Characterization of Escherichia coli d -Cycloserine Transport and Resistant Mutants

Abstract

b 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 (316, 444, D -Cycloserine (DCS) is a broad-spectrum antibiotic produced by Streptomyces garyphalus, Streptomyces orchidaceus, andStreptomyces lavendulae. DCS is a cyclic, structural analog of D-alanine that inhibits alanine racemase and D-alanine ligase activity (1, 2). Inactivation of these enzymes results in a failure to produce mature peptidoglycan and an increased susceptibility to osmotic lysis. DCS is occasionally used as a second-line drug in the treatment of multidrug-resistant Mycobacterium tuberculosis infections (3, 4).DCS is not commonly used in chemotherapy regimens due to its adverse neurological side effects when administered at an effective dose (5). Unfortunately, physicians are increasingly being forced to use drugs like DCS to combat antibiotic-resistant bacterial infections. The development of new drugs is in high demand. The continued characterization of drugs like DCS could play a pivotal role in the development of novel drugs. The identification of additional DCS targets and the characterization of additional DCS resistance mechanisms could contribute to the development of new drugs with novel targets that possess less adverse effects.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 (6-10). 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). 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. As a result, the role cycA ...

Global gene expression in Escherichia coli, isolated from the diseased ocular surface of the human eye with a potential to form biofilm

“…In addition, RfaH a virulence regulator, which regulates expression of several virulent genes in E. coli is significantly up regulated in ABU strains during biofilm phase [43]. None of the above genes was differentially regulated in ocular E. coli but up regulation of gene ECs3276 a virulence protein populating the cluster O80301 [60] was observed (Additional file 3: Table S2). Apart from this, upregulation of several genes encoding for toxin production and secretions ( sat, yjg K, chp S, chp B and ygj N) was observed in ocular E. coli biofilm cells which is an important factor that governs the virulence in pathogenic bacterial cells.…”

A Gestalt approach to Gram-negative entry

“…Some of these natural product compounds are known to use transporters but have characteristics that might seem to allow their CM diffusion. And indeed, it is known that although D-cycloserine (MW=102, CLogD=-1.9) and negamycin (MW=248, CLogD=-5.9) can be taken up by specific protein carriers in certain media, they may also enter by diffusion 71,77 .…”

Physiology and Substrate Specificity of Two Closely Related Amino Acid Transporters, SerP1 and SerP2, of Lactococcus lactis

“…Growth was not restored in the SerP2 deletion mutant, demonstrating that SerP2 was solely responsible for the uptake of D-alanine in the wild-type strain. Both in E. coli and M. tuberculosis it is believed that the CycA transporter is also responsible for the uptake of D-cycloserine into the cell, even though some doubt was raised recently (34,35). The SerP2 deletion mutant was equally sensitive to D-cycloserine, suggesting that at least in this mutant of L. lactis, another transporter is responsible for the uptake of the drug.…”