Amplification products were visualized following electrophoresis in agarose gels. Inc-group-specific PCR fragments were purified with the Wizard SV and PCR Clean-up System (Promega) and sequenced at the Department of Genetics, CINVESTAV, Irapuato, México. For colony assays, bacteria were inoculated on LB agar plates, and after overnight

growth, colonies were lysed with 10% sodium dodecyl sulfate, debris were removed, and DNA was alkali-denatured. DNA was then transferred to nitrocellulose membranes (Hybond-N+; Amersham) and fixed by UV-light exposure. DNA for Southern blot assays was isolated by the alkaline lysis procedure described above, separated by agarose gel electrophoresis, and transferred to nitrocellulose membranes by capillarity. The coding Afatinib clinical trial KU-57788 mw region of the chrA gene was utilized as a probe for chromate-resistance (CrR) genes; a 1.25-kb fragment was PCR-amplified from the pEPL1 plasmid (7.7 kb), which contains a BamHI-PstI 3.8-kb fragment bearing the pUM505 chrA gene cloned in the pUCP20 vector

(Ramírez-Díaz et al., 2011). PCR was conducted employing forward oligonucleotide 1D (5′-GAGCGTTGCGAATGAAGAGTCG-3′) and reverse oligonucleotide 1R (5′-GGAAGCATGAAACCGAGTCCC-3′). As a probe for mercury-resistance (HgR) genes, a 1.18-kb fragment comprising most of the merA gene was amplified from pUM505 using forward oligonucleotide MerA-2D (5′-CATATCGCCATCATTGGCAGC-3′) and reverse oligonucleotide MerA-2R (5′-CCTCGATGACCAGCTTGATGAAG-3′). PCRs were carried out with Accuprime Super Mix II (Invitrogen) with an initial denaturation for 5 min at 95 °C succeeded by 30 cycles as follows: a denaturation step at 95 °C for 1 min; an annealing step at 60 °C for 45 s, and an elongation step at 72 °C for 1 min, with a final extension at 72 °C for 10 min. PCR products were purified as described previously and labeled with the Gene Images AlkPhos Direct Labeling kit (Amersham). Conditions for labeling, hybridization, and signal detection were as recommended by the provider at high stringency (63 °C). To investigate the presence of CrR genes in nosocomial bacteria, MG-132 a collection of 109 antibiotic-resistant

enterobacterial isolates from Mexican hospitals was utilized. This bacterial group was previously characterized by its resistance to multiple antibiotics, including beta-lactams, third-generation cephalosporins, and carbapenems (Miranda et al., 2004; Silva-Sánchez et al., 2011). MIC distribution curves demonstrated different levels of chromate susceptibility for each bacterial species (Fig. 1). A clear bimodal distribution of E. coli and K. pneumoniae allowed us to separate CrS from CrR isolates (Fig. 1a and c); for E. cloacae, where a single susceptibility group was found, an arbitrary separation was employed (Fig. 1b). Thus, for E. coli and E. cloacae, species exhibiting a low level of CrR isolates separated from the CrS predominant group, a cutoff value of ≥ 1.

The median (range) gestation at delivery was 40 (27–42) weeks and the median (range) birthweight was 3.1 (1.2–4.5) kg. There were no HIV-positive infants. Antepartum and postpartum LPV and RTV pharmacokinetic data from 46 patients are summarized in Table 2. Geometric mean (95% CI) total LPV concentrations were comparable during the first, second [3525 (2823–4227) ng/mL] and third trimesters [3346 (2813–3880) ng/mL; P=0.910], but were ∼35% lower relative to LPV

concentrations selleck compound library observed during the postpartum period [5136 (3693–6579) ng/mL; P=0.006; all comparisons]. Equally, RTV Ctrough values were significantly reduced antepartum vs. postpartum (P=0.017; all comparisons). Inter-subject variation in LPV Ctrough was moderately high both antepartum (24–45%) and postpartum (44%). The time of post-dose sampling was consistent across the trimesters of pregnancy and postpartum, at approximately 13 h (P=0.924). Overall, six of 46 patients (13%)

had LPV concentrations below the proposed MEC (<1000 ng/mL) in pregnancy; one patient (8%) in the second trimester and five patients (12%) in the third trimester (LPV=<73–831 ng/mL; 14.5–26 h post-dose); all were receiving standard dosing of the LPV/r tablet at baseline. All 12 patients at postpartum had plasma concentrations in excess of the LPV MEC. A single patient below target in the second trimester (LPV Ctrough=790ng/mL; 29 weeks; 15 h post-dose) was dose-adjusted to three tablets (600/150 mg) twice daily at 32 weeks which achieved above-target SCH772984 solubility dmso concentrations (LPV=4575 ng/mL; 34 weeks; 12.7 h). She was later reduced back to two tablets twice daily post-delivery and remained therapeutic at 6 weeks postpartum. Of the five patients below target in the third trimester, one patient had an LPV Ctrough of 831 ng/mL (32 weeks; 17 h post-dose); no changes were made to the LPV/r dose, and she underwent no further TDM sampling having

O-methylated flavonoid delivered elsewhere. Another had an LPV Ctrough of 647 ng/mL (26 weeks; 15.7 h post-dose). No dose adjustments were made and an additional TDM was performed at 32 weeks, in which she remained below target (641 ng/mL). Both patients discontinued ART post-delivery. The remaining three patients had LPV concentrations below our predefined cut-off for adherence (<384 ng/mL) and were therefore excluded from subsequent statistical analyses. These subjects were suspected by the study personnel as being nonadherent to treatment with one patient admitting to having missed doses one day. In two instances the time of pharmacokinetic sampling was greater than 20 h and this may also have contributed to the low LPV concentrations observed. Of the six patients who were below the MEC during pregnancy, five had undetectable pVL (<50 copies/mL) at the time of TDM sampling. The remaining subject had a pVL of 209 copies/ml in the third trimester. LPV unbound trough concentrations (Table 2) were lower in the first, second and third trimesters relative to postpartum (P=0.

Number of patients with an undetectable VL on current regimen and documented previous NRTI resistance who have switched a PI/r to either an NNRTI or INI as the third agent. Number of patients on PI/r monotherapy as ART maintenance strategy in virologically suppressed patients and record of rationale. Record in patient’s notes of resistance result

at ART initiation (if available) and at first VL >400 copies/mL and/or before switch. Record in patient’s notes of adherence assessment and tolerability/toxicity to ART, in patients experiencing SGI-1776 mouse virological failure or repeated viral blips. Number of patients experiencing virological failure on current ART regimen. Proportion of patients experiencing virological failure switched to a new suppressive ALK phosphorylation regimen within 6 months. Proportion of patients on ART with previous documented HIV drug resistance with VL <50 copies/mL. Record of patients with three-class virological failure with or without three-class resistance referred/discussed in multidisciplinary team with expert advice. Proportion of patients with TB and CD4 cell count <100 cells/μL started on ART within 2 weeks of starting TB therapy. Proportion of patients with active TB on anti-TB therapy started on ART containing EFV, TDF and FTC. Proportion of patients with a CD4 cell count ≥500 cells/μL and an HBV DNA ≥2000 IU/mL and/or evidence of more than minimal

fibrosis commencing ART inclusive of anti-HBV antivirals. Proportion of patients with a CD4 cell count <500 cells/μL receiving TDF/FTC or TDF/3TC as part of a fully suppressive combination ART regimen. Proportion of patients receiving 3TC or FTC as the sole active drug against HBV in ART. Proportion of patients with a CD4 cell count <500 cells/μL commencing ART. Among patients receiving DAAs for HCV genotype 1 with ART for wild type HIV, the percentage on a recommended regimen,

i.e. RAL with TDF plus FTC with boceprevir; or RAL or boosted ATV with standard dose telaprevir; or EFV with increased dose 1125 mg tds telaprevir. Proportion of patients with an AIDS-defining malignancy on ART. Proportion of patients with a non-AIDS-defining malignancy on ART. Record in patient’s notes of potential pharmacokinetic drug interactions Resveratrol between ARVs and systemic anticancer therapy. Proportion of patients with symptomatic HIV-associated NC disorders on ART. Proportion of patients with HIV-associated NC disorders on ART containing two NRTIs and one of the following: NNRTI, or PI/r or INI. Proportion of patients with HIVAN started on ART within 2 weeks of diagnosis of CKD. Number of patients with CKD stages 3–5 on ARVs that are potentially nephrotoxic and record of rationale. Record in patient’s notes of the calculated dose of renally cleared ARVs in patients with CKD stage 3 or greater.

(C) CQ402 What should we tell the patient when prescribing oral contraceptives (OC)? Answer Provide information based on the ‘Guidelines concerning the use of low-dose oral contraceptives (year 2007 revision)’. 1 Efficacy and safety: OC is the most effective reversible method of contraception available. It is also very safe. (B) CQ403 What should we inform the patient when an intrauterine device (IUD) (including the intrauterine system) is chosen for contraception? Answer Provide information as below.

1 It does not prevent pregnancy without fail. (A) CQ404 How do we manage Turner’s syndrome? Answer 1 For patients diagnosed before puberty, growth hormone may be needed for treatment. Management of patient can be carried out in coordination with a pediatrician/endocrinologist. (A) CQ405 How should we provide care for XY female patients? Answer 1 After definitive diagnosis is made, provide appropriate BAY 80-6946 chemical structure counseling C646 cost for both the patient and her parents. (B) CQ406 How do we provide care for patients with Mayer–Rokitansky–Küster (–Hauser) syndrome? Answer 1 Provide information for the patient regarding her medical condition in

a timely and approachable manner. (A) CQ407 What are the important points when we perform medical examinations on an adolescent? Answer 1 Medical interviews are very important, and can be conducted with or without the accompaniment of a family member. (B) CQ408 What are the important points when treating a female adolescent? Answer 1 For amenorrhea, use cyclic progestins therapy or cyclic estrogen–progestin therapy once every 2–3 months. (C) CQ409 What should we do when we encounter a sexual assault

victim? Answer 1 Victims who have not reported their ordeal to the law enforcement authorities should be reported to the police after obtaining their consent before any medical examination takes place. (A) CQ410 How do we help patients modify their menstrual cycle? Answer 1 To shorten the menstrual cycle, administer combined estrogen–progestin (EP) or norethisterone from the 3rd to 7th day of the menstrual cycle for 10–14 days. (B) CQ411 What are the important points in the diagnosis of Diflunisal climacteric disorder? Answer 1 Suspect climacteric disorder in a woman who has already undergone menopause that comes with a myriad of complaints. (A) CQ412 How should we treat climacteric disorder? Answer 1 Hormone replacement therapy is effective for symptoms caused by autonomous nervous system dysregulation, such as flushing, sweating, insomnia etc. (B) CQ413 How should we provide information regarding the side-effects of hormone replacement therapy and the corresponding strategies for treatment? Answer 1 The minor side-effects are: (A) Abnormal vaginal bleeding, mastalgia (breast pain), breast swelling. Breast cancer, ovarian cancer, lung cancer, coronary vascular disease, ischemic cerebral stroke, thromboembolism.

Ninety-three patients

had taken at least one PI in their treatments: 11 of them showed no resistance; 12 displayed resistance to one class of drug (eight to NNRTIs, two to NRTIs and two to PIs); 34 patients showed resistance to two classes of drug (23 to NRTIs+NNRTIs, 10 to NRTIs+PIs and one to NNRTIs+PIs), and 37 showed resistance to three classes of drug. Figure 1 shows the resistance mutations that were observed in the study population. At least one thymidine-associated mutation (TAM), that is a mutation at position 41, 67, 210, 215 or 219 in RT, was seen in 60% of patients, and the lamivudine/emtricitabine resistance mutation M184I/V was observed in 62% of the patients. Multi-nucleoside resistance mutations, Selleck Bioactive Compound Library such as Q151M, were rare and such a mutation was only observed in one patient. The K103N mutation was the most frequently observed (30%) of the NNRTI resistance mutations. A smaller proportion of the study subjects (32%) had at least one major PI resistance mutation; for example, a mutation at position 30, 46, 82, 84, 88 or 90 of PR. The present study describes the prevalence of genotypic resistance to antiretroviral drugs in clinical samples from 138 Honduran patients who were failing ART. It was found that the prevalence of resistance was high

(81%) in our study population. Thus, resistance to at least one drug class was found in 11% of the patients, dual class resistance was found BLZ945 cost in 43% of the patients and triple class resistance was found in 27% of the patients. The proportion of individuals with resistance was higher among children (98%) than among adults (74%). The type of treatment failure (virological, immunological or clinical) was the strongest predictor of resistance, but route of transmission and years on therapy were also independently associated

with the presence of genotypic Glutamate dehydrogenase resistance. Our study revealed that there are considerable problems with resistance to antiretroviral drugs in Honduras. However, it is important to stress that our results do not reflect the prevalence of resistance among all HIV-infected patients in Honduras, because the study subjects were selected on the basis of treatment failure. Nevertheless, it is worrying that dual- and triple-class resistance was very common. Furthermore, we observed that treatment changes were common and associated with a higher prevalence of resistance, as was years on therapy. Our review of the patient records revealed that many of the treatment changes were not driven by laboratory results indicating treatment failure, primarily because access to plasma HIV-1 RNA and CD4 quantification was irregular during the study period. Instead, treatment changes had often been initiated as a consequence of clinical progression or interrupted access to specific antiretroviral drugs.

Ninety-three patients

had taken at least one PI in their treatments: 11 of them showed no resistance; 12 displayed resistance to one class of drug (eight to NNRTIs, two to NRTIs and two to PIs); 34 patients showed resistance to two classes of drug (23 to NRTIs+NNRTIs, 10 to NRTIs+PIs and one to NNRTIs+PIs), and 37 showed resistance to three classes of drug. Figure 1 shows the resistance mutations that were observed in the study population. At least one thymidine-associated mutation (TAM), that is a mutation at position 41, 67, 210, 215 or 219 in RT, was seen in 60% of patients, and the lamivudine/emtricitabine resistance mutation M184I/V was observed in 62% of the patients. Multi-nucleoside resistance mutations, buy PF-562271 such as Q151M, were rare and such a mutation was only observed in one patient. The K103N mutation was the most frequently observed (30%) of the NNRTI resistance mutations. A smaller proportion of the study subjects (32%) had at least one major PI resistance mutation; for example, a mutation at position 30, 46, 82, 84, 88 or 90 of PR. The present study describes the prevalence of genotypic resistance to antiretroviral drugs in clinical samples from 138 Honduran patients who were failing ART. It was found that the prevalence of resistance was high

(81%) in our study population. Thus, resistance to at least one drug class was found in 11% of the patients, dual class resistance was found selleck screening library in 43% of the patients and triple class resistance was found in 27% of the patients. The proportion of individuals with resistance was higher among children (98%) than among adults (74%). The type of treatment failure (virological, immunological or clinical) was the strongest predictor of resistance, but route of transmission and years on therapy were also independently associated

with the presence of genotypic Methocarbamol resistance. Our study revealed that there are considerable problems with resistance to antiretroviral drugs in Honduras. However, it is important to stress that our results do not reflect the prevalence of resistance among all HIV-infected patients in Honduras, because the study subjects were selected on the basis of treatment failure. Nevertheless, it is worrying that dual- and triple-class resistance was very common. Furthermore, we observed that treatment changes were common and associated with a higher prevalence of resistance, as was years on therapy. Our review of the patient records revealed that many of the treatment changes were not driven by laboratory results indicating treatment failure, primarily because access to plasma HIV-1 RNA and CD4 quantification was irregular during the study period. Instead, treatment changes had often been initiated as a consequence of clinical progression or interrupted access to specific antiretroviral drugs.

Moreover, the in vitro functions of MycE and MycF proteins were characterized using the purified MycE and MycF proteins overexpressed in E. coli cells (Li et al., 2009; Fig. 1). The purified MycE and MycF proteins methylated the C2″-OH group of mTOR inhibitor 6-deoxyallose in mycinamicin VI (M-VI) and the C3″-OH group of

javose (i.e. C2″-methylated 6-deoxyallose) in M-III, respectively. Here, we have demonstrated the isolation and characterization of mycE and mycF disruption mutants obtained from M. griseorubida A11725, which would not possess the φC31 attB site on the chromosome, by the disruption cassette FRT-neo-oriT-FRT-attB and the genetic complemented strains, in which plasmids including each OMT gene –mycE or mycF– were inserted into the artificially inserted attB site. The strains used in this study are shown in Table

1. The culture conditions of M. griseorubida and E. coli were according to our previous report (Anzai et al., 2004a). FMM broth containing 7% dextrin, 0.5% glucose, 0.5% yeast extract, 0.5% soybean meal (Ajinomoto, Japan), 0.5% CaCO3, 0.1% K2HPO4, 0.4% MgSO4·7H2O, and 0.0002% CoCl2·6H2O was used for fermentation of M. griseorubida. The vectors used in this study are shown in Table 1. TaKaRa ExTaq® (TaKaRa, Japan) and PfuTurbo® (Stratagene) DNA polymerase used for the DNA fragment were amplified by PCR. Plasmid and genomic DNA amplification, restriction enzyme digestion, RG7420 research buy fragment isolation, cloning, and DNA fragment amplification were performed according to standard procedures. Southern blot analysis was performed according to our previous procedure (Anzai et al., 2004a). Using pIJ776 containing FRT-neo-oriT-FRT as the template, the gene disruption cassette FRT-neo-oriT-FRT-attB was amplified by PfuTurbo® DNA polymerase

with the primers FRTF+attB containing the sequence of the bacteriophage φC31 attB attachment site and FRTR (Table 2). The PCR fragment was cloned into the EcoRV site of pLITMUS38 to generate pMG501. The mycE-disrupted plasmid, pMG502, was constructed using three restriction fragments (3.2 kb BamHI–MluI, 0.7 kb MluI–EcoRI, and 3.8 kb StuI–BamHI) derived from pMR01, and the 1.5-kb EcoRV fragment containing the disruption cassette FRT-neo-oriT-FRT-attB derived from pMG501. The 9.5-kb DNA ifenprodil fragment linking these three restriction fragments and the disruption cassette together was inserted into the BglII and EcoRI sites on pSAN-lac to create pMG502. To generate pMG503 whose neo gene was in the opposite direction from the mycinose biosynthesis gene cluster, the 1.3-kb XbaI fragment including neo and oriT derived from pMG501 was ligated with the 15-kb XbaI fragment derived from pMG502. To construct pMG504 containing myrB, mycG, mycF, mycCI, and mycCII, the 2.4-kb BsiWI–StuI and 3.8-kb StuI–MluI fragments obtained from pMR01 were cloned into pLITMUS28 and pLITMUS38, respectively; then, the 2.4-kb BglII–StuI and 3.

1. Motamedi SM, Posadas-Calleja J, Straus S, et al. (2011) The efficacy of computer-enabled discharge communication interventions: a systematic review. BMJ Qual Saf, 20(5), 403–415. 2. Scottish Intercollegiate Guidelines Network (SIGN). 128 The SIGN discharge document. (2012) Edinburgh: SIGN. Available from www.sign.ac.uk Date accessed 30/07/2012 J. Sowtera, P. Knappc, L. Dyea, F. Astinb, P. Marshalla aUniversity of Leeds, Leeds, West Yorkshire, UK, bUniversity CX-5461 of Salford, Salford, Greater Manchester, UK, cUniversity of York, York, North Yorkshire,

UK This exploratory study assessed the quality of a purposive sample of 39 commercial and non-commercial websites containing information about herbal remedies for menopausal symptoms. Commercial websites were the most prevalent and scored lower for quality than non-commercial sites using the http://www.selleckchem.com/products/Cisplatin.html DISCERN tool. Coverage of information about specific herbal remedies was poor across all websites. There is room for improvement in quality and coverage of website information about herbal remedies for menopausal symptoms. The internet is increasingly used as a source of health information for consumers despite concerns about the quality

of health information on the internet, particularly about herbal remedies. The study aim was to analyse the content of a sample of commercial and non-commercial websites with information about herbal remedies for menopausal symptoms, to determine their quality and the extent to which Fludarabine cell line they met women’s identified information needs. This exploratory study used a purposive sample of websites for analysis. The sample included websites used by women or recommended by service providers, supplemented by websites identified via a series of searches conducted in Google using search terms volunteered by women. Inclusion criteria were that they contained information about herbal remedies for menopausal symptoms and had a key purpose for providing information about treatment. Research ethics approval was not required. The websites were assessed for quality using validated tools for: Information quality (using the DISCERN

tool1) Coverage of information specific to needs identified by a sample of women with menopausal symptoms (e.g. range of treatment choices, clinical effects of products, combining products for optimal effect and real life experiences) Accessibility (assessed by readability scores using the SMOG tool2) Thirty-nine websites were analysed. The majority of websites were for commercial providers. There was a statistically significant difference between commercial and non-commercial (e.g. charities and government) websites, with commercial websites scoring lower than non-commercial for the DISCERN tool (p = 0.014). There was no statistical difference between the types of website provider for the SMOG readability test (p = 0.324) or for the tool assessing coverage of specific information (p = 0.60).

aureus virulence in silkworms. The LD50 values of the hla-disrupted mutant, hlb-disrupted mutant, hla/hlb double-disrupted mutant, psmα-deleted mutant and psmβ-deleted mutant were similar to those of the PD-1 inhibitor parent strain (Table 4). Thus, hla, hlb, psmα and psmβ encoding hemolysins do not contribute to S. aureus virulence in silkworms. In contrast, the LD50 of the agr mutant was 2.5-fold higher than that of the parent strain (Table 4). This confirms previous findings that the agr locus

contributes to S. aureus virulence in silkworms, and suggests that the agr locus functions in silkworms via hla-, hlb-, psmα- and psmβ-independent pathways. Staphylococcus aureus possesses 16 two-component regulatory systems (Cheung et al., 2004). Among them, arlRS and saeRS broadly regulate the expression of virulence genes (Fournier et al., 2001; Liang et al., 2005, 2006). The arlRS-deleted mutant exhibited attenuated virulence in a mouse systemic infection model (Benton et al., 2004). The saeRS-deleted mutant showed attenuated virulence in a mouse pyelonephritis infection model (Liang et al., 2006). We examined whether the arlS Doxorubicin mw and saeS genes of S. aureus contribute to virulence against silkworms. The LD50 values of the arlS- and saeS-disrupted mutants were 2.7- and 1.8-fold higher than

that of the parent strain, respectively (Table 4). This indicates that arlS and saeS contribute to virulence of S. aureus against silkworms. Cell-wall-anchored proteins of S. aureus are reported to contribute to virulence by facilitating bacterial attachment to host tissues or escape from immune systems (Foster Inositol monophosphatase 1 & Hook, 1998). Sortase A is required for anchoring of various proteins to the cell wall (Mazmanian et al., 1999). A gene-disrupted mutant of srtA encoding sortase A had attenuated virulence in mouse infection models (Table 3) (Jonsson et al., 2002, 2003; Weiss et al., 2004). We tested whether the srtA-disrupted mutant showed decreased virulence in silkworms.

The LD50 of the srtA-disrupted mutant was 3.1-fold higher than that of the parent strain (Table 4). This suggests that the anchoring of cell-wall proteins by sortase A is required for S. aureus virulence in silkworms. Mouse pneumonia (Bubeck Wardenburg et al., 2007) Rabbit corneal infection (O’Callaghan et al., 1997) psmα1 psmα2 psmα3 psmα4 PSMα1, PSMα2, PSMα3, PSMα4 Mouse systemic infection (Wang et al., 2007) Mouse skin infection (Wang et al., 2007) psmβ1 psmβ2 PSMβ1, SMβ2 AgrA, AgrB, AgrC, AgrD, RNAIII SA1842 SA1843 SA1844 Mouse pneumonia (Heyer et al., 2002) Rabbit corneal infection (O’Callaghan et al., 1997) Silkworm (Kaito et al., 2005) C. elegans (Sifri et al., 2003) Manduca sexta (Fleming et al., 2006) NA in Drosophila (Needham et al., 2004) SA1246 SA1247 SA1248 SA0660 SA0661 C. elegans (Bae et al.

coli was due to the absence of essential genes

that are not linked to the cloned pqq operon, but are present in the P. ananatis chromosome, and whose products are responsible for enhancement of the PQQ pool in the latter microorganism. To distinguish between these possibilities, additional investigations are necessary. It should especially be mentioned, as well, that the homologous pqq operon from K. pneumoniae earlier cloned into the E. coli could lead to the production of visible amounts of PQQ only being amplified in multicopy-number recombinant plasmids (Meulenberg et al., 1990; Sode et al., 1996). It is possible that new E. coli strains that grow efficiently on glucose using the PQQ-mGDH-mediated pathway could be constructed in further studies. At minimum, these strains have to grow on glucose no worse than in the presence of PQQ added to the minimal cultivation medium. These Alpelisib strains could have some Gefitinib advantages for applied biotechnology. It has been shown that strains with a PTS−/glucose+ phenotype could be useful for biotechnological applications in which large quantities of phosphoenolpyruvate have to be consumed for biosynthesis of the target product (Flores et al., 1996; Hernández-Montalvo et al., 2003). By decoupling glucose transport from phosphoenolpyruvate consumption,

the metabolic availability of this intermediate molecule is significantly increased when compared with a PTS+ strain. The production of other metabolites with phosphoenolpyruvate as a precursor should therefore be enhanced in a PTS−/glucose+ strain. This expectation has been confirmed using strains designed to direct carbon flow to the common aromatic pathway (Báez-Viveros et al., 2004). It goes without saying that the construction of such glucose-oxidizing strains for biotechnology is a complex task. At minimum, in addition to the optimization of P. ananatis pqq operon Ribonucleotide reductase expression

in E. coli, it seems necessary to make the expression of some genes CRP-independent (gcd, gntKU, for example), to perhaps increase the expression level of the E. coli pgl gene (Thomason et al., 2004; Zimenkov et al., 2005) for the efficient conversion of glucono-1,5-lactone into gluconate. At the final stage, it seems necessary to balance the rate of gluconic acid production and its further utilization preventing the acidification of a growth media. We wish to thank Irina L. Tokmakova and Natalia V. Gorshkova for helpful discussion and participation in determining GDH activity and the accumulated extracellular PQQ level. Participation of a postgraduate student (I.G. Andreeva) in this work was supported in part by grant NK127P-4 from the Russian Federation Education Agency. Table S1. Primers used for PCR in this study. Fig. S1. Scheme for in vivo cloning of the Pantoea ananatis pqq operon. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors.