Assembly of Escherichia coli cytochrome bd and periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type and cydD mutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in the cydD periplasm. Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner. New pleiotropic cydD phenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation. Exogenous cysteine reversed these phenotypes and affected levels of periplasmic c-type cytochromes in cydD and wild-type strains but did not restore cytochrome d. Consistent with CydDC being a cysteine exporter, cydD mutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective in orf299, encoding a transporter of the major facilitator superfamily. A cydD orf299 double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm.

Nitric oxide (NO) is a key signaling and defense molecule in biological systems. The bactericidal effects of NO produced, for example, by macrophages are resisted by various bacterial NO-detoxifying enzymes, the best understood being the flavohemoglobins exemplified by Escherichia coli Hmp. However, many bacteria, including E. coli, are reported to produce NO by processes that are independent of denitrification in which NO is an obligatory intermediate. We demonstrate using an NOspecific electrode that E. coli cells, grown anaerobically with nitrate as terminal electron acceptor, generate significant NO on adding nitrite. The periplasmic cytochrome c nitrite reductase (Nrf) is shown, by comparing Nrf ؉ and Nrf ؊ mutants, to be largely responsible for NO generation. Surprisingly, an hmp mutant did not accumulate more NO but, rather, failed to produce detectable NO. Anaerobic growth of the hmp mutant was not stimulated by nitrate, and the mutant failed to produce periplasmic cytochrome ( Nitric oxide (nitrogen monoxide, NO)1 is a molecule of major importance in biological systems where it plays signaling, vasodilatory, and cytotoxic roles. Recent attention has focused on NO synthesis from the sequential oxidation of L-arginine by NO synthases in eukaryotic cells (1), their mitochondria (2), and certain bacteria (3). NO is also an obligate intermediate in denitrification, the process by which certain bacteria sequentially reduce nitrate ion to dinitrogen (4, 5). However, several representatives of the "non-denitrifying" Enterobacteriaceae, including Escherichia coli, grown anaerobically with nitrate, were shown to produce up to one-twentieth of the NO produced by denitrifiers. NO production from nitrite, measured by the nitrosation of 2,3-diaminonaphthalene (DAN) (6) was proposed to involve enzymatic reduction of nitrite to NO followed by oxygen-dependent DAN nitrosation. NO production has also been shown in Serratia marcescens (7), Bacillus cereus (8), three species of methanotrophic bacteria (9), and the green micro alga Scenedesmus obliquus (10). Ji and Hollocher (11) concluded that nitrite-dependent NO production by E. coli was due to the activity of the membrane-associated (dissimilatory) nitrate reductase. Nitrate reductase exhibited at all stages of its purification a nitrite reductase activity, which was strongly inhibited by nitrate and azide.More recent evidence for NO production by E. coli has come from expression of the Paracoccus denitrificans transcription factor NNR in E. coli. This protein is activated by NO, and transcription of a target melR-lacZ promoter in E. coli was attributed to formation of NO (or related species) from nitrate by molybdenum-dependent nitrite reductase (12). NO production from nitrite, however, was not dependent on molybdenum cofactor biosynthesis.Since the initial reports of NO production by E. coli (6, 13), advances have been made that prompt a reinvestigation. First, sensitive NO electrodes with markedly improved selectivity have been developed (14). Second, several prote...

Role of flavohemoglobin in combating nitrosative stress in uropathogenic Escherichia coliimplications for urinary tract infection. During the course of urinary tract infection (UTI) nitric oxide (NO) is generated as part of host response. The aim of this study was to investigate the significance of the NO-detoxifying enzymes flavohemoglobin (Hmp) and flavorubredoxin (Norv) in protection of uropathogenic Escherichia coli (UPEC) against nitrosative stress. Hmp (J96∆hmp) and norV (J96∆norV) knockout mutants of UPEC strain J96 were constructed using a single-gene deletion strategy. Bacterial tolerance and expression of hmp and norV in response to the NO-donor DETA/NO was evaluated in Luria broth and urine from healthy volunteers. Bacterial NO consumption and respiratory inhibition were assessed when exposed to NO. Expression of hmp and norV from E. coli originating from patients with UTI was evaluated using real-time PCR. The colonizing ability of J96 wild-type (wt) compared to an hmp-deficient mutant was assessed using a competition-based mouse UTI model. The viability of J96∆hmp and J96∆norV was significantly reduced compared to the wildtype strain after exposure to DETA/NO. The hmp expression in DETA/NO-exposed cultures was similar in J96wt and J96∆norV, while J96∆hmp showed an increased norV expression compared to J96wt. The NO consumption in J96∆hmp, but not in J96∆norV, was significantly impaired compared to J96wt. An up-regulation of hmp expression was found in E. coli isolated from all UTIpatients while norV expression increased in 50% of the patients. In the mouse UTI model, the hmp-mutant strain was significantly out-competed by the wild-type strain in the bladder and kidney. Hmp and NorV contribute to the protection of UPEC against NOmediated toxicity in vitro. Screening UPEC isolates from UTI patients revealed an increased hmp expression in all patients which confirms that hmp expression occurs in vivo in the infected human urinary tract. The ability to colonize the mouse urinary tract was impaired in the hmp-deficient mutant compared to the wild-type strain. NO-detoxification by Hmp is suggested to be an important characteristic for UPEC in protection against nitrosative stress and may be a virulence-facilitating factor.

Escherichia coli Hmp is a homologue of Ralstonia eutropha FHP, the first reported bacterial flavohaemoglobin, and functions in NO detoxification. Photolysis of CO-ligated Hmp in the presence of oxygen gave a photodissociable oxy species with k(on) 2.82x10(7) M(-1) s(-1) and k(off) 4.49x10(3) s(-1). The dissociation constant of the primary O(2) compound was 160 microM (25 degrees C, pH 7.0). In order to detect superoxide formation, ferric horseradish peroxidase was used. Hmp formed the oxy compound within milliseconds, followed by formation of compound III, arising from superoxide formation. The rate of superoxide formation was independent of oxygen concentration between 0.05 and 0.7 mM oxygen, suggesting a K(m) <0.05 mM. During prolonged oxidation of NADH, the spectral signals of Hmp decayed and iron was released in a process prevented by superoxide dismutase or catalase. NADH oxidation by purified Hmp was characterised by progressive slowing of oxygen uptake. Inclusion of NO, superoxide dismutase or catalase during NADH oxidation partially protected oxygen uptake, consistent with the formation, in the absence of NO, of reactive oxygen species that inhibit Hmp function. The results are discussed in relation to the tight control exerted on Hmp synthesis in vivo.

Azotobacter vinelandii cydAB mutants lacking cytochrome bd lost viability in stationary phase, irrespective of temperature, but microaerobiosis or iron addition to stationary phase cultures prevented viability loss. Growth on solid medium was inhibited by a diffusible factor from neighbouring cells, and by iron chelators, In(III) or Ga(III); microaerobic growth overcame inhibition by the extracellular factor. Siderophore production and total Fe(III)-chelating activity were not markedly affected in Cyd(-) mutants, and remained responsive to iron repression. Cyd(-) mutants were hypersensitive to Cu(II), Zn(II), and compounds exerting oxidative stress. Failure to synthesise haemoproteins does not explain the complex phenotype since mutants retained significant catalase activity. We hypothesise that Cyd(-) mutants are defective in maintaining the near-anoxic cytoplasm required for reductive iron metabolism and nitrogenase activity.