Primary structure of porin from <i>Rhodobacter capsulatus</i>

Schiltz, Emile; Kreusch, Andreas; Nestel, Uwe; Schulz, Georg E.

doi:10.1111/j.1432-1033.1991.tb16158.x

Cited by 84 publications

(34 citation statements)

References 26 publications

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“…The eyelet is lined by positively charged groups at the channel wall and by negatively charged groups being part of two loops (Weiss et al, 1991 b). A very similar arrangement has been shown to exist in the porins OmpF and PhoE of E. coli although they seem to be different regarding details like the tip of the channel-size-defining loop which is conserved amongst them and 12 other porin molecules but not in the R. capsulatus porin (Cowan et al, 1992;Weiss et al, 1991 b;Schiltz et al, 1991 ;Jeanteur et al, 1991). A loop structure corresponding to the large loop of R. capsulatus has also been proposed for Omp32 of C. acidovorans, which is closely related to A. delafeldii (GerblRieger et al, 1992).…”

Section: Voltage-dependent Gating Of Omp34mentioning

confidence: 99%

Asymmetry of orientation and voltage gating of the Acidovorax delafieldii porin Omp34 in lipid bilayers

Brunen

Engelhardt

1993

European Journal of Biochemistry

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The functional properties of the major outer-membrane protein of Acidovorax delafieldii, the anion-selective porin Omp34, were investigated in artificial membranes. Detergent-solubilized porin incorporates into the membrane in a unidirectional orientation solely determined by protein features. This enabled us to characterize the vectorial properties of the porin channels. Omp34 is electrostatically asymmetric regarding both the ion conductivity of a single trimer and the macroscopic ion conductance of multiple porin molecules. Voltage-dependent closing occured at negative potentials ; 50% of the channels were already closed at -10 mV (switching voltage). Our experimental results suggest that protein charges situated on flexible parts inside the channel are involved in the gating mechanism. A simple model is proposed illustrating the mechanism of voltage-dependent opening and closing of the porin channels. This model explains the functional characteristics of Omp34 and the dependence of the switching voltage on the electrolyte concentration in particular. Further factors influencing voltage gating include the buffer concentration as well as the technique used for membrane formation. Altogether these factors may explain the relatively high voltages needed to obtain voltage gating with other porins.In recent years a wealth of new information on the structure and function of porins of bacterial outer membranes has been accumulated. A major achievement was the determination of porin structures at atomic resolution, in particular of the Rhodobacter capsulatus porin (Weiss et al., 1991 a) and porins from Escherichia coli (Jap et al., 1991 ;Cowan et al., 1992). These structures provided for the first time an insight into the channel morphology and the charge distribution inside the channel.Many functional investigations confirmed the initial finding of Schindler and Rosenbusch (1978) that porin channels open and close in a voltage-dependent manner. Interestingly the voltages required for channel closing were different even for identical porins in the various studies. This applies to OmpF (Lakey and Pattus, 1989;Morgan et al., 1990) and OmpC (Lakey et al., 1991 ; Biihler, 1991) of E. coli as well as to protein I of Neisseria gonorrhoeae (Young et al., 1983 ;Mauro et al., 1988). A revealing comparison of the effects resulting from the different techniques of membrane formation was given by Lakey and Pattus (1989), but the prerequisites for channel closing at low potentials have not been investigated systematically so far. It is shown here for the porin Omp34 of Acidovorax delafieldii that functional experiments together with the structural information available provides further insight into the mechanisms allowing closing and opening of porin channels.Omp34 of A. delafieldii is a strongly anion-selective porin, and recently its biochemical and some of its functional characteristics have been described (Brunen et al., 1991). The Correspondence to M. Brunen,

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Section: Voltage-dependent Gating Of Omp34mentioning

confidence: 99%

Asymmetry of orientation and voltage gating of the Acidovorax delafieldii porin Omp34 in lipid bilayers

Brunen

Engelhardt

1993

European Journal of Biochemistry

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“…For determination of the N terminus of the subunits, the subunits were blotted onto a Sequi-Blot polyvinylidene difluoride membrane (BioRad) following the manufacturer's instructions. The N-terminal sequences were determined as described earlier (18).…”

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confidence: 99%

Characterization of the Chlorate Reductase from Pseudomonas chloritidismutans

Wolterink¹,

Schiltz

Hagedoorn

et al. 2003

J Bacteriol

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A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P. chloritidismutans reduced only chlorate and bromate. Differences were also found in N-terminal sequences, molecular weight, and subunit composition. Metal analysis and electron paramagnetic resonance measurements showed the presence of iron and molybdenum, which are also found in other dissimilatory oxyanion reductases.Pseudomonas chloritidismutans (strain AW-1) is a recently isolated facultative anaerobic chlorate-reducing bacterium (22). In chlorate-reducing bacteria, chlorate is reduced to chlorite by a chlorate reductase, and in a second reaction, chlorite is disproportionated to chloride and oxygen by a chlorite dismutase. Two closely related strains of P. chloritidismutans, Pseudomonas stutzeri DSM 50227 and DSM 5190 T (with 16S ribosomal DNA similarities of 100% and 98.6%, respectively), were not able to grow by dissimilatory chlorate reduction. Accordingly, both P. stutzeri strains lack chlorite dismutase activity. However, in addition to nitrate reductase activity, these strains also showed chlorate reductase activity, which has been observed before for other denitrifying bacteria (6, 15). P. chloritidismutans differed from other (per)chlorate-reducing bacteria in that it was only able to use chlorate as a terminal electron acceptor. Other chlorate-reducing bacteria can also couple the reduction of perchlorate or nitrate to growth (1, 13). Although cell extracts also showed bromate reductase activity, P. chloritidismutans could not grow on bromate (22).Up to now, two enzymes that can reduce chlorate and/or perchlorate have been purified and characterized. A chlorate reductase C has been purified from the denitrifying strain Proteus mirabilis, as well as two nitrate reductases (15). The only known substrate of chlorate reductase C is chlorate, which was reduced to chlorite. It was not demonstrated that Proteus mirabilis was able to couple the reduction of chlorate to growth. A second (per)chlorate reductase has been purified from strain GR-1 (10). Experiments showed that one enzyme is responsible for both chlorate and perchlorate reduction activity. Besides (per)chlorate, nitrate, iodate, and bromate were also reduced by the (per)chlorate reductase of strain GR-1. Perchlorate-grown cells were unable to oxidize nitrate or nitrite, indicating that another nitrate reductase may be involved in nitrate-grown cells (17). The purified chlorate reductase of P. chloritidismutans reported here is the first chlorate reductase derived from a chlorate-reducing bacterium that is capable of only dissimilatory chlorate reduction.P. chloritidismutans (DSM 13592) was grown under strictly anaerobic conditions at 30°C, as described before (1). Strain GR-1 (DSM 11199) was grown as described previously (10, 17). Chlorate (10 mM) was used as the electron acceptor, while acetate (10 mM) was used as the e...

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“…It can also be seen that in all strains, there is cross-reactivity to other higher-Mr proteins, perhaps due to nonspecific binding of the IgG. One in particular had an apparent Mr of 31,000, similar to that of the outer membrane porin (36 (Fig. 4, lane 6), the putative porin band also exhibited a much higher reactivity toward the antibody, possibly indicating a strong hydrophobic association between the porin and the PufQ protein.…”

Section: Resultsmentioning

confidence: 85%

Identification of the PufQ protein in membranes of Rhodobacter capsulatus

et al. 1994

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The PufQ protein has been detected in vivo for the first time by Western blot (immunoblot) analyses of the chromatophore membranes of Rhodobacter capsulatus. The PufQ protein was not visible in Western blots of membranes of a mutant (ARC6) lacking the puf operon but appeared in membranes of the same mutant to which the pufQ gene had been added in trans. It was also detected in elevated amounts in a mutant (CB1200) defective in two bch genes and unable, therefore, to make bacteriochlorophyll. The extremely hydrophobic nature of the PufQ protein was also apparent in these studies since it was not extracted from chromatophores by 3% (wt/vol) n-octyl-p-D-glucopyranoside, a procedure which solubilized the reaction center and lightharvesting complexes. During adaptation of R. capsulatus from aerobic to semiaerobic growth conditions (during which time the synthesis of bacteriochlorophyll was induced), the PufQ protein was observed to increase to the level of detection in the developing chromatophore fraction approximately 3 h after the start of the adaptation. The enzyme, S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase, also increased in amount in the developing chromatophore fraction but was present in a cell membrane fraction at the start of the adaptation as well.The classic study by Cohen-Bazire et al. (14) first demonstrated the regulatory role of oxygen and light on pigment synthesis in purple nonsulfur bacteria. In heterotrophic cultures of Rhodobacter sphaeroides grown with vigorous aeration for several successive transfers, bacteriochlorophyll (Bchl) was barely detectable (14). Even so, Gorchein et al. (26) showed that an enzyme of the magnesium branch of Bchl synthesis, S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase (MT), was still present in low but measurable amounts in oxygen-grown cultures. After subsequent incubation under anaerobic or semiaerobic conditions in the light, the cells underwent a period of adaptation during which time no growth occurred, but the level of the MT increased approximately sevenfold, followed by the appearance of Bchl and phototrophic growth (26). Gorchein (25) found a similar effect on the induction of magnesium chelatase activity in whole cells of R sphaeroides during adaptation from aerobic to phototrophic growth conditions.Although no assays for any other enzymes of the magnesium branch of Bchl synthesis have been developed, genes for these enzymes (the bch genes) have been mapped and cloned in both Rhodobacter capsulatus (38, 44) and R. sphaeroides (15). High oxygen tension resulted in a two-to fourfold repression of the transcription of bch genes (2,8,28,33,43), whereas high light intensity had no such effect (5). However, a much more severe repression by oxygen occurred on the transcription of the genes of Bchl-binding proteins, including the a and 13 subunits of the B870 light-harvesting I (LH I) complex and L and M subunits of the reaction center (RC) complex encoded by the puf operon, and the ao and 13 subunits of the B800-850 * C...

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Primary structure of porin from Rhodobacter capsulatus

Cited by 84 publications

References 26 publications

Asymmetry of orientation and voltage gating of the Acidovorax delafieldii porin Omp34 in lipid bilayers

Asymmetry of orientation and voltage gating of the Acidovorax delafieldii porin Omp34 in lipid bilayers

Characterization of the Chlorate Reductase from Pseudomonas chloritidismutans

Identification of the PufQ protein in membranes of Rhodobacter capsulatus

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