For the culture, a cysteine production medium [composition (per liter): a quantity of 12 g of ammonium chloride, 1.5 g of potassium dihydrogenphosphate, 1 g of magnesium sulfate heptahydrate, 0.1 mg of thiamine hydrochloride, 1.7 mg of ferrous sulfate heptahydrate, 0.15 mg of sodium molybdate dihydrate, 0.7 mg of cobalt chloride

hexahydrate, 1.6 mg of manganese chloride tetrahydrate, 0.3 mg of zinc sulfate heptahydrate, 0.25 mg of copper sulfate pentahydrate, 0.6 g of tryptone, 0.3 g of yeast extract, 0.6 g of sodium chloride, 20 g of calcium carbonate, 135 mg of l-histidine monohydrochloride Galunisertib chemical structure monohydrate, 4 g of sodium thiosulfate, 2 mg of pyridoxine hydrochloride, 40 g of glucose, 12.5 mg of tetracycline] (Nonaka, 2010) was used. For the cultivation with thiosulfate or sulfite as a sulfur source, 8 g L−1 of sodium thiosulfate or 2.6 g L−1 of sodium sulfite was added, respectively.

For the cultivation with sulfate PLX-4720 cell line as a sulfur source, 15 g L−1 of ammonium sulfate was added instead of ammonium chloride. The BW26424/pACYC-DES and the BW25113/pACYC-DES strains were each applied and spread onto LB agar medium containing tetracycline, and precultured overnight at 34 °C. The streak cells corresponding to about 7 cm on the plate were scraped with an inoculating loop of 10 μL size (NUNC Blue Loop), and inoculated into 2 mL of the cysteine production medium. The culture was grown at 32 °C with shaking for 42 h, and the amount of cysteine accumulated in the medium was quantified. The quantification of cysteine was performed by the method described by Gaitonde (1967). The experiment was performed in hexaplicate for both the strains, and averages and standard

deviations of the accumulated cysteine amounts were calculated. Escherichia coli cells grown in 10 mL of LB medium MYO10 were harvested by centrifugation and resuspended in 0.2 mL 8 M urea/lysis buffer (8 M urea, 50 mM Tris-HCl, pH 8.0 at 4 °C, and 100 mM NaCl), and sonicated. Cell extracts (10 μg) were subjected to 10% SDS-PAGE and blotted on to polyvinylidene difluoride (PVDF) membranes using iBlot semi-dry transfer apparatus (Invitrogen). Membranes were first immuno-detected with anti-β-galactosidase (Progema) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Nacalai tesque) antibodies and then developed with a chemiluminescence kit (Nacalai tesque). The image was analyzed with a LAS-4000 IR multi color (Fuji Film). The transcriptome analysis of E. coli response to metal-shock indicated that the genes for cysteine biosynthesis including cysK are regulated by several metals (Yamamoto & Ishihama, 2005a, b; Hobman et al., 2007). Since a set of the metal stimulon genes are regulated by some of two-component system (TCS) (Yamamoto & Ishihama, 2006; Yamamoto et al., 2008), we tested possible influence of TCS knock-out on cysK expression. For this purpose, we used the collection of TCS deficient E. coli mutants (Oshima et al.

Groups fed with PY79 and CotB-VP28 spores at day 7 had increased SOD activities of 29% and increased phenoloxidase activities of 15% and 33%, respectively, compared to those of the control group. Fourteen days

postchallenge, 35% of vaccinated shrimps had died compared to 49% of those fed naked spores (PY79) and 66% untreated, unchallenged animals. These data suggest that spores expressing VP28 have potential as a prophylactic treatment of WSS. “
“Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin biosynthesized by various Fusarium fungi. These fungal buy Everolimus species frequently infest grains; therefore, ZEN represents a common contaminant in cereal products. The biotransformation of ZEN differs significantly from species to species, and several metabolites are known to be formed by animals, plants, and microorganisms. The aim of the present study was to investigate the microbial conversion of ZEN by species of the genera Rhizopus and Selleck AZD6244 Aspergillus representing relevant fungi for food processing (e.g. fermentation). To monitor the ZEN metabolism, ZEN was added to liquid cultures of the different fungal species. After a period of 3 days, the media were analyzed by HPLC-MS/MS for metabolite formation. Two

Aspergillus oryzae strains and all seven Rhizopus species were able to convert ZEN into various metabolites, including ZEN-14-sulfate as well as ZEN-O-14- and ZEN-O-16-glucoside. Microbial transformation of ZEN into the significantly more estrogenic α-zearalenol (α-ZEL) was also observed. Additionally, a novel fungal metabolite, α-ZEL-sulfate, was detected. Semi-quantification of the main metabolites indicates

that more than 50% of initial ZEN may be modified. The results show that fungal strains have the potential to convert ZEN into various metabolites leading to a masking of the toxin, for example in fermented food. “
“Salmonella enterica serotype Paratyphi B is a globally distributed human-specific pathogen causing paratyphoid fever. The aim of 5-Fluoracil clinical trial this study was to develop a rapid and reliable polymerase chain reaction (PCR) assay for its detection in food. The SPAB_01124 gene was found to be unique to S. Paratyphi B using comparative genomics. Primers for fragments of the SPAB_01124 gene and the Salmonella-specific invA gene were used in combination to establish a multiplex PCR assay that showed 100% specificity across 45 Salmonella strains (representing 34 serotypes) and 18 non-Salmonella strains. The detection limit was 2.2 CFU mL−1 of S. Paratyphi B after 12-h enrichment in pure culture. It was shown that co-culture with S. Typhimurium or Escherichia coli up to concentrations of 3.6 × 105 CFU and 3.3 × 104 CFU, respectively, did not interfere with PCR detection of S. Paratyphi B. In artificially contaminated milk, the assay could detect as few as 62 CFU mL−1 after 8 h of enrichment.

The NR114 mutant had similar levels of both KatA and CatE when compared to wild-type NTL4 (data not shown). These results indicate

that the H2O2-hypersensitive phenotype of NR114 is not because of a reduction in the catalase activity. MbfA-mediated H2O2 resistance was further assessed in the wild-type NTL4, the catalase-deficient strain (KC05, katA and catE double mutation) (Prapagdee et al., 2004) and the rhizobial iron regulator (rirA) mutant strain (PN094) (Ngok-ngam et al., 2009) containing the plasmid vector pBBR1MCS-4 (pBBR) or expressing MbfA from the plasmid pNR114C. The catalase-deficient strain KC05/pBBR was 103-fold Dactolisib manufacturer more sensitive than the wild-type NTL4/pBBR strain to 200 μM H2O2 (Fig. 3a). The KC05/pNR114C strain had slightly increased resistance (< 10-fold) to 200 μM H2O2 compared with the KC05/pBBR strain (Fig. 3a). In contrast, the KC05 strain complemented with a functional KatA from the plasmid pKatA (KC05/pKatA) showed similar levels of H2O2 resistance to wild-type NTL4/pBBR (Prapagdee et al., 2004). These data suggest that MbfA plays a role in H2O2 resistance, but to a lesser extent than catalase, which directly degrades H2O2. Agrobacterium tumefaciens rirA is a repressor of iron uptake systems. Inactivation of the rirA gene leads to derepression of iron uptake and to an increase in intracellular Erastin mw free iron (Ngok-ngam et al.,

2009). The PN094 mutant has increased sensitivity to H2O2, which is likely

due to increased intracellular free iron-mediated H2O2 from toxicity, and this H2O2-hypersensitive phenotype can be reversed by an iron chelator (Ngok-ngam et al., 2009). Multicopy mbfA was able to complement the H2O2-hypersensitive phenotype of PN094 (Fig. 3b). Moreover, multicopy mbfA in strains NTL4/pNR114C and PN094/pNR114C conferred higher resistance levels (10-fold and 102-fold, respectively) to 350 μM H2O2 than in their parental strains, NTL4/pBBR and PN094/pBBR (Fig. 3b). These data support the view that MbfA helps to protect A. tumefaciens from H2O2 killing, possibly by sequestering iron and inhibiting the oxidative damage mediated by the Fenton reaction. Expression of mbfA in response to iron and H2O2 was determined. Exponential-growth phase cells of wild-type NTL4 and the NR114 mutant were treated with 50 μM FeCl3, 200 μM Dipy or 250 μM H2O2 for 15 min. RT-PCR analysis showed that expression of mbfA in the wild-type NTL4 strain was responsive to iron levels. Under low-iron conditions (Dipy), mbfA was repressed compared to high-iron conditions (Fe) (Fig. 4a). Furthermore, expression of mbfA was increased when the wild-type NTL4 strain was treated with 250 μM H2O2 (Fig. 4a). The induction of mbfA expression during exposure to iron and H2O2 further supports the view that A. tumefaciens mbfA is involved in the iron and H2O2 stress responses. The A. tumefaciens mbfA gene is predicted to be regulated by irr (Rodionov et al., 2006). An A.

A decline in toxicity to this magnitude may infer that receptor binding event was affected or proteolytic check details degradation in the gut lumen. Alternatively, loss of toxicity may be attributed to the disruption of the membrane insertion event and should be considered (Nair et al., 2008). We thank Dr Xinyan Sylvia Liu, Dr Manoj Nair, Dr Dan Zeigler, Carol Zeigler, Sharnise

Mitchell and Yoshio Ikeda for their contributions, as well as stimulating talks that shed some insight on analysing the results. We thank Dr Hansjuerg Alder for giving us access to the Nucleic Acid Shared Resource to utilize the Personal densitometer SI. We also thank the Biochemistry Department for providing access to the departmental CD spectrometer. NIH (R01-AI 29092) funding to D.H.D. supported this research. “
“φEf11 is a temperate Siphoviridae bacteriophage isolated by induction from a lysogenic Enterococcus faecalis strain. The φEf11 DNA was completely sequenced and found to be 42 822 bp in length, with a G+C mol% of 34.4%. Genome analysis revealed 65 ORFs, accounting for 92.8% of the DNA content. All except for seven of the ORFs displayed sequence similarities to previously characterized proteins. The Pirfenidone genes were arranged in functional

modules, organized similar to that of several other phages of low GC Gram-positive bacteria; however, the number and arrangement of lysis-related genes were atypical of these bacteriophages. A 159 bp noncoding region between predicted cI and cro genes is highly similar to the functionally characterized early promoter region of lactococcal temperate phage TP901-1, and selleck chemicals possessed a

predicted stem-loop structure in between predicted PL and PR promoters, suggesting a novel mechanism of repression of these two bacteriophages from the λ paradigm. Comparison with all available phage and predicted prophage genomes revealed that the φEf11 genome displays unique features, suggesting that φEf11 may be a novel member of a larger family of temperate prophages that also includes lactococcal phages. Trees based on the blast score ratio grouped this family by tail fiber similarity, suggesting that these trees are useful for identifying phages with similar tail fibers. Enterococcus faecalis is a facultatively anaerobic, Gram-positive coccus, commonly growing in short chains or clusters. Although these bacteria have long been considered to be ubiquitous, commensal organisms commonly isolated from the mammalian alimentary canal as well as from water and soil (Facklam et al., 2002), more recently, they have emerged as opportunistic pathogens associated with a variety of medical and dental infectious diseases. These organisms are among the most frequent causes of nosocomial infections (Moellering, 1992; Edgeworth et al., 1999; Richards et al.

A decline in toxicity to this magnitude may infer that receptor binding event was affected or proteolytic selleck chemicals llc degradation in the gut lumen. Alternatively, loss of toxicity may be attributed to the disruption of the membrane insertion event and should be considered (Nair et al., 2008). We thank Dr Xinyan Sylvia Liu, Dr Manoj Nair, Dr Dan Zeigler, Carol Zeigler, Sharnise

Mitchell and Yoshio Ikeda for their contributions, as well as stimulating talks that shed some insight on analysing the results. We thank Dr Hansjuerg Alder for giving us access to the Nucleic Acid Shared Resource to utilize the Personal densitometer SI. We also thank the Biochemistry Department for providing access to the departmental CD spectrometer. NIH (R01-AI 29092) funding to D.H.D. supported this research. “
“φEf11 is a temperate Siphoviridae bacteriophage isolated by induction from a lysogenic Enterococcus faecalis strain. The φEf11 DNA was completely sequenced and found to be 42 822 bp in length, with a G+C mol% of 34.4%. Genome analysis revealed 65 ORFs, accounting for 92.8% of the DNA content. All except for seven of the ORFs displayed sequence similarities to previously characterized proteins. The KU-57788 in vitro genes were arranged in functional

modules, organized similar to that of several other phages of low GC Gram-positive bacteria; however, the number and arrangement of lysis-related genes were atypical of these bacteriophages. A 159 bp noncoding region between predicted cI and cro genes is highly similar to the functionally characterized early promoter region of lactococcal temperate phage TP901-1, and selleck kinase inhibitor possessed a

predicted stem-loop structure in between predicted PL and PR promoters, suggesting a novel mechanism of repression of these two bacteriophages from the λ paradigm. Comparison with all available phage and predicted prophage genomes revealed that the φEf11 genome displays unique features, suggesting that φEf11 may be a novel member of a larger family of temperate prophages that also includes lactococcal phages. Trees based on the blast score ratio grouped this family by tail fiber similarity, suggesting that these trees are useful for identifying phages with similar tail fibers. Enterococcus faecalis is a facultatively anaerobic, Gram-positive coccus, commonly growing in short chains or clusters. Although these bacteria have long been considered to be ubiquitous, commensal organisms commonly isolated from the mammalian alimentary canal as well as from water and soil (Facklam et al., 2002), more recently, they have emerged as opportunistic pathogens associated with a variety of medical and dental infectious diseases. These organisms are among the most frequent causes of nosocomial infections (Moellering, 1992; Edgeworth et al., 1999; Richards et al.

A decline in toxicity to this magnitude may infer that receptor binding event was affected or proteolytic LGK-974 degradation in the gut lumen. Alternatively, loss of toxicity may be attributed to the disruption of the membrane insertion event and should be considered (Nair et al., 2008). We thank Dr Xinyan Sylvia Liu, Dr Manoj Nair, Dr Dan Zeigler, Carol Zeigler, Sharnise

Mitchell and Yoshio Ikeda for their contributions, as well as stimulating talks that shed some insight on analysing the results. We thank Dr Hansjuerg Alder for giving us access to the Nucleic Acid Shared Resource to utilize the Personal densitometer SI. We also thank the Biochemistry Department for providing access to the departmental CD spectrometer. NIH (R01-AI 29092) funding to D.H.D. supported this research. “
“φEf11 is a temperate Siphoviridae bacteriophage isolated by induction from a lysogenic Enterococcus faecalis strain. The φEf11 DNA was completely sequenced and found to be 42 822 bp in length, with a G+C mol% of 34.4%. Genome analysis revealed 65 ORFs, accounting for 92.8% of the DNA content. All except for seven of the ORFs displayed sequence similarities to previously characterized proteins. The Caspase-independent apoptosis genes were arranged in functional

modules, organized similar to that of several other phages of low GC Gram-positive bacteria; however, the number and arrangement of lysis-related genes were atypical of these bacteriophages. A 159 bp noncoding region between predicted cI and cro genes is highly similar to the functionally characterized early promoter region of lactococcal temperate phage TP901-1, and Glutathione peroxidase possessed a

predicted stem-loop structure in between predicted PL and PR promoters, suggesting a novel mechanism of repression of these two bacteriophages from the λ paradigm. Comparison with all available phage and predicted prophage genomes revealed that the φEf11 genome displays unique features, suggesting that φEf11 may be a novel member of a larger family of temperate prophages that also includes lactococcal phages. Trees based on the blast score ratio grouped this family by tail fiber similarity, suggesting that these trees are useful for identifying phages with similar tail fibers. Enterococcus faecalis is a facultatively anaerobic, Gram-positive coccus, commonly growing in short chains or clusters. Although these bacteria have long been considered to be ubiquitous, commensal organisms commonly isolated from the mammalian alimentary canal as well as from water and soil (Facklam et al., 2002), more recently, they have emerged as opportunistic pathogens associated with a variety of medical and dental infectious diseases. These organisms are among the most frequent causes of nosocomial infections (Moellering, 1992; Edgeworth et al., 1999; Richards et al.

Fractions exhibiting QPO activity that eluted with 0.4–0.5 M potassium phosphate were pooled and loaded onto a 1-mL AF-Red-560M column (Tosoh Corp., Tokyo, Japan). QPO did not bind to the column. The flow-through ERK inhibitor cell line was pooled and concentrated by the addition of PEG6000 (Yamada et al., 2007). QPO activity was measured by a previously described method (Yamada et al., 2007), with slight modification. This activity was measured at 25 °C in a buffer containing 100 mM Tris-HCl (pH, 7.5), 0.1% (w/v) SM-1200 (Nacalai Tesque Inc.), and ubiquinol-1 (20, 50, 70, 100, 200, and 300 μM). Ubiquinone-1 was kindly gifted by Eisai (Tokyo, Japan), and the reduced form (ubiquinol-1) was

prepared by the method described previously (Rieske, 1967). The reaction was initiated by the addition of 80 μM H2O2. Oxidation of ubiquinol-1 was assessed at 278 nm using an extinction coefficient of 10 mM−1 cm−1. The kinetic

parameters were calculated using graphpad prism (Graphpad software, San Diego, CA) and a nonlinear Ruxolitinib molecular weight least-squares analysis. Redox titrations were performed using a platinum electrode (Radiomater, Copenhagen, Denmark). The titration was carried out at 25 °C in 100 mM Tris-HCl buffer (pH, 7.5) in the presence of several electron mediators as follows: 50 μM ferrocyanide, 10 μM 2-OH-1,4 naphtoquinone, 20 μM phenazine methosulfate, 20 μM phenazine ethosulfate, 20 μM 2,3,5,6-tetramethyl-p-phenylene diamine, and 20 μM duroquinone (Matsushita et al., 1999). The buffer also contains 0.5% SM-1200 to improve the stability of the measurement system. The course of reduction of heme c was recorded at its α-band maximum at 556.6 nm using MultiSpec-1500 (Shimadzu, Kyoto, Japan). Midpoint potentials were calculated using the Nernst equation for three components

(n=1) with unknown redox potentials with igor pro (WaveMetrics, Lake Oswego, OR) and a nonlinear least-squares analysis. Heterogenous expression of cytochrome c increased by the overexpression of ccm genes and the deletion of degP protease, which is one of the major proteases Casein kinase 1 in the periplasmic region of E. coli (Brige et al., 2001). In order to obtain active rQPO, we introduced pET101QPO into Keio:JW0157(DE3)/pCCM, a λDE3-lysogenized strain lacking degP protease and harboring the plasmid pCCM that constitutively expresses ccm genes. We tested several production protocols and found that the highest activity of rQPO was obtained in cultures grown without induction of isopropyl thio-β-d-galactoside. Unfortunately, the His-tag that was introduced into the C-terminus of QPO resulted in the production of inactive rQPO. rQPO with a His-tag at the N-terminus was actively expressed, however, this enzyme was highly unstable upon solubilization (not shown). Because membrane-bound enzymes are difficult to handle, we also attempted to express QPO that lacked the single N-terminal transmembrane region in order to obtain a soluble form of rQPO.

These findings are consistent with earlier work carried out by other researchers. Lima et al. [12]. found that individuals CHIR-99021 cost on boosted PI-based regimens were less likely to develop resistance than those on NNRTI-based regimens (AOR 0.42; 95% CI 0.28–0.62) and Riddler et al. [13] found that those on efavirenz-based regimens were more prone to the development of drug resistance mutations than those on lopinavir/ritonavir-based therapies (9 vs. 6%, respectively).

The two comparison drug classes were equally efficacious, as evidenced by proportions of participants who achieved virological suppression (plasma viral load <50 copies/mL) in the first year of therapy (66% for the NNRTI group and 67% for the boosted PI group). Such a similarity in virological response and other clinical outcomes has been documented in other studies [25,26]. This rate of response occurred despite lower adherence selleck chemicals in

the NNRTI group. This kind of response to NNRTI was also demonstrated by Nachenga et al., who found that moderate levels of adherence to these drugs often led to viral suppression among patients [27]. These results may also suggest that, despite adequate virological response, patients still remain at a greater risk of developing resistance to NNRTIs. The generalization of these findings to RLSs, where NNRTI-based ART is primarily used for first-line treatment, may be limited by the fact that this study was carried out in a developed country where most of the social demographic features are different from those in developing countries. Furthermore, most participants in this cohort had HIV-1 subtype B, which accounts for only 10% of HIV infections world-wide, and recent evidence suggests that different HIV genetic variants have different biological properties, including susceptibility and response to antiretroviral Ureohydrolase drugs [28]. In addition, the way in which ART is managed in the face of drug resistance is very different in BC from

RLSs. However, we believe that concerns regarding NNRTI-induced resistance mutations require greater study in RLSs. The potential for the development of mutations is probably even greater in these settings, where individuals may have prolonged periods of uncontrolled viraemia prior to switching the class of their third drug. Our results suggest that evaluating the strategy of NNRTI- versus boosted PI-based HAART in RLSs should be a main priority. This should be coupled with documentation of the impact of these mutations on subsequent virological suppression and clinical outcomes among patients who are failing ART in RLSs. Advocacy targeted at reduction in prices for boosted PIs and licensing of generic products can help to increase the availability of these drugs in RLSs. The authors would like to thank the participants in the BC HIV/AIDS DTP and the nurses, physicians, social workers and volunteers who support them.

We have previously observed that thioridazine reduces the oxacillin-dependent induction of the resistance genes mecA and blaZ (Klitgaard et al., 2008). Besides the acquisition of mecA, the resistance level of MRSA strains is influenced by the expression

levels of several housekeeping genes that are either directly or indirectly involved in cell wall biosynthesis and cell wall turnover (Fig. 1). These include the bifunctional native PBP2 of which the transglycosylase domain was shown to be necessary to retain high-level resistance in the MRSA strain COL (Pinho et al., 2001) and the femAB operon, which encodes two peptidyl transferases that are necessary for the formation of pentaglycine bridges and, hence, the cross-linking of peptidoglycan layers (Stranden et al., 1997). Additionally, the two-component system VraSR is an important factor in the tolerance of S. aureus to a broad range of antibiotics targeting the cell wall selleckchem (Kuroda et al., 2003). Upon perturbation of cell wall synthesis, VraSR induces the transcription of GSK2126458 mouse a number of genes including murZ (a redundant MurA isozyme) (Blake et al., 2009), pbpB (PBP2), sgtB (a soluble transglycosylase), and fmtA (an accessory PBP with low affinity to β-lactams)(Utaida et al., 2003; McAleese et al., 2006; Fan et al., 2007), all of which

are involved in murein monomer synthesis or peptidoglycan polymerization. The involvement of another native PBP, PBP4 (encoded by the pbpD gene), in β-lactam resistance is not as certain and might be strain specific. PBP4, which possesses transpeptidase, carboxypeptidase, and β-lactamase activity (Kozarich & Strominger, 1978), is necessary to achieve a highly cross-linked cell wall (Leski & Tomasz, 2005) and was shown to play a prominent role in the β-lactam resistance of community-acquired MRSA (Memmi et al., 2008). However, in the highly resistant MRSA strain COL, PBP4 seems to be largely dispensable (Katayama et al., 2003; Memmi et al., 2008). The pbpD gene shares an overlapping promoter region with the divergently transcribed AZD9291 concentration abcA gene, encoding an ATP-dependent

transporter of the ABC superfamily (Domanski et al., 1997). The abcA gene product functions as an efflux pump (Truong-Bolduc & Hooper, 2007) and its expression is controlled by the global regulatory agr system (Schrader-Fischer & Berger-Bachi, 2001). AbcA is involved in control of autolytic activities, offering a protective role against β-lactams (Schrader-Fischer & Berger-Bachi, 2001). Concordantly, overproduction of AbcA was shown to result in increased β-lactam resistance (Truong-Bolduc & Hooper, 2007). The agr quorum-sensing system is induced at high cell densities in response to the extracellular cell-density-dependent accumulation of autoinducer. The agr locus regulates the production of virulence factors by repressing expression of cell surface associated factors and activating expression of secreted toxins and enzymes (Vandenesch et al., 1991; Saravia-Otten et al., 1997; Ziebandt et al.

cereus KF1, a strain isolated from artisanal kefir produced in Vietnam, were included. For flagellin extraction,

we set up a protocol involving the use of 5 M LiCl. LiCl is a chaotropic agent which destabilizes both hydrophobic/electrostatic interactions and hydrogen bonds, leading to the extraction of noncovalent surface-associated proteins (Sánchez et al., 2008). LiCl (5 M) solution was always supplemented with EDTA 5 mM and PMSF 1 mM, the absence of the latter leading to complete proteolysis of the flagellins (data not shown). As shown in Fig. 1a, incubations of 30 min at 4 °C led to the extraction of prominent bands with observed molecular masses of 28–60 kDa. All bands were identified as Bacillus flagellins by MS, or by tandem MS (MS/MS) in cases that needed a more powerful analytic technique (Table 2). A single flagellin product was

Ganetespib ic50 detected in all the cases except for B. cereus ATCC see more 14579 and B. cereusN, in which two bands identified as flagellins were observed. These flagellins could be extracted from SDS gels and renaturalized as described (Peant & LaPointe, 2004); they were able to bind mucin, as shown in a previous work (data not shown) (Sánchez et al., 2009a). The size of the different flagellins varied from approximately 30 to 60 kDa (Fig. 1). In the B. cereus group, flagellins comprise a set of variable proteins in terms of sequence and molecular masses. This is due to Branched chain aminotransferase the fact that the domains D2 and D3 are highly variable, producing potentially infinite variants (Beatson et al., 2006). The gene coding from the B. cereus CH flagellin of 45 kDa was amplified using its total DNA as template. Primer sequences were deduced from protein sequence AAZ22698, to which B. cereus CH flagellin showed the higher degree of identity. This gene was cloned into the blunt-end NaeI site of pNZ8110, the resulting ligation being electroporated in L. lactis NZ9000. In this way we isolated a Lactococcus clone producing the recombinant flagellin, which was denominated L. lactis ssp. cremoris NZ9000-CH. Surface-associated protein and secreted protein profiles were obtained and, surprisingly, two bands of around 30 and 25 kDa were identified in

the supernatant fraction of the induced cultures (data not shown). After MS analysis, these bands were properly identified as the B. cereus CH flagellin (Table 1). These results suggested that flagellin was proteolyzed on the Lactococcus surface, an aberrant form of the flagellin being released into the bacterial environment. It is known that the housekeeping protease HtrA from L. lactis, is targeted to the cell surface, where it degrades abnormal proteins, somehow monitoring the quality of surface proteins (Lyon & Caparon, 2004). We thought of the possibility that the recombinant protein was recognized as aberrant, being degraded by the action of this enzyme (or other surface-associated proteases) and released to the surrounding media.