Therefore, cryo beads

of E. coli and P. fluorescens were pre-cultivated over night at 37°C (E. coli) or 30°C (P. fluorescens) in filtrated #Selleck Ro 61-8048 randurls[1|1|,|CHEM1|]# Nutrient Broth (NB) medium. For this pre-culture, approx. 106 cells per ml were used to inoculate 100 ml fresh and NB medium. These cultures were incubated for 10 h at the respective optimal growth temperature to obtain the working culture. C. thermocellum cells were cultivated in GS2 nutrient solution [43] at anaerobic conditions at 55°C for 30 h. M. barkeri and P. acne were cultivated in mixed culture in DSM medium 120 (Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures, Germany) at anaerobic conditions at 37°C for 48 h. All culture media were sterilized by autoclaving process before use. Operation and sampling of the biogas reactor The design and operation of the upflow anaerobic solid state (UASS) reactor connected with a downstream anaerobic filter (AF) reactor was described in detail by Pohl et al. (2012) [44].

For this study, chopped wheat straw was used as substrate at an organic loading rate (OLR) of 2.5 g volatile substances (VS) per liter and day. The UASS reactor was operated at mesophilic temperatures (37°C). Two liquid samples were taken from the effluent of the UASS reactor at various times (hereafter referred to as UASS-1 und UASS-2). Samples were processed immediately after sampling for further analyses. Sample fixation Sample fixation was carried out immediately after sampling according to a protocol after Kepner and Pratt (1994) [45]. Therefore, 10 ml of pure cultures or liquid samples from the UASS reactor, respectively, were fixed with 30 ml selleck chemical of a 3.7% formaldehyde solution (diluted in 1× PBS pH 7.4) for 4 h at 4°C. Protein kinase N1 After fixation, the samples were centrifuged at 8,000 × g for 20 min at room temperature (RT). The supernatant was discarded and the pellet was washed twice in 1× PBS using same centrifugation conditions

as before. The 1× PBS was prepared of 140 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, and 1.8 mM KH2PO4. The pH was adjusted to 7.4 with HCl (all reagents were provided by Carl Roth GmbH & Co. KG, Germany). After washing the pellet was re-suspended in 5 ml 1× PBS, mixed with 5 ml 96% ethanol p.a. and stored until further use at −20°C. Alternatively, a fixation with 50% ethanol (diluted in 1× PBS pH 7.4) was performed for Gram-positive prokaryotes. In this case, the samples were centrifuged at 8,000 × g for 20 min. The pellets were re-suspended in 5 ml 1× PBS, mixed with 5 ml 96% ethanol p.a. and stored until further use at −20°C. Sample pre-treatment for Flow-FISH analyses Six different pre-treatment techniques for sample purification taken from the recent literature (in the following denominated as procedure 1 to procedure 6) were tested on both, pure cultures and UASS biogas reactor samples. An overview about all pre-treatment procedures and their modifications is given in Table 1.

Human molecular genetics 2004,13(16):1785–1791.CrossRefPubMed #selleck chemicals llc randurls[1|1|,|CHEM1|]# 35. Bogerd HP, Doehle BP, Wiegand HL, Cullen BR: A single amino acid difference in the host APOBEC3G protein controls the primate species specificity of HIV type 1 virion infectivity factor. Proc Natl Acad Sci USA 2004,101(11):3770–3774.CrossRefPubMed 36. Schrofelbauer B, Chen D, Landau NR: A single amino acid of APOBEC3G controls

its species-specific interaction with virion infectivity factor (Vif). Proc Natl Acad Sci USA 2004,101(11):3927–3932.CrossRefPubMed 37. Takeuchi H, Matano T: Host factors involved in resistance to retroviral infection. Microbiology and immunology 2008,52(6):318–325.CrossRefPubMed 38. Brass AL, Dykxhoorn DM, Benita Y, Yan N, Engelman A, Xavier RJ, Lieberman J, Elledge SJ: Identification of host proteins required for HIV infection through a functional LB-100 mw genomic screen. Science 2008,319(5865):921–926.CrossRefPubMed 39. Zhang S, Feng Y, Narayan O, Zhao LJ: Cytoplasmic retention of HIV-1 regulatory protein Vpr by protein-protein interaction with a novel human cytoplasmic protein VprBP. Gene 2001,263(1–2):131–140.CrossRefPubMed 40. Sims AC, Burkett SE, Yount B, Pickles RJ: SARS-CoV replication and pathogenesis in an in vitro model of the human conducting airway epithelium. Virus Res 2008,133(1):33–44.CrossRefPubMed 41. Frieman

M, Heise M, Baric R: SARS coronavirus and innate immunity. Virus Res 2008,133(1):101–112.CrossRefPubMed 42. Peiris M: Pathogenesis of avian flu H5N1 and SARS. Novartis Found Symp 2006, 279:56–60. discussion 60–55, 216–219.CrossRefPubMed 43. Freeze HH: Genetic defects in the human glycome. Nat Rev Genet 2006,7(7):537–551.CrossRefPubMed 44. Walsh CT, Garneau-Tsodikova S, Gatto GJ Jr: Protein posttranslational modifications: the chemistry of proteome diversifications. Angew Chem Int Ed Engl 2005,44(45):7342–7372.CrossRefPubMed 45. Kim A, Pettoello-Mantovani

M, Goldstein H: Decreased susceptibility of peripheral blood mononuclear cells from individuals heterozygous for a mutant CCR5 allele to HIV Galeterone infection. J Acquir Immune Defic Syndr Hum Retrovirol 1998,19(2):145–149.PubMed Authors’ contributions FCC conceived the project. FKL, CLP, and JMY analyzed the data. FKL and CLP constructed the interface. FCC, FKL and TJC drafted the manuscript. All authors read and approved the manuscript.”
“Background L-arabinose and D-xylose are two of the most abundant monosaccharides in nature. They are components of the plant cell wall polysaccharides xylan, xyloglucan and pectin [1] and therefore an important carbon source for microorganisms growing on plants or plant matter. In fungi, L-arabinose and D-xylose are catabolised through the pentose catabolic pathway [2]. L-arabinose is converted to xylitol in 3 steps by the enzymes L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase, while D-xylose reductase converts D-xylose in a single step to xylitol.

Multimode interference Multimode interference (MMI) is a waveguide effect which waveguide modes are interfered and self-imaged in a multimode waveguide. MMI is used for the plasmonic couplers due to its large fabrication tolerance and integrated size [16, 17]. Several works have presented plasmonic

MMI couplers to split AZD5363 clinical trial SPP intensities and filter wavelengths. Multimode interference couplers were often studied using calculation methods, such as finite-element method [18] (FEM), beam propagation method [17] (BPM), and finite-difference time-domain (FDTD) method [19]. By using these methods, the functions of MMI devices can be theoretically demonstrated. However, MMI patterns are hard to be directly visualized. To show MMI in DLSPPW experimentally, Bafilomycin A1 we studied a wide

DLSPPW with 300-nm-high, 4.6-μm-wide strip on a 100-nm-thick silver film. The waveguide length was longer than 100 μm. The incident wavelength was 830 nm owing to the good SPP propagation length and quantum efficiency of CCD. This waveguide provided TM00 ~ TM06 in 830-nm wavelength and gave rise to multimode interference along the waveguide. The interference effect can be express by (1) where m is the number of guided mode, a m is superposition constant and u m (y) is complex amplitude depended with incident field. The MMI pattern changed with the incident field u m (y).The incident field was changed by varying the launching position of the fiber tip. In the experiment, the near-field excitation location was moved from the north corner to south corner by using move-mode in NFES. Figures 3 show the leakage radiation Sitaxentan images that correspond the fiber tips located at corners and middle of the waveguide. Figure 3a was a LY2874455 research buy chain-like MMI pattern. The field intensity was splitting to 50:50

with a gap of 2.237 μm (red arrow). Figure 3b,c shows the LRM images when input field was launched at the corner. Both of them showed zigzag bright dashed lines and symmetric to each other. Some inconspicuous illuminations were observed between these bright patterns. The angle of refraction is about 40°. Figure 3 A multimode waveguide excited by NFES. (a) Leakage radiation image when the fiber tip was at the center of the waveguide. The red arrow shows the location of intensity was spitted into 50:50. (b, c) Leakage radiation images when the fiber tip was located at two different corners of the waveguide. (d to f) The calculated optical field distributions (E z ) for near-field excitation at different positions, (d) at the center of waveguide, (e, f) and at two different corners. To understand these properties, we calculated the plasmonic modes (E z ) by using 3D-FDTD method. The calculation fields were shown in Figure 3d,e,f). In these simulations, a 300-nm-hight, 4.6-μm-width, and 30-μm-length dielectric stripe with a refractive index of 1.61 was placed on 100-nm-thick silver film coated on a glass substrate.

F-ratios of all three models were highly significant in all the age groups, Mocetinostat ic50 except the first model (control variables only) in the youngest age group. Standardized coefficients (Beta) and the percentages of explained variance of each model are shown in Table 3 for each age group separately. The models show a rather good fit: between 53 and 65% of the variance in job satisfaction was explained. The job demands explain about 15% of the variance in job

satisfaction in all the age groups. Addition of the job resources yields an increase of on average 35% of the variance explained. The second model (control variables and job demands) shows that more problems with workload and more conflicts at work are associated with lower job PXD101 concentration satisfaction in all the age groups. In the final models (control variables, job demands and job resources), problems with workload is no longer associated with job satisfaction. Especially, skill discretion and to a lesser extent relation with colleagues are associated with job satisfaction. More skill discretion (i.e. the possibility to use all

ones knowledge and skills at work) and better relation with colleagues are associated with more job satisfaction. NVP-HSP990 order Among 45- to 54-year olds, more autonomy is also associated with more job satisfaction, while in the oldest age group also opportunities for further education and support from supervisor show a significant positive association. Discussion The purpose of the present study was to explore differences and similarities in work characteristics [i.e. job demands, job resources and other (work) characteristics] between employees divided into four different age groups. In addition, by applying regression analyses, determinants of job satisfaction were investigated as job satisfaction is known to be one of the variables associated with early retirement (Sibbald et al. 2003) and intention to drop-out (Irvine and Evans 1995; Karatepe 2007; McCarthy 2007). Both research questions are

discussed separately below. Differences Vorinostat concentration and similarities in work characteristics The answer to the first research question is not straightforward. It depends on the way the results are looked at. In 17 out of the 20 work characteristics analysed, mean scores were either satisfying or disappointing in all the age groups (see Table 2). So, concordance was found regarding the mean scores using the chosen cut-offs (>3.5 for positively phrased variables and ≤2.5 for negatively phrased variables, respectively). Nonetheless, the results showed small but statistically significant differences between the four age groups with regard to many work characteristics. In addition, the higher standard errors in both the youngest and the oldest age group indicate larger in-group differences among the youngest and the oldest respondents.

A single crossover between the regions of homology leads to a functional tetA gene. Plasmids pYA4463 and pYA4590 were constructed to test intraplasmid recombination (Figure 1 panel A). Plasmid pYA4463 carries two truncated tetA genes (5′ end and 3′end), which have CH5183284 price 466-bp of tandemly repeated sequence. An intramolecular recombination event can delete one of the repeats resulting in an intact tetA gene, thereby recreating the structure of plasmid pACYC184 (Figure 1 panel A). Theoretically, intermolecular recombination may occur between two pYA4463 molecules to form a plasmid dimer with a functional tetA gene (Figure 1 panel C). Plasmid pYA4590 contains a 602-bp tetA sequence duplication separated by a

1041-bp kan cassette. The intramolecular recombination product is equivalent to pACYC184. The intermolecular recombination product is a dimer plasmid containing an intact tetA gene (Figure 1 panel C). Plasmids pYA4464 and pYA4465 carry the 3′tet gene and 5′tet gene, respectively (Figure 1). The Rec+ Salmonella strain χ3761 carrying either plasmid individually was sensitive to tetracycline. There is 751-bp of tetA DNA in common between the two truncated tetA genes. Recombination between the two plasmids creates a hybrid plasmid containing an intact selleck compound tetA gene (Figure 1 panel C). Intraplasmid recombination products To verify the recombination products, plasmid DNA was prepared

from tetracycline resistant (TcR) single colonies derived from χ3761(pYA4463), χ3761(pYA4590) and χ3761(pYA4464, pYA4465). Plasmids extracted from TcR clones of χ3761(pYA4463) were digested with XbaI and SalI. Theoretically, XbaI/SalI digestion of pYA4463 will yield two fragments (3524 bp and 1187 bp), pACYC184 will yield two fragments (3524 bp and 721 bp) and pYA4463 dimer will yield four fragments (3524 bp, 3524 bp, 1653 bp and 721 bp). The results (Figure 3A) showed that digestion of all 16 TcR clones yielded a 721-bp band, indicating either a pYA4463 dimer or a plasmid equivalent to Nintedanib (BIBF 1120) pACYC184. Three clones (lane 1, 5 and 10) yielded the pYA4463 dimer-specific 1653-bp band. Therefore, we conclude that the other 13 clones recombined to form the pACYC184-like

structure. Of note, several clones (2, 13-16) also yielded the 1187-bp pYA4463-specific band, suggesting that the original plasmid (pYA4463) and its recombination product (pACYC184-like) could coexist in the same bacterial cell. Figure 3 Verification of plasmid recombination product by agarose gel separation. (A) Plasmid DNA was p38 MAPK activation isolated from TcR clones derived from χ3761(pYA4463) and digested by XbaI and SalI. (B) Plasmid DNA was isolated from TcR clones of χ3761(pYA4590) and digested by KpnI and EcoRI. (C) Plasmid DNA was isolated from TcR or TcS clones of χ3761(pYA4464, pYA4465). The purified plasmids were digested with NcoI and BglII. Plasmids extracted from TcR clones of χ3761(pYA4590) were digested with KpnI and EcoRI.

They include type II PKS classes such as keto synthase (KS), chain length factor (CLF), acyl carrier protein (ACP), keto reductase (KR), aromatase (ARO), cyclase (CYC), keto synthase III (KSIII), acyl CoA ligase (AL), acyl transferase (AT), malonyl-CoA: ACP transacylase (MCAT), and thioesterase (TE). We performed homology based clustering analysis for the sequences of each type II PKS class based on sequence similarity and biosynthetic function because several classes of type II PKSs such as KR, ARO and CYC have various

different types of subclasses [4, 14] and the Pfam search tool [15] and the Conserved Domain Napabucasin cost Database (CDD) server of NCBI [16] often failed to identify domains in type II PKS protein sequences (see Additional file 1: Table S3). The sequences of each type II PKS class were grouped into clusters using the BLASTCLUST from the BLAST software package [17]. The number of cluster is determined when type

II PKSs with different biosynthetic function were accurately separated. The subclasses determined by the sequence clustering analysis matched well with the known functional subclasses reported in literature for KR, ARO, and CYC. There was no evidence showing separate why functional groups in KS III class yet but our analysis showed see more that the sequence-based subclasses of KS III have discriminating patterns

as significant as the subclasses of other PKS domains. We maintain these subclasses of KS III as the potential subgroups of KS III in our study. We could confirm that the pattern of sequence conservation in C7 KR cluster is different from that of C9 KR cluster. We also could confirm that ARO clusters agreed well with previously known subgroups such as a monodomain and two didomain types. The N-terminal and C-terminal domain types of didomain aromatase and monodomain types of aromatases from literature are mapped to ARO subclasses a, b, and c, respectively [18]. In addition, CYC clusters well correspond to previously reported phylogenetic analysis PF-3084014 mouse result of type II PKS tailoring enzymes, which shows that the ring topology of aromatic polyketide correlates well with the types of cyclases [4]. As a result, we identified that 11 type II PKS classes were clustered into a total of 20 types of subclasses with distinct biosynthetic function and different average length of domain sequences as shown in Table 1 (see Additional file 1: Table S4).

Br.001/002. This sub-group is a major presence in relationship to our world-wide collection since 70% of all the isolates and most of the diversity for this sub-group were in this Chinese collection. These results suggest that the

A.Br.001/002 cluster may have CB-839 cost originated in China. Finally, the Ames and Ames-like strains in Texas are descended from common ancestors in Inner Mongolia in China as an extension of this sub-group. It is curious that this lineage would become established in Texas, and perhaps Louisiana, and not in Europe. This leaves behind a missing historical gap within the phylogeography of the Ames lineage. Methods B. anthracis isolates The 191 B. anthracis isolates from China used in this study were previously isolated from a variety of Selleckchem Screening Library sources and provinces in China (see Additional file 1). One hundred and fifteen isolates were from Xinjiang Province in western China including 107 isolates from soil samples. selleckchem The remainder of the isolates were recovered from the following provinces with the number of isolates in parenthesis: Hebei (10), Gansu (8), Henan (2), Inner Mongolia (10), Jiangxi (1), Liaoning (26), Sichuan (1) and 18 isolates where the province of origin was not known. In addition to the 107 soil samples from Xinjiang Province isolates were obtained from the following sources: soil (15 additional), air (4), bovine (3), buffalo (1) fur (2), human (25), laboratory (1), marmot (1), sheep (3), swine

(3) and unknown sources (26). In addition to the Chinese isolates there are 6 isolates that were used to describe Figure 4[9, 10] and an additional 5 isolates that were obtained from the CDC as part of the “”Brachman Collection”" (CDC ID # 34064, 34279, 402, 482, 490). All 11 of these isolates belong to the Ames sub-lineage and all were isolated in Texas between

1959–2007. This analysis also includes the original Ames strain that was isolated in 1981 from bovine in Jim Hogg County. All isolates were initially genotyped Adenosine for a B. anthracis species-specific plcR nonsense mutation that has been suggested as being necessary for stabilization of the virulence plasmids [18]. This single nucleotide polymorphism appears to be diagnostic for B. anthracis [19]. In this study the ancestral State for this marker was used to root the B. anthracis SNP tree to the older and more diverse B. cereus/B. thuringiensis tree. DNA was isolated from each of the 191 isolates as previously described [5]. CanSNP Genotyping TaqMan™ -Minor Groove Binding (MGB) allelic discrimination assays were designed for each of 13 canSNPs and have been described in great detail by Van Ert et al. [5]. The genomic positions for each canSNP and the primer sequences and probes for each site can be found in Supplemental Tables 4 and 5 in the Van Ert et al. [5]. MLVA Genotyping Multiple Locus Variable Number Tandem Repeat (VNTR) Analysis (MLVA) was used to determine the overall diversity of the isolates within each sub-group and sub-lineage.

In this study, we hypothesize that the direct intra-tumoral injection of zinc could be a safe and efficacious treatment for prostate cancer. To our knowledge, this is the first examination of intra-tumoral zinc delivery as a treatment strategy for prostate cancer, and we feel that these data form powerful preliminary evidence indicating that such a minimally invasive strategy could be efficacious. Furthermore, because of the preferential accumulation of learn more zinc in prostate tissue, it is conceivable that such a strategy could be entirely free of the debilitating and dose-limiting side effects typical of other cancer chemotherapeutics. Methods Cell lines

PC3, DU148, LNCaP cells were originally obtained from ATCC (Rockville, Maryland, USA). Cells were maintained at 37°C, 5% CO2 and 95% humidity in DMEM (CellGro, Herndon, Virginia, USA)

supplemented with 10% (v/v) heat inactivated fetal bovine serum (BioWhittaker, Walkersville, Maryland, www.selleckchem.com/products/selonsertib-gs-4997.html USA), 2 mM L-glutamine and 100 units/ml penicillin and 1000 ug/ml streptomycin (Invitrogen, Carlsbad, California, USA). Animals NOD/SCID mice at 8 weeks of age were purchased from Charles River Laboratories (Wilmington, Massachusetts, USA) and were housed at the Saint Louis University comparative medicine facility. Animals were allowed to acclimate for 2 weeks prior to experimentation. The animals were under the care of a staff veterinarian and managed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Xenografts PC3 cells grown to 70% confluence were harvested and injected in the dorsum of animals subcutaneously. Each inoculum consisted of 100 μL of cell suspension at a concentration of 107 cells/ml in phosphate-buffered saline. Tumors were allowed to grow to a size of 300 mm3 prior to intra-tumoral Flavopiridol (Alvocidib) injection. Tumors were injected with 200 μL of 3 mM zinc acetate solution every 48 hours. Tumors were measured every 2–3 days with digital calipers. Tumor volume was determined using the following formula: Volume = Length × Width2. Zinc Measurements

Zinc was quantified in serum and tissues using the TSQ fluorophore (Invitrogen, Carlsbad, California, USA). 50 mM TSQ was prepared in 10 mM Tris buffer (ph = 8.0). TSQ was added to samples and standard zinc solutions to a final concentration of 10 μM in black round-bottom 96 well plates. Endpoint fluorescence was read on a Spectfluor with excitation wavelength of 360 nm and emission wavelength of 535 nm. Tissue zinc levels were measured similarly, after weighing and homogenizing tissue in water by repeated freeze/thaw cycles. MTT Assay Cell viability was determined via MTT assay. Briefly, media was aspirated from cells grown in 6 well Selleck mTOR inhibitor plates and 1 ml of MTT (1 mg/ml) solution was added. After 1 hour incubation, MTT solution was aspirated and 0.04 N HCL was added to solubilize the cells and absorbance at 540 nM was measured.

Follow-up The total cohort was followed for mortality until 30 April 2006. By means of the Dutch Municipal Population Registries, information was collected on the vital status of each study subject. For deceased workers, the underlying cause of death

was obtained from the Central Bureau of Statistics. Ascertainment of vital status and causes of death The procedures that were applied to obtain the vital status and the causes of death were similar to the previous study. The municipal population registries (about 460 in The Netherlands in 2006) were requested to provide information on the whereabouts of the workers that were included in this study. For workers who had moved from one municipality to another, the new municipality was requested to provide vital status information on that particular worker. This process was repeated after each notification PF-3084014 that a person had moved. In this way, all of the 570 ex-workers were traced. Vorinostat supplier Another route for identification of vital status was by consulting a special registry for persons

who had left The Netherlands by means of emigration. It was noted that quite a lot of people who had emigrated during some time in their lives returned to The Netherlands after retirement. Checking the data provided by this registry revealed additional information on former workers. As a result, these Androgen Receptor Antagonist datasheet persons were no longer considered lost to follow-up and their person years were calculated and added to the total person years of follow-up. (More detailed information on vital status is shown in Table 1.) Table 1 Vital status ascertainment on 1 May 2006 for 570 workers exposed the dieldrin and aldrin between 1 January 1954 and 1 January 1970 Vital status at end date of follow-up Follow-up until 1 January 1993 Follow-up until 1 January 2001 Follow-up until 1 May 2006 N (%) N (%) N (%) Alive 402 70.5 335 58.8 297 52.1 Emigrated 35 6.2 47 8.2 38 6.7 Lost to follow-up 15 2.6 17 3.0 9 1.6

Deceased 118 20.7 171 30.0 226 39.6 Number of person-years at risk 16,297.28   19,704.56   21,702.0   Total group 570 100 570 100 570 100 In the last step in identifying the individual causes of death for all the deceased former employers death certificate data was Buspirone HCl retrieved from the Central Bureau of Statistics (CBS). The CBS receives a copy of all Dutch death certificates after a person’s death. After the receipt of the death certificates, the causes of death are coded by trained nosologists and computerized to accumulate the annual vital statistics, which are presented by causes of death. For all deceased workers, the cause of death was identified in this database. Statistics The observed cause-specific mortality of the cohort was compared with the expected number based on age and time interval cause-specific mortality rates of the total male Dutch population.

The main oxidases for the culture conditions we used (LB broth, 37°C, aerobic growth) include cytochrome bo oxidase, cytochrome bd I and II oxidases [18]. To determine if and which oxidase or oxidases contribute to the ATP detected in the culture supernatant, we obtained a panel of mutants that each contained a deletion mutation in one of the subunits encoding the terminal oxidases [19] [Coli Genetic Stock Center, Yale University]. The growth properties and ATP levels in the culture

supernatant from each mutant were determined (Table 3). All strains of terminal oxidase mutants grew normally under the assay condition, and the only exception was the cytochrome bd-I oxidase mutant ∆cydB that displayed a growth delay in the log phase (Table 4 selleck kinase inhibitor and data not selleck inhibitor shown). The peak extracellular ATP level of the ∆cydB mutant at 6 hours of incubation was very low at 1.3 ± 2.2% of that of the see more parental strain. However; because of the growth defect of the ∆cydB mutant it was not possible to distinguish if the decreased ATP level was caused directly by the lack of the cytochrome bd I oxidase activity or indirectly by the slow growth of the ∆cydB mutant. Therefore the ∆cydB mutant was not analyzed further. In contrast to the cytochrome bd-I oxidase mutant ∆cydB, all mutants of the cytochrome bo oxidase and the cytochrome bd II oxidase grew normally (data not shown). The peak extracellular ATP levels

in mutants lacking one of the subunits of cytochrome bo oxidase (∆cyoA, ∆cyoC and ∆cyoD mutants) ranged from 26.1% to 36.6% of that of the wild type level (p < 0.05, Student’s t-test). The peak ATP level from the mutant lacking cytochrome bd II oxidase (∆appC) was 94.8 ± 2.5% of that of the parental strain; the difference is small but is statistically significant (p < 0.05, Student’s t-test) (Table 4). Table 4 Peak ATP levels

Loperamide in culture supernatant of terminal oxidase mutants of E. coli Enzyme Mutant Growth property % of the WT level p, student’s t-test Cytochrome bd-I oxidase ∆cydB Defective 1.3 ± 2.2 < 0.05 Cytochrome bd-II oxidase ∆appC Normal 95.0 ± 2.5 < 0.05 Cytochrome bo oxidase ∆cyoA Normal 25.0 ± 3.7 < 0.05   ∆cyoC Normal 36.6 ± 1.5 < 0.05   ∆cyoD Normal 26.1 ± 5.4 < 0.05 Results are the average of three assays with standard deviations. The cytochrome bo oxidase mutants of E. coli were analyzed further to characterize the extracellular ATP levels during growth. While the extracellular ATP levels in the ∆cyo mutants displayed time courses similar to that of the parental strain, the peak levels were significantly lower than that observed in the parental strain (Figure 4A). These results suggest that cytochrome bo oxidase contributes to the extracellular ATP even though it had no significant influence on the growth of E. coli under the conditions used for the assay (LB broth, 37°C, with shaking).