A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli.

The envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measure Escherichia coli envelope permeability to a ß-galactosidase chromogenic substrate. The signal produced by cytoplasmic ß-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds and E. coli gene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinct E. coli strains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 µM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R > 0.5 µg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

The structure and synthetic capabilities of a catalytic peptide formed by substrate-directed mechanism--implications to prebiotic catalysis.

Previously, we have shown that a small substrate may serve as a template in the formation of a specific catalytic peptide, a phenomenon which might have had a major role in prebiotic synthesis of peptide catalysts. This was demonstrated experimentally by the formation of a catalytic metallo-dipeptide, Cys2-Fe2+, around o-nitrophenyl beta-D-galactopyranoside (ONPG), by dicyandiamide (DCDA)-assisted condensation under aqueous conditions. This dipeptide was capable of hydrolyzing ONPG at a specific activity lower only 1000 fold than that of beta galactosidase. In the present paper we use molecular modeling techniques to elucidate the structure of this catalyst and its complex with the substrate and propose a putative mechanism for the catalyst formation and its mode of action as a "mini enzyme". This model suggests that interaction of Fe2+ ion with ONPG oxygens and with two cysteine SH groups promotes the specific formation of the Cys2-Fe2+ catalyst. Similarly, the interaction of the catalyst with ONPG is mediated by its Fe2+ with the substrate oxygens, leading to its hydrolysis. In addition, immobilized forms of the catalyst were synthesized on two carriers--Eupergit C and amino glass beads. These preparations were capable of catalyzing the formation of ONPG from beta-D-galactose and o-nitrophenol (ONP) under anhydrous conditions. The ability of the catalyst to synthesize the substrate that mediates its own formation creates an autocatalytic cycle where ONPG catalyzes the formation of a catalyst which, in turn, catalyzes ONPG formation. Such autocatalytic cycle can only operate by switching between high and low water activity conditions, such as in tidal pools cycling between wet and dry environments. Implications of the substrate-dependent formation of catalytically active peptides to prebiotic processes are discussed.

Aromatic stacking in the sugar binding site of the lactose permease.

Major determinants for substrate recognition by the lactose permease of Escherichia coli are at the interface between helices IV (Glu126, Ala122), V (Arg144, Cys148), and VIII (Glu269). We demonstrate here that Trp151, one turn of helix V removed from Cys148, also plays an important role in substrate binding probably by aromatic stacking with the galactopyranosyl ring. Mutants with Phe or Tyr in place of Trp151 catalyze active lactose transport with time courses nearly the same as wild type. In addition, apparent K(m) values for lactose transport in the Phe or Tyr mutants are only 6- or 3-fold higher than wild type, respectively, with a comparable V(max). Surprisingly, however, binding of high-affinity galactoside analogues is severely compromised in the mutants; the affinity of mutant Trp151-->Phe or Trp151-->Tyr is diminished by factors of at least 50 or 20, respectively. The results demonstrate that Trp151 is an important component of the binding site, probably orienting the galactopyranosyl ring so that important H-bond interactions with side chains in helices IV, V, and VIII can be realized. The results are discussed in the context of a current model for the binding site.

Molecular cloning and sequencing of two phospho-beta-galactosidase I and II genes of Lactobacillus gasseri JCM1031 isolated from human intestine.

Lactobacillus (Lb.) gasseri JCM1031, which is classified into the B1 subgroup of the Lb. acidophilus group of lactic acid bacteria, characteristically produces two different phospho-beta-galactosidases (P-beta-gal) I and II in the same cytosol as reported in our previous papers [Biosci. Biotech. Biochem., 60, 139-141, 708-710 (1996)]. To clarify the functional and genetic properties of the two enzymes, the structural genes of P-beta-gal I and II were cloned and sequenced. The structural gene of P-beta-gal I had 1,446 bp, encoding a polypeptide of 482 amino acid residues. The structural gene of P-beta-gal II had 1,473 bp, encoding a polypeptide of 491 amino acid residues. The deduced relative molecular masses of 55,188 and 56,243 agreed well with the previous value obtained from the purified P-beta-gal I and II protein, respectively. Multiple alignment of the protein sequence of P-beta-gal I and II with those of P-beta-gals from 5 microorganisms had 30-35% identity on the amino acid level, but those with phospho-beta-glucosidases from 5 microorganisms had the relatively high identity of about 50%. Considering that this strain grows on lactose medium and shows no beta-galactosidase activity, and that purified P-beta-gal I and II can obviously hydrolyze o-nitrophenyl-beta-D-galactopyranoside 6-phosphate (substrate), and also the conservation of a cysteine residue in the molecule, the P-beta-gal I and II were each confirmed as a novel P-beta-gal enzyme.