Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli.

The alaE gene in Escherichia coli encodes an l-alanine exporter that catalyzes the active export of l-alanine using proton electrochemical potential. In our previous study, alaE expression was shown to increase in the presence of l-alanyl-l-alanine (Ala-Ala). In this study, the global regulator leucine-responsive regulatory protein (Lrp) was identified as an activator of the alaE gene.

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.

Isolation of a mutant auxotrophic for L-alanine and identification of three major aminotransferases that synthesize L-alanine in Escherichia coli.

For Escherichia coli, it has been assumed that L-alanine is synthesized by alanine-valine transaminase (AvtA) in conjunction with an unknown alanine aminotransferase(s). We isolated alanine auxotrophs from a prototrophic double mutant deficient in AvtA and YfbQ, a novel alanine aminotransferase, by chemical mutagenesis. A shotgun cloning experiment identified two genes, uncharacterized yfdZ and serC, that complemented the alanine auxotrophy.

Inducible L-alanine exporter encoded by the novel gene ygaW (alaE) in Escherichia coli.

We previously isolated a mutant hypersensitive to L-alanyl-L-alanine from a non-L-alanine-metabolizing Escherichia coli strain and found that it lacked an inducible l-alanine export system. Consequently, this mutant showed a significant accumulation of intracellular L-alanine and a reduction in the L-alanine export rate compared to the parent strain. When the mutant was used as a host to clone a gene(s) that complements the dipeptide-hypersensitive phenotype, two uncharacterized genes, ygaW and ytfF, and two characterized genes, yddG and yeaS, were identified.

Identification of an L-alanine export system in Escherichia coli and isolation and characterization of export-deficient mutants.

An Escherichia coli strain that exhibits a double auxotrophy for L-alanine and D-alanine was constructed. During growth in the presence of the dipeptide L-alanyl-L-alanine (Ala-Ala), this was fully consumed with concomitant extracellular accumulation of l-alanine in a twofold molar concentration compared with the dipeptide. This finding indicates that the strain not only can hardly degrade L-alanine but has an export system(s) for L-alanine.

Tertiary structure-related activity of tick defensin (persulcatusin) in the taiga tick, Ixodes persulcatus.

Defensins are small cysteine-rich cationic proteins found in both vertebrates and invertebrates constituting the front line of host innate immunity. To examine the importance of the tertiary structure of tick defensin in its antimicrobial activity, we synthesized two types of the peptides with tertiary structure or primary one on basis of the information of the sequence in the defensin originated from the taiga tick, Ixodes persulcatus. Chemically synthesized peptides were used to investigate the activity spectrum against Staphylococcus aureus, Borrelia garinii and flora-associated bacteria.

Tat pathway-mediated translocation of the sec pathway substrate protein MexA, an inner membrane component of the MexAB-OprM xenobiotic extrusion pump in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is equipped with the Sec and Tat protein secretion systems, which translocate the xenobiotic transporter MexAB-OprM and the pathogenic factor phospholipase C (PlcH), respectively. When the signal sequence of MexA was replaced with that of PlcH, the hybrid protein was successfully expressed and recovered from the periplasmic fraction, suggesting that the hybrid protein had been translocated across the inner membrane. MexA-deficient cells harboring the plasmid carrying the plcH-mexA fusion gene showed antibiotic resistance comparable to that of the wild-type cells.