Tasuke Ando

Inducible l-Alanine Exporter Encoded by the Novel Gene ygaW (alaE) in Escherichia coli

ABSTRACT 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. Overexpression of each gene in the mutant resulted in a decrease in the intracellular l-alanine level and enhancement of the l-alanine export rate in the presence of the dipeptide, suggesting that their products function as exporters of l-alanine. Since ygaW exhibited the most striking impact on both the intra- and the extracellular l-alanine levels among the four genes identified, we disrupted the ygaW gene in the non-l-alanine-metabolizing strain. The resulting isogenic mutant showed the same intra- and extracellular l-alanine levels as observed in the dipeptide-hypersensitive mutant obtained by chemical mutagenesis. When each gene was overexpressed in the wild-type strain, which does not intrinsically excrete alanine, only the ygaW gene conferred on the cells the ability to excrete alanine. In addition, expression of the ygaW gene was induced in the presence of the dipeptide. On the basis of these results, we concluded that YgaW is likely to be the physiologically most relevant exporter for l-alanine in E. coli and proposed that the gene be redesignated alaE for alanine export.

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. To obtain access to the system, we chemically mutagenized the L-alanine-nonmetabolizing strain and isolated mutants with increased Ala-Ala sensitivity. Two such mutants accumulated L-alanine up to 150-190 mM in the cytoplasm with a reduced rate of L-alanine export relative to the parent strain in the presence of Ala-Ala. Furthermore, when chloramphenicol was added together with Ala-Ala, the parent strain accumulated L-alanine in the cytoplasm to a level similar to that observed in the mutants in the absence of chloramphenicol. In contrast, the intracellular l-alanine level in the mutants did not change irrespective of chloramphenicol treatment. From these results, we conclude that E. coli has an inducible l-alanine export carrier, together with a second, as yet unidentified, mechanism of alanine export.

Antimicrobial activity of a bovine myeloid antimicrobial peptide (BMAP-28) against methicillin-susceptible and methicillin-resistant Staphylococcus aureus.

A bovine myeloid antimicrobial peptide (BMAP-28) is a member of the cathelicidin family which is included in the innate immune system of mammals. Recently, there have been many studies about antimicrobial peptides. This study aims to clarify whether BMAP-28 has bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA) and compares its activity against methicillin-susceptible S. aureus (MSSA) and MRSA. We found that the peptide was effective in killing MRSA (minimal inhibitory concentration (MIC) range; 5-20 µg/mL). It was also revealed that MSSA (MIC range; 1.25-20 µg/mL) had two levels of susceptibility to BMAP-28. We also examined the effect of BMAP-28 on bacterial shape to visually show its activity. After exposure to the peptide, both MSSA and MRSA cells showed the morphological changes on their surfaces. Our results indicate that BMAP-28 is a promising candidate for medicine against drug-resistant bacteria.

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. When the yfdZ- or serC-mutation was introduced into the double mutant, one triple mutant (avtA yfbQ yfdZ) showed alanine auxotrophy, and another (avtA yfbQ serC), prototrophy. In addition, we found that four independent alanine auxotrophs possessed a point mutation in yfdZ but not in serC. We also found that yfdZ expression was induced in minimal medium. Furthermore, yfbQ-bearing plasmid conferred the ability to excrete alanine on the mutant lacking D-amino acid dehydrogenase-encoding gene, dadA. From these results, we concluded that E. coli synthesizes L-alanine by means of three aminotransferases, YfbQ, YfdZ, and AvtA.

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 [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.

A bovine myeloid antimicrobial peptide (BMAP-28) kills methicillin-resistant Staphylococcus aureus but promotes adherence of the bacteria.

The cathelicidin family is one of the several families of antimicrobial peptides (AMPs). A bovine myeloid antimicrobial peptide (BMAP-28) belongs to this family. Recently, the emergence of drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) has become a big problem. AMPs are expected to be leading compounds of new antibiotics against drug-resistant bacteria. In this study, we focused on the activity of BMAP-28 against bacterial cell surfaces. First, we observed morphological change of MRSA caused by BMAP-28 using a scanning probe microscope. We also studied activities of BMAP-28 against adherence of S. aureus to fibronectin, collagen type I, collagen type IV. We confirmed whether BMAP-28 can bind to lipoteichoic acid (LTA) of S. aureus. BMAP-28 was indicated as damaging the cell surface of MRSA. In a particular range of concentrations, BMAP-28 promoted adherence of S. aureus against fibronectin and collagens. It was revealed that BMAP-28 and LTA of S. aureus bound with each other. Our study showed the potential of BMAP-28 which can damage MRSA and interact with LTA of S. aureus but promote its adherence in some concentrations. This study provides new points of which to take notice when we use AMPs as medicines.

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.

ABSTRACT 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. This result suggested that MexA secreted via the Tat machinery was properly assembled and functioned as a subunit of the MexAB-OprM efflux pump. A mutation was introduced into the chromosomal tatC gene encoding an inner membrane component of the Tat protein secretion machinery in mexA-deficient cells, and they were transformed with the plasmid carrying the plcH-mexA fusion gene. The transformants showed antibiotic susceptibility comparable to that of mexA-deficient cells, indicating that the hybrid protein was not transported to the periplasm. Whole-cell lysate of the mexA-tatC double mutant harboring the plcH-mexA plasmid produced mainly unprocessed PlcH-MexA. The periplasmic fraction showed no detectable anti-MexA antibody-reactive material. On the basis of these results, we concluded that MexA could be translocated across the inner membrane through the Tat pathway and assembled with its cognate partners, MexB and OprM, and that this complex machinery was fully functional. This hybrid protein translocation system has the potential to be a powerful screening tool for antimicrobial agents targeting the Tat system, which is not present in mammalian cells.

Secretion of d-alanine by Escherichia coli.

Escherichia coli has an l-alanine export system that protects the cells from toxic accumulation of intracellular l-alanine in the presence of l-alanyl-l-alanine (l-Ala-l-Ala). When a DadA-deficient strain was incubated with 6.0 mM l-Ala-l-Ala, we detected l-alanine and d-alanine using high-performance liquid chromatography (HPLC) analysis at a level of 7.0 mM and 3.0 mM, respectively, after 48 h incubation. Treatment of the culture supernatant with d-amino acid oxidase resulted in the disappearance of a signal corresponding to d-alanine. Additionally, the culture supernatant enabled a d-alanine auxotroph to grow without d-alanine supplementation, confirming that the signal detected by HPLC was authentic d-alanine. Upon introduction of an expression vector harbouring the alanine racemase genes, alr or dadX, the extracellular level of d-alanine increased to 11.5 mM and 8.5 mM, respectively, under similar conditions, suggesting that increased metabolic flow from l-alanine to d-alanine enhanced d-alanine secretion. When high-density DadA-deficient cells preloaded with l-Ala-l-Ala were treated with 20 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), secretion of both l-alanine and d-alanine was enhanced ~twofold compared with that in cells without CCCP treatment. In contrast, the ATPase inhibitor dicyclohexylcarbodiimide did not exert such an effect on the l-alanine and d-alanine secretion. Furthermore, inverted membrane vesicles prepared from DadA-deficient cells lacking the l-alanine exporter AlaE accumulated [3H]D-alanine in an energy-dependent manner. This energy-dependent accumulation of [3H]D-alanine was strongly inhibited by CCCP. These results indicate that E. coli has a transport system(s) that exports d-alanine and that this function is most likely modulated by proton electrochemical potential.

Antimicrobial activity of tea catechin against canine oral bacteria and the functional mechanisms

Epigallocatechin gallate (EGCG) is the major polyphenolic compound of green tea. Polyphenolic compounds were extracted from the leaf of Camellia sinensis (Japanese green tea), and the minimum inhibitory concentration against canine oral bacteria was measured. Subsequently, we investigated the inhibitory effects of polyphenolic compounds and EGCG on the growth of canine oral bacteria. EGCG showed antimicrobial activity against a model bacterium, Streptococcus mutans. Our results indicate that EGCG can inhibit the growth and biofilm formation of S. mutans and that EGCG does not interact with streptococcal lipoteichoic acid (LTA). Furthermore, our findings suggest that EGCG interacts with other component(s) of the bacterial membrane aside from streptococcal LTA to inhibit biofilm formation and damage biofilms.

Susceptibility difference between methicillin-susceptible and methicillin-resistant Staphylococcus aureus to a bovine myeloid antimicrobial peptide (BMAP-28)

A bovine myeloid antimicrobial peptide antimicrobial peptide (BMAP-28) is a member of the cathelicidin family and acts as a component of innate immunity. There are few reports of susceptibility difference of methicillin-resistant Staphylococcus aureus (MRSA) and susceptible strains (MSSA) against BMAP-28. This study aims to clarify how a few amino acid substitutions of BMAP-28 are related to its antimicrobial activity using four analog peptides of BMAP-28. We also compared cellular fatty acid components of MSSA and MRSA using gas chromatography. We found that a few amino acid substitutions of BMAP-28 do not change antimicrobial activity. It was also revealed that the percentage of cis-11-eicosenoic acid in total detected fatty acids of MRSA was significantly higher than that of MSSA. In addition, the percentage of palmitic acid in total detected fatty acids of MRSA tended to be lower than that of MSSA. Our results will provide new information to deal with the question of differences in bacterial susceptibility against BMAP-28.