Non-Equilibrium Fractionation Factors for D/H and 18O/16O During Oceanic Evaporation in the North-West Atlantic Region.

Ocean isotopic evaporation models, such as the Craig-Gordon model, rely on the description of nonequilibrium fractionation factors that are, in general, poorly constrained. To date, only a few gradient-diffusion type measurements have been performed in ocean settings to test the validity of the commonly used parametrization of nonequilibrium isotopic fractionation during ocean evaporation. In this work, we present 6 months of water vapor isotopic observations collected from a meteorological tower located in the northwest Atlantic Ocean (Bermuda) with the objective of estimating nonequilibrium fractionation factors (k, ‰) for ocean evaporation and their wind speed dependency. The Keeling Plot method and Craig-Gordon model combination were sensitive enough to resolve nonequilibrium fractionation factors during evaporation resulting into mean values of k 18 = 5.2 ± 0.6‰ and k 2 = 4.3 ± 3.4‰. Furthermore, we evaluate the relationship between k and 10-m wind speed over the ocean. Such a relationship is expected from current evaporation theory and from laboratory experiments made in the 1970s, but observational evidence is lacking. We show that (a) in the observed wind speed range [0-10 m s-1], the sensitivity of k to wind speed is small, in the order of -0.2‰ m-1 s for k 18, and (b) there is no empirical evidence for the presence of a discontinuity between smooth and rough wind speed regime during isotopic fractionation, as proposed in earlier studies. The water vapor d-excess variability predicted under the closure assumption using the k values estimated in this study is in agreement with observations over the Atlantic Ocean.

Transcriptomic Analysis of the Dual Response of Rhodococcus aetherivorans BCP1 to Inorganic Arsenic Oxyanions.

Bacterial strains belonging to the genus Rhodococcus are able to degrade various toxic organic compounds and tolerate high concentrations of metal(loid)s. We have previously shown that Rhodococcus aetherivorans BCP1 is resistant to various levels of the two arsenic inorganic species, arsenite [As(III)] and arsenate [As(V)]. However, while arsenite showed toxic effects at concentrations as low as 5 mM, arsenate at 30 mM boosted the growth rate of BCP1 cells and was toxic only at concentrations of >100 mM. Since such behavior could be linked to peculiar aspects of its metabolism, the transcriptomic analysis of BCP1 cells exposed to 5 mM As(III) and 30 mM As(V) was performed in this work. The aim was to clarify the mechanisms underlying the arsenic stress response of the two growth phenotypes in the presence of the two different oxyanions. The results revealed that As(III) induced higher activity of reactive oxygen species (ROS)-scavenging enzymes than As(V) in relation to the expression of enzymes involved in cellular damage recovery and redox buffers/cofactors (ergothioneine, mycofactocin, and mycothiol). Further, As(III) downregulated pathways related to cell division, while both oxyanions downregulated genes involved in glycolysis. Notably, As(V) induced the expression of enzymes participating in the synthesis of metallophores and rearranged the central and energetic metabolism, also inducing alternative pathways for ATP synthesis and glucose consumption. This study, in providing transcriptomic data on R. aetherivorans exposed to arsenic oxyanions, sheds some light on the plasticity of the rhodococcal response to arsenic stress, which may be important for the improvement of biotechnological applications. IMPORTANCE Members of the genus Rhodococcus show high metabolic versatility and the ability to tolerate/resist numerous stress conditions, including toxic metals. R. aetherivorans BCP1 is able to tolerate high concentrations of the two inorganic arsenic oxyanions, arsenite [As(III)] and arsenate [As(V)]. Despite the fact that BCP1 intracellularly converts As(V) into As(III), this strain responds very differently to the presence of these two oxyanions in terms of cell growth and toxic effects. Indeed, while As(III) is highly toxic, exposure to specific concentrations of As(V) seems to boost cell growth. In this work, we investigated the transcriptomic response, ATP synthesis, glucose consumption, and H2O2 degradation in BCP1 cells exposed to As(III) and As(V), inducing two different growth phenotypes. Our results give an overview of the transcriptional rearrangements associated with the dual response of BCP1 to the two oxyanions and provide novel insights into the energetic metabolism of Rhodococcus under arsenic stress.

Methods for radioactivity measurements in drinking water using gamma spectrometry.

In this study, three methods to measure activity concentrations of radionuclides through high resolution gamma spectrometry are developed, optimized, and tested on drinking water samples. Two pre-concentration methods (partial evaporation and ion-exchange resins) were optimized for accuracy, precision, detection limits, costs, preparation, and measurements times. A new sampling method for 222Rn was designed and optimized to directly sample water from the tap, reducing and minimizing losses of radon during the sampling. A total number of 85 water samples were collected between 2017 and 2019 in collaboration with two drinking water suppliers in a wide area (~2000 km2) of the Veneto region, northeast Italy. These are the first results of radionuclides activity concentration in drinking water concerning a large extension in the foothill Veneto region. Finally, this study provides a first attempt of determining the spatial distribution and seasonal variations of radon activity concentration in drinking water in the study area.

Genome Sequence of Rhodococcus sp. Strain BCP1, a Biodegrader of Alkanes and Chlorinated Compounds.

Rhodococcus sp. strain BCP1 cometabolizes chlorinated compounds and mineralizes a broad range of alkanes, as it is highly tolerant to them. The high-quality draft genome sequence of Rhodococcus sp. strain BCP1, consisting of 6,231,823 bp, with a G+C content of 70.4%, 5,902 protein-coding genes, and 58 RNA genes, is presented here.

Microbial degradation of chloroform.

Chloroform (CF) is largely produced by both anthropogenic and natural sources. It is detected in ground and surface water sources and it represents the most abundant halocarbon in the atmosphere. Microbial CF degradation occurs under both aerobic and anaerobic conditions. Apart from a few reports describing the utilization of CF as a terminal electron acceptor during growth, CF degradation was mainly reported as a cometabolic process. CF aerobic cometabolism is supported by growth on short-chain alkanes (i.e., methane, propane, butane, and hexane), aromatic hydrocarbons (i.e., toluene and phenol), and ammonia via the activity of monooxygenases (MOs) operatively divided into different families. The main factors affecting CF cometabolism are (1) the inhibition of CF degradation exerted by the growth substrate, (2) the need for reductant supply to maintain MO activity, and (3) the toxicity of CF degradation products. Under anaerobic conditions, CF degradation was mainly associated to the activity of methanogens, although some examples of CF-degrading sulfate-reducing, fermenting, and acetogenic bacteria are reported in the literature. Higher CF toxicity levels and lower degradation rates were shown by anaerobic systems in comparison to the aerobic ones. Applied physiological and genetic aspects of microbial cometabolism of CF will be presented along with bioremediation perspectives.

Analyses of both the alkB gene transcriptional start site and alkB promoter-inducing properties of Rhodococcus sp. strain BCP1 grown on n-alkanes.

Rhodococcus sp. strain BCP1, known for its capacity to grow on short-chain n-alkanes (C(2) to C(7)) and to cometabolize chlorinated solvents, was found to also utilize medium- and long-chain n-alkanes (C(12) to C(24)) as energy and carbon sources. To examine this feature in detail, a chromosomal region which includes the alkB gene cluster encoding a non-heme di-iron monooxygenase (alkB), two rubredoxins, and one rubredoxin reductase was cloned from the BCP1 genome. Furthermore, the activity of the alkB gene promoter (P(alkB)) was examined in the presence of gaseous, liquid, and solid n-alkanes along with intermediates of the putative n-alkane degradation pathway. A recombinant plasmid, pTP(alkB)LacZ, was constructed by inserting the lacZ gene downstream of P(alkB), and it was used to transform Rhodococcus sp. strain BCP1. Measurements of ß-galactosidase activity showed that P(alkB) is induced by C(6) to C(22) n-alkanes. Conversely, C(2) to C(5) and >C(22) n-alkanes and alkenes, such as hexene, were not inducers of alkB expression. The effects on P(alkB) expression induced by alternative carbon sources along with putative products of n-hexane metabolism were also evaluated. This report highlights the great versatility of Rhodococcus sp. strain BCP1 and defines for the first time the alkB gene transcriptional start site and the alkB promoter-inducing capacities for substrates different from n-alkanes in a Rhodococcus strain.

A histidine-kinase cheA gene of Pseudomonas pseudoalcaligens KF707 not only has a key role in chemotaxis but also affects biofilm formation and cell metabolism.

A histidine-kinase cheA gene in Pseudomonas pseudoalcaligenes KF707 plays a central role in the regulation of metabolic responses as well as in chemotaxis. Non-chemotactic mutants harboring insertions into the cheA gene were screened for their ability to form biofilms in the Calgary biofilm device. Notably, ≥95% decrease in the number of cells attached to the polystyrene surface was observed in cheA mutants compared to the KF707 wild-type biofilm phenotype. The ability to form mature biofilms was restored to wild-type levels, providing functional copies of the KF707 cheA gene to the mutants. In addition, phenotype micro-arrays and proteomic analyses revealed that several basic metabolic activities and a few periplasmic binding proteins of cheA mutant cells differed compared to those of wild-type cells. These results are interpreted as evidence of a strong integration between chemotactic and metabolic pathways in the process of biofilm development by P. pseudoalcaligenes KF707.

Reduction of potassium tellurite to elemental tellurium and its effect on the plasma membrane redox components of the facultative phototroph Rhodobacter capsulatus.

Anaerobically light-grown cells of Rhodobacter capsulatus B100 are highly resistant to the toxic oxyanion tellurite (TeO(3)(2-); minimal inhibitory concentration, 250 microg/ml). This study examines, for the first time, some structural and biochemical features of cells and plasma membrane fragments of this facultative phototroph grown in the presence of 50 microg of K(2)TeO(3) per ml. Through the use of transmission microscopy and X-ray microanalysis we show that several "needlelike" shaped granules of elemental tellurium are accumulated into the cytosol near the intracytoplasmic membrane system. Flash-spectroscopy, oxygen consumption measurements, and difference spectra analysis indicated that membrane vesicles (chromatophores) isolated from tellurite-grown cells are able to catalyze both photosynthetic and respiratory electron transport activities, although they are characterized by a low c-type cytochrome content (mostly soluble cytochrome c(2)). This feature is paralleled by a low cytochrome c oxidase activity and with an NADH-dependent respiration which is catalyzed by a pathway leading to a quinol oxidase (Qox) inhibited by high (millimolar) concentrations of cyanide (CN(-)). Conversely, membranes from R. capsulatus B100 cells grown in the absence of tellurite are characterized by a branched respiratory chain in which the cytochrome c oxidase pathway (blocked by CN(-) in the micromolar range) accounts for 35-40% of the total NADH-dependent oxygen consumption, while the remaining activity is catalyzed by the quinol oxidase pathway. These data have been interpreted to show that tellurite resistance of R. capsulatus B100 is characterized by the presence of a modified plasma-membrane-associated electron transport system.

Polychlorinated biphenyl degradation activities and hybridization analyses of fifteen aerobic strains isolated from a PCB-contaminated site.

Fifteen bacterial strains using biphenyl as sole carbon and energy source, obtained from different positions and depths of a polychlorinated biphenyl (PCB)-contaminated area, were analyzed for their basic metabolic phenotypes and subjected to genomic DNA hybridization screening for the presence of well characterized bph operons such as those of Pseudomonas pseudoalcaligenes KF707 and Rhodococcus globerulus P6. Most of the isolates belonged to the gamma-subdivision (Pseudomonas stutzeri, P. plutida, P. fluorescens and Vibrio logei species) and to the beta-subdivision (genera Alcaligenes, Comamonas, Ralstonia) of the Proteobacteria. All the isolates were able to cometabolize different low chlorinated PCB congeners. Among the dichlorinated biphenyls tested, a lower degradation capacity was observed for the di-ortho substituted congeners, whereas high levels of degradation were observed for the di-meta and di-para isomers, whether they were chlorinated on one or on both rings. The PCB congeners nonsubstituted in the 2,3 or 2,3 and 3,4 positions were also degraded by most of the isolated strains, which were, however, unable to significantly metabolize PCBs with more than 3 chlorine atoms. Five of the isolated strains were also able to degrade some of the tri- and tetrachlorobiphenyls tested. Southern hybridization analysis showed a strong homology between four of the fifteen isolated strains and the bph operon obtained from P. pseudoalcaligenes strain KF707. Conversely, none of the isolates here examined showed homology with the bph operon of R. globerulus strain P6. In line with this, the KF707 bph probe strongly hybridized with DNA of a significant number of bacterial colonies obtained from selected locations in the contaminated area using biphenyl-supplemented minimal medium agar plates.

Respiratory electron transport and light-induced energy transduction in membranes from the aerobic photosynthetic bacterium Roseobacter denitrificans.

Membrane fragments isolated from the aerobic phototrophic bacterium Roseobacter denitrificans were examined. Ninety-five percent of the total NADH-dependent oxidative activity was inhibited either by antimycin A or myxothiazol, two specific inhibitors of the cytochrome bc1 complex, which indicates that the respiratory electron transport chain is linear. In agreement with this finding, light-induced oxygen uptake, an electron transport activity catalyzed by the "alternative quinol oxidase pathway" in membranes of several facultative phototrophic species, was barely detectable in membranes of Rsb. denitrificans. Redox titrations at 561-575 nm, 552-540 nm, and 602-630 nm indicated the presence of three b-type cytochromes (Em,7 of +244 +/- 8, +24 +/- 3, -163 +/- 11 mV), four c-type cytochromes (Em,7 of +280 +/- 10, +210 +/- 5, +125 +/- 8, and 20 +/- 3 mV) and two a-type cytochromes (Em,7 of +335 +/- 15, +218 +/- 18 mV). The latter two a-type hemes were shown to be involved in cytochrome c oxidase activity, which was inhibited by both cyanide (I50 = 2 microM) and azide (I50 = 1 mM), while a soluble cytochrome c (c551, Em,7 = +217 +/- 2 mV) was shown to be the physiological electron carrier connecting the bc1 complex to the cytochrome c oxidase. A comparison of the ATP synthesis generated by continuous light in membranes of Rsb. denitrificans and Rhodobacter capsulatus showed that in both bacterial species photophosphorylation requires a membrane redox poise at the equilibrium (Eh > or = +80 < or = +140 mV), close to the oxidation-reduction potential of the ubiquinone pool. These data, taken together, suggest that, although the photosynthetic apparatus of Rsb. denitrificans is functionally similar to that of typical anoxygenic phototrophs, e.g. Rba. capsulatus, the in vivo requirement of a suitable redox state at the ubiquinone pool level restricts the growth capacity of Rsb. denitrificans to oxic conditions.

Plasmid transfer and susceptibility to antibiotics in the halophilic phototrophs Rhodovibrio salinarum and Rhodothalassium salexigens.

The present study defines a series of genetic procedures to be used for molecular studies in photosynthetic halophilic species such as Rhodovibrio salinarum and Rhodothalassium salexigens. In both species, the minimal inhibitory concentrations for the antibiotics tetracycline, rifampicin, chloramphenicol, spectinomycin, streptomycin, and kanamycin were determined. In addition, conjugal transfer of IncP and IncQ plasmids from Escherichia coli was demonstrated and the resistance markers expressed in these halophiles were determined. Finally, Rth. salexigens growth dependence on variable salt concentrations was measured: maximal growth rates were seen at 6% and 4% NaCl under phototrophic and chemotrophic conditions, respectively. To the best of our knowledge, this is the first report analyzing the genetic properties of two representative species of halophilic purple non-sulfur phototrophs.

Mobile cytochrome c2 and membrane-anchored cytochrome cy are both efficient electron donors to the cbb3- and aa3-type cytochrome c oxidases during respiratory growth of Rhodobacter sphaeroides.

We have recently established that the facultative phototrophic bacterium Rhodobacter sphaeroides, like the closely related Rhodobacter capsulatus species, contains both the previously characterized mobile electron carrier cytochrome c2 (cyt c2) and the more recently discovered membrane-anchored cyt cy. However, R. sphaeroides cyt cy, unlike that of R. capsulatus, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt bc1 complex during photosynthetic growth. Nonetheless, R. sphaeroides cyt cy can act at least in R. capsulatus as an electron carrier between the cyt bc1 complex and the cbb3-type cyt c oxidase (cbb3-Cox) to support respiratory growth. Since R. sphaeroides harbors both a cbb3-Cox and an aa3-type cyt c oxidase (aa3-Cox), we examined whether R. sphaeroides cyt cy can act as an electron carrier to either or both of these respiratory terminal oxidases. R. sphaeroides mutants which lacked either cyt c2 or cyt cy and either the aa3-Cox or the cbb3-Cox were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt bc1 complex and the cyt c oxidases. They were characterized with respect to growth phenotypes, contents of a-, b-, and c-type cytochromes, cyt c oxidase activities, and kinetics of electron transfer mediated by cyt c2 or cyt cy. The findings demonstrated that both cyt c2 and cyt cy are able to carry electrons efficiently from the cyt bc1 complex to either the cbb3-Cox or the aa3-Cox. Thus, no dedicated electron carrier for either of the cyt c oxidases is present in R. sphaeroides. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt bc1 complex appears to be mediated via the cyt c2-to-cbb3-Cox and cyt cy-to-cbb3-Cox subbranches. The presence of multiple electron carriers and cyt c oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of R. sphaeroides are more complex than those of R. capsulatus.

Assay of ATP in intact cells of the facultative phototroph Rhodobacter capsulatus expressing recombinant firefly luciferase.

This study reports on the construction, calibration and use of recombinant cells of Rhodobacter capsulatus expressing the luciferase gene of the North American firefly Photinus pyralis to detect, by bioluminescence, variations of endogenous ATP levels under various physiological conditions. We show that the antibiotic polymyxin B allows luciferin to rapidly move into cell cytosol, but does not make external ATP freely accessible to intracellular luciferase. Notably, in toluene:ethanol-permeabilized cells, the apparent K(mATP) for luciferase (50 microM) is similar to that measured in soluble cell fractions. This finding limits the applicability of the firefly luciferase for monitoring intracellular maximal ATP concentration because dark/aerobic-grown recombinant cells of Rba. capsulatus contain approximately 1.3-2.6+/-0.5 mM ATP. Therefore, the effects of chemical and physical factors such as oxygen, light, carbonyl cyanide m-chlorophenyl hydrazone and antimycin A on ATP synthesis were examined in cells subjected to different starvation periods to reduce the endogenous ATP pool below the luciferase ATP saturation level (< or =0.2 mM). We conclude that the amount of endogenous ATP generated by light is maximal in the presence of oxygen, which is required to optimize the membrane redox poise.

The membrane-attached electron carrier cytochrome cy from Rhodobacter sphaeroides is functional in respiratory but not in photosynthetic electron transfer.

Rhodobacter species are useful model organisms for studying the structure and function of c type cytochromes (Cyt c), which are ubiquitous electron carriers with essential functions in cellular energy and signal transduction. Among these species, Rhodobacter capsulatus has a periplasmic Cyt c2Rc and a membrane-bound bipartite Cyt cyRc. These electron carriers participate in both respiratory and photosynthetic electron-transfer chains. On the other hand, until recently, Rhodobacter sphaeroides was thought to have only one of these two cytochromes, the soluble Cyt c2Rs. Recent work indicated that this species has a gene, cycYRs, that is highly homologous to cycYRc, and in the work presented here, functional properties of its gene product (Cyt cyRs) are defined. It was found that Cyt cyRs is unable to participate in photosynthetic electron transfer, although it is active in respiratory electron transfer, unlike its R. capsulatus counterpart, Cyt cyRc. Chimeric constructs have shown that the photosynthetic incapability of Cyt cyRs is caused, at least in part, by its redox active subdomain, which carries the covalently bound heme. It, therefore, seems that this domain interacts differently with distinct redox partners, like the photochemical reaction center and the Cyt c oxidase, and allows the bacteria to funnel electrons efficiently to various destinations under different growth conditions. These findings raise an intriguing evolutionary issue in regard to cellular apoptosis: why do the mitochondria of higher organisms, unlike their bacterial ancestors, use only one soluble electron carrier in their respiratory electron-transport chains?

Characterization of DorC from Rhodobacter capsulatus, a c-type cytochrome involved in electron transfer to dimethyl sulfoxide reductase.

The dorC gene of the dimethyl sulfoxide respiratory (dor) operon of Rhodobacter capsulatus encodes a pentaheme c-type cytochrome that is involved in electron transfer from ubiquinol to periplasmic dimethyl sulfoxide reductase. DorC was expressed as a C-terminal fusion to an 8-amino acid FLAG epitope and was purified from detergent-solubilized membranes by ion exchange chromatography and immunoaffinity chromatography. The DorC protein had a subunit Mr = 46,000, and pyridine hemochrome analysis indicated that it contained 5 mol heme c/mol DorC polypeptide, as predicted from the derived amino acid sequence of the dorC gene. The reduced form of DorC exhibited visible absorption maxima at 551.5 nm (alpha-band), 522 nm (beta-band), and 419 nm (Soret band). Redox potentiometry of the heme centers of DorC identified five components (n = 1) with midpoint potentials of -34, -128, -184, -185, and -276 mV. Despite the low redox potentials of the heme centers, DorC was reduced by duroquinol and was oxidized by dimethyl sulfoxide reductase.

On the role of high-potential iron-sulfur proteins and cytochromes in the respiratory chain of two facultative phototrophs

The capability of high potential iron-sulfur proteins (HiPIPs) and soluble cytochromes to shuttle electrons between the bc1 complex and the terminal oxidase in aerobically grown cells of Rhodoferax fermentans and Rhodospirillum salinarum, two facultative phototrophs, was evaluated. In Rs. salinarum, HiPIP and a c-type cytochrome (alpha-band at 550 nm, Em,7=+290 mV) are both involved in the electron transfer step from the bc1 complex to the terminal oxidase. Kinetic studies indicate that cytochrome c550 is more efficient than HiPIP in oxidizing the bc1 complex, and that HiPIP is a more efficient reductant of the terminal oxidase as compared to cytochrome c550. Rs. salinarum cells contain an additional c-type cytochrome (asymmetric alpha-band at 556 nm, Em,7=+180 mV) which is able to reduce the terminal oxidase, but unable to oxidize the bc1 complex. c-type cytochromes could not be isolated from Rf. fermentans, in which HiPIP, the most abundant soluble electron carrier, is reduced by the bc1 complex (zero-order kinetics) and oxidized by the terminal oxidase (first-order kinetics), respectively. These data, taken together, indicate for the first time that HiPIPs play a significant role in bacterial respiratory electron transfer.

The respiratory chain of the halophilic anoxygenic purple bacterium Rhodospirillum sodomense.

The halophilic purple nonsulfur bacterium Rhodospirillum sodomense has been previously described as an obligate phototroph that requires yeast extract and a limited number of organic compounds for photoheterotrophic growth. In this work, we report on chemoheterotrophic growth of R. sodomense in media containing either acetate or succinate supplemented with 0.3-0.5% yeast extract. Plasma membranes isolated from cells grown aerobically in the dark contained three b-type and three c-type membrane-bound cytochromes with Em,7 of +171 +/- 10, +62 +/- 10 and -45 +/- 13 mV (561-575 nm), and +268 +/- 6, +137 +/- 10 and -43 +/- 12 mV (551-540 nm). A small amount of a soluble c-type cytochrome with a mol. mass of 15 kDa (Em, 7 >/= +150 mV) was identified. Spectroscopic and immunological methods excluded the presence of cytochrome of the c2 class and high-potential iron-sulfur proteins. Inhibitory studies indicated that only 60-70% of the respiratory activity was blocked by low concentrations of cyanide, antimycin A, and myxothiazol (10, 0.1, and 0.2 microM, respectively). These results were interpreted to show that the oxidative electron transport chain of R. sodomense is branched, leads to a quinol oxidase that is fully blocked by 1 mM cyanide and that is involved in light-dependent oxygen reduction, and leads to a cytochrome c oxidase that is inhibited by 10 microM cyanide. These features taken together suggest that R. sodomense differs from the closely related species Rhodospirillum salinarum and from other species of the genus Rhodospirillum in that it contains multiple membrane-bound cytochromes c.

The electron transport system of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum. 1. A functional and thermodynamic analysis of the respiratory chain in aerobically and photosynthetically grown cells.

Plasma membranes isolated from cells of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum grown in light or in the dark were examined. Membranes isolated from cells grown aerobically in the dark contained three b-type and two c-type membrane-bound cytochromes with Em,7 of +180, +72 and -5 mV (561-575 nm), and +244 and +27 mV (551-540 nm), respectively. Conversely, membranes isolated from cells grown anaerobically in the light contained two b-type and five c-type haems with Em,7 of +60 and -45 mV and +290, +250, +135, -20 and -105 mV, respectively. In addition to haems of the b- and c-type, two haems of the a-type (Em,7 of +325 and +175 mV) were present only in cells grown in the dark. Four soluble cytochromes of the c type, but not cytochrome c2, along with two high-potential iron-sulfur proteins (HiPIP iso-1 and iso-2) were also identified in cells grown aerobically. Inhibitory studies showed that 85-90% of the respiratory activity was blocked by very low concentrations of cyanide, antimycin A and myxothiazol (50, 0.1 and 0.2 mM, respectively). These results taken together were interpreted to show that the oxidative electron transport chain of Rsp. salinarum is linear, leading to a membrane-bound oxidase of the aa3 type in cells grown in the dark, while no significant cytochrome oxidase activity is catalyzed by photosynthetic membranes. These features suggest that this halophilic species is unique among the genus Rhodospirillum and that it also differs from other facultative phototrophs (e.g., Rhodobacter species) in that it does not contain either cytochrome c2 or a branched respiratory chain.

Functional and structural role of the cytochrome b subunit of the membrane-bound hydrogenase complex of Alcaligenes eutrophus H16.

This study shows that the product of the hoxZ gene of Alcaligenes eutrophus H16 is a b-type cytochrome (cytochrome b(z)), which is essential for anchoring the membrane-bound hydrogenase (MBH) complex to the periplasmic side of the membrane and for H2-coupled respiration. The hoxZ product is not required for MBH translocation and H2-dependent reduction of the redox dye, 2,3,5-triphenyl-2-tetrazolium chloride. The lack of cytochrome b(z) does not affect the electron-transport activities linked to oxidation of succinate and NADH, although it enhances the electron-flow rate through the cytochrome-c oxidase pathway in hoxZdelta membranes. We show that the hoxZ product is a dihaem cytochrome b (haems with E(m7.0) of +10 mV and +166 mV) involved in H2-dependent electron transfer. We conclude that cytochrome b(z) of the A. eutrophus MBH complex is the link necessary for transfer of electrons from H2 to the ubiquinone pool and that it is required for attachment of MBH to the membrane.

The effect of respiration on the phototactic behavior of the purple nonsulfur bacterium Rhodospirillum centenum.

The effect of respiration on the positive phototactic movement of swarming agar colonies of the facultative phototroph Rhodospirillum centenum was studied. When the electron flow was blocked at the bc1 complex level by myxothiazol, the oriented movement of the colonies was totally blocked. Conversely, inhibition of respiration via the cytochrome c oxidase stimulated the phototactic response. No phototaxis was observed in a photosynthesis deficient mutant (YB707) lacking bacteriochlorophylls. Analyses of the respiratory activities as monitored by a oxygen microelectrode in single agar colonies during light/dark transitions showed a close functional correlation between the photosynthetic and respiratory apparatuses. The respiratory chain of Rsp. centenum was formed by two oxidative pathways: one branch leading to a cytochrome c oxidase inhibited by low cyanide concentrations and a second pathway formed by an oxidase less-sensitive to cyanide that also catalyzes the light-driven respiration. These results were interpreted to indicate that (1) there is a cyclic electron transport, and (2) photoinduced cyclic electron flow is required for the phototactic response of Rsp. centenum. Furthermore, under oxic conditions in the light, reducing equivalents may switch from photosynthetic to respiratory components so as to reduce both the membrane potential and the rate of locomotion.