Hydrogen oxidation by Fdh-N and Fdh-O is dependent on the accessory proteins FdhD and FdhE The fdoGHI operon encoding Fdh-O is flanked by fdhD and fdhE, both of which encode accessory enzymes required for the synthesis

of active Fdh enzymes [22, 23]. To demonstrate the dependence of the H2-oxidizing activities of both Fdhs on FdhD and FdhE, individual mutants lacking either the fdhD or the fdhE gene were analyzed under the same conditions as described above for the wild type and fdoG and fdnG mutants. All three activities were absolutely dependent on both FdhD and FdhE (Figure 4). Complementation experiments revealed that while FdhD on a plasmid fully complemented the fdhD mutation, plasmid-encoded FdhE only partially complemented

the fdhE mutation. Discussion We demonstrate here Selumetinib in vivo that both of the respiratory formate dehydrogenases Fdh-N and Fdh-O have hydrogen-oxidizing enzyme activity. Together with the three characterized [NiFe]-hydrogenases, these are the only two enzymes in E. coli crude extracts that had this activity. These results suggest that the Fdh-N and Fdh-O enzymes show a degree of non-specificity with regard to the electron donor they can use. Notably, formate and dihydrogen (CO2/formate, Eo’ = -432 mV [24]) and (H+/hydrogen, Eo’ = -414 mV) are both strong reductants. Previous studies have demonstrated that E. coli can couple hydrogen oxidation Rucaparib mouse to nitrate reduction and Hyd-1 and Hyd-2 participate in this process [25]. However, attempts to demonstrate significant hydrogen-dependent nitrate reduction in the absence of Hyd-1 and Hyd-2 did not deliver reproducible Selonsertib in vivo levels of hydrogen oxidation, presumably due to the limited

hydrogen-oxidizing Staurosporine datasheet activity of Fdh-N and Fdh-O. Nevertheless, the findings reported here might have physiological relevance in other microorganisms. For example, enzymes with subunits orthologous to FdnG are found in the obligate dehalorespiring and hydrogen-oxidizing Dehalococcoides spp., e.g. strain CBDB1, and have an associated subunit with similarity to hydrogenase membrane-anchoring subunits [26]. Rather than having a selenocysteinyl residue in their presumptive active site they have a seryl residue. It is established that in E. coli replacement of selenocysteine with serine abolishes the formate-oxidizing activity of Fdh-H [27]. Moreover, it is also clear that Dehalococcoides strain CBDB1 cannot use formate as a substrate, suggesting that this formate dehydrogenase-like enzyme might have another function. One possibility based on the findings presented here might be that it accepts H2 as substrate. As both Fdh enzymes are selenium-dependent, impaired co-translational insertion of selenocysteine prevented synthesis of either enzyme and concomitantly abolished the [NiFe]-hydrogenase-independent H2: BV oxidoreductase activity.

Microbiol Inmmunol 2004, 48:791–805. 41. Deng X, Xiao Y, Lan

L, Zhou JM, Tang X: Pseudomonas Capmatinib syringae pv. Phaseolicola mutants compromised for type III secretion system gene induction. Mol Plant Microbe Int 2009, 22:964–976.CrossRef XMU-MP-1 ic50 42. Burch AY, Shimada BK, Mullin SWA, Dunlap CA, Bowman MJ, Lindow SE: Pseudomonas syringae coordinates production of a motility-enabling surfactant with flagellar assembly. J Bacteriol 2012, 194:1287–1298.PubMedCrossRef 43. Kong HS, Roberts DP, Patterson CD, Kuehne SA, Heeb S, Lakshman DK, Lydon J: Effect of overexpressing rsmA from Pseudomonas aeruginosa on virulence of select phytotozin-producing strains of P. syringae. Phytopatol 2012, 102:575–587.CrossRef 44. Braun V, Hantke K, Koster W: Bacterial

iron transport:mechanisms, genetics and regulation. Metal Ions Biol Syst 1998, 35:67–145. 45. Visca P, Imperi F, Lamont LL: Pyoverdine siderophores:from biogenesis to biosignificance. Trends Microbiol 2006. doi:10.1016/j.tim.2006.11.004. 46. Swingle B, Thete D, Moll M, Myers CR, Schneider DJ, Cartinhour S: Characterization of the PvdS-regulated promoter motif in Pseudomonas syringae pv. tomato DC3000 reveals regulon members and insights regarding PvdS function in other pseudomonads. Mol Microbiol 2008, 68:871–889.PubMedCrossRef 47. Vasil ML: How we learnt about iron acquisition in Pseudomonas aeruginosa: a series of very fortunate events. Biometals 2007, 20:587–601.PubMedCrossRef 48. Chattopadhyay MK, Raghu G, Sharma YVRK, Rajasehharan MV, C646 manufacturer Shivaji S: Increase in oxidative stress at low temperature in an Antarctic bacterium. Curr Microbiol 2011, 62:544–546.PubMedCrossRef 49. Smirnova GV, Zakirova ON, Oktyabrskii ON: The role of antioxidant systems in the cold stress response of Escherichia coli . Microbiol

2001, 70:45–50.CrossRef 50. Palma M, DeLuca D, Worgall S, Quadri LEN: Transcriptome analysis of the response of Pseudomonas aeruginosa to hydrogen peroxide. J Bacteriol 2004, 186:248–252.PubMedCrossRef Adenosine triphosphate 51. Outten FW, Djaman O, Storz G: A suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli . Mol Microbiol 2004, 52:861–872.PubMedCrossRef 52. Zheng M, Wang X, Templeton LJ, Smulski DR, LaRosa RA, Storz G: DNA microarray mediated transcriptional profiling of the Escherichia coli response to hydrogen peroxide. J Bacteriol 2001, 183:4562–4570.PubMedCrossRef 53. Patriquin GM, Banin E, Gilmour C, Tuchman R, Greenberg EP, Poole K: Influence of quorum sensing and iron on twitching motility and biofilm formation in Pseudomonas aeruginosa . J Bacteriol 2008, 190:662–671.PubMedCrossRef 54. May TB, Shinabarger D, Maharaj R, Kato J, Chu L, Devault JD, Roychoudhury S, Zielinski NA, Berry A, Rothmel RK, Misra TK, Chakrabarty AM: Alginate synthesis by Pseudomonas aeruginosa : a key pathogenic factor in chronic pulmonary infections of cystic fibrosis patients. Clinical Microbiol Rev 1991, 4:191–206. 55.

Ishiga Y, Uppalapati SR, Ishiga T, Elavarthi MK0683 S, Martin B, Bender CL: Involvement of coronatine-inducible reactive oxygen species in bacterial speck disease of tomato. Plant Signaling and Behavior 2009, 4:237–239.PubMedCrossRef 21. Henkle-Dührsen K, Kampkötter A: Antioxidant enzyme families in parasitic nematodes. Mol Biochem Parasitol 2001, 114:129–142.PubMedCrossRef 22. Molinari S: Changes of catalase and SOD activities in the early response of tomato to Meloidogyne attack. Nematol Mediterr 1998, 26:167–172. 23. Robertson L, Robertson WM, Sobczak M, Helder J, Tetaud E, Ariyanayagam MR, Ferguson MAJ, Fairlamb A, Jones

JT: Cloning, expression and functional characterisation of a peroxiredoxin from the potato cyst GSI-IX nematode Globodera rostochiensis . Mol Biochem Parasitol 2000, 111:41–49.PubMedCrossRef 24. Jones J, Reavy B, Smant G, Prior A: Glutathione peroxidases of the potato cyst nematode Globodera Rostochiensis . Gene 2004, 324:47–54.PubMedCrossRef 25. Bellafiore S, Shen Z, Rosso MN, Abad P, Shih P, Briggs SP: Direct identification of the Meloidogyne incognita secretome reveals proteins with host cell reprogramming potential. PLoS pathogens 2008, 4:e1000192.PubMedCentralPubMedCrossRef 26. Hirao T, Fukatsu E, Watanabe A: Characterization of resistance to pine wood nematode SN-38 datasheet infection in Pinus thunbergii using suppression subtractive hybridization. BMC plant biology 2012, 12:13.PubMedCentralPubMedCrossRef 27. Santos CSS, Vascocelos MW: Identification

of genes differentially expressed in Pinus pinaster and Pinus pinea after infection with pine wood nematode. Eur J Plant Pathol 2012, 132:407–418.CrossRef 28. Shinya R, Morisaka H, Takeuchi Y, Futai K, Ueda M: Making headway in understanding pine wilt disease: What do we perceive in the postgenomic era? J Biosci Bioeng 2013, 116:1–8.PubMedCrossRef 29. Molinari S: Antioxidant enzymes in (a)virulent populations of root-knot nematodes. Nematology 2009, 11:689–697.CrossRef 30. Kikuchi T, Cotton JA, Dalzell JJ, Hasegawa K, Kanzaki N, McVeigh P, Takanashi T, Tsai IJ, Aseffa SA, Cock PJA, Otto TD, Hunt M, Reid AJ, Sanchez-Flores A, Tsuchihara K, Yokoi T, Larsson MC, Miwa J, Maule AG, Sahashi N, Jones

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Similarly, we mapped their agricultural and animal husbandry activities and the annual distribution of on- and off-farm incomes and then combined

the participatory exercise results from all four communities into a generalized seasonal calendar. While individual factors, such as the incidence of diseases and food costs differed between communities, a similar pattern of hardship could be identified in all study locations for a typical year. The core of the calendars thus reflects farmers’ general consensus of a ‘conventional’ bimodal rainy season, irrespective of the observed and perceived changes in rainfall dynamics in recent years. The ‘wheel of DMXAA datasheet hardship’, seen in Fig. 6, is a summary of these findings indicating that livelihood conditions and activities differ considerably throughout the year, rendering farmer households more or less exposed and sensitive to climate-induced stressors and with more or less capacity to cope with impacts. Interestingly, comparisons of data from the four sites show that conditions

differ more throughout the year than between locations. When integrating the results two key periods of severe livelihood hardship can be identified; January–March and October–November. Within these, January and February are the worst hardship MRT67307 purchase months because climate exposure coincides with increased sensitivity to diseases and limited buffers, due chiefly to lack of food and income

opportunities imposed by high expenditures for food, school fees, medical needs, renting of grazing land and hiring of agricultural labor. Similar conditions apply to the months of October and November but are usually less severe since households still have staple crops left from the previous harvest and can also sell newly harvested vegetables. Fig. 6 ‘Wheel of hardship’—a generalized seasonal calendar illustrating livelihood conditions Carnitine palmitoyltransferase II and stress based on participatory exercises with smallholder farmers from four communities in the LVB Fortunately, periods of recovery also exist, the main occurring between May and August. From data we learn that crops have matured and fish are abundant in lakes and selleck products streams, which means that caloric (and protein) needs are met while crops can be sold and possibly even stored. Grazing land is also lush and green, so there is no problem of extra costs for animal feed. Subsequently, families who can afford them make major household investments, including purchases of livestock, house-building materials, clothes, agricultural tools and seeds. Medical check-ups and veterinary visits are also common.

Saline was added to make the volume to 11 ml to which 2 ml diethyl ether was added and centrifuged at 10,000 rpm for 10 minutes. This procedure was repeated twice after which the supernatant was discarded and the sediment preserved in sterile containers in normal saline. Ether is classed as an extremely flammable reagent requiring storage in suitable flammable-liquid storage cabinets; therefore, we used ethyl acetate

as an alternative. Formalin was not used as it leads to a reduction in the fluorescence intensity of stained spores and being a this website Polymerase Chain Reaction (PCR) inhibitor, it may interfere with the molecular study of the parasites to be conducted later [4]. After concentration the saline and iodine Compound C buy Trichostatin A preparations of the samples were microscopically observed as above. Staining The concentrated samples were used for staining. Thin smears from all the samples were prepared on two different slides. The first slide was stained by Modified safranin technique [3]. In this method 3% acid alcohol was used for fixation. Safranin was used for staining and counterstaining was done by Malachite green. Kinyoun’s staining was used to stain the second slide [3]. The smear was fixed with absolute methanol and stained with Kinyoun’s carbol fuschin. Destaining was done by

10% alcohol and the smear was counterstained by Malachite green. At least 200 oil immersion fields of the above smears were examined for the parasites. Fluorescence microscopy A wet mount preparation of the concentrated samples was made and checked for autoflourescence of Cyclospora cayetanensis at 200× magnification with a 330 to 380 nm UV filter. The use of Calcoflour White (Sigma, USA) for fluorescent labeling of Microsporidia spores based on the presence of α-chitin in the inner endospore layer of the spore wall was first introduced by Vávra et al [5]. Calcoflour White stain (10 μl)

was added to the same amount of concentrated samples taken on clean, dry slides. The working solution was prepared by making 1:10 dilution of the stock (1%) and adding 0.05% Evan’s Blue dye. Slides were examined with the help Cyclin-dependent kinase 3 of UV fluorescence microscope at an excitation wavelength of 405 nm. A modification of the above method was performed by using a fluorescent probe 4, 6-diamidine-2-phenylindole (DAPI). Equal quantities (10 μl) of stool sample and DAPI (Sigma, USA) were put on a slide and left for 5 minutes. Thereafter, 10 μl of Calcoflour White was added and the slides were air dried. The slides were screened with the help of a fluorescence microscope using 435-485 BA filter. Antigen detection The third part of the unconcentrated stool samples was subjected to sandwich ELISA for Cryptosporidium parvum antigen detection. The procedure was performed as per the instructions given in the commercially available kit (IVD Research Inc. CA, USA).

Methylation has implications in gene expression [66], and H. pylori associates with several pathologies that may result from different sets of expressed genes [67]. For instance, DNA methylation by M. HpyAIV

was shown to alter transcription of the catalase gene (katA) in H. pylori [68]. Further evidence is needed to understand if the high number Lazertinib in vivo and diversity of MTases expressed among H. pylori strains is beneficial for the bacteria and/or plays any role in pathogenicity. Conclusion In conclusion, there is a clear association of some MTases with geographic groups of H. pylori strains, making them useful as geomarkers (Table 2). Indeed, other genes, as cagA or vacA, have allelic forms with particular geographic distributions [6, 8, 69]. Similar results are now observed for the majority strain-specific genes. M. HhaI and M. NaeI are common to all tested strains, which is consistent with the co-evolution theory of man and H. pylori [2, 3] and suggests that their presence in bacterial genome preceded the human diaspora out of Africa. Finally, the association of MTases with geographical bacterial clusters may be observed in other bacterial species, and may reveal to be this website good geographic markers to trace bacterial evolution.

Methods H. pylori strains 221 H. pylori strains were isolated from different regions (Africa, America, Asia and Europe) (Table 4). Strains belong to the collections of the Helicobacter and Selleck Selinexor Campylobacter Reference Center of the Portuguese National Institute of Health (INSA), the Helicobacter and Campylobacter National Reference Center (Victor Segalen University, Bordeaux, France) and the Medical Microbiology Institute of Hannover (Germany). Strains belonging to INSA and Helicobacter and Campylobacter National Reference Center were randomly selected, except in cases in which all strains available for each sub-sample group were analysed (strains with African origin). DNA from H. pylori Singapore strains was randomly selected from Histone demethylase East Asia

H. pylori strain collection of the Medical Microbiology Institute of Hannover. Except for the country of origin, there is no further information about the ethnic group or ancestry of the human host providing the strain. Due to difficulty in obtaining strains from different geographic origins the number of strains from each continent is uneven. Table 4 Geographic origin of H. pylori strains. Continent Country Number of strains Percentage Europe   146 66.1   Portugal 106 48.0   France 11 5.0   United Kingdom a) 8 3.6   Germany 6 2.7   Sweden 9 4.1   Norway 6 2.7 Africa   38 17.2   Portuguese with African origin b) 20 9.0   Egypt 7 3.2   Burkina Faso 11 5.0 America   27 12.2   USA c) 1 0.5   Costa Rica 6 2.7   Mexico 6 2.7   Argentina 14 6.3 Asia   10 4.5   Singapore 10 4.5 Total   221 100.

PLoS Pathog 2011, 7:e1002355.PubMedCrossRef 22. Levy P505-15 molecular weight O: Antimicrobial proteins and peptides of blood: templates for novel antimicrobial agents. Blood 2000, 96:2664–2672.PubMed

23. Radek K, Gallo R: Antimicrobial peptides: natural effectors of the innate immune system. Semin Immunopathol 2007, 29:27–43.PubMedCrossRef 24. Blair P, Flaumenhaft R: Platelet alpha-granules: basic biology and clinical correlates. Blood Rev 2009, 23:177–189.PubMedCrossRef 25. Tohidnezhad M, Varoga D, Podschun R, Wruck CJ, Seekamp A, Brandenburg LO, Pufe T, Lippross S: Thrombocytes are effectors of the innate immune system releasing human beta defensin-3. Injury 2011, 42:682–686.PubMedCrossRef 26. Tohidnezhad M, Varoga D, Wruck CJ, Podschun R, Sachweh BH, Bornemann JAK inhibitor J, Bovi M, Sönmez TT, Slowik A, Houben A, Seekamp A, Brandenburg LO, Pufe T, Lippross S: Platelets display potent antimicrobial activity and release human beta-defensin 2. Platelet 2012, 23:217–223.CrossRef 27. Tohidnezhad M, Varoga D, Wruck CJ, Brandenburg LO, Seekamp A, Shakibaei M, Sönmez TT, Pufe T, Lippross S: Platelet-released growth factors can accelerate tenocyte proliferation and activate the anti-oxidant response element.

Histochem Cell Biol 2011, 135:453–460.PubMedCrossRef 28. Schnabel LV, Mohammed HO, Miller BJ, et al.: Platelet rich plasma (PRP) enhances anabolic gene expression patterns in flexor digitorum superficialis tendons. J Orthop Res 2007, 25:230–240.PubMedCrossRef 29. Falanga V, Grinnell F, Gilchrest B, Maddox YT, Moshell

A: Workshop on the pathogenesis of chronic wounds. J Invest Dermatol 1994, 102:125–127.PubMedCrossRef 30. Steed DL, Donohoe D, Webster MW, Lindsley L: Effect of extensive debridement and treatment on the healing of diabetic foot ulcers. Diabetic Ulcer Study Group. J Am Coll Surg 1996, 183:61–64.PubMed 31. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB: Enterococcus faecalis: its role in root canal treatment Torin 1 failure and current concepts in retreatment. J Endod 2006, 32:93–98.PubMedCrossRef 32. Farah CS, Lynch N, McCullough MJ: Oral fungal infections: an update Pyruvate dehydrogenase for the general practitioner. Aust Dent J 2010, 55:48–54.PubMedCrossRef Competing interests The authors declare that they have no financial or non-financial competing interest. Authors’ contributions LD: Conceived the study, participated in its design and coordination and revised the manuscript. BM: Acquired data, participated in their analysis and interpretation and drafted the manuscript. CV: Acquired data, participated in their analysis and interpretation and drafted the manuscript. ST: Revised the manuscript. MdF: Conceived the study, participated in its design and coordination and revised the manuscript. All authors read and approved the final manuscript.”
“Background Operons are multigene arrangements transcribed as a single mRNA and are one of the defining features found in bacterial and archaeal genomes.

DNA extraction was carried out by mechanical disruption of the microbial cell wall using beads (Lysing matrix E, MP Biomedicals, Spain). The disruption was performed by shaking the mixture using the Bead-Beater-8 (BioSpec, USA) at a medium speed of about 1500 oscillations/min for 3 minutes, followed by 3 minutes in ice and again followed by 5 minutes at a medium speed of about 1500 oscillations/min. Finally, nucleic acids were recovered from clear lysates by alcohol precipitation. To evaluate the effect of Selleckchem BTSA1 stool water

content and a bead-beating step, aliquots of samples were homogenised with various volumes of PBS (final weight of 250 mg) and with or without beads, as described in Table 1. They were then processed the same way as described above. In samples in which beads were not used, Cilengitide purchase the bead-beater step was also omitted. After genomic DNA extraction, an equivalent of 1 mg of each sample was used for DNA quantification using a NanoDrop ND-1000 Spectrophotometer (Nucliber). DNA integrity was examined by microcapillary electrophoresis using an Agilent 2100 Bioanalyzer with the DNA 12000 kit, which resolves the distribution of double-stranded DNA fragments up to 17,000 bp in length. Microbial community analyses 454 pyrosequencing of the V4 variable

KPT-8602 region of the 16 S rRNA gene To analyse bacterial composition, we subjected extracted genomic DNA to PCR-amplification of the V4 hyper-variable region Acetophenone of the 16S rRNA gene. On the basis of our analysis done using PrimerProspector software [16], the V4 primer pairs used in this study were expected to amplify almost 100% of the Archaea and Bacteria domains. The 5’ ends of the forward primer V4F_517_17 (5′-GCCAGCAGCCGCGGTAA-3′) [17] and the reverse primer V4R_805_19 (5′-GACTACCAGGGTATCTAAT-3′) [18] were tagged with specific sequences for pyrosequencing as follows: 5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-MID-GCCAGCAGCCGCGGTAA-3′ and 5′ CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-GACTACCAGGGTATCTAAT-3′. Tag pyrosequencing was performed using multiplex identifiers (MIDs) (Roche Diagnostics) of 10 bases, which were specified upstream of the forward primer sequence (V4F_517_17). Standard PCR amplification

was run in a Mastercycler gradient (Eppendorf) at 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec, 56°C for 20 sec, 72°C for 40 sec, and a final cycle of 72°C for 7 min. PCR products were purified using a PCR Purification kit (Qiagen, Spain) and subsequently sequenced on a 454 Life Sciences (Roche) FLX system (Scientific and Technical Support Unit, Vall d’Hebron Research Institute, Barcelona, Spain), following standard 454 platform protocols. 16S rRNA sequence data analysis A total of 1.47 million sequence reads from 96 samples were analysed using the default settings in the Quantitative Insights Into Microbial Ecology (QIIME) package of software tools [19]. The 16S rRNA sequences were quality-filtered and demultiplexed.

The level of Lux induction with pBAD-aphB compared to pBAD24 in E. coli in the presence of 0.01% arabinose is given in the table. Alignment of putative AphB binding sites in tcpP, aphB, and toxR promoter region is given. (B) Gel shift assays using BKM120 datasheet purified MBP-AphB and DNA containing various lengths of the regulatory regions of the toxR promoter. Protein concentrations used in the gel shift assay (shown as shaded triangles) were 0, 20, 40, 80 ng/reaction (5 μl). The selleck effects of AphB on ToxR-regulated genes In addition to regulation

of toxT, ToxR has been previously shown to alter the porin levels in V. cholerae by activating expression of ompU and repressing ompT [26, 27]. Since we showed that AphB affects ToxR levels, we hypothesized that AphB might thus indirectly modulate the expression of ompU and ompT as well. We performed SDS-PAGE on total protein extracts of wild type V. cholerae as well as toxR and aphB mutants. As expected, DNA Damage inhibitor the toxR strain had significantly lower OmpU and higher OmpT levels than in the wild-type strain. Interestingly, the aphB mutant strain produced slightly higher levels of OmpT than wild type, though OmpU

levels did not seem to change (Fig. 6A). In addition, Provenzano et al. showed that ToxR-dependent modulation of outer membrane proteins enhances V. cholerae resistance to antimicrobial compounds such as bile salts and sodium dodecyl sulfate (SDS) [28]. We confirmed that the toxR mutant strain had a reduced minimum bactericidal concentration (MBC) Progesterone of SDS compared to wild type strains, but AphB did not affect the MBC of SDS (Fig. 6A). Thus, AphB may only subtly modulate outer membrane porin expression through its effect on toxR expression. This may be another downstream effect of AphB on the virulence capabilities of V. cholerae in addition to its better characterized influences on ToxT levels. Moreover, as both

ToxR and TcpP are required to activate toxT expression and AphB is required to activate tcpP expression (Fig. 1) [19, 29], we tested whether AphB effects on toxR expression affect toxT expression under the AKI virulence induction condition [22]. As expected, toxT expression in aphB mutants was significantly reduced as compared to that of wild type (Fig. 6B), however, bypassing the AphB regulation of tcpP by constitutively expressing tcpPH (pBAD-tcpPH induced with 0.01% arabinose) restored toxT expression in aphB mutants. These data suggest that AphB modulation of toxR expression has minor effects on virulence gene expression as compared to that of AphB regulation of tcpP under the condition we tested. Figure 6 The influence of AphB on V. cholerae outer membrane composition, SDS resistance, and toxT expression. (A). Analysis of outer membrane preparations of V. cholerae derivatives. SDS-PAGE gel stained with Coomassie blue. OmpT and OmpU are indicated at the right.

cv. Frisson) seeds were surface-disinfected, pregerminated on agar plates, sown in Leonard jar-type assemblies, and inoculated with R. leguminosarum bv. viciae strains, as previously described [45]. Plants were grown for 21 days under bacteriologically controlled conditions with a nitrogen-free plant nutrient solution in a greenhouse adjusted to 18/25°C(night/day) temperatures. Nitrogen-free plant nutrient solution was supplemented with 170 μM NiCl2 on day 10 after seedling inoculation.

Bacteroid suspensions were obtained from nodules as previously described [40]. Hydrogenase activity assays Hydrogenase activity in bacteroid suspensions and Cell Cycle inhibitor in free-living microaerobic cell cultures was measured by an amperometric method using a Clark-type electrode with oxygen as PRI-724 electron acceptor [45]. find more Hydrogenase activity in vegetative cells was induced in 40-ml cultures grown under continuous bubbling with a gas mixture containing O2 concentrations of 1 or 3% in N2. Strains were aerobically grown

in YMB medium to an optical density at 600 nm (OD600) of ca. 0.4. From these cultures a 1:4 dilution was made in fresh YMB medium. Flasks were capped with a stoppered-tube system adapted to continuous flushing with 1% or 3% O2 on N2, and incubated at 28°C for 20 h. For HupL stability studies, bacteri-al cultures were maintained in a bottle with continuous bubbling with either 1% O2 or air

during 3 hours after standard microaerobic induction (1% O2). Cell cultures were centrifuged and suspended in 5 ml Dixon buffer (32 mM K2HPO4, 24 mM KH2PO4 and 0.24 mM MgCl2) before amperometric determinations. To prevent dam-age of hydrogenase due to O2 exposition, extracts were bubbled with argon during preparation. Protein contents of vegetative cells and bacteroids were determined by the bicinchoninic acid method SPTBN5 [46] after alkaline digestion of cells at 90°C in NaOH for 10 min, with bovine serum albumin as the standard. DNA manipulation techniques and mutant construction DNA manipulations, including purification, restriction, ligation, agarose gel electrophoresis, PCR amplification, and transformation into E. coli cells were carried out by standard methods [47]. In-frame deletions of hupF, hupK and hypC genes were generated in plasmid pALPF1 as described by Manyani et al. [19], resulting in plasmids pALPF5, pALPF10, and pALPF14, respectively. Primers used for deletions and plasmid generation are included in Table  4.