Characteristics of Antibiotic Resistance and Tolerance of Environmentally Endemic Pseudomonas aeruginosa.

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.

Importance of transmembrane helix 4 of l-alanine exporter AlaE in oligomer formation and substrate export activity in Escherichia coli.

AlaE is the smallest amino acid exporter identified in Escherichia coli. It exports l-alanine using the proton motive force and plays a pivotal role in maintaining intracellular l-alanine homeostasis by acting as a safety valve. However, our understanding of the molecular mechanisms of substrate export by AlaE is still limited because structural information is lacking. Due to its small size (149 amino acid residues), it has been speculated that AlaE functions by forming an oligomer. In this study, we performed chemical cross-linking and pull-down assays and showed that AlaE indeed generates homo-oligomers as a functional unit. Previous random mutagenesis experiments identified three loss-of-function AlaE point mutations in the predicted transmembrane helix 4 (TM4) region, two of which are present in the GxxxG motif. When alanine-scanning mutagenesis was applied to the TM4 region, the AlaE derivatives that had amino acid substitutions around the GxxxG motif showed low l-alanine export activities, indicating that the GxxxG motif in TM4 plays an important role in substrate export. However, these AlaE variants with low activity could still form oligomers. We therefore concluded that AlaE forms homo-oligomers and that the GxxxG motif in the TM4 region plays an essential role in AlaE activity but is not involved in AlaE oligomer formation.

Impact of charged amino acid substitution in the transmembrane domain of L-alanine exporter, AlaE, of Escherichia coli on the L-alanine export.

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.

Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives.

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 [(3) H]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 [(3) H]l-alanine accumulation occurred under either condition. This energy-dependent uphill accumulation of [(3) H]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.