Two different Escherichia coli proP promoters respond to osmotic and growth phase signals

Mellies, Jay L.; Wise, Arlene A.; Villarejo, Merna

doi:10.1128/jb.177.1.144-151.1995

Cited by 67 publications

(79 citation statements)

References 38 publications

(52 reference statements)

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“…We have demonstrated in this in vitro study the existence of two promoters, P1 and P2, for proU. We have also confirmed the participation of E S in proU transcription, which has been documented for a variety of other osmoresponsive operons in E. coli (17,18,24,35,58). Three effects of potassium glutamate on proU transcription in vitro were identified, namely, (i) direct activation of P1 transcription by E S (and P2 transcription by E 70 ), (ii) inhibition of P1 transcription by E 70 (both together leading to the increase in selectivity for E S at P1), and (iii) antagonism of inhibition of H-NS.…”

supporting

confidence: 65%

Effects of H-NS and potassium glutamate on sigmaS- and sigma70-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli

et al. 1996

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We have used supercoiled DNA templates in this study to demonstrate that transcription in vitro from the P1 and P2 promoters of the osmoresponsive proU operon of Escherichia coli is preferentially mediated by the S -and 70 -bearing RNA polymerase holoenzymes, respectively. Addition of potassium glutamate resulted in the activation of transcription from both P1 and P2 and also led to a pronounced enhancement of S selectivity at the P1 promoter. Transcription from P2, and to a lesser extent from P1, was inhibited by the nucleoid protein H-NS but only in the absence of potassium glutamate. This study validates the existence of dual promoters with dual specificities for proU transcription. Our results also support the proposals that potassium, which is known to accumulate in cells grown at high osmolarity, is at least partially responsible for effecting the in vivo induction of proU transcription and that it does so through two mechanisms, directly by the activation of RNA polymerase and indirectly by the relief of repression imposed by H-NS.The proU operon in Escherichia coli and Salmonella typhimurium encodes a binding-protein-dependent transporter that mediates the osmoprotective effects of exogenous glycine betaine and L-proline when these organisms are grown in media of elevated osmolarity. proU transcription is markedly induced (more than 100-fold) in high-osmolarity media, and the mechanism by which this is brought about has been the subject of intensive, but as yet inconclusive, genetic and biochemical studies (for reviews, see references 6, 16, and 29).With regard to the cis elements mediating proU osmoresponsivity, there is a consensus on the existence of (i) a promoter whose transcription start site is approximately 60 nucleotides upstream of the initiation codon of the first structural gene (proV) (10,15,41,46,55) and (ii) a negative regulatory element (NRE) situated in a region overlapping the proximal (5Ј) end of proV whose deletion leads to a 25-fold derepression of proU expression at low osmolarity (8,13,30,40,42). This promoter is recognized in vitro by the 70 -RNA polymerase holoenzyme (E 70 ) (10, 55). Our group has also identified, by in vivo studies, another promoter located 250 nucleotides upstream of proV in E. coli (8,15) and has shown recently that this promoter is both RpoS ( S ) dependent and stationary-phase inducible (31). We have designated the two (proV-proximal and proV-distal) promoters P2 and P1, respectively. The role of the upstream P1 promoter and S -RNA polymerase (E S ) in proU regulation is still uncertain, however, for the following reasons: (i) proU expression in vivo is not affected by deletion of P1 or by mutations in rpoS (28, 31), whereas a mutation in rpoD (encoding 70 ) results in nearly complete abolition of proU expression (58); (ii) proU expression in vivo is not significantly induced in stationaryphase cultures (31); (iii) Overdier et al. (41) have failed to identify an equivalent promoter during subcloning experiments with S. typhimurium proU; and (iv) in vi...

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confidence: 65%

Effects of H-NS and potassium glutamate on sigmaS- and sigma70-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli

et al. 1996

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“…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. Instead, we found a sequence similar to the consensus for oB of Bacillus subtilis (Fig.…”

Section: Ectoine Synthesizing Capacity Of Deletion Derivatives Of Posmllmentioning

confidence: 99%

“…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 the molecular level have so far focused on the various solute uptake systems of E. coli (Altendorf & Epstein, 1993;Mellies et al, 1995;Gowrishankar & Manna, 1996). The only investigations concerned with the biosynthesis of compatible solutes covered choline oxidation and trehalose synthesis (Lamark et al, 1996;Strarm & Kaasen, 1993).…”

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confidence: 99%

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

1997

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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...

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“…1A). Transcription from the P2 promoter is dependent on s and Fis (32,47). During late exponential phase, s levels are increasing while Fis levels are decreasing (1,21,27,36).…”

mentioning

confidence: 99%

“…The ProP transporter appears to function as a (compatible solute ϩ H ϩ )/K ϩ antiporter (29). ProP activity can be rapidly stimulated by osmotic shock (17,18,30) by enhancing transcription (13,22,32,35) or by posttranslational mechanisms (34).…”

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confidence: 99%

Cyclic AMP receptor protein functions as a repressor of the osmotically inducible promoter proP P1 in Escherichia coli

Johnson

1997

J Bacteriol

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Transcription of the proP gene, encoding a transporter of the osmoprotectants proline and glycine betaine, is controlled from two promoters, P1 and P2, that respond primarily to osmotic and stationary-phase signals, respectively. The P1 promoter is normally expressed at a very low level under low or normal medium osmolarity. We demonstrate that the binding of the cyclic AMP (cAMP) receptor protein (CRP) to a site centered at ؊34.5 within the promoter is responsible for the low promoter activity under these conditions. A brief period of reduced CRP binding in early log phase corresponds to a transient burst of P1 transcription upon resumption of growth in Luria-Bertani broth. A CRP binding-site mutation or the absence of a functional crp gene leads to high constitutive expression of P1. We show that the binding of CRP-cAMP inhibits transcription by purified RNA polymerase in vitro at P1, but this repression is relieved at moderately high potassium glutamate concentrations. Likewise, open-complex formation at P1 in vivo is inhibited by the presence of CRP under low-osmolarity conditions. Because P1 expression can be further induced by osmotic upshifts in a ⌬crp strain or in the presence of the CRP binding-site mutation, additional controls exist to osmotically regulate P1 expression.

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Two different Escherichia coli proP promoters respond to osmotic and growth phase signals

Cited by 67 publications

References 38 publications

Effects of H-NS and potassium glutamate on sigmaS- and sigma70-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli

Effects of H-NS and potassium glutamate on sigmaS- and sigma70-directed transcription in vitro from osmotically regulated P1 and P2 promoters of proU in Escherichia coli

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

Cyclic AMP receptor protein functions as a repressor of the osmotically inducible promoter proP P1 in Escherichia coli

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