Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli

Petra Louis, Erwin A. Galinski

Research output: Contribution to journalArticle

124 Citations (Scopus)

Abstract

The genes of the biosynthetic pathway of ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) from the Gram-positive moderate halophile Marinococcus halophilus were cloned by functional expression in Escherichia coli. These genes were not only expressed, but also osmoregulated in E. coli, as demonstrated by increasing cytoplasmic ectoine concentration in response to medium salinity. Sequencing of a 4 4 kb fragment revealed four major ORFs, which were designated ectA, ectB, ectC and orfA. The significance of three of these genes for ectoine synthesis was proved by sequence comparison with known proteins and by physiological experiments. Several deletion derivatives of the sequenced fragment were introduced into E. coli and the resulting clones were investigated for their ability to synthesize ectoine or one of the intermediates in its biosynthetic pathway. It was demonstrated that ectA codes for L-2,4-diaminobutyric acid acetyltransferase, ectB for L-2,4-diaminobutyric acid transaminase and ectC for L-ectoine synthase. A DNA region upstream of ectA was shown to be necessary for the regulated expression of ectoine synthesis in response to the osmolarity of the medium.

Original languageEnglish
Pages (from-to)1141-1149
Number of pages9
JournalMicrobiology
Volume143
Issue number4
DOIs
Publication statusPublished - Apr 1997

Keywords

  • Marinococcus halophilus
  • compatible solutes
  • ectoine genes
  • osmoregulation
  • salt stress
  • bacillus-subtilis
  • ectothiorhodospira-halochloris
  • transport-system
  • glycine betaine
  • tetrahydropyrimidines
  • trehalose
  • stress
  • operon
  • prou
  • transcription

Cite this

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title = "Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli",
abstract = "The genes of the biosynthetic pathway of ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) from the Gram-positive moderate halophile Marinococcus halophilus were cloned by functional expression in Escherichia coli. These genes were not only expressed, but also osmoregulated in E. coli, as demonstrated by increasing cytoplasmic ectoine concentration in response to medium salinity. Sequencing of a 4 4 kb fragment revealed four major ORFs, which were designated ectA, ectB, ectC and orfA. The significance of three of these genes for ectoine synthesis was proved by sequence comparison with known proteins and by physiological experiments. Several deletion derivatives of the sequenced fragment were introduced into E. coli and the resulting clones were investigated for their ability to synthesize ectoine or one of the intermediates in its biosynthetic pathway. It was demonstrated that ectA codes for L-2,4-diaminobutyric acid acetyltransferase, ectB for L-2,4-diaminobutyric acid transaminase and ectC for L-ectoine synthase. A DNA region upstream of ectA was shown to be necessary for the regulated expression of ectoine synthesis in response to the osmolarity of the medium.",
keywords = "Marinococcus halophilus, compatible solutes, ectoine genes, osmoregulation, salt stress, bacillus-subtilis, ectothiorhodospira-halochloris, transport-system, glycine betaine, tetrahydropyrimidines, trehalose, stress, operon, prou, transcription",
author = "Petra Louis and Galinski, {Erwin A.}",
year = "1997",
month = "4",
doi = "10.1099/00221287-143-4-1141",
language = "English",
volume = "143",
pages = "1141--1149",
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TY - JOUR

T1 - Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli

AU - Louis, Petra

AU - Galinski, Erwin A.

PY - 1997/4

Y1 - 1997/4

N2 - The genes of the biosynthetic pathway of ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) from the Gram-positive moderate halophile Marinococcus halophilus were cloned by functional expression in Escherichia coli. These genes were not only expressed, but also osmoregulated in E. coli, as demonstrated by increasing cytoplasmic ectoine concentration in response to medium salinity. Sequencing of a 4 4 kb fragment revealed four major ORFs, which were designated ectA, ectB, ectC and orfA. The significance of three of these genes for ectoine synthesis was proved by sequence comparison with known proteins and by physiological experiments. Several deletion derivatives of the sequenced fragment were introduced into E. coli and the resulting clones were investigated for their ability to synthesize ectoine or one of the intermediates in its biosynthetic pathway. It was demonstrated that ectA codes for L-2,4-diaminobutyric acid acetyltransferase, ectB for L-2,4-diaminobutyric acid transaminase and ectC for L-ectoine synthase. A DNA region upstream of ectA was shown to be necessary for the regulated expression of ectoine synthesis in response to the osmolarity of the medium.

AB - The genes of the biosynthetic pathway of ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) from the Gram-positive moderate halophile Marinococcus halophilus were cloned by functional expression in Escherichia coli. These genes were not only expressed, but also osmoregulated in E. coli, as demonstrated by increasing cytoplasmic ectoine concentration in response to medium salinity. Sequencing of a 4 4 kb fragment revealed four major ORFs, which were designated ectA, ectB, ectC and orfA. The significance of three of these genes for ectoine synthesis was proved by sequence comparison with known proteins and by physiological experiments. Several deletion derivatives of the sequenced fragment were introduced into E. coli and the resulting clones were investigated for their ability to synthesize ectoine or one of the intermediates in its biosynthetic pathway. It was demonstrated that ectA codes for L-2,4-diaminobutyric acid acetyltransferase, ectB for L-2,4-diaminobutyric acid transaminase and ectC for L-ectoine synthase. A DNA region upstream of ectA was shown to be necessary for the regulated expression of ectoine synthesis in response to the osmolarity of the medium.

KW - Marinococcus halophilus

KW - compatible solutes

KW - ectoine genes

KW - osmoregulation

KW - salt stress

KW - bacillus-subtilis

KW - ectothiorhodospira-halochloris

KW - transport-system

KW - glycine betaine

KW - tetrahydropyrimidines

KW - trehalose

KW - stress

KW - operon

KW - prou

KW - transcription

U2 - 10.1099/00221287-143-4-1141

DO - 10.1099/00221287-143-4-1141

M3 - Article

VL - 143

SP - 1141

EP - 1149

JO - Microbiology

JF - Microbiology

SN - 1350-0872

IS - 4

ER -