Protein A-Carboxylate beads (0.981 μm diameter) were purchased from Polysciences Inc. (Warrington, PA). The beads were coupled with MAb 3/1 or 26/1 by incubation of 1 mL of cell culture supernatant containing 15 μg IgG3 mL−1 with 108 beads for LY294002 research buy 2 h at 4 °C at 150 r.p.m. on an orbital shaker. After washing three times by centrifugation at 10 000 g for 2 min with RPMI 1640 containing 40 μg 

bovine serum albumin (BSA) mL−1 (Sigma-Aldrich, Munich, Germany), the beads were resuspended in 1 mL of the same medium. Legionella pneumophila was inoculated in YE broth and incubated at 37 °C on an orbital shaker at 300 r.p.m. for 12 and 24 h to obtain cells in the E- and PE-phases, respectively. The cells were pelleted by centrifugation at 18 000 g for 10 min. The culture supernatant was then filtered through a membrane with 0.2-μm pores (VWR, sterile syringe filter) to exclude bacterial cells, but include parts of the OMV that have a diameter of 186±83 nm (Fernandez-Moreira et al., 2006). To determine whether inhibitory activity was mediated only by OMV or also by LPS species <300 kDa, these two fractions were prepared by Vivaspin filtration with an exclusion size of 300 kDa (Vivascience, Sartorius Group, Stonehouse, UK). For this culture, supernatants were centrifuged

at 200 g until the volume of fractions >300 kDa was reduced to 10% v/v. In the following details, we refer to the fraction >300 kDa MG-132 in vivo as OMV and the filtrate as LPS species <300 kDa. Quantification of LPS in the fractions was not carried out. The comparison between LPS fractions of both strains derived from the E- and the PE-phases was still ensured on the basis of bacterial ability to shed LPS in the corresponding liquid cultures. For this,

the volume of the OMV fractions was refilled with YE broth to the same volume as the LPS fractions <300 kDa in order to avoid a concentration step of OMV. Using this step, the Meloxicam concentration of shed LPS components in the broth reflects simultaneously the accumulated LPS during the growth phases depending on the strains. Subsequently, 2 × 106 MAb-coated beads were added to 1 mL of the LPS fractions and incubated at 37 °C on an orbital shaker for 90 min at 150 r.p.m., then washed once with phosphate-buffered saline (PBS) containing 40 μg BSA mL−1 and centrifuged at 10 000 g for 3 min. After removal of the supernatant, the beads were resuspended with 100 μL PBS containing 40 μg BSA mL−1 and used for phagocytosis experiments. To label lysosomes by endocytosis, host cells were incubated at 37 °C for 1 h with fluorescein-dextran with a molecular weight of 10 000 (FDx) as described elsewhere (Fernandez-Moreira et al., 2006). Acanthamoeba castellanii was stained with 4 mg anionic FDx (Invitrogen, Karlsruhe, Germany) per milliliter PYG 712 and monocytic cells with 5 mg anionic lysine fixable FDx (Invitrogen) per milliliter RPMI containing 10% v/v FCS.

Additionally,

bifidobacteria were enumerated on modified Navitoclax mw de Man–Rogosa–Sharpe agar (MRS; Difco) modified with 0.05% cysteine and 100 mg mL−1 mupirocin (Oxoid Ltd, Hampshire, UK). Lacticin 3147 production in the kefir fermentation was also examined using agar well diffusion assays as described previously (Ryan et al., 1996). Briefly, 20 mL of sterile LM17 containing 1.5% (w/v) agar was seeded with 100 μL of the lacticin 3147-sensitive indicator strain, L. lactis ssp. cremoris HP and poured into a sterile Petri dish. Lactococcus lactis ssp. cremoris HP (pMRC01), a lacticin 3147-insensitive derivative of L. lactis HP was also used as an indicator in order to confirm that inhibition of the target strain was solely due to the production of lacticin 3147. Once solidified, wells AZD2281 of uniform diameter were then bored into the medium and 50 μL of the fermented kefir milk was then added to each well. Plates were incubated overnight aerobically at 30 °C and examined for zones of clearing. For 16S compositional

sequencing analysis, genomic DNA from a single kefir fermentation was collected from duplicate samples (∼50 mg) from both the starter grain (interior and exterior surfaces) and kefir milk (2 mL) using the protocols of Garbers et al. (2004) and Lipkin et al. (1993), respectively, and used as a template for PCR amplification of the V4 variable region of the 16S rRNA gene with universal primers [i.e. forward primer F1 (5′-AYTGGGYDTAAAGNG) and R1 (5′-TACCRGGGTHTCTAATCC), R2 (5′-TACCAGAGTATCTAATTC), R3 (5′-CTACDSRGGTMTCTAATC) and R4 (5′-TACNVGGGTATCTAATC)]. Unique molecular identifier (MID) tags were attached between the 454 adaptor sequences and the forward primers. Amplicons generated from two PCR reactions per sample were pooled and cleaned using the AMPure purification system (Beckman Coulter Genomics, Takeley, UK). Sequencing was performed using a 454 Genome Sequencer FLX platform (Roche Diagnostics Ltd) according to 454 protocols. Raw sequencing reads were quality trimmed using the RDP Pyrosequencing Pipeline, applying criteria as outlined previously (Rea et al., 2010). Clustering and statistical analysis of sequence data were performed using the mothur software

package (Schloss & Handelsman, 2008). Trimmed FASTA sequences were then subjected Adenosine triphosphate to blast analysis using a previously published 16S rDNA gene-specific database and default parameters (Altschul et al., 1997; Urich et al., 2008). blast outputs were parsed using megan (Huson et al., 2007). A bit-score of 86, as previously employed for 16S ribosomal sequence data, was used from within megan for filtering the results before tree construction and summarization (Urich et al., 2008). Over the course of the fermentation, lactococci proved to be the dominant microorganism within the kefir fermentation (Fig. 2a). An approximate 5-log increase in presumptive lactococci was observed over the 24 h fermentation period from 7.6 × 104 to 1.1 × 109 CFU mL−1.

Panel A of Fig. 3 shows the topography of the differential alpha-band (8–14 Hz) oscillatory activity between all attend-auditory and all attend-visual trials (auditory – visual) at 1000 ms (i.e. where switch and repeat trials are collapsed together). The parieto-occipital focus of differential alpha power was highly consistent with our previous findings (Foxe et al., 1998; Fu et al., 2001; Gomez-Ramirez et al., 2007). Panel B of Fig. 3 depicts

the alpha-band (8–14 Hz) TSE waveforms derived from the three highlighted parieto-occipital electrode sites (central head; panel A). A sustained divergence in TSE amplitude is seen starting at ~600 ms post-cue, Fulvestrant some 750 ms before the onset of the S2 task stimulus, which occurs at 1350 ms. Alpha-band activity was greater when subjects had

been cued to attend selectively to impending auditory stimulation (i.e. to ignore or suppress concurrent visual inputs). In panel C of Fig. 3, ABT-737 research buy the TSE waveforms for attend-auditory (red traces) and attend-visual (black traces) are further distinguished according to trial type [i.e. switch trials (dotted traces) vs. repeat trials (solid traces)]. If participants were required to reconfigure the task-set on switch trials, the divergence in TSE waveforms was seen to start ~200 ms earlier at ~400 ms post-cue and reached a maximum just before the S2 stimulus onset. Figure 4 depicts the TSE waveforms for attend-auditory and attend-visual trials at six representative electrodes over frontopolar and parieto-occipital scalp regions, broken out for Mannose-binding protein-associated serine protease switch trials (panel A) and repeat trials (panel B). The extended electrode representation reveals that the modulation of alpha-band activity showed a considerably broader topographic distribution from the more typical focus over the parieto-occipital

region, with clear divergence seen over frontal and frontopolar scalp regions when participants were preparing for a switch of task (panel A). Early and widespread TSE modulation for switch compared to repeat trials is also depicted in the SCP (far right column). For repeat trials there was one main cluster of activation starting at ~1100 ms post-cue and this was distributed over both frontal and parieto-occipital scalp regions. For switch trials, two main clusters of differential activation were evident, an early one starting at ~600 ms and a later one starting at ~1100 ms. Both the early and late clusters showed widespread scalp distributions over parieto-occipital, central and frontopolar scalp regions. Topographical mapping shows maximal distributions over the parieto-occipital region starting at ~700 ms and over more frontal regions starting at ~1000 ms; both were enhanced on switch trials (panel C).

It is also a useful measure when they are asked at the end of the therapy to do the same and will often choose a different card. This again demonstrates the movement that has occurred during the course of counselling. Consent was obtained from all those referred by the counsellor for anonymised data to be used for evaluation of the service. We performed a retrospective Veliparib solubility dmso analysis of data obtained for people referred to the service between June 2007 and June 2010, using measurements

made pre- and post-attendance at the course of counselling. We looked at effects on HbA1c as a measure of glycaemic control, and changes in scores from the Clinical Outcomes in Routine Evaluation (CORE) outcome measure questionnaire,7 a measure of feelings of anxiety and risk, to assess the effectiveness of the counselling. This system was chosen over specific diabetes evaluation measures because it related

to the person as a whole rather than their diabetes alone. As life events that result in anxiety have a detrimental effect on the ability to self-care, we used a measure encompassing their anxieties as a whole rather than focusing purely on the diabetes. Comparison of pre- and post-counselling Copanlisib purchase values were made using chi-squared test for gender, Wilcoxon signed rank test for non-parametric data (HbA1c) and paired t-test for normally distributed data (age, CORE scores), with a 5% level of probability denoting significance. There were 79 people referred to the type 1 diabetes counselling service. The Nintedanib (BIBF 1120) average age was 40.1 years (SD 15.3), with 21 males and 58 females. Glycaemic control in the full cohort was sub-optimal (HbA1c pre-counselling [median (range)] 9.7% [5.8, 17.8]), and CORE scores revealed high levels of anxiety in these patients about their diabetes (CORE score pre-counselling [mean ± SD] 1.63±0.74). Of the 79 people referred, 17 did not complete the course of counselling. There was a trend towards these being more likely to be male (seven males and 10 females did not complete the counselling course; p=0.059), but there was no difference in age (completers [mean ± SD] 39.9±15.6 years, non-completers 39.3±13.8 years; p=0.883), glycaemic control (completers [median

(range)] 9.5% [6.2, 17.8], non-completers 10.6% [7.8, 13.7]; p=0.164) or CORE score (completers [mean ± SD] 1.60±0.71, non-completers 1.90±1.00; p=0.283). Of this group, seven did not start their counselling course despite referral, four did not complete the course after discussion with the counsellor, and six missed one or more sessions, so were not re-appointed. We did not explore the specific reasons why they did not complete the course, and the small numbers preclude further analysis of the different groups of non-completers. Data from the 62 people who completed the course were analysed to assess the impact of counselling. There was a reduction observed in both glycaemic control (HbA1c pre-counselling [median (range)] 9.5% [6.2, 17.8], post-counselling 9.3% [5.

We present strong evidence that HbpS belongs to the small set of proteins, which do not use histidine to coordinate the metal in the haem group. Further spectroscopic Bleomycin mw evidence strongly indicates that threonine 113 is actively involved in coordination of haem. Subsequent protein/haem titration experiments show a 1 : 2, protein/haem stoichiometry. We also present data showing the degradation of haem by HbpS in vivo. Because HbpS is conserved in many Actinobacteria, the presented results are applicable to related species. “
“Endoglucanase CelJ (Cel9D-Cel44A) is the largest

multi-enzyme subunit of the Clostridium thermocellum cellulosome and is composed of glycoside hydrolase (GH) families 9 and 44 (GH9 and GH44) and carbohydrate-binding module (CBM) families 30 and http://www.selleckchem.com/products/BIBF1120.html 44 (CBM30 and CBM44). The study of CelJ has been hampered by the inability to isolate full-length CelJ from recombinant Escherichia coli cells. Here, full-length CelJ and its N- and C-terminal segments, CBM30-GH9 (Cel9D) and GH44-CBM44 (Cel44A), were synthesized using a wheat germ cell-free protein synthesis system and then were purified to homogeneity. Analysis of the substrate specificities of CelJ and its derivatives demonstrated that the fusion of Cel9D and Cel44A results in threefold synergy for the degradation of xyloglucan,

one of the major structural polysaccharides of plant cell walls. Because CelJ displayed broad substrate specificity including significant carboxymethylcellulase (CMCase) and xylanase activities in addition Ribose-5-phosphate isomerase to high xyloglucanase activity, CelJ may play an important role in the degradation of plant cell walls, which are composed of highly heterogeneous polysaccharides. Furthermore, because Cel9D, but not Cel44A, acts as a semi-processive endoglucanase, the different modes of action between Cel9D and Cel44A may be responsible for the observed synergistic effect on the activity of CelJ (Cel9D-Cel44A). “
“Ophiobolin A is sesterterpenoid-type phytotoxin and may be an important candidate for

development of new crop protection and pharmaceutical products. The restriction enzyme-mediated integration (REMI) method was used to introduce the plasmid pSH75 into the ophiobolin A-producing filamentous fungus Bipolaris eleusines. A total of 323 stable transformants were obtained, all of which were capable of growing on potato-dextrose agar medium containing 200 μg mL−1 hygromycin B. The transformation frequency was about 4–5 transformants μg−1 plasmid DNA. An ophibolin A-deficient transformant (B014) was assessed and the presence of the hph gene in this transformant was confirmed by PCR. The cell-free cultural filtrates of this transformant showed significantly less inhibition on mycelial growth of the fungal pathogen Rhizoctoni solani but little effect on barnyard grass as opposed to that of the wild-type B.

Data regarding the 131I content in these 28 women and relevant information released by the citizens group on April 21 and May 18 were obtained from their website (‘Radioactivity in breast milk’, cited September 15, 2011; available from URL: http://bonyuutyousa.net/). Air pollution with radioactive materials occurred over a geographically wide area within 300 to 400 km of the FNP in the morning of March 15, 2011 (Fig. 2). Although the air radiation

dose rate was <0.07 µGy/h before the FNP accident in the areas shown in Figure 1, it increased sharply to 19 µGy/h in Fukushima city on March 15, then decreased to 1.6 µGy/h by the end of May. In Tokyo, located 230 km south of the FNP, the highest radiation dose rate of 0.81 µGy/h on March 15 decreased to <0.07 µGy/h by mid-April. The amount Cyclopamine chemical structure of 131I radioactivity in fallout per day reached a peak level of 93 000 MBq/km2 in Hitachinaka

city, located 130 km south of the FNP, on March 20, while it reached a peak level of 38 000 MBq/km2 in Tokyo on March 22 (Fig. 3). Consequently, vegetables such as spinach, cows milk and chicken eggs were also contaminated with 131I (Fig. 4). The highest content of 131I was 24 000 Bq/kg, found in spinach on March 18 in Kitaibaraki city, located 75 km south of the FNP. The 131I content in spinach decreased over time; for example, a level of 3500 Bq/kg was recorded in Utsunomiya city on March 19, decreasing to 480 Bq/kg on April 13, 120 Bq/kg on April 20, 12 Bq/kg on April 26, and became undetectable on May 3 (Fig. 4). Among the three foods, see more the 131I content was lowest in chicken eggs. It rained on March 20 and 21 in these areas, and the rain accelerated the pollution of water with 131I (Fig. 5). In Tokyo, 131I radioactivity in tap water from the Kanamachi water

purification plant reached a peak level of 210 Bq/kg on March 22. The content of 131I in the tap water decreased and became undetectable in many cities by mid-April (Fig. 5). Seven of 23 women (30.4%) who were tested in April secreted a detectable level of 131I in their breast milk (Table 1). The concentrations ranged from 2.2 to 8.0 Bq/kg and appeared to be higher than those in tap water Bcl-w available for these seven women at the same time points. As expected from the data on 131I radioactivity in the fallout, vegetables and water (Figs 3 to 5), the radioactivity of 131I in the breast milk became undetectable by May 15 in these seven women (Table 1). None of the remaining 96 women tested in May exhibited a detectable amount of 131I in their breast milk samples with detection limits of 1.6 ± 0.3 Bq/kg (data not shown). The present study demonstrated that environmental pollution with 131I causes the contamination of breast milk with 131I.

The survey was completely anonymous, but collected information

on the medical level of the provider (i.e., physician, physician assistant or nurse, medic), branch of service, any specialty training, deployment experience, current assignment location, and any recent education in the area of TD. The survey included multiple types of question formats including ranking, multiple choice, and Likert-type scale. Multiple-choice selleck kinase inhibitor questions on deployment-related diagnosis and management were scenario-based and designed to have a step-wise increase in complexity and/or severity and included: loose motions (unformed stools that did not meet TD definition), mild TD (three loose stools in 24 h with no activity limitations), mild TD with limitations (two loose stools in 8 h with some

activity limitations), moderate to severe TD (six loose stools in 24 h with no activity limitations), moderate TD with limitations (three loose stools in 24 h with some activity limitations), severe inflammatory TD (two loose stools in 8 h with fever and activity limitations), dysentery (three loose stools in 24 h with blood streaks), viral GSK2126458 molecular weight gastroenteritis (two loose stools in 8 h with vomiting predominate illness), and persistent diarrhea (14 d loose stools). The choices of treatment and management, from oral and/or IV rehydration and follow-up to management with antibiotics and nonantibiotic antidiarrheal medications (i.e, bismuth subsalicylate, diphenoxylate/atropine, and loperamide), were identical for each of the clinical scenarios, and one or more treatment modalities could be selected for any given scenario. In addition to univariate analyses describing provider characteristics and knowledge, attitude and practices outcomes, multiple-choice questions were scored

as correct/incorrect based on consensus among three clinicians (D. R. T, J. W. S., M. S. R.) with greater than 30 years of combined experience in research and clinical management of TD during deployment and from referenced published treatment guidelines.6,15 For each of nine TD management scenarios, a score was assigned based on a provider’s selection from Florfenicol among 10 various treatment options which included: fluid therapy [rehydration (oral), rehydration (IV)], nonantibiotic antidiarrheal medications (bismuth subsalicylate, diphenoxylate/atropine, and loperamide), and antibiotic antidiarrheal medications (ciprofloxacin, azithromycin, trimethoprim/sulfamethoxazole, rifaximin, and metronidazole). A provider could select any single or combination of therapy for each scenario. Specific to each scenario, a particular therapy could be scored as 1 (well evidenced), 0 (acceptable, not optimal), or −1 (not recommended).

The survey was completely anonymous, but collected information

on the medical level of the provider (i.e., physician, physician assistant or nurse, medic), branch of service, any specialty training, deployment experience, current assignment location, and any recent education in the area of TD. The survey included multiple types of question formats including ranking, multiple choice, and Likert-type scale. Multiple-choice selleckchem questions on deployment-related diagnosis and management were scenario-based and designed to have a step-wise increase in complexity and/or severity and included: loose motions (unformed stools that did not meet TD definition), mild TD (three loose stools in 24 h with no activity limitations), mild TD with limitations (two loose stools in 8 h with some

activity limitations), moderate to severe TD (six loose stools in 24 h with no activity limitations), moderate TD with limitations (three loose stools in 24 h with some activity limitations), severe inflammatory TD (two loose stools in 8 h with fever and activity limitations), dysentery (three loose stools in 24 h with blood streaks), viral GKT137831 ic50 gastroenteritis (two loose stools in 8 h with vomiting predominate illness), and persistent diarrhea (14 d loose stools). The choices of treatment and management, from oral and/or IV rehydration and follow-up to management with antibiotics and nonantibiotic antidiarrheal medications (i.e, bismuth subsalicylate, diphenoxylate/atropine, and loperamide), were identical for each of the clinical scenarios, and one or more treatment modalities could be selected for any given scenario. In addition to univariate analyses describing provider characteristics and knowledge, attitude and practices outcomes, multiple-choice questions were scored

as correct/incorrect based on consensus among three clinicians (D. R. T, J. W. S., M. S. R.) with greater than 30 years of combined experience in research and clinical management of TD during deployment and from referenced published treatment guidelines.6,15 For each of nine TD management scenarios, a score was assigned based on a provider’s selection from www.selleck.co.jp/products/tenofovir-alafenamide-gs-7340.html among 10 various treatment options which included: fluid therapy [rehydration (oral), rehydration (IV)], nonantibiotic antidiarrheal medications (bismuth subsalicylate, diphenoxylate/atropine, and loperamide), and antibiotic antidiarrheal medications (ciprofloxacin, azithromycin, trimethoprim/sulfamethoxazole, rifaximin, and metronidazole). A provider could select any single or combination of therapy for each scenario. Specific to each scenario, a particular therapy could be scored as 1 (well evidenced), 0 (acceptable, not optimal), or −1 (not recommended).

“
“Immunocytochemistry

shows that purinergic receptors (P1Rs) type A1 and A2A (A1R and A2AR, respectively) are present in the nerve endings at the P6 and P30 Levator auris longus (LAL) mouse neuromuscular junctions (NMJs). As described elsewhere, 25 μm adenosine reduces (50%) acetylcholine release in high Mg2+ or d-tubocurarine paralysed muscle. We hypothesize that in more preserved neurotransmission machinery conditions (blocking the voltage-dependent sodium channel of the muscle Veliparib order cells with μ-conotoxin GIIIB) the physiological role of the P1Rs in the NMJ must be better observed. We found that the presence of a non-selective P1R agonist (adenosine) or antagonist (8-SPT) or selective modulators of A1R or A2AR subtypes (CCPA and DPCPX, or CGS-21680 and SCH-58261, respectively) does not result in any changes in the evoked release. However, P1Rs seem to be involved in spontaneous release (miniature endplate potentials MEPPs) because MEPP frequency is increased by non-selective block but decreased by non-selective stimulation, with A1Rs playing the main role. We assayed the role of P1Rs in presynaptic short-term plasticity during imposed synaptic activity (40 Hz for 2 min of supramaximal stimuli). Depression is reduced by micromolar adenosine but increased by blocking P1Rs with 8-SPT. Synaptic depression is not affected by the presence of selective A1R and A2AR modulators, which suggests that both receptors

need to collaborate. Thus, A1R and A2AR might have no real effect on neuromuscular transmission in resting conditions. However, these receptors can conserve resources by limiting spontaneous quantal leak of Pexidartinib acetylcholine and may protect synaptic function by reducing the magnitude of depression during repetitive activity. “
“Morphine remains one of the most potent analgesic compounds used to control chronic pain despite its known adverse effects. It binds to the opioid receptors mu, delta and kappa, which are involved in aspects of neuronal fate such as cell proliferation, neuroprotection and neuronal differentiation.

However, the effect of morphine on these processes is controversial and in vitro studies, as well as in vivo Low-density-lipoprotein receptor kinase studies on adults and neonates in mammalian models, have not been able to clarify the diverse roles of morphine in the central nervous system. We have used zebrafish embryos to determine in vivo how morphine affects neuronal fate and opioid receptor gene expression and to elucidate if there is a link between these processes. Our results show that at 24 and 48 h post fertilization (hpf) morphine enhances cell proliferation, although it has opposing effects as an inducer of neuronal differentiation at these two stages, increasing the number of certain neuronal populations at 24 hpf and decreasing it at 48 hpf. The present study also demonstrates that in 24-hpf embryos morphine acts as a neuroprotector against glutamate damage in motor neurons and Pax-6-positive neurons.

9 kDa GlnR protein in the mutant strain compared with the wild type (Supporting information, Fig. S2). A phenotypic BIRB 796 chemical structure growth effect was observed by OD and CFU mL−1 for WT M. smegmatis cells grown in nitrogen-limiting media when compared with nitrogen-excess media (Fig. 1a and b). At the time of external nitrogen run-out, as determined by Aquaquant analysis (Fig. 1e), a reduction in growth rate between the two conditions is evident (Fig. 1a and b). Growth rate in the nitrogen-limiting media was restored to

the same rate as the nitrogen-excess media with the addition of an exogenous nitrogen source to the nitrogen-limiting media at this time point (Fig. 1a and b); no effect on growth rate was seen when exogenous nitrogen was added to the nitrogen-excess

media (data not shown). Further confirmation that our media was nitrogen-limiting and inducing a nitrogen-stress response in WT M. smegmatis cells was obtained by analysing the transcriptomic levels of genes known to be up-regulated in conditions of nitrogen limitation: amt1, amtB and glnA1 (Amon et al., 2008). The transcript levels LBH589 cost of amt1, amtB and glnA1 were all significantly induced in the nitrogen-limiting media, but not induced in the nitrogen-excess conditions at 13 h, 2 h after nitrogen run-out in the limiting media (Fig. 2). We therefore confirmed that our nitrogen-limiting conditions were stimulating a genetic response to nitrogen limitation in M. smegmatis. Growth kinetics of the M. smegmatis mutant strains in nitrogen-limiting Megestrol Acetate and nitrogen-excess medium were performed. Although the M. smegmatis strains grew similarly in nitrogen-excess conditions (data not shown), the GlnR and GlnR_D48A mutants exhibited a reduced growth

rate when compared with the wild type under nitrogen-limiting conditions (Fig. 1c and d). However, no major growth defect was noted for either mutant strain; this is intriguing, suggesting that the M. smegmatis GlnR-mediated transcriptomic response is not essential for growth during nitrogen limitation. Another interesting observation was the reduced uptake of ammonium from the medium by both mutants. Two ammonium transporters (amtB and amt1) have previously been shown to be up-regulated under nitrogen limitation by GlnR (Amon et al., 2008). The inability of the GlnR mutant strains to induce expression of ammonium transporters could explain the observed reduction in growth rate, suggesting ammonium uptake in these mutants is by diffusion alone. The transcriptomic response of the wild type and mutants during nitrogen limitation was therefore investigated further.