, 2009; Semrau et al, 2010) The latter issue may play a role in

, 2009; Semrau et al., 2010). The latter issue may play a role in the ability of some methanotrophs to utilize multicarbon compounds, with alphaproteobacterial and verrucomicrobial methanotrophs utilizing the serine pathway for carbon assimilation, while Gammaproteobacteria methanotrophs utilize the ribulose monophosphate (RuMP) Anti-cancer Compound Library datasheet pathway (as discussed in more detail below). As comprehensively reported in several recent reviews (Trotsenko & Murrell, 2008; Op den Camp et al., 2009; Semrau et al., 2010), methanotrophs were initially characterized over 100 years ago, and subsequent studies in the 1950s

and 1960s indicated that these strains could only utilize methane or methanol for growth (Dworkin & Foster, 1956; Leadbetter & Foster, 1958; Brown et al., 1964; Foster & Davis, 1966). In 1970, however, a first indication that methanotrophs could utilize multicarbon compounds to accentuate growth was reported (Whittenbury et al., 1970). In this classic manuscript

describing the isolation and characterization of methanotrophs from sites around the world, a wide variety of methanotrophs were reported to show enhanced growth on methane when malate, acetate, or succinate was also present in the culture medium. Such findings suggested that facultative methanotrophs may exist, i.e., strains that could utilize multicarbon compounds as well as methane as a sole growth substrate. Carfilzomib Shortly thereafter, the first facultative methanotrophic isolates from freshwater lake sediments and water were reported. These could utilize a wide range of multicarbon compounds as growth substrates, including many organic acids (malate, succinate, fumarate, and acetate) and sugars (glucose, galactose, sucrose, lactose, and ribose) (Patt et al., 1974). One strain, later described as Methylobacterium organophilum (belonging to the Alphaproteobacteria), was further characterized, and had the complete tricarboxylic

acid (TCA) cycle (Patt et al., 1976). This strain, however, lost the ability to oxidize methane when grown repeatedly on glucose, and other workers subsequently did not succeed in growing the strain Grape seed extract on methane (Green & Bousfield, 1983; Urakami et al., 1993). Collectively, these findings suggested that these isolates were not facultative methanotrophs as originally surmised. Other early studies reported the isolation of facultative methanotrophs from a rice paddy in South China, as well as from soils collected from an oil refinery in the Northeastern United States (Patel et al., 1978; Zhao & Hanson, 1984a, b). These strains were found to have the complete TCA cycle and two of them, strains R6 and 761H, were able to grow solely on glucose, but not with other sugars such as fructose, galactose, or sucrose. In addition, a variant of strain 761H, strain 761M, could not grow on glucose as the sole carbon source, but glucose, as well as acetate and malate, were reported to enhance its growth on methane.

To overexpress these proteins, salicylate (SAL) can be used to bl

To overexpress these proteins, salicylate (SAL) can be used to block the activity of MarR (Martin & Rosner, 1995) and paraquat (PQ) can oxidize and hence activate SoxR (Demple, 1996). Alternatively, 2,2′- or 4,4′-dipyridyl (DIP) enhances post-translational activation of Rob (Rosner et al., 2002). As a result of the homology in their DNA binding domains, these proteins activate overlapping regulons leading to two major phenotypes: (1) the superoxide resistance phenotype, which depends upon increasing the expression of the sodA, fpr, acnA, zwf, and fumC genes,

among others; and (2) the multiple antibiotic or multidrug resistance (MDR) phenotype, which mostly depends on activation of the acrAB, tolC, and micF genes (Pomposiello et al., 2001; Martin & Rosner, 2002). However, these activators CP-868596 nmr differ in the extents to which they activate particular promoters, for example, SoxS activates fpr to a www.selleckchem.com/products/PD-0325901.html much greater extent than MarA does. According to these differences, overexpression of SoxS leads to greater superoxide resistance than overexpression of MarA. The primary basis of these effects is because of small differences in the binding affinities of the proteins to the DNA, particularly to the binding sequences termed

soxbox, when SoxS is the primary activator, or marbox, when all three activators can bind and activate the downstream genes (Fawcett & Wolf, 1995; Martin et al., 2000; Martin & Rosner, 2011). Mutations within marR (leading to a lack of repressor function) and soxR (leading to a constitutively active state) have been found to overexpress the corresponding activators, MarA and SoxS, and hence show an MDR phenotype in addition to organic solvent tolerance associated with the overexpression of the efflux pump AcrAB/TolC (Oethinger et al., 1998; Kern et al., 2000; Koutsolioutsou et al., 2005). In a previous study of our group (Fabrega et al., 2010), the science differences in gene expression between an MDR

E. coli selected in vitro and its susceptible parental clinical isolate were analyzed. Several genes were found to be up-regulated in the resistant mutant, for example, soxS, marA, acrAB, and ompN, and a mutation within soxR, leading to a truncated form of the protein and thus to a constitutively active state, was detected as the most likely explanation for the MDR phenotype. This work has focused on the study of the increased expression of the ompN gene and its possible link with the resistance phenotype. OmpN, like OmpX and OmpW, is one of the minor porins present in E. coli that are poorly expressed and it is closely related to other quiescent porins such as the OmpS1 of Salmonella Typhi and OmpK36 of Klebsiella pneumonia. Moreover, it displays functional properties (single-channel conductance) that closely resemble those of the OmpC porin (Prilipov et al., 1998). However, the physiological role of OmpN is yet to be determined. The bacterial strains and plasmids used in this study are listed in Table 1.

, 2006) Thus, we defined an extended consensus sequence (CG-N-TA

, 2006). Thus, we defined an extended consensus sequence (CG-N-TAT-N2-G-N6-CTA-N-ATA-N-CG) based on the three strongly repressed Mo-boxes upstream of the morA, mopA, and anfA genes (Fig. 1a; Consensus R). The major difference between MopA/MopB-repressed

Mo-boxes and the MopA-activated mop-Mo-box seems to lie in the right half-site. Therefore, we investigated whether this region of the mop-Mo-box either selectively facilitates binding of MopA and/or discriminates against binding GSK1120212 manufacturer of MopB. Several rationally designed single-base substitutions were introduced to convert the anfA-Mo-box into the mop-Mo-box and vice versa (Materials and methods). Specifically, mutations T3A, A7G, T17C, A18T, A23T, and C24T converted the anfA-Mo-box toward the mop-Mo-box (Fig. 1b), while mutations A3T, T16C, C17T, T18A, C19T, T23A, and T24C made the mop-Mo-box more similar to the anfA-Mo-box (Fig. 1c). Mutations A18G, T21C, and C24A probed for the principal importance of highly conserved nucleotides, which were exchanged for nucleotides not occurring in any of the Mo-boxes. To prove that the mop-Mo-box was essential for MopA-dependent mop

gene activation, the triple mutation T4A-A5T-G7C was constructed to destroy the conserved left half-site of the mop-Mo-box (Fig. 1c). In addition to these single-base substitutions, the anfA- and mop-Mo-boxes were GDC-0941 cell line exchanged against each other (anfAmop and mopanfA). In anfAmop, the entire 25-bp anfA-Mo-box was replaced Carnitine palmitoyltransferase II by the mop-Mo-box (Fig. 1b). In contrast, in mopanfA, only the first 22 nucleotides were replaced, because nucleotides 23–25 of the mop-Mo-box overlap with the −35 region of the mop promoter and are thus essential for mop gene expression (Fig. 1c). The effects of Mo-box mutations on anfA transcription were examined by lacZ reporter fusions. For this purpose, wild-type and mutant anfA promoter fragments were cloned into the low-copy broad-host-range vector pML5, thus creating transcriptional

fusions to the promoterless lacZ reporter gene (Fig. 1b; Table 1; Materials and methods). These reporter plasmids were transferred into R. capsulatus wild-type and mutant strains defective for mopA, mopB, or both. The resulting reporter strains were grown in minimal medium under Mo-limiting and Mo-replete conditions before determination of β-galactosidase activities (Fig. 2). In addition to these in vivo studies, the in vitro effects of selected anfA-Mo-box mutations on binding by MopA and MopB were analyzed by DNA mobility shift assays (Fig. 3). For this purpose, 209-bp anfA promoter fragments (PanfA; Fig. 1b) were PCR amplified and used for gel-shift assays with increasing amounts of the regulators (Materials and methods). The effects of Mo-box mutations on anfA gene expression and regulator binding may be summarized as follows: (1) In the R.

1 There had been an increasing number of cases involving bird-to-

1 There had been an increasing number of cases involving bird-to-human transmission of H5N1,

with resultant severe and fatal human infections,2 heightening concerns that potential reassortment of influenza virus genes could give rise to a human pandemic influenza A virus. In response to this, Australian hostelers indicated moderate concern about acquiring avian influenza,3 which was higher than the level of concern regarding terrorism while traveling abroad, but lower than the level of general concern for personal safety.4 In 2009, both the global financial crisis http://www.selleckchem.com/products/ve-821.html (GFC) and Pandemic (H1N1) 2009 impacted on travel, with global travel decreasing 4% to 880 million international arrivals.5 The GFC and Pandemic (H1N1) 2009 may well have had some impact on tourism in Australia. Seasonally adjusted estimates demonstrated that there were monthly decreases in short-term visitor arrivals of 0.2% for April, 1.7% for May, 5.1% for June, 1.2% for July, and 3.3% for August during the height of Pandemic (H1N1) 2009.6 Seasonally adjusted estimates of short-term resident departures from Australia appeared to be less affected with a 10% increase

for April, virtually no change for May, a 0.4% decrease for June, and a 9.7% increase for July 2009.6 Information on trends on short-term resident departures were suspended thereafter.6 During the evolving Pandemic (H1N1) 2009, the Exoribonuclease Australian Government introduced a number of measures that were directed at both Staurosporine cost in-coming and out-going travelers.7 In-coming travelers were subject to increased screening for influenza. Australian travel advisories briefed outgoing travelers on Pandemic (H1N1) 2009 precautions before, during, and after travel. They also detailed what travelers may be subjected to if they were suspected of having Pandemic (H1N1)

abroad and to consider postponing travel if they had influenza-like symptoms.8 Little is known about the extent to which Pandemic (H1N1) 2009 created concern among Australian travelers and how this may have impacted on their travel plans, particularly if they had influenza-like symptoms themselves. The objective of this study was to examine Australian’s level of concern regarding travel during the height of Pandemic (H1N1) 2009 and how this impacted on their travel. Data for this study were collected as part of the Queensland Social Survey (QSS) 2009. QSS is an annual state-wide survey conducted by the Population Research Laboratory (PRL) in Central Queensland (CQ) University’s Institute for Health and Social Science Research. Through a cost-sharing arrangement, QSS enables researchers and policy-makers to incorporate questions into the survey.

The study was approved by the University of East Anglia Ethics Co

The study was approved by the University of East Anglia Ethics Committee. An introductory e-mail was sent out to 10,000 e-mail addresses held by the Centre for Pharmacy Postgraduate Education

containing a link to an online survey with a follow up e-mail after two weeks. It was estimated that 1/3rd of e-mail addresses may be no longer active and that only 50 % of the remaining e-mail addresses were for practicing community pharmacists. Participants were asked to enter how many consultations (one to one discussions in the consultation room) they had held with patients in their last standard working week. STATA® 12 SE was ERK inhibitor clinical trial used to conduct a backward stepwise elimination linear regression model for the number of consultations as the dependent variable. A total of 700 responses (42% of predicted potential LGK 974 respondents) with 595 responses eligible for inclusion.

Descriptive results have been reported previously2. The median (quartiles) for the number consultations performed in a standard week was 5 (3, 10), these include Medicine Use Reviews, New Medicine Service and additional enhanced services such as emergency contraception. The statistically significant predictors of number of consultations in the final model were: working in a multiple pharmacy, having received consultation skills training during preregistration, male gender,

requesting further consultation skills training, and greater confidence in consultation skills. Confidence in consultations skills had the highest positive relationship with number of consultations. Participants had to rate their how confident they were in their consultation skills on a scale where 1 was not confident and 5 was fully confident. A value of 3 on the confidence scale was modelled Methane monooxygenase as having an increase of 34% in the number of consultations compared to the reference group of confidence 1 or 2 (p = 0.025); a value of 4 an increase of 56% (p < 0.001) and a confidence rating of 5 an 81% increase on the reference group (p < 0.001). The model explained 27.2% of the variance in the number of consultations. This exploratory analysis suggests that the more confident a participant is in their consultation skills, the more consultations they conduct. Previous research suggests that training is important in increasing confidence3. While there are many changes in pharmacy education to include consultation skills training during undergraduate and pre-registration year, there are still a large number of registered pharmacists for whom further training in consultation skills could help increase the delivery of more patient facing services.

melbae and C columbae samples, respectively We thank

th

melbae and C. columbae samples, respectively. We thank

the Slovak Academy of Science and IAEA for tsetse pupae. We acknowledge the funding support of NASA NNX07AL53A, NIH R03AI081701 and NSF-REU DBI-0849917. Table S1. Primers, annealing temperatures (Ta), and resulting amplicon sizes. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials PD-0332991 nmr supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“Genetic transformation is an indispensable tool for basic fungal research, as well as a useful technique for directed improvement of industrial strains. Here we describe a simple and reproducible transformation system for the filamentous fungus Hypocrea jecorina. The system is based on hxk1 (encoding hexokinase) as selectable marker, a hexokinase-negative strain and d-mannitol, which is used as selective carbon source and osmotic stabilizer. Following transformation with the hxk1 gene, the obtained transformants were able to grow on d-mannitol as sole carbon source. Transformation efficiency achieved using d-mannitol as carbon source and osmotic stabilizer was roughly five

times higher than that using d-sorbitol. The utility of this system was further demonstrated by transformation of H. jecorina with the egfp (encoding the enhanced green fluorescent protein) gene. Fluorescence microscopy revealed EGFP fluorescence Ergoloid in positive transformants. Our results demonstrated the feasibility of exploiting a carbon source metabolic selleck chemicals llc pathway for the development of promising fungal transformation systems, which provides a new molecular toolbox for genetic modifications of the cell factory H. jecorina. Hypocrea jecorina (anamorph Trichoderma reesei) is one of the workhorse organisms for production of a wide spectrum of polysaccharide-hydrolyzing enzymes, including

cellulases and xylanases, which are applied today in the food, pharmaceutical, textile and pulp industries. Hypocrea jecorina is also recognized as a model cellulolytic microorganism and research efforts today are focused on understanding and improving cellulase efficiency and productivity (Hartl et al., 2007; Seiboth et al., 2007; Fekete et al., 2008; Martinez et al., 2008; Stricker et al., 2008). Moreover, due to its enormous secretion potential and its generally regarded as safe status, H. jecorina is considered an attractive cell factory for large-scale production of homologous and heterologous proteins (Nyyssonen et al., 1993; Nevalainen et al., 2005). The genetic transformation of filamentous fungi is a crucial prerequisite for manipulations at the molecular level. Techniques suitable for H. jecorina transformation, such as protoplast transformation (Gruber et al., 1990), biolistic transformation (Te’o et al., 2002) and Agrobacterium tumefaciens-mediated transformation (Zhong et al.

Most of these requests were ordered from the hospital’s emergency

Most of these requests were ordered from the hospital’s emergency department for suspected insufficient serum concentrations. Antiepileptic drug monotherapy is still the most frequently employed therapeutic strategy in adult patients with epilepsy in keeping

with the standard therapeutic guidelines. Sodium valproate is commonly used for different types of seizures reflecting its wide spectrum of anticonvulsant potential. Newer AED utilizations are becoming increasingly popular in our subjects particularly as add-on with other standard AEDs. “
“Objectives  To determine statin usage pattern and evaluate whether new generation statins are actually needed by the patients receiving them. Methods  Cisplatin price This retrospective cohort included patients receiving first-time statins at a tertiary care hospital in Thailand. Using electronic medical records from 2005, its indication was determined based on history of coronary heart disease (CHD) and CHD-risk equivalents. The lipid profiles tested within 30 days prior to the first date of statins prescription were analysed. Each patient was assessed as

selleck kinase inhibitor to whether statin was needed based on low-density lipoprotein cholesterol (LDL-C) reduction capacity and lipid goals. Results  A total of 2479 first-time statin users was included. Ninety percent of the users received simvastatin, while 8% and 2% received atorvastatin and pravastatin respectively. More than half (58.0%) used statins for primary prevention, although all usage of atorvastatin was considered not needed. Considering the use of statin for secondary prevention to achieve the LDL-C goal of <130 mg/dl (3.37 mmol/l), more than 80% of atorvastatin users could be switched to simvastatin. Only 8% of simvastatin Megestrol Acetate usage would not be able to achieve this target. When

the LDL-C goal was <70 mg/dl (1.81 mmol/l), 40.2% simvastatin users was considered appropriate, while 58.6% needed atorvastatin to be prescribed. Conclusion  A substantial proportion of patients did not need statins therapy, particularly for primary prevention. In addition, atorvastatin use is mostly not needed except in patients requiring statins for secondary prevention to achieve the LDL-C goal of <70 mg/dl (1.81 mmol/l). The findings should prompt hospital policy makers to develop measures to ensure the proper use of statins in their clinical settings. "
“Objective  Most epilepsies are managed with anti-epileptic drugs (AEDs), but medication non-adherence has been frequently reported. Satisfying patient information needs has demonstrated improved adherence. Multi-professional working has been encouraged to provide cost-effective health services by using the most appropriate healthcare professional. Research has demonstrated that pharmacist-led consultations are acceptable to patients with other medical conditions and therefore may be appropriate for patients with epilepsy.

cerevisiae and Aspergillus fumigatus) revealed the presence of tw

cerevisiae and Aspergillus fumigatus) revealed the presence of two distinct regions. The one

located at the 5′- region showed high homology with the Spe genes, whereas the one present at the 3′-region was homologous to the Sdh genes; both were linked through a region of approximately 60 nucleotides without Selleck Selumetinib homology (not shown). As expected, the alignment of amino acid sequences encoded by these genes showed the same pattern of homology, demonstrating the high preservation of the gene in the Basidiomycota (not shown). With these data we designed degenerate primers to be used for PCR amplification of the chimeric genes. The forward primer was selected at the 3′-end of the region with homology to Spe, and the reverse primer was designed from the homologous region

at the 5′-end of the Sdh, in such a way that the amplification fragment covered the nonhomologous region that separates both coding regions (see Fig. 1a). Using the PCR conditions described above and selleck chemical the designed degenerate primers, it was possible to amplify DNA fragments of the predicted size from genomic DNA of all the Basidiomycota species tested (see Materials and methods), whose genomes have been sequenced or not, that represented the three subphyla from Basidiomycota. The size of the fragments (around 1300 bp) coincided with the expected values. On the other hand, and as expected, no such amplification occurred when DNA from Ascomycota or Zygomycota species was used as template (Fig. 1b). The PCR products corresponding to the Basidiomycota species analyzed in this work were sequenced. Alignment of the encoded sequences revealed their high conservation (Fig. 2). Additionally, the encoded sequences

of the amplified fragments from Basidiomycota species whose genomes had been previously sequenced were compared with those existing in their corresponding data banks. The results obtained confirmed the fidelity of the PCR amplification Enzalutamide clinical trial (Table 1). The differences observed can be explained by the fact that different isolates were used in these studies. The sequences of the fragments were deposited in GenBank, with the following accession numbers: Ustilago cynodontis, FN646089; Tilletia foetida, FN646090; Bjerkandera adusta, FN646091; Rhizoctonia solani, FN822770; Schizophyllum commune, FN822771; Ustilago hordei, FN822772; Ustilago maydis, FN822773; Coprinus cinerea, FN822774; Pleurotus ostreatus, FN822775; Ganoderma lucidum, FN822776; Agaricus bisporus, FN827330; and Ganoderma sp., FN827329. The sequences of the regions corresponding to the fragments amplified by PCR from the Spe-Sdh genes obtained in this study, and those reported in the databases, were used for the construction of a phylogenetic tree. The results obtained showed the phylogenetic relationship (Fig.

The spectral width in the carbon dimension was 170 ppm and 180

The spectral width in the carbon dimension was 170 p.p.m. and 180 p.p.m., respectively. All spectra were processed and analyzed using Bruker’s topspin

(v3.0) software. Usually, zero-filling was applied to double the number of real points in each dimension. Chemical shifts were referenced to the HDO resonance at 4.7 p.p.m. Chemical shift assignments for 13C were determined indirectly from HSQC and HMBC spectra. Pseudomonas sp. strain Chol1 was subjected to random transposon mutagenesis Y-27632 by insertion of the transposon mini-Tn5 Km1 and screened for transposon mutants showing altered growth with cholate as described previously (Birkenmaier et al., 2007). One mutant, strain G12, was analyzed further. Strain G12 could not grow with cholate as the sole substrate, but it could grow with succinate in the presence of cholate. HPLC analysis of supernatants from these cultures revealed that strain G12 did not transform cholate at all. We then checked this website whether strain G12 could grow with intermediates of cholate degradation. With supernatants containing DHADD (VIII), strain G12 could grow after a long lag phase. Notably, cells of strain G12 induced for growth with DHADD were also induced for cholate transformation during growth with succinate in

the presence of cholate. HPLC analysis revealed that cholate was transformed into several compounds with an absorption maximum at 244 nm, which is indicative of steroids with a 3-keto-1,4-diene structure of the A-ring (Philipp et al., 2006). In the next step, we identified the gene in strain G12, in which the mini-Tn5 Km1 had been inserted. The transposon Cyclooxygenase (COX) was inserted into an

ORF of 1212 bp at bp 333. The predicted protein had 403 amino acids and showed high identity to nonspecific lipid transfer proteins from various bacteria. Among these were two bacteria, for which growth with cholate had been demonstrated, namely Pseudoalteromonas haloplanktis strain TAC125 (Birkenmaier et al., 2007) and Comamonas testosteroni strain KF-1 (Rösch et al., 2008). The nonspecific lipid transfer proteins from strains TAC125 and KF-1 showed 80% and 68% identity, respectively, to the gene product from strain Chol1 (Fig. 2). This gene was named skt (for steroid β-ketothiolase) for reasons that will be described below. To investigate the function of skt for cholate degradation further, we decided to construct a defined mutant of this gene by subjecting strain Chol1 to insertional mutagenesis with the suicide vector pKnockoutG. The resulting strain Chol1-KO[skt] could not grow with cholate; growth with cholate was restored when an intact copy of skt was provided in trans on the vector pBBR1MCS-5 (Fig. 3a). This complementation clearly showed that the phenotype of this mutant was caused by the inactivation of skt. Strain Chol1-KO[skt] could grow with succinate in the presence of cholate (Fig. 3b).

The spectral width in the carbon dimension was 170 ppm and 180

The spectral width in the carbon dimension was 170 p.p.m. and 180 p.p.m., respectively. All spectra were processed and analyzed using Bruker’s topspin

(v3.0) software. Usually, zero-filling was applied to double the number of real points in each dimension. Chemical shifts were referenced to the HDO resonance at 4.7 p.p.m. Chemical shift assignments for 13C were determined indirectly from HSQC and HMBC spectra. Pseudomonas sp. strain Chol1 was subjected to random transposon mutagenesis p38 MAPK inhibitors clinical trials by insertion of the transposon mini-Tn5 Km1 and screened for transposon mutants showing altered growth with cholate as described previously (Birkenmaier et al., 2007). One mutant, strain G12, was analyzed further. Strain G12 could not grow with cholate as the sole substrate, but it could grow with succinate in the presence of cholate. HPLC analysis of supernatants from these cultures revealed that strain G12 did not transform cholate at all. We then checked selleck products whether strain G12 could grow with intermediates of cholate degradation. With supernatants containing DHADD (VIII), strain G12 could grow after a long lag phase. Notably, cells of strain G12 induced for growth with DHADD were also induced for cholate transformation during growth with succinate in

the presence of cholate. HPLC analysis revealed that cholate was transformed into several compounds with an absorption maximum at 244 nm, which is indicative of steroids with a 3-keto-1,4-diene structure of the A-ring (Philipp et al., 2006). In the next step, we identified the gene in strain G12, in which the mini-Tn5 Km1 had been inserted. The transposon Paclitaxel order was inserted into an

ORF of 1212 bp at bp 333. The predicted protein had 403 amino acids and showed high identity to nonspecific lipid transfer proteins from various bacteria. Among these were two bacteria, for which growth with cholate had been demonstrated, namely Pseudoalteromonas haloplanktis strain TAC125 (Birkenmaier et al., 2007) and Comamonas testosteroni strain KF-1 (Rösch et al., 2008). The nonspecific lipid transfer proteins from strains TAC125 and KF-1 showed 80% and 68% identity, respectively, to the gene product from strain Chol1 (Fig. 2). This gene was named skt (for steroid β-ketothiolase) for reasons that will be described below. To investigate the function of skt for cholate degradation further, we decided to construct a defined mutant of this gene by subjecting strain Chol1 to insertional mutagenesis with the suicide vector pKnockoutG. The resulting strain Chol1-KO[skt] could not grow with cholate; growth with cholate was restored when an intact copy of skt was provided in trans on the vector pBBR1MCS-5 (Fig. 3a). This complementation clearly showed that the phenotype of this mutant was caused by the inactivation of skt. Strain Chol1-KO[skt] could grow with succinate in the presence of cholate (Fig. 3b).