Different foci for the regulation of the activity of the KefB and KefC glutathione-gated K+ efflux systems

KefB and KefC are glutathione-gated K+ efflux systems in Escherichia coli, and the proteins exhibit strong similarity at the level of both primary sequence and domain organization. The proteins are maintained closed by glutathione and are activated by binding of adducts formed between glutathione and electrophiles. By construction of equivalent mutations in each protein, this study has analyzed the control over inactive state of the proteins. A UV-induced mutation in KefB, L75S, causes rapid spontaneous K+ efflux but has only a minor effect on K+ efflux via KefC. Similarly amino acid substitutions that cause increased spontaneous activity in KefC have only small effects in KefB. Exchange of an eight amino acid region from KefC (HALESDIE) with the equivalent sequence from KefB (HELETAID) has identified a role for a group of acidic residues in controlling KefC activity. The mutations HELETAID and L74S in KefC act synergistically, and the activity of the resultant protein resembles that of KefB. We conclude that, despite the high degree of sequence similarity, KefB and KefC exhibit different sensitivities to the same site-specific mutations.     

Protection of the DNA during the exposure of Escherichia coli cells to a toxic metabolite: the role of the KefB and KefC potassium channels

The effect of the toxic metabolite methylglyoxal on the DNA of Escherichia coli cells has been investigated. Exposure of E. coli cells to methylglyoxal reduces the transformability of plasmid DNA and results in the degradation of genomic DNA. The activity of the KefB and KefC potassium channels protects E. coli cells against methylglyoxal and limits the amount of DNA damage. In mutants lacking KefB and KefC, methylglyoxal-induced DNA damage was reduced by incubation with a weak acid that lowers the pHi to the same extent as through KefB and KefC activation. This provides evidence that acidification of the cytoplasm protects E. coli DNA against methylglyoxal. By the analysis of cells lacking UvrA, we demonstrate that this repair protein is required for the degradation of the DNA upon methylglyoxal exposure. However, protection by KefB and KefC occurred independently of UvrA. Although we present evidence that exposure of E. coli cells to methylglyoxal results in DNA degradation, our results suggest this event is not essential for methylglyoxal-induced death. The implications of these findings will be discussed.     

KefF, the regulatory subunit of the potassium efflux system KefC, shows quinone oxidoreductase activity

Escherichia coli and many other Gram-negative pathogenic bacteria protect themselves from the toxic effects of electrophilic compounds by using a potassium efflux system (Kef). Potassium efflux is coupled to the influx of protons, which lowers the internal pH and results in immediate protection. The activity of the Kef system is subject to complex regulation by glutathione and its S conjugates. Full activation of KefC requires a soluble ancillary protein, KefF. This protein has structural similarities to oxidoreductases, including human quinone reductases 1 and 2. Here, we show that KefF has enzymatic activity as an oxidoreductase, in addition to its role as the KefC activator. It accepts NADH and NADPH as electron donors and quinones and ferricyanide (in addition to other compounds) as acceptors. However, typical electrophilic activators of the Kef system, e.g., N-ethyl maleimide, are not substrates. If the enzymatic activity is disrupted by site-directed mutagenesis while retaining structural integrity, KefF is still able to activate the Kef system, showing that the role as an activator is independent of the enzyme activity. Potassium efflux assays show that electrophilic quinones are able to activate the Kef system by forming S conjugates with glutathione. Therefore, it appears that the enzymatic activity of KefF diminishes the redox toxicity of quinones, in parallel with the protection afforded by activation of the Kef system.     

Characterization of an insertion sequence, IS12528, from Gluconobacter suboxydans.

A novel insertion sequence element, IS12528, was found to be associated with inactivation of the alcohol dehydrogenase by insertion in the adhA gene, which encodes the primary dehydrogenase subunit of the three-component membrane-bound alcohol dehydrogenase complex in Gluconobacter suboxydans. Cloning and sequencing analyses revealed that IS12528 was 905 bp in length and had a terminal inverted repeat of 18 bp. In addition, IS12528 was found to generate a 3-bp duplication (TMA, where M represents C or A) at the inserted site upon transposition. IS12528 encoded one long product of 274 amino acids that was rich in basic amino acids. This protein showed significant homology with putative transposases of the IS1031 family isolated from Acetobacter xylinum, which belongs to another genus of acetic acid bacteria. IS12528-like sequences were distributed in a wide variety of acetic acid bacteria, as determined by Southern hybridization and PCR. These observations suggest that IS12528 is one of the insertion sequences that are responsible for genetic instability leading to deficiencies in various physiological properties in a variety of acetic acid bacteria.     

Survival during exposure to the electrophilic reagent N-ethylmaleimide in Escherichia coli: role of KefB and KefC potassium channels.

The role of the KefB and KefC potassium efflux systems in protecting Escherichia coli cells against the toxic effects of the electrophile N-ethylmaleimide has been investigated. Activation of KefB and KefC aids the survival of cells exposed to high concentrations (> 100 microM) of NEM. High potassium concentrations reduce the protection afforded by activation of KefB and KefC, but the possession of these systems is still important under these conditions. The Kdp system, which confers sensitivity to the electrophile methylglyoxal, did not affect the survival of cells exposed to NEM. Survival is correlated with the reduction of the cytoplasmic pH upon activation of the channels. In particular, the kinetics of the intracellular pH (pHi) change are crucial to the retention of viability of cells exposed to NEM; slow acidification does not protect cells as effectively as rapid lowering of pHi. Cells treated with low levels of NEM (10 microM) recover faster if they activate KefB and KefC, and this correlates with changes in pHi. The pHi does not significantly alter the rate of NEM metabolism. The possible mechanisms by which protection against the electrophile is mediated are discussed.     

The role of glyoxalase I in the detoxification of methylglyoxal and in the activation of the KefB K+ efflux system in Escherichia coli

The glyoxalase I gene ( gloA ) of Escherichia coli has been cloned and used to create a null mutant. Cells overexpressing glyoxalase I exhibit enhanced tolerance of methylglyoxal (MG) and exhibit elevated rates of detoxification, although the increase is not stoichiometric with the change in enzyme activity. Potassium efflux via KefB is also enhanced in the overexpressing strain. Analysis of the physiology of the mutant has revealed that growth and viability are quite normal, unless the cell is challenged with MG either added exogenously or synthesized by the cells. The mutant strain has a low rate of detoxification of MG, and cells rapidly lose viability when exposed to this electrophile. Activation of KefB and KefC is diminished in the absence of functional glyoxalase I. These data suggest that the glutathione-dependent glyoxalase I is the dominant detoxification pathway for MG in E . coli and that the product of glyoxalase I activity, S-lactoylglutathione, is the activator of KefB and KefC.     

Insertion sequence 1 of muscle-specific calpain, p94, acts as an internal propeptide

The physiological role of the skeletal muscle-specific calpain 3, p94, is presently unknown, but defects in its gene cause limb girdle muscular dystrophy type 2A. This calcium-dependent cysteine protease resembles the large subunit of m-calpain but with three unique additional sequences: an N-terminal region (NS), and two insertions (IS1 and IS2). The latter two insertions have been linked to the chronic instability of the whole enzyme both in vivo and in vitro. We have shown previously that the core of p94 comprising NS, domains I and II, and IS1 is stable as a recombinant protein in the absence of Ca(2+) and undergoes autolysis in its presence. Here we show that p94I-II cannot hydrolyze an exogenous substrate before autolysis but is increasingly able to do so when autolysis proceeds for several hours. This gain in activity is caused by cleavage of IS1 during autolysis because a deletion mutant lacking the NS region (p94I-II DeltaNS) shows the same activation profile. Similarly, the calpain inhibitors E-64 and leupeptin have almost no inhibitory effect on substrate hydrolysis by p94I-II soon after calcium addition but cause complete inhibition when autolysis progresses for several hours. As autolysis proceeds, there is release of the internal IS1 peptide, but the two portions of the core remain tightly associated. Modeling of p94I-II suggests that IS1 contains an amphipathic alpha-helix flanked by extended loops. The latter are the targets of autolysis and limited digestion by exogenous proteases. The presence and location of the alpha-helix in recombinant IS1 were confirmed by circular dichroism and by the introduction of a L286P helix-disrupting mutation. Within p94I-II, L286P caused premature autoproteolysis of the enzyme. IS1 is an elaboration of a loop in domain II near the active site, and it acts as an internal autoinhibitory propeptide, blocking the active site of p94 from substrates and inhibitors.     

Survival of Escherichia coli cells exposed to iodoacetate and chlorodinitrobenzene is independent of the glutathione-gated K+ efflux systems KefB and KefC.

The KefB and KefC systems of Escherichia coli cells are activated by iodoacetate (IOA) and chlorodinitrobenzene (CDNB), leading to a rapid drop in the intracellular pH. However, survival of exposure to IOA or CDNB was found to be essentially independent of KefB and KefC activation. No correlation was found between the toxicity of the compound and its ability to elicit protective acidification via activation of KefB and KefC.     

Transposon Tn10: genetic organization, regulation, and insertion specificity

Transposon Tn10 is a composite element in which two individual insertion sequence (IS)-like sequences cooperate to mediate transposition of the intervening material. The two flanking IS10 elements are not identical; IS10-right is responsible for functions required to promote transposition, and IS10-left is defective in transposition functions. We suggest that the two IS10 elements were originally identical in sequence and have subsequently diverged. IS10-right is compactly organized with structural gene(s), promoters, and sites important for transposition and (presumably) its regulation all closely linked and, in some cases, overlapping. IS10 has a single major coding region that almost certainly encodes an essential transposition function. A pair of opposing promoters flank the start of this coding region. One of these promoters is responsible for expression in vivo of transposon-encoded transposition functions. We propose that the second promoter is involved in modulation of Tn10 transposition. Genetic analysis suggests that transposon-encoded function(s) may be preferentially cis-acting. Insertion of Tn10 into particular preferred target sites is due primarily to the occurrence of a particular six-base pair target DNA sequence. The properties of this sequence suggest that symmetrically disposed subunits of a single protein may be responsible for both recognition and cleavage of target DNA during insertion.     

Functional organization of the ends of IS 1: specific binding site for an IS1-encoded protein

The IS 1-encoded protein InsA binds specifically to both ends of IS1, and acts as a repressor of IS1 gene expression and may be a direct inhibitor of the transposition process. We show here, using DNasel 'foot-printing' and gel retardation, that the InsA binding sites are located within the 24/25 bp minimal active ends of IS1 and that InsA induces DNA bending upon binding. Conformational modification of the ends of IS1 as a result of binding of the host protein integration host factor (IHF) to its site within the minimal ends has been previously observed. Using a collection of synthetic mutant ends we have mapped some of the nucleotide sequence requirements for InsA binding and for transposition activity. We show that sequences necessary for InsA binding are also essential for transposition activity. We demonstrate that InsA and IHF binding sites overlap since some sequence determinants are shared by both InsA and IHF. The data suggest that these ends contain two functional domains: one for binding of InsA and IHF, and the other for transposition activity. A third region, when present, may enhance transposition activity with an intact right end. This 'architecture' of the ends of IS1 is remarkably similar to that of IS elements IS10, IS50 and IS903.     

Identification of a Shiga-toxin type I variant containing an IS1203-like element, from Shiga-toxin producing Escherichia coli O157:H7

We found two Shiga toxin producing Escherichia coli O157:H7 strains isolated from humans carrying the stx(1) gene with an IS1203-like element (designated as IS1203v(1)). The IS1203v(1) was inserted into the coding region of the A subunit 7 bp upstream from the TGA termination codon, resulting in a loss of two amino acid residues (Ser-Ser) from its C terminus. Toxicity of the Stx1 was confirmed by Vero cell assay. IS1203v(1) hardly affected the stx(1) gene in either its expression or the toxicity of its product.     

Role of amino acid residues in transmembrane segments IS6 and IIS6 of the Na+ channel alpha subunit in voltage-dependent gating and drug block

Alanine-scanning mutagenesis of transmembrane segments IS6 and IIS6 of the rat brain Na(v)1.2 channel alpha subunit identified mutations N418A in IS6 and L975A in IIS6 as causing strong positive shifts in the voltage dependence of activation. In contrast, mutations V424A in IS6 and L983A in IIS6 caused strong negative shifts. Most IS6 mutations opposed inactivation from closed states, but most IIS6 mutations favored such inactivation. Mutations L421C and L983A near the intracellular ends of IS6 and IIS6, respectively, exhibited significant sustained Na(+) currents at the end of 30-ms depolarizations, indicating a role for these residues in Na(+) channel fast inactivation. These residues, in combination with residues at the intracellular end of IVS6, are well situated to form an inactivation gate receptor. Mutation I409A in IS6 reduced the affinity of the local anesthetic etidocaine for the inactivated state by 6-fold, and mutations I409A and N418A reduced use-dependent block by etidocaine. No IS6 or IIS6 mutations studied affected inactivated-state affinity or use-dependent block by the neuroprotective drug sipatrigine (compound 619C89). These results suggest that the local anesthetic receptor site is formed primarily by residues in segments IIIS6 and IVS6 with the contribution of a single amino acid in segment IS6.     

Glutathione-dependent conversion of N-ethylmaleimide to the maleamic acid by Escherichia coli: an intracellular detoxification process

The electrophile N-ethylmaleimide (NEM) elicits rapid K(+) efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K(+) efflux systems. The fate of the ESG has not previously been investigated. In this report we demonstrate that NEM and N-phenylmaleimide (NPM) are rapidly detoxified by E. coli. The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released. N-ethylmaleamic acid is not toxic to E. coli cells even at high concentrations. The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm. The detoxification is independent of new protein synthesis and NAD(+)-dependent dehydrogenase activity and entirely dependent upon glutathione. The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K(+) efflux systems.     

IS10 transposition is regulated by DNA adenine methylation

We show that dam- mutants are a major class of E. coli mutants with increased IS10 activity. IS10 has two dam methylation sites, one within the transposase promoter and one within the inner terminus where transposase presumably binds. Absence of methylation results in increased activity of both promoter and terminus, and completely accounts for increased transposition in dam- strains. Transposition of Tn903 and Tn5 are also increased in dam- strains, probably for analogous reasons. Transposition is also increased when IS10 is hemimethylated. One hemimethylated species is much more active than the other and is estimated to be at least 1000 times more active than a fully methylated element. Evidence is presented that the promoter and inner terminus of IS10 are coordinately activated in a dam-dependent fashion, presumably because they are hemimethylated at the same time. Thus, in dam+ strains, IS10 will transpose preferentially when DNA is hemimethylated. We suggest specifically that IS10 transposition may preferentially occur immediately after passage of a chromosomal replication fork.     

Importance of RpoS and Dps in Survival of Exposure of Both Exponential- and Stationary-Phase Escherichia coli Cells to the Electrophile N-Ethylmaleimide

The mechanisms by which Escherichia coli cells survive exposure to the toxic electrophile N-ethylmaleimide (NEM) have been investigated. Stationary-phase E. coli cells were more resistant to NEM than exponential-phase cells. The KefB and KefC systems were found to play an important role in protecting both exponential- and stationary-phase cells against NEM. Additionally, RpoS and the DNA-binding protein Dps aided the survival of both exponential- and stationary-phase cells against NEM. Double mutants lacking both RpoS and Dps and triple mutants deficient in KefB and KefC and either RpoS or Dps had an increased sensitivity to NEM in both exponential- and stationary-phase cells compared to mutants missing only one of these protective mechanisms. Stationary- and exponential-phase cells of a quadruple mutant lacking all four protective systems displayed even greater sensitivity to NEM. These results indicated that protection by the KefB and KefC systems, RpoS and Dps can each occur independently of the other systems. Alterations in the level of RpoS in exponentially growing cells correlated with the degree of NEM sensitivity. Decreasing the level of RpoS by enriching the growth medium enhanced sensitivity to NEM, whereas a mutant lacking the ClpP protease accumulated RpoS and gained high levels of resistance to NEM. A slower-growing E. coli strain was also found to accumulate RpoS and had enhanced resistance to NEM. These data emphasize the multiplicity of pathways involved in protecting E. coli cells against NEM.     

A novel IS element,IS621, of the IS110/IS492 family transposes to a specific site in repetitive extragenic palindromic sequences in Escherichia coli

An Escherichia coli strain, ECOR28, was found to have insertions of an identical sequence (1,279 bp in length) at 10 loci in its genome. This insertion sequence (named IS621) has one large open reading frame encoding a putative protein that is 326 amino acids in length. A computer-aided homology search using the DNA sequence as the query revealed that IS621 was homologous to the piv genes, encoding pilin gene invertase (PIV). A homology search using the amino acid sequence of the putative protein encoded by IS621 as the query revealed that the protein also has partial homology to transposases encoded by the IS110/IS492 family elements, which were known to have partial homology to PIV. This indicates that IS621 belongs to the IS110/IS492 family but is most closely related to the piv genes. In fact, a phylogenetic tree constructed on the basis of amino acid sequences of PIV proteins and transposases revealed that IS621 belongs to the piv gene group, which is distinct from the IS110/IS492 family elements, which form several groups. PIV proteins and transposases encoded by the IS110/IS492 family elements, including IS621, have four acidic amino acid residues, which are conserved at positions in their N-terminal regions. These residues may constitute a tetrad D-E(or D)-D-D motif as the catalytic center. Interestingly, IS621 was inserted at specific sites within repetitive extragenic palindromic (REP) sequences at 10 loci in the ECOR28 genome. IS621 may not recognize the entire REP sequence in transposition, but it recognizes a 15-bp sequence conserved in the REP sequences around the target site. There are several elements belonging to the IS110/IS492 family that also transpose to specific sites in the repeated sequences, as does IS621. IS621 does not have terminal inverted repeats like most of the IS110/IS492 family elements. The terminal sequences of IS621 have homology with the 26-bp inverted repeat sequences of pilin gene inversion sites that are recognized and used for inversion of pilin genes by PIV. This suggests that IS621 initiates transposition through recognition of their terminal regions and cleavage at the ends by a mechanism similar to that used for PIV to promote inversion at the pilin gene inversion sites.     

Identification of an unknown promoter,OUTIIp, within the IS10R element

A novel promoter in IS10R (OUTIIp) has been found in one of its ends in an inverted position relative to promoter pOUT. OUTIIp shows characteristics similar to those of rpoS-dependent promoters such as a gearbox expression pattern. It is under catabolite repression and positively regulated by ppGpp or conditioned media. This opens new challenges in IS10R transposition.