Sham rTMS stimulation (n = 3) followed the exact same procedure PLX3397 in vivo described above, except the coil surface was held at 90° perpendicular to the surface of the scalp to direct the magnetic field away from the skull. Animals received a total

of seven rounds of real or sham rTMS [Round (R)1 to R7], which were defined each as a total of 10 days of stimulation (5 days on, 2 days off, repeated once more before the next rTMS round started) delivered across 2 weeks. During the 2 weeks prior to rTMS procedures, all felines were acclimated to the sound of rTMS pulses and accustomed to remain in a veterinary bag to ensure no distress during stimulation. No signs of abnormal behavior (e.g., aggression, anxiety, stress, reductions in agility or increases in reclusiveness) were noted during or after the stimulation. The study follow-up was divided into five phases:

VE-821 pre-lesion (Phase I), immediate post-lesion (days 1–2 post injury; Phase II), spontaneous recovery (days 2–70 post-injury; Phase III), rTMS treatment (R1 to R7); (Phase IV), and Post-rTMS treatment follow-up of at least 6 weeks (Phase V). Visuospatial performances assessed at the end of those five phases were taken as milestones to define the status of the animals’ behavioral recovery. The day of the surgically induced focal brain injury served as a zero-point time reference. The peak of spontaneous recovery level right before the onset of the rTMS therapy (i.e., before the first rTMS session of R1) is referred as pre-rTMS performance. Measurements gauged at the end of the seven rounds of rTMS are titled ‘rTMS R7 performance’. Finally, measurements recorded after the discontinuation of the treatment are termed ‘post-rTMS performance’. Throughout the rTMS phase each daily session of stimulation was immediately followed by a 15-min testing session composed of a single block of trials for each of the three above-mentioned visuospatial tasks (Static, Moving 2 and Moving 1). Every 7 days and prior to the next rTMS stimulation session, animals received three blocks of trials for each of the three above-mentioned paradigms. In total, animals completed a total of seven rounds

of rTMS, i.e., seventy daily sessions of stimulation, across a total of 14 weeks of treatment. At the ID-8 end of the rTMS phase, the durability of the rTMS effects in the absence of treatment was assessed in all animals for 6 weeks following the last stimulation session. This was done through weekly evaluations identical to those performed during the rTMS treatment phase (Valero-Cabré et al., 2005, 2006). The evaluation of the rTMS effects was made against the backdrop of results from our laboratory (Rushmore et al., 2010) and from other studies (Huxlin & Pasternak, 2001, 2004; Sherk & Fowler, 2002; Das et al., 2012) which show that unilateral ibotenic acid lesion-induced deficits are consistent and robust, and spontaneous recovery is observed only if intensive specific training is instituted.

94±0.52 g/dL; P<0.038). The rate of BMI≥28 kg/m2 was significantly higher in the HIV-monoinfected group than in the HIV/HCV-coinfected group (21%vs. 4.48%, respectively; P=0.05). All statistical differences between the groups remained significant after controlling for age, gender, CD4 cell count, viral load, injecting of illicit drugs and race, using manova. There

were no significant differences in Selleck GDC 941 the use of ART, BMI, haemoglobin, haematocrit or bilirubin between these two groups. Table 1 shows the proportion of patients using alcohol, cigarettes and illicit drugs, including injected drugs, in the two groups. Alcohol was habitually consumed by 54.7% of participants, but there were no significant differences between the two groups in the proportion of participants who used alcohol, either

by answering ‘yes’ or ‘no’ to a question about consuming alcohol (57.9% in the coinfected group answered yes vs. 54.7% in the HIV-monoinfected group; P=0.562) or answering ‘yes’ to a question about consuming >2 alcoholic drinks daily (17.5% in the coinfected group answered yes vs. 12.6% in the HIV-monoinfected group; P=0.367). Cigarette smoking was reported by 83.3% of the participants, with frequent cigarette smoking (>1 pack daily) reported by 70.2%; there was also no difference between the HIV/HCV-coinfected and the HIV-monoinfected groups in the proportion of participants smoking cigarettes. There were no significant differences in use of illicit drugs between the two groups, with the exception of injected drugs. There was a small number of injecting drug users

selleck products (n=4), and all of them were in the HIV/HCV-coinfected group (P=0.045). We adjusted for this variable in the regression models. Random subsamples of the two groups were selected, one including 40 HIV/HCV-coinfected and the other 38 HIV-monoinfected participants, for more detailed studies. Oxidative stress was represented by the plasma level of MDA. MDA levels were significantly elevated in those with triglycerides ≥150 mg/dL (β=0.47, P=0.0029) compared with those with normal triglyceride levels, and showed a strong, but nonsignificant, trend towards being elevated in those who were obese (BMI≥28 kg/m2; β=0.28, P=0.07) compared with those with BMI<28 kg/m2. ioxilan As shown in Table 3, the mean MDA in both the HIV/HCV-coinfected and the HIV-monoinfected groups were higher than the normal reference value of <1.3 nmol/mL. MDA was significantly higher in HIV/HCV-coinfected participants (1.897±0.835 nmol/mL) than in those who were HIV-monoinfected (1.344±0.223 nmol/mL; P=0.006). The HIV/HCV-coinfected group also had significantly lower levels of plasma antioxidants, including vitamin A (39.5±14.1 vs. 52.4±16.2 μg/dL in the monoinfected group; P=0.0004), vitamin E (8.29±2.1 vs. 9.89±4.5 μg/mL, respectively; P=0.043) and plasma zinc (0.61±0.14 vs. 0.67±0.15mg/L, respectively; P=0.016), than the HIV-monoinfected group.

Such cooperative catabolism has been reported for the microbial degradation of chloronitrobenzenes and atrazine (Park et al., 2002; Smith et al., 2005). Further analysis of the enrichment culture media in this study could lead to the isolation of novel microorganisms

PF-02341066 mouse that promoted metabolism in the latter half of the DON-degradation pathway. The second difference is the ability to express DON-degradation activities under preincubation conditions. The Gram-positive strains needed preincubating in DON-containing media for maximal expression of degradation activities, suggesting that they possess some regulatory system for the expression of DON-degrading enzyme or DON-uptake machineries. Together with the finding that Gram-positive

strains can assimilate DON, we postulate that the Gram-positive strains are native DON-degraders whose DON-assimilating abilities play a key role in their survival in nature. By contrast, the Gram-negative strains might be casual degraders, given that they did not assimilate DON or need preincubating in DON-containing media for expression of DON-degrading activities. Note that it was not on mineral media containing DON as carbon source but on complete media such as diluted NA and R2A agar plates that we isolated DDBs. Use of the complete media in our study resulted in the successful isolation of the casual DON-degraders. The third difference is the DON metabolites produced. HPLC analysis revealed that the two Gram types produced different DON metabolites, suggesting differences in the DON-degradation GDC-0068 mw pathways. The identification of DON metabolites is necessary for understanding the bacterial DON-degradation pathways. We found that all the strains produced P-type ATPase 3-epi-DON as an intermediate of DON degradation, suggesting that the DON-degradation pathways of the two Gram types of bacteria were in part identical. We are particularly

interested in the enzyme responsible for the transformation of DON to 3-epi-DON. This unique enzyme, which only the aerobic DDBs possess, might be one of the reasons why the bacteria belong to phylogenetically restricted groups. Our results that the aerobic DDBs form two phylogenetically distant bacterial groups and that the degradation phenotypes differ between the Gram types suggest the independent evolution of two aerobic DON-degradation mechanisms. Our findings may also be useful for analysing the divergency of DON-metabolizing enzyme genes as well as their significance in evolution. This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Research project for ensuring food safety from farm to table MT-3209). We thank M. Imai for technical assistance. We also thank H. Nakagawa (National Food Research Institute) for technical advice about the enrichment medium. “
“In Actinomyces oris T14V, sortase SrtC1 mediates the assembly of type 1 fimbriae.

Such cooperative catabolism has been reported for the microbial degradation of chloronitrobenzenes and atrazine (Park et al., 2002; Smith et al., 2005). Further analysis of the enrichment culture media in this study could lead to the isolation of novel microorganisms

Target Selective Inhibitor Library nmr that promoted metabolism in the latter half of the DON-degradation pathway. The second difference is the ability to express DON-degradation activities under preincubation conditions. The Gram-positive strains needed preincubating in DON-containing media for maximal expression of degradation activities, suggesting that they possess some regulatory system for the expression of DON-degrading enzyme or DON-uptake machineries. Together with the finding that Gram-positive

strains can assimilate DON, we postulate that the Gram-positive strains are native DON-degraders whose DON-assimilating abilities play a key role in their survival in nature. By contrast, the Gram-negative strains might be casual degraders, given that they did not assimilate DON or need preincubating in DON-containing media for expression of DON-degrading activities. Note that it was not on mineral media containing DON as carbon source but on complete media such as diluted NA and R2A agar plates that we isolated DDBs. Use of the complete media in our study resulted in the successful isolation of the casual DON-degraders. The third difference is the DON metabolites produced. HPLC analysis revealed that the two Gram types produced different DON metabolites, suggesting differences in the DON-degradation BMS-907351 in vivo pathways. The identification of DON metabolites is necessary for understanding the bacterial DON-degradation pathways. We found that all the strains produced Selleck Decitabine 3-epi-DON as an intermediate of DON degradation, suggesting that the DON-degradation pathways of the two Gram types of bacteria were in part identical. We are particularly

interested in the enzyme responsible for the transformation of DON to 3-epi-DON. This unique enzyme, which only the aerobic DDBs possess, might be one of the reasons why the bacteria belong to phylogenetically restricted groups. Our results that the aerobic DDBs form two phylogenetically distant bacterial groups and that the degradation phenotypes differ between the Gram types suggest the independent evolution of two aerobic DON-degradation mechanisms. Our findings may also be useful for analysing the divergency of DON-metabolizing enzyme genes as well as their significance in evolution. This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Research project for ensuring food safety from farm to table MT-3209). We thank M. Imai for technical assistance. We also thank H. Nakagawa (National Food Research Institute) for technical advice about the enrichment medium. “
“In Actinomyces oris T14V, sortase SrtC1 mediates the assembly of type 1 fimbriae.

The remaining patients had undergone one or several treatment changes. The majority of these treatment changes (49%) were made rationally (e.g. because of suspected treatment failure or drug toxicity), in 12% of the cases the treatment changes were irrational (e.g. because of cost or interrupted drug supplies) and 17% of the changes involved treatment interruption (often because of cost or interrupted drug supplies) (Table 2). CDC stage and self-reported adherence levels were not significantly correlated to resistance, whereas CD4 cell counts and plasma HIV RNA levels were PI3K inhibitor significantly correlated to resistance. However, it should

be pointed out that these CD4 and HIV RNA levels frequently were not obtained concomitantly with the resistance test and often not even while the patient was Selleck isocitrate dehydrogenase inhibitor on the same therapy as when the resistance test was carried out. Multiple logistic regression was used to identify variables that were independently associated with the presence of genotypic resistance. The final model includes as categorical variables: route of infection, start of therapy within the national treatment programme (yes/no) and type of virological failure (virological, immunological or clinical). Number of treatment changes and years on therapy were included as continuous variables. Age (adult vs. child) was

not included as a variable because it largely overlapped with route of infection. CD4 cell counts and HIV RNA were not included because results were not available for all patients and often were obtained long before the sample used for resistance testing. The multivariable analysis identified the following variables as independently associated with resistance: type of treatment failure [virological failure (OR=1) vs. immunological failure (OR=0.11; 95% CI 0.030–0.43) vs. clinical failure (OR=0.037; 95% CI 0.0063–0.22)]; route of transmission (OR=42.8; 95% CI 3.73–491); http://www.selleck.co.jp/products/Fludarabine(Fludara).html and years on therapy (OR=1.81;

95% CI 1.11–2.93). This indicates that VL testing was needed to correctly identify patients with treatment failure attributable to resistance. As shown in Table 3, genotypes predicted to have reduced susceptibility to at least one NRTI were observed in 98 of 138 patients (71%; 95% CI 63–78%); to at least one NNRTI in 96 patients (70%; 95% CI 61–77%); and to at least one PI in 51 patients (37%; 95% CI 29–45%). Dual and triple class resistance was very common. Thus, triple-class drug resistance was documented in 37 of the 138 study subjects (27%; 95% CI 20–35%) and dual-class drug resistance was detected in 59 patients (43%; 95% CI 34–51%), whereas only 16 (12%; 95% CI 7–18) of the patients showed single-class resistance.

Although preliminary, these results suggest that genetic modification of a Mesorhizobium strain can improve its symbiotic performance under salt stress and indicate that ACC deaminase can play an important role in facilitating plant–rhizobium interaction under salinity conditions. “
“Quorum sensing (QS) is a system of cell-to-cell

communication by means of intercellular signaling molecules to coordinate a set of targeted gene expression or repression in many Gram-negative bacteria; it plays important roles for bacteria in adaptation to adverse environmental conditions. GDC-0199 mouse In this study, we first demonstrated that Microcystis aeruginosa PCC-7820 could produce QS-related signal acylated homoserine lactones (AHLs) among the metabolite of axenic M. aeruginosa, based on bioassay and liquid chromatography–mass spectrometry (LC-MS) analysis. The concentration of the AHLs in the culture medium was cell density dependent and reached a maximum of 18 nM at 1.03 × 107 cells mL−1, 30 days after inoculation. The regulation mechanism of QS in M. aeruginosa and its possible role in bloom formation are discussed. Quorum sensing (QS) is a system of stimulus and response that is correlated to population density by means of inter- or intracellular

signaling molecules (autoinducers) (Kaplan & Greenberg, 1985). Many species of bacteria use QS to coordinate sets of targeted gene expression or repression that relies on the density of their local population, which experiences a concentration threshold of signal molecules such as N-acyl-homoserine selleck lactone (AHL), cyclic thiolactone, furanosylborate, methyl dodecenoic acid, hydroxypalmitic acid methyl ester, and farnesoic acid (Dong & Zhang, 2005; Williams, 2007; von Bodman et al., Non-specific serine/threonine protein kinase 2008). QS and its mediated signals have been described in more than 70 different Gram-negative species of bacteria. However, to date, there have been a few investigations of its occurrence in cyanobacteria, which are photosynthetic Gram-negative prokaryotes. Sharif et al. (2008) indicated that the epilithic colonial cyanobacterial species Gloeothece PCC6909 had a QS system that was

mediated by a signal molecule of C8-AHL. It was suspected that QS was able to improve Gloeothece’s adaptation to environmental stress and acquire species competition advantages in the natural ecosystems. Cyanobacteria, a group of photosynthetic prokaryotes, are the dominant bloom-forming species because of its strong adaptation to environmental stress by utilization of various sensing mechanisms and intracellular signaling systems. The involvement of signal transduction and cell-to-cell cooperation indicates that a role for autoinducer-like compounds may exist in such responses (Sharif et al., 2008). In fact, such QS molecules have been reported in cyanobacterial assemblages (Bachofen & Schenk, 1998; Braun & Bachofen, 2004).

Rumen bacterium R-25, which was isolated from the rumen of sheep and classified in the group U2 (Koike et al., 2010), was used in this study. Fibrobacter succinogenes S85, which is a fibrolytic bacterium, was purchased from American Type Culture Collection. Selenomonas ruminantium S137, which was isolated from sheep rumen (Sawanon et al., 2011), was used as a metabolite (lactate and succinate) utilizer. Basal medium was prepared anaerobically according to the method of Bryant (1972) to have the following composition (100 mL−1):

7.5 mL of mineral MEK inhibitor solutions I and II (Bryant & Burkey, 1953), 0.1 mL of 0.1% resazurin, 40 mL of clarified rumen fluid, 39 mL of distilled water, 1 mL of 5% l-cysteine-HCl·H2O, and 5 mL of 8% Na2CO3. Rice straw was air-dried in an oven at 60 °C, ground to pass through a 1-mm sieve, and used as a substrate to measure fiber digestion. Strain R-25 and S. ruminantium S137 were grown to the end of log phase at 39 °C in basal medium containing cellobiose and glucose [0.5% (w/v) each] as carbon sources. After three passages, the cultures were centrifuged

(2300 g, 4 °C, 10 min) to pellet the bacteria. Anaerobic dilution solution (Bryant & Burkey, 1953) was added to the pellet to resuspend the bacteria to an optical density at 660 nm (OD660 nm) of 0.2. Fibrobacter succinogenes S85 was grown for 48 h in basal medium containing 1.0% (w/v) rice straw as the sole carbon source. After three passages, the culture was centrifuged (377 g, 4 °C, 1 min) to separate the rice straw particles and supernatant Selleck Tofacitinib containing bacterial cells (Minato & Suto, 1978). The supernatant was collected in another sterile tube and centrifuged (2300 g, 4 °C, 10 min) to pellet the bacteria. The pellet was resuspended as above, and these OD-adjusted cell suspensions were used as inocula. Each inoculum was added at 0.1 mL to 10 mL of basal medium containing 0.1 g of rice straw as the sole carbon source. Six test tubes were prepared for respective mono- and cocultures and incubated at 39 °C under anaerobic condition. On the basis of a previous

study (Shinkai et al., Non-specific serine/threonine protein kinase 2009), samples were collected at three time points after incubation; 0 h (corresponding to inocula), 48 h (middle of digestion) and 96 h (endpoint of digestion). Samples were collected from three of six test tubes at 48 h, and the rest of three test tubes were incubated until 96 h. Tubes with no inocula were prepared as a blank and treated in the same manner. After 96 h incubation, the cultures were cooled on ice for 30 min to detach bacterial cells from fiber particles (Minato & Suto, 1978) and centrifuged (377 g, 4 °C, 10 min), and the supernatant containing bacterial cells was collected. The residue was washed with 10 mL of 0.1 M potassium phosphate buffer and re-centrifuged (2300 g, 4 °C, 10 min). The washed residue was dried at 105 °C for 48 h and weighed to calculate dry matter (DM) digestion.

Of 467 participants enrolled, 361 (77.3%) completed questionnaires and had sufficient paired pre- and post-travel serum for testing; 58 (12.4%) were lost to follow-up; 21 had insufficient blood for testing; and 27 were excluded. There were 214 females (59.3%) and 147 males (40.7%). Pre- and post-travel specimens were collected at a median of 29 days prior to travel (range 0–265 days) and a median Talazoparib manufacturer of 6 days following return to Australia (range 0–31 days). The

median travel duration was 21 days (range 7–326 days) with 74% <30 days. The major reasons for travel were tourism (73.1%), business (17.7%), and visiting friends and relatives (VFRs, 4.71%). Table 1 shows the demographic data and total traveler-days for the top 10 countries visited. Four of the 361 travelers (1.1%) demonstrated serological evidence of HCV infection. Two were past infections and two travelers had evidence of seroconversion, representing an incidence density of 1.8 new infections per 10,000 traveler-days (95% CI: 0.22–6.53). Both travelers with seroconversion were asymptomatic, and likely acquired PD-166866 datasheet their infection in Vietnam (n = 1) or Thailand (n = 1) during short-term travel (14 days duration each). The traveler to Thailand was a 24-year-old female tourist who visited Koh Samui and Bangkok. The traveler to Vietnam (a 50-year-old male) traveled to the cities of Hanoi and Ho Chi Minh. None of the

four HCV seropositive travelers were viremic on testing of either pre- or post-travel sera. Six of the 361 travelers (1.77%) were anti-HBc antibody positive, consistent with evidence of HBV infection. Five of these infections were present before travel. One traveler showed evidence of seroconversion [pre-travel serum negative for anti-HBc immunoglobulin G (IgG) and IgM, anti-HBs, anti-HBe, HBsAg, and HBV DNA; post-travel anti-HBc IgG positive

but IgM negative, anti-HBs positive, HBsAg, HBeAg, anti-HBe, and HBV DNA negative]. The serological profile was consistent with self-limited primary infection. This traveler, a male aged 40, had evidence of seroconversion consistent with acquisition of HBV during his short business trip to China. He had his pre-travel blood collected 31 days prior to departure, traveled through China for 22 days, and ID-8 had post-travel bloods taken 8 days post return to Australia. HBV PCR testing of sera from the entire cohort was negative; 56% of travelers (202/361) were HBV immune (anti-HBs ≥10 mIU/mL). The incidence density of HBV infection in nonimmune travelers was calculated as 2.19 per 10,000 traveler-days (95% CI: 0.07–12.19). This retrospective cohort study demonstrates that travelers are at risk of both HBV and HCV infection, and is the first to quantify the risk of HCV infection in travelers. While the number of seroconversions was small the identification of two HCV and one HBV seroconversion is notable and indicates potential exposure to other blood and bodily fluid-borne infections such as HIV.

Of 467 participants enrolled, 361 (77.3%) completed questionnaires and had sufficient paired pre- and post-travel serum for testing; 58 (12.4%) were lost to follow-up; 21 had insufficient blood for testing; and 27 were excluded. There were 214 females (59.3%) and 147 males (40.7%). Pre- and post-travel specimens were collected at a median of 29 days prior to travel (range 0–265 days) and a median Selleckchem Ceritinib of 6 days following return to Australia (range 0–31 days). The

median travel duration was 21 days (range 7–326 days) with 74% <30 days. The major reasons for travel were tourism (73.1%), business (17.7%), and visiting friends and relatives (VFRs, 4.71%). Table 1 shows the demographic data and total traveler-days for the top 10 countries visited. Four of the 361 travelers (1.1%) demonstrated serological evidence of HCV infection. Two were past infections and two travelers had evidence of seroconversion, representing an incidence density of 1.8 new infections per 10,000 traveler-days (95% CI: 0.22–6.53). Both travelers with seroconversion were asymptomatic, and likely acquired see more their infection in Vietnam (n = 1) or Thailand (n = 1) during short-term travel (14 days duration each). The traveler to Thailand was a 24-year-old female tourist who visited Koh Samui and Bangkok. The traveler to Vietnam (a 50-year-old male) traveled to the cities of Hanoi and Ho Chi Minh. None of the

four HCV seropositive travelers were viremic on testing of either pre- or post-travel sera. Six of the 361 travelers (1.77%) were anti-HBc antibody positive, consistent with evidence of HBV infection. Five of these infections were present before travel. One traveler showed evidence of seroconversion [pre-travel serum negative for anti-HBc immunoglobulin G (IgG) and IgM, anti-HBs, anti-HBe, HBsAg, and HBV DNA; post-travel anti-HBc IgG positive

but IgM negative, anti-HBs positive, HBsAg, HBeAg, anti-HBe, and HBV DNA negative]. The serological profile was consistent with self-limited primary infection. This traveler, a male aged 40, had evidence of seroconversion consistent with acquisition of HBV during his short business trip to China. He had his pre-travel blood collected 31 days prior to departure, traveled through China for 22 days, and Thymidylate synthase had post-travel bloods taken 8 days post return to Australia. HBV PCR testing of sera from the entire cohort was negative; 56% of travelers (202/361) were HBV immune (anti-HBs ≥10 mIU/mL). The incidence density of HBV infection in nonimmune travelers was calculated as 2.19 per 10,000 traveler-days (95% CI: 0.07–12.19). This retrospective cohort study demonstrates that travelers are at risk of both HBV and HCV infection, and is the first to quantify the risk of HCV infection in travelers. While the number of seroconversions was small the identification of two HCV and one HBV seroconversion is notable and indicates potential exposure to other blood and bodily fluid-borne infections such as HIV.

azotoformans failed to complement the SP8 phenotype (Fig. 3a). When these plasmids were introduced into SP7 (ΔrpoN2::kan), we observed that only rpoN2 from R. azotoformans was able to restore the swimming defect of this strain (Fig. 3b). To further evaluate the ability of the different rpoN genes to complement the phenotype of the SP8 strain, we determined their capacity to restore the wild-type level of the transcriptional activity of the nifU promoter (nifUp) in the SP8 background. It has been previously shown that the activity of this promoter is mainly

dependent on RpoN1 and the bEBP NifA (Poggio et al., 2002, 2006). For this, a plasmid carrying a transcriptional fusion of the uidA gene (encoding the β-glucuronidase selleck kinase inhibitor enzyme) with nifUp was introduced into the SP8 derivative strains expressing the different rpoN genes. As expected, the rpoN genes that complemented the phenotype of the SP8 strain allowed a wild-type level of activity http://www.selleckchem.com/products/17-AAG(Geldanamycin).html of nifUp, while in the strains expressing the noncomplementing rpoNs, the activity of the nifUp was reduced

approximately 100 times (data not shown). This result suggests that the strains that showed a growth defect under diazotrophic growth conditions are unable to induce the genes required for nitrogen fixation. Together, these results suggest that rpoN1 and rpoN2 of R. azotoformans are also specialized to transcribe a particular set of genes, as occurs in R. sphaeroides. In addition, the fact that rpoN3 of R. azotoformans cannot express the σ54-dependent fli and nif genes of R. sphaeroides strengthens the notion Thiamet G that in these species rpoN3 may be specialized to transcribe a different subset of genes. With the exception of rpoN3 from R. azotoformans, all the other rpoN genes complemented either SP7 or SP8 strains, indicating that the tested rpoN1 and rpoN2 genes were being expressed in at least that condition. Nevertheless, it could be argued that the rpoN genes cloned in pRK415 could be

conditionally expressed, and R. azotoformans rpoN3 not expressed at all. To discard these possibilities, we cloned the rpoN gene from R. blasticus, and rpoN1 and rpoN3 from R. azotoformans in a construct that added a 6His-tag at the carboxy terminus of the protein. This tag allowed us to detect the resulting proteins by western blot. All the proteins were present when the cells were grown under aerobic or anaerobic N-limiting conditions (Fig. 4a and b), supporting our previous conclusions. Our results show that the orthologues of rpoN1Rs and rpoN2Rs can complement the mutants in these genes, suggesting that following duplication, a fast process of specialization occurred, after which each of the copies has maintained the characteristics that allow them to transcribe their particular set of genes. Given that rpoN1 from R.