Introduction of the flhD4131 allele into YK410 (λPmcb-lacZ) by transduction with phage P1vir also did not change the levels of β-galactosidase expression. The difference in Pmcb-lacZ expression between YK410 and YK4131 is not dependent on the thyA allele present (data not shown). Using Hfr mapping, we have localized the region in YK4131 that is responsible for decreased stationary-phase activity of Pmcb to between 9 and 36 min on the

E. coli chromosome. Our results suggest that more than one mutation may be needed for the phenotype as we recover Napabucasin chemical structure three classes of exconjugants. In addition to recombinants that have the expected high and low levels of β-galactosidase activity, we recovered recombinants with intermediate levels of β-galactosidase activity. We plan to sequence the genomes of YK410 and YK4131 in order to identify the mutation(s). In addition to the mcb operon, five E. coli genes or operons have been reported to be regulated by FlhD independent of FlhC (Prüßet al., 2003). Because these genes were identified using YK410, YK4131, and YK4136 (an flhC derivative of YK410), the observed effects on gene expression may also be due to the same unidentified mutation(s) in strain YK4131 that affects expression from Pmcb. Further study is needed to answer this

question. This work was supported in part by a National Institutes of Health James A. Shannon Director’s Award (GM49770) to D.A.S. The authors thank Philip Matsumura and Birgit Prüß for strains, Mike Manson and

Susan Van Way for strains Carfilzomib price and advice on swarm assays, Daren Zentz, Yen Hoang Nong, Sylvia Ontiveros, and Rami Weaver for help performing growth assays, and Jim Hu and Matt Sachs for critical comments on the manuscript. “
“Captive snakes, that is, a Jamaican boa (Epicrates subflavus) a yellow anaconda (Eunectes notaeus) and a corn snake (Pantherophis guttatus guttatus), died with signs of bacteraemia including the presence of petechial haemorrhages in the mouth and gums and haemorrhages in the lung, spleen and intestines. The abdomen and anus were swollen with bloody-tinged mucus in the colon. Aeromonas hydrophila was recovered in dense virtually pure culture growth from the internal organs. Characterization of the isolates was by phenotyping and sequencing of the 16S rRNA gene (sequence homology of 99% with A. hydrophila) with outputs confirming Tideglusib the identity as A. hydrophila. Pathogenicity experiments confirmed virulence to frogs (Rana esculenta) and rainbow trout (Oncorhynchus mykiss). The genus Aeromonas comprises Gram-negative, oxidase and catalase-positive, heterotrophic, nonhalophilic and facultative anaerobic bacilli, which are widely distributed in natural waters (Holmes et al., 1996). The group is often associated with aquatic animals, and several species are primary or opportunistic pathogens of invertebrates and vertebrates, including humans (Martin Carnahan & Joseph, 2005).

4 g dry biomass mol−1 fructose (20.4 g dry biomass mol−1 substrate carbon). Genes encoding the enzymes required for both pathways of glycolysis are present in the genome of S. stellata. The Ymax observed during fructose-limited growth (64.2 g dry biomass mol−1 fructose) is closer to that predicted for dissimilation via the Enter–Doudoroff pathway, suggesting that it is probably in use in this case; however, it must be noted that many

bacteria use multiple pathways of glycolysis simultaneously during growth on hexoses (Wood & Kelly, 1977). The Ymax in the presence of DMS (73.2 g dry biomass mol−1 fructose, a 14% increase in Ymax from growth on fructose alone) is closer to the theoretical Ymax, indicative of a tighter coupling Roscovitine order of fructose oxidation to growth in the presence of DMS, with less dissimilation to carbon dioxide to meet the energy requirements of growth and maintenance. The oxidation of DMS to DMSO is catalyzed by DMS dehydrogenase in R. sulfidophilum (McDevitt et al., 2002): The subunits of DMS dehydrogenase have been shown to be encoded by the operon ddhABDC (McDevitt et al., 2002). Searching the S. stellata genome using the blastp algorithm reveals predicted proteins with >55% identity to DdhABC, clustered together

and annotated as components of a nitrate reductase NarYZV (EBA07058–EBA07060). It is worth noting that González et al. (1997) did not observe nitrate reduction during heterotrophic growth of S. stellata under anoxic conditions. Additionally, genes annotated as a DMSO reductase-like molybdopterin-containing dehydrogenase are also present

in the genome selleck chemicals of S. stellata (EBA06368–EBA06370); a tblastx search against the GenBank™ database confirms the annotation. The oxidation of DMS to DMSO could potentially be catalyzed by this enzyme performing its reverse reaction (Adams et al., 1999). Enzyme assays were many conducted for DMS dehydrogenase and DMSO reductase on cell-free extracts prepared from cells obtained from succinate-limited chemostats (D=0.03 h−1) grown with DMS (Table 2). It can be seen that DMS dehydrogenase (DCPIP or ferricyanide linked) activity was absent, although it could be assayed in the control organism R. sulfidophilum; DMSO reductase activity was also absent, but could be assayed in the positive control (H. sulfonivorans). It is, of course, possible that a DMS dehydrogenase is present in S. stellata grown under these conditions, but is either too unstable in cell-free extracts to assay or does not couple to DCPIP or ferricyanide in vitro. Additional assays were conducted in the presence of 1 mM NAD+ and NADP+, but no activity was observed. The ATP content of whole cells obtained from a succinate-limited chemostat (D=0.03 h−1) grown in the presence of DMS was monitored over time after the addition of DMS to 1 mM and the results are shown in Fig. 1. It can be seen that ATP is produced in the presence of DMS by cells of S.

Taking life-long treatment with a high adherence demand may also have emotional effects. Some compounds exacerbate mental health symptoms [7], while others may be associated with side effects (e.g. lipodystrophy) with mental health sequelae [8]. Poor mental health or heavy mental health burden is associated with reduced adherence, which in turn is associated with poorer outcome [6-9]. Therefore, incorporating assessment of mental health

into the routine follow-up of patients at all stages is important but is particularly critical at first presentation in order to establish a baseline. It is also important prior to commencement of ART (see 6.2 Monitoring of ART-naïve patients) and in those individuals with suboptimal adherence and/or virological failure, or signs of mental health symptoms (such Rucaparib datasheet as depressed mood, heightened anxiety, relationship concerns, memory or functioning concerns). Cognitive symptoms have been noted from the early days of the

epidemic, ranging from mild cognitive symptoms to more severe memory loss, executive functioning difficulties and cognitive impairment [10]. The advent of treatment has clearly reduced the prevalence of severe cognitive disorders [11, 12], while milder forms have continued in a proportion of patients. There is currently much debate about the prevalence, risk factors for, and prognosis of, mild-to-moderate cognitive impairment in persons taking effective ART Ruxolitinib research buy (full virological suppression). Joint psychological support standards are currently being consulted on and it is anticipated that these will make recommendations about screening [13], although there is not yet consensus about easy-to-administer and effective measurements. The finalized standards will be available late in 2011. Standardized monitoring of psychological wellbeing at baseline, at annual follow-up and at change points (such as treatment initiation and treatment switching) (III). Having good referral mechanisms to psychological services in place and clear criteria for referral (see BHIVA guidelines on psychological support

[13]) (IV). Inclusion of psychological next consideration in relation to fertility, drug use, treatment change, side effects, adherence, relationships and doctor–patient interaction (IV). There is no high-grade evidence for what is the optimal frequency at which to measure CD4 T cells in well-resourced health environments. We have considered three different scenarios: initial HIV diagnosis; monitoring ART-naïve patients; and CD4 T-cell counts in patients on ART. Recommendations for how often we should be measuring CD4 T-cell counts are mainly based on expert opinion [1-3]. For ART-naïve patients, we used data from a cost-effectiveness analysis using an HIV simulation model incorporating CD4 T-cell count and plasma HIV-1 RNA load as predictors of disease progression [4].

All media contained 20 g agar and 1 L of seawater, and were adjusted to pH 7.0. For bacterial isolation, 0.05 g L−1 streptomycin and potassium dichromate (50 mL of 1 g L−1 sterilized potassium dichromate in 1 L sterilized media) was added to the

bacterial isolation basic media to inhibit the growth of fungi. For fungal isolation, to inhibit the growth of bacteria, 0.5 g L−1 benzylpenicillin HDAC inhibitor and 0.03 g L−1 Rose bengal were added to the fungal isolation basic media. For bacterial DNA extraction, the selected bacterial isolates were inoculated into 7-mL centrifuge tubes containing 1 mL M2-broth medium (removed 20 g agar from M2) and cultured at 30 °C with shaking at 150 r.p.m. for 3–5 days. Total genomic DNA was extracted from all selected strains as described by Li & De (1995). From the genomic DNA, nearly full-length 16S rRNA gene sequences were amplified by polymerase chain reaction using primers 27°F (5′-GAGTTTGATCCTGGCTCAG-3′)

and 1525R (5′-AGAAAGGAGGTGATCCAGCC-3′; Warneke et al., 2006). All of the primers were synthesized by SBS Genetech (China). The polymerase chain reaction mixtures Selumetinib mw consisted of 12.5 μL Taq premix (TakaRa, China), 1 μL (10 μM) of each primer (TakaRa), 1.5 μL DMSO, 8 μL water and 1 μL of template DNA. After denaturation at 94 °C for 6 min, amplification was performed with 30 cycles of 40 s at 94 °C, 40 s at 53 °C, 2 min at 72 °C and a final extension at 72 °C for 10 min (Lee et al., 2003). Detailed information of fungal DNA extraction and fungal identification are given by Zhang et al. (2012). DNA sequencing of the selected bacterial and fungal isolates was carried out by Invitrogen (China). Sequences were corrected using sequencher, and the most similar sequences in GenBank were found using Basic Local Alignment Search Tool (blast) searches. When the top three matching blast hits were from the same species and

were ≥ 98% similar to the query sequence, this species name was assigned to the selected isolate (Toledo-Hernandez et al., 2008). The antimicrobial activities of bacterial and fungal isolates were determined by a Methocarbamol double-layer technique (Wu et al., 2009). Selected bacterial and fungal isolates were grown on M2 at 30 °C and M7 at 26 °C, respectively, for 5–14 days depending upon the growth rate of the various isolates. Two marine bacteria (Micrococcus luteus and Pseudoaltermonas piscida) and two marine coral pathogenic fungi [A. versicolor (AV) and A. sydowii (AS)] are the indicator microorganisms for the double-layer assay. Detailed information of the antimicrobial activity test is given by Zhang et al. (2012).

, 2000). To date, a number of SEs have been identified, including SEA-E, SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, and SEO (Omoe et al., 2002). Although their exact mechanisms of action have not been fully elucidated, these enterotoxins are believed to stimulate an enteric-vagus nerve reflex, triggering the vomiting centres of the brain (Sears & Kaper, 1996). Licochalcone A is one of the many flavonoids present in Chinese liquorice root, which has been used for centuries in traditional Chinese medicine. It has been demonstrated that licochalcone A possesses a variety of biological activities, including antimicrobial (Fukai et al., 2002), selleck chemicals llc anti-inflammatory (Kwon et

al., 2008), antiprotozoal (Chen et al., 2001), antitumour (Shibata, 2000), and antioxidative (Haraguchi et al., 1998) activities. Strikingly, previous studies have shown that licochalcone A was potent against methicillin-sensitive S. aureus Selleck ABT 199 (MSSA) and methicillin-resistant S. aureus (MRSA), with minimum inhibitory concentrations (MICs) ranging from 3 to 16 μg mL−1 depending on the strain (Hatano et al., 2000; Fukai et al., 2002). These results indicate that licochalcone A could be a potentially effective antimicrobial against S. aureus and could be used to treat patients infected with drug-resistant bacteria. Furthermore, in our previous study, we reported that subinhibitory concentrations of licochalcone A significantly

decreased α-toxin production in both MSSA and MRSA isolates (Qiu et al., 2009). However, there were no data on enterotoxin secretion by S. aureus exposed to licochalcone A obtained in this study. The present study click here was aimed to investigate the influence of subinhibitory concentrations of licochalcone A on

the production of enterotoxins A and B by S. aureus. The clinical isolate MRSA 2985 was isolated at the First Hospital of Jilin University from a blood sample from an infected patient. The MSSA ATCC 29213 isolate was obtained from the American Type Culture Collection (ATCC). Licochalcone A was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and stock solutions at various concentrations were prepared in dimethyl sulphoxide (DMSO) (Sigma-Aldrich, St. Louis, MO). The MIC in Mueller–Hinton broth (BD Biosciences Inc., Sparks, MD) was evaluated in triplicate using a broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (2005). The licochalcone A MIC values for S. aureus strain ATCC 29213 and MRSA strain 2985 were 4 μg mL−1. Furthermore, the MICs of the strains vs. licochalcone A in Luria-Bertani (LB) broth (BD Biosciences Inc.) were also 4 μg mL−1. Staphylococcus aureus strain ATCC 29213 was grown to an OD600 nm value of 0.3 in LB, and 100-mL volumes of the culture were placed into five 250-mL Erlenmeyer flasks.

The phylogenetic potential of the 23S ribosomal RNA marker has previously been exploited for Legionella and Coxiella (Afseth et al., 1995; Grattard et al., 2006), but has not yet been explored for Rickettsiella bacteria. Moreover, in attempts to go beyond ribosomal phylogenies,

several protein-encoding genes have been investigated as possible phylogenetic markers within the Coxiellaceae (Sekeyová et al., 1999; Leclerque & Kleespies, 2008a, b; Mediannikov et al., 2010), but often with rather limited success. The systematic taxonomic analysis of the first Rickettsiella genome sequence (Leclerque, 2008a) has revealed a set of protein-encoding markers that operate reasonably well above the genus

level; however, the suitability of these markers for generic and infra-generic taxonomic assignments has not been studied check details previously. Independently, the ftsY gene, which encodes the bacterial homolog of the eukaryotic signal click here recognition particle receptor subunit alpha involved in protein translocation and has previously been identified as the most appropriate single gene marker for the estimation of the G+C content in prokaryotic genomes (Fournier et al., 2006), has recently been introduced as a phylogenetic marker for the characterization of Rickettsiella-like bacteria (Mediannikov et al., 2010; Kleespies et al., 2011). In the study presented here, a partial sequence of the 23S rRNA-encoding gene, an MLST marker set consisting

of six protein-encoding genes selected on the basis of previous data-mining of the R. grylli genome, and the ftsY gene together with the virtually complete 16S rRNA-encoding sequence as a reference were compared for their phylogenetic potential with respect to the generic and infra-generic classification of Rickettsiella bacteria. For this purpose, the orthologous sequences from the R. popilliae-synonymized pathotypes ‘R. melolonthae’ and ‘R. tipulae’ were determined and analyzed together oxyclozanide with the corresponding R. grylli sequences by a methodological approach combining phylogenetic reconstruction with likelihood-based significance testing. Genomic DNA of Rickettsiella strains BBA1806 (pathotype ‘R. melolonthae’) and BBA296 (pathotype ‘R. tipulae’) was extracted by a standard protocol (Walsh et al., 1991) based on the Chelex 100 resin (Bio-Rad) from, respectively, infected fat body tissue of diseased Melolontha grubs collected in the Lorsch area, Germany, and L3–L4 larvae of the crane fly, T. paludosa, collected near Burscheid, Germany.

The highest prevalence rates (3.8%) were mainly in the lake and marshland

regions. It is estimated that there are 726,112 human cases and the number of people at risk in endemic areas was 13,937,235.[10] China is a nonendemic area for S haematobium infection. However, this website with the increase of the workers and tourists to endemic countries, schistosomiasis haematobium has made its entry as an imported disease.[11] Even a single exposure (eg, from swimming, bathing, paddling, or rafting) can cause infection.[12] At present, thousands of persons only from Henan Province are employed in building, water supply, and irrigation projects in Africa. Additionally, the number CDK inhibitors in clinical trials of travelers going to Africa has increased along with the increase in income and development of tourism. It is estimated that some of the people returning from Africa might be infected with S haematobium

and the infected patients probably remain undiagnosed, because schistosomiasis haematobium is rare in China, and the patients and their physicians are unfamiliar with its clinical manifestations and unaware of the possibility of schistosomiasis. Hence, this imported disease may be neglected, undiagnosed, or misdiagnosed. The delay in diagnosis will put these patients at risk of complications, even development of bladder cancer.[13] The reported two patients received inappropriate treatment for 6 months. Additionally, an American patient with loiasis presented with migratory facial edema 21 years after visiting an endemic area in Africa for only 4 days, and was misdiagnosed as “suspicious for lymphoma” for 2 years.[14]

Furthermore, during this period of time, the patients were potentially at risk of contaminating the environment; thus, the parasitic disease imported from Africa might be introduced into China and could spread in the case of presence of appropriate intermediate snail hosts. In the event of this happening, control and elimination of schistosomiasis in China would become more complicated and difficult. The emergence and misdiagnosis of S haematobium infection is the consequence of poor knowledge of African diseases in China. These Adenylyl cyclase two cases indicated the gaps in knowledge and awareness among the general public and authorities of the risk of schistosomiasis from freshwater exposure in Africa. Heath education is the prerequisite of all preventive measures for S haematobium infection. Comprehensive public health education to avoid exposure to contaminated freshwater should be provided to all travelers going to endemic areas. Before workers are sent to Africa, the international labor export companies and public health authorities should adopt a clear policy outlining the risk of schistosomiasis and forbidding exposure to freshwater through swimming, bathing, washing, paddling, and so on.

For each ‘yes’ response, patients were asked if the test result had been communicated (response options: ‘yes’, ‘no’ and ‘I do not remember’). If no result was communicated, patients were asked if they believed the test result to be normal (response options: ‘yes’, ‘no’ and ‘I do not know’). Roxadustat They were then informed that, of the blood tests mentioned, only clotting function is performed regularly prior to orthopaedic surgery. In the second section of the questionnaire, patients were asked if they would be agreeable, in principle, to routine preoperative

testing for diabetes, HIV and cholesterol (response options: ‘I would agree’ or ‘I would disagree’). Using jmp 8.0.1 software (SAS Institue Inc., Cary, NC, USA), we employed a χ2 test or Fisher’s exact test to compare categorical variables in contingency tables and Student’s t-test to analyse continuous data. We expressed data as mean ± standard deviation (SD) or as a percentage. A total of 1330 patients were eligible for inclusion in the study, of whom 991 (75%) completed the questionnaire (Fig. 1). Of these, 50% were male and the mean age was 49 ± 15 years. Age categories were represented as follows: 16–29 years, 16%; 30–39 years, 11%; 40–49 years, 17%; 50–59 years, 25%; 60–70 years, 31%. The most common surgical procedures were foot surgery (28%), arthroplasty (21%), shoulder surgery (18%) and anterior selleck cruciate ligament reconstruction (15%). None of the study patients Cyclin-dependent kinase 3 had been tested for HIV

as part of their preoperative work-up. Three hundred and seventy-five of 991 patients (38%) believed that they had been tested for HIV preoperatively. Of this group, 70 patients (7%) were informed of blood test results prior to the operation. Of

the remaining 305 patients in this group who received no results, 293 (96%) interpreted the lack of result communication as a negative HIV test. Younger age was associated with a higher rate of belief that an HIV test had been performed (mean age 46 years vs. 50 years for those who did not believe that a test had been performed; P < 0.0001) (Table 1). Older age was associated with a higher rate of belief that tests had been performed for diabetes (mean age 51 years vs. 46 years for those who did not believe that a test had been performed) and high cholesterol (mean age 53 years vs. 43 years, respectively) (P < 0.0001 in both cases) (Table 1). Questionnaire responses did not differ significantly between male and female patients. Younger patients were more likely to state that they would accept routine HIV testing prior to future surgery (mean age 47 years for those who would agree vs. 56 years for those who would not; P < 0.0001) (Table 1). More men than women were in favour of routine preoperative HIV testing (85% of men vs. 78% of women; P < 0.009) (Table 1), with the highest proportion among 16–29-year-old men (98%; data not shown). This study demonstrates an incomplete patient understanding of preoperative blood tests.

One defense mechanism used by plant cells is the release of reactive oxygen species (ROS), produced in part by a NOX (NADPH oxidase) complex whose catalytic subunit MK-8669 price shares sequence homology with mammalian NOX enzymes. The plant’s oxidative burst is thought to inhibit the progress of the invader. Furthermore, ROS provide a signal to promote programmed death of neighboring cells, a hallmark of the hypersensitive response (HR). The complete picture is more complex, because ROS also provide signals in addition to those for the HR (Torres & Dangl, 2005). Necrotrophic fungal pathogens that kill host tissue appear to thrive in an oxidant environment,

as shown for the gray mold pathogen Botrytis cinerea (Govrin & Levine, 2000). They produce their own ROS in addition to those originating from the host (see Heller & Tudzynski, 2011). To establish infection, the pathogen must be able to cope

with oxidative stress. Cochliobolus heterostrophus, a necrotrophic foliar pathogen of maize, counteracts oxidative stress by several pathways. The redox-sensitive selleckchem transcription factor ChAP1 is responsible for induction of a set of genes with predicted functions in detoxifying ROS, for example glutathione reductase (GLR1) and thioredoxin (TRX2); loss-of-function mutants in ChAP1 are hypersensitive to oxidants (Lev et al., 2005). Loss of the stress-activated MAPK ChHog1, its upstream two-component system response regulator Ssk1, and the response regulator Skn7 also result in hypersensitivity to oxidants (Izumitsu et al., 2007; Igbaria et al., 2008; Oide et al., 2010). Although Δchap1 and Δskn7 mutants are sensitive to oxidants in culture, no difference

in virulence to maize was reported (Lev et al., 2005; Oide et al., 2010). If the pathways mediated by these two transcription factors act in an additive, rather than sequential manner, a double mutant would be expected to show a more severe phenotype than either single mutant. Two independent stress response pathways would, in this way, act together to provide tolerance to oxidants. To address this question, we generated double mutants in which the coding sequences of both ChAP1 and Skn7 were replaced by selectable antibiotic resistance markers and tested their virulence and tolerance to stresses. Etofibrate Wild-type C4 (MAT1-2 Tox1+), Δchap1 and Δskn7 strains of C. heterostrophus were described previously (Turgeon et al., 1987; Lev et al., 2005; Oide et al., 2010). Standard growth medium was complete xylose medium (CMX, see Turgeon et al., 2010). The Δchap1-Δskn7 mutant was constructed starting with Δskn7. Linear DNA for double-crossover integration was amplified using the split-marker method (Catlett et al., 2003). A linear DNA construct was made, consisting of the neomycin selectable marker flanked on both sides with ChAP1 UTR`s, targeting the integration to the ChAP1 locus in the Δskn7 genome.

One defense mechanism used by plant cells is the release of reactive oxygen species (ROS), produced in part by a NOX (NADPH oxidase) complex whose catalytic subunit selleck kinase inhibitor shares sequence homology with mammalian NOX enzymes. The plant’s oxidative burst is thought to inhibit the progress of the invader. Furthermore, ROS provide a signal to promote programmed death of neighboring cells, a hallmark of the hypersensitive response (HR). The complete picture is more complex, because ROS also provide signals in addition to those for the HR (Torres & Dangl, 2005). Necrotrophic fungal pathogens that kill host tissue appear to thrive in an oxidant environment,

as shown for the gray mold pathogen Botrytis cinerea (Govrin & Levine, 2000). They produce their own ROS in addition to those originating from the host (see Heller & Tudzynski, 2011). To establish infection, the pathogen must be able to cope

with oxidative stress. Cochliobolus heterostrophus, a necrotrophic foliar pathogen of maize, counteracts oxidative stress by several pathways. The redox-sensitive Ku-0059436 purchase transcription factor ChAP1 is responsible for induction of a set of genes with predicted functions in detoxifying ROS, for example glutathione reductase (GLR1) and thioredoxin (TRX2); loss-of-function mutants in ChAP1 are hypersensitive to oxidants (Lev et al., 2005). Loss of the stress-activated MAPK ChHog1, its upstream two-component system response regulator Ssk1, and the response regulator Skn7 also result in hypersensitivity to oxidants (Izumitsu et al., 2007; Igbaria et al., 2008; Oide et al., 2010). Although Δchap1 and Δskn7 mutants are sensitive to oxidants in culture, no difference

in virulence to maize was reported (Lev et al., 2005; Oide et al., 2010). If the pathways mediated by these two transcription factors act in an additive, rather than sequential manner, a double mutant would be expected to show a more severe phenotype than either single mutant. Two independent stress response pathways would, in this way, act together to provide tolerance to oxidants. To address this question, we generated double mutants in which the coding sequences of both ChAP1 and Skn7 were replaced by selectable antibiotic resistance markers and tested their virulence and tolerance to stresses. Ribonucleotide reductase Wild-type C4 (MAT1-2 Tox1+), Δchap1 and Δskn7 strains of C. heterostrophus were described previously (Turgeon et al., 1987; Lev et al., 2005; Oide et al., 2010). Standard growth medium was complete xylose medium (CMX, see Turgeon et al., 2010). The Δchap1-Δskn7 mutant was constructed starting with Δskn7. Linear DNA for double-crossover integration was amplified using the split-marker method (Catlett et al., 2003). A linear DNA construct was made, consisting of the neomycin selectable marker flanked on both sides with ChAP1 UTR`s, targeting the integration to the ChAP1 locus in the Δskn7 genome.