DsdX Is the Second d -Serine Transporter in Uropathogenic Escherichia coli Clinical Isolate CFT073

Abstract

D-Serine is an amino acid present in mammalian urine that is inhibitory toEscherichia coli is a normal resident of the vertebrate large intestine, and certain pathogenic strains are capable of infecting sites outside of the intestine. The sequencing of multiple E. coli genomes has allowed for a better insight into what genes may permit success in niches outside the intestine. Comparison of an E. coli K-12 isolate (MG1655) to an O157:H7 enterohemorrhagic E. coli isolate (EDL933) and a uropathogenic E. coli (UPEC) isolate (CFT073) showed numerous genetic differences between the strains. Less than 50% of the apparent genes were common to all three strains; these common genes represent what can be thought of as the core chromosome of E. coli. The variable regions specific to each isolate include genes potentially important to disease or growth in different environments (38). One such variable site is a genetic island starting 3Ј to the argW tRNA gene and ending with the dsdCXA locus. The dsdCXA locus is intact in MG1655 and CFT073 but is truncated in EDL933 (28). In fact, the dsdCXA locus is intact in most extraintestinal pathogenic E. coli (ExPEC) strains but is truncated in the vast majority of diarrheagenic E. coli strains (R. L. Moritz and R. A. Welch, submitted for publication). This region of the E. coli chromosome frequently replaces dsdC and dsdX with csrRAKB genes responsible for sucrose catabolism (12,28).The E. coli dsdCXA locus permits growth on D-serine as a sole carbon and nitrogen source. The E. coli K-12 dsdCXA locus was extensively studied by McFall and coworkers (23). The DNA sequence of the locus was originally described by this group, but apparent sequence assembly problems and the inability at the time to create targeted, site-specific mutations prevented proper identification and functional analysis of dsdX. The dsdC gene encodes a Lys-R-type transcriptional regulator that induces transcription of dsdX and dsdA in the presence of D-serine and inhibits its own transcription in the absence of D-serine (23). DsdX has been hypothesized to act as a D-serine transporter (23). The dsdA gene encodes a pyridoxal phosphate-dependent D-serine deaminase (DsdA) that degrades D-serine to ammonia and pyruvate (18). D-Serine catabolism is biologically important because D-serine is available in some environments as a readily utilizable nutrient source, but it can also have also inhibitory effects on growth. D-Serine is bacteriostatic to cells lacking DsdA grown in minimal medium (16). D-Serine toxicity on minimal medium can be reversed with expression of functional DsdA or by the addition of pantothenate or ␤-alanine to the medium; this suggests that the inhibitory effect of D-serine is associated with pantothenate biosynthesis due to the structural similarity between D-serine and ␤-alanine (2, 4, 7, 15). A D-serine deaminase gene can serve as a selective marker on par with antibiotic resistance genes for bacteria (16), yeast (35), or plant transformations (8) due to the toxicity of D-serine. Despite the fact t...

Characterization of Escherichia coli d -Cycloserine Transport and Resistant Mutants

“…We hypothesize instead that the lack of DCS transport through CycA most likely is due to competition from other CycA substrates present in LB and human urine. CycA is capable of transporting D/L/␤-alanine, D-serine, glycine, and DCS (6)(7)(8)(9)(10). Uptake experiments performed on E. coli CFT073 show that glycine and D-alanine are capable of outcompeting D-serine for transport through CycA (6).…”

“…A mutation in the cycA gene in the E. coli K-12 and the uropathogenic E. coli (UPEC) CFT073 strains results in increased DCS resistance when grown in a minimal medium (6,11,12). The resistance to and transport of DCS in a complex medium, like Luria Bertani (LB), or a biologically relevant medium, such as human urine, has not been reported for E. coli.…”

“…Glycine accumulation assay. A modified version of the Anfora and Welch transport assay was used in this glycine uptake study (6). Ten ml of the appropriate broth was inoculated with an overnight culture to a starting optical density at 600 nm (OD 600 ) of 0.03 and incubated at 37°C with shaking.…”

“…The Escherichia coli cycA gene codes for a permease that transports the antibiotic D-cycloserine and the amino acids ␤-/L-/Dalanine, glycine, and D-serine when grown in minimal glucose or glycerol media (6)(7)(8)(9)(10). A mutation in the cycA gene in the E. coli K-12 and the uropathogenic E. coli (UPEC) CFT073 strains results in increased DCS resistance when grown in a minimal medium (6,11,12).…”

Uropathogenic Escherichia coli CFT073 Is Adapted to Acetatogenic Growth but Does Not Require Acetate during Murine Urinary Tract Infection

“…Previous work in our laboratory focused on serine metabolism of extraintestinal pathogenic E. coli, using the urosepsis UPEC isolate, CFT073 (3,19,33,44). We determined that approximately 85% of UPEC pyelonephritis and urosepsis isolates carry an intact D-serine deaminase operon (dsdCXA) (33).…”