacted with Alt a 1 in the IgE-dot blot assay (data not shown). IgE binding to Alt a 1 and its recombinant counterpart was quantitatively evaluated by ELISA inhibition experiments performed by coating wells with nAlt a 1 and comparing the IgE binding capacity of nAlt a 1 and rAlt a 1.

Colonies were counted after 24-h incubation at 37 °C and the number

of CFU was calculated. A suspension of C. albicans ATCC 14053 containing 1 × 106 cells mL−1 in RPMI 1640 was mixed with different dilutions of allicin and fluconazole (1/2 × MIC, 1 × MIC and 10 × MIC) and incubated at 35 °C for 24 h. Cells were fixed in 2% v/v glutaraldehyde in phosphate-buffered saline (pH 7.2) and washed with sodium cacodylate buffer. For postfixation, samples were rinsed in 1% osmium tetroxide for 2 h at 4 °C, washed again with sodium cacodylate buffer and then dehydrated with ascending ethanol series. After that, samples Vemurafenib on coverslips were put into a critical point dryer and then stuck onto the stub. The specimens were coated with gold and observed through a JEOL JSM 6400 scanning electron microscope. For the experiments on the animal model of systemic candidiasis, 4–6-week-old female BALB/c mice were infected intravenously through the tail vein with 200 μL per mouse of C. albicans ATCC 14053 (5 × 106 yeast cells mL−1). The mice were divided into five experimental groups of 12 mice each. In the first two groups of mice, allicin (200 μL per mouse) was administered intravenously once daily for 5 days beginning 1 h after Candida injection (postinfection) at 1 and 5 mg kg−1 day−1, respectively (Shadkchan et al., 2004). For the third and fourth

groups of mice, fluconazole (200 μL per mouse) was administered via the intraperitoneal route once daily for 5 days starting 1-h postinfection at 1 and 5 mg kg−1 day−1, respectively (Rex et al., 1998). For the untreated control group, 200 μL of normal saline was injected into each mouse at 1-h postinfection. Idelalisib The infection was followed up for 28 days and evaluated in terms of mortality and morbidity. For studies of tissue burden, two randomly chosen mice were sacrificed from each experimental group on days 2, 4, 7, 10, 14 and 28 after infection (Shadkchan et al., 2004). Kidneys, liver and spleen from each mouse were aseptically removed and homogenized in 1 mL of sterile normal

saline and cultured on Sabouraud dextrose agar Urocanase plates as explained in the time–kill study, and assessed by determination of fungal colonization of viscera. Histopathologic analyses were performed for a qualitative confirmation of the result. Tissues were fixed in 10% formalin, then blocked by paraffin wax and cut with a microtome (Leica, model RM2025) in 4-μm thickness. Hematoxylin and eosin and periodic acid-Schiff staining were used to observe the tissue and presence of fungal elements. All animal care procedures were supervised and approved by the University of Putra Malaysia Animal Ethics Committee (ACUC NO.: UPM/FPSK/PADS/BRUUH/00278). For quantitative statistical analysis of inhibitory effects of drugs in vitro and also reduction of fungal load in tissues of mice, data were analyzed in terms of normality and one-way anova was carried out.

Its termini contain the inverted repeat sequence 5′-CCTGC … GCAGG-3′, and its 5′-ends are covalently capped with protein (Overhage et al., 2005; Parschat et al., 2007). Our previous sequence analysis of pAL1 and predictions of possible secondary structures formed by potential telomeric 3′-overhangs indicated significant differences of the ‘left’ and ‘right’ terminus of pAL1, raising the question of whether each terminus of pAL1 is recognized, or even capped, by a specific protein (Parschat et al., Ivacaftor price 2007). Rhodococcal plasmids pHG201 and pHG205 are other examples of actinomycete

linear plasmids that do not show striking homology between their ‘left’ and ‘right’ telomere sequences (Kalkus et al., 1998), but their TPs have not been described. In contrast, the

ends of Streptomyces find more linear replicons usually contain well-conserved terminal palindromic sequences (Zhang et al., 2006). The gene product of pAL1.102 is the only protein exhibiting a weak similarity to known (Streptomyces) TPs; however, due to the low sequence similarity, its annotation as a ‘putative terminal protein’ was tentative (Parschat et al., 2007). As a first step toward characterizing the telomere complex of pAL1, we identified the protein attached to both termini of pAL1 and demonstrated its specific deoxynucleotidylation in vitro. The strains and plasmids used in this study are listed in Table 1. For isolation of total DNA, A. nitroguajacolicus Rü61a [pAL1] was grown in a mineral salts medium (Parschat et al., 2003) on 8 mM sodium benzoate at 30 °C. Arthrobacter nitroguajacolicus Rü61a [pAL1, pART2malE-ORF102 or pART2malE-ORF103] was cultivated in a mineral salts medium supplemented with 4 mM 4-hydroxyquinaldine Chloroambucil and 140 μg mL−1 kanamycin. Cells were harvested by centrifugation at an OD600 nm of approximately 2.5. Escherichia coli DH5α clones containing derivatives of pMal-c2x or pART2 were grown in lysogeny broth (LB) (Sambrook & Russell, 2001) at 37 °C in the presence of 100 μg mL−1 ampicillin or 50 μg mL−1 kanamycin, respectively. For the synthesis of fusion

proteins of maltose-binding protein (MBP) and the protein encoded by pAL1.102 (termed pORF102), E. coli K12 ER2508 [pLysSRARE] harboring pMal-c2x-ORF102 was grown in LB with ampicillin (100 μg mL−1), chloramphenicol (34 μg mL−1), and auto induction solutions ‘5052’ and ‘M’ (Studier, 2005) at 30 °C. Cells were harvested by centrifugation at an OD600 nm of ∼5 and stored at −80 °C before use. Total DNA of A. nitroguajacolicus Rü61a [pAL1] was isolated according to Rainey et al. (1996). Plasmid DNA was isolated using the EZNA Plasmid Miniprep kit (Peqlab, Erlangen, Germany). Gel extraction of DNA fragments from agarose gels was performed with the Perfectprep gel cleanup kit (Eppendorf, Hamburg, Germany). For cloning purposes, DNA fragments were purified using the High Pure PCR Product Purification kit (Roche Diagnostics GmbH, Mannheim, Germany).

In a retrospective assessment of HIV-infected patients see more initiating ATV/r-containing ART, using logistic regression we determined factors associated with UTrI, the prevalence of emergent resistance mutations and virological response after ART reinitiation. A total of 202 patients [median age 33 years (interquartile range (IQR) 29–40 years); 52% female; median CD4 count 184 cells/μL (IQR 107–280

cells/μL); median HIV RNA 4.6 log10 HIV-1 RNA copies/mL (IQR 3.2–5.1 copies/mL)] initiated ATV/r between 2004 and 2009; 80 (43%) were ART naïve. One hundred and ten patients (55%) underwent 195 UTrIs after a median (IQR) 25 (10–52) weeks on ART, with a median (IQR) UTrI duration of 10 (3–31) weeks. Fifty-four of 110 patients (49%) underwent more than one UTrI. The commonest reasons for UTrI were nonadherence (52.7%) and drug intolerance (20%). Baseline HIV RNA > 100 000 copies\mL [odds ratio (OR) 3.6; 95% confidence interval (CI) 1.3–9.95] and being HCV positive, an injecting drug user or on methadone (OR 2.4; 95% CI 1.3–4.4) were independently associated with UTrI. In 39 patients with at least two resistance assays during UTrIs, 72 new mutations emerged; four nucleoside reverse transcriptase inhibitor (NRTI), two nonnucleoside reverse transcriptase inhibitor (NNRTI) and 66 protease inhibitor (PI) resistance mutations.

All emergent PI resistance mutations were minor mutations. At least 65% of patients were re-suppressed on ATV/r

reinitiation. In this PI-treated cohort, UTrIs are common. All emergent PI resistance mutations were minor buy Everolimus and ATV/r retained activity and efficacy most when reintroduced, even after several UTrIs, raising questions regarding the need for routine genotypic resistance assays in PI/r-treated patients prior to ART reinitiation after UTrI. “
“Infection with hepatitis C virus (HCV) is a major cause of chronic liver disease. High HCV RNA levels have been associated with poor treatment response. This study aimed to examine the natural history of HCV RNA in chronically HCV/HIV-coinfected individuals. Mixed models were used to analyse the natural history of HCV RNA changes over time in HIV-positive patients with chronic HCV infection. A total of 1541 individuals, predominantly White (91%), male (73%), from southern (35%) and western central Europe (23%) and with HCV genotype 1 (58%), were included in the analysis. The median follow-up time was 5.0 years [interquartile range (IQR) 2.8 to 8.3 years]. Among patients not on combination antiretroviral therapy (cART), HCV RNA levels increased by a mean 27.6% per year [95% confidence interval (CI) 6.1−53.5%; P = 0.0098]. Among patients receiving cART, HCV RNA levels were stable, increasing by a mean 2.6% per year (95% CI −1.1 to 6.5%; P = 0.17). Baseline HCV RNA levels were 25.5% higher (95% CI 8.8 to 39.1%; P = 0.

, 2007), hydrophobins (Wosten Han, 2001) and lectins (Wang et al., 1995; Yagi et al., 1997) are some examples. Laccases (Yaropolov et al., 1994) and hydrophobins have biotechnological applications (Janssen

et al., 2002; Scholtmeijer et al., 2004), mushroom antifungal proteins have potential applications in agriculture (Wang et al., 2004), and mushroom lectins and RNAses inhibit tumor growth and tumor cell proliferation (Wang et al., 1995; Guan et al., 2007). Moreover, mushroom-forming fungi have been implicated in the industrial production of homologous (Alves Selleck BTK inhibitor et al., 2004) and heterologous proteins (Berends et al., 2009). Hemolysins have been reported from mushroom species including Pleurotus ostreatus, Agrocybe cylindracea (Berne et al., 2002), Pleurotus

eryngii (Ngai & Ng, 2006), Flammulina velutipes (Bernheimer & Oppenheim, 1987) and Pleurotus nebrodensis (Lv et al., 2009). However, no hemolysin has been isolated from the split gill mushroom Schizophyllum commune. Schizophyllum commune Vorinostat cost is a model system for mushroom production. It is the only fungus in which genes have been reported to be inactivated by homologous recombination. Moreover, its genome has recently been sequenced. So far, several proteins of S. commune have been characterized, including a 5′-aldehyde-forming enzyme (Chen & McCormick, 1997), a β-glucosidase (Desrochers et al., 1981), a cellobiose dehydrogenase (Fang et al., 1998), a cholesterol oxidase (Fukuyama & Miyake, 1979), a lectin (Han et al.,

2005), several hydrophobins (Wosten Han, 2001), a squalene synthase inhibitor (Tanimoto et al., 1996) and a trehalose phosphorylase (Eis & Nidetzky, 1999). Here we report the isolation of a hemolysin from S. commune. Schizophyllum commune strain 0805, isolated from wild S. commune, was grown at 25 °C in the dark on medium composed of 85% cotton seed husk and 15% wheat bran, with a moisture content of 70%. After about 4 weeks, mycelia were transferred to bags and incubated in a growth chamber with a constant temperature of 16 °C, PLEK2 in an atmosphere of 90–95% air humidity, >0.001 g g−1 CO2 and scattering light. The humidity was decreased to 85–90% after the primordia had developed. The mushroom was harvested when the diameter of fruit bodies had reached 4 cm. Fresh fruiting bodies (100 g) were collected and homogenized in 1000 mL 0.15 M NaCl. Following centrifugation at 14 000 g for 25 min at 4 °C, proteins in the supernatant were precipitated with 30–80% (NH4)2SO4. The precipitate was dissolved in distilled water and dialyzed against distilled water. NH4HCO3 buffer (pH 9.4, 1.0 M) was then added until a concentration of 10 mM was reached. After centrifugation, the supernatant (S2) was applied on 2.5 × 20 cm of DEAE-cellulose (Sigma) which was eluted with 10 mM NH4HCO3 buffer (pH 9.4). After removal of unadsorbed proteins (fraction D1), the column was eluted sequentially with 10 mM NH4HCO3 buffer (pH 9.

The resultant plasmids pAK3-0664, pAK3-2684, and pAK3-3876 were conjugated into this website AMB0101, AMB0102, and AMB0103 mutant strains, respectively, to generate the complementary strains AMB0105, AMB0106, and AMB0107. Transmission electron microscopic (TEM) observations of AMB-1 cells and magnetosomes were performed with JEM-100CXII

at an acceleration voltage of 120 kV. The average magnetosome number per cell was determined directly by counting magnetosome particles in at least 130 individual cells. Strains of M. magneticum AMB-1, AMB0101, AMB0102, and AMB0103 were cultured to the stationary phase in a 300-mL sealed serum bottle containing 250 mL liquid medium before being transferred to initiate another round of subculture. This process was repeated at least 30 times. Cells were collected at the indicated rounds of subcultures

and genomic DNA was extracted using a Bacterial DNA Kit (Omega). Nonmagnetic cells were examined for the existence of a genomic MAI region by amplifying the four marker genes Trametinib indicated in Fig. 4a using primers 16–19 listed in Table S1. Quantitative real-time PCR was performed in a Bio-Rad Sequence Detection System with a total volume of 20 μL, containing 250 nM of primers, 10 μL of SYBR Green PCR mix (Takara), 0.4 μL of ROX, 6.8 μL of ddH2O, and 2 μL DNA template under the following conditions: 3 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 15 s at 60 °C, and 15 s at 72 °C. All samples were also analyzed with primers specific for the 16S rRNA gene and this result was used as a ‘loading control’ to normalize results from for the marker genes inside the genomic MAI (amb0956). The calibration standard curve of each

gene was run in triplicate from a series of 10-fold dilutions of genomic DNA of M. magneticum AMB-1. The ratio of signals from amb0956 and 16S rRNA gene starting the subculture was set to 100%, and other time points show the level relative to the starting point. Magnetospirillum magneticum AMB-1, AMB0101, AMB0102, and AMB0103 cells sampled at the indicated time intervals were in the meantime spread on enriched MSGM plates and incubated for 7 days. Each colony was transferred into a liquid culture in a 1.5-mL tube to test magnetic sensitivity by a bar magnet and microscope in an applied magnetic field. The percentage of magnetic-sensitive cells was calculated by counting at least 200 colonies. An extensive analysis of the genome of M. magneticum AMB-1 revealed the existence of three peroxiredoxin-like genes (Matsunaga et al., 2005). The gene encoding AhpC-like protein (amb0664, Prx1) is located upstream of two other genes encoding a hypothetical protein (amb0662) and a thioredoxin reductase (amb0663), respectively, with the downstream amb0665 transcribed in the opposite direction and amb0663 in the same orientation as amb0664 (Fig. S1).

Continuous-time, multi-state Markov models CDK inhibitors in clinical trials were applied to the status data on HPV detection, VL and CD4 cell count. The following four states

were defined for the VL model (Fig. 1a) to describe the high-risk HPV detection and clearance rates with time-varying VL: 1 = none of the high-risk HPV types (HPV negative) and HIV VL >400 copies/mL; 2 = at least one of the high-risk HPV types detected (HPV positive) and VL > 400 copies/mL; 3 = HPV negative and VL ≤ 400 copies/mL, and 4 = HPV positive and VL ≤ 400 copies/mL. A multi-state model describes a process where an individual is in one of the specified states at any time. An individual’s status at any time can be categorized as one of the four states above, and changes in the state can be followed. The choice of 400 copies/mL was based on the lower limit of quantification of the Roche assay at the time of A5029. To illustrate the assumed state structure, suppose a woman begins in state 1. She may acquire HPV without improvement of VL (transition to state

2), or she may remain HPV negative with improvement find more of VL (transition to state 3). State 4 may be reached from state 1 via state 2 (change in HPV status first) or state 3 (change in VL first), but not directly from state 1; that is, we assume that simultaneous changes in HPV status and VL do not occur biologically. (However, pheromone state 4 may be observed after state 1 in the previous visit.) From state 2, the woman may clear HPV and return to state 1, or she may retain HPV and transition to state 4 with decreased VL. From state 3, she may acquire HPV while

maintaining VL status (transition to state 4), or her VL may increase while she remains HPV negative (transition to state 1). From state 4, she may clear HPV and transition to state 3, or remain HPV positive while her VL increases (transition to state 2). She may also remain in any of the states for the remainder of the study. The analysis methods of Kalbfleisch and Lawless [12] were applied to account for the lack of exact times of HPV detection and clearance. The methods also account for differences in visit times, numbers of visits and initial states among the study participants. The transition rates, or cause-specific hazard rates, are denoted by λs in Figure 1. For instance, λ12 represents the hazard rate for acquiring HPV when VL remains >400 copies/mL and λ34 represents the rate when VL remains ≤400 copies/mL. For HPV clearance rates, λ21 represents the rate for clearing HPV when VL remains >400 copies/mL, and λ43 represents the rate for clearing HPV when VL remains ≤400 copies/mL. To describe the changes in HIV-related status, λ13 represents the rate from VL > 400 to ≤ 400 copies/mL without HPV, and λ24 that with HPV.

3c). These differences were not the consequence of different growth rates as both strains showed similar growth curves in minimal medium (data not shown). The M. loti triple mutant also showed a significantly lower competitive ability when co-inoculated with the rhcN mutant strain (Fig. 3a). Different independent experiments (Fig. 3a) indicated a positive role for the protein encoded in mlr6316 in the symbiotic competitiveness on Lo. tenuis cv. Esmeralda: The wt strain showed a slightly higher competitiveness than the mlr6316 mutant, and the same difference was observed when the double mutant mlr6358/mlr6361 was co-inoculated with the triple mutant. The comparison between the results obtained when the wt strain

was co-inoculated with the mlr6316 mutant and those obtained when the wt strain was co-inoculated with the triple mutant indicates CX-5461 molecular weight that the triple mutation affects competitiveness more drastically than the single mutation in mlr6316 (Fig. 3a and b). This suggests the possibility that the protein encoded in mlr6358 and/or the protein encoded in mlr6361 play a positive role in the symbiotic

competitiveness. Consistent with this, the double mutant mlr6358/mlr6361 was less competitive than the wt strain (Fig. 3a). The triple mutation in mlr6358, mlr6361, and mlr6316 also GSK-3 beta phosphorylation caused a more drastic effect on competitiveness than the combined mlr6316/mlr6361 mutation (Fig. 3a and b). This indirectly indicates that Mlr6358 has a positive effect on competitiveness on Lo. tenuis. No statistically significant differences were observed in competitiveness on Lo. tenuis cv. Esmeralda between the wt and the mlr6361 mutant or between the wt and the double mutant mlr6331/mlr6361 (Fig. 3a). However, the mutant affected in both Mlr6361

and Mlr6331 showed decreased competitiveness compared with the wt strain on Lo. japonicus selleck chemicals llc MG-20 (Fig. 3c). To determine which of the two proteins are responsible for the positive effect on this plant, co-inoculation assays of the double mutant with each of the single mutants were performed. Results indicate that the double mutant was less competitive than the single mutant affected in mlr6361 but more competitive than the single mutant affected in mlr6331 (Fig. 3c). This indicates that Mlr6331 has a positive effect and that Mlr6361 has a negative effect on the competitiveness on Lo. japonicus MG-20. We determined the nodulation kinetics for the M. loti wt, the rhcN mutant, and the triple mutant on Lo. tenuis cv. Esmeralda (Fig. 4). Although the rhcN mutant showed greater competitive ability on this plant (Fig. 3a), its nodulation kinetics was negatively affected when compared with the wt strain. On the other hand, in concordance with the competitiveness results, the M. loti triple mutant presented a kinetic phenotype significantly negatively affected compared with the wt strain and a delayed nodulation kinetics compared with the rhcN mutant strain (Fig. 4).

In all cases, killing curves were performed with two different spore preparations, and these yielded essentially similar (±20%) results. MK-1775 cost Survivors of wet heat treatment were transferred onto either minimal medium or sporulation agar plates and incubated for 24–48 h to assess the percentage of survivors that had acquired auxotrophic or asporogenous mutations as described previously (Fairhead et al., 1993). We decided to use the strong PsspB promoter

to overexpress Nfo, because PsspB has yielded high-level expression of several proteins in spores (Paidhungat & Setlow, 2001; Cabrera et al., 2003). To confirm that PsspB in our construct was indeed forespore-specific, we used this promoter to drive GFP expression, and examined sporulating cells of the PsspB-gfp strain (PERM751) by fluorescence microscopy (Fig. 1a). The results showed that in around 30% of analyzed sporangia, GFP was clearly accumulated to significant levels in developing

spores (Fig. 1a, arrows), and there was no noticeable fluorescence in the mother cell compartment of sporulating cells. The above results indicated that the PsspB we planned to use to overexpress Nfo is indeed forespore-specific. SDS-PAGE of extracts of spores of strains with or without nfo under PsspB control (Fig. 1b) showed that spores of a B. subtilis strain (PERM641) with PsspB-nfo contained a prominent band at 33 kDa, the expected molecular mass of Nfo (Salas-Pacheco et al., 2003), buy Fulvestrant while this band was not prominent in extracts from spores of strains in which nfo was not controlled by PsspB (PERM450 and PS832) (Fig. 1b). These results indicate that PsspB directs forespore-specific overexpression of nfo in strain PERM641, and densitometry indicated that Nfo was overexpressed ∼50-fold in the spores of this strain (Fig. 1b, bottom). A similar level of Nfo overexpression was observed in spore extracts of the wild-type strain containing the

PsspB-nfo construct (Fig. 1b, bottom). Previous work has suggested that it is generation of AP sites in α−β−, but not wild-type spore DNA that sensitizes α−β− spores to wet heat (Setlow, 2006). With α−β− spores, only the absence of two AP endonucleases, ExoA and Nfo, decreased these spores’ resistance to wet heat ROS1 (Salas-Pacheco et al., 2005). Therefore, the exoA nfoα−β− genetic background was used to investigate the effects of elevated Nfo levels on spore resistance to wet heat and other treatments. As found previously (Salas-Pacheco et al., 2005), spores of the exoA nfoα−β− strain were very sensitive to wet heat (Fig. 2a and b). However, overexpression of Nfo decreased the rate of wet heat killing of nfo exoAα−β− spores significantly, and the LD90 value, the time for 90% wet heat killing at 90 °C, increased from 7.5 min for nfo exoAα−β− spores to ∼45 min for the nfo exoAα−β− spores overexpressing Nfo (Fig. 2a and b). Indeed, the wet heat resistance of the latter spores was slightly higher than that of wild-type PS832 spores (Fig.