Search

The dcuC gene of Escherichia coli encodes an alternative C4-dicarboxylate carrier (DcuC) with low transport activity. The expression of dcuC was investigated. dcuC was expressed only under anaerobic conditions; nitrate and fumarate caused slight repression and stimulation of expression, respectively. Anaerobic induction depended mainly on the transcriptional regulator FNR. Fumarate stimulation was independent of the fumarate response regulator DcuR. The expression of dcuC was not significantly inhibited by glucose, assigning a role to DcuC during glucose fermentation. The inactivation of dcuC increased fumarate-succinate exchange and fumarate uptake by DcuA and DcuB, suggesting a preferential function of DcuC in succinate efflux during glucose fermentation. Upon overexpression in a dcuC promoter mutant (dcuC*), DcuC was able to compensate for DcuA and DcuB in fumarate-succinate exchange and fumarate uptake.

In Escherichia coli, two carriers (DcuA and DcuB) for the transport of C4 dicarboxylates in anaerobic growth were known. Here a novel gene dcuC was identified encoding a secondary carrier (DcuC) for C4 dicarboxylates which is functional in anaerobic growth. The dcuC gene is located at min 14.1 of the E. coli map in the counterclockwise orientation. The dcuC gene combines two open reading frames found in other strains of E. coli K-12. The gene product (DcuC) is responsible for the transport of C4 dicarboxylates in DcuA-DcuB-deficient cells. The triple mutant (dcuA dcuB dcuC) is completely devoid of C4-dicarboxylate transport (exchange and uptake) during anaerobic growth, and the bacteria are no longer capable of growth by fumarate respiration. DcuC, however, is not required for C4-dicarboxylate uptake in aerobic growth. The dcuC gene encodes a putative protein of 461 amino acid residues with properties typical for secondary procaryotic carriers. DcuC shows sequence similarity to the two major anaerobic C4-dicarboxylate carriers DcuA and DcuB. Mutants producing only DcuA, DcuB, or DcuC were prepared. In the mutants, DcuA, DcuB, and DcuC were each able to operate in the exchange and uptake mode.

FNR, the O2-responsive gene regulator of anaerobic respiratory genes in Escherichia coli, contains an N-terminal cluster of four cysteine residues (Cys16-X3-Cys20-X2-Cys23-X5-Cys29), three of which are thought to be involved in the binding of an iron cofactor. The accessibility of the cysteine residues for iodoacetate is known to increase upon switch from the active (anaerobic) to the inactive (aerobic or metal depleted) state. It was analyzed which residues become accessible under either condition. Up to four modified forms, FNR-I, FNR-II, FNR-III, and FNR-IV, containing approximately 1, 2, 3.5, and 5 carboxymethyl groups, were obtained either by reaction in vivo and in vitro under conditions of aerobiosis, anaerobiosis, or iron limitation. By N-terminal sequencing, the carboxymethylated cysteine residues were identified. The amount of label in each of the four cysteine residues increased rather uniformly and gradually from FNR-I to FNR-IV irrespective of the condition of labeling; only Cys16 was preferentially labeled to some extent. It is concluded that the four essential cysteine residues change their accessibility in a similar way in the switch from active to inactive (aerobic or metal depleted) FNR, without specific differences in their reaction or function. Potential modes of Fe-binding by the cysteine residues are discussed. In addition, a different type of interaction of Fe(II) with FNR is described. The interaction occurred also in FNR carboxymethylated at approximately three cysteine residues.

A new binding-protein-dependent transport system of Escherichia coli specific for L-arginine was characterized by genetic and biochemical means. The system is encoded by five adjacent genes, artPIQMJ (art standing for arginine transport), which are organized in two transcriptional units (artPIQM and artJ). The artl and artJ gene products (Artl and ArtJ) are periplasmic binding proteins with sequence similarity to binding proteins for polar (basic) amino acids. The artQ, artM and artP products are similar to the transmembraneous proteins and the ATPase of binding-protein-dependent carriers. The mature Artl and J proteins were localized in the periplasm and lacked signal peptides of 19 amino acid residues. Artl and ArtJ were isolated from overproducing strains. ArtJ specifically binds L-arginine with high affinity and overproduction of ArtJ stimulated L-arginine uptake by the bacteria. The substrate for Artl is not known, and isolated Artl did not bind common amino acids, various basic uncommon amino acids or amines. It is concluded that the artPIQM artJ genes encode a third arginine-uptake system in addition to the known argT hisJQMP system of Salmonella typhimurium and E. coli and the arginine (-ornithine) carrier (aps) of E. coli.

Availability of O2 is one of the most important regulatory signals in facultatively anaerobic bacteria. Various two- or one-component sensor/regulator systems control the expression of aerobic and anaerobic metabolism in response to O2. Most of the sensor proteins contain heme or Fe as cofactors that interact with O2 either by binding or by a redox reaction. The ArcA/ArcB regulator of aerobic metabolism in Escherichia coli may use a different sensory mechanism. In two-component regulators, the sensor is located in the cytoplasmic membrane, whereas one-component regulators are located in the cytoplasm. Under most conditions, O2 can readily reach the cytoplasm and could provide the signal in the cytoplasm. The transcriptional regulator FNR of E. Coli controls the expression of many genes required for anaerobic metabolism in response to O2. Functional homologs of FNR are present in facultatively anaerobic Proteobacteria and presumably also in gram-positive bacteria. The target genes of FNR are mostly under multiple regulation by FNR and other regulators that respond to O2, nitrate, or glucose. FNR represents a 'one-component' sensor/regulator and contains Fe for signal perception. In response to O2 availability, FNR is converted reversibly from the aerobic (inactive) state to the anaerobic (active) state. Experiments suggest that the Fe cofactor is bound by four essential cysteine residues. The O2-triggered transformation between active and inactive FNR presumably is due to a redox reaction at the Fe cofactor, but other modes of interaction cannot be excluded. O2 seems to affect the site-specific DNA binding of FNR at target genes or the formation of an active transcriptional complex with RNA polymerase.

The nucleotide sequences of two Escherichia coli genes, dcuA and dcuB (formerly designated genA and genF), have been shown to encode highly homologous products, M(r) 45,751 and 47,935 (434 and 446 amino acid residues) with 36% sequence identity (63% similarity). These proteins have a high proportion (approximately 61%) of hydrophobic residues and are probably members of a new group of integral inner membrane proteins. The locations of the dcu genes, one upstream of the aspartase gene (dcuA-aspA) and the other downstream of the anaerobic fumarase gene (fumB-dcuB), suggested that they may function in the anaerobic transport of C4-dicarboxylic acids. Growth tests and transport studies with mutants containing insertionally inactivated chromosomal dcuA and dcuB genes show that their products perform analogous and mutually complementary roles as anaerobic dicarboxylate carriers. The anaerobic dicarboxylate transport systems (Dcu) are genetically and functionally distinct from the aerobic system (Dct).

FNR is a transcriptional regulator mediating the activation or repression of a variety of Escherichia coli genes in response to anoxia. The FNR protein resembles CRP (the cyclic-AMP receptor protein) except for the presence of a cysteine-rich N-terminal segment which may form part of an iron-binding redoxsensing domain. The FNR protein was purified by a new procedure. It was monomeric (Mr = 30,000) and contained as much as 1.1 mol of iron per monomer when purified in the presence of added iron. This iron was associated with cysteine residues, because there was an inverse relation between iron content and titratable sulphydryl groups. Other physical and chemical properties are reported including evidence for a potential disulphide group or analogous modification. The interaction between FNR protein and target DNA appeared weak and non-specific in gel-retardation assays, but specific binding to the proposed DNA-binding site was shown for the first time in footprinting studies. A role for iron in FNR-mediated gene expression was confirmed by using cultures in which FNR was inactivated by growth in the presence of the specific chelator, ferrozine, but protected by ferrous iron.