Phenethyl Alcohol as a Suppressor of the EnvA Phenotype Associated with the envA Gene in Escherichia coli K-12

In Escherichia coli K-12 the envA gene was previously shown to mediate chain formation and a decreased tolerance to several antibacterial agents. Phenethyl alcohol at low concentrations has now been found to increase the tolerance to actinomycin D, ampicillin, rifampin, and gentian violet in strains containing envA. The increased tolerance to gentian violet was correlated to a decreased uptake of the dye. A phenotype suppression of chain formation and colony morphology in envA mutants was also obtained. Except for an increase in palmitic acid, chemical analysis revealed no differences between an envA and its wild-type strain in the lipopolysaccharide part of the envelope. However, a decrease in the amount of phosphatidylglycerol and a C18: 1 fatty acid was observed in the extractable lipids of a strain containing envA. Growth in the presence of phenethyl alcohol reversed the changes in fatty acid and the phospholipid composition. Phenethyl alcohol was found to cause an immediate but transient inhibition of ribonucleic acid synthesis. It is suggested that this inhibition affects the penetrability barrier of the outer cell envelope layers in strains containing envA.     

Correlation of genes in the pap gene cluster to expression of globoside-specific adhesin by uropathogenic Escherichia coli

Abstract Expression of globoside-specific pilus adhesin of Escherichia coli is the virulence factor most commonly associated with pyelonephritis. In the clinical isolate J96 (O4:K6:H5) expression of globoside binding pili require the proteins encoded by the papE, papF , and papG genes in the pap gene cluster. Probes derived from these genes were used in dot blot hybridization analysis of E. coli urinary tract isolates obtained from patients with significant bacteriuria. Fecal E. coli isolates from healthy individuals were also analyzed. The probe encompassing the papF and papF J96 genes hybridized to all urinary tract infectious (UTI) isolates expressing globoside-specific adhesin, whereas papG J96 only hybridized to the strain from which the fragment was cloned. In contrast, a papG -specific probe from the O:6 strain IA2 hybridized to all but one of the UTI isolates that expressed the adhesin. In both materials, but especially among the fecal isolates, strains were found that hybridized to the probes but did not express the adhesin. The data shows that papEF -specific DNA can be used for the diagnosis of potentially pyelonephritic E. coli .     

Overproduction of outer membrane protein suppresses envA-induced hyperpermeability.

A quantitative study on outer membrane components was performed in a number of envelope mutants of Escherichia coli K-12 exhibition different permeability properties for antimicrobial agents. The envA1 allele causing an increased influx for both hydrophobic and hydrophilic drugs was found to be associated with a deficiency in the amount of lipopolysaccharides. The sefA1 envA1 double mutant was found to have a higher outer membrane buoyant density, apparently due to an increase in protein content. This double mutant was still low in lipopolysaccharide content.     

A relationship between plasmid structure, structural lability, and sensitivity to site-specific endonucleases in Neisseria gonorrhoeae

Summary Nearly all gonococcal strains carry a small phenotypically cryptic plasmid of approximately 4,200 basepairs. A detailed physical map of this plasmid has been constructed, revealing the presence of numerous putative inverted repeats. These studies also revealed the presence on the plasmid of recognition sequences for several site-specific endonucleases (particularly HpaII, MspI and AluI) that are particularly resistant to cleavage, and confirmed previous reports of structural lability. Both the sites that are resistant to cleavage, and the observed structural variation are associated with the inverted repetitive sequences.     

Nucleotide sequence of the papA gene encoding the Pap pilus subunit of human uropathogenic Escherichia coli

The papA gene of the uropathogenic strain Escherichia coli J96, coding for the Pap pili subunit, was subjected to DNA sequencing, and found to code for an 185-amino acid-long polypeptide with a 22-amino acid-long signal peptide. Here we present the primary sequence, the hydrophilicity profile, and the predicted polypeptide secondary structure of the Pap pili subunit.     

Sensitivity of Escherichia coli to various β-lactams is determined by the interplay of outer membrane permeability and degradation by periplasmic β-lactamases: a quantitative predictive treatment

In Gram-negative bacteria, β-lactam antibiotics must overcome two barriers, the outer membrane and the periplasmic β-lactamase, before they reach the targets of their action, penicillin-binding proteins. Although the barrier property of the outer membrane and catalytic property of the β-lactamases have been studied and their significance in creating β-lactam resistance emphasized, the interaction between these two barriers has not been treated quantitatively. Such treatment shows that the sensitivity, to a variety of β-lactams, of the Escherichia coli K-12 cells containing very different levels of chromosomally coded AmpC β-lactamase, or a plasmid-coded TEM-type β-lactamase, can be predicted rather accurately from the penetration rate through the outer membrane and the hydrolysis rate in the periplasm. We further propose a new parameter,‘target access Index', which is a quantitative expression of the result of interaction between the two barriers, and reflects the probability of success for the antibiotic to reach the targets.     

Sequence and comparative analysis of three Enterobacter cloacae ampC beta-lactamase genes and their products.

Changes in cyclic AMP concentrations were studied in intact PC12 pheochromocytoma cells exposed to a variety of treatments. A marked increase was triggered by N-(L-2-phenylisopropyl)adenosine, the activator of an adenosine receptor, whereas a decrease (observed even after phosphodiesterase blockade) was induced by carbachol, working through a muscarinic receptor inhibited by the selective muscarinic blocker pirenzepine, only at high concentration (Ki 450 nM). A decrease in cyclic AMP was also induced by clonidine, an alpha 2-adrenergic-receptor agonist. Both the alpha 2-adrenergic and the muscarinic inhibitions were prevented by pretreatment of the cells with pertussis toxin, and were unaffected by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. The latter drug caused a decrease in the resting cyclic AMP concentrations, and a potentiation of the increase induced by adenosine-receptor activation. Except for clonidine, all these treatments were found to be effective in both growing PC12 cells and, although to a smaller degree, in cells that had stopped growing and had acquired a neuron-like phenotype after prolonged treatment with nerve growth factor (NGF). Neither forskolin (a direct activator of adenylate cyclase) nor the activation of adenosine and alpha-adrenergic receptors was able to modify the resting cytosolic Ca2+ concentration [Ca2+]i in PC12 cells. Likewise, the K+-induced [Ca2+]i transients were unchanged after these treatments, whereas the transients induced by carbachol through the activation of a muscarinic receptor highly sensitive to pirenzepine were moderately potentiated by forskolin (and, to a lesser degree, by the adenosine analogue) and attenuated by clonidine. These results characterize in further detail the spectrum and the mutual interrelationships of the intracellular signals induced by receptor activation in PC12 cells, also as a function of the NGF-induced differentiation.     

PapD, a periplasmic transport protein in P-pilus biogenesis.

The product of the papD gene of uropathogenic Escherichia coli is required for the biogenesis of digalactoside-binding P pili. Mutations within papD result in complete degradation of the major pilus subunit, PapA, and of the pilinlike proteins PapE and PapF and also cause partial breakdown of the PapG adhesin. The papD gene was sequenced, and the gene product was purified from the periplasm. The deduced amino acid sequence and the N-terminal sequence obtained from the purified protein revealed that PapD is a basic and hydrophilic peripheral protein. A periplasmic complex between PapD and PapE was purified from cells that overproduced and accumulated these proteins in the periplasm. Antibodies raised against this complex reacted with purified wild-type P pili but not with pili purified from a papE mutant. In contrast, anti-PapD serum did not react with purified pili or with the culture fluid of piliated cells. However, this serum was able to specifically precipitate the PapE protein from periplasmic extracts, confirming that PapD and PapE were associated as a complex. It is suggested that PapD functions in P-pilus biogenesis as a periplasmic transport protein. Probably PapD forms complexes with pilus subunits at the outer surface of the inner membrane and transports them in a stable configuration across the periplasmic space before delivering them to the site(s) of pilus polymerization.     

The effect of iron starvation on the outer membrane protein composition of Neisseria gonorrhoeae

Abstract: A dipeptidase activity on ms -A₂pm- d -Ala and l -Lys- d -Ala was found in sporulating cells of Bacillus sphaericus 9602. The specific activity of this enzyme was low during the growth cycle and showed a rapid increase throughout sporulation. Values in the dormant spores were 1.3-fold higher than those found in cells late in sporulation and 20-fold higher than those found in log-phase cells.