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d-Cycloserine (DCS) is a broad-spectrum antibiotic that inhibits d-alanine ligase and alanine racemase activity. When Escherichia coli K-12 or CFT073 is grown in minimal glucose or glycerol medium, CycA transports DCS into the cell. E. coli K-12 cycA and CFT073 cycA mutant strains display increased DCS resistance when grown in minimal medium. However, the cycA mutants exhibit no change in DCS sensitivity compared to their parental strains when grown in LB (CFT073 and K-12) or human urine (CFT073 only). These data suggest that cycA does not participate in DCS sensitivity when strains are grown in a non-minimal medium. The small RNA GvcB acts as a negative regulator of E. coli K-12 cycA expression when grown in LB. Three E. coli K-12 gcvB mutant strains failed to demonstrate a change in DCS sensitivity when grown in LB. This further suggests a limited role for cycA in DCS sensitivity. To aid in the identification of E. coli genes involved in DCS sensitivity when grown on complex media, the Keio K-12 mutant collection was screened for DCS-resistant strains. dadA, pnp, ubiE, ubiF, ubiG, ubiH, and ubiX mutant strains showed elevated DCS resistance. The phenotypes associated with these mutants were used to further define three previously characterized E. coli DCS-resistant strains (χ316, χ444, and χ453) isolated by Curtiss and colleagues (R. Curtiss, III, L. J. Charamella, C. M. Berg, and P. E. Harris, J. Bacteriol. 90:1238-1250, 1965). A dadA mutation was identified in both χ444 and χ453. In addition, results are presented that indicate for the first time that DCS can antagonize d-amino acid dehydrogenase (DadA) activity.

d-Serine is an amino acid present in mammalian urine that is inhibitory to Escherichia coli strains lacking a functional dsdA gene. Counterintuitively, a dsdA strain of E. coli clinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake of d-serine in CFT073. d-Serine enters E. coli K-12 via CycA, the d-alanine transporter and d-cycloserine sensitivity locus. CFT073 cycA can grow on minimal medium with d-serine as a sole carbon source. The dsdX gene of the dsdCXA locus is a likely candidate for an additional d-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073 dsdX can grow on d-serine as a sole carbon source; however, CFT073 dsdX cycA cannot. Additionally, CFT073 dsdXA cycA is not sensitive to inhibitory concentrations of d-serine during growth on glycerol and d-serine minimal medium. d-[(14)C]serine uptake experiments with CFT073 dsdX cycA harboring dsdX or cycA recombinant plasmids confirm that d-serine is able to enter E. coli cells via CycA or DsdX. In whole-cell d-[(14)C]serine uptake experiments, DsdX has an apparent K(m) of 58.75 microM and a V(max) of 75.96 nmol/min/mg, and CycA has an apparent K(m) of 82.40 microM and a V(max) of 58.90 nmol/min/mg. Only d-threonine marginally inhibits DsdX-mediated d-serine transport, whereas d-alanine, glycine, and d-cycloserine inhibit CycA-mediated d-serine transport. DsdX or CycA is sufficient to transport physiological quantities of d-serine, but DsdX is a d-serine-specific permease.

Whole cells of Bacillus subtilis transported d-alanine and l-alanine by two different systems. The high-affinity system (K(m) of 1 muM and V(max) of 0.6 to 0.8 nmol/min per mg of protein) was specific for the two stereoisomers of alanine. The low-affinity system (K(m) of 10 muM for l-alanine and 20 muM for d-alanine and glycine) had a V(max) of 5 to 12 nmol/min per mg of protein. This system transported glycine, d-cycloserine, and d-serine, in addition to d- and l-alanine. Azide inhibited the uptake of these amino acids and caused the efflux of d-alanine from preloaded cells. These data suggest that transport of these amino acids is energized by the electron transport chain.

The accumulation of d-alanine, l-alanine, glycine, and d-cycloserine in Escherichia coli was found to be mediated by at least two transport systems. The systems for d-alanine and glycine are related, and are separate from that involved in the accumulation of l-alanine. d-Cycloserine appears to be primarily transported by the d-alanine-glycine system. The accumulation of d-alanine, glycine, and d-cycloserine was characterized by two line segments in the Lineweaver-Burk analysis, whereas the accumulation of l-alanine was characterized by a single line segment. d-Cycloserine was an effective inhibitor of glycine and d-alanine accumulation, and l-cycloserine was an effective inhibitor of l-alanine transport. The systems were further differentiated by effects of azide, enhancement under various growth conditions, and additional inhibitor studies. Since the primary access of d-cycloserine in E. coli is via the d-alanine-glycine system, glycine might be expected to be a better antagonist of d-cycloserine inhibition than l-alanine. Glycine and d-alanine at 10(-5)m antagonized the effect of d-cycloserine in E. coli, whereas this concentration of l-alanine had no effect.

The accumulation of d-alanine and the accumulation of glycine in Escherichia coli are related and appear to be separate from the transport of l-alanine. The analysis of four d-cycloserine-resistant mutants provides additional support for this conclusion. The first-step mutant from E. coli K-12 that is resistant to d-cycloserine was characterized by the loss of the high-affinity line segment of the d-alanine-glycine transport system in the Lineweaver-Burk plot. This mutation, which is linked to the met(1) locus, also resulted in the loss of the ability to transport d-cycloserine. The second-step mutation that is located 0.5 min from the first-step mutation resulted in the loss of the low-affinity line segment for the d-alanine-glycine transport system. The transport of l-alanine was decreased only 20 to 30% in each of these mutants. A multistep mutant from E. coli W that is 80-fold resistant to d-cycloserine lost >90% of the transport activity for d-alanine and glycine, whereas 75% of the transport activity for l-alanine was retained. E. coli W could utilize either d- or l-alanine as a carbon source, whereas the multistep mutant could only utilize l-alanine. Thus, a functioning transport system for d-alanine and glycine is required for both d-cycloserine action and growth on d-alanine.

The d-serine transport system in Escherichia coli K-12 was studied by use of a mutant unable to form d-serine deaminase, yet resistant to d-serine. The mutant is greatly impaired in its ability to accumulate d-serine, d-alanine, and glycine. Transport of l-alanine is partially affected but transport of l-serine is unaffected. The mutant is also resistant to d-cycloserine, indicating that d-serine is transported by the system responsible for uptake of d-cycloserine. The d-serine transport system is not inducible, but appears to be formed constitutively, as are the transport systems of most amino acids. The transport mutation appears to be multistep and maps to the right of malB on the E. coli linkage map.