Regulation of enterobactin iron transport in Escherichia coli: characterization of ent::Mu d(Apr lac) operon fusions

144

189

Summary The TonB‐dependent energy transduction system couples cytoplasmic membrane proton motive force to active transport of iron–siderophore complexes across the outer membrane in Gram‐negative bacteria. In Escherichia coli, the primary players known in this process to date are: FepA, the TonB‐gated transporter for the siderophore enterochelin; TonB, the energy‐transducing protein; and two cytoplasmic membrane proteins with less defined roles, ExbB and ExbD. In this study, we report the per cell numbers of TonB, ExbB, ExbD and FepA for cells grown under iron‐replete and iron‐limited conditions. Under iron‐replete conditions, TonB and FepA were present at 335 ± 78 and 504 ± 165 copies per cell respectively. ExbB and ExbD, despite being encoded from the same operon, were not equimolar, being present at 2463 ± 522 and 741 ± 105 copies respectively. The ratio of these proteins was calculated at one TonB:two ExbD:seven ExbB under all four growth conditions tested. In contrast, the TonB:FepA ratio varied with iron status and according to the method used for iron limitation. Differences in the method of iron limitation also resulted in significant differences in cell size, skewing the per cell copy numbers for all proteins.

Section: Discussionmentioning

confidence: 86%

Quantification of known components of the Escherichia coli TonB energy transduction system: TonB, ExbB, ExbD and FepA

Higgs¹,

Larsen²,

Postle³

2002

144

189

“…Under anaerobic conditions, Fe" predominates and can be transported by a TonB-independent mechanism (Hantke, 1987;Kammler etai, 1993). Previous studies of tonS transcription have been performed in tonB strains and consequently detected only the anaerobic repression of tonB (Hantke, 1981;Dorman etai., 1988) or no regulation at all (Fleming et al, 1983). Increased negative supercoiling was proposed to mediate anaerobic repression of tonB, again using tonB strains (Dorman et ai, 1988).…”

Section: Introductionmentioning

confidence: 99%

Repression of tonB transcription during anaerobic growth requires Fur binding at the promoter and a second factor binding upstream

Young

Postle

1994

Although iron is an essential nutrient, its toxicity at high levels necessitates regulated transport. In Gram-negative bacteria a central target for regulation is the TonB protein, an energy transducer that couples the cytoplasmic membrane proton motive force to active transport of (FeIII)-siderophore complexes across the outer membrane. We have previously demonstrated the threefold repression of tonB transcription by excess iron in the presence of Fur repressor protein under aerobic conditions. In this report, we examine tonB regulation under anaerobic conditions where the solubility of iron is not a limiting factor and, presumably, siderophore-mediated transport is not required. Under these conditions, tonB transcription is repressed at least 10-fold by excess iron in the presence of Fur, but can be fully derepressed in the absence of Fur. Based on several lines of evidence, this anaerobic repression is not due to increased negative supercoiling as previously postulated. Our results rule out both supercoiling-mediated decreased promoter function and increased Fur binding as mediators of anaerobic repression. Under iron-limiting anaerobic conditions tonB expression is as high or higher than under iron-limiting aerobic conditions, suggesting that promoter function has not decreased anaerobically. Furthermore, under anaerobic conditions in tonB+ strains, tonB promoter function is insensitive to the gyrase inhibitor novobiocin and to changes in medium osmolarity and temperature, three conditions known to change levels of supercoiling. We also rule out effects of mutations in arcA or fnr as mediators of anaerobic repression. Results from in vivo dimethyl sulphate protection foot-printing indicate that Fur binds to an operator site between the -10 and -35 regions of the promoter, but not to a less homologous operator site centered at +26. The binding is, if anything, weaker under anaerobic conditions, indicating that anaerobic repression is not mediated through Fur. Additional changes in the in vivo footprint upstream from the promoter implicate a second factor in tonB anaerobic repression. Together, these results suggest that the mechanism responsible for this regulation (and, by analogy, that of other anaerobically repressed, iron-regulated genes such as cir, exbB, and fhuA) is a novel one.

“…1970). The genes specifying enterobactin biosynthesis and transport functions map to minute 13 of the chromosome (Bachmann, 1983), and are expressed coordinately in response to the intracellular iron concentration (Fleming et al, 1983). Enterobactin biosynthesis begins with the conversion of chorismate, an intermediate of aromatic amino acid biosynthesis, to 2,3-dihydroxybenzoate (DHBA) by the activities encoded by entC, entB, and entA (Young et al, 1971).…”

Section: Introductionmentioning

confidence: 99%

The Escherichia coli enterobactin biosynthesis gene, entD: nucleotide sequence and membrane localization of its protein product

Armstrong

Pettis

Forrester

et al. 1989

The nucleotide sequence of the Escherichia coli enterobactin biosynthesis gene entD has been determined. entD specifies a predicted 23579 Dalton protein containing several helical regions, a transmembrane segment and one positively charged domain. The EntD polypeptide was overexpressed and identified in electrophoretic gels as a membrane protein. Although results of conventional membrane fractionation techniques were inconclusive, protease accessibility studies provided evidence that EntD domains are exposed on the inner leaflet of the cytoplasmic membrane. The presence of repetitive extragenic palindromic (REP) sequences within the fepA-entD intercistronic region was confirmed. Lack of a canonical promoter and an iron control region 5' to entD, along with RNA hybridization data, suggest that an iron-regulated transcript contains both fepA and entD.