Directed evolution of cellobiose utilization in Escherichia coli K12

The cellobiose catabolic system of Escherichia coli K12 is being used to study the role of cryptic genes in evolution of new functions. Escherichia coli does not use beta-glucoside sugars; however, mutations in several loci can activate the cryptic bgl operon and permit growth on the beta-glucoside sugars arbutin and salicin. Such Bgl+ mutants do not use cellobiose, which is the most common beta-glucoside in nature. We have isolated a Cel+ (cellobiose-utilizing) mutant from a Bgl+ mutant of E. coli K12. The Cel+ mutant grows well on cellobiose, arbutin, and salicin. Genes for utilization of these beta-glucosides are located at 37.8 min on the E. coli map. The genes of the bgl operon are not involved in cellobiose utilization. Introduction of a deletion covering bgl does not affect the ability to utilize cellobiose, arbutin, or salicin, indicating that the new Cel+ genes provide all three functions. Spontaneous cellobiose negative mutants also become arbutin and salicin negative. Analysis of beta-glucoside positive revertants of these mutants indicates that there are separate loci for utilization of each of the beta-glucoside sugars. The genes are closely linked and may be activated from a single locus. A fourth gene at an unknown location increases the growth rate on cellobiose. The cel genes constitute a second cryptic system for beta-glucoside utilization in E. coli K12.      

Antimicrobial activities of components of the glandular secretions of leaf cutting ants of the genus <Emphasis Type="Italic">Atta</Emphasis>

The secretions of the mandibular and metapleural glands of leaf cutting ants contain antimicrobial substances that protect the mutualistic fungal colony within the nest from attack by parasitic micro-organisms. The major constituents of these secretions (citral, 4-methyl-3-heptanol, 2-heptanone, 3-octanone, 4-methyl-2-heptanone, β-citronellol, geraniol, phenylacetic, indolacetic, hexanoic and octanoic acids were tested against resistant strains of the human pathogens, Escherichia coli, Staphylococcus aureus and Candida albicans. Assays were carried out using filter paper discs impregnated with either hexane or water solutions of the analytes in the concentration range 250–6,000 ng/μl. Although most of the tested compounds presented strong antibacterial and antifungal activities, citral, geraniol, 4-methyl-3-heptanol, hexanoic and octanoic acids were the most effective, particularly against C. albicans. The results suggest that these compounds may be of potential value as antibiotics in the treatment of human candidiasis.     

A new esterase EstD2 isolated from plant rhizosphere soil metagenome

Soil metagenome constitutes a reservoir for discovering novel enzymes from the unculturable microbial diversity. From three plant rhizosphere metagenomic libraries comprising a total of 142,900 members of recombinant plasmids, we obtained 14 recombinant fosmids that exhibited lipolytic activity. A selected recombinant plasmid, pFLP-2, which showed maximum lipolytic activity, was further analyzed. DNA sequence analysis of the subclone in pUC119, pELP-2, revealed an open reading frame of 1,191 bp encoding a 397-amino-acid protein. Purified EstD2 exhibited maximum enzymatic activity towards p-nitrophenyl butyrate, indicating that it is an esterase. Purified EstD2 showed optimal activity at 35 °C and at pH 8.0. The K _m and K _cat values were determined to be 79.4 μM and 120.5/s, respectively. The esterase exhibited an increase in enzymatic activity in the presence of 15% butanol and 15% methanol. Phylogenetic analysis revealed that the lipolytic protein EstD2 may be a member of a novel family of lipolytic enzymes. Several hypothetical protein homologs of EstD2 were found in the database. A hypothetical protein from Phenylobacterium zucineum HLK1, a close homolog of EstD2, displayed lipolytic activity when the corresponding gene was expressed in Escherichia coli. Our results suggest that the other hypothetical protein homologs of EstD2 might also be members of this novel family.     

Metabolic biomarkers of prenatal disorders: an exploratory NMR metabonomics study of second trimester maternal urine and blood plasma

This work describes an exploratory NMR metabonomic study of second trimester maternal urine and plasma, in an attempt to characterize the metabolic changes underlying prenatal disorders and identify possible early biomarkers. Fetal malformations have the strongest metabolic impact in both biofluids, suggesting effects due to hypoxia (leading to hypoxanthine increased excretion) and a need for enhanced gluconeogenesis, with higher ketone bodies (acetone and 3-hydroxybutyric acid) production and TCA cycle demand (suggested by glucogenic amino acids and cis-aconitate overproduction). Choline and nucleotide metabolisms also seem affected and a distinct plasma lipids profile is observed for mothers with fetuses affected by central nervous system malformations. Urine from women who subsequently develop gestational diabetes mellitus exhibits higher 3-hydroxyisovalerate and 2-hydroxyisobutyrate levels, probably due to altered biotin status and amino acid and/or gut metabolisms (the latter possibly related to higher BMI values). Other urinary changes suggest choline and nucleotide metabolic alterations, whereas lower plasma betaine and TMAO levels are found. Chromosomal disorders and pre-preterm delivery groups show urinary changes in choline and, in the latter case, in 2-hydroxyisobutyrate. These results show that NMR metabonomics of maternal biofluids enables the noninvasive detection of metabolic changes associated to prenatal disorders, thus unveiling potential disorder biomarkers.