PhoU Is a Persistence Switch Involved in Persister Formation and Tolerance to Multiple Antibiotics and Stresses in Escherichia coli

ABSTRACT When a bactericidal antibiotic is added to a growing bacterial culture, the great majority of the bacterial population is killed but a small number of metabolically quiescent bacteria called persisters survive antibiotic treatment. The mechanism of this bacterial persistence is poorly understood. In Escherichia coli, we identified a new persistence gene, phoU, whose inactivation leads to a generalized higher susceptibility than that of the parent strain to a diverse range of antibiotics, including ampicillin, norfloxacin, and gentamicin, and stresses, such as starvation, acid pH, heat, peroxide, weak acids, and energy inhibitors, especially in stationary phase. The PhoU mutant phenotype could be complemented by a functional phoU gene. Mutation in PhoU leads to a metabolically hyperactive status of the cell, as shown by an increased expression of energy production genes, flagella, and chemotaxis genes and a defect in persister formation. PhoU, whose expression is regulated by environmental changes like nutrient availability and age of culture, is a global negative regulator beyond its role in phosphate metabolism and facilitates persister formation by the suppression of many important cellular metabolic processes. A new model of persister formation based on PhoU as a persister switch is proposed. PhoU may be an ideal drug target for designing new drugs that kill persister bacteria for more effective control of bacterial infections.

Structure and Function Analysis of CaMdr1p, a Major Facilitator Superfamily Antifungal Efflux Transporter Protein of Candida albicans: Identification of Amino Acid Residues Critical for Drug/H+ Transport

ABSTRACT We have cloned and overexpressed multidrug transporter CaMdr1p as a green fluorescent protein-tagged protein to show its capability to extrude drug substrates. The drug extrusion was sensitive to pH and energy inhibitors and displayed selective substrate specificity. CaMdr1p has a unique and conserved antiporter motif, also called motif C [G(X6)G(X3)GP(X2)GP(X2)G], in its transmembrane segment 5 (TMS 5). Alanine scanning of all the amino acids of the TMS 5 by site-directed mutagenesis highlighted the importance of the motif, as well as that of other residues of TMS 5, in drug transport. The mutant variants of TMS 5 were placed in four different categories. The first category had four residues, G244, G251, G255, and G259, which are part of the conserved motif C, and their substitution with alanine resulted in increased sensitivity to drugs and displayed impaired efflux of drugs. Interestingly, first category mutants, when replaced with leucine, resulted in more dramatic loss of drug resistance and efflux. Notwithstanding the location in the core motif, the second category included residues which are part of the motif, such as P260, and those which were not part of the motif, such as L245, W248, P256, and F262, whose substitution with alanine resulted in a severe loss of drug resistance and efflux. The third category included G263, which is a part of motif C, but unlike other conserved glycines, its replacement with alanine or leucine showed no change in the phenotype. The replacement of the remaining 11 residues of the fourth category did not result in any change. The putative helical wheel projection showed clustering of functionally critical residues to one side and thus suggests an asymmetric nature of TMS 5.

Evidence for the Presence of an Alternative Glucose Transport System in Clostridium beijerinckii NCIMB 8052 and the Solvent-Hyperproducing Mutant BA101

ABSTRACT The effects of substrate analogs and energy inhibitors on glucose uptake and phosphorylation by Clostridium beijerinckii provide evidence for the operation of two uptake systems: a previously characterized phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a non-PTS system probably energized by the transmembrane proton gradient. In both wild-type C. beijerinckii NCIMB 8052 and the butanol-hyperproducing mutant BA101, PTS activity declined at the end of exponential growth, while glucokinase activity increased in the later stages of fermentation. The non-PTS uptake system, together with enhanced glucokinase activity, may provide an explanation for the ability of the mutant to utilize glucose more effectively during fermentation despite the fact that it is partially defective in PTS activity.

Chromate Efflux by Means of the ChrA Chromate Resistance Protein from Pseudomonas aeruginosa

ABSTRACT Everted membrane vesicles of Pseudomonas aeruginosaPAO1 harboring plasmid pCRO616, expressing the ChrA chromate resistance protein, accumulated four times more51CrO4 2− than vesicles from plasmidless cells, indicating that a chromate efflux system functions in the resistant strain. Chromate uptake showed saturation kinetics with an apparent Km of 0.12 mM chromate and aV max of 0.5 nmol of chromate/min per mg of protein. Uptake of chromate by vesicles was dependent on NADH oxidation and was abolished by energy inhibitors and by the chromate analog sulfate. The mechanism of resistance to chromate determined by ChrA appears to be based on the active efflux of chromate driven by the membrane potential.

Communication of post-Golgi elements with early endocytic pathway: regulation of endoproteolytic cleavage of Semliki Forest virus p62 precursor

A number of cellular proteins and viral spike proteins are cleaved at a basic recognition sequence. To characterize the membrane traffic step at which this proteolysis occurs we have studied the intracellular processing site of Semliki Forest virus (SFV) spike precursor p62 in BHK21 cells. The p62 is endoproteolytically cleaved at a tetrabasic Arg-His-Arg-Arg recognition sequence. Previously, it has been shown that the SFV p62 remains uncleaved when accumulated to the trans-Golgi network (TGN/20 degrees C block site). We show here that exit from the trans-Golgi is required for the cleavage of p62. Proteolytic processing was inhibited in synchronized assays when the 20 degrees C transport block was released in the presence of brefeldin A, energy inhibitors (azide and deoxyglucose; carbonyl cyanide m-chlorophenylhydrazone, CCCP) or an effector of trimeric G proteins, AlFn. Endocytosed antibodies against the SFV spike glycoproteins or antibodies against a peptide corresponding to the enzymatically active motif of furin inhibited cleavage of p62 at a post-TGN location. The results indicate a post-TGN communication step between exocytic and endocytic elements. Kinetic experiments suggested that this communication may involve an early compartment of the endocytic pathway.

Influence of ionophores and energy inhibitors on peptide metabolism by rumen bacteria

SUMMARYIonophores and inhibitors of bacterial energy metabolism were added to mixed rumen bacteria prepared from sheep receiving grass hay plus concentrate diets, and their influence on the rate of metabolism of alanine (Ala) peptides was determined. Dicyclohexylcarbodiimide (DCCD) had no influence on the rate of breakdown of Ala2, Ala3, Ala4or Ala5, indicating that the metabolism of these peptides did not require ATP. The protonophores tetrachlorosalicylanilide (TCS) and carbonyl cyanidem-chlorophenyl-hydrazone (CCCP) inhibited peptide breakdown to a minor extent (< 15%), whereas the ionophores monensin and tetronasin had greater, but still small (12–31%), inhibitory effects. Toluene stimulated peptide breakdown, consistent with a permeability barrier having been removed. Thus, at least some peptide metabolism depends on transport into bacteria; transport appears not to be ATP-linked, and may well be coupled to the uptake of mineral cations rather than protons. Rumen fluid from sheep receiving a similar diet with added monensin (33 mg/kg) or tetronasin (10 mg/kg) hydrolysed Ala3and Ala4at rates that did not differ significantly from controls. Nevertheless, the peak concentration of free peptides in rumen fluid after feeding was more than doubled in ionophore-fed sheep, and peptides persisted for longer than in control animals.

Cellular and lysosomal uptake of methylamine in isolated rat hepatocytes

Upon addition of methylamine to intact cells, this lysosomotropic weak base accumulates intracellularly as the result of at least two different mechanisms: (1) facilitated diffusion across the plasma membrane, i.e. a process which is carrier-mediated and subject to both trans-stimulation (accelerative exchange) and cis-inhibition (competition) by other amines (e.g. ammonia, methylamine and triethylamine); this transport process is furthermore non-concentrative, energy-independent, and (although moderately temperature-sensitive) operative even at 0 degrees C; (2) active uptake, i.e. an energy-dependent concentrative process which is inhibited by anoxia and energy inhibitors. With time, methylamine accumulates in lysosomes and gives rise to a lysosomal swelling which is easily visible by optical microscopy, and which causes the cells to appear coarsely granular. After a 1h incubation with 10mM-methylamine, the total cell volume is increased by about 12%. Under anoxic conditions or in the presence of energy inhibitors, lysosomal swelling is abolished regardless of there being a high concentration of methylamine intracellularly (taken up by facilitated diffusion). The continuous accumulation of methylamine in lysosomes therefore seems to depend on an energy-requiring process (such as continuous proton pumping), and not only on trapping by Donnan-equilibrium-generated protons.

Epidermal growth factor-induced centrosomal separation: mechanism and relationship to mitogenesis.

Using a rabbit antibody to MAP1 to stain centrosomes we have studied the mechanism by which epidermal growth factor (EGF) induces centrosomal separation in HeLa cells. The response is rapid, being detectable within 20 min after EGF (100 ng/ml) addition and by 4 h 40% of logarithmically growing cells and greater than 70% of cells synchronized at G1/S with 1 mM hydroxyurea show centrosomes separated by more than one diameter. A concentration of 0.05 ng/ml of EGF induces significant separation in synchronized cells (5-9% control vs. 20% with EGF at 0.05 ng/ml) and 0.1 to 0.5 ng/ml induces a half maximal response. Centrosomal separation is blocked by energy inhibitors, trifluoperazine, chlorpromazine, and W-7, cytochalasins B and D, and taxol, and is stimulated or enhanced by A23187, colchicine, and oncodazole. Trifluoperazine, W-7, cytochalasin D, and taxol also block DNA synthesis in response to EGF as measured by autoradiography using [3H]thymidine. Our hypothesis based upon these results is that EGF, by raising the free calcium level, activates calmodulin, which stimulates contraction of microfilaments attached to the centrosome, pulling the daughter centrosome apart. EGF may also induce depolymerization or detachment of microtubules in the vicinity of the centrosome which ordinarily serve to maintain its position and inhibit separation. Centrosomal separation may be a key event in triggering DNA synthesis in response to EGF and colchicine.