, 2008). Therefore, to characterize the histone acetylation activity of LdHAT1, in vitro assays were carried out using a peptide substrate derived from the N-terminus of L. donovani histone H4. To identify the lysine residue that was specifically acetylated this website by LdHAT1, three antibodies were raised against L. donovani histone H4–derived peptides acetylated on K4, K10 or K14 residue, respectively. Specificities of the antibodies were ensured by dot blot analysis, which showed no cross-reactivity (Fig. 3a).

Once the specificities were confirmed, the antibodies were used to identify the lysine residue on the peptide derived from N-terminus of L. donovani histone H4 acetylated by LdHAT1. As shown in Fig. 3b, the peptide acetylated by LdHAT1 could be detected only by anti-H4K10Ac antibody, but not with other two antibodies (data not shown), suggesting that the acetyltransferase from L. donovani specifically acetylates H4K10 residue. As LdHAT1 was shown to be phosphorylated by S-phase kinase LdCyc1-CRK3, it would be interesting to investigate

any possible effect of such phosphorylation on its histone acetylation activity. To explore such a possibility, H4K10 acetylation activity of non-phosphorylated LdHAT1 was compared with that of LdHAT1 phosphorylated by LdCyc1-CRK3. As depicted in Fig. 3c, the phosphorylated form of LdHAT1 did not show Protein Tyrosine Kinase inhibitor any H4K10 acetylation activity, suggesting the regulation of histone H4 acetylation by S-phase cell cycle kinase. Intriguingly, LdHAT1ΔCy and LdHAT1-T394A mutants also did not show any acetylation activity (Fig. 3d) implicating the contribution of the mutated residues in the enzymatic activity. Previous studies demonstrated the acetylation Dichloromethane dehalogenase of K4, K10 and K14 residues of the N-terminal tail of histone H4 from T. brucei and T. cruzi (da

Cunha et al., 2006; Janzen et al., 2006; Mandava et al., 2007). Moreover, cell cycle-dependent post-S-phase enhancement of K4, K10 and K14 acetylation of histone H4 was observed in T. cruzi (Nardelli et al., 2009). Therefore, the observed inhibition of histone H4K10 acetylation by LdHAT1 owing to its phosphorylation by S-phase kinase in the present studies could correlate such cell cycle–specific periodic acetylation. It will be interesting to study the effect of inhibition of the HAT activity on the S-phase events. The work was partially supported by the project grant [37(1044)/00/EMR-II] from Council of Scientific and Industrial Research (CSIR), India and intramural grant from Department of Atomic Energy, Government of India. The authors have no conflict of interest to declare. “
“Metagenomic DNA libraries constructed from the Dagong Ancient Brine Well were screened for genes with Na+/H+ antiporter activity on the antiporter-deficient Escherichia coli KNabc strain. One clone with a stable Na+-resistant phenotype was obtained and its Na+/H+ antiporter gene was sequenced and designated as m-nha.

1 and the possible significance of the histidine-rich C-terminal tail in selecting these polypeptide substrates. In

GroEL, the C-terminal tail is highly flexible and thus undefined in the crystal structures (Hartl & Hayer-Hartl, 2002; Machida et al., 2008). However, a detailed genetic analysis of the final 23 residues assessing the ability of C-terminal-truncated, double- and single-ring mutants to assist the refolding of rhodanese and malate dehydrogenase showed that this domain defines the environment within the central cavity and in particular its hydropathicity, features that would impact on both the size and nature of the substrate protein folded by the chaperonin (Tang et al., 2006; Machida et al., 2008). This is consistent with a role for the mycobacterial Cpn60.1 Doxorubicin ic50 chaperonins in the folding Bioactive Compound Library in vivo of a distinct class of proteins, possibly unique to mycobacteria or actinomyces. Although a distinct DNA-bound function in the assembly of the nucleoid has recently been proposed for Cpn60.1 (Basu et al., 2009) this is unlikely to involve the C-terminal tail sequence, as the mitochondrial Hsp60 chaperonin for which nucleotide binding has also been reported does not have a histidine-rich C-terminal tail (Kaufman et al., 2003; Basu et al., 2009). A database search with the histidine-rich C-terminal sequence of Cpn60.1 reveals highly homologous proteins across

all mycobacterial species, as well as Corynebacteria, Nocardia and Rhodococcus (C. Colaco, unpublished data). A common feature of all these Actinobacteria is their synthesis of a complex cell wall containing mycolic acid derivatives, and this suggests the intriguing possibility that the biological role of the mycobacterial Cpn60.1 may be to chaperone the folding of key enzymes involved in the synthesis Dichloromethane dehalogenase of mycolic acid. Such a role for Cpn60.1 is also consistent with the defects

in mycolates and biofilm formation observed in the cpn60.1 knockouts in M. smegmatis, where the protein was also found to be associated with KasA and SMEG4308, both key enzymes implicated in biofilm formation and involved in fatty acid synthesis (Tang et al., 2006; Kumar et al., 2009). In this respect, it is interesting to note that the oligomerisation of Cpn60.1 has been shown to be facilitated by phosphorylation (Canova et al., 2009), which is thought to be mediated by the serine threonine protein kinases that have also been implicated in biofilm formation (Gopalaswamy et al., 2008). Finally, as KasA has been identified as an important drug target for the development of new drugs against TB (Brown et al., 2009), the most interesting implication of the suggested role of Cpn60.1 is that this novel mycobacterial chaperonin may present an upstream target for drug development. Thus, therapeutics that target Cpn60.

In general, inhibition of fatty acid biosynthesis by the addition of cerulenin to the medium caused an increase in the polyhydroxyalkanoates and glycogen content in cells. The mutants affected in triacylglycerol accumulation used in this study also produced increased amounts of glycogen and eventually of polyhydroxyalkanoates during cultivation on gluconate in comparison

with the wild type. This effect was more evident in the mutant PDM41 than in the atf1ΩKm mutant. When the biosynthesis of triacylglycerols is impaired by inhibition of the de novo fatty acid biosynthesis GSK2126458 pathway or the disruption of a gene involved in triacylglycerol accumulation, carbon distribution through metabolism changes and intermediates become more available for the synthesis of glycogen and polyhydroxyalkanoates in cells. These approaches contribute toward a better understanding of storage compound metabolism and the interaction of pathways in Rhodococcus species, which could be of interest for planning further metabolic manipulation of cells for biotechnological purposes. We are grateful to Dr Alexander Steinbüchel for providing R. opacus mutant PDM41.

This study was financially supported by the Agencia Nacional de Promoción Científica y Tecnológica, Argentina (Project PME no. 216) and SCyT of the University of Patagonia San Juan Bosco. H.M. Alvarez is a career investigator and M.A. Hernández a scholarship holder of the Consejo Nacional de Investigaciones this website Científicas y Técnicas (CONICET), Argentina. “
“Antisense oligonucleotides (AS-ODN) target genes in a sequence-specific manner inhibit gene function

and have potential use as antimicrobial agents. Cell barriers, such as peptidoglycan, cell surface proteins and lipopolysaccharide membranes, prevent delivery of AS-ODN into the bacterial cell, limiting their use as an effective treatment option. The β-lactam antibiotic penicillin was examined for its ability to deliver phosphorothioate oligodeoxyribonucleotides (PS-ODNs) and γ32 P-ODN into Streptococcus mutans OMZ175. Treatment of lag-phase S. mutans OMZ175 cells with penicillin and FBA (PS-ODN targeting the fructose-biphosphate aldolase gene), resulted in prolonged suppression of growth (> 24 h) and fba expression (656.9 ± 194.4-fold Rebamipide decrease at 5 h). Suppression of both cell growth and fba expression corresponded with a greater amount of γ32 P-ODN becoming cell associated, with a maximum γ32 P-ODN concentration per cell achieved 5 h after penicillin treatment (6.50 ± 1.39 × 108 molecules per CFU). This study confirms that for S. mutans OMZ175, the peptidoglycan layer acts as a major barrier preventing AS-ODN penetration and suggests that the use of agents such as penicillin that interfere with peptidoglycan integrity can significantly increase the uptake of PS-ODN by these cells.

All tests were conducted

in triplicate and controls were included. Sigmoidal curves were fitted to each set of triplicate growth data (Microsoft Excel) and the equation for each curve selleck chemical used to calculate the time taken for that culture to reach an initial OD+0.1 (lag phase). Differences between lag phase values were analysed for statistical significance using the Tukey multiple comparison test (prism Software). Each bacterial strain was incubated in the presence of increasing concentrations of zoocin A. The zoocin A concentration selected as sublethal was one that significantly (P<0.001) increased lag phase without decreasing the OD of the culture at 18 h in comparison with the untreated control. The sublethal concentrations used in this study are given in Table 1. Streptococcus oralis 34 and Actinomyces viscosus T14AV were resistant to all concentrations of zoocin A tested and a concentration of 50 μg mL−1 was arbitrarily chosen for use with these strains as a control for possible toxic effects resulting from the combination of zoocin A and PS-ODNs. Streptococcus mutans OMZ175 Epigenetic inhibitors library was incubated with zoocin

A at 0.1 μg mL−1 and FABM at 1, 5, 8, 10, and 20 μM. Streptococcus mutans OMZ175 was incubated with FABM at 10 μM and zoocin A at 0.05, 0.1, 0.125, and 0.15 μg mL−1. Unless otherwise stated, PS-ODNs were diluted to attain a final concentration of 50 μM for Streptococcus sobrinus 6715 and Streptococcus sanguinis K11 and 10 μM for all other strains. Zoocin A was diluted to reach the sublethal concentrations

given in Table 1. The levels of mRNA transcript of fba, 16sRNA. and gyrA in S. mutans OMZ175 were determined using quantitative reverse transcriptase PCR (qRT-PCR). A 5% inoculum of S. mutans OMZ175 in THB was incubated until an OD of 0.4 was obtained, at which point 8-mL volumes of the culture were treated with either THB, 0.4 μg mL−1 zoocin A, 10 μM FBA, 10 μM ATS, 0.4 μg mL−1 zoocin A+10 μM FBA, or 0.4 μg mL−1 zoocin A+10 μM ATS. Samples for selleckchem RNA extraction were removed at times 0, 0.5, 5, and 16 h, post addition of zoocin A and PS-ODNs. This experiment was repeated three times. Cells were harvested by centrifugation at 18 000 g for 10 min at 4 °C, and the RNA was extracted using TRIZOL™ (Invitrogen) according to the manufacturer’s instructions. The RNA was dissolved in molecular biology grade water (5 Prime) and treated for DNA contamination with the QIAgen RNeasy mini kit and DNase I, according to the manufacturer’s instructions. Viable counts were performed using the drop plate method and blood agar. The sequences of fba, 16sRNA, and gyrA were identified within the S. mutans UA159 genome sequence (NC004350) by blast, and PCR primers designed to amplify each gene. PCR products amplified from S.

Thus, our method would be useful to reveal the functional find more microarchitecture of the brain. We did not observe different movement of whiskers when stimulating various points in the endoscopic field of view. The endoscopic field of view may be too small to cover areas for multiple whisker movement, because whisker area occupies a large portion of the rodent primary motor cortex. In our experiments, fluorescently-labeled neurons (presumably cell bodies) could be observed through the probe (Fig. 2G), but the region where neural activities were detected did not always correspond to the fluorescent signals (Fig. 4A and D). This is primarily due to the

fact that ChR2-expressing neurons were only a subset of EGFP-labeled neurons (Fig. 2H). Another possible reason is that the optical fiber bundle-based endoscope has limited spatial resolution and depth of field (Vincent et al., 2006), therefore thin structures such as axons and dendrites are often not visualized. In addition, neurons distant from the endoscope tip cannot be visualized, because the working distance of this optical fiber bundle-based endoscope is nearly zero (Vincent et al., 2006). Stimulating ChR2 located in these non-visualized neurons can also be activated by light (Fig. S3A). This might have caused the poor correlation between fluorescence signal and neural activity generating areas. The widespread subcellular distribution of ChR2 could also interfere with spatially restricted

C59 price photostimulation. In most neural tissues, long-range axons are intermingled. Thus, targeted photostimulation on the somatodendritic region of a neuron often also excites buy STA-9090 colocalized axons of distant neurons. Electrical microstimulation suffers the same problem – stimulating current excites both the soma and axon of neurons; therefore, electrical microstimulation activates neurons around the electrode, sometimes as far as millimeters away (Histed et al., 2009). However, in the case of optical stimulation, this problem would be overcome by molecular biological techniques. A recent report has shown that ChR2 fused with the myosin-binding domain from Melanophilin is targeted to the somatodendritic compartment

of neurons in vivo (Lewis et al., 2009). This kind of technology for anchoring ChR2 to specific subcellular regions will aid in achieving high spatial resolution when one uses the method presented here or other optical stimulation techniques for controlling neural activity. This work was supported by PRESTO (to Y.H.) and CREST (to K.F.), Japan Science and Technology Agency. We thank members of the Nakanishi Laboratory for helpful advice, M. Okazawa for help in preparing plasmid DNAs, H. Mizuno for assistance with in utero electroporation, and T. Yoshida for critical comments on this work. The authors declare that there are no conflicts of interest. Abbreviations ChR2 channelrhodopsin-2 EGFP enhanced green fluorescent protein EYFP enhanced yellow fluorescent protein Data S1.

Thus, our method would be useful to reveal the functional Ion Channel Ligand Library microarchitecture of the brain. We did not observe different movement of whiskers when stimulating various points in the endoscopic field of view. The endoscopic field of view may be too small to cover areas for multiple whisker movement, because whisker area occupies a large portion of the rodent primary motor cortex. In our experiments, fluorescently-labeled neurons (presumably cell bodies) could be observed through the probe (Fig. 2G), but the region where neural activities were detected did not always correspond to the fluorescent signals (Fig. 4A and D). This is primarily due to the

fact that ChR2-expressing neurons were only a subset of EGFP-labeled neurons (Fig. 2H). Another possible reason is that the optical fiber bundle-based endoscope has limited spatial resolution and depth of field (Vincent et al., 2006), therefore thin structures such as axons and dendrites are often not visualized. In addition, neurons distant from the endoscope tip cannot be visualized, because the working distance of this optical fiber bundle-based endoscope is nearly zero (Vincent et al., 2006). Stimulating ChR2 located in these non-visualized neurons can also be activated by light (Fig. S3A). This might have caused the poor correlation between fluorescence signal and neural activity generating areas. The widespread subcellular distribution of ChR2 could also interfere with spatially restricted

click here photostimulation. In most neural tissues, long-range axons are intermingled. Thus, targeted photostimulation on the somatodendritic region of a neuron often also excites Belnacasan colocalized axons of distant neurons. Electrical microstimulation suffers the same problem – stimulating current excites both the soma and axon of neurons; therefore, electrical microstimulation activates neurons around the electrode, sometimes as far as millimeters away (Histed et al., 2009). However, in the case of optical stimulation, this problem would be overcome by molecular biological techniques. A recent report has shown that ChR2 fused with the myosin-binding domain from Melanophilin is targeted to the somatodendritic compartment

of neurons in vivo (Lewis et al., 2009). This kind of technology for anchoring ChR2 to specific subcellular regions will aid in achieving high spatial resolution when one uses the method presented here or other optical stimulation techniques for controlling neural activity. This work was supported by PRESTO (to Y.H.) and CREST (to K.F.), Japan Science and Technology Agency. We thank members of the Nakanishi Laboratory for helpful advice, M. Okazawa for help in preparing plasmid DNAs, H. Mizuno for assistance with in utero electroporation, and T. Yoshida for critical comments on this work. The authors declare that there are no conflicts of interest. Abbreviations ChR2 channelrhodopsin-2 EGFP enhanced green fluorescent protein EYFP enhanced yellow fluorescent protein Data S1.

3% (mutation at codon

70) and no significant increase in the risk of transmission was observed after adjusting for viral load at delivery (OR 4.8; with wide 95% CI 0.2–131; P = 0.35) [142]. High-level resistance was not reported and the median CD4 cell count in the women was 540 cells/μL. In retrospective cohort studies from France [277] and the USA [140], 20% and 8.3%, respectively, of HIV-positive newborns had zidovudine-resistance mutations after maternal zidovudine prophylaxis. In the WITS, lower CD4 cell check details count and higher HIV viral load at delivery were associated with increased risk of transmission while in the multivariate analysis, the presence of at least one mutation associated with zidovudine resistance was also associated with an increased risk of transmission (OR 5.15; 95% CI 1.4–18.97) [141]. With infant feeding patterns, it is difficult to separate drug dosing www.selleckchem.com/products/epacadostat-incb024360.html from feeds, so drugs without food restrictions are preferred, an advantage of zidovudine. Important in this age group, where therapeutic options are more limited than in older children and adults, should transmission occur multidrug resistance is avoided. However, some clinicians prefer to choose another antiretroviral, with no history of maternal resistance, for

infant post-exposure monotherapy. The established alternatives, nevirapine and lamivudine, have potent antiretroviral effect but a low (single-point mutation) barrier to resistance. The dosing and safety issues with newer therapies, such as lopinavir/ritonavir, are outlined below. It is therefore suggested that neonatal zidovudine monotherapy remains a reasonable approach for infants born to mothers with a HIV viral load < 50 HIV RNA copies/mL plasma, even if there is a history over of zidovudine resistance. Further investigation of the national cohort data to address this question is under way. Where

a low transmission-risk mother (see Section 5: Use of antiretroviral therapy in pregnancy) chooses zidovudine monotherapy plus PLCS, the infant should receive zidovudine monotherapy [4]. There are two situations where triple combination PEP for neonates is advised: Post-delivery infant-only prophylaxis: mother found to be HIV positive after delivery, this is only effective if given within 48–72 hours of birth Detectable maternal viraemia (> 50 HIV RNA copies/mL) at delivery, mother may be on cART or not: delivery before complete viral suppression is achieved: e.g. starting cART late or delivery premature viral rebound with or without resistance, with or without poor adherence unplanned delivery: e.g. premature delivery prior to starting ART; or late presentation when maternal HIV parameters may be unknown 8.1.2 Infants < 72 hours old, born to untreated HIV-positive mothers, should immediately initiate three-drug antiretroviral therapy for 4 weeks.

Plant insect mite dermatitis may become chronic or recur on indoor or outdoor mite reexposure on the heads, limbs, and trunks of backpackers, campers, and resort vacationers during peak mite-feeding and breeding seasons in the spring and summer. Only biting larvae of Asian scrub typhus chiggers (Leptotrombidium species) transmit scrub typhus caused by O tsutsugamushi (formerly Rickettsia tsutsugamushi),

and only biting house-mouse mites (Liponyssoides sanguineus) transmit rickettsialpox BIBW2992 ic50 caused by R akari. Although these two mite-transmitted infectious diseases do share mites as vectors, their preferred mite vectors, disease ecologies, and clinical presentations are different, when compared with Table 3. Although initially classified in the genus Rickettsia, O tsutsugamushi was reclassified into a separate genus based on molecular

evidence that its cell wall ultrastructure differed significantly from Rickettsia species. 25 Both scrub typhus and rickettsialpox respond to treatment with oral tetracycline, oral doxycycline, or intravenous chloramphenicol, which is not recommended due to its bone marrow toxicity. 25 Both scrub typhus mites and house-mouse mites are, like ticks, capable of inheriting bacterial infections by transovarial transmission GSK J4 price and maintaining infections in several mite generations, because bacteria are passed from adults to juveniles (nymphs and larvae) by transstadial transmission. 25 Scrub typhus chiggers are the main environmental reservoirs of O tsutsugamushi in endemic regions with much smaller secondary reservoirs in wild rodents. 1,25 Common house mice are the zoonotic reservoirs of R akari, not only in crowded urban apartment buildings in the United States but also in all mice-infested buildings and sheds in more rural locations worldwide. Among the scrub typhus-carrying Leptotrombidium larval chigger mites, Leptotrombidium deliense, the Asian rodent chigger, is a principal vector throughout eastern Asia

and Eurasia. before 25,26 Following scrub typhus-infected chigger bites, there is an 8- to 10-day incubation period before onset of classical clinical manifestations including bite-eschar, regional lymphadenopathy, conjunctival injection, hearing loss, and centrifugal rash. 25,26 In the temperate regions of Eurasia, there is a definite scrub typhus seasonal transmission cycle determined by peaking temperatures and humidity during weeks of marked seasonal change between spring and summer and fall and winter. 27 In the tropics, scrub typhus transmission occurs year-round. 25 In Asia, the most common endemic rickettsioses include scrub typhus, murine typhus, and Q fever, which may be difficult to differentiate clinically and also serologically due to cross-reacting antigens.

Furthermore, we demonstrated that the eGFP-PilACt fusion protein specifically labeled similar EPS structures as the WGA in starvation biofilms, trail structures and selleck chemicals developmental

fruiting bodies, evidence for a direct interaction between pilin and EPS of M. xanthus under native conditions. At the same time, the eGFP-tagged truncated pilin could be utilized to visualize EPS distribution in M. xanthus. The novel approach developed in this study can be applied in future studies of M. xanthus cell behaviors involving EPS and TFP. We thank Drs Mitch Singer and Dale Kaiser for providing bacterial strains, and Aida Kaplan and Dr Howard Kuramitsu for editing the manuscript. This work was supported by the GSK126 nmr US National Institutes of Health Grant GM54666 (to W.S), International Science and Technology Cooperation Program of China 2011DFA30940 (to W.S.) and the Chinese National Natural Science Foundation Grant 30870020 (to W.H.). W.H. and Z.Y. contributed equally to this work. “
“Lahey Clinic Medical Center, Burlington, MA, USA The marRAB operon is conserved in seven genera of enteric bacteria (Escherichia, Shigella, Klebsiella, Enterobacter, Salmonella, Cronobacter,

and Citrobacter). MarA is a transcriptional regulator affecting many genes involved in resistance to stresses, and MarR is an autorepressor of the operon, but a role for the marB gene has been unclear. A recent work reported that deletion of marB causes resistance to certain stresses and increases the amount of marA transcript. We show here that the small (216 bp) marB gene encodes a protein, not an sRNA, because two different stop codons within the predicted open reading frame of marB prevented plasmid-borne marB from complementing Unoprostone ΔmarB::Kan.

The ΔmarB::Kan mutation did not increase the stability of the marA transcript, suggesting that MarB does not destabilize the marA transcript but rather reduces its rate of transcription. Placing the putative signal sequence of MarB upstream of signal-sequence-less alkaline phosphatase guided the phosphatase to its normal periplasmic location. We conclude that MarB is a small periplasmic protein that represses the marRAB promoter by an indirect mechanism, possibly involving a signal to one of the cytoplasmic regulators of that promoter. “
“Group B streptococci (GBS) are a major cause of neonatal meningitis, and sialic acid is a determinant of the development of meningitis. The transcription level of the neuD gene, used as a marker of neu gene expression and capsular production, was significantly higher in serotype III GBS strains isolated from meningitis than from vaginal carriage. This was irrespective both of the phylogenetic position of strains and of the presence of a thymine at position 264 in the neuD gene. Differences in neuD gene transcription may explain in part why particular isolates among the GBS strains colonizing the vagina can cause meningitis.

For the culture, a cysteine production medium [composition (per liter): a quantity of 12 g of ammonium chloride, 1.5 g of potassium dihydrogenphosphate, 1 g of magnesium sulfate heptahydrate, 0.1 mg of thiamine hydrochloride, 1.7 mg of ferrous sulfate heptahydrate, 0.15 mg of sodium molybdate dihydrate, 0.7 mg of cobalt chloride

hexahydrate, 1.6 mg of manganese chloride tetrahydrate, 0.3 mg of zinc sulfate heptahydrate, 0.25 mg of copper sulfate pentahydrate, 0.6 g of tryptone, 0.3 g of yeast extract, 0.6 g of sodium chloride, 20 g of calcium carbonate, 135 mg of l-histidine monohydrochloride AG-14699 monohydrate, 4 g of sodium thiosulfate, 2 mg of pyridoxine hydrochloride, 40 g of glucose, 12.5 mg of tetracycline] (Nonaka, 2010) was used. For the cultivation with thiosulfate or sulfite as a sulfur source, 8 g L−1 of sodium thiosulfate or 2.6 g L−1 of sodium sulfite was added, respectively.

For the cultivation with sulfate Epacadostat mw as a sulfur source, 15 g L−1 of ammonium sulfate was added instead of ammonium chloride. The BW26424/pACYC-DES and the BW25113/pACYC-DES strains were each applied and spread onto LB agar medium containing tetracycline, and precultured overnight at 34 °C. The streak cells corresponding to about 7 cm on the plate were scraped with an inoculating loop of 10 μL size (NUNC Blue Loop), and inoculated into 2 mL of the cysteine production medium. The culture was grown at 32 °C with shaking for 42 h, and the amount of cysteine accumulated in the medium was quantified. The quantification of cysteine was performed by the method described by Gaitonde (1967). The experiment was performed in hexaplicate for both the strains, and averages and standard

deviations of the accumulated cysteine amounts were calculated. Escherichia coli cells grown in 10 mL of LB medium ADAM7 were harvested by centrifugation and resuspended in 0.2 mL 8 M urea/lysis buffer (8 M urea, 50 mM Tris-HCl, pH 8.0 at 4 °C, and 100 mM NaCl), and sonicated. Cell extracts (10 μg) were subjected to 10% SDS-PAGE and blotted on to polyvinylidene difluoride (PVDF) membranes using iBlot semi-dry transfer apparatus (Invitrogen). Membranes were first immuno-detected with anti-β-galactosidase (Progema) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Nacalai tesque) antibodies and then developed with a chemiluminescence kit (Nacalai tesque). The image was analyzed with a LAS-4000 IR multi color (Fuji Film). The transcriptome analysis of E. coli response to metal-shock indicated that the genes for cysteine biosynthesis including cysK are regulated by several metals (Yamamoto & Ishihama, 2005a, b; Hobman et al., 2007). Since a set of the metal stimulon genes are regulated by some of two-component system (TCS) (Yamamoto & Ishihama, 2006; Yamamoto et al., 2008), we tested possible influence of TCS knock-out on cysK expression. For this purpose, we used the collection of TCS deficient E. coli mutants (Oshima et al.