OpuA, an Osmotically Regulated Binding Protein-dependent Transport System for the Osmoprotectant Glycine Betaine in Bacillus subtilis

Kempf, Bettina; Bremer, Erhard

doi:10.1074/jbc.270.28.16701

Cited by 175 publications

(241 citation statements)

References 59 publications

Supporting

Mentioning

230

Contrasting

Order By: Relevance

“…The transcription initiation site of the gbsA gene was determined by primer extension analysis with a synthetic primer (5Ј-TGCAATTTCTTCTTGATTAA ATGGG-3Ј) complementary to the gbsA mRNA (bp 1358 to 1334 [see Fig. 3]) and avian myeloblastosis virus reverse transcriptase as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes

et al. 1996

View full text Add to dashboard Cite

Synthesis of the osmoprotectant glycine betaine from the exogenously provided precursor choline or glycine betaine aldehyde confers considerable osmotic stress tolerance to Bacillus subtilis in high-osmolarity media. Using an Escherichia coli mutant (betBA) defective in the glycine betaine synthesis enzymes, we cloned by functional complementation the genes that are required for the synthesis of the osmoprotectant glycine betaine in B. subtilis. The DNA sequence of a 4.1-kb segment from the cloned chromosomal B. subtilis DNA was established, and two genes (gbsA and gbsB) whose products were essential for glycine betaine biosynthesis and osmoprotection were identified. The gbsA and gbsB genes are transcribed in the same direction, are separated by a short intergenic region, and are likely to form an operon. The deduced gbsA gene product exhibits strong sequence identity with members of a superfamily of specialized and nonspecialized aldehyde dehydrogenases. This superfamily comprises glycine betaine aldehyde dehydrogenases from bacteria and plants with known involvement in the cellular adaptation to high-osmolarity stress and drought. The deduced gbsB gene product shows significant similarity to the family of type III alcohol dehydrogenases. B. subtilis mutants with defects in the chromosomal gbsAB genes were constructed by marker replacement, and the growth properties of these mutant strains in high-osmolarity medium were analyzed. Deletion of the gbsAB genes destroyed the cholineglycine betaine synthesis pathway and abolished the ability of B. subtilis to deal effectively with high-osmolarity stress in choline-or glycine betaine aldehyde-containing medium. Uptake of radiolabelled choline was unaltered in the gbsAB mutant strain. The continued intracellular accumulation of choline or glycine betaine aldehyde in a strain lacking the glycine betaine-biosynthetic enzymes strongly interfered with the growth of B. subtilis, even in medium of moderate osmolarity. A single transcription initiation site for gbsAB was detected by high-resolution primer extension analysis. gbsAB transcription was initiated from a promoter with close homology to A -dependent promoters and was stimulated by the presence of choline in the growth medium.

show abstract

Section: Methodsmentioning

confidence: 99%

“…Glycine betaine can be accumulated directly from the environment through three osmotically controlled uptake systems (25): OpuD, a single-component transporter, and OpuA and OpuC (ProU), multicomponent transport systems that are related to the binding protein-dependent transport system ProU from Escherichia coli (25,26,34).…”

mentioning

confidence: 99%

Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes

et al. 1996

View full text Add to dashboard Cite

show abstract

“…3). A search for consensus sequences of the osmoresponsive promoters for the compatible solute transport systems proU, prop and o p u A (Mellies et al, 1994(Mellies et al, ,1995Kempf & Bremer, 1995) revealed no matches. As several osmoregulated genes in non-halophiles, including the genes for the biosynthesis of trehalose, are known to be under the control of as-dependent promoters (Strram & Kaasen, 1993;Gordia & Gutierrez, 1996;Manna & Gowrishankar, 1994;Mellies et al, 1995), we also conducted a search for consensus sequences of these (Strram & Kaasen, 1993), but were unable to find any matches.…”

Section: Ectoine Synthesizing Capacity Of Deletion Derivatives Of Posmllmentioning

confidence: 99%

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

1997

View full text Add to dashboard Cite

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, ectf?, ectC and odA. 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 ~-2,4-diaminobutyric acid acetyltransferase, ectf? for ~-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. INTROD JCTIONKeywords : Marinococcus halophilus, compatible solutes, ectoine genes, osmoregulation, salt stress Saline environments are characterized by high osmotic strength (low water potential). Most halophilic eubacteria cope with these conditions by accumulating small, highly water-soluble organic compounds, the so-called compatible solutes (Brown, 1976). These osmolytes enable organisms to adapt to a wide range of salt concentrations by adjusting the cytoplasmic solute pool to the osmolarity of the surrounding environment. Ectoines represent the predominant class of osmolytes in aerobic chemoheterotrophic eubacteria (Severin et al., 1992;Frings et al., 1993;Galinski, 1995). The biosynthetic pathway for ectoine has been elucidated at the enzymological level in Gram-negative eubacterial halophiles (Peters et al., 1990;Tao et al., 1992;Galinski & Truper, 1994). It comprises three steps, the first being the conversion of aspartate semialdehyde, an intermediate in amino acid metabolism, to ~-2,4-diaminobutyric acid. This is followed by acetylation to NYThe GenBank accession number for the sequence reported in this paper is U66614 acetyldiaminobutyric acid. The last step consists of a cyclic condensation reaction to form the tetrahydropyrimidine ectoine (Fig. 1).Expression and regulation of genes involved in osmoadaptation has thus far been investigated almost exclusively in non-halophilic bacteria, especially Escherichia coli (for recent reviews see Csonka & Hanson, 1991; Lucht & Bremer, 1994;Galinski, 1995). This organism responds to increased salinity with the rapid accumulation of potassium and concomitant synthesis of glutamate as a counter anion. Subsequently, these charged solutes are partially replaced by endogenous trehalose or compatible solutes accumulated from the medium, if present (Dinnbier et al., 1988). Most studies at...

show abstract

“…Comparisons with sequences deposited in GenBank indicated that clone pUC-AS17 contained about one-third of the 5' end of the f i ORF at the erm-proximal end of Tn917, and, at the em-distal end of the transposon, the complete ylxM gene. Upstream from ylxM, sequences were found that were homologous to the proU/opuA operon of B. subtilis (Kempf & Bremer, 1995), specifically to the 3' half of the proV/opuAA gene and to unlinked segments of the complete downstream proW/opuAB and proX/opuAC genes. The proU/opuA operon encodes an osmoregulated ABC-dependent transport system involved in the uptake of compatible solutes including glycine betaine.…”

Section: Cloning and Sequence Analysis Of The Sat Locusmentioning

confidence: 99%

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress

et al. 1999

View full text Add to dashboard Cite

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, t o tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of 5. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS1 7, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology t o the y/xM-ffh operon of Bacillus subtik The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an 5. mutans genomic library. Sequencing of the complementing clone identified the intact yIxM and #%i genes as well as a partial ORF with homology t o the proU/opuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat=l::Tn917 mutation on ffh expression. Upon acid shock (pH 5)# wild-type ffh mRNA levels were found t o increase approximately four-to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptiveholerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acidAshocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable t o ferment sorbitol although it was able t o grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans t o changing environmental conditions. Abbreviations: ATR, acid tolerance response; GSP, general secretory pathway; SRP, signal recognition particle. KeywordsThe CenBank accession number for the DNA sequence of the 3 kb DNA fragment containing the y/xM-ffh genes is U88582. INTRODUCTIONStreptococcus mutans plays a major role in the aetiology of human dental caries. This bacterium is able to demineralize dental enamel by generation of a localized acidic environment in plaque by rapid glycolysis of dietary sugars to lactic acid. In addition to its acidogenicity, S. mutans is able to tolerate exposure to continual and rapid cycles of acid shock. In vivo pH measurements have demonstrated that the pH of plaque can drop from pH 7.0 to values ranging from 3.0 to 4.0 in less than 20 min following the intake of carbohydrates (Imfeld & Lutz, 1980;Yamada et al., 1980;Mormann & Muhlemann, 1981 ; Schachtele & Harlander, 1984;Jensen & Wefel, 1989). E...

show abstract

OpuA, an Osmotically Regulated Binding Protein-dependent Transport System for the Osmoprotectant Glycine Betaine in Bacillus subtilis

Cited by 175 publications

References 59 publications

Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes

Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes

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

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress

Contact Info

Product

Resources

About