Expression, Purification, and Characterization of Recombinant Forms of Membrane-Bound Cytochromec-550nm fromBacillus subtilis

David, Pamela S.; Morrison, Megan R.; Wong, Sui‐Lam; Hill, Bruce C.

doi:10.1006/prep.1998.1001

Cited by 9 publications

(4 citation statements)

References 29 publications

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“…Remarkably, the His ' -tagged protein was produced in lower concentrations than the untagged protein. The same results were obtained by David et al [39] : the overall yield of the untagged form of cytochrome c-552 of Bacillus subtilis per volume of culture was also higher than the tagged form, although purification of the latter was easier. Despite the different expression levels, the reduced, oxidized and ' reduced-minus-oxidized ' spectra of the tagged and untagged forms of NapB were identical, indicating that the tagged protein retained its redox activity.…”

Section: Figure 1 Haem-stained Sds/15 % (W/v) Polyacrylamide Gel Of Ssupporting

confidence: 83%

Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae

Brigé

Cole

Hagen

et al. 2001

Biochemical Journal

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The napB gene of the pathogenic bacterium Haemophilus influenzae encodes a dihaem cytochrome c, the small subunit of a heterodimeric periplasmic nitrate reductase similar to those found in other bacteria. In order to obtain sufficient protein for biophysical studies, we aimed to overproduce the recombinant dihaem protein in Escherichia coli. Initial expression experiments indicated that the NapB signal peptide was not cleaved by the leader peptidase of the host organism. Apocytochrome was formed under aerobic, semi-aerobic and anaerobic growth conditions in either Luria–Bertani or minimal salts medium. The highest amounts of apo-NapB were produced in the latter medium, and the bulk was inserted into the cytoplasmic membrane. The two haem groups were covalently attached to the pre-apocytochrome only under anaerobic growth conditions, and with 2.5mM nitrite or at least 10mM nitrate supplemented to the minimal salts growth medium. In order to obtain holocytochrome, the gene sequence encoding mature NapB was cloned in-frame with the E. coli ompA (outer membrane protein A) signal sequence. Under anaerobic conditions, NapB was secreted into the periplasmic space, with the OmpA signal peptide being correctly processed and with both haem c groups attached covalently. Unless expressed in the DegP-protease-deficient strain HM125, some of the recombinant NapB polypeptides were N-terminally truncated as a result of proteolytic activity. Under aerobic growth conditions, co-expression with the E. coli ccm (cytochrome c maturation) genes resulted in a higher yield of holocytochrome c. The pure recombinant NapB protein showed absorption maxima at 419, 522 and 550nm in the reduced form. The midpoint reduction potentials of the two haem groups were determined to be −25mV and −175mV. These results support our hypothesis that the Nap system fulfils a nitrate-scavenging role in H. influenzae.

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Section: Figure 1 Haem-stained Sds/15 % (W/v) Polyacrylamide Gel Of Ssupporting

confidence: 83%

Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae

Brigé

Cole

Hagen

et al. 2001

Biochemical Journal

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“…This protein (PedF, 153 amino acids; Mr, 16.30 kDa) shows strong homology with a cytochrome c ‐type protein located in the periplasm of different bacteria (Schobert and Görisch, 1999; Stover et al ., 2000; Nelson et al ., 2002; Paulsen et al ., 2005). It contains a CccA domain that includes a haem‐binding motif (CAACH) (David et al ., 1999; Schobert and Görisch, 1999). Knocking out this gene (see Experimental procedures ) revealed that this mutant ( P. putida U Δ pedF ::pK18 mob ) was unable to grow in chemically defined media containing PhEtOH, hexanol, heptanol, octanol, nonanol or decanol, although like the wild type ( P. putida U) it was able to catabolize phenylacetate, PhEtNH 2 , 4‐OH‐PhAc and other carbon sources.…”

Section: Resultsmentioning

confidence: 99%

Genetic analyses and molecular characterization of the pathways involved in the conversion of 2‐phenylethylamine and 2‐phenylethanol into phenylacetic acid in Pseudomonas putida U

Arias

Olivera

Arcos

et al. 2007

Environmental Microbiology

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In Pseudomonas putida U two different pathways (Pea, Ped) are required for the conversion of 2-phenylethylamine and 2-phenylethanol into phenylacetic acid. The 2-phenylethylamine pathway (PeaABCDEFGHR) catalyses the transport of this amine, its deamination to phenylacetaldehyde by a quinohaemoprotein amine dehydrogenase and the oxidation of this compound through a reaction catalysed by a phenylacetaldehyde dehydrogenase. Another catabolic route (PedS(1)R(1)ABCS(2)R(2)DEFGHI) is needed for the uptake of 2-phenylethanol and for its oxidation to phenylacetic acid via phenylacetaldehyde. This implies the participation of two different two-component signal-transducing systems, two quinoprotein alcohol dehydrogenases, a cytochrome c, a periplasmic binding protein, an aldehyde dehydrogenase, a pentapeptide repeat protein and an ABC efflux system. Additionally, two accessory sets of elements (PqqABCDEF and CcmABCDEFGHI) are necessary for the operation of the main pathways (Pea and Ped). PqqABCDEF is required for the biosynthesis of pyrroloquinoline quinone (PQQ), a prosthetic group of certain alcohol dehydrogenases that transfers electrons to an independent cytochrome c; whereas CcmABCDEFGHI is required for cytochrome c maturation. Our data show that the degradation of phenylethylamine and phenylethanol in P. putida U is quite different from that reported in Escherichia coli, and they demonstrate that PeaABCDEFGHR and PedS(1)R(1)ABCS(2)R(2)DEFGHI are two upper routes belonging to the phenylacetyl-CoA catabolon.

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“…Several other c-type cytochromes have known or postulated regulatory roles. Cyt c 561 of Bacillus subtilis has a midpoint potential of ∼190 mV (39) and a role in initiation of sporulation (40). Involvement of c 6 -like cytochromes in redox or oxygen sensing related to heterocyst differentiation may be an intriguing possibility.…”

Section: Discussionmentioning

confidence: 99%

Deeply Branching c₆-like Cytochromes of Cyanobacteria

et al. 2008

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The cyanobacterium Synechococcus sp. PCC 7002 carries two genes, petJ1 and petJ2, for proteins related to soluble, cytochrome c6 electron transfer proteins. PetJ1 was purified from the cyanobacterium, and both cytochromes were expressed with heme incorporation in Escherichia coli. The expressed PetJ1 displayed spectral and biochemical properties virtually identical to those of PetJ1 from Synechococcus. PetJ1 is a typical cytochrome c6 but contains an unusual KDGSKSL insertion. PetJ2 isolated from E. coli exhibited absorbance spectra characteristic of cytochromes, although the alpha, beta, and gamma bands were red-shifted relative to those of PetJ1. Moreover, the surface electrostatic properties and redox midpoint potential of PetJ2 (pI 9.7; E(m,7) = 148 +/- 1.7 mV) differed substantially from those of PetJ1 (pI 3.8; E(m,7) = 319 +/- 1.6 mV). These data indicate that the PetJ2 cytochrome could not effectively replace PetJ1 as an electron acceptor for the cytochrome bf complex in photosynthesis. Phylogenetic comparisons against plant, algal, bacterial, and cyanobacterial genomes revealed two novel and widely distributed clusters of previously uncharacterized, cyanobacterial c 6-like cytochromes. PetJ2 belongs to a group that is distinct from both c6 cytochromes and the enigmatic chloroplast c 6A cytochromes. We tentatively designate the PetJ2 group as c6C cytochromes and the other new group as c6B cytochromes. Possible functions of these cytochromes are discussed.

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Expression, Purification, and Characterization of Recombinant Forms of Membrane-Bound Cytochromec-550nm fromBacillus subtilis

Cited by 9 publications

References 29 publications

Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae

Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae

Genetic analyses and molecular characterization of the pathways involved in the conversion of 2‐phenylethylamine and 2‐phenylethanol into phenylacetic acid in Pseudomonas putida U

Deeply Branching c₆-like Cytochromes of Cyanobacteria

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Expression, Purification, and Characterization of Recombinant Forms of Membrane-Bound Cytochromec-550nm fromBacillus subtilis

Cited by 9 publications

References 29 publications

Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae

Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae

Genetic analyses and molecular characterization of the pathways involved in the conversion of 2‐phenylethylamine and 2‐phenylethanol into phenylacetic acid in Pseudomonas putida U

Deeply Branching c6-like Cytochromes of Cyanobacteria

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Deeply Branching c₆-like Cytochromes of Cyanobacteria