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Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli.

ABSTRACT: L-cysteine is an important amino acid in terms of its industrial applications. We previously found a marked production of L-cysteine from glucose in recombinant Escherichia coli cells expressing an altered cysE gene encoding feedback inhibition-insensitive serine acetyltransferase. Also, a lower level of cysteine desulfhydrase (CD) activity, which is involved in L-cysteine degradation, increased L-cysteine productivity in E. coli. The use of an L-cysteine efflux system could be promising for breeding L-cysteine overproducers. In addition to YdeD and YfiK, which have been reported previously as L-cysteine exporter proteins in E. coli, we analyzed the effects of 33 putative drug transporter genes in E. coli on L-cysteine export and overproduction. Overexpression of the acrD, acrEF, bcr, cusA, emrAB, emrKY, ybjYZ, and yojIH genes reversed the growth inhibition of tnaA (the major CD gene)-disrupted E. coli cells by L-cysteine. We also found that overexpression of these eight genes reduces intracellular L-cysteine levels after cultivation in the presence of L-cysteine. Amino acid transport assays showed that Bcr overexpression conferring bicyclomycin and tetracycline resistance specifically promotes L-cysteine export driven by energy derived from the proton gradient. When a tnaA-disrupted E. coli strain expressing the altered cysE gene was transformed with a plasmid carrying the bcr gene, the transformant exhibited more L-cysteine production than cells carrying the vector only. A reporter gene assay suggested that the bcr gene is constitutively expressed at a substantial level. These results indicate that the multidrug transporter Bcr in the major facilitator family is involved in L-cysteine export and overproduction in genetically engineered E. coli cells.

SUBMITTER: Yamada S

PROVIDER: S-EPMC1489377 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli.

Yamada Satoshi S Awano Naoki N Inubushi Kyoko K Maeda Eri E Nakamori Shigeru S Nishino Kunihiko K Yamaguchi Akihito A Takagi Hiroshi H

Applied and environmental microbiology 20060701 7

L-cysteine is an important amino acid in terms of its industrial applications. We previously found a marked production of L-cysteine from glucose in recombinant Escherichia coli cells expressing an altered cysE gene encoding feedback inhibition-insensitive serine acetyltransferase. Also, a lower level of cysteine desulfhydrase (CD) activity, which is involved in L-cysteine degradation, increased L-cysteine productivity in E. coli. The use of an L-cysteine efflux system could be promising for bre ...[more]

PMID: 16820466

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Project description:Genes encoding the photoreactive protein proteorhodopsin (PR) have been found in a wide range of marine bacterial species, reflecting the significant contribution that PR makes to energy flux and carbon cycling in ocean ecosystems. PR can also confer advantages to enhance the ability of marine bacteria to survive periods of starvation. Here, we investigate the effect of heterologously produced PR on the viability of Escherichia coli Quantitative mass spectrometry shows that E. coli, exogenously supplied with the retinal cofactor, assembles as many as 187,000 holo-PR molecules per cell, accounting for approximately 47% of the membrane area; even cells with no retinal synthesize ?148,000 apo-PR molecules per cell. We show that populations of E. coli cells containing PR exhibit significantly extended viability over many weeks, and we use single-cell Raman spectroscopy (SCRS) to detect holo-PR in 9-month-old cells. SCRS shows that such cells, even incubated in the dark and therefore with inactive PR, maintain cellular levels of DNA and RNA and avoid deterioration of the cytoplasmic membrane, a likely basis for extended viability. The substantial proportion of the E. coli membrane required to accommodate high levels of PR likely fosters extensive intermolecular contacts, suggested to physically stabilize the cell membrane and impart a long-term benefit manifested as extended viability in the dark. We propose that marine bacteria could benefit similarly from a high PR content, with a stabilized cell membrane extending survival when those bacteria experience periods of severe nutrient or light limitation in the oceans.IMPORTANCE Proteorhodopsin (PR) is part of a diverse, abundant, and widespread superfamily of photoreactive proteins, the microbial rhodopsins. PR, a light-driven proton pump, enhances the ability of the marine bacterium Vibrio strain AND4 to survive and recover from periods of starvation, and heterologously produced PR extends the viability of nutrient-limited Shewanella oneidensis We show that heterologously produced PR enhances the viability of E. coli cultures over long periods of several weeks and use single-cell Raman spectroscopy (SCRS) to detect PR in 9-month-old cells. We identify a densely packed and consequently stabilized cell membrane as the likely basis for extended viability. Similar considerations are suggested to apply to marine bacteria, for which high PR levels represent a significant investment in scarce metabolic resources. PR-stabilized cell membranes in marine bacteria are proposed to keep a population viable during extended periods of light or nutrient limitation, until conditions improve.

Project description:BACKGROUND:Avian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T). RESULTS:Following whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (?speB, ?speC, ?speF, ?speB/C and ?speD/E), and in polyamines transport systems (?potE, ?yeeF, ?potABCD and ?potFGHI). Contrary to what was observed for MG1655, the ?potE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ?speB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ?speB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed. CONCLUSIONS:Deletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.

Dataset Information

Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli.

Publications

Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli.

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