The multiplex-PCR-based detection and genotyping of diarrhoeagenic Escherichia coli in diarrhoeal stools.

In several hospitals in Beirut, Lebanon, 77 isolates of Escherichia coli were successfully derived from the stools of patients with diarrhoeal diseases, by culture on MacConkey or MacConkey-sorbitol agar. When the isolates were screened, using a multiplex PCR, 14 (from 14 different patients) were each found positive for one of the various genes defining the enterotoxigenic (five), enteroinvasive (four), enteroaggregative (three) or enteropathogenic (two) groups. Genotyping of these 14 diarrhoeagenic isolates, by pulsed-field gel electrophoresis, indicated that all were genomically distinct with the exception of two of the enteroaggregative isolates (which were of the same genotype). The E. coli apparently involved in diarrhoeal disease in Beirut therefore belong to at least four different diarrhoeagenic groups and show strain variation within each group. Diarrhoea in the absence of diarrhoeagenic E. coli may be the result of infection with bacteria other than E. coli or viral or parasitic enteropathogens.

Periplasmic aggregation limits the proteolytic maturation of the Escherichia coli penicillin G amidase precursor polypeptide.

The Escherichia coli penicillin G amidase (PGA), which is a key enzyme in the production of penicillin G derivatives is generated from a precursor polypeptide by an unusual internal maturation process. We observed the accumulation of the PGA precursor polypeptide in the insoluble material recovered after sonication of recombinant E. coli JM109 cells grown at 26 degrees C. The aggregated nature of the accumulated molecules was demonstrated using detergents and chaotropic agents in solubilization assays. The periplasmic location of the aggregates was shown by trypsin-accessibility experiments performed on the spheroplast fraction. Finally, we showed that addition of sucrose or glycerol in the medium strongly reduces this periplasmic aggregation and as a consequence PGA production is substantially increased. Thus, periplasmic aggregation of the PGA precursor polypeptide limits PGA production by recombinant E. coli and this limitation can be overcome by addition in the medium of a non-metabolizable sugar, such as sucrose, or of glycerol.

Improved penicillin amidase production using a genetically engineered mutant of Escherichia coli ATCC 11105.

Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, we constructed various recombinant E. coli HB101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic and (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the Hindlll fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene.