Iron uptake and iron limited growth of Escherichia coli K-12

Cells of Escherichia coli K-12 could grow aerobically at an iron concentration as low as 0.05 M without any of the known iron ionophores present. The growth rate increased between 0.05 and 2 M iron. Supplementation with the iron ligands ferrichrome and citrate resulted in optimal growth already at 0.05 M iron. Under certain conditions iron uptake preceded growth of cells by more than an hour. During logarithmic growth the rate of iron uptake matched the growth rate. The radioactive tracer method revealed a cellular iron content of 4 nmol/mg dry weight.After consumption of the iron in the medium cells continued to grow with high rate for 1–2 generations. The iron uptake activity was increased during iron starvation.     

Iron transport in Escherichia coli K-12

The study of iron uptake promoted by 2,3-dihydroxybenzoate (DHB) into Escherichia coli K-12 aroB mutants allowed some dissection of outer and cytoplasmic membrane functions. These strains are unable to produce the iron-transporting chelate enterochelin, unless fed with a precursor such as DHB. When added to the medium, enterochelin and its natural breakdown products, the linear dimer and trimer of 2,3-dihydroxybenzoylserine (DBS), efficiently transported iron via the feuB, tonB and fep gene products. Thus mutants in these genes were defective in transport of the above chelates. However, feuB and tonB mutants were able to take up iron when DHB was added to the medium. Thus DHB-promoted iron uptake bypassed two functions required for the transport of ferric-enterochelin from the medium. One of these functions, feuB, has been shown to be an outer membrane protein. In contrast to three other iron transport systems including ferric-enterochelin uptake, DHB-promoted iron uptake was little affected by the uncoupler 2,4-dinitrophenol. Dissipation of the energized state of the cytoplasmic membrane apparently only affects those iron transport systems which require an outer membrane protein. Since DHB-promoted iron uptake bypasses the feuB outer membrane protein and the tonB function, it is concluded that, in ferricenterochelin transport, the tonB gene may function in coupling the energized state of the cytoplasmic membrane to the protein-dependent outer membrane permeability. DHB-promoted iron uptake required the synthesis and enzymatic breakdown of enterochelin as judged by the effects of the entF and fesB mutations. A fep mutant was not only deficient in the transport of the ferric chelates of enterochelin and its breakdown products, but was also deficient in DHB-promoted iron uptake. A scheme is presented in which iron diffuses as DHB-complex through the outer membrane, and is subsequently captured by enterochelin or DBS dimer or trimer and translocated across the cytoplasmic membrane.List of Abbreviations DHB 2,3-dihydroxybenzoate - DBS 2,3-dihydroxybenzoylserine - NTA nitrilotriacetate - DNP 2,4-dinitrophenol     

Iron uptake in pseudorevertants of Escherichia coli K-12 mutants with multiple defects in the enterochelin system

Iron uptake in pseudorevertants of Escherichia coli K-12 strains which lack the ability to synthesize enterochelin, 2,3-dihydroxybenzoate, and the ferrienterochelin receptor protein was characterized. In four independent pseudorevertants, the suppressor mutations which permitted growth in iron-poor environments appeared to be located in ompB, the regulatory locus for the porin proteins. Unlike wild-type cells, the pseudorevertants were unable to utilize ferrienterochelin and could acquire iron from citrate without induction by prior growth in citrate. The energy requirements of the pseudorevertant system appeared to be identical to those of the enterochelin system. Evidence that loss of the porin proteins results in the secretion by the pseudorevertants of a molecule with siderophore activity is presented; this siderophore is able to remove iron from the non-biological iron chelators nitrilotriacetic acid and , -dipyridyl but not from the siderophores ferrichrome and enterochelin.     

Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12

Summary In the course of an attempt to identify genes that encode Escherichia coli dihydropteridine reductase (DHPR) activities, a chromosomal DNA fragment that directs synthesis of two soluble polypeptides of M_r 44000 and 46000 was isolated. These proteins were partially purified and were identified by determination of their N-terminal amino acid sequences. The larger was serine hydroxymethyltransferase, encoded by the glyA gene, while the smaller was the previously described product of an unnamed gene closely linked to glyA, and transcribed in the opposite direction. Soluble extracts of E. coli cells that overproduced the 44 kDa protein had elevated DHPR activity, and were yellow in colour. Their visible absorption spectra were indicative of a CO-binding b-type haemoprotein that is high-spin in the reduced state. The sequence of the N-terminal 139 residues of the protein, deduced from the complete nucleotide sequence of the gene, had extensive homology to almost all of Vitreoscilla haemoglobin. We conclude that E. coli produces a soluble haemoglobin-like protein, the product of the hmp gene (for haemoprotein). Although the protein has DHPR activity, it is distinct from the previously purified E. coli DHPR.     

The Electro-Optical Investigation of Suspensions of Escherichia coli K-12 Cells Metabolizing Glucose,Lactose, and Galactose

The electro-optical characteristics of suspensions of Escherichia coli K-12 cells metabolizing glucose, lactose, and galactose were studied by measuring the suspension turbidity as a function of cell alignment in an orienting electric field whose frequency was varied from 10 kHz to 10 MHz. In a frequency range of 10 kHz to 1 MHz, the orientational spectra of E. coli K-12 cells grown on glucose and lactose considerably changed after their incubation in the presence of the sugars. These changes likely reflect alterations in the polarizability of the cells induced by sugar metabolism.     

Role of respiration and glutathione in cadmium-induced oxidative stress in Escherichia coli K-12

Cadmium is a widespread pollutant that has been associated with oxidative stress, but the mechanism behind this effect in prokaryotes is still unclear. In this work, we exposed two glutathione deficient mutants (ΔgshA and ΔgshB) and one respiration deficient mutant (ΔubiE) to a sublethal concentration of cadmium. The glutathione mutants show a similar increase in reactive oxygen species as the wild type. Experiments performed using the ΔubiE strain showed that this mutant is more resistant to cadmium ions and that Cd-induced reactive oxygen species levels were not altered. In the light of these facts, we conclude that the interference of cadmium with the respiratory chain is the cause of the oxidative stress induced by this metal and that, contrary to previously proposed models, the reactive oxygen species increase is not due to glutathione depletion, although this peptide is crucial for cadmium detoxification.     

Evidence for cAMP-mediated control of isoleucyl-tRNA synthetase formation in Escherichia coli K-12

The ability of cAMP to inhibit isoleucyl-tRNA synthetase (IRS) formation has been demonstrated in wild type K-12 Escherichia coli and two adenyl-cyclase (cya) mutants. cAMP appeared not to have any effect on either the valyl- or arginyl-tRNA synthetase (VRS and ARS respectively). Addition of cAMP led to a reduction in rate of IRS synthesis but not VRS or ARS. Furthermore, derepression of IRS and VRS by isoleucine limitation was completely prevented by cAMP.Abbreviations IRS isoleucyl-tRNA synthetase - VRS valyl-tRNA synthetase - ARS arginyl-tRNA synthetase - cAMP cyclic adenosine-3,5-monophosphate - Cya adenyl cyclase Gene - CRP cAMP receptor protein - O.D. optical density     

Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P.

The 2.1 A resolution crystal structure of flavin reductase P with the inhibitor nicotinamide adenine dinucleotide (NAD) bound in the active site has been determined. NAD adopts a novel, folded conformation in which the nicotinamide and adenine rings stack in parallel with an inter-ring distance of 3.6 A. The pyrophosphate binds next to the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine moiety faces away from the flavin. The observed NAD conformation is quite different from the extended conformations observed in other enzyme/NAD(P) structures; however, it resembles the conformation proposed for NAD in solution. The flavin reductase P/NAD structure provides new information about the conformational diversity of NAD, which is important for understanding catalysis. This structure offers the first crystallographic evidence of a folded NAD with ring stacking, and it is the first enzyme structure containing an FMN cofactor interacting with NAD(P). Analysis of the structure suggests a possible dynamic mechanism underlying NADPH substrate specificity and product release that involves unfolding and folding of NADP(H).     

Reduction of methylglyoxal in Escherichia coli K12 by an aldehyde reductase and alcohol dehydrogenase

Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K₁₂ cells. Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively. The K_m for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme. Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes. In phosphate buffer, both the enzymes are active in the pH range of 5.8–6.6 with no sharp pH optimum. Molecular weight of both the enzymes were found to be 100,000 ± 3,000 by gel filtration on a Sephacryl S-200 column. Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents.     

Identification of the recR locus of Escherichia coli K-12 and analysis of its role in recombination and DNA repair

Summary A new recombination gene called recR has been identified and located near dnaZ at minute 11 on the current linkage map of Escherichia coli. The gene was detected after transposon mutagenesis of a recB sbcB sbcC strain and screening for insertion mutants that had a reduced efficiency of recombination in Hfr crosses. The recR insertions obtained conferred a recombination deficient and extremely UV sensitive phenotype in both recB recC sbcA and recB recC sbcB sbcC genetic backgrounds. recR derivatives of recBC ⁺ sbc ⁺ strains were proficient in conjugational and transductional recombination but deficient in plasmid recombination and sensitive to UV light. Strains carrying recR insertions combined with mutations uvrA and other rec genes revealed that the gene is involved in a recombinational process of DNA repair that relies also on recF and recO, and possibly recJ, but which is independent of recB, recC and recD. The properties of two other insertions, one located near pyrE and the other near guaA, are discussed in relation to their proximity to recG and xse (the gene for exonuclease VII), respectively.     

Uptake of the Herbicidal Glyphosate by Escherichia coli K-12

The uptake of the aminoacid biosynthesis inhibitor, used as the broad-spectrum herbicide ingredient, glyphosate (N-[phosphonomethyl]-glycine) was investigated in E. coli as a model to study mechanisms of cell resistance to antimetabolites as drugs and pesticides. Unlike the glyphosate-degrading Arthrobacter sp. strain for which the first successful measurement of glyphosate uptake and its inhibition by orthophosphate was reported [15], E. coli K-12 cannot take up this inhibitor either in the presence of orthophosphate, or after a prolonged starvation for it. However, cells made competent after an overnight cold CaCl₂ exposure followed by dimethyl sulfoxide (DMSO) treatment could take up this compound (K _m for glyphosate uptake, 274 M). Neither amino acids, belonging to a single transport system, nor orthophosphate gave essential inhibition of glyphosate uptake by these cells.     

Acid response of exponentially growing Escherichia coli K-12

Induction of acid tolerance response (ATR) of exponential-phase Escherichia coli K-12 cells grown and adapted at different conditions was examined. The highest level of protection against pH 2.5 challenges was obtained after adaptation at pH 4.5-4.9 for 60 min. To study the genetic systems, which could be involved in the development of log-phase ATR, we investigated the acid response of E. coli acid resistance (AR) mutants. The activity of the glutamate-dependent system was observed in exponential cells grown at pH 7.0 and acid adapted at pH 4.5 in minimal medium. Importantly, log-phase cells exhibited significant AR when grown in minimal medium pH 7.0 and challenged at pH 2.5 for 2 h without adaptation. This AR required the glutamate-dependent AR system. Acid protection was largely dependent on RpoS in unadapted and adapted cells grown in minimal medium. RpoS-dependent oxidative, glutamate and arginine-dependent decarboxylase AR systems were not involved in triggering log-phase ATR in cells grown in rich medium. Cells adapted at pH 4.5 in rich medium showed a higher proton accumulation rate than unadapted cells as determined by proton flux assay. It is clear from our study that highly efficient mechanisms of protection are induced, operate and play the main role during log-phase ATR.     

Alternative gene for biotin sulfoxide reduction inEscherichia coli K-12

A gene at 42 min on theEscherichia coli chromosome, identified as the locus of pseudoreversion of knockout mutations in the biotin sulfoxide reductase gene,bisC, has 64% base sequence identity withbisC. This makes it a member of a multigene family of molybdopterin enzymes that includes genes for anaerobic reduction of trimethylamine oxide (torA) and dimethylsulfoxide (dmsA). Disruption of this gene eliminates the background activity of biotin sulfoxide reduction observed inbisC mutants. Sequence comparison of the new gene (bisZ) withbisC indicates that certaints mutants ofbisC arise by gene conversion between the two loci.     

Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12

Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g. sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist.     

Comparison between Escherichia coli K-12 strains W3110 and MG1655 and wild-type E. coli B as platforms for xylitol production

Escherichia coli W3110 was previously engineered to produce xylitol from a mixture of glucose plus xylose by expressing xylose reductase (CbXR) and deleting xylulokinase (DeltaxylB), combined with either plasmid-based expression of a xylose transporter (XylE or XylFGH) (Khankal et al., J Biotechnol, 2008) or replacing the native crp gene with a mutant (crp*) that alleviates glucose repression of xylose transport (Cirino et al., Biotechnol Bioeng 95:1167-1176, 2006). In this study, E. coli K-12 strains W3110 and MG1655 and wild-type E. coli B were compared as platforms for xylitol production from glucose-xylose mixtures using these same strategies. The engineered strains were compared in fed-batch fermentations and as non-growing resting cells. Expression of CRP* in the E. coli B strains tested was unable to enhance xylose uptake in the presence of glucose. Xylitol production was similar for the (crp*, DeltaxylB)-derivatives of W3110 and MG1655 expressing CbXR (average specific productivities of 0.43 g xylitol g cdw(-1 )h(-1) in fed-batch fermentation). In contrast, results varied substantially between different DeltaxylB-derivative strains co-expressing either XylE or XylFGH. The differences in genetic background between these host strains can therefore profoundly influence metabolic engineering strategies.     

Close genetic relationship between Nitrobacter hamburgensis nitrite oxidoreductase and Escherichia coli nitrate reductases

The nitrite oxidoreductase (NOR) from the facultative nitrite-oxidizing bacterium Nitrobacter hamburgensis X14 was investigated genetically. In order to develop a probe for the gene norB, the N-terminal amino acid sequence of the NOR -subunit (NorB) was determined. Based on that amino acid sequence, an oligo-nucleotide was derived that was used for the identification and cloning of gene norB. Sequence analysis of DNA fragments revealed three adjacent open reading frames in the order norA, norX, norB. The DNA sequences of norX and norB represented complete genes while the open reading frame of norA was truncated by the cloning site. The deduced amino acid sequence of protein NorB contained four cysteine clusters with striking homology to those of iron-sulfur centers of bacterial ferredoxins. NorB shares significant sequence similarity to the -subunits (NarH, NarY) of the two dissimilatory nitrate reductases (NRA, NRZ) of Escherichia coli. Additionally, the derived amino acid sequence of the truncated open reading frame of norA showed striking resemblance to the -subunits (NarG, NarZ) of the E. coli nitrate reductases.     

Light producing reporter plasmids for Escherichia coli K-12 that can be integrated into the chromosome

Plasmid vectors using the Photorhabdus luminescenslux operon can be used for real time measurements of promoter activity. We have generated a series of lux vectors that have a conditional origin of replication, different selectable markers and the attP sequence from λ. Single copies of these plasmids can be integrated into the λ attachment site in the Escherichia coli chromosome. We constructed reporter derivatives and compared light production when the plasmids were present in multiple copies and in single copies. We also demonstrated that expression could be induced under the appropriate conditions.     

Copper sensitivity of cueO mutants of Escherichia coli K-12 and the biochemical suppression of this phenotype

The cueO gene of Escherichia coli encodes a multi-copper oxidase, which contributes to copper tolerance in this bacterium. It was observed that a cueO mutant was highly sensitive to killing by copper ions when cells were grown on defined minimal media. Copper sensitivity was correlated with accumulation of copper in the mutant strain. Growth of the cueO mutant in the presence of copper could be restored by addition of divalent zinc and manganese ions or ferrous iron but not by other first row transition metal ions or magnesium ions. Copper toxicity towards a cueO mutant could also be suppressed by addition of the superoxide quencher 1,2-dihydroxybenzene-3,5-disulfonic acid (tiron), suggesting that a primary cause of copper toxicity is the copper-catalyzed production of superoxide anions in the cytoplasm.     

Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives

Summary We describe mutants of Escherichia coli that decrease the plasmid copy number of pBR322 derivatives. One mutant was partially characterized genetically and its mutation, designated pcnB for plasmid copy number, was mapped to approximately 3 min on the E. coli chromosome. This locus is distinct from other genes whose products are known to affect plasmid replication or stable plasmid maintenance. The pcnB mutant strain should be useful for cloning genes into pBR322 that have aberrant or deleterious effects on the cell when present in high copy number.