Linkage between catecholate siderophores and the multicopper oxidase CueO in Escherichia coli

The multicopper oxidase CueO had previously been demonstrated to exhibit phenoloxidase activity and was implicated in intrinsic copper resistance in Escherichia coli. Catecholates can potentially reduce Cu(II) to the prooxidant Cu(I). In this report we provide evidence that CueO protects E. coli cells by oxidizing enterobactin, the catechol iron siderophore of E. coli, in the presence of copper. In vitro, a mixture of enterobactin and copper was toxic for E. coli cells, but the addition of purified CueO led to their survival. Deletion of fur resulted in copper hypersensitivity that was alleviated by additional deletion of entC, preventing synthesis of enterobactin. In addition, copper added together with 2,3-dihydroxybenzoic acid or enterobactin was able to induce a Phi(cueO-lacZ) operon fusion more efficiently than copper alone. The reaction product of the 2,3-dihydroxybenzoic acid oxidation by CueO that can complex Cu(II) ions was determined by gas chromatography-mass spectroscopy and identified as 2-carboxymuconate.     

一株高锰氧化活性大肠杆菌的分离鉴定和多铜氧化酶基因的克隆与结构特性

锰氧化物是Mn(Ⅱ)经生物和化学氧化后形成的矿物成分,在元素生物地球化学循环过程中起着重要作用,而不同种类的细菌对Mn(Ⅱ)的氧化作用是自然界中氧化锰矿物形成的主要成因.从山东崅峪采集的铁锰结核棕壤中分离得到一株具有高锰氧化活性的土壤杆菌,其对Mn(Ⅱ)的氧化作用活性明显高于其它分离菌株,达到65 μmol/L.通过个...     

Transformation of Rickettsia prowazekii to erythromycin resistance encoded by the Escherichia coli ereB gene

Rickettsia prowazekii, the etiologic agent of epidemic typhus, is an obligate, intracytoplasmic, parasitic bacterium. Recently, the transformation of this bacterium via electroporation has been reported. However, in these studies identification of transformants was dependent upon either selection of an R. prowazekii rpoB chromosomal mutation imparting rifampin resistance or expression of the green fluorescent protein and flow cytometric analysis. In this paper we describe the expression in R. prowazekii of the Escherichia coli ereB gene. This gene codes for an erythromycin esterase that cleaves erythromycin. To the best of our knowledge, this is the first report of the expression of a nonrickettsial, antibiotic-selectable gene in R. prowazekii. The availability of a positive selection for rickettsial transformants is an important step in the characterization of genetic analysis systems in the rickettsiae.     

A novel membrane-associated threonine permease encoded by the tdcC gene of Escherichia coli.

A novel L-threonine transport system is induced in Escherichia coli cells when incubated in amino acid-rich medium under anaerobic conditions. Genetic and biochemical analyses with plasmids harboring mutations in the anaerobically expressed tdcABC operon indicated that the tdcC gene product was responsible for L-threonine uptake. Competition experiments revealed that the L-threonine transport system is also involved in L-serine uptake and is partially shared for L-leucine transport; L-alanine, L-valine, and L-isoleucine did not affect L-threonine uptake. Transport of L-threonine was inhibited by the respiratory chain inhibitors KCN and carbonyl cyanide m-chlorophenylhydrazone and was Na+ independent. These results identify for the first time an E. coli gene encoding a permease specific for L-threonine-L-serine transport that is distinct from the previously described threonine-serine transport systems. A two-dimensional topological model predicted from the amino acid composition and hydropathy plot showed that the TdcC polypeptide appears to be an integral membrane protein with several membrane-spanning domains exhibiting a striking similarity with other bacterial permeases.     

Expression of hydroxamate and phenolate siderophores by Shigella flexneri.

Shigella flexneri strains were assayed for the ability to synthesize and utilize phenolate and hydroxamate siderophores. The hydroxamate aerobactin was synthesized by all isolates tested, whereas phenolates were only rarely produced. Expression of aerobactin was accompanied by production of a single iron-regulated outer membrane protein (Mr = 74,000). This protein was not produced by a mutant defective in aerobactin utilization and may serve as the aerobactin receptor. Phenolate (enterobactin)-producing strains synthesized three additional outer membrane proteins (Mr = 74,000, 81,000, and 83,000) in response to iron starvation. These proteins are the same apparent size as those produced by Escherichia coli K-12 strains. Ent sequences are apparently present in strains which do not synthesize this compound. Although normally silent, ent genes can be activated in Ent- strains to produce Ent+ variants. These laboratory variants are phenotypically indistinguishable from clinical Ent+ isolates.     

Isolation and characterization of an aldehyde dehydrogenase encoded by the aldB gene of Escherichia coli

An aldehyde dehydrogenase was detected in crude cell extracts of Escherichia coli DH5alpha. Growth studies indicated that the aldehyde dehydrogenase activity was growth phase dependent and increased in cells grown with ethanol. The N-terminal amino acid sequence of the purified enzyme identified the latter as an aldehyde dehydrogenase encoded by aldB, which was thought to play a role in the removal of aldehydes and alcohols in cells that were under stress. The purified enzyme showed an estimated molecular mass of 220 +/- 8 kDa, consisting of four identical subunits, and preferred to use NADP and acetaldehyde. MgCl2 increased the activity of the NADP-dependent enzyme with various substrates. A comparison of the effect of Mg2+ ions on the bacterial enzyme with the effect of Mg2+ ions on human liver mitochondrial aldehyde dehydrogenase revealed that the bacterial enzyme shared kinetic properties with the mammalian enzyme. An R197E mutant of the bacterial enzyme appeared to retain very little NADP-dependent activity on acetaldehyde.     

The RNase Z homologue encoded by Escherichia coli elaC gene is RNase BN

In eukaryotes, archaea, and in some eubacteria, removal of 3' precursor sequences during maturation of tRNA is catalyzed by an endoribonuclease, termed RNase Z. In contrast, in Escherichia coli, a variety of exoribonucleases carry out final 3' maturation. Yet, E. coli retains an RNase Z homologue, ElaC, whose function is under active study. We have overexpressed and purified to homogeneity His-tagged ElaC and show here that it is, in fact, the previously described enzyme, RNase BN. Thus, purified ElaC displays structural and catalytic properties identical to those ascribed to RNase BN. In addition, an elaC mutant strain behaves identically to a known RNase BN- strain, CAN. Finally, we show that wild type elaC can complement the mutation in strain CAN and that the elaC gene in strain CAN carries a nonsense mutation that results in loss of RNase BN activity. These data correct a previous misassignment for the gene encoding RNase BN. Based on the fact that the original RNase BN mutation has now been identified, we propose that the elaC gene be renamed rbn.     

Analysis of the multicopper oxidase gene regulatory network of Aeromonas hydrophila

Multicopper oxidase (MCO) is an enzyme which involves in reducing the oxygen in a four electron reduction to water with concomitant one electron oxidation of reducing the substrate. We have generated the 3-D structure of MCO by homology modeling and validated on the basis of free energy while 90.4 % amino acid residues present in allowed regions of Ramachandran plot. The screening of potential hazardous aromatic compounds for MCO was performed using molecular docking. We obtained Sulfonaphthal, Thymolphthalein, Bromocresol green and Phloretin derivatives of phenol and aromatic hydrocarbon were efficient substrates for MCO. The phylogeny of MCO reveals that other bacteria restrain the homologous gene of MCO may play an important role in biodegradation of aromatic compounds. We have demonstrated the gene regulatory network of MCO with other cellular proteins which play a key role in gene regulation. These findings provide a new insight for oxidization of phenolic and aromatic compounds using biodegradation process for controlling environmental pollution.     

Globoside-specific adhesins of uropathogenic Escherichia coli are encoded by similar trans-complementable gene clusters.

Uropathogenic Escherichia coli frequently express globoside-specific adhesins, shown to mediate binding to uroepithelial cells. For one gene cluster pap, it recently has been demonstrated that globoside binding is not dependent on expression of the pilus subunit gene papA. Instead, two other pap genes papF and papG are specifically required for globoside binding (F. P. Lindberg et al., EMBO J. 3:1167-1173, 1984). By restriction enzyme mapping, DNA hybridization, DNA sequencing, and protein expression in minicells, we show that three gene clusters encoding globoside binding have a very similar structure and gene organization, although they were cloned from different E. coli isolates. Major differences between the adhesin clones were restricted to the central part of the pilin gene (papA) and to one of the two adhesin gene (papG). The three functional units required for biogenesis of globoside-binding pili, i.e., pilin synthesis, pilin export, and pilin assembly, as well as expression of adhesion function, were all trans complementable among the gene clusters.     

Inducible L-alanine exporter encoded by the novel gene ygaW (alaE) in Escherichia coli

We previously isolated a mutant hypersensitive to L-alanyl-L-alanine from a non-L-alanine-metabolizing Escherichia coli strain and found that it lacked an inducible l-alanine export system. Consequently, this mutant showed a significant accumulation of intracellular L-alanine and a reduction in the L-alanine export rate compared to the parent strain. When the mutant was used as a host to clone a gene(s) that complements the dipeptide-hypersensitive phenotype, two uncharacterized genes, ygaW and ytfF, and two characterized genes, yddG and yeaS, were identified. Overexpression of each gene in the mutant resulted in a decrease in the intracellular l-alanine level and enhancement of the L-alanine export rate in the presence of the dipeptide, suggesting that their products function as exporters of L-alanine. Since ygaW exhibited the most striking impact on both the intra- and the extracellular L-alanine levels among the four genes identified, we disrupted the ygaW gene in the non-L-alanine-metabolizing strain. The resulting isogenic mutant showed the same intra- and extracellular L-alanine levels as observed in the dipeptide-hypersensitive mutant obtained by chemical mutagenesis. When each gene was overexpressed in the wild-type strain, which does not intrinsically excrete alanine, only the ygaW gene conferred on the cells the ability to excrete alanine. In addition, expression of the ygaW gene was induced in the presence of the dipeptide. On the basis of these results, we concluded that YgaW is likely to be the physiologically most relevant exporter for L-alanine in E. coli and proposed that the gene be redesignated alaE for alanine export.