Consistent with this idea is the

previous observation that overexpression of glpD and plsB involved in energy production caused increased persister formation (Spoering et al., 2006). GSK-3 beta pathway The mechanism by which bacteria form persisters is not well understood and is the topic of considerable recent interest. It is quite likely that multiple mechanisms of varying hierarchy and importance are involved in persister formation. It is interesting to note that the phoU mutation identified in our previous work seems to increase the cellular metabolism so the bacteria are defective in forming persisters and thus remain susceptible to antibiotics even in the stationary phase. In contrast to the phoU mutation, the sucB and ubiF mutations interfere with energy production and appear to affect the persister survival and exit from dormancy by decreasing the metabolism. The energy metabolism-related click here mechanism of persister formation mediated by UbiF and SucB may be located somewhere downstream of a primary sensor switch mechanism such as PhoU in coordinating persister formation. Further studies are needed to determine how the different mechanisms cooperate to mediate persister formation in response to environmental cues. Because SucB and

UbiF are involved in persister survival and because they are widely present in different bacterial species, they may serve as attractive persister drug and vaccine targets for more effective control of bacterial infections. We thank Hirotada Mori for providing the E. coli Keio deletion mutant library. C.M. was sponsored by the China Scholarship Council. Y.Z. was supported by NIH grant AI44063 and Changjiang Scholars Program.

“
“Genes involved in the 4-aminobenzenesulfonate (4-ABS) degradation pathway of Hydrogenophaga sp. PBC were identified using transposon mutagenesis. The screening of 10 000 mutants for incomplete 4-ABS biotransformation identified four mutants with single transposon insertion. Genes with insertions that impaired the ability to utilize 4-ABS for growth included (1) 4-sulfocatechol mafosfamide 1,2-dioxygenase β-subunit (pcaH2) and 3-sulfomuconate cycloisomerase involved in the modified β-ketoadipate pathway; (2) 4-aminobenzenesulfonate 3,4-dioxygenase component (sadA) involved in aromatic ring hydroxylation; and (3) transposase gene homolog with a putative cis-diol dehydrogenase gene located downstream. The pcaH2 mutant strain accumulated brown metabolite during growth on 4-ABS which was identified as 4-sulfocatechol through thin layer chromatography and HPLC analyses.

3E–E6) suggests that these cells first migrate caudally in the

lateral subpallium before turning, and migrating in the lateral-to-medial direction within the EA. In sum, our analysis revealed that scgn+ cells cytoarchitecturally resembling migrating neurons formed a continuous stream along the palliosubpallial boundary before reaching their final destinations in the OB or EA (Fig. 4). Next, we analyzed the distribution of scgn+ neurons click here in neonatal mouse brain. We observed that the migration of scgn+ cells concluded by birth and scgn+ neurons inhabited, in an anterior-to-posterior direction, the spatially interrelated nuclei of the BST, interstitial nucleus of the posterior limb of the anterior commissure (IPAC), ventral pallidum (VP), dorsal substantia innominata (SI), and the central and medial amygdaloid nuclei (CA and MA; Fig. 5A–A7). Morphometric analysis revealed that scgn identifies divergent neuron

subpopulations with different somatic diameters in the VP and EA (Fig. 5B). By using genetically tagged reporter mice we demonstrated that scgn+ neurons either adopted a GABA phenotype along the longitudinal axis of the EA (Fig. 5D and D1), similar to scgn+/GABA+ neurons in DAPT in vitro the embryonic OB (Fig. 5C and C1), or co-express ChAT when found in small-diameter cholinergic neurons of the dorsal SI (Fig. 5E and E1). Collectively, our data suggest that by E18 scgn+ neurons can acquire a distribution pattern resembling that in the adult brain, and differentiate into neurochemically distinct subtypes of EA

neurons. Systematic analysis along the longitudinal axis of the fetal primate brain revealed the first contingent of scgn+ neurons in the granular and glomerular layers of the OB (Fig. 6A). However, unlike in the adult primate brain (Mulder et al., 2009b), neuroblasts migrating in the prenatal rostral migratory stream (Pencea & Luskin, 2003) did not harbour scgn expression (Fig. 6A). Pallial areas were devoid of discernible Montelukast Sodium scgn immunoreactivity. In the basal forebrain, scgn+ neurons were seen in the horizontal diagonal band, nucleus accumbens, medial septum, VP, GP and SI (Fig. 6A1–A7). In contrast to scgn distribution in the neonatal rodent brain, scgn+ cells were only infrequently found in either the CA or MA. In the hypothalamus, substantial scgn+ neuron populations occupied the paraventricular and periverticular nuclei and the supraoptic nucleus (Fig. 6A5–A7). A morphological dichotomy of scgn+ neurons was evident in the basal telencephalon (Fig. 6B): small-to-medium-sized scgn+ neurons populated the horizontal diagonal band, SI and CA. In contrast, large-diameter scgn+ neurons were found in the IPAC and GP.

After incubation, the supernatant was separated for extracellular oxidative and nitrosative stress assay and the plate was rinsed with phosphate-buffered solution (PBS, pH 7.2). After drying, staining for adherent biofilms was performed using CV (1%). Then, the CV was removed and cells were rinsed three times with 300 μL PBS (pH 7.2) before drying for 24 h at room temperature. A quantitative assessment of the biofilm formation was obtained

by extracting the CV with 200 μL per well of the bleaching solution: ethanol/glacial acetone (70 : 30). The intensity of the coloration was determined at 595 nm using a microplate reader (Model 680 BioRad, Hercules, CA). All strains were tested in three independent experiments on different days. The average OD595 nm value was determined by three replicates and was interpreted by the following scale: positive (>0.24), weak (>0.12 and <0.24) or negative (<0.12) (Deighton et see more al., 2001). The biofilm biomass unit (BBU) was arbitrarily defined with 0.1 OD595 nm=1 BBU. Biofilm formation Tanespimycin concentration was investigated at 12, 24 and 48 h, and the effect of temperature was evaluated at 25, 30 or 37 °C. Static conditions were used at 37 °C for 24 h at different pH values (5–8). The influence of the reduction conditions was assayed in thioglycolate broth and microaerobic conditions with TSB or thioglycolate broth were

also studied. Three wells with 200 μL TSB or thioglycolate were added to serve as negative controls and to obtain a background value, which was then subtracted from the values obtained from the cells in the wells. The intra- (iROS) and extracellular (eROS) production of ROS was detected by the reduction of nitro-blue

tetrazolium (NBT, Sigma) to nitroblue diformazan. The Selleckchem MG132 supernatant was separated by measuring the eROS. Then the biofilms in individual wells of sterile 96-well polystyrene microtiter plates were treated with 0.05 mL dimethyl sulfoxide (Merck) to extract the reduced NBT using 0.1 mL NBT (1 mg mL−1) and 0.1 mL TSB (for the final volume) at 37 °C for 30 min, followed by the addition of 0.02 mL hydrochloric acid (0.1 M) to stop the reaction and measure iROS. The reaction is detectable by the byproducts of the assay, which are proportional to the ROS generated in biofilm and were measured by OD at 540 nm (Paraje et al., 2009; Aiassa et al., 2010; Páez et al., 2010). The supernatant under different conditions was separated for extracellular nitrosative stress assay and incubated for NO measurement. The NO was evaluated as nitrite by a microplate assay method using Griess reagent (Paraje et al., 2009). One hundred microliter aliquots of supernatant were mixed with 200 μL Griess reagent [sulfanilamide 1.5% in 1 N HCL and N-1-naphthyl ethylenediamide dihidrochloride 0.13% in sterile distilled water (dH2O)].

Because cystine appears to be an important nutrient for S. mutans growth, understanding the genetic pathways required for its acquisition satisfies an important step in attempts to modulate the growth and virulence of S. mutans. We thank Dr Joyce Azavedo for help with preparation of this manuscript. This study was supported by NIH grant R01DE013230-03 and CIHR grant MT-15431 to D.G.C. D.G.C. is a recipient of a Canada Research Chair. “
“State Key Laboratory of Microbial Resources, selleck kinase inhibitor Institute of Microbiology, Chinese Academy

of Sciences, Beijing, China Increasing evidence has shown that antibiotics function as intermicrobial signaling molecules instead of killing weapons. However, mechanisms and key factors that are involved in such functions remain poorly understood. Earlier findings have HKI-272 cell line associated antibiotic signaling with quorum sensing (QS); however, results varied among experiments, antibiotics, and bacterial strains. In this study, we found that antibiotics at subinhibitory concentrations improved the violacein-producing ability of Chromobacterium violaceum ATCC 12472. Quantitative real-time polymerase chain reaction of QS-associated gene transcripts and bioassay of violacein

production in a QS mutant strain demonstrated that antibiotics enhanced the production of N-acyl-l-homoserine lactones (AHLs; QS signaling molecules) and increased AHL-inducing QS-mediated virulence, including chitinase production and biofilm formation. Moreover, a positive flagellar activity and an increased Fluorouracil datasheet bacterial

clustering ability were found, which are related to the antibiotic-induced biofilm formation. Our findings suggested that antibiotic-mediated interspecific signaling also occurs in C. violaceum, thereby expanding the knowledge and language of cell-to-cell communication. “
“The study compared images of mature Streptococcus mutans biofilms captured at increasing magnification to determine which microscopy method is most acceptable for imaging the biofilm topography and the extracellular polymeric substance (EPS). In vitro S. mutans biofilms were imaged using (1) scanning electron microscopy (SEM), which requires a dehydration process; (2) SEM and ruthenium red (SEM-RR), which has been shown to support the EPS of biofilms during the SEM dehydration; and (3) variable pressure scanning electron microscopy (VPSEM), which does not require the intensive dehydration process of SEM. The dehydration process and high chamber vacuum of both SEM techniques devastated the biofilm EPS, removed supporting structures, and caused cracking on the biofilm surface. The VPSEM offered the most comprehensive representation of the S. mutans biofilm morphology.

GPP   We recommend following discussion, if a patient with a CD4 cell count >350 cells/μL wishes to start ART to reduce the risk of transmission to partners, this decision is respected and ART is started. GPP a Abacavir is contraindicated if HLA-B*57:01 positive. 5.3 We recommend therapy-naïve patients start combination ART containing tenofovir (TDF) and emtricitabine (FTC) Target Selective Inhibitor Library as the NRTI backbone. 1A   We suggest abacavir (ABC) and lamivudine (3TC) is an acceptable alternative NRTI backbone in therapy-naïve patients who, before starting ART, have baseline

viral load (VL) of ≤100 000 copies/mL. 2A   ABC must not be used in patients who are HLA-B*57:01 positive. 1A 5.4 We recommend therapy-naïve patients start combination ART containing one of the following as the third agent: atazanavir/ritonavir (ATV/r), darunavir/ritonavir (DRV/r), efavirenz (EFV), raltegravir (RAL) or elvitegravir (ELV)/cobicistat (COBI). 1A   We suggest that in therapy-naïve patients lopinavir/ritonavir (LPV/r) and fosamprenavir/ritonavir (FPV/r) are acceptable alternative PIs, and nevirapine (NVP) is an acceptable alternative NNRTI. 2A 5.5 We recommend against the use of PI monotherapy as initial therapy for treatment-naïve

patients. 1C   We recommend against the use of PI-based dual ART with a single NRTI, NNRTI, C–C chemokine receptor type 5 (CCR5) receptor antagonist or INI as initial therapy for treatment-naïve patients. 1C 6.1.1 We recommend adherence and RG7204 clinical trial potential barriers to it are assessed and discussed with the patient whenever ART is prescribed or dispensed. GPP   We recommend adherence support should address both perceptual barriers (e.g. beliefs and preferences) and/or practical barriers (e.g. limitations in capacity and resources) to adherence. GPP 6.2.1 We recommend that potential adverse pharmacokinetic interactions between ARV drugs and other concomitant medications are checked before administration (with tools such as http://www.hiv-druginteractions.org). GPP 6.2.2 We recommend against the unselected use of therapeutic drug monitoring (TDM). GPP 6.2.3 We recommend patients stopping ART containing an NNRTI in combination with an NRTI backbone

replace all drugs with a PI (LPV/r) for 4 weeks. 1C   We recommend patients stopping a PI-containing regimen stop all drugs simultaneously and no replacement TCL is required. 1C 6.3.2 We recommend in patients on suppressive ART regimens, consideration is given to differences in side effect profile, drug–drug interaction (DDIs) and drug resistance patterns before switching any ARV component. GPP   We recommend, in patients with previous NRTI resistance mutations, against switching a PI/r to either an NNRTI or an INI as the third agent. 1B 6.3.3 We recommend continuing standard combination ART as the maintenance strategy in virologically suppressed patients. There are insufficient data to recommend PI/r monotherapy in this clinical situation. 1C 6.

, 2010). Although integrons are transposition defective, they can be mobilized in association with functional transposons and/or conjugative plasmids (Cambray et al., 2010). Despite their relevance in HGT processes, the association of integrons with conjugative plasmids has been poorly addressed in aquatic environments. Wastewater treatment plants (WWTPs) are important reservoirs of resistance determinants and favourable places for HGT, due to high microbial abundance, high nutrient concentrations and intense selective pressures imposed by antibiotics, detergents and other pollutants

(Schlüter et al., 2007). Moreover, it has been shown that natural conjugative plasmids may induce the development of biofilms, which might also increase the chances of cell-to-cell contact and the occurrence of HGT events (Ghigo, 2001). As a result, WWTPs may favour the www.selleckchem.com/products/azd9291.html persistence of plasmids through the treatment Ceritinib solubility dmso process, contributing to the dissemination

of integrons and undesirable genetic traits, such as those coding for antibiotic resistance and virulence determinants, to natural waters, soils and eventually the food chain. Previously, the presence and distribution of integron-carrying bacteria was investigated at different stages of the treatment process in two WWTPs, one treating urban discharges and the other treating wastewaters from a slaughterhouse (Moura et al., 2007, 2012). The present study was performed PTK6 to investigate the diversity of plasmids in integron-positive strains retrieved from wastewaters, providing data pertaining to the contribution of these environments to the spread of integrons and antibiotic resistance determinants through HGT. Sixty-six integron-positive (intI+) strains belonging to Aeromonas sp. (n = 48) and Enterobacteriaceae (n = 18) previously isolated from urban and slaughterhouse wastewaters (Moura et al., 2007, 2012) were included as donors in mating assays using rifampicin- and kanamycin-resistant Escherichia coli CV601-GFP

and Pseudomonas putida KT2442-GFP as recipient strains (Smalla et al., 2006). Liquid cultures of donor and recipient strains were prepared separately in 10 mL Luria–Bertani broth (LB) and grown overnight with gentle shaking at 28 °C. Recipient and donor strains were mixed (ratio 1 : 1) and centrifuged for 5 min at 6700 g to precipitate cells. Supernatants were discarded and replaced by 1 mL fresh LB. Mixtures were incubated overnight at 28 °C without shaking. Cells were then precipitated by centrifugation (5 min, 6700 g) and washed in 0.9% NaCl solution. Serial dilutions were prepared in 0.9% NaCl and aliquots of 100 μL were spread on Plate Count Agar plates supplemented with rifampicin (50 mg L−1) and streptomycin (50 mg L−1) or with rifampicin (50 mg L−1) and tetracycline (50 mg L−1). Putative transconjugants were grown at 28 °C for 48 h. Assays were run in duplicate.

1d), indicating the cells had acquired ability to grow with glucose as the sole carbon source. The strains able to use glucose (EH1-3) were passed PD0332991 chemical structure four times through MM (L), following the diauxic growth analysis. They were then reinoculated into medium with glucose as the sole carbon source. All three strains followed a similar

growth pattern as previously seen in glucose medium (Fig. 1b and d). To verify glucose assimilation and/or respiration, two independent techniques were employed. The HPLC results shown in Fig. 2a confirm that glucose disappeared from the culture medium (from 18 mM to < 2 mM during 91 h) as OD600 nm increased. The glucose incorporation/respiration experiment (Fig. 2b) revealed that the majority of glucose was respired to CO2 by the S. oneidensis strains EH1-3 rather than being incorporated into biomass. Glucose incorporation and respiration in the wild-type S. oneidensis MR-1 grown in MM (L) were significantly lower than those in EH1-3; however, like the EH1-3 strains, respiration instead of assimilation was the dominant utilization pathway for glucose (Fig. 2b). Preliminary studies using EH1 in a MFC showed it was able to utilize lactate and glucose to generate current, but the response was delayed for glucose (data not shown). This result confirms that what most likely occurred in our previous complex media MR-1 MFC experiments (Biffinger et al., 2008, BGB324 datasheet 2009) was

the growth advantage of glucose-utilizing mutants over time, resulting in a delayed current-generating response to the addition of glucose. The traditional concept that a characteristic of Shewanella spp. is the inability to use glucose as a growth substrate has diminished with the emergence of new studies demonstrating utilization of glucose by many Shewanella species (Bowman et al., 1997; Nogi et al., 1998; Leonardo et al., Thalidomide 1999; Brettar et al., 2002; Gao et al., 2006; Zhao et al., 2006; Xiao et al., 2007; Rodionov et al., 2010). The current study shows growth, incorporation, and respiration of glucose by S. oneidensis (Figs 1b and 2), an organism previously considered

unable to use glucose as a growth substrate (Myers & Nealson, 1988; Venkateswaran et al., 1999; Rodionov et al., 2010). These results indicate that S. oneidensis uses glucose primarily as an energy source and less so as a building block for biomass (Fig. 2b). The use of S. oneidensis in MFCs with glucose has interesting implications including dual-carbon source systems where the primary carbon source gives immediate current, while the glucose can extend the usefulness of the MFC, delivering delayed current or sustainment of the microbial catalyst during limited optimal electron donor periods. The most successful applications of MFCs include environmental deployment (e.g., ocean, seafloor, marsh, rice fields) and wastewater treatment, including biomass conversion to electricity.

, 1976; Mani et al., 1993). Briefly, 100 mL cultures of S. aureus growing exponentially (OD620 nm≈0.6) in TSB medium at 37 °C with aeration were pelleted, washed twice in cold 0.05 M Tris-HCl (pH 7.2) and then resuspended in 50 mL of 0.05 M Tris-HCl (pH 7.2) containing 0.05% (v/v) Triton X-100 (Sigma Chemical Co., St. Louis, MO). The cells were incubated at 37 °C with shaking and the OD620 nm was measured at 30-min intervals for 5 h. Values reported are averages of at least three

independent experiments. The statistical significance of the data was evaluated using a Student’s t-test. To proactively examine resistance to ramoplanin, we generated a resistant strain by serial passage of S. aureus NCTC 8325-4 in the presence of sub-MICs of ramoplanin. Selleck CP-868596 The results from each passage of NCTC 8325-4 are shown in Table 1. In general, multiple passages were required for S. aureus to be able grow in the next higher concentration of ramoplanin. During the 16th IDH targets passage, growth was observed in a culture containing 5 μg mL−1 ramoplanin. A sample from this culture was plated on TSA. An isolated colony was selected and passed twice on TSA, and then a colony

was selected and named RRSA16 for ‘ramoplanin-resistant S. aureus 16th series.’ The nucleotide sequence of the 16s rRNA genes of RRSA16 were identical to those of its S. aureus NCTC 8325-4 progenitor. The susceptibility of RRSA16 to a panel of antimicrobials focused on cell wall active compounds was determined by broth microdilution (Table 2). The ramoplanin MIC increased from 0.75 μg mL−1 for NCTC 8325-4 to 8 μg mL−1 for RRSA16. Interestingly, RRSA16 had reduced susceptibility to two other antimicrobials Thymidylate synthase that act by binding peptidoglycan lipid intermediate II, vancomycin and nisin. The vancomycin MIC increased from 1.25 μg mL−1 for NCTC 8325-4 to 9 μg mL−1 for RRSA16, a level classified as VISA. The nisin MIC increased from 10 μg mL−1 for NCTC 8325-4 to >32 μg mL−1 for RRSA16. The MIC for oxacillin, which inhibits peptidoglycan at the transpeptidation step, increased slightly from 0.25 μg mL−1 for NCTC 8325-4 to 0.5 μg mL−1 for RRSA16. No changes in the

susceptibility were observed for bacitracin, phosphomycin, d-cycloserine, ciprofloxacin, erythromycin or rifampcin. The resistant RRSA16 was passed in an antibiotic-free medium for 18 days, generating R16-18d, a strain that was more sensitive to ramoplanin and vancomycin than RRSA16 (Table 2). These values are still higher than the MICs observed for NCTC 8325-4. The nisin MIC of R16-18d remained higher than 32 μg mL−1, the highest concentration tested. We next wished to examine RRSA16 for altered growth characteristics when grown in rich media. The doubling time of RRSA16 was 41 min, almost twice as long as the 23-min doubling time observed for NCTC 8325-4. The R16-18d doubling time of 26 min was similar to the doubling time of NCTC 8325-4.

Patients receiving 24 weeks of early cART more often reported tingling in the hands or feet (P = 0.02) and a numb feeling in the fingers or toes (P = 0.01) than patients receiving 60 weeks of early cART or no treatment. Patients receiving no treatment more often reported itchiness (P = 0.001) and skin changes (P = 0.04)

than patients receiving 24 or 60 weeks of early cART. At week 8, patients receiving 24 or 60 weeks of early cART more often reported nausea (P = 0.002), diarrhoea (P < 0.001), abdominal pain (P = 0.02), stomach pain (P = 0.049) and dizziness (P = 0.01) than patients receiving no treatment (Fig. 2). These differences had disappeared at week 24. No differences in patient characteristics and HRQL at baseline www.selleckchem.com/products/17-AAG(Geldanamycin).html H 89 purchase and during follow-up were seen between the randomized (n = 16) and nonrandomized (n = 12) untreated patients, except that the randomized patients were more often born in the Netherlands [15 of 16 (94%) versus seven of 12 (58%); P = 0.02]. When we repeated the mixed linear models including only the RCT patients, the significant differences in HRQL among the three groups

disappeared for cognitive functioning and mental health, although the trend remained similar. The differences in pain, physical functioning, role functioning and the PHS score remained significant. For these scales, patients receiving 60 weeks of early cART had a significantly better HRQL than patients this website receiving 24 weeks of early cART. The differences seen in reported symptoms remained the same. The present study was set up as a substudy of the Primo-SHM RCT, which demonstrated a clinical benefit of 24 and 60 weeks of cART initiated during PHI [1]. This substudy provides the first data on the effects on HRQL of temporary treatment during PHI. Early cART did not have a negative impact on patients’ HRQL over a study period of 96 weeks as compared with no treatment. Overall, patients receiving 60 weeks of cART showed a better HRQL than patients in whom treatment was deferred. Although the patients on early cART initially suffered more from physical symptoms,

which were probably related to drug toxicity, this seemed to have minor effects on their HRQL perception. This is in agreement with a previous study in which persons with chronic HIV infection on cART made distinctions between symptoms caused by HIV itself and those caused by drug toxicity when evaluating HRQL. Disease-related symptoms, but not side effects, were related to perceptions of general health [14]. Regardless of cART intervention, social functioning, health distress, overall quality of life, energy/fatigue and the MHS score improved significantly during the 96 weeks of follow-up in all groups. This might be explained by initial psychological distress as a consequence of being diagnosed with PHI and its acceptance over time. In addition, the symptoms occurring during PHI will also diminish without early treatment over time.

Sera from 42 patients allergic to A. alternata (18 female and 24 male; mean age, 20.5 years; age range, 10–33 years) and 17 control subjects (11 female and six male; mean age, 32.3 years; age range, 14–70 years) were included in the study. Diagnosis of A. alternata allergy was based on a clinical history of recurrent rhinitis (four patients), asthma (four patients), rhinoconjunctivitis (12 patients), rhinitis and asthma (12 patients), rhinoconjunctivitis and asthma (10 patients); a positive cutaneous response to a commercial A. alternata extract (Bial-Arístegui, Bilbao, Spain); and specific IgE to A. alternata extract > 0.35 IU mL−1 according ImmunoCAP (Thermo-Fisher,

Uppsala, Sweden). Eight Selleck Omipalisib healthy subjects and nine allergic individuals selleckchem sensitized to different allergenic sources unrelated

to A. alternata, as demonstrated by negative SPT responses and lack of A. alternata-specific IgE, were used as controls. Standard molecular genetic techniques were used (Sambrook et al., 1989). For Southern blotting, genomic DNA was prepared from recombinant yeasts as previously described (Barth & Gaillardin, 1996). Afterwards, DNA was digested, separated on a 0.8% agarose gel, and transferred onto Hybond-N+ nylon membranes (GE-Healthcare, Little Chalfont, Buck, UK). Probes were labeled with [32P]-dCTP using the MegaPrime Kit (GE-Healthcare). The autosomal vector pMM4 was used to express the Alt a 1 allergen. The YlMETII promoter was obtained from plasmid pSG70 (García, 1993; Domínguez et al., 2003) and cloned between the EcoRI-BamHI restriction sites of the pBluescript-SK. The Alt a 1-coding gene sequence (Asturias et al., 2003) MycoClean Mycoplasma Removal Kit was cloned after this promoter into the BamHI site, resulting in the pMMR2 vector. As the insertion of the target gene between yeast promoter and terminator sequences produces more efficient expression of heterologous genes in Y. lipolytica (Franke et al., 1998), the YlSTE7 terminator was amplified using specific primers and cloned into the SpeI and XbaI-restriction sites of pMMR2, resulting in the pMMR3 plasmid. This plasmid

was digested with ClaI and XbaI and the 1.8 kb-fragment containing the fusion of the YlMTPII promoter-Alt a 1-YlSTE7-terminator was purified and inserted into the pINA240 plasmid (Barth & Gaillardin, 1996), giving rise to pMMR4. The correct construction was verified by sequencing. The construction of the integrative plasmid pMMR10 was performed as follows. To create the pMMR10 plasmid, the 1.8-kb ClaI-SphI fragment from pMMR4 carrying the YlMTPII promoter-Alt a 1-YlSTE7-terminator fusion was cloned into the pINA62 plasmid. The correct construction was verified by sequencing. Plasmid maps of pMMR4 and pMMR10 and sequences of the specific primers used for YlSTE7 terminator amplification are available as Supporting Information (Fig. S1, Table S1). nAlt a 1 was purified from A. alternata CBS 603.78 spent culture medium after 3 weeks of static growth in Czapeck broth at 25 °C.