Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad

Abstract

Proteus, Providencia, and Morganella species produce deaminases that generate ␣-keto acids from amino acids. The ␣-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5␣ for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.Among the most common human infections are those of the urinary tract. After Escherichia coli, Proteus mirabilis is a frequent etiological agent particularly in catheterized patients or individuals with structural abnormalities of the urinary tract (46). P. mirabilis appears to localize in the kidney in higher numbers than other uropathogens (13, 43), and there it can cause serious complications including acute pyelonephritis, bladder and kidney stones, and bacteremia (36, 40). Testing of isogenic mutants in a CBA mouse model of ascending urinary tract infection has demonstrated that urease (18, 19), mannose-resistant/Proteus-like fimbriae (3-5), and P. mirabilis fimbriae (25) contribute significantly to the ability of this organism to establish infection. Other putative virulence determinants include hemolysin (28,32,33,44), flagella associated with swarming motility (6, 31), a uroepithelial cell adhesin (47), ambient temperature fimbriae (24), and an immunoglobulin A-degrading protease (38, 39).The role of iron in infections has been well established (2,7,8,48), and most bacterial species produce phenolate-and/or hydroxamate-type siderophores to compete with the host for iron (7,8,30). Most genera of the family Enterobacteriaceae produce either enterobactin (a phenolate siderophore) or aerobactin (a mixed phenolate-hydroxamate siderophore) (7, 30). These traditional-type siderophores are biosynthesized by the action of a number of enzymes whose genes exist in an operon (30). Despite evidence that the urinary tract is an iron-limited niche (41) and that iron limitation reduces susceptibility of animals to developme...

Cloning and Functional Expression of a Mammalian Gene for a Peroxisomal Sarcosine Oxidase

“…Lower identities (14.9, 14.6, and 12.3%) were found with the ␤-subunit of the heterotetrameric sarcosine oxidase from Corynebacterium sp. P-1 (49), with the N terminus of dimethylglycine dehydrogenase from rat liver (50), and with the amino acid deaminase from Proteus mirabilis (51). High identities occurred with a not yet identified gene product (accession number U23529) from Caenorhabditis elegans.…”

Iron-regulated excretion of alpha-keto acids by Salmonella typhimurium

“…They are also produced by transaminases and deaminases and in other metabolic processes. In species such as P. mirabilis (21), degradation of amino acids is due to specific amino acid deaminases; indeed, the production of ␤-phenylpyruvic acid by the action of phenylalanine deaminase, detectable as a colored ferric complex, is a key diagnostic tool for the Proteus-Providencia-Morganella group (15). Salmonella, however, is phenylalanine deaminase negative, producing only trace amounts of ␤-phenylpyruvic acid and other amino acid degradation products primarily by aminotransferase-catalyzed removal of amino groups to KGA.…”

Proteus sp. – an opportunistic bacterial pathogen – classification, swarming growth, clinical significance and virulence factors

“…It was shown that amino acid deaminase (Aad) produces α-keto acids which serves as siderophores involved in iron acquisition (DRECHSEL et al 1993, MASSAD et al 1995. Recently, HIMPSI et al (2010) described proteobactin and yersiniabactin related siderophore function in P. mirabilis.…”