We previously reported that the alaE gene of Escherichia coli encodes the l-alanine exporter AlaE. The objective of this study was to elucidate the mechanism of the AlaE exporter. The minimum inhibitory concentration of l-alanine and l-alanyl-l-alanine in alaE-deficient l-alanine-nonmetabolizing cells MLA301ΔalaE was 4- and >4000-fold lower, respectively, than in the alaE-positive parent cells MLA301, suggesting that AlaE functions as an efflux pump to avoid a toxic-level accumulation of intracellular l-alanine and its derivatives. Furthermore, the growth of the alaE-deficient mutant derived from the l-alanine-metabolizing strain was strongly inhibited in the presence of a physiological level of l-alanyl-l-alanine. Intact MLA301ΔalaE and MLA301ΔalaE/pAlaE cells producing plasmid-borne AlaE, accumulated approximately 200% and 50%, respectively, of the [3H]l-alanine detected in MLA301 cells, suggesting that AlaE exports l-alanine. When 200 mmol/L l-alanine-loaded inverted membrane vesicles prepared from MLA301ΔalaE/pAlaE were placed in a solution containing 200 mmol/L or 0.34 μmol/L l-alanine, energy-dependent [3H]l-alanine accumulation occurred under either condition. This energy-dependent uphill accumulation of [3H]l-alanine was strongly inhibited in the presence of carbonyl cyanide m-chlorophenylhydrazone but not by dicyclohexylcarbodiimide, suggesting that the AlaE-mediated l-alanine extrusion was driven by proton motive force. Based on these results, physiological roles of the l-alanine exporter are discussed.
The Escherichia coli alaE gene encodes the L-alanine exporter, AlaE, that catalyzes active export of L-alanine using proton electrochemical potential. The transporter comprises only 149 amino acid residues and four predicted transmembrane domains (TMs), which contain three charged amino acid residues. The AlaE-deficient L-alanine non-metabolizing cells (ΔalaE cells) appeared hypersusceptible to L-alanyl-L-alanine showing a minimum inhibitory concentration (MIC) of 2.5 µg/ml for the dipeptide due to a toxic accumulation of L-alanine. To elucidate the mechanism by which AlaE exports L-alanine, we replaced charged amino acid residues in the TMs, glutamic acid-30 (TM-I), arginine-45 (TM-II), and aspartic acid-84 (TM-III) with their respective charge-conserved amino acid or a net neutral cysteine. The ΔalaE cells producing R45K or R45C appeared hypersusceptible to the dipeptide, indicating that arginine-45 is essential for AlaE activity. MIC of the dipeptide in the ΔalaE cells expressing E30D and E30C was 156 µg/ml and >10,000 µg/ml, respectively, thereby suggesting that a negative charge at this position is not essential. The ΔalaE cells expressing D84E or D84C showed an MIC >10,000 and 78 µg/ml, respectively, implying that a negative charge is required at this position. These results were generally consistent with that of the L-alanine accumulation experiments in intact cells. We therefore concluded that charged amino acid residues (R45 and D84) in the AlaE transmembrane domain play a pivotal role in L-alanine export. Replacement of three cysteine residues at C22, C28 (both in TM-I), and C135 (C-terminal region) with alanine showed only a marginal effect on L-alanine export.
Antibiotic-resistant bacteria remain a serious public health threat. In order to determine the percentage of antibiotic-resistant and -tolerant Pseudomonas aeruginosa cells present and to provide a more detailed infection risk of bacteria present in the environment, an isolation method using a combination of 41 °C culture and specific primers was established to evaluate P. aeruginosa in the environment. The 50 strains were randomly selected among 110 isolated from the river. The results of antibiotic susceptibility evaluation showed that only 4% of environmental strains were classified as antibiotic-resistant, while 35.7% of clinical strains isolated in the same area were antibiotic-resistant, indicating a clear difference between environmental and clinical strains. However, the percentage of antibiotic-tolerance, an indicator of potential resistance risk for strains that have not become resistant, was 78.8% for clinical strains and 90% for environmental strains, suggesting that P. aeruginosa, a known cause of nosocomial infections, has a high rate of antibiotic-tolerance even in environmentally derived strains. It suggested that the rate of antibiotic-tolerance is not elicited by the presence or absence of antimicrobial exposure. The combination of established isolation and risk analysis methods presented in this study should provide accurate and efficient information on the risk level of P. aeruginosa in various regions and samples.
The purpose of this paper is to critically examine the representation of people with autistic spectrum disability in video media from the perspective of cultural studies, and to present the desirable tasks of disability and inclusion in disability studies through a comparative study on the representation of people with autistic spectrum disability in the Korean movie Marathon and the Japanese drama Marathon based on the same original work. Therefore, the narrative of growth through communication and perception of disability and inclusion were compared between them. The one is progressive in that it emphasizes self-reliance and nothing about us without us, seeks communication and change in society, and highlights an active female image. But It shows the task of inclusion through the reality of the prejudice and conflicts of society towards people with the autistic disabled and education or vocational training that is separated from people without disabilities. The other reproduces a social foundation that is more advanced than Korea in terms of harmony with people with autistic spectrum disability and integrated workplaces, but emphasizes family love and self-denial, revealing the limitations of the individual model that disabilities must be overcome through individual efforts.
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