Urinary catheter use is prevalent in health care settings, and polymicrobial colonization by urease-positive organisms, such as Proteus mirabilis and Providencia stuartii, commonly occurs with long-term catheterization. We previously demonstrated that coinfection with P. mirabilis and P. stuartii increased overall urease activity in vitro and disease severity in a model of urinary tract infection (UTI). In this study, we expanded these findings to a murine model of catheter-associated UTI (CAUTI), delineated the contribution of enhanced urease activity to coinfection pathogenesis, and screened for enhanced urease activity with other common CAUTI pathogens. In the UTI model, mice coinfected with the two species exhibited higher urine pH values, urolithiasis, bacteremia, and more pronounced tissue damage and inflammation compared to the findings for mice infected with a single species, despite having a similar bacterial burden within the urinary tract. The presence of P. stuartii, regardless of urease production by this organism, was sufficient to enhance P. mirabilis urease activity and increase disease severity, and enhanced urease activity was the predominant factor driving tissue damage and the dissemination of both organisms to the bloodstream during coinfection. These findings were largely recapitulated in the CAUTI model. Other uropathogens also enhanced P. mirabilis urease activity in vitro, including recent clinical isolates of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. We therefore conclude that the underlying mechanism of enhanced urease activity may represent a widespread target for limiting the detrimental consequences of polymicrobial catheter colonization, particularly by P. mirabilis and other urease-positive bacteria.KEYWORDS CAUTI, Enterococcus, Proteus mirabilis, Providencia stuartii, UTI, catheterassociated urinary tract infection, polymicrobial, urease, urinary tract infection U rinary catheters are common in health care settings and are utilized by over 60% of critically ill patients, 20% of patients in medical and surgical units, and 5 to 10% of residents in nursing homes (1-3). The incidence of bacteria in urine (bacteriuria) is 3 to 8% per day of catheterization, and long-term catheterization (Ͼ30 days) results in continuous bacteriuria (1). The microbial composition of urine colonization changes over time, initially involving Escherichia coli, Klebsiella pneumoniae, Serratia spp., Citrobacter spp., Enterobacter spp., Pseudomonas aeruginosa, and/or Gram-positive cocci and

The enteric bacterium Proteus mirabilis is a common cause of complicated urinary tract infections. In this study, microarrays were used to analyze P. mirabilis gene expression in vivo from experimentally infected mice. Urine was collected at 1, 3, and 7 days postinfection, and RNA was isolated from bacteria in the urine for transcriptional analysis. Across nine microarrays, 471 genes were upregulated and 82 were downregulated in vivo compared to in vitro broth culture. Genes upregulated in vivo encoded mannose-resistant Proteus-like (MR/P) fimbriae, urease, iron uptake systems, amino acid and peptide transporters, pyruvate metabolism enzymes, and a portion of the tricarboxylic acid (TCA) cycle enzymes. Flagella were downregulated. Ammonia assimilation gene glnA (glutamine synthetase) was repressed in vivo, while gdhA (glutamate dehydrogenase) was upregulated in vivo. Contrary to our expectations, ammonia availability due to urease activity in P. mirabilis did not drive this gene expression. A gdhA mutant was growth deficient in minimal medium with citrate as the sole carbon source, and loss of gdhA resulted in a significant fitness defect in the mouse model of urinary tract infection. Unlike Escherichia coli, which represses gdhA and upregulates glnA in vivo and cannot utilize citrate, the data suggest that P. mirabilis uses glutamate dehydrogenase to monitor carbon-nitrogen balance, and this ability contributes to the pathogenic potential of P. mirabilis in the urinary tract.

Escherichia coli glycogen synthase (EcGS, EC 2.4.1.21) is a retaining glycosyltransferase (GT) that transfers glucose from adenosine diphosphate glucose to a glucan chain acceptor with retention of configuration at the anomeric carbon. EcGS belongs to the GT-B structural superfamily. Here we report several EcGS x-ray structures that together shed considerable light on the structure and function of these enzymes. The structure of the wild-type enzyme bound to ADP and glucose revealed a 15.2°o verall domain-domain closure and provided for the first time the structure of the catalytically active, closed conformation of a glycogen synthase. The main chain carbonyl group of His-161, Arg-300, and Lys-305 are suggested by the structure to act as critical catalytic residues in the transglycosylation. Glu-377, previously thought to be catalytic is found on the ␣-face of the glucose and plays an electrostatic role in the active site and as a glucose ring locator. This is also consistent with the structure of the EcGS(E377A)-ADP-HEPPSO complex where the glucose moiety is either absent or disordered in the active site.

We conclude that P. mirabilis and P. stuartii coinfection promotes urolithiasis and bacteremia in a urease-dependent manner, at least in part through synergistic induction of urease activity. These data provide a possible explanation for the high incidence of bacteremia resulting from polymicrobial CaUTI.

Benzoylformate decarboxylase from Pseudomonas putida (PpBFDC) is a thiamin diphosphate-dependent enzyme that carries out the nonoxidative decarboxylation of aromatic 2-keto acids. The x-ray structure of PpBFDC suggested that Ser-26, His-70, and His-281 would play important roles in its catalytic mechanism, and the S26A, H70A, and H281A variants all exhibited greatly impaired catalytic activity. Based on stopped-flow studies with the alanine mutants, it was proposed that the histidine residues acted as acid-base catalysts, whereas Ser-26 was involved in substrate binding and played a significant, albeit less well defined, role in catalysis. While developing a saturation mutagenesis protocol to examine residues involved in PpBFDC substrate specificity, we tested the procedure on His-281. To our surprise, we found that His-281, which is thought to be necessary for protonation of the carbanion/enamine intermediate, could be replaced by phenylalanine with only a 5-fold decrease in k cat. Even more surprising were our subsequent observations (i) that His-70 could be replaced by threonine or leucine with approximately a 30-fold decrease in kcat/Km compared with a 4,000-fold decrease for the H70A variant and (ii) that Ser-26, which forms hydrogen bonds with the substrate carboxylate, could be replaced by threonine, leucine, or methionine without significant loss of activity. These results call into question the assigned roles for Ser-26, His-70, and His-281. Further, they demonstrate the danger in assigning catalytic function based solely on results with alanine mutants and show that saturation mutagenesis is a valuable tool in assessing the role and relative importance of putative catalytic residues. catalytic mechanism ͉ thiamin diphosphate

Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg 2+ as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 Å (Protein Data Bank entry 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.

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Benzoylformate decarboxylase (BFDC) is a thiamin diphosphate (ThDP)-dependent enzyme acting on aromatic substrates. In addition to its metabolic role in the mandelate pathway, BFDC shows broad substrate specificity coupled with tight stereocontrol in the carbon-carbon bond-forming reverse reaction, making it a useful biocatalyst for the production of chiral α-hydroxyketones. The reaction of methyl benzoylphosphonate (MBP), an analogue of the natural substrate benzoylformate, with BFDC results in the formation of a stable analogue (C2α-phosphonomandelylThDP) of the covalent ThDP-substrate adduct C2α-mandelylThDP. Formation of the stable adduct is confirmed both by formation of a circular dichroism band characteristic of the 1',4'-iminopyrimidine tautomeric form of ThDP (commonly observed when ThDP forms tetrahedral complexes with its substrates), and by high resolution mass spectrometry of the reaction mixture. In addition, the structure of BFDC with the MBP inhibitor was solved by X-ray crystallography, to a spatial resolution of 1.37 Å. The electron density clearly shows formation of a tetrahedral adduct between the C2 atom of ThDP and the carbonyl carbon atom of the MBP. This adduct resembles the intermediate from the penultimate step of the carboligation reaction between benzaldehyde and acetaldehyde. The combination of realtime kinetic information via stopped-flow circular dichroism with steady-state data from equilibrium circular dichroism measurements and X-ray crystallography reveals details of the first step of the reaction catalyzed by BFDC. The MBP-ThDP adduct on BFDC is compared to the recently solved structure of the same adduct on benzaldehyde lyase, another ThDP-dependent enzyme capable of catalyzing aldehyde condensation with high stereospecificity.Characterizing the discrete steps of a chemical reaction remains an exceptionally difficult task. One strategy for enzyme-catalyzed reactions has been to obtain the structure of the protein catalyst in the presence of a reaction intermediate. In most cases, though, structural determination is relatively slow and the lifetime of relevant intermediates fleeting. The use of molecules that resemble the intermediate but that are longer lasting is an established method for obtaining detailed information on the molecular species formed during an enzymecatalyzed reaction pathway(1-6).*To whom correspondence should be addressed: e-mail: ringe@brandeis.edu; tel: 781-736-4902; fax: 781-736-2405; frjordan@newark.rutgers.edu; tel: 973-353-5470; fax: 973-353-1264 Benzoylformate decarboxylase (BFDC, EC 4.1.1.7) catalyzes the decarboxylation of benzoylformate to benzaldehyde (Scheme 1a). A thiamin diphosphate (ThDP)-dependent enzyme, BFDC plays a fundamental role in mandelate metabolism (7,8). One of several ThDPdependent enzymes that catalyze the decarboxylation of 2-oxo acids, it has received considerable interest in studies aimed at understanding the substrate specificity in enzymes of this class(9-12), because of the enzyme's ability to catalyze stereospec...