Structural Diversity of Class 1 Integrons in Multiresistant Strains of Escherichia coli Isolated from Patients in a Hospital in Mexico City.

Since a decade, Escherichia coli has been considered an important nosocomial pathogen due to the high number of isolates multiresistant to antimicrobials reported worldwide. In clinical and environmental strains, transposons, plasmids, and integrons are currently considered the principal genetic elements responsible for the acquisition of antibiotic resistance through horizontal transfer. The objective of this research was to correlate the resistance to antibiotics of E. coli clinical strains with the presence class I integrons. In the present study, one hundred E. coli strains were isolated and tested for susceptibility and resistance to antimicrobials. Class 1 integrons were detected by PCR, and the arrangement of gene cassettes was determined by sequencing. Twenty two strains were found to carry Class 1 integrons. Sequence analysis of the variable regions revealed the presence of several gene cassettes, such as dihydrofolate reductases (dfr2d, dfrA17, and dhfrXVb), adenylyl transferases (aadA2, addA5, and addA22), and chloramphenicol efflux pump (cmlA), and oxacillinase (bla OXA-1 ). The dfrA17-addA5 arrangement prevailed upon other integrons in the study. This is the first report of the presence of the dfr2d and dhfrXVb-aadA2 cassette arrangements in a Class 1 integrons from clinical strains of E. coli. In most of the strains, it was found a direct relationship between genetic arrangements and resistance phenotypes. Four integrons were detected in plasmids that might be involved in the resistance genes transfer to other bacteria of clinical importance. Our results confirm the presence of Class 1 integrons and their essential role in the dissemination of resistance cassettes among E. coli strains.

Identification of α-amylase by random and specific mutagenesis of Texcoconibacillus texcoconensis 13CCT strain isolated from extreme alkaline-saline soil of the former Lake Texcoco (Mexico).

The alkaline α-amylase produced by Texcoconibacillus texcoconensis 13CC(T) strain was identified by random mutagenesis and confirmed by directed mutagenesis. A transposon mutagenesis approach was taken to identify the gene responsible for the degradation of starch in T. texcoconensis 13CC(T) strain. The deduced amino acids of the amy gene had a 99% similarity with those of Bacillus selenitireducens MLS10 and 97% with those of Paenibacillus curdlanolyticus YK9. The enzyme showed a maximum activity of 131.1 U/mL at 37 °C and pH 9.5 to 10.5. In situ activity of the enzyme determined by polyacrylamide gel electrophoresis showed only one band with amylolytic activity. This is the first report of a bacterium isolated from the extreme alkaline-saline soil of the former Lake Texcoco (Mexico) with amylolytic activity in alkaline conditions while its potential as a source of amylases for the industry is discussed.

Production and characterization of extracellular α-amylase produced by Wickerhamia sp. X-Fep.

A yeast isolate able to produce high levels of extracellular α-amylase was selected from a collection of 385 yeasts and identified as Wickerhamia sp. by the sequence of the D1/D2 domain of the 26 S rDNA gene. Part of the nucleotide sequence of the amy1-W gene was cloned, and a sequence of 191 amino acids deduced from this gene was analyzed. The peptide contains three characteristic well-conserved regions in the active sites of α-amylases (EC 3.2.1.1). The enzyme was purified and in situ activity showed only one band with amylolytic activity. The molecular mass of the α-amylase was estimated at 54 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enzymatic activity on soluble starch as substrate was optimal at pH 5-6 and 50 °C. This thermostable enzyme was inhibited by EDTA-Na(2) and 1,10-phenanthroline; the activity of the dialyzed enzyme was reactivated with Ca(2+) and Mg(2+) cations, which indicates that the α-amylase is a metalloenzyme. α-Amylase production was induced by starch and maltose and repressed by glucose. The high yield and productivity found in this work makes this Wickerhamia sp. strain a promising candidate for the biotechnological production of α-amylase.

The intracellular proteolytic system of Yarrowia lipolytica and characterization of an aminopeptidase.

Intracellular proteases of Yarrowia lipolytica have been scarcely studied. These enzymes may play an important role in nitrogen metabolism, posttranslational processing, nutritional stress, dimorphism, etc.; biochemical and genetic control of these enzymes can help in obtaining high-level expression of recombinant proteins in heterologous systems. In this study, we report the presence of three proteases: aminopeptidase yylAPE, carboxypeptidase yylCP and dipeptidyl aminopeptidase yylDAP, measured under several nutritional conditions. Yarrowia lipolytica produced the highest level of intracellular proteolytic enzymes, i.e. yylAPE, yylCP and yylDAP, in media with peptone during stationary growth phase. When soluble extracts were subjected to PAGE, and the three activities were revealed in gels with the corresponding substrates, only one band of activity was detected for each one. The three enzymes were affected by serine protease inhibitors. Chelating agents affected mainly APE activity. The aminopeptidase was purified by selective fractionation with ammonium sulfate and three chromatographic steps (anion exchange, hydrophobic interaction and gel filtration chromatography). The enzyme had a molecular mass of 97 kDa; optimal pH and temperature were 7.0 and 37 degrees C, respectively. The aminopeptidase showed a preference for lysine in the N-position. The K(m) value was 0.86 microM and V(max) value was 990.8 micromoL min(-1) mg(-1) for Lys-pNA.