Comparison of Substrate Specificity of <em>Escherichia Coli</em>p-Aminobenzoyl-Glutamate Hy-drolase with <em>Pseudomonas</em>Carboxypeptidase G

Reduced folic acid derivatives support biosynthesis of DNA, RNA and amino acids in bacteria as well as in eukaryotes, including humans. While the genes and steps for bacterial folic acid synthesis are known, those associated with folic acid catabolism are not well understood. A folate catabolite found in both humans and bacteria is p-aminobenzoyl-glutamate (PABA-GLU). The enzyme p-aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, the abg region, in E. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from Pseudomonas species. A comparison of the subunit sequences and activity of PGH, relative to carboxypeptidase enzymes, may lead to a better understanding of bacterial physiology and pathway evolution. We first compared the amino acid sequences of AbgA, AbgB and carboxypeptidase G2 from Pseudomonas sp. RS-16, which has been crystallized. Then we compared the enzyme activities of E. coli PGH and commercially available Pseudomonas carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The Km and Vmax values for the folate and anti-folate substrates of PGH could not be determined, because the instrument reached its limit before the enzyme was saturated. Therefore, activity of PGH was compared to the activity of CPG, or normalized to PABA-GLU (nmole/min/μg). Relative to its activity with 10 μM PABA-GLU (100%), PGH cleaved glutamate from methotrexate (48%), aminopterin (45%) and folate (9%). Reduced folates leucovorin (5-formyltetrahydrofolate) and 5-methyltetrahydrofolate were not cleaved by PGH. Our data suggest that E. coli PGH is specific for PABA-GLU as its activity with natural folates (folate, 5-methyltetrahydrofolate, and leucovorin) was very poor. It does, however, have some ability to cleave anti-folates which may have clinical applications in treatment of chemotherapy overdose.

Effect of Human Insulin on the Formation of Catheter-Associated <i>E. coli</i>Biofilms

Biofilm formation is essential for the survival and growth of Escherichia coli?in catheter-associated infections. Individuals with type 2 diabetes mellitus can excrete insulin and/or glucose in their urine. This population also has an increased incidence of urinary tract infections. The focus of this study was to determine if the composition of Foley catheter material affects biofilm formation by E. coli in a model system for type 2 diabetes mellitus. Rubber (lubricious-coated), silicon-coated, silver-coated and nitrofurazone-coated catheter segments (5 mm;n = 6) were tested. Catheter segments were added to E. coli ATCC25922 (104 CFU/ml, final concentration) in artificial urine alone, or with insulin (40 μU/ml) and/or glucose (0.1%). After incubation (18 h, 37?C, in air and anaerobically) the level of catheter-associated biofilm was determined by crystal violet staining (Abs550nm). Statistical analysis was done by ANOVA with post-hoc analysis (Tukey). Neither nitrofurazone-coated nor silver-coated catheters supported the formation of E. coli biofilm, regardless of growth condition tested. In contrast, under aerobic biofilm formation on silicon catheters was significantly higher (p E. coli controls. Biofilm formation was also significantly increased