Identification of the formate dehydrogenases and genetic determinants of formate-dependent nitrite reduction by Escherichia coli K12

Abstract

The formate dehydrogenases of Escherichia coli involved in electron transfer from formate to nitrite (Nrf activity : nitrite reduction by formate) have been identified. No previously undescribed selenoprotein was detected in bacteria grown under conditions optimal for the expression of Nrf activity. The Nrf activities of single mutants defective in either FdhN or FdhH were between 50 and 60% that of the parental strain. A double mutant defective in both FdhN and FdhH retained less than 10% of the activity of the FdhN+FdhH+ strain. No Nrf activity was detected in a triple mutant defective in FdhN, FdhH and FdhO or in the selC strain. It is concluded that all three of the known formate dehydrogenases of E. coli can contribute to the transfer of electrons from formate to the Nrf pathway. Mutants defective in Nrf activity and cytochrome c552 synthesis were isolated by insertion mutagenesis or identified amongst strains received from the E. coli Genetic Stock Center. The mutations were located in at least three regions of the chromosome, including the 92 to 94 minute region which includesfdhF, the gene encoding FdhH required for formate hydrogenlyase activity. Fine structure mapping by P1 transduction established that the nrf mutations in thefdhF region were due to defects in three separable loci, all of which were independent of but close to fdhF. Clones were isolated from a cosmid library that complemented a deletion extending from fdhF into a region essential for Nrf activity. From these clones, plasmids were isolated that complemented only some of the N r f mutations in the 92 to 94 minute region, confirming the presence of different operons essential for Nrf activity and cytochrome c552 synthesis in this region. Suggested reasons for this genetic complexity include the need for proteins involved in electron transfer from the various formate dehydrogenases to cytochrome c552, for the attachment of the haem group to the apocytochrome and for cytochrome ~5 5 2 export into the periplasm.

Mutants of Escherichia coli lacking disulphide oxdoreductases DsbA and DsbB cannot synthesise an exogenous monohaem c‐type cytochrome except in the presence of disulphide compounds

“…These E. coli strains were transformed by the plasmid pKPD1 [9] which carries the wild-type P. denitrificans cytochrome c550 gene under the control of tac promoter of the original vector pKK223-3 (ampicillin resistance, Pharmacia). The transformed cells were grown in 2 ml of LB media overnight, and then 50 ~tl of fresh cells were inoculated into 15 ml conical tube filled with anaerobic minimal media [15] supplemented with 2.5 mM nitrite and 40 mM fumarate as electron acceptors, 0.4% (w/v) glycerol as a carbon source, and thiamine (1 ~tg/ml). To demonstrate the effects of disulphide compounds, freshly made stocks of these compounds in the same liquid media were diluted into the tubes.…”

Specific thiol compounds complement deficiency in c‐type cytochrome biogenesis in Escherichia coli carrying a mutation in a membrane‐bound disulphide isomerase‐like protein

“…The E. coli K-12 strain JCB606, which is deficient in both endogenous and exogenous periplasmic and membrane c-type cytochromes [1], and its parental strain JCB387 [3], which can produce c-type cytochromes normally, were used in this study. These strains were transformed by the plasmid pKPD1 which carries the complete gene for P denitrificans cytochrome c550 [4].…”

“…alkaline phosphatase (AP) translational fusion gene described below. The cells were grown at 370C anaerobically in minimal media [3] in the presence of glycerol, fumarate and nitrite supplemented with various chemical compounds described in section 3. The antibiotics ampiciUin (50 #g/ml) and kanamycin (25/.tg/ml) were added to the media.…”

Membrane Potential as a Regulation Mechanism of Periplasmic Nitrite Reductase Activity: A Mathematical Model

“…Ряд экспериментальных данных [8,14,6,13,16,17] позволяет сделать предположение, что в формировании протонного градиента в условиях анаэробного дыхания на нитрите может участвовать Fdh-H форматдегидрогеназа, активные сайты которой расположены в цитоплазме. Предпосылкой служат данные [6], что в условиях хемостата Fdh-H дегидрогеназа является единственной форматдегидрогеназой, экспрессия которой зависит от концентрации нитрита в среде, и профиль этой экспрессии удивительно напоминает таковой для nrf оперона, кодирующего структуру NrfА нитритредуктазы [7].…”

“…Ряд экспериментальных данных [6,8,13,14,16,17] позволил нам предположить, что в условиях, близких к экспериментам Ванга и коллег [15] по культивированию культуры бактерий в хемостате на нитрите, в формировании протонного градиента может участвовать Fdh-H форматдегидрогеназа, активные сайты которой расположены в цитоплазме. В связи с предполагаемой ролью Fdh-H фермента в формировании дыхательной цепи, были рассмотрены два альтернативных сценария возможных механизмов передачи электронов от формата к нитриту с участием этого фермента и промежуточных посредников.…”