Ian R. Booth

A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli

The proU locus encodes an osmotically inducible glycine betaine transport system that is important in the adaptation to osmotic stress. We present evidence that DNA supercoiling plays a key role in the osmotic induction of proU transcription. An increase in extracellular osmolarity increases in vivo DNA supercoiling, and the expression of proU is highly sensitive to these changes. Furthermore, topA mutations can mimic an increase in osmolarity, facilitating proU expression even in media of low osmolarity in which it is not normally expressed. Selection for trans-acting mutations that affect proU expression has yielded only mutations that alter DNA supercoiling, either in topA or a new genetic locus, osmZ, which strongly influences in vivo supercoiling. Mutations in osmZ are highly pleiotropic, affecting expression of a variety of chromosomal genes including ompF, ompC, fimA, and the bgl operon, as well as increasing the frequency of site-specific DNA inversions that mediate fimbrial phase variation.

Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity

Mechanosensitive channels are ubiquitous amongst bacterial cells and have been proposed to have major roles in the adaptation to osmotic stress, in particular in the management of transitions from high to low osmolarity environments. Electrophysiological measurements have identified multiple channels in Escherichia coli cells. One gene, mscL, encoding a large conductance channel has previously been described, but null mutants were without well‐defined phenotypes. Here, we report the characterization of a new gene family required for MscS function, YggB and KefA, which has enabled a rigorous test of the role of the channels. The channel determined by KefA does not appear to have a major role in managing the transition from high to low osmolarity. In contrast, analysis of mutants of E.coli lacking YggB and MscL shows that mechanosensitive channels are designed to open at a pressure change just below that which would cause cell disruption leading to death.

Methylglyoxal production in bacteria : suicide or survival?

Abstract Methylglyoxal is a toxic electrophile. In Escherichia coli cells, the principal route of methylglyoxal production is from dihydroxyacetone phosphate by the action of methylglyoxal synthase. The toxicity of methylglyoxal is believed to be due to its ability to interact with the nucleophilic centres of macromolecules such as DNA. Bacteria possess an array of detoxification pathways for methylglyoxal. In E. coli, glutathione-based detoxification is central to survival of exposure to methylglyoxal. The glutathione-dependent glyoxalase I-II pathway is the primary route of methylglyoxal detoxification, and the glutathione conjugates formed can activate the KefB and KefC potassium channels. The activation of these channels leads to a lowering of the intracellular pH of the bacterial cell, which protects against the toxic effects of electrophiles. In addition to the KefB and KefC systems, E. coli cells are equipped with a number of independent protective mechanisms whose purpose appears to be directed at ensuring the integrity of the DNA. A model of how these protective mechanisms function will be presented. The production of methylglyoxal by cells is a paradox that can be resolved by assigning an important role in adaptation to conditions of nutrient imbalance. Analysis of a methylglyoxal synthase-deficient mutant provides evidence that methylglyoxal production is required to allow growth under certain environmental conditions. The production of methylglyoxal may represent a high-risk strategy that facilitates adaptation, but which on failure leads to cell death. New strategies for antibacterial therapy may be based on undermining the detoxification and defence mechanisms coupled with deregulation of methylglyoxal synthesis.

Inhibition of Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity

The mechanism by which methionine relieves the growth inhibition of Escherichia coli K-12 that is caused by organic weak acid food preservatives was investigated. In the presence of 8 mM acetate the specific growth rate of E. coli Frag1 (in MacIlvaines minimal medium pH 6.0) is reduced by 50%. Addition of methionine restores growth to 80% of that observed in untreated controls. Similar relief was seen with cultures treated with either benzoate or propionate. Mutants with an elevated intracellular methionine pool were almost completely resistant to the inhibitory effects of acetate, suggesting that the methionine pool becomes limiting for growth in acetate-treated cells. Measurement of the intracellular concentrations of pathway intermediates revealed that the homocysteine pool is increased dramatically in acetate-treated cells, suggesting that acetate inhibits a biosynthetic step downstream from this intermediate. Supplementation of the medium with homocysteine inhibits the growth of E. coli cells. Acetate inhibition of growth arises from the depletion of the intracellular methionine pool with the concomitant accumulation of the toxic intermediate homocysteine and this augments the effect of lowering cytoplasmic pH.

Two Families of Mechanosensitive Channel Proteins

SUMMARY Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 α-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.

Stress and the single cell: intrapopulation diversity is a mechanism to ensure survival upon exposure to stress.

We traditionally celebrate the capacity of bacteria for growth in a diverse range of environmental niches. As the attention has switched to their survival, we are no less impressed by the diversity of mechanisms that aid survival upon exposure to a variety of stresses. Mechanistically, we usually measure adaptation by the changes that occur upon rapid transfer from condition A to B. Implicit in such analyses is the homogeneity of the population of cells in terms of their biochemistry and responsiveness. In contrast, the literature contains many reports of heterogeneity within bacterial populations. A practical importance of such heterogeneity is the ability of a small fraction of any population to survive exposure to stresses that kill the majority of the population. The origins and properties of such organisms have been receiving renewed attention. This brief review considers some of the routes by which heterogeneity is generated in bacterial populations and suggests that such inherent transient diversity in phenotype of individual cells is a survival aid.

The Structure of an Open Form of an E. coli Mechanosensitive Channel at 3.45 Å Resolution

How ion channels are gated to regulate ion flux in and out of cells is the subject of intense interest. The Escherichia coli mechanosensitive channel, MscS, opens to allow rapid ion efflux, relieving the turgor pressure that would otherwise destroy the cell. We present a 3.45 angstrom–resolution structure for the MscS channel in an open conformation. This structure has a pore diameter of ∼13 angstroms created by substantial rotational rearrangement of the three transmembrane helices. The structure suggests a molecular mechanism that underlies MscS gating and its decay of conductivity during prolonged activation. Support for this mechanism is provided by single-channel analysis of mutants with altered gating characteristics.

Managing hypoosmotic stress: Aquaporins and medianosensitive channels in Escherichia coli

When Escherichia coli cells are subject to hypoosmotic shock they are subject to substantial flows of water that can be equivalent to a 4-5-fold increase in the pressure exerted from the cytoplasm on the membrane and peptidoglycan wall. The recently described aquaporin that facilitates rapid water movement across the cytoplasmic membrane is repressed during growth at high osmolarity. This may enable the cell to reduce the rate of pressure build up during transitions from high to low osmolarity. The presence of multiple mechanosensitive channels in the E. coli cell membrane is well documented. The recent identification of genes that inactivate the MscL and MscS channels has established their role in releasing the pressure built up by hypoosmotic shock. The isolation of specific mutations and the structural studies on MscL now pave the way to a molecular understanding of the mechanism of activation of mechanosensitive channels.

Glutathione Is Involved in Environmental Stress Responses in Rhizobium tropici, Including Acid Tolerance

The isolation of rhizobial strains which exhibit an intrinsic tolerance to acidic conditions has been reported and has facilitated studies on the basic mechanisms underlying acid tolerance. Rhizobium tropici strain CIAT899 displays a high intrinsic tolerance to acidity and therefore was used in this work to study the molecular basis of bacterial responses to acid conditions and other environmental stresses. We generated a collection of R. tropici CIAT899 mutants affected in acid tolerance using Tn5-luxAB mutagenesis, and one mutant strain (CIAT899-13T2), which fails to grow under acid conditions, was characterized in detail. Strain CIAT899-13T2 was found to contain a single Tn5-luxAB insertion in a gene showing a high degree of similarity with the Escherichia coli gshB gene, encoding the enzyme glutathione synthetase. Intracellular potassium pools and intracellular pH levels were found to be lower in the mutant than in the parent. The glutathione-deficient mutant was shown to be sensitive to weak organic acids, osmotic and oxidative stresses, and the presence of methylglyoxal. Glutathione restores responses to these stresses almost to wild-type levels. Our data show that in R. tropici the production of glutathione is essential for growth in extreme environmental conditions. The mutant strain CIAT899-13T2 induced effective nodules; however, it was found to be outcompeted by the wild-type strain in coinoculation experiments.

Ionic regulation of MscK, a mechanosensitive channel from Escherichia coli

Three gene products that form independent mechanosensitive channel activities have been identified in Escherichia coli. Two of these, MscL and MscS, play a vital role in allowing the cell to survive acute hypotonic stress. Much less is known of the third protein, MscK (KefA). Here, we characterize the MscK channel activity and compare it with the activity of its structural and functional homologue, MscS. While both show a slight anionic preference, MscK appears to be more sensitive to membrane tension. In addition, MscK, but not MscS activity appears to be regulated by external ionic environment, requiring not only membrane tension but also high concentrations of external K+, NH4+, Rb+ or Cs+ to gate; no activity is observed with Na+, Li+ or N‐methyl‐D‐glucamine (NMDG). An MscK gain‐of‐function mutant gates spontaneously in the presence of K+ or similar ions, and will gate in the presence of Na+, Li+ and NMDG, but only when stimulated by membrane tension. Increased sensitivity and the highly regulated nature of MscK suggest a more specialized physiological role than other bacterial mechanosensitive channels.