Melvin Midgley

Substrate specificity and energetics of antiseptic and disinfectant resistance in Staphylococcus aureus

Clinical isolates of Staphylococcus aureus carry various antiseptic and disinfectant resistance determinants (qac genes) on a variety of plasmids. The biochemistry and specificity of these resistance genes in S. aureus is the subject of this report. The qac genes were separated into two families on the basis of resistance profiles and DNA homology. Isotopic and fluorimetric assays demonstrated that the qac genes encode efflux systems that rely on proton motive force.

Physical and biochemical characterization of the qacA gene encoding antiseptic and disinfectant resistance in Staphylococcus aureus.

We have previously cloned a 3.5 kb fragment from the Staphylococcus aureus multiresistance plasmid pSK1 which carries the qacA determinant responsible for linked resistance to acriflavine (Acr), ethidium bromide (Ebr), quaternary ammonium compounds (Qar), propamidine isethionate (Pir), and diamidinodiphenylamine dihydrochloride (Ddr). This report presents a biochemical and physical analysis of qacA and shows the widespread carriage of this gene on S. aureus resistance plasmids. Tn5 insertion mutagenesis defined the extent of qacA to within 2.40 kb of pSK1 DNA. Examination of the expression of insertion and deletion mutants of the cloned qacA sequences in both maxicells and minicells led to the association of a 50 kDa protein, designated QacA, with the AcrEbrQarPirDdr phenotype. Based on fluorimetric and isotopic assays used to determine the extent of accumulation of ethidium bromide by S. aureus strains harbouring pSK1, we propose that the basis of AcrEbrQarPirDdr in S. aureus is a qacA-mediated efflux system.

THE ALD6 GENE OF SACCHAROMYCES CEREVISIAE ENCODES A CYTOSOLIC, MG2+-ACTIVATED ACETALDEHYDE DEHYDROGENASE

The deduced translation product of an open reading frame on the left arm of chromosome XVI of Saccharomyces cerevisiae, with the systematic name of YPL061w, is 500 amino acids in length and shares significant homology with aldehyde dehydrogenases. Amino acids 2 to 16 of the protein encoded by YPL061w were found to be identical to the N‐terminal 15 amino acids of the purified cytosolic, Mg2+‐activated acetaldehyde dehydrogenase (ACDH) of S. cerevisiae. This enzyme is thought to be involved in the production of acetate from which cytosolic acetyl‐CoA is then synthesized. Deletion of YPL061w was detrimental to the growth of haploid strains of yeast; an analysis of one deletion mutant revealed a maximum specific growth rate (in complex medium containing glucose) of one‐third of that displayed by the wild‐type strain. Mutants deleted in YPL061w were also unable to use ethanol as a carbon source. As expected, the cytosolic, Mg2+‐activated ACDH activity had been lost from the mutants, although the mitochondrial, K+‐activated ACDH was readily detected. YPL061w has been registered with the name of ALD6 in the Saccharomyces Genome Database and the nucleotide sequence submitted to GenBank as part of accession number U39205.

The Phosphonium Ion Efflux System of Escherichia coli: Relationship to the Ethidium Efflux System and Energetic Studies

The extent of accumulation of methyltriphenylphosphonium ion by Escherichia coli was shown to be dependent on the permeability of the outer membrane and the activity of an efflux system for this compound. Evidence consistent with the operation of a single efflux system for compounds such as phosphonium ions, phenanthridiniums and flavines is presented. Studies on the energy coupling mechanism for this efflux system indicated that it was driven by the transmembrane proton electrochemical gradient.

Cloning of the ethidium efflux gene from Escherichia coli

The gene specifying the ethidium efflux system of Escherichia coli has been cloned on a 3.2 kbp HindIII fragment and located on a 1.2 kbp fragment within this. Cross-resistance studies indicate that the system has a broad specificity for monovalent cations and the gene shows no hybridisation with similar genes found in Staphylococci.

Effect of starvation on transport, membrane potential and survival of Staphylococcus epidermidis under anaerobic conditions.

When washed suspensions of Staphylococcus epidermidis were starved under anaerobic conditions the viability declined to less than 10% within 12 h. Although RNA was slightly degraded during this period the principal substrate for endogenous metabolism was protein and the intracellular amino acid pool. The adenylate energy charge and the ability to transport serine declined markedly within the first 6 h of starvation. With the majority of batches of organism investigated the membrane potential, as measured by the accumulation of Cs+ by valinomycin-treated organisms, also decreased significantly during this period. Addition of glucose or serine during starvation reversed these effects to varying extents provided that feeding took place during an early phase (2 h) of starvation. There was no apparent correlation between the magnitude of the membrane potential and viability.

The interaction of phosphonium ions with Acinetobacter calcoaceticus: evidence for the operation of an efflux system

Abstract Experiments with washed cell suspensions of Acinetobacter calcoaceticus NCIB 8250 indicated that this organism possesses an energy-linked efflux system for phosphonium ions and related compounds. Evidence is presented to show that the apparent impermeability to low concentrations of methyltriphenylphosphonium ion is a result of the combination of the outer membrane acting as a partial permeability barrier and the activity of the efflux system.

Energy Transduction in Chlorobium limicola: Role of Membrane-bound Adenosine Triphosphatase and the Proton Electrochemical Gradient

Photophosphorylation in vivo by Chlorobium limicola was inhibited by lipophilic cations and the energy-transfer inhibitors diphenylphosphorylazide, Dio-9, 4-chloro-7-nitrobenzofurazan and chlorhexidene. Membrane-bound ATPase activity was also inhibited by these energy-transfer inhibitors. The formation of a membrane potential was stimulated approximately 1.7-fold on illumination, rising to a value between −110 and −150 mV. The sensitivity of the processes producing this membrane potential to uncouplers, energy-transfer inhibitors and 2-heptyl-4-hydroxyquinoline-N-oxide was measured in the light and the dark.

Anaerobic transport of serine and 2-aminoisobutyric acid by Staphylococcus epidermidis.

A membrane-bound ATPase detected in extracts of anaerobically grown Staphylococcus epidermidis was inhibited by a variety of compounds which inhibit ATPases in other organisms. Serine and 2-aminoisobutyric acid (AIB) were shown to enter the organism via the same transport system. The transport of AIB, the membrane potential and the transmembrane pH gradient were partially or completely abolished by the same inhibitors and also by uncoupling agents and lipid-soluble ions. It is proposed therefore that this ATPase generates and maintains an electrochemical gradient of protons across the cytoplasmic membrane of S. epidermidis capable of driving AIB uptake. Studies of AIB-induced proton movements suggested that AIB enters via a proton symport mechanism.

The Roles of Acetaldehyde Dehydrogenases in Saccharomyces Cerevisiae

This work was undertaken to try to understand the routes that are used for synthesis of acetyl-CoA in Saccharomyces cerevisaeunder a variety of nutritional conditions. It is a topic of interest in connection with the production of flavour compounds in beer and lager. Distinct physiological roles have been proposed for the Mg2+ activated, NADP+-dependent acetaldehyde dehydrogenase (Mg-ACDH) and the K+-activated NAD+/NADP+ dependent mitochrial enzyme (K-ACDH) (Llorente&de Castro 1977). The former enzyme was thought to function in acetate formation from acetaldehyde derived from pyruvate through the action of pyruvate decarboxylase. The latter enzyme has been implicated in acetate formation from acetaldehyde derived from ethanol. On this basis, disruption mutants for each of the enzymes would be expected to have markedly different potential when grown on 5% glucose, where mitochondrial function is severely repressed and on 1 % ethanol where mitochondrial function is developed.