Jorge Membrillo-Hernández

Zinc(II) tolerance in Escherichia coli K-12: evidence that the zntA gene (o732) encodes a cation transport ATPase.

A transposon (Tn10dCam) insertion mutant of Escherichia coli K‐12 was isolated that exhibited hypersensitivity to zinc(II) and cadmium(II) and, to a lesser extent, cobalt(II) and nickel (II). The mutated gene, located between 75.5 and 76.2 min on the chromosome, is named zntA (for Zn(II) transport or tolerance). The metal‐sensitive phenotype was complemented by a genomic DNA clone mapping at 3677.90–3684.60 kb on the physical map. Insertion of a kanamycin resistance (KnR) cassette at a Sal I site in a subcloned fragment generated a plasmid that partially complemented the zinc(II)‐sensitive phenotype. DNA sequence analysis revealed that the KnR cassette was located within the putative promoter region of an ORF (o732 or yhhO) predicted to encode a protein of 732 amino acids, similar to cation transport P‐type ATPases in the Cpx‐type family. Inverse PCR and sequence analysis revealed that the Tn10dCam element was located within o732 in the genome of the zinc(II)‐sensitive mutant. The zntA mutant had elevated amounts of intracellular and cell surface‐bound Zn(II), consistent with the view that zntA+ encodes a zinc(II) efflux protein. Exposure of the zntA mutant to cobalt(II) and cadmium(II) also resulted in elevated levels of intracellular and cell surface‐bound metal ions.

The Flavohemoglobin of Escherichia coli Confers Resistance to a Nitrosating Agent, a “Nitric Oxide Releaser,” and Paraquat and Is Essential for Transcriptional Responses to Oxidative Stress

Escherichia coli possesses a flavohemoglobin (Hmp), product of hmp, the first microbial globin gene to be sequenced and characterized at the molecular level. Although related proteins occur in numerous prokaryotes and eukaryotic microorganisms, the function(s) of these proteins have been elusive. Here we report construction of a defined hmp mutation and its use to probe Hmp function. As anticipated from up-regulation ofhmp expression by nitric oxide (NO),S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP), the hmp mutant is hypersensitive to these agents. Thehmp promoter is more sensitive to SNP andS-nitroso-N-penicillamine (SNAP) than is thesoxS promoter, consistent with the role of Hmp in protection from reactive nitrogen species. Additional functions for Hmp are indicated by (a) parallel sensitivity of thehmp mutant to the redox-cycling agent, paraquat, (b) inability of the mutant to up-regulate fully thesoxS and sodA promoters in response to oxidative stress caused by paraquat, GSNO and SNP, and (c) failure of the mutant to accumulate reduced paraquat radical after anoxic growth. We conclude that Hmp plays a role in protection from nitrosating agents and NO-related species and oxidative stress. This protective role probably involves direct detoxification of those species and sensing of NO-related and oxidative stress.

Genome-wide Transcriptional Profile of Escherichia coli in Response to High Levels of the Second Messenger 3′,5′-Cyclic Diguanylic Acid

Cyclicdiguanylicacid(c-di-GMP;cGpGp)isaglobalsecondmessenger controlling motility and adhesion in bacterial cells. Intracellular concentrations of c-di-GMP depend on two opposite activities: diguanylate cyclase, recently assigned to the widespread GGDEF domain, and c-di-GMP-specific phosphodiesterase, associated with proteins harboring the EAL domain. To date, little is known about the targets of c-di-GMP in the cell or if it affects transcriptional regulation of certain genes. In order to expand our knowledge of the effect of this molecule on the bacterial metabolism, here we report on the Escherichia coli transcriptional profile under high levels of c-di-GMP. We show that an important number of genes encoding cell surface and membrane-bound proteins are altered in their transcriptional activity. On the other hand, genes encoding several transcriptional factors, such as Fur, RcsA, SoxS, and ZraR, are up-regulated, and others, such as GadE, GadX, GcvA, and MetR, are down-regulated. Transcription of motility and cell division genes were altered, and consistent with this was the physiological analysis of cells overexpressing yddV,adiguanylatecyclase;thesecellsdisplayedanabnormalcelldivision process when high levels of c-di-GMP were present. We also show evidence that the diguanylate cyclase gene yddV is co-transcribed with dos,a heme base oxygen sensor with c-di-GMP-specific phosphodiesterase activity. A Δdos::kan mutation rendered the cells unable to divide properly,suggestingthatdosandyddVmaybepartofafine-tuningmechanism for regulating the intracellular levels of c-di-GMP.

A novel mechanism for upregulation of the Escherichia coli K-12 hmp (flavohaemoglobin) gene by the 'NO releaser', S-nitrosoglutathione: nitrosation of homocysteine and modulation of MetR binding to the glyA-hmp intergenic region

The flavohaemoglobin gene, hmp, of Escherichia coli is upregulated by nitric oxide (NO) in a SoxRS‐independent manner. We now show that hmp expression is also upregulated by S‐nitrosoglutathione (GSNO, widely used as an NO releaser) and sodium nitroprusside (SNP, which is a NO+ donor). Elevated homocysteine (Hcy) levels, achieved either by adding Hcy extracellularly or using metE mutants, decreased hmp expression. Conversely, metC mutants (defective in Hcy synthesis) had higher levels of hmp expression. Mutations in metR abolished hmp induction by GSNO and SNP, and hmp expression became insensitive to Hcy. We propose that the previously documented modulation by Hcy of MetR binding to the glyA‐hmp intergenic regulatory region regulates hmp transcription. Although two MetR binding sites are present in this region, only the higher affinity site proximal to hmp is required for hmp induction by GSNO and SNP. GSNO and SNP react with Hcy in vitro under physiologically relevant conditions of pH and temperature generating S‐nitrosohomocysteine, although in the latter case this would be co‐ordinated to the Fe in SNP as a stable species. The free S‐nitrosocysteine generated in the reaction with GSNO breaks down to release NO more readily than via homolysis of GSNO. As GSNO and SNP upregulate hmp similarly, the NO released in the former case on reaction with homocysteine cannot be involved in hmp regulation.

Evolution of the adhE Gene Product ofEscherichia coli from a Functional Reductase to a Dehydrogenase GENETIC AND BIOCHEMICAL STUDIES OF THE MUTANT PROTEINS

The multifunctional AdhE protein ofEscherichia coli (encoded by the adhE gene) physiologically catalyzes the sequential reduction of acetyl-CoA to acetaldehyde and then to ethanol under fermentative conditions. The NH2-terminal region of the AdhE protein is highly homologous to aldehyde:NAD+ oxidoreductases, whereas the COOH-terminal region is homologous to a family of Fe2+-dependent ethanol:NAD+oxidoreductases. This fusion protein also functions as a pyruvate formate lyase deactivase. E. coli cannot grow aerobically on ethanol as the sole carbon and energy source because of inadequate rate of adhE transcription and the vulnerability of the AdhE protein to metal-catalyzed oxidation. In this study, we characterized 16 independent two-step mutants with acquired and improved aerobic growth ability on ethanol. The AdhE proteins in these mutants catalyzed the sequential oxidation of ethanol to acetaldehyde and to acetyl-CoA. All first stage mutants grew on ethanol with a doubling time of about 240 min. Sequence analysis of a randomly chosen mutant revealed an Ala-267 → Thr substitution in the acetaldehyde:NAD+ oxidoreductase domain of AdhE. All second stage mutants grew on ethanol with a doubling time of about 90 min, and all of them produced an AdhEA267T/E568K. Purified AdhEA267T and AdhEA267T/E568K showed highly elevated acetaldehyde dehydrogenase activities. It therefore appears that when AdhE catalyzes the two sequential reactions in the counter-physiological direction, acetaldehyde dehydrogenation is the rate-limiting step. Both mutant proteins were more thermosensitive than the wild-type protein, but AdhEA267T/E568K was more thermal stable than AdhEA267T. Since both mutant enzymes exhibited similar kinetic properties, the second mutation probably conferred an increased growth rate on ethanol by stabilizing AdhEA267T.

Roles of respiratory oxidases in protecting Escherichia coli K12 from oxidative stress

Isogenic strains of Escherichia coli that were defective in either of the two major aerobic terminal respiratory oxidases (cytochromes bo′ and bd) or in the putative third oxidase (cytochrome bd-II) were studied to elucidate role(s) for oxidases in protecting cells from oxidative stress in the form of H2O2 and paraquat. Exponential phase cultures of all three oxidase mutants exhibited a greater decline in cell viability when exposed to H2O2 stress compared to the isogenic parent wild-type strain. Cytochrome bo′ mutants showed the greatest sensitivity to H2O2 under all conditions studied indicating that this oxidase was crucial for protection from H2O2 in E. coli. Cell killing of all oxidase mutants by H2O2 was by an uncharacterized mechanism (mode 2 killing) with cell growth rate affected. The expression of Φ(katG-lacZ), an indicator of intracellular H2O2, was 2-fold higher in a cydAB::kan mutant compared to the wild-type strain at low H2O2 concentrations (< 100 μM) suggesting that cytochrome bd mutants were experiencing higher intracellular levels of H2O2. Protein fusions to the three oxidase genes demonstrated that expression of genes encoding cytochrome bd, but not cytochrome bo′ or cytochrome bd-II was increased in the presence of external H2O2. This increase in expression of Φ(cydA-lacZ) by H2O2 was further enhanced in a cyo::kan mutant. The level of cytochrome bd determined spectrally and Φ(cydA-lacZ) expression was 5-fold and 2-fold higher respectively in an rpoS mutant compared to isogenic wild-type cells suggesting that RpoS was a negative regulator of cytochrome bd. Whether the effect of RpoS is direct or indirect remains to be determined.

DnaK dependence of mutant ethanol oxidoreductases evolved for aerobic function and protective role of the chaperone against protein oxidative damage in Escherichia coli

The adhE gene of Escherichia coli encodes a multifunctional ethanol oxidoreductase (AdhE) that catalyzes successive reductions of acetyl-CoA to acetaldehyde and then to ethanol reversibly at the expense of NADH. Mutant JE52, serially selected for acquired and improved ability to grow aerobically on ethanol, synthesized an AdhEA267T/E568K with two amino acid substitutions that sequentially conferred improved catalytic properties and stability. Here we show that the aerobic growth ability on ethanol depends also on protection of the mutant AdhE against metal-catalyzed oxidation by the chaperone DnaK (a member of the Hsp70 family). No DnaK protection of the enzyme is evident during anaerobic growth on glucose. Synthesis of DnaK also protected E. coli from H2O2 killing under conditions when functional AdhE is not required. Our results therefore suggest that, in addition to the known role of protecting cells against heat stress, DnaK also protects numerous kinds of proteins from oxidative damage.

Plasmid DNA Supercoiling and Gyrase Activity in Escherichia coli Wild-Type and rpoS Stationary-Phase Cells

Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.

Multiple regulators of the Flavohaemoglobin (hmp) gene of Salmonella enterica serovar Typhimurium include RamA, a transcriptional regulator conferring the multidrug resistance phenotype

Microbial flavohaemoglobins are proteins with homology to haemoglobins from higher organisms, but clearly linked to nitric oxide (NO) metabolism by bacteria and yeast. hmp mutant strains of several bacteria are hypersensitive to NO and related compounds and hmp genes are up-regulated by the presence of NO. The regulatory mechanisms involved in hmp induction by NO and the superoxide-generating agent, methyl viologen (paraquat; PQ), are complex, but progressively being resolved. Here we show for the first time that, in Salmonella enterica serovar Typhimurium, hmp transcription is increased on exposure to PQ and demonstrate that RamA, a homologue of MarA is responsible for most of the hmp paraquat regulation. In addition we demonstrate NO-dependent elevation of Salmonella hmp transcription and Hmp accumulation. In both Escherichia coli and Salmonella modest transcriptional repression of hmp is exerted by the iron responsive transcriptional repressor Fur. Finally, in contrast to previous reports, we show that in E. coli and Salmonella, hmp induction by both paraquat and sodium nitroprusside is further elevated in a fur mutant background, indicating that additional regulators are implicated in this control process.

Roles of RpoS (σS), IHF and ppGpp in the expression of the hmp gene encoding the flavohemoglobin (Hmp) of Escherichia coli K-12

Abstract The Escherichia coli K-12 gene hmp encodes the flavohemoglobin Hmp. A hmp promoter Φ(hmp-lacZ)-operon fusion was constructed in the chromosome and its activity measured during the growth cycle. Logarithmically growing cultures had low levels of Φ(hmp-lacZ) expression, which increased two-fold at the onset of stationary phase in rich medium. The effect was abolished in a strain carrying a null allele of the gene rpoS encoding the stationary phase-specific sigma subunit of RNA polymerase σS. A himA mutation resulted in a 1.5-fold increase in expression of Φ(hmp-lacZ), but did not affect growth phase-dependent regulation. A single transcriptional start site was found for hmp, located 38 bp upstream of the initiation codon. Putative Fnr boxes at positions −2 to +11 occur in the hmp promoter region.