YddG from<i>Escherichia coli</i>promotes export of aromatic amino acids

Doroshenko, V. G.; Airich, L. G.; Vitushkina, Maria V.; Kolokolova, A. V.; Livshits, Vitaliy A.; Mashko, Sergey V.

doi:10.1111/j.1574-6968.2007.00894.x

Cited by 90 publications

(63 citation statements)

References 17 publications

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“…2), (ii) disruption of the ygaW gene in the non-L-alanine-metabolizing mutant MLA301 resulted in intracellular accumulation and a reduced export rate of L-alanine in the presence of extracellular Ala-Ala (Fig. 3), (iii) yddG (9) and yeaS (26) have been characterized to encode exporters for L-aromatic amino acids and L-leucine, respectively, but not for L-alanine, (iv) disruption of the ytfF gene in MLA301 did not lead to a change in the The non-L-alanine-metabolizing strain MLA301 was incubated in minimal medium with (lanes 3, 5, 7, 9, and 11) or without (lanes 2, 4, 6, 8, and 10) 6 mM Ala-Ala. After a 5-min incubation, total RNA isolation and RT-PCR were performed as described in Materials and Methods. Lane 1, molecular size marker in base pairs; lanes 2 and 3, ygaW; lanes 4 and 5, ytfF; lanes 6 and 7, yddG; lanes 8 and 9, yeaS; lanes 10 and 11, gapA.…”

Section: Discussionmentioning

confidence: 99%

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

Hori

Yoneyama

Tobe

et al. 2011

Appl Environ Microbiol

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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.Bacteria are known to export xenobiotic substances, such as heavy metals (34), antibiotics (35), or organic solvents (49), to survive under harsh circumstances. In the last 15 years, it has been shown that in addition to harmful substances, normal metabolites, such as amino acids (10), purine ribonucleosides (13), and sugars (28), are exported by specific exporters. However, a physiological function of the exporters remains obscure. In regard to amino acids, after the identification of LysE as the exporter for lysine in Corynebacterium glutamicum (47), more than 10 transporters have been shown to export amino acids and their analogues. In C. glutamicum, BrnFE (20), NCgl1221 (32), and ThrE (43) were found to mediate the efflux of L-isoleucine, L-glutamic acid, and L-threonine, respectively. In Escherichia coli, exporters for L-cysteine (YdeD, YfiK, CydDC, and Bcr) (7, 12, 37, 50), L-aromatic amino acids (YddG) (9), L-leucine (YeaS) (26), L-threonine (RhtA and RhtC) (29, 51), Larginine (YggA) (33), L-valine (YgaZH) (36), and L-homoserine (RhtB) (51) were identified.The specific exporter of alanine has not been identified so far in E. coli and other bacteria except for a peculiar case in Tetragenococcus halophilus, where AspT has been found to function as an L-asparate:L-alanine exchanger (1). On the one hand, a wide range of wild-type and metabolically engineered bacterial strains (...

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Section: Discussionmentioning

confidence: 99%

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

Hori

Yoneyama

Tobe

et al. 2011

Appl Environ Microbiol

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“…Protein structure prediction using the protein homology/analogy recognition engine (PHYRE) search tool suggests that STM1530 is a porin similar to Klebsiella pneumoniae OmpK36, E. coli PhoE, and E. coli OmpF. It has been reported that in S. enterica serovar Typhimurium, the porin OmpD together with a putative inner membrane efflux pump, YddG, is involved in resistance to methyl viologen and aromatic amino acids (8,32). In addition, it has been suggested that OmpD in S. enterica serovar Typhimurium interacts with the periplasmic protein YdeI/ OmdA to increase resistance to antimicrobial peptides (29).…”

Section: Discussionmentioning

confidence: 99%

The Expression Levels of Outer Membrane Proteins STM1530 and OmpD, Which Are Influenced by the CpxAR and BaeSR Two-Component Systems, Play Important Roles in the Ceftriaxone Resistance of Salmonella enterica Serovar Typhimurium

Hu¹,

Chen²,

Zhang³

et al. 2011

Antimicrob Agents Chemother

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Significant increases in STM3031, STM1530, and AcrD protein levels and significant decreases in OmpC and OmpD protein levels are present when the ceftriaxone-resistant Salmonella enterica serovar Typhimurium R200 strain is compared with the ceftriaxone-susceptible strain 01-4. AcrD is known to be involved in drug export, and STM3031 seems to play a key role in ceftriaxone resistance. Here, we examine the roles of STM1530, OmpC, and OmpD in ceftriaxone resistance. An ompD gene deletion mutant showed 4-fold higher ceftriaxone resistance than 01-4. An ompC gene deletion mutant showed 4-fold higher cephalothin and erythromycin resistance than 01-4, but there was no effect on ceftriaxone resistance. However, a stm1530 deletion mutant did show >64-fold lower ceftriaxone resistance than R200. Moreover, the STM3031 protein was significantly decreased in R200(⌬stm1530) compared to R200. STM3031 expression has been shown to be influenced by the two-component system regulator gene baeR. CpxR seems to modulate BaeR. A cpxA-cpxR gene deletion mutant showed >2,048-fold lower ceftriaxone resistance than R200. The outer membrane protein profile of R200(⌬cpxAR) showed significant decreases in STM3031 and STM1530 compared to R200, while OmpD had returned to the level found in 01-4. Furthermore, the stm3031, stm1530, and ompD mRNA levels were correlated with their protein expression levels in these strains, while decreases in the mRNA levels of the efflux pump acrB, acrD, and acrF genes were found in R200(⌬cpxAR). Findings similar to those for R200(⌬cpxAR) were found for R200(⌬baeSR). These results, together with those for STM3031 and the fact that STM1530 is an outer membrane protein, suggest that STM1530 and OmpD are influenced by the CpxAR and BaeSR two-component systems and that this contributes to S. enterica serovar Typhimurium ceftriaxone resistance.

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“…The plasmids containing the P nlp -driven cysM gene and chloramphenicol resistance marker flanked by the attachment sites of the Mu phage, designed for inducing mini Mu-mediated integration, were introduced by electroporation into strain SC17, which harbored the helper plasmid pMH10 (51) that expressed Mu transposase. Mumediated chromosomal integration was induced according to previously described methods (34,52). Strains with the chromosomal P nlp -cysM cassettes were selected on an agar plate containing chloramphenicol.…”

Section: Plasmid Name Vectormentioning

confidence: 99%

Fermentative Production of Cysteine by Pantoea ananatis

Takumi

Ziyatdinov

Samsonov

et al. 2017

Appl Environ Microbiol

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Cysteine is a commercially important amino acid; however, it lacks an efficient fermentative production method. Due to its cytotoxicity, intracellular cysteine levels are stringently controlled via several regulatory modes. Managing its toxic effects as well as understanding and deregulating the complexities of regulation are crucial for establishing the fermentative production of cysteine. The regulatory modes include feedback inhibition of key metabolic enzymes, degradation, efflux pumps, and the transcriptional regulation of biosynthetic genes by a master cysteine regulator, CysB. These processes have been extensively studied using Escherichia coli for overproducing cysteine by fermentation. In this study, we genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to identify key factors required for cysteine production. According to this and our previous studies, we identified a major cysteine desulfhydrase gene, ccdA (formerly PAJ_0331), involved in cysteine degradation, and the cysteine efflux pump genes cefA and cefB (formerly PAJ_3026 and PAJ_p0018, respectively), which may be responsible for downregulating the intracellular cysteine level. Our findings revealed that ccdA deletion and cefA and cefB overexpression are crucial factors for establishing fermentative cysteine production in P. ananatis and for obtaining a higher cysteine yield when combined with genes in the cysteine biosynthetic pathway. To our knowledge, this is the first demonstration of cysteine production in P. ananatis, which has fundamental implications for establishing overproduction in this microbe.IMPORTANCE The efficient production of cysteine is a major challenge in the amino acid fermentation industry. In this study, we identified cysteine efflux pumps and degradation pathways as essential elements and genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to establish the fermentative production of cysteine. This study provides crucial insights into the design and construction of cysteine-producing strains, which may play central roles in realizing commercial basis production.

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YddG fromEscherichia colipromotes export of aromatic amino acids

Cited by 90 publications

References 17 publications

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

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

The Expression Levels of Outer Membrane Proteins STM1530 and OmpD, Which Are Influenced by the CpxAR and BaeSR Two-Component Systems, Play Important Roles in the Ceftriaxone Resistance of Salmonella enterica Serovar Typhimurium

Fermentative Production of Cysteine by Pantoea ananatis

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